Silvia Musio PhDMassimo Costanza PhDPietro Luigi Poliani MD

PhDElena Fontana PhDManuela Cominelli PhDGabriella Abolafio MScLawrence Steinman MDRosetta Pedotti MD

PhD

Correspondence toDr Pedottirosettapedottiistituto-bestait

Treatment with anti-FceRIa antibodyexacerbates EAE and T-cell immunityagainst myelin

ABSTRACT

Objective To investigate the effects of targeting the high-affinity receptor for immunoglobu-lin E (FceRI) that plays a central role in allergic responses and is constitutively expressedon mast cells and basophils in clinical disease and autoimmune T-cell response in experi-mental MS

Methods Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL6 mice byimmunization with myelin oligodendrocyte glycoprotein 35ndash55 Anti-FceRI a-chain antibodywas administered intraperitoneally CNS immunohistochemistry flow cytometry analysis ofimmune cell populations IgE and histamine serum concentration immune cell proliferation andcytokine measurement were performed In BALBc mice EAE was induced by immunization withmyelin proteolipid protein 185ndash206

Results Treatment with anti-FceRIa antibody resulted in exacerbation of EAE and increasedCNS inflammation in C57BL6 mice Treated mice displayed long-lasting complete depletionof basophils in the blood stream and peripheral lymphoid organs and increased antigen-induced immune cell proliferation and production of interferon-g interleukin (IL)-17 IL-6and granulocyte-macrophage colony-stimulating factor In BALBc mice which are T-helper(Th) 2 prone and resistant to EAE treatment with anti-FceRIa antibody restored susceptibilityto EAE

Conclusion Our observations that anti-FceRIa antibody increases Th1 and Th17 responsesagainst myelin antigen and exacerbates EAE suggest that FceRI basophils and possibly otherFceRI-bearing cells that might be affected by this antibody play important roles in influencing theseverity of CNS autoimmunity Neurol Neuroimmunol Neuroinflamm 20174e342 doi 101212

NXI0000000000000342

GLOSSARYCFA 5 complete Freund adjuvant EAE 5 experimental autoimmune encephalomyelitis FceRI 5 high-affinity receptor forimmunoglobulin E GM-CSF 5 granulocyte-macrophage colony-stimulating factor iDC 5 inflammatory dendritic cell IFN 5interferon IL 5 interleukin LNC 5 lymph node cell MAR-1 5 anti-FceRIa monoclonal antibody MBP 5 myelin basic proteinMC5mast cellMOG5myelin oligodendrocyte glycoprotein PLP5myelin proteolipid protein PTX5 pertussis toxin Th5 Thelper

MS is an autoimmune disease of the CNS leading to demyelination and neurodegeneration1

CD41 T cells reactive against myelin antigens and secreting T-helper (Th) 1 and Th17 cyto-kines are regarded as critical in MS and its mouse model experimental autoimmune encepha-lomyelitis (EAE)2 Several lines of evidence suggest that immune components and mechanismsof Th2 immunity associated with a set of cytokines such as interleukin (IL)-4 IL-5 and IL-13can modulate the autoimmune response in EAE3ndash5 Mast cells (MCs) and basophils are regardedas major effector cells in Th2 immunity and can contribute to the development of allergic

From the Department of Clinical Neuroscience (SM M Costanza RP) Foundation Neurological Institute IRCCS C Besta Milan Departmentof Molecular and Translational Medicine (PLP EF M Cominelli) Pathology Unit University of Brescia Department of ExperimentalOncology and Molecular Medicine (GA) Fondazione IRCCS ldquoIstituto Nazionale dei Tumorirdquo Milan Italy and Department of Neurology andNeurological Sciences (LS) Stanford University School of Medicine CA

Funding information and disclosures are provided at the end of the article Go to Neurologyorgnn for full disclosure forms The Article ProcessingCharge was paid by the Fondazione Italiana Sclerosi Multipla FISMAISM

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CCBY-NC-ND) which permits downloading and sharing the work provided it is properly cited The work cannot be changed in any way or usedcommercially without permission from the journal

Neurologyorgnn Copyright copy 2017 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

disorders clearance of parasites and immunedefense against venoms6 These cells can alsoinfluence immune responses by releasinga plethora of mediators and cytokines7

The high-affinity receptor for immuno-globulin E (FceRI) plays a key role inIgE-mediated allergic reactions and is con-stitutively expressed on the surface of bothMCs and basophils8 Crosslinking of FceRI-bound IgE by antigen induces in these cellsthe release of several cytokines and inflam-matory mediators such as histamine hepa-rin and serotonin8 The expression of FceRIin mice can be induced during certainimmune responses also in a subset of den-dritic cells (inflammatory dendritic cells[iDCs])9 and in eosinophils

The potential role of MCs in CNS auto-immunity has been extensively studiedalthough their exact function in this disorderis still controversial10ndash15 Unlike MCs therole of basophils in MS and EAE has neverbeen directly investigated Basophils are cir-culating granulocytes accounting for less than1 of blood leukocytes On activation thesecells can release several effector moleculessuch as histamine platelet-activating factorleukotrienes and Th2 cytokines IL-4 and IL-1316ndash18 Basophils are an important innatesource of Th2 cytokines required for Th2immunity187 In mouse models of autoim-mune disorders such as lupus nephritis1920

and collagen-induced arthritis basophilshave been suggested to play a detrimentalrole2122 Conversely in a mouse model ofautoimmune chronic colitis basophils ap-peared to exert a beneficial role23

In this study we attempted to evaluate thepotential effects of targeting FceRI in CNSautoimmunity using a monoclonal antibodyagainst the a-chain of FceRI (FceRIa) inchronic EAE and in an EAE model inducedin BALBc mice which are Th2 prone and areresistant against the disease

METHODS Mice peptides and EAE induction C57BL6 (H-2b) and BALBc (H-2d) female mice 8ndash12 weeks old were

purchased from Charles River Laboratories Inc (Calco Italy)

FceRI a-chain 22 mice24 (backcrossed to C57BL6 for more

than 8 generations) were kindly provided by Dr SJ Galli

(Stanford University Stanford CA) All mice were housed and

maintained in specific pathogen-free conditions at the animal

facility of the Foundation IRCCS Neurological Institute

Carlo Besta Myelin oligodendrocyte glycoprotein (MOG) 35ndash

55 (MEVGWYRSPFSRVVHLYRNGK) (synthetized by Dr R

Longhi Consiglio Nazionale delle RicerchemdashCNR Milan

Italy) and myelin proteolipid protein (PLP) 186ndash204

(SIAFPSKTSASIGSLCADARMY) (Caslo ApS Denmark) were

synthesized using standard 9-fluorenylmethoxycarbonyl chemis-

try (Applied Biosystems Foster City CA) and purified as

previously described25 EAE was induced in C57BL6 mice with

100 mg of MOG 35ndash55 peptide in complete Freund adjuvant

(CFA) (Difco BD Bioscience Buccinasco Italy) subcutaneously

and 50 ng of pertussis toxin (PTX List Biological Lab Campbell

CA) intravenously on day 0 and 12 postimmunization as

described25 In BALBc mice EAE was induced with 200 mg of PLP

186ndash204 peptide in CFA containing 10 mgmL of heat-killed

Mycobacterium tuberculosis (Difco) subcutaneously and 500 ng of

PTX intravenously on days 0 and 2 postimmunization26 Mice were

assessed daily for clinical signs of EAE with a 5-point scale25 by an

observer masked to treatment assignment All procedures involving

animals were approved by the Ethical Committee of the Foundation

Neurological Institute Carlo Besta and by the Italian General

Direction for Animal Health at the Ministry of Health Animal

studies were performed in accordance with the institutional guidelines

and national law (DL11692) and carried out according to the

Principles of Laboratory Animal Care (European Communities

Council Directive 201063EU)

Treatments A monoclonal antibody against mouse a-chain of

FceRI (anti-FceRIa clone MAR-1 eBioscience San Diego CA)

or isotype control antibody (clone eBio299Arm eBioscience) was

administered intraperitoneally at a dose of 5mgmouse twice daily for

3 consecutive days from day 27 to day 25 before EAE induction

Flow cytometry Single-cell suspensions were incubated with

blocking monoclonal antibody anti-CD16CD32 (24G2)

(1100) and then stained with APC anti-CD200R3 FITC

anti-CD200R3 PECy7 anti-CD49b PE anti-CD49b PerCP

anti-CD45 PE-Vio770 anti-CD11b FITC anti-CD3 APC-

Vio770 anti-CD4 PE-vio770 anti-CD45B220 PE anti-

CD19 PE anti-CD115 and APC anti-FceRIa (MAR1) (all

from Miltenyi Bergisch Gladbach Germany) Cells were

analyzed by flow cytometry on a FACSCanto (BD Bio-

sciences) and analyses were performed using FlowJo software

(Treestar Inc Ashland OR)

Tissue pathology and immunohistochemistry studiesBrains and spinal cords were removed and fixed in 10 formalin

Paraffin-embedded sections (4 mm) were used for hematoxylin

and eosin staining Sections were blindly and independently

analyzed by 2 pathologists (EF and PLP) The degree of

inflammation was expressed by measuring the percentage of area

infiltrated with immune cells over the total spinal cord section

For immunohistochemistry studies sections were stained with rat

antindashmyelin basic protein (MBP) antibody (150 dilution Mil-

lipore Temecula CA) for myelin rabbit anti-CD3 monoclonal

antibody (SP-7 1100 dilution Thermo Scientific Fremont

CA) for T cells rat anti-B220 antibody (supernatant) for B cells

and rabbit anti-IBA1 antibody (1300 dilution Wako Chemicals

Neuss Germany) for microglia cells and macrophages CD3 and

IBA-1 were revealed with EnVision1System-HRP labeled poly-

mer anti-rabbit (Dako Glostrup Denmark) and B220 and MBP

with rat HRP-Polymer Kit (Biocare Medical) 339-

diaminobenzidine was used as a chromogen according to the

manufacturerrsquos protocol (Dako Glostrup Denmark) Sections

were counterstained with hematoxylin Giemsa staining was

performed for highlighting MCs

2 Neurology Neuroimmunology amp Neuroinflammation

T-cell proliferation Axillary and inguinal lymph node cells

(LNCs) were isolated from mice and cultured in vitro as

described25 in 96-well plates (5 3 105 cellswell) in 200 mL

enRPMI 1640 (EuroClone Pero Milan Italy) supplemented

with 10 fetal bovine serum (Gibco Thermo Fisher Waltham

MA) Cells were stimulated for 72 hours with MOG 35ndash55

Con A (2 mgmL) or medium alone and pulsed for 18 hours

with [3H]-thymidine before harvesting Proliferation was

Figure 1 Treatment with antibody against the a-chain of the high affinity receptor for immunoglobulin E inC57BL6 mice exacerbates experimental autoimmune encephalomyelitis

(A) Clinical experimental autoimmune encephalomyelitis (EAE) scores of myelin oligodendrocyte glycoprotein 35-55ndashimmunized mice treated with antibody against the a-chain of the high affinity receptor for immunoglobulin E (FceRIa)(white squares) or isotype control antibody (black squares) (n 5 26 mice per group) Data (mean 6 SEM) are pooled from 2consecutive experiments that gave similar results p 001 by analysis of variance test (B) Representative hematoxylinand eosin (HampE) staining of spinal cord sections from EAE mice treated with isotype control (Ba and Bc) or anti-FceRIa (Bband Bd) 6 weeks after disease induction Asterisks highlight immune cell infiltration Original magnification 203 (Ba andBb) and 403 (Bc and Bd) Graph (Be) shows comparative analysis of infiltrated area ( of total area) in the spinal cordEach dot represents an individual mouse Mean 6 SEM are also depicted p 005 Student t test 2 tailed (C) Repre-sentative antindashmyelin basic protein staining of spinal cord sections from EAE mice treated with isotype control (Ca) or anti-FceRIa (Cb) 6 weeks after disease induction as in B Asterisks highlight demyelinated areas Magnification 603

Neurology Neuroimmunology amp Neuroinflammation 3

measured from triplicate wells on a beta counter (Perkin-Elmer

Waltham MA)

Cytokine analysis LNCs were cultured in the same condi-

tions as described above25 and cytokines analyzed in cells super-

natants by sandwich ELISA (anti-mouse OptEIA ELISA Set for

interferon [IFN]-g IL-6 IL-10 and IL-4 BD Pharmingen

San Jose CA Mouse IL-17 Duoset and Mouse granulocyte-

macrophage colony-stimulating factor [GM-CSF] Duoset

RampD Systems Minneapolis MN) according to the manu-

facturerrsquos instructions

Serum histamine and IgE measurement Serum samples

were obtained from the tail vein and stored at280degC until tested

Total IgE antibody concentration was measured on serum

samples at a 150 dilution by ELISA (IgE OptEIA kit BD

Pharmingen)25 Histamine concentration was measured with an

enzyme immunoassay kit (ImmunoTech Beckman Coulter

Brea CA) according to the manufacturerrsquos instructions25

Statistical analysis Two-way analysis of variance followed by

the Bonferroni post hoc test was used for comparison of EAE

scores between groups For all other analyses unpaired Student

t test 2 tailed was used Analysis was performed using SPSS

software (IBM SPSS Statistics Armonk NY) In all tests p values005 were considered statistically significant

RESULTS Anti-FceRIa antibody treatment increased

the severity of EAE To evaluate the potential effects oftargeting FceRI in CNS autoimmunity we treatedC57BL6 female mice with a monoclonal antibodyagainst FceRIa (MAR-1) or an isotype control anti-body Treatment (5 mg) was administered to miceintraperitoneally twice daily for 3 consecutive daysOne week after treatment EAE was induced by activeimmunization with MOG 35ndash55 and PTX In 2consecutive experiments treatment with anti-FceRIaantibody resulted in higher incidence of EAE andsignificant exacerbation of disease severity comparedwith isotype controlndashtreated mice (figure 1A andtable) In line with these clinical findings neuro-pathologic analysis of the spinal cord obtained frommice 6 weeks after EAE induction revealed signifi-cantly increased inflammation in the white matter ofanti-FceRIa antibodyndashtreated mice compared withisotype controlndashtreated mice (figure 1B) In bothgroups infiltrating cells were mainly composed ofmacrophages with a minority of cells represented byT cells and very rare B cells (data not shown)Granulocytes were barely detected in both control

and treated mice In Giemsa-stained sections of thespinal cord rare cells with the morphologicalappearance of MCs were observed in perivascularareas in both treatment groups (data not shown) Inparallel with the higher extent of inflammationobserved in anti-FceRIa antibodyndashtreated mice inspinal cord sections of these mice demyelination wasmore prominent compared with that observed insections of isotype controlndashtreated mice (figure 1C)

We then analyzed by flow cytometry the effects ofanti-FceRIa antibody treatment on the frequency ofimmune cell subsets in blood and peripheral lym-phoid organs Because an experimental group wastreated with anti-FceRIa antibody we could notuse the same fluorochrome-labeled monoclonal anti-body clone MAR-1 for the identification of FceRI-expressing cells As previously reported2728 weobserved that anti-FceRIa antibody induced completedepletion of basophils identified as CD200R31CD49b1 CD32 CD45B2202 cells in the bloodlymph node and spleen (figure 2A) In these treatedmice basophils were completely depleted at the timeof EAE induction during priming and acute phase ofEAE and began to reappear in blood circulation 3weeks after immunization (figure 2C) The frequencyof other leukocytes known to play important roles inEAE including T cells B cells and monocytes wasnot significantly modified by the treatment (figure2B) Anti-FceRIa antibody (MAR-1) staining of leu-kocytes disappeared in FceRI a-chain 22 miceconfirming that this antibody binds uniquely to itstarget the a-chain of the receptor (figure 2D)

Anti-FceRIa antibodyndashtreated mice exhibited enhanced

Th1 and Th17 responses against myelin antigen Wenext evaluated whether the exacerbated EAE pheno-type observed in mice treated with anti-FceRIa anti-body was associated with an alteration of T-cellactivation and Th regulation We isolated LNCs fromanti-FceRIa antibodyndashtreated and isotype controlndashtreated mice with EAE during the priming phase ofthe disease (7ndash10 days after immunization) andexamined the recall response to peptide stimulationin vitro LNCs from anti-FceRIa antibodyndashtreatedmice displayed increased proliferation in response toMOG 35ndash55 compared with isotype controlndashtreatedmice (figure 3A) Moreover in LNCs of anti-FceRIaantibodyndashtreated mice the secretion of proin-flammatory cytokines IFN-g IL-17 and IL-6known to play a critical role in EAE was signifi-cantly increased (figure 3A) GM-CSF consideredessential in CNS autoimmune inflammation29 wasalso increased in the supernatant of antigen-stimulated LNCs from anti-FceRIa antibodyndashtreated mice compared to that from isotypecontrolndashtreated mice (figure 3A) We did not observe

Table Experimental autoimmune encephalomyelitis features in C57BL6 micetreated with antibody against the a-chain of the high affinity receptor forimmunoglobulin E

Group Incidence () Onset d Mean maximum score Cumulative disease score

Anti-FceRIa 2626 (100) 101 6 04 33 6 02a 462 6 42a

Isotype 2226 (846) 103 6 04 25 6 03 307 6 42

Data are shown as mean 6 SEM of pooled data from 2 consecutive independent experi-ments each with 13 mice per group that gave similar resultsap 005

4 Neurology Neuroimmunology amp Neuroinflammation

Figure 2 Flow cytometry analysis of basophils and immune cell subsets in C57BL6 mice treated withantibody against the a-chain of the high affinity receptor for immunoglobulin E

(A and B) Lymph nodes spleens and blood were obtained at the time of experimental autoimmune encephalomyelitis (EAE) induc-tion 7 days after treatment with antibody against the a-chain of the high affinity receptor for immunoglobulin E or isotype controlantibody and analyzed by flow cytometry (A) Representative flow cytometry plots of basophils in blood lymph node cells (LNCs)and splenocytes (SPL) of treatedmice Live cellswere gated onCD32 andCD45B220- cells Numbers representmeanpercentageof total leukocytes6 SEM (n55mice per group) from1of3 independent experiments that gave similar results (B) Representativeflow cytometry plots of B cellsmonocytes T cells andCD41 T cells in blood cells of antibody treatedmice Live cellswere gated onexpression of CD45 Data represent mean percentage 6 SEM (n 5 5 mice per group) and are representative of 2 independentexperiments that gave similar results (C) Quantification of basophils in the blood of C57BL6mice treated with anti-FceRIa (whitebars) or isotype control antibody (black bars) beforeEAE induction (day0) and at different timepoints during disease Data representmean6 SEM of 3ndash6 animals per group for each time point and are representative of 2 consecutive experiments that gave similarresults (D) Representative flow cytometry plots of APC-conjugated anti-FceRIa (MAR-1) antibody staining in blood leukocytes ofnaive FceRI a-chain11 and22 mice Data represent mean percentage6 SEM of 3 mice per group and are representative of 2independent experiments Similar datawere obtained by stainingwith APC-MAR-1 antibody LNCs and splenocytes of FceRI a-chain11 and 22 mice p 005 Student t test 2 tailed FSC 5 forward-scattered light SSC 5 side-scattered light

Neurology Neuroimmunology amp Neuroinflammation 5

significant differences in IL-10 production in antigen-stimulated LNCs from anti-FceRIa antibodyndashtreatedmice vs those from isotype controlndashtreated mice(figure 3A) while IL-4 was below the detection limit(data not shown) We next repeated the analysis of therecall response in LNCs from mice 6 weeks after EAEinduction at a time where the frequency of basophils inthe blood of anti-FceRIa antibodyndashtreated mice hadreturned almost to normal values (figure 2C) No dif-ferences were observed at this time point in either pro-liferation (figure 3B) or cytokine production betweenanti-FceRIa antibodyndashtreated mice and isotypecontrolndashtreated mice with the exception of the sup-pressor cytokine IL-10 which was significantly higher inanti-FceRIa antibodyndashtreated mice (figure 3B)

Effects of anti-FceRIa treatment on serum concentration

of histamine and total IgE Histamine has been shownto modulate the immune response in EAE30ndash32 andhistidine decarboxylasendashdeficient mice (22 mice)that are histamine deficient displayed exacerbatedEAE and increased Th1 responses2533 Because ba-sophils which represent an important source of his-tamine were completely depleted by anti-FceRIaantibody in our model we measured the concentra-tion of histamine in mouse sera during EAE Bothanti-FceRIa antibody and isotype controlndashtreatedgroups displayed similar levels of serum histamineconcentrations (figure 3C) Of note histamine levelsdid not change significantly in either group duringthe course of EAE (figure 3C) These findings suggestthat the increased severity of EAE that we observed inanti-FceRIa antibodyndashtreated mice is unlikely to berelated with an effect of the treatment on blood his-tamine levels These findings also suggest that thereare substantial sources of blood histamine in thesemice in addition to basophils

Basophils are a predominant source of IL-4 inallergen and parasite-activated blood leukocytes34

This cytokine induces in B-cell antibody class switchtoward IgE and IgG1 We previously reported thatIgE increases in mouse serum during EAE25 althoughthe biological significance of the increase of antibod-ies of this class is yet to be defined We thereforemeasured IgE concentrations in serum and found thatIgE concentrations were significantly decreased inanti-FceRIa antibodyndashtreated mice during thepriming phase of EAE (day 7) compared with isotypecontrolndashtreated mice These differences in IgE con-centrations disappeared 6 weeks after EAE inductionat the time when basophil frequency had returned toalmost normal levels (figure 3D)

Treatment with anti-FceRIa induced susceptibility to

EAE in resistant BALBc mice The results reportedabove show that anti-FceRIa antibody treatmentboth exacerbated EAE in C57BL6 mice and depleted

basophils suggesting that depletion of these cells andperhaps additional effects of anti-FceRIa antibodytreatment contributed to increase the severity ofEAE in this model To further explore this hypothe-sis we used anti-FceRIa antibody in another modelof EAE obtained in BALBc mice which are Th2prone and resistant against EAE35 We attempted toinduce EAE in this strain by immunization with theself-antigen PLP 186ndash204 in CFA and PTX26 Saline-treated mice were completely resistant to EAE (none of5 mice immunized developed the disease) and amongisotype controlndashtreated mice only 2 of 5 (40)displayed minimal signs of EAE (score 1 5 tail paral-ysis) that lasted only 1 day (figure 4A) Conversely themajority of BALBc mice (4 of 5 80) treated withanti-FceRIa antibody developed EAE (figure 4A) withmaximum scores of 1 2 (hind-limb paralysis) and 5(death) Similar to what we observed in C57BL6 miceflow cytometry analysis conducted on blood cells ofanti-FceRIa antibody and isotype controlndashtreatedBALBc mice revealed that this treatment inducedcomplete depletion of basophils (figure 4B)

DISCUSSION Th1 and Th17 cells play key roles inthe pathogenesis of EAE and MS and it is widelyaccepted that Th2 responses hinder the developmentand pathobiology of these diseases However emerg-ing evidence suggests that in addition to Th2 polar-ized cells that counteract Th1 and Th17 responsesalso effector cells and mediators of the Th2 immunitycan limit the severity of EAE For example we andothers have shown that EAE is more severe in theabsence of histamine2533 and similar findings wereobserved in MCndashdeficient c-kit mutant mice undercertain experimental conditions1415 In this report tofurther test the hypothesis that mechanisms of theTh2 immunity can influence the development inEAE we targeted with a monoclonal antibody FceRIthat plays a crucial role in IgE-mediated allergic re-actions and is constitutively expressed on MCs andbasophils We found that treatment with this anti-body before the induction of the disease exacerbatedMOG 35ndash55 EAE in C57BL6 mice and increasedCNS neuroinflammation Moreover anti-FceRIaantibody increased Th1 and Th17 responses againstmyelin antigen Last treatment induced susceptibilityto EAE in BALBc mice which are Th2 prone andresistant to the development of EAE

The mechanisms involved in disease exacerbationcaused by anti-FceRIa antibody treatment are yet tobe fully understood Complete depletion of basophilsandor alteration of the frequency and function ofother FceRI-bearing cells associated with the treat-ment can be postulated as one potential mechanismIndeed anti-FceRIa antibody has been previouslyshown to deplete basophils in vivo and to reduce

6 Neurology Neuroimmunology amp Neuroinflammation

Figure 3 Effects of antibody against the a-chain of the high affinity receptor for immunoglobulin E treatment onantigen-induced T-cell response and on serum concentration of histamine and IgE antibody

(A and B) Draining lymph nodes were obtained from antibody against the a-chain of the high affinity receptor for immunoglobulin E(FceRIa) or isotype control antibodyndashtreated mice (n5 5mice per group) 7 days (A) and 42 days (B) after experimental autoimmuneencephalomyelitis (EAE) induction Proliferation (mean 6 SEM from triplicate wells) and cytokine production (mean 6 SEM fromduplicate wells) were analyzed in lymph node cells stimulated in vitro with serial concentrations of myelin oligodendrocyte glyco-protein (MOG) 35ndash55 or medium alone Data are representative of 2 independent experiments that gave similar results (C and D)serumsampleswere obtained frommice treatedwith anti-FceRIa (white bars) or isotype control antibody (black bars) 7 and 42daysafter the induction of EAE Histaminewasmeasured by enzyme immunoassay in duplicatedwells (n5 3 to 6mice per group in eachtime point) (C) and total IgE was measured by ELISA in duplicated wells (n 5 8 to 16 mice per group in each time point) (D) Datarepresent mean6 SEM from 2 different experiments PI 5 postimmunization p 005 p 001 p 0005 by Studentt test 2 tailed GM-CSF 5 granulocyte-macrophage colony-stimulating factor IFN 5 interferon IL 5 interleukin

Neurology Neuroimmunology amp Neuroinflammation 7

the frequency of peritoneal MCs27 In addition to thepotential effects of anti-FceRIa antibody on baso-phils and MCs this antibody can also deplete a pop-ulation of dendritic cells namely iDC that canexpress FceRI after immunization with an allergen9

In this regard in the model of CNS autoimmunitythat we used we failed to detect by flow cytometrysuch immune cells (non-T and non-B cells expressingFceRI and lacking CD49b) in lymph nodes spleensor blood of mice during the course of EAE (data notshown) It is possible that the generation of these cellscritically depends on the type of antigen andor immu-nization protocol used and that such FceRI 1 iDCsmight play a marginal or no role in the development ofEAE Thus it is unlikely that depletion of iDCs con-tributed to the results that we observed

We found that anti-FceRIa antibodyndashtreatedmice exhibited enhanced recall responses againstMOG 35ndash55 antigen in their LNCs compared withisotype controlndashtreated mice with increased T-cellproliferation and secretion of Th1 and Th17 cyto-kines such as IFN-g IL-17 IL-6 and GM-CSFThese cytokines are known to play important rolesin the development of EAE1229 Thus immune mod-ulation might represent an important mechanism bywhich anti-FceRIa antibody treatment induces EAEexacerbation In line with our results in anothermodel of T-cell autoimmunity Gomez et al haveshown that depletion of basophils by monoclonalantibodies in a mouse model of autoimmune colitisresulted in enhanced production of Th1 and Th17cytokines and exacerbated colitis23

Among the mechanisms potentially contributingto disease exacerbation observed in anti-FceRIaantibodyndashtreated mice depletion of basophils andpossibly MCs as important sources of mediators andcytokines that can counteract Th1Th17 autoim-munity can be postulated For example IL-4 of whichthese cells represent important sources plays a key rolein counteracting CNS autoimmunity and limits theseverity of EAE2 Of interest in anti-FceRIaantibodyndashtreated mice serum concentration of IgEwhose production depends on IL-4 was significantlylower during the priming phase of EAE at a time whenbasophils were completely depleted This differencedisappeared at a later time point when basophil fre-quency had returned to normal level

Among other mediators secreted by basophilsand MCs that may potentially induce immunemodulation histamine which is synthetized andsecreted by these cells in high amounts is also con-sidered to play a role in limiting the severity of EAEHowever we did not find significant differences inhistamine serum concentrations between mice trea-ted with anti-FceRIa antibody and those treatedwith isotype control antibody and thus it is

unlikely that the effects of this treatment are relatedto histamine

Although it is generally accepted that immunedeviation toward Th2 protects against EAE Lafailleand colleagues reported that transfer of Th2 lympho-cytes specific for MBP failed to protect against Th1cellndashmediated EAE Moreover these Th2 lympho-cytes can cause themselves EAE when transferred intoimmune-deficient RAG-1 knockout recipient micethat lack mature T and B cells3 An unusually highnumber of polymorphonuclear cells and MCs wereobserved in CNS inflammatory infiltrates of Th2cellndashinduced EAE3 In our study we have used anactive EAE model obtained in immune competentmice to evaluate the potential effects of targetingFceRI constitutively expressed on mouse MCs andbasophils We show that treatment with anti-FceRIaantibody induced an exacerbation of Th1 and Th17responses with mechanisms likely involving immunecells such as basophils and MCs both importantsources of IL-4 and other key cytokines and involvedin Th2 immunity Understanding how Th2 lympho-cytes and effector cells of Th2 immunity such asbasophils and MCs intervene in the complex patho-biology of CNS autoimmunity remains an open fieldof investigation

Of interest in the context of CNS autoimmunityit has been recently reported that subacute infectionwith intestinal parasites is associated with reduced dis-ease flares in relapsing-remitting patients with MSwith reduced disability progression and fewer MRIchanges compared with uninfected patients withMS3637 Although an increased production of IL-10from B cells and an induction of regulatory T cellswere observed in these parasite-infected patients withMS it is also possible that activation of basophilsoccurring during helminth infection could haveinduced beneficial effects and reduced encephalito-genic responses in these patients Of note increasingevidence suggests that basophil activation can con-tribute to the induction or intensity of Th2 re-sponses38 Such effects might be beneficial in thecontext of EAE and MS

The findings that we report here are associatedwith the effects of a treatment with a monoclonalantibody We confirmed that MAR-1 antibody bindsuniquely to its target the a-chain of FceRI (figure2D) and that it completely depletes basophils how-ever this antibody may also reduce the numbers andor affect the functions of other FceRI-bearing cellsAlternatively it is possible that MAR-1 antibodyresults in some activation of basophils (andor otherFceRI-bearing cells) at the time of treatmentadministration as well as depleting this populationIn that case such MAR-1ndashdependent cell activation(instead of or in addition to MAR-1ndashdependent cell

8 Neurology Neuroimmunology amp Neuroinflammation

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

T-cell proliferation Axillary and inguinal lymph node cells

(LNCs) were isolated from mice and cultured in vitro as

described25 in 96-well plates (5 3 105 cellswell) in 200 mL

enRPMI 1640 (EuroClone Pero Milan Italy) supplemented

with 10 fetal bovine serum (Gibco Thermo Fisher Waltham

MA) Cells were stimulated for 72 hours with MOG 35ndash55

Con A (2 mgmL) or medium alone and pulsed for 18 hours

with [3H]-thymidine before harvesting Proliferation was

Figure 1 Treatment with antibody against the a-chain of the high affinity receptor for immunoglobulin E inC57BL6 mice exacerbates experimental autoimmune encephalomyelitis

(A) Clinical experimental autoimmune encephalomyelitis (EAE) scores of myelin oligodendrocyte glycoprotein 35-55ndashimmunized mice treated with antibody against the a-chain of the high affinity receptor for immunoglobulin E (FceRIa)(white squares) or isotype control antibody (black squares) (n 5 26 mice per group) Data (mean 6 SEM) are pooled from 2consecutive experiments that gave similar results p 001 by analysis of variance test (B) Representative hematoxylinand eosin (HampE) staining of spinal cord sections from EAE mice treated with isotype control (Ba and Bc) or anti-FceRIa (Bband Bd) 6 weeks after disease induction Asterisks highlight immune cell infiltration Original magnification 203 (Ba andBb) and 403 (Bc and Bd) Graph (Be) shows comparative analysis of infiltrated area ( of total area) in the spinal cordEach dot represents an individual mouse Mean 6 SEM are also depicted p 005 Student t test 2 tailed (C) Repre-sentative antindashmyelin basic protein staining of spinal cord sections from EAE mice treated with isotype control (Ca) or anti-FceRIa (Cb) 6 weeks after disease induction as in B Asterisks highlight demyelinated areas Magnification 603

Neurology Neuroimmunology amp Neuroinflammation 3

measured from triplicate wells on a beta counter (Perkin-Elmer

Waltham MA)

Cytokine analysis LNCs were cultured in the same condi-

tions as described above25 and cytokines analyzed in cells super-

natants by sandwich ELISA (anti-mouse OptEIA ELISA Set for

interferon [IFN]-g IL-6 IL-10 and IL-4 BD Pharmingen

San Jose CA Mouse IL-17 Duoset and Mouse granulocyte-

macrophage colony-stimulating factor [GM-CSF] Duoset

RampD Systems Minneapolis MN) according to the manu-

facturerrsquos instructions

Serum histamine and IgE measurement Serum samples

were obtained from the tail vein and stored at280degC until tested

Total IgE antibody concentration was measured on serum

samples at a 150 dilution by ELISA (IgE OptEIA kit BD

Pharmingen)25 Histamine concentration was measured with an

enzyme immunoassay kit (ImmunoTech Beckman Coulter

Brea CA) according to the manufacturerrsquos instructions25

Statistical analysis Two-way analysis of variance followed by

the Bonferroni post hoc test was used for comparison of EAE

scores between groups For all other analyses unpaired Student

t test 2 tailed was used Analysis was performed using SPSS

software (IBM SPSS Statistics Armonk NY) In all tests p values005 were considered statistically significant

RESULTS Anti-FceRIa antibody treatment increased

the severity of EAE To evaluate the potential effects oftargeting FceRI in CNS autoimmunity we treatedC57BL6 female mice with a monoclonal antibodyagainst FceRIa (MAR-1) or an isotype control anti-body Treatment (5 mg) was administered to miceintraperitoneally twice daily for 3 consecutive daysOne week after treatment EAE was induced by activeimmunization with MOG 35ndash55 and PTX In 2consecutive experiments treatment with anti-FceRIaantibody resulted in higher incidence of EAE andsignificant exacerbation of disease severity comparedwith isotype controlndashtreated mice (figure 1A andtable) In line with these clinical findings neuro-pathologic analysis of the spinal cord obtained frommice 6 weeks after EAE induction revealed signifi-cantly increased inflammation in the white matter ofanti-FceRIa antibodyndashtreated mice compared withisotype controlndashtreated mice (figure 1B) In bothgroups infiltrating cells were mainly composed ofmacrophages with a minority of cells represented byT cells and very rare B cells (data not shown)Granulocytes were barely detected in both control

and treated mice In Giemsa-stained sections of thespinal cord rare cells with the morphologicalappearance of MCs were observed in perivascularareas in both treatment groups (data not shown) Inparallel with the higher extent of inflammationobserved in anti-FceRIa antibodyndashtreated mice inspinal cord sections of these mice demyelination wasmore prominent compared with that observed insections of isotype controlndashtreated mice (figure 1C)

We then analyzed by flow cytometry the effects ofanti-FceRIa antibody treatment on the frequency ofimmune cell subsets in blood and peripheral lym-phoid organs Because an experimental group wastreated with anti-FceRIa antibody we could notuse the same fluorochrome-labeled monoclonal anti-body clone MAR-1 for the identification of FceRI-expressing cells As previously reported2728 weobserved that anti-FceRIa antibody induced completedepletion of basophils identified as CD200R31CD49b1 CD32 CD45B2202 cells in the bloodlymph node and spleen (figure 2A) In these treatedmice basophils were completely depleted at the timeof EAE induction during priming and acute phase ofEAE and began to reappear in blood circulation 3weeks after immunization (figure 2C) The frequencyof other leukocytes known to play important roles inEAE including T cells B cells and monocytes wasnot significantly modified by the treatment (figure2B) Anti-FceRIa antibody (MAR-1) staining of leu-kocytes disappeared in FceRI a-chain 22 miceconfirming that this antibody binds uniquely to itstarget the a-chain of the receptor (figure 2D)

Anti-FceRIa antibodyndashtreated mice exhibited enhanced

Th1 and Th17 responses against myelin antigen Wenext evaluated whether the exacerbated EAE pheno-type observed in mice treated with anti-FceRIa anti-body was associated with an alteration of T-cellactivation and Th regulation We isolated LNCs fromanti-FceRIa antibodyndashtreated and isotype controlndashtreated mice with EAE during the priming phase ofthe disease (7ndash10 days after immunization) andexamined the recall response to peptide stimulationin vitro LNCs from anti-FceRIa antibodyndashtreatedmice displayed increased proliferation in response toMOG 35ndash55 compared with isotype controlndashtreatedmice (figure 3A) Moreover in LNCs of anti-FceRIaantibodyndashtreated mice the secretion of proin-flammatory cytokines IFN-g IL-17 and IL-6known to play a critical role in EAE was signifi-cantly increased (figure 3A) GM-CSF consideredessential in CNS autoimmune inflammation29 wasalso increased in the supernatant of antigen-stimulated LNCs from anti-FceRIa antibodyndashtreated mice compared to that from isotypecontrolndashtreated mice (figure 3A) We did not observe

Table Experimental autoimmune encephalomyelitis features in C57BL6 micetreated with antibody against the a-chain of the high affinity receptor forimmunoglobulin E

Group Incidence () Onset d Mean maximum score Cumulative disease score

Anti-FceRIa 2626 (100) 101 6 04 33 6 02a 462 6 42a

Isotype 2226 (846) 103 6 04 25 6 03 307 6 42

Data are shown as mean 6 SEM of pooled data from 2 consecutive independent experi-ments each with 13 mice per group that gave similar resultsap 005

4 Neurology Neuroimmunology amp Neuroinflammation

Figure 2 Flow cytometry analysis of basophils and immune cell subsets in C57BL6 mice treated withantibody against the a-chain of the high affinity receptor for immunoglobulin E

(A and B) Lymph nodes spleens and blood were obtained at the time of experimental autoimmune encephalomyelitis (EAE) induc-tion 7 days after treatment with antibody against the a-chain of the high affinity receptor for immunoglobulin E or isotype controlantibody and analyzed by flow cytometry (A) Representative flow cytometry plots of basophils in blood lymph node cells (LNCs)and splenocytes (SPL) of treatedmice Live cellswere gated onCD32 andCD45B220- cells Numbers representmeanpercentageof total leukocytes6 SEM (n55mice per group) from1of3 independent experiments that gave similar results (B) Representativeflow cytometry plots of B cellsmonocytes T cells andCD41 T cells in blood cells of antibody treatedmice Live cellswere gated onexpression of CD45 Data represent mean percentage 6 SEM (n 5 5 mice per group) and are representative of 2 independentexperiments that gave similar results (C) Quantification of basophils in the blood of C57BL6mice treated with anti-FceRIa (whitebars) or isotype control antibody (black bars) beforeEAE induction (day0) and at different timepoints during disease Data representmean6 SEM of 3ndash6 animals per group for each time point and are representative of 2 consecutive experiments that gave similarresults (D) Representative flow cytometry plots of APC-conjugated anti-FceRIa (MAR-1) antibody staining in blood leukocytes ofnaive FceRI a-chain11 and22 mice Data represent mean percentage6 SEM of 3 mice per group and are representative of 2independent experiments Similar datawere obtained by stainingwith APC-MAR-1 antibody LNCs and splenocytes of FceRI a-chain11 and 22 mice p 005 Student t test 2 tailed FSC 5 forward-scattered light SSC 5 side-scattered light

Neurology Neuroimmunology amp Neuroinflammation 5

significant differences in IL-10 production in antigen-stimulated LNCs from anti-FceRIa antibodyndashtreatedmice vs those from isotype controlndashtreated mice(figure 3A) while IL-4 was below the detection limit(data not shown) We next repeated the analysis of therecall response in LNCs from mice 6 weeks after EAEinduction at a time where the frequency of basophils inthe blood of anti-FceRIa antibodyndashtreated mice hadreturned almost to normal values (figure 2C) No dif-ferences were observed at this time point in either pro-liferation (figure 3B) or cytokine production betweenanti-FceRIa antibodyndashtreated mice and isotypecontrolndashtreated mice with the exception of the sup-pressor cytokine IL-10 which was significantly higher inanti-FceRIa antibodyndashtreated mice (figure 3B)

Effects of anti-FceRIa treatment on serum concentration

of histamine and total IgE Histamine has been shownto modulate the immune response in EAE30ndash32 andhistidine decarboxylasendashdeficient mice (22 mice)that are histamine deficient displayed exacerbatedEAE and increased Th1 responses2533 Because ba-sophils which represent an important source of his-tamine were completely depleted by anti-FceRIaantibody in our model we measured the concentra-tion of histamine in mouse sera during EAE Bothanti-FceRIa antibody and isotype controlndashtreatedgroups displayed similar levels of serum histamineconcentrations (figure 3C) Of note histamine levelsdid not change significantly in either group duringthe course of EAE (figure 3C) These findings suggestthat the increased severity of EAE that we observed inanti-FceRIa antibodyndashtreated mice is unlikely to berelated with an effect of the treatment on blood his-tamine levels These findings also suggest that thereare substantial sources of blood histamine in thesemice in addition to basophils

Basophils are a predominant source of IL-4 inallergen and parasite-activated blood leukocytes34

This cytokine induces in B-cell antibody class switchtoward IgE and IgG1 We previously reported thatIgE increases in mouse serum during EAE25 althoughthe biological significance of the increase of antibod-ies of this class is yet to be defined We thereforemeasured IgE concentrations in serum and found thatIgE concentrations were significantly decreased inanti-FceRIa antibodyndashtreated mice during thepriming phase of EAE (day 7) compared with isotypecontrolndashtreated mice These differences in IgE con-centrations disappeared 6 weeks after EAE inductionat the time when basophil frequency had returned toalmost normal levels (figure 3D)

Treatment with anti-FceRIa induced susceptibility to

EAE in resistant BALBc mice The results reportedabove show that anti-FceRIa antibody treatmentboth exacerbated EAE in C57BL6 mice and depleted

basophils suggesting that depletion of these cells andperhaps additional effects of anti-FceRIa antibodytreatment contributed to increase the severity ofEAE in this model To further explore this hypothe-sis we used anti-FceRIa antibody in another modelof EAE obtained in BALBc mice which are Th2prone and resistant against EAE35 We attempted toinduce EAE in this strain by immunization with theself-antigen PLP 186ndash204 in CFA and PTX26 Saline-treated mice were completely resistant to EAE (none of5 mice immunized developed the disease) and amongisotype controlndashtreated mice only 2 of 5 (40)displayed minimal signs of EAE (score 1 5 tail paral-ysis) that lasted only 1 day (figure 4A) Conversely themajority of BALBc mice (4 of 5 80) treated withanti-FceRIa antibody developed EAE (figure 4A) withmaximum scores of 1 2 (hind-limb paralysis) and 5(death) Similar to what we observed in C57BL6 miceflow cytometry analysis conducted on blood cells ofanti-FceRIa antibody and isotype controlndashtreatedBALBc mice revealed that this treatment inducedcomplete depletion of basophils (figure 4B)

DISCUSSION Th1 and Th17 cells play key roles inthe pathogenesis of EAE and MS and it is widelyaccepted that Th2 responses hinder the developmentand pathobiology of these diseases However emerg-ing evidence suggests that in addition to Th2 polar-ized cells that counteract Th1 and Th17 responsesalso effector cells and mediators of the Th2 immunitycan limit the severity of EAE For example we andothers have shown that EAE is more severe in theabsence of histamine2533 and similar findings wereobserved in MCndashdeficient c-kit mutant mice undercertain experimental conditions1415 In this report tofurther test the hypothesis that mechanisms of theTh2 immunity can influence the development inEAE we targeted with a monoclonal antibody FceRIthat plays a crucial role in IgE-mediated allergic re-actions and is constitutively expressed on MCs andbasophils We found that treatment with this anti-body before the induction of the disease exacerbatedMOG 35ndash55 EAE in C57BL6 mice and increasedCNS neuroinflammation Moreover anti-FceRIaantibody increased Th1 and Th17 responses againstmyelin antigen Last treatment induced susceptibilityto EAE in BALBc mice which are Th2 prone andresistant to the development of EAE

The mechanisms involved in disease exacerbationcaused by anti-FceRIa antibody treatment are yet tobe fully understood Complete depletion of basophilsandor alteration of the frequency and function ofother FceRI-bearing cells associated with the treat-ment can be postulated as one potential mechanismIndeed anti-FceRIa antibody has been previouslyshown to deplete basophils in vivo and to reduce

6 Neurology Neuroimmunology amp Neuroinflammation

Figure 3 Effects of antibody against the a-chain of the high affinity receptor for immunoglobulin E treatment onantigen-induced T-cell response and on serum concentration of histamine and IgE antibody

(A and B) Draining lymph nodes were obtained from antibody against the a-chain of the high affinity receptor for immunoglobulin E(FceRIa) or isotype control antibodyndashtreated mice (n5 5mice per group) 7 days (A) and 42 days (B) after experimental autoimmuneencephalomyelitis (EAE) induction Proliferation (mean 6 SEM from triplicate wells) and cytokine production (mean 6 SEM fromduplicate wells) were analyzed in lymph node cells stimulated in vitro with serial concentrations of myelin oligodendrocyte glyco-protein (MOG) 35ndash55 or medium alone Data are representative of 2 independent experiments that gave similar results (C and D)serumsampleswere obtained frommice treatedwith anti-FceRIa (white bars) or isotype control antibody (black bars) 7 and 42daysafter the induction of EAE Histaminewasmeasured by enzyme immunoassay in duplicatedwells (n5 3 to 6mice per group in eachtime point) (C) and total IgE was measured by ELISA in duplicated wells (n 5 8 to 16 mice per group in each time point) (D) Datarepresent mean6 SEM from 2 different experiments PI 5 postimmunization p 005 p 001 p 0005 by Studentt test 2 tailed GM-CSF 5 granulocyte-macrophage colony-stimulating factor IFN 5 interferon IL 5 interleukin

Neurology Neuroimmunology amp Neuroinflammation 7

the frequency of peritoneal MCs27 In addition to thepotential effects of anti-FceRIa antibody on baso-phils and MCs this antibody can also deplete a pop-ulation of dendritic cells namely iDC that canexpress FceRI after immunization with an allergen9

In this regard in the model of CNS autoimmunitythat we used we failed to detect by flow cytometrysuch immune cells (non-T and non-B cells expressingFceRI and lacking CD49b) in lymph nodes spleensor blood of mice during the course of EAE (data notshown) It is possible that the generation of these cellscritically depends on the type of antigen andor immu-nization protocol used and that such FceRI 1 iDCsmight play a marginal or no role in the development ofEAE Thus it is unlikely that depletion of iDCs con-tributed to the results that we observed

We found that anti-FceRIa antibodyndashtreatedmice exhibited enhanced recall responses againstMOG 35ndash55 antigen in their LNCs compared withisotype controlndashtreated mice with increased T-cellproliferation and secretion of Th1 and Th17 cyto-kines such as IFN-g IL-17 IL-6 and GM-CSFThese cytokines are known to play important rolesin the development of EAE1229 Thus immune mod-ulation might represent an important mechanism bywhich anti-FceRIa antibody treatment induces EAEexacerbation In line with our results in anothermodel of T-cell autoimmunity Gomez et al haveshown that depletion of basophils by monoclonalantibodies in a mouse model of autoimmune colitisresulted in enhanced production of Th1 and Th17cytokines and exacerbated colitis23

Among the mechanisms potentially contributingto disease exacerbation observed in anti-FceRIaantibodyndashtreated mice depletion of basophils andpossibly MCs as important sources of mediators andcytokines that can counteract Th1Th17 autoim-munity can be postulated For example IL-4 of whichthese cells represent important sources plays a key rolein counteracting CNS autoimmunity and limits theseverity of EAE2 Of interest in anti-FceRIaantibodyndashtreated mice serum concentration of IgEwhose production depends on IL-4 was significantlylower during the priming phase of EAE at a time whenbasophils were completely depleted This differencedisappeared at a later time point when basophil fre-quency had returned to normal level

Among other mediators secreted by basophilsand MCs that may potentially induce immunemodulation histamine which is synthetized andsecreted by these cells in high amounts is also con-sidered to play a role in limiting the severity of EAEHowever we did not find significant differences inhistamine serum concentrations between mice trea-ted with anti-FceRIa antibody and those treatedwith isotype control antibody and thus it is

unlikely that the effects of this treatment are relatedto histamine

Although it is generally accepted that immunedeviation toward Th2 protects against EAE Lafailleand colleagues reported that transfer of Th2 lympho-cytes specific for MBP failed to protect against Th1cellndashmediated EAE Moreover these Th2 lympho-cytes can cause themselves EAE when transferred intoimmune-deficient RAG-1 knockout recipient micethat lack mature T and B cells3 An unusually highnumber of polymorphonuclear cells and MCs wereobserved in CNS inflammatory infiltrates of Th2cellndashinduced EAE3 In our study we have used anactive EAE model obtained in immune competentmice to evaluate the potential effects of targetingFceRI constitutively expressed on mouse MCs andbasophils We show that treatment with anti-FceRIaantibody induced an exacerbation of Th1 and Th17responses with mechanisms likely involving immunecells such as basophils and MCs both importantsources of IL-4 and other key cytokines and involvedin Th2 immunity Understanding how Th2 lympho-cytes and effector cells of Th2 immunity such asbasophils and MCs intervene in the complex patho-biology of CNS autoimmunity remains an open fieldof investigation

Of interest in the context of CNS autoimmunityit has been recently reported that subacute infectionwith intestinal parasites is associated with reduced dis-ease flares in relapsing-remitting patients with MSwith reduced disability progression and fewer MRIchanges compared with uninfected patients withMS3637 Although an increased production of IL-10from B cells and an induction of regulatory T cellswere observed in these parasite-infected patients withMS it is also possible that activation of basophilsoccurring during helminth infection could haveinduced beneficial effects and reduced encephalito-genic responses in these patients Of note increasingevidence suggests that basophil activation can con-tribute to the induction or intensity of Th2 re-sponses38 Such effects might be beneficial in thecontext of EAE and MS

The findings that we report here are associatedwith the effects of a treatment with a monoclonalantibody We confirmed that MAR-1 antibody bindsuniquely to its target the a-chain of FceRI (figure2D) and that it completely depletes basophils how-ever this antibody may also reduce the numbers andor affect the functions of other FceRI-bearing cellsAlternatively it is possible that MAR-1 antibodyresults in some activation of basophils (andor otherFceRI-bearing cells) at the time of treatmentadministration as well as depleting this populationIn that case such MAR-1ndashdependent cell activation(instead of or in addition to MAR-1ndashdependent cell

8 Neurology Neuroimmunology amp Neuroinflammation

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

measured from triplicate wells on a beta counter (Perkin-Elmer

Waltham MA)

Cytokine analysis LNCs were cultured in the same condi-

tions as described above25 and cytokines analyzed in cells super-

natants by sandwich ELISA (anti-mouse OptEIA ELISA Set for

interferon [IFN]-g IL-6 IL-10 and IL-4 BD Pharmingen

San Jose CA Mouse IL-17 Duoset and Mouse granulocyte-

macrophage colony-stimulating factor [GM-CSF] Duoset

RampD Systems Minneapolis MN) according to the manu-

facturerrsquos instructions

Serum histamine and IgE measurement Serum samples

were obtained from the tail vein and stored at280degC until tested

Total IgE antibody concentration was measured on serum

samples at a 150 dilution by ELISA (IgE OptEIA kit BD

Pharmingen)25 Histamine concentration was measured with an

enzyme immunoassay kit (ImmunoTech Beckman Coulter

Brea CA) according to the manufacturerrsquos instructions25

Statistical analysis Two-way analysis of variance followed by

the Bonferroni post hoc test was used for comparison of EAE

scores between groups For all other analyses unpaired Student

t test 2 tailed was used Analysis was performed using SPSS

software (IBM SPSS Statistics Armonk NY) In all tests p values005 were considered statistically significant

RESULTS Anti-FceRIa antibody treatment increased

the severity of EAE To evaluate the potential effects oftargeting FceRI in CNS autoimmunity we treatedC57BL6 female mice with a monoclonal antibodyagainst FceRIa (MAR-1) or an isotype control anti-body Treatment (5 mg) was administered to miceintraperitoneally twice daily for 3 consecutive daysOne week after treatment EAE was induced by activeimmunization with MOG 35ndash55 and PTX In 2consecutive experiments treatment with anti-FceRIaantibody resulted in higher incidence of EAE andsignificant exacerbation of disease severity comparedwith isotype controlndashtreated mice (figure 1A andtable) In line with these clinical findings neuro-pathologic analysis of the spinal cord obtained frommice 6 weeks after EAE induction revealed signifi-cantly increased inflammation in the white matter ofanti-FceRIa antibodyndashtreated mice compared withisotype controlndashtreated mice (figure 1B) In bothgroups infiltrating cells were mainly composed ofmacrophages with a minority of cells represented byT cells and very rare B cells (data not shown)Granulocytes were barely detected in both control

and treated mice In Giemsa-stained sections of thespinal cord rare cells with the morphologicalappearance of MCs were observed in perivascularareas in both treatment groups (data not shown) Inparallel with the higher extent of inflammationobserved in anti-FceRIa antibodyndashtreated mice inspinal cord sections of these mice demyelination wasmore prominent compared with that observed insections of isotype controlndashtreated mice (figure 1C)

We then analyzed by flow cytometry the effects ofanti-FceRIa antibody treatment on the frequency ofimmune cell subsets in blood and peripheral lym-phoid organs Because an experimental group wastreated with anti-FceRIa antibody we could notuse the same fluorochrome-labeled monoclonal anti-body clone MAR-1 for the identification of FceRI-expressing cells As previously reported2728 weobserved that anti-FceRIa antibody induced completedepletion of basophils identified as CD200R31CD49b1 CD32 CD45B2202 cells in the bloodlymph node and spleen (figure 2A) In these treatedmice basophils were completely depleted at the timeof EAE induction during priming and acute phase ofEAE and began to reappear in blood circulation 3weeks after immunization (figure 2C) The frequencyof other leukocytes known to play important roles inEAE including T cells B cells and monocytes wasnot significantly modified by the treatment (figure2B) Anti-FceRIa antibody (MAR-1) staining of leu-kocytes disappeared in FceRI a-chain 22 miceconfirming that this antibody binds uniquely to itstarget the a-chain of the receptor (figure 2D)

Anti-FceRIa antibodyndashtreated mice exhibited enhanced

Th1 and Th17 responses against myelin antigen Wenext evaluated whether the exacerbated EAE pheno-type observed in mice treated with anti-FceRIa anti-body was associated with an alteration of T-cellactivation and Th regulation We isolated LNCs fromanti-FceRIa antibodyndashtreated and isotype controlndashtreated mice with EAE during the priming phase ofthe disease (7ndash10 days after immunization) andexamined the recall response to peptide stimulationin vitro LNCs from anti-FceRIa antibodyndashtreatedmice displayed increased proliferation in response toMOG 35ndash55 compared with isotype controlndashtreatedmice (figure 3A) Moreover in LNCs of anti-FceRIaantibodyndashtreated mice the secretion of proin-flammatory cytokines IFN-g IL-17 and IL-6known to play a critical role in EAE was signifi-cantly increased (figure 3A) GM-CSF consideredessential in CNS autoimmune inflammation29 wasalso increased in the supernatant of antigen-stimulated LNCs from anti-FceRIa antibodyndashtreated mice compared to that from isotypecontrolndashtreated mice (figure 3A) We did not observe

Table Experimental autoimmune encephalomyelitis features in C57BL6 micetreated with antibody against the a-chain of the high affinity receptor forimmunoglobulin E

Group Incidence () Onset d Mean maximum score Cumulative disease score

Anti-FceRIa 2626 (100) 101 6 04 33 6 02a 462 6 42a

Isotype 2226 (846) 103 6 04 25 6 03 307 6 42

Data are shown as mean 6 SEM of pooled data from 2 consecutive independent experi-ments each with 13 mice per group that gave similar resultsap 005

4 Neurology Neuroimmunology amp Neuroinflammation

Figure 2 Flow cytometry analysis of basophils and immune cell subsets in C57BL6 mice treated withantibody against the a-chain of the high affinity receptor for immunoglobulin E

(A and B) Lymph nodes spleens and blood were obtained at the time of experimental autoimmune encephalomyelitis (EAE) induc-tion 7 days after treatment with antibody against the a-chain of the high affinity receptor for immunoglobulin E or isotype controlantibody and analyzed by flow cytometry (A) Representative flow cytometry plots of basophils in blood lymph node cells (LNCs)and splenocytes (SPL) of treatedmice Live cellswere gated onCD32 andCD45B220- cells Numbers representmeanpercentageof total leukocytes6 SEM (n55mice per group) from1of3 independent experiments that gave similar results (B) Representativeflow cytometry plots of B cellsmonocytes T cells andCD41 T cells in blood cells of antibody treatedmice Live cellswere gated onexpression of CD45 Data represent mean percentage 6 SEM (n 5 5 mice per group) and are representative of 2 independentexperiments that gave similar results (C) Quantification of basophils in the blood of C57BL6mice treated with anti-FceRIa (whitebars) or isotype control antibody (black bars) beforeEAE induction (day0) and at different timepoints during disease Data representmean6 SEM of 3ndash6 animals per group for each time point and are representative of 2 consecutive experiments that gave similarresults (D) Representative flow cytometry plots of APC-conjugated anti-FceRIa (MAR-1) antibody staining in blood leukocytes ofnaive FceRI a-chain11 and22 mice Data represent mean percentage6 SEM of 3 mice per group and are representative of 2independent experiments Similar datawere obtained by stainingwith APC-MAR-1 antibody LNCs and splenocytes of FceRI a-chain11 and 22 mice p 005 Student t test 2 tailed FSC 5 forward-scattered light SSC 5 side-scattered light

Neurology Neuroimmunology amp Neuroinflammation 5

significant differences in IL-10 production in antigen-stimulated LNCs from anti-FceRIa antibodyndashtreatedmice vs those from isotype controlndashtreated mice(figure 3A) while IL-4 was below the detection limit(data not shown) We next repeated the analysis of therecall response in LNCs from mice 6 weeks after EAEinduction at a time where the frequency of basophils inthe blood of anti-FceRIa antibodyndashtreated mice hadreturned almost to normal values (figure 2C) No dif-ferences were observed at this time point in either pro-liferation (figure 3B) or cytokine production betweenanti-FceRIa antibodyndashtreated mice and isotypecontrolndashtreated mice with the exception of the sup-pressor cytokine IL-10 which was significantly higher inanti-FceRIa antibodyndashtreated mice (figure 3B)

Effects of anti-FceRIa treatment on serum concentration

of histamine and total IgE Histamine has been shownto modulate the immune response in EAE30ndash32 andhistidine decarboxylasendashdeficient mice (22 mice)that are histamine deficient displayed exacerbatedEAE and increased Th1 responses2533 Because ba-sophils which represent an important source of his-tamine were completely depleted by anti-FceRIaantibody in our model we measured the concentra-tion of histamine in mouse sera during EAE Bothanti-FceRIa antibody and isotype controlndashtreatedgroups displayed similar levels of serum histamineconcentrations (figure 3C) Of note histamine levelsdid not change significantly in either group duringthe course of EAE (figure 3C) These findings suggestthat the increased severity of EAE that we observed inanti-FceRIa antibodyndashtreated mice is unlikely to berelated with an effect of the treatment on blood his-tamine levels These findings also suggest that thereare substantial sources of blood histamine in thesemice in addition to basophils

Basophils are a predominant source of IL-4 inallergen and parasite-activated blood leukocytes34

This cytokine induces in B-cell antibody class switchtoward IgE and IgG1 We previously reported thatIgE increases in mouse serum during EAE25 althoughthe biological significance of the increase of antibod-ies of this class is yet to be defined We thereforemeasured IgE concentrations in serum and found thatIgE concentrations were significantly decreased inanti-FceRIa antibodyndashtreated mice during thepriming phase of EAE (day 7) compared with isotypecontrolndashtreated mice These differences in IgE con-centrations disappeared 6 weeks after EAE inductionat the time when basophil frequency had returned toalmost normal levels (figure 3D)

Treatment with anti-FceRIa induced susceptibility to

EAE in resistant BALBc mice The results reportedabove show that anti-FceRIa antibody treatmentboth exacerbated EAE in C57BL6 mice and depleted

basophils suggesting that depletion of these cells andperhaps additional effects of anti-FceRIa antibodytreatment contributed to increase the severity ofEAE in this model To further explore this hypothe-sis we used anti-FceRIa antibody in another modelof EAE obtained in BALBc mice which are Th2prone and resistant against EAE35 We attempted toinduce EAE in this strain by immunization with theself-antigen PLP 186ndash204 in CFA and PTX26 Saline-treated mice were completely resistant to EAE (none of5 mice immunized developed the disease) and amongisotype controlndashtreated mice only 2 of 5 (40)displayed minimal signs of EAE (score 1 5 tail paral-ysis) that lasted only 1 day (figure 4A) Conversely themajority of BALBc mice (4 of 5 80) treated withanti-FceRIa antibody developed EAE (figure 4A) withmaximum scores of 1 2 (hind-limb paralysis) and 5(death) Similar to what we observed in C57BL6 miceflow cytometry analysis conducted on blood cells ofanti-FceRIa antibody and isotype controlndashtreatedBALBc mice revealed that this treatment inducedcomplete depletion of basophils (figure 4B)

DISCUSSION Th1 and Th17 cells play key roles inthe pathogenesis of EAE and MS and it is widelyaccepted that Th2 responses hinder the developmentand pathobiology of these diseases However emerg-ing evidence suggests that in addition to Th2 polar-ized cells that counteract Th1 and Th17 responsesalso effector cells and mediators of the Th2 immunitycan limit the severity of EAE For example we andothers have shown that EAE is more severe in theabsence of histamine2533 and similar findings wereobserved in MCndashdeficient c-kit mutant mice undercertain experimental conditions1415 In this report tofurther test the hypothesis that mechanisms of theTh2 immunity can influence the development inEAE we targeted with a monoclonal antibody FceRIthat plays a crucial role in IgE-mediated allergic re-actions and is constitutively expressed on MCs andbasophils We found that treatment with this anti-body before the induction of the disease exacerbatedMOG 35ndash55 EAE in C57BL6 mice and increasedCNS neuroinflammation Moreover anti-FceRIaantibody increased Th1 and Th17 responses againstmyelin antigen Last treatment induced susceptibilityto EAE in BALBc mice which are Th2 prone andresistant to the development of EAE

The mechanisms involved in disease exacerbationcaused by anti-FceRIa antibody treatment are yet tobe fully understood Complete depletion of basophilsandor alteration of the frequency and function ofother FceRI-bearing cells associated with the treat-ment can be postulated as one potential mechanismIndeed anti-FceRIa antibody has been previouslyshown to deplete basophils in vivo and to reduce

6 Neurology Neuroimmunology amp Neuroinflammation

Figure 3 Effects of antibody against the a-chain of the high affinity receptor for immunoglobulin E treatment onantigen-induced T-cell response and on serum concentration of histamine and IgE antibody

(A and B) Draining lymph nodes were obtained from antibody against the a-chain of the high affinity receptor for immunoglobulin E(FceRIa) or isotype control antibodyndashtreated mice (n5 5mice per group) 7 days (A) and 42 days (B) after experimental autoimmuneencephalomyelitis (EAE) induction Proliferation (mean 6 SEM from triplicate wells) and cytokine production (mean 6 SEM fromduplicate wells) were analyzed in lymph node cells stimulated in vitro with serial concentrations of myelin oligodendrocyte glyco-protein (MOG) 35ndash55 or medium alone Data are representative of 2 independent experiments that gave similar results (C and D)serumsampleswere obtained frommice treatedwith anti-FceRIa (white bars) or isotype control antibody (black bars) 7 and 42daysafter the induction of EAE Histaminewasmeasured by enzyme immunoassay in duplicatedwells (n5 3 to 6mice per group in eachtime point) (C) and total IgE was measured by ELISA in duplicated wells (n 5 8 to 16 mice per group in each time point) (D) Datarepresent mean6 SEM from 2 different experiments PI 5 postimmunization p 005 p 001 p 0005 by Studentt test 2 tailed GM-CSF 5 granulocyte-macrophage colony-stimulating factor IFN 5 interferon IL 5 interleukin

Neurology Neuroimmunology amp Neuroinflammation 7

the frequency of peritoneal MCs27 In addition to thepotential effects of anti-FceRIa antibody on baso-phils and MCs this antibody can also deplete a pop-ulation of dendritic cells namely iDC that canexpress FceRI after immunization with an allergen9

In this regard in the model of CNS autoimmunitythat we used we failed to detect by flow cytometrysuch immune cells (non-T and non-B cells expressingFceRI and lacking CD49b) in lymph nodes spleensor blood of mice during the course of EAE (data notshown) It is possible that the generation of these cellscritically depends on the type of antigen andor immu-nization protocol used and that such FceRI 1 iDCsmight play a marginal or no role in the development ofEAE Thus it is unlikely that depletion of iDCs con-tributed to the results that we observed

We found that anti-FceRIa antibodyndashtreatedmice exhibited enhanced recall responses againstMOG 35ndash55 antigen in their LNCs compared withisotype controlndashtreated mice with increased T-cellproliferation and secretion of Th1 and Th17 cyto-kines such as IFN-g IL-17 IL-6 and GM-CSFThese cytokines are known to play important rolesin the development of EAE1229 Thus immune mod-ulation might represent an important mechanism bywhich anti-FceRIa antibody treatment induces EAEexacerbation In line with our results in anothermodel of T-cell autoimmunity Gomez et al haveshown that depletion of basophils by monoclonalantibodies in a mouse model of autoimmune colitisresulted in enhanced production of Th1 and Th17cytokines and exacerbated colitis23

Among the mechanisms potentially contributingto disease exacerbation observed in anti-FceRIaantibodyndashtreated mice depletion of basophils andpossibly MCs as important sources of mediators andcytokines that can counteract Th1Th17 autoim-munity can be postulated For example IL-4 of whichthese cells represent important sources plays a key rolein counteracting CNS autoimmunity and limits theseverity of EAE2 Of interest in anti-FceRIaantibodyndashtreated mice serum concentration of IgEwhose production depends on IL-4 was significantlylower during the priming phase of EAE at a time whenbasophils were completely depleted This differencedisappeared at a later time point when basophil fre-quency had returned to normal level

Among other mediators secreted by basophilsand MCs that may potentially induce immunemodulation histamine which is synthetized andsecreted by these cells in high amounts is also con-sidered to play a role in limiting the severity of EAEHowever we did not find significant differences inhistamine serum concentrations between mice trea-ted with anti-FceRIa antibody and those treatedwith isotype control antibody and thus it is

unlikely that the effects of this treatment are relatedto histamine

Although it is generally accepted that immunedeviation toward Th2 protects against EAE Lafailleand colleagues reported that transfer of Th2 lympho-cytes specific for MBP failed to protect against Th1cellndashmediated EAE Moreover these Th2 lympho-cytes can cause themselves EAE when transferred intoimmune-deficient RAG-1 knockout recipient micethat lack mature T and B cells3 An unusually highnumber of polymorphonuclear cells and MCs wereobserved in CNS inflammatory infiltrates of Th2cellndashinduced EAE3 In our study we have used anactive EAE model obtained in immune competentmice to evaluate the potential effects of targetingFceRI constitutively expressed on mouse MCs andbasophils We show that treatment with anti-FceRIaantibody induced an exacerbation of Th1 and Th17responses with mechanisms likely involving immunecells such as basophils and MCs both importantsources of IL-4 and other key cytokines and involvedin Th2 immunity Understanding how Th2 lympho-cytes and effector cells of Th2 immunity such asbasophils and MCs intervene in the complex patho-biology of CNS autoimmunity remains an open fieldof investigation

Of interest in the context of CNS autoimmunityit has been recently reported that subacute infectionwith intestinal parasites is associated with reduced dis-ease flares in relapsing-remitting patients with MSwith reduced disability progression and fewer MRIchanges compared with uninfected patients withMS3637 Although an increased production of IL-10from B cells and an induction of regulatory T cellswere observed in these parasite-infected patients withMS it is also possible that activation of basophilsoccurring during helminth infection could haveinduced beneficial effects and reduced encephalito-genic responses in these patients Of note increasingevidence suggests that basophil activation can con-tribute to the induction or intensity of Th2 re-sponses38 Such effects might be beneficial in thecontext of EAE and MS

The findings that we report here are associatedwith the effects of a treatment with a monoclonalantibody We confirmed that MAR-1 antibody bindsuniquely to its target the a-chain of FceRI (figure2D) and that it completely depletes basophils how-ever this antibody may also reduce the numbers andor affect the functions of other FceRI-bearing cellsAlternatively it is possible that MAR-1 antibodyresults in some activation of basophils (andor otherFceRI-bearing cells) at the time of treatmentadministration as well as depleting this populationIn that case such MAR-1ndashdependent cell activation(instead of or in addition to MAR-1ndashdependent cell

8 Neurology Neuroimmunology amp Neuroinflammation

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

Figure 2 Flow cytometry analysis of basophils and immune cell subsets in C57BL6 mice treated withantibody against the a-chain of the high affinity receptor for immunoglobulin E

(A and B) Lymph nodes spleens and blood were obtained at the time of experimental autoimmune encephalomyelitis (EAE) induc-tion 7 days after treatment with antibody against the a-chain of the high affinity receptor for immunoglobulin E or isotype controlantibody and analyzed by flow cytometry (A) Representative flow cytometry plots of basophils in blood lymph node cells (LNCs)and splenocytes (SPL) of treatedmice Live cellswere gated onCD32 andCD45B220- cells Numbers representmeanpercentageof total leukocytes6 SEM (n55mice per group) from1of3 independent experiments that gave similar results (B) Representativeflow cytometry plots of B cellsmonocytes T cells andCD41 T cells in blood cells of antibody treatedmice Live cellswere gated onexpression of CD45 Data represent mean percentage 6 SEM (n 5 5 mice per group) and are representative of 2 independentexperiments that gave similar results (C) Quantification of basophils in the blood of C57BL6mice treated with anti-FceRIa (whitebars) or isotype control antibody (black bars) beforeEAE induction (day0) and at different timepoints during disease Data representmean6 SEM of 3ndash6 animals per group for each time point and are representative of 2 consecutive experiments that gave similarresults (D) Representative flow cytometry plots of APC-conjugated anti-FceRIa (MAR-1) antibody staining in blood leukocytes ofnaive FceRI a-chain11 and22 mice Data represent mean percentage6 SEM of 3 mice per group and are representative of 2independent experiments Similar datawere obtained by stainingwith APC-MAR-1 antibody LNCs and splenocytes of FceRI a-chain11 and 22 mice p 005 Student t test 2 tailed FSC 5 forward-scattered light SSC 5 side-scattered light

Neurology Neuroimmunology amp Neuroinflammation 5

significant differences in IL-10 production in antigen-stimulated LNCs from anti-FceRIa antibodyndashtreatedmice vs those from isotype controlndashtreated mice(figure 3A) while IL-4 was below the detection limit(data not shown) We next repeated the analysis of therecall response in LNCs from mice 6 weeks after EAEinduction at a time where the frequency of basophils inthe blood of anti-FceRIa antibodyndashtreated mice hadreturned almost to normal values (figure 2C) No dif-ferences were observed at this time point in either pro-liferation (figure 3B) or cytokine production betweenanti-FceRIa antibodyndashtreated mice and isotypecontrolndashtreated mice with the exception of the sup-pressor cytokine IL-10 which was significantly higher inanti-FceRIa antibodyndashtreated mice (figure 3B)

Effects of anti-FceRIa treatment on serum concentration

of histamine and total IgE Histamine has been shownto modulate the immune response in EAE30ndash32 andhistidine decarboxylasendashdeficient mice (22 mice)that are histamine deficient displayed exacerbatedEAE and increased Th1 responses2533 Because ba-sophils which represent an important source of his-tamine were completely depleted by anti-FceRIaantibody in our model we measured the concentra-tion of histamine in mouse sera during EAE Bothanti-FceRIa antibody and isotype controlndashtreatedgroups displayed similar levels of serum histamineconcentrations (figure 3C) Of note histamine levelsdid not change significantly in either group duringthe course of EAE (figure 3C) These findings suggestthat the increased severity of EAE that we observed inanti-FceRIa antibodyndashtreated mice is unlikely to berelated with an effect of the treatment on blood his-tamine levels These findings also suggest that thereare substantial sources of blood histamine in thesemice in addition to basophils

Basophils are a predominant source of IL-4 inallergen and parasite-activated blood leukocytes34

This cytokine induces in B-cell antibody class switchtoward IgE and IgG1 We previously reported thatIgE increases in mouse serum during EAE25 althoughthe biological significance of the increase of antibod-ies of this class is yet to be defined We thereforemeasured IgE concentrations in serum and found thatIgE concentrations were significantly decreased inanti-FceRIa antibodyndashtreated mice during thepriming phase of EAE (day 7) compared with isotypecontrolndashtreated mice These differences in IgE con-centrations disappeared 6 weeks after EAE inductionat the time when basophil frequency had returned toalmost normal levels (figure 3D)

Treatment with anti-FceRIa induced susceptibility to

EAE in resistant BALBc mice The results reportedabove show that anti-FceRIa antibody treatmentboth exacerbated EAE in C57BL6 mice and depleted

basophils suggesting that depletion of these cells andperhaps additional effects of anti-FceRIa antibodytreatment contributed to increase the severity ofEAE in this model To further explore this hypothe-sis we used anti-FceRIa antibody in another modelof EAE obtained in BALBc mice which are Th2prone and resistant against EAE35 We attempted toinduce EAE in this strain by immunization with theself-antigen PLP 186ndash204 in CFA and PTX26 Saline-treated mice were completely resistant to EAE (none of5 mice immunized developed the disease) and amongisotype controlndashtreated mice only 2 of 5 (40)displayed minimal signs of EAE (score 1 5 tail paral-ysis) that lasted only 1 day (figure 4A) Conversely themajority of BALBc mice (4 of 5 80) treated withanti-FceRIa antibody developed EAE (figure 4A) withmaximum scores of 1 2 (hind-limb paralysis) and 5(death) Similar to what we observed in C57BL6 miceflow cytometry analysis conducted on blood cells ofanti-FceRIa antibody and isotype controlndashtreatedBALBc mice revealed that this treatment inducedcomplete depletion of basophils (figure 4B)

DISCUSSION Th1 and Th17 cells play key roles inthe pathogenesis of EAE and MS and it is widelyaccepted that Th2 responses hinder the developmentand pathobiology of these diseases However emerg-ing evidence suggests that in addition to Th2 polar-ized cells that counteract Th1 and Th17 responsesalso effector cells and mediators of the Th2 immunitycan limit the severity of EAE For example we andothers have shown that EAE is more severe in theabsence of histamine2533 and similar findings wereobserved in MCndashdeficient c-kit mutant mice undercertain experimental conditions1415 In this report tofurther test the hypothesis that mechanisms of theTh2 immunity can influence the development inEAE we targeted with a monoclonal antibody FceRIthat plays a crucial role in IgE-mediated allergic re-actions and is constitutively expressed on MCs andbasophils We found that treatment with this anti-body before the induction of the disease exacerbatedMOG 35ndash55 EAE in C57BL6 mice and increasedCNS neuroinflammation Moreover anti-FceRIaantibody increased Th1 and Th17 responses againstmyelin antigen Last treatment induced susceptibilityto EAE in BALBc mice which are Th2 prone andresistant to the development of EAE

The mechanisms involved in disease exacerbationcaused by anti-FceRIa antibody treatment are yet tobe fully understood Complete depletion of basophilsandor alteration of the frequency and function ofother FceRI-bearing cells associated with the treat-ment can be postulated as one potential mechanismIndeed anti-FceRIa antibody has been previouslyshown to deplete basophils in vivo and to reduce

6 Neurology Neuroimmunology amp Neuroinflammation

Figure 3 Effects of antibody against the a-chain of the high affinity receptor for immunoglobulin E treatment onantigen-induced T-cell response and on serum concentration of histamine and IgE antibody

(A and B) Draining lymph nodes were obtained from antibody against the a-chain of the high affinity receptor for immunoglobulin E(FceRIa) or isotype control antibodyndashtreated mice (n5 5mice per group) 7 days (A) and 42 days (B) after experimental autoimmuneencephalomyelitis (EAE) induction Proliferation (mean 6 SEM from triplicate wells) and cytokine production (mean 6 SEM fromduplicate wells) were analyzed in lymph node cells stimulated in vitro with serial concentrations of myelin oligodendrocyte glyco-protein (MOG) 35ndash55 or medium alone Data are representative of 2 independent experiments that gave similar results (C and D)serumsampleswere obtained frommice treatedwith anti-FceRIa (white bars) or isotype control antibody (black bars) 7 and 42daysafter the induction of EAE Histaminewasmeasured by enzyme immunoassay in duplicatedwells (n5 3 to 6mice per group in eachtime point) (C) and total IgE was measured by ELISA in duplicated wells (n 5 8 to 16 mice per group in each time point) (D) Datarepresent mean6 SEM from 2 different experiments PI 5 postimmunization p 005 p 001 p 0005 by Studentt test 2 tailed GM-CSF 5 granulocyte-macrophage colony-stimulating factor IFN 5 interferon IL 5 interleukin

Neurology Neuroimmunology amp Neuroinflammation 7

the frequency of peritoneal MCs27 In addition to thepotential effects of anti-FceRIa antibody on baso-phils and MCs this antibody can also deplete a pop-ulation of dendritic cells namely iDC that canexpress FceRI after immunization with an allergen9

In this regard in the model of CNS autoimmunitythat we used we failed to detect by flow cytometrysuch immune cells (non-T and non-B cells expressingFceRI and lacking CD49b) in lymph nodes spleensor blood of mice during the course of EAE (data notshown) It is possible that the generation of these cellscritically depends on the type of antigen andor immu-nization protocol used and that such FceRI 1 iDCsmight play a marginal or no role in the development ofEAE Thus it is unlikely that depletion of iDCs con-tributed to the results that we observed

We found that anti-FceRIa antibodyndashtreatedmice exhibited enhanced recall responses againstMOG 35ndash55 antigen in their LNCs compared withisotype controlndashtreated mice with increased T-cellproliferation and secretion of Th1 and Th17 cyto-kines such as IFN-g IL-17 IL-6 and GM-CSFThese cytokines are known to play important rolesin the development of EAE1229 Thus immune mod-ulation might represent an important mechanism bywhich anti-FceRIa antibody treatment induces EAEexacerbation In line with our results in anothermodel of T-cell autoimmunity Gomez et al haveshown that depletion of basophils by monoclonalantibodies in a mouse model of autoimmune colitisresulted in enhanced production of Th1 and Th17cytokines and exacerbated colitis23

Among the mechanisms potentially contributingto disease exacerbation observed in anti-FceRIaantibodyndashtreated mice depletion of basophils andpossibly MCs as important sources of mediators andcytokines that can counteract Th1Th17 autoim-munity can be postulated For example IL-4 of whichthese cells represent important sources plays a key rolein counteracting CNS autoimmunity and limits theseverity of EAE2 Of interest in anti-FceRIaantibodyndashtreated mice serum concentration of IgEwhose production depends on IL-4 was significantlylower during the priming phase of EAE at a time whenbasophils were completely depleted This differencedisappeared at a later time point when basophil fre-quency had returned to normal level

Among other mediators secreted by basophilsand MCs that may potentially induce immunemodulation histamine which is synthetized andsecreted by these cells in high amounts is also con-sidered to play a role in limiting the severity of EAEHowever we did not find significant differences inhistamine serum concentrations between mice trea-ted with anti-FceRIa antibody and those treatedwith isotype control antibody and thus it is

unlikely that the effects of this treatment are relatedto histamine

Although it is generally accepted that immunedeviation toward Th2 protects against EAE Lafailleand colleagues reported that transfer of Th2 lympho-cytes specific for MBP failed to protect against Th1cellndashmediated EAE Moreover these Th2 lympho-cytes can cause themselves EAE when transferred intoimmune-deficient RAG-1 knockout recipient micethat lack mature T and B cells3 An unusually highnumber of polymorphonuclear cells and MCs wereobserved in CNS inflammatory infiltrates of Th2cellndashinduced EAE3 In our study we have used anactive EAE model obtained in immune competentmice to evaluate the potential effects of targetingFceRI constitutively expressed on mouse MCs andbasophils We show that treatment with anti-FceRIaantibody induced an exacerbation of Th1 and Th17responses with mechanisms likely involving immunecells such as basophils and MCs both importantsources of IL-4 and other key cytokines and involvedin Th2 immunity Understanding how Th2 lympho-cytes and effector cells of Th2 immunity such asbasophils and MCs intervene in the complex patho-biology of CNS autoimmunity remains an open fieldof investigation

Of interest in the context of CNS autoimmunityit has been recently reported that subacute infectionwith intestinal parasites is associated with reduced dis-ease flares in relapsing-remitting patients with MSwith reduced disability progression and fewer MRIchanges compared with uninfected patients withMS3637 Although an increased production of IL-10from B cells and an induction of regulatory T cellswere observed in these parasite-infected patients withMS it is also possible that activation of basophilsoccurring during helminth infection could haveinduced beneficial effects and reduced encephalito-genic responses in these patients Of note increasingevidence suggests that basophil activation can con-tribute to the induction or intensity of Th2 re-sponses38 Such effects might be beneficial in thecontext of EAE and MS

The findings that we report here are associatedwith the effects of a treatment with a monoclonalantibody We confirmed that MAR-1 antibody bindsuniquely to its target the a-chain of FceRI (figure2D) and that it completely depletes basophils how-ever this antibody may also reduce the numbers andor affect the functions of other FceRI-bearing cellsAlternatively it is possible that MAR-1 antibodyresults in some activation of basophils (andor otherFceRI-bearing cells) at the time of treatmentadministration as well as depleting this populationIn that case such MAR-1ndashdependent cell activation(instead of or in addition to MAR-1ndashdependent cell

8 Neurology Neuroimmunology amp Neuroinflammation

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

significant differences in IL-10 production in antigen-stimulated LNCs from anti-FceRIa antibodyndashtreatedmice vs those from isotype controlndashtreated mice(figure 3A) while IL-4 was below the detection limit(data not shown) We next repeated the analysis of therecall response in LNCs from mice 6 weeks after EAEinduction at a time where the frequency of basophils inthe blood of anti-FceRIa antibodyndashtreated mice hadreturned almost to normal values (figure 2C) No dif-ferences were observed at this time point in either pro-liferation (figure 3B) or cytokine production betweenanti-FceRIa antibodyndashtreated mice and isotypecontrolndashtreated mice with the exception of the sup-pressor cytokine IL-10 which was significantly higher inanti-FceRIa antibodyndashtreated mice (figure 3B)

Effects of anti-FceRIa treatment on serum concentration

of histamine and total IgE Histamine has been shownto modulate the immune response in EAE30ndash32 andhistidine decarboxylasendashdeficient mice (22 mice)that are histamine deficient displayed exacerbatedEAE and increased Th1 responses2533 Because ba-sophils which represent an important source of his-tamine were completely depleted by anti-FceRIaantibody in our model we measured the concentra-tion of histamine in mouse sera during EAE Bothanti-FceRIa antibody and isotype controlndashtreatedgroups displayed similar levels of serum histamineconcentrations (figure 3C) Of note histamine levelsdid not change significantly in either group duringthe course of EAE (figure 3C) These findings suggestthat the increased severity of EAE that we observed inanti-FceRIa antibodyndashtreated mice is unlikely to berelated with an effect of the treatment on blood his-tamine levels These findings also suggest that thereare substantial sources of blood histamine in thesemice in addition to basophils

Basophils are a predominant source of IL-4 inallergen and parasite-activated blood leukocytes34

This cytokine induces in B-cell antibody class switchtoward IgE and IgG1 We previously reported thatIgE increases in mouse serum during EAE25 althoughthe biological significance of the increase of antibod-ies of this class is yet to be defined We thereforemeasured IgE concentrations in serum and found thatIgE concentrations were significantly decreased inanti-FceRIa antibodyndashtreated mice during thepriming phase of EAE (day 7) compared with isotypecontrolndashtreated mice These differences in IgE con-centrations disappeared 6 weeks after EAE inductionat the time when basophil frequency had returned toalmost normal levels (figure 3D)

Treatment with anti-FceRIa induced susceptibility to

EAE in resistant BALBc mice The results reportedabove show that anti-FceRIa antibody treatmentboth exacerbated EAE in C57BL6 mice and depleted

basophils suggesting that depletion of these cells andperhaps additional effects of anti-FceRIa antibodytreatment contributed to increase the severity ofEAE in this model To further explore this hypothe-sis we used anti-FceRIa antibody in another modelof EAE obtained in BALBc mice which are Th2prone and resistant against EAE35 We attempted toinduce EAE in this strain by immunization with theself-antigen PLP 186ndash204 in CFA and PTX26 Saline-treated mice were completely resistant to EAE (none of5 mice immunized developed the disease) and amongisotype controlndashtreated mice only 2 of 5 (40)displayed minimal signs of EAE (score 1 5 tail paral-ysis) that lasted only 1 day (figure 4A) Conversely themajority of BALBc mice (4 of 5 80) treated withanti-FceRIa antibody developed EAE (figure 4A) withmaximum scores of 1 2 (hind-limb paralysis) and 5(death) Similar to what we observed in C57BL6 miceflow cytometry analysis conducted on blood cells ofanti-FceRIa antibody and isotype controlndashtreatedBALBc mice revealed that this treatment inducedcomplete depletion of basophils (figure 4B)

DISCUSSION Th1 and Th17 cells play key roles inthe pathogenesis of EAE and MS and it is widelyaccepted that Th2 responses hinder the developmentand pathobiology of these diseases However emerg-ing evidence suggests that in addition to Th2 polar-ized cells that counteract Th1 and Th17 responsesalso effector cells and mediators of the Th2 immunitycan limit the severity of EAE For example we andothers have shown that EAE is more severe in theabsence of histamine2533 and similar findings wereobserved in MCndashdeficient c-kit mutant mice undercertain experimental conditions1415 In this report tofurther test the hypothesis that mechanisms of theTh2 immunity can influence the development inEAE we targeted with a monoclonal antibody FceRIthat plays a crucial role in IgE-mediated allergic re-actions and is constitutively expressed on MCs andbasophils We found that treatment with this anti-body before the induction of the disease exacerbatedMOG 35ndash55 EAE in C57BL6 mice and increasedCNS neuroinflammation Moreover anti-FceRIaantibody increased Th1 and Th17 responses againstmyelin antigen Last treatment induced susceptibilityto EAE in BALBc mice which are Th2 prone andresistant to the development of EAE

The mechanisms involved in disease exacerbationcaused by anti-FceRIa antibody treatment are yet tobe fully understood Complete depletion of basophilsandor alteration of the frequency and function ofother FceRI-bearing cells associated with the treat-ment can be postulated as one potential mechanismIndeed anti-FceRIa antibody has been previouslyshown to deplete basophils in vivo and to reduce

6 Neurology Neuroimmunology amp Neuroinflammation

Figure 3 Effects of antibody against the a-chain of the high affinity receptor for immunoglobulin E treatment onantigen-induced T-cell response and on serum concentration of histamine and IgE antibody

(A and B) Draining lymph nodes were obtained from antibody against the a-chain of the high affinity receptor for immunoglobulin E(FceRIa) or isotype control antibodyndashtreated mice (n5 5mice per group) 7 days (A) and 42 days (B) after experimental autoimmuneencephalomyelitis (EAE) induction Proliferation (mean 6 SEM from triplicate wells) and cytokine production (mean 6 SEM fromduplicate wells) were analyzed in lymph node cells stimulated in vitro with serial concentrations of myelin oligodendrocyte glyco-protein (MOG) 35ndash55 or medium alone Data are representative of 2 independent experiments that gave similar results (C and D)serumsampleswere obtained frommice treatedwith anti-FceRIa (white bars) or isotype control antibody (black bars) 7 and 42daysafter the induction of EAE Histaminewasmeasured by enzyme immunoassay in duplicatedwells (n5 3 to 6mice per group in eachtime point) (C) and total IgE was measured by ELISA in duplicated wells (n 5 8 to 16 mice per group in each time point) (D) Datarepresent mean6 SEM from 2 different experiments PI 5 postimmunization p 005 p 001 p 0005 by Studentt test 2 tailed GM-CSF 5 granulocyte-macrophage colony-stimulating factor IFN 5 interferon IL 5 interleukin

Neurology Neuroimmunology amp Neuroinflammation 7

the frequency of peritoneal MCs27 In addition to thepotential effects of anti-FceRIa antibody on baso-phils and MCs this antibody can also deplete a pop-ulation of dendritic cells namely iDC that canexpress FceRI after immunization with an allergen9

In this regard in the model of CNS autoimmunitythat we used we failed to detect by flow cytometrysuch immune cells (non-T and non-B cells expressingFceRI and lacking CD49b) in lymph nodes spleensor blood of mice during the course of EAE (data notshown) It is possible that the generation of these cellscritically depends on the type of antigen andor immu-nization protocol used and that such FceRI 1 iDCsmight play a marginal or no role in the development ofEAE Thus it is unlikely that depletion of iDCs con-tributed to the results that we observed

We found that anti-FceRIa antibodyndashtreatedmice exhibited enhanced recall responses againstMOG 35ndash55 antigen in their LNCs compared withisotype controlndashtreated mice with increased T-cellproliferation and secretion of Th1 and Th17 cyto-kines such as IFN-g IL-17 IL-6 and GM-CSFThese cytokines are known to play important rolesin the development of EAE1229 Thus immune mod-ulation might represent an important mechanism bywhich anti-FceRIa antibody treatment induces EAEexacerbation In line with our results in anothermodel of T-cell autoimmunity Gomez et al haveshown that depletion of basophils by monoclonalantibodies in a mouse model of autoimmune colitisresulted in enhanced production of Th1 and Th17cytokines and exacerbated colitis23

Among the mechanisms potentially contributingto disease exacerbation observed in anti-FceRIaantibodyndashtreated mice depletion of basophils andpossibly MCs as important sources of mediators andcytokines that can counteract Th1Th17 autoim-munity can be postulated For example IL-4 of whichthese cells represent important sources plays a key rolein counteracting CNS autoimmunity and limits theseverity of EAE2 Of interest in anti-FceRIaantibodyndashtreated mice serum concentration of IgEwhose production depends on IL-4 was significantlylower during the priming phase of EAE at a time whenbasophils were completely depleted This differencedisappeared at a later time point when basophil fre-quency had returned to normal level

Among other mediators secreted by basophilsand MCs that may potentially induce immunemodulation histamine which is synthetized andsecreted by these cells in high amounts is also con-sidered to play a role in limiting the severity of EAEHowever we did not find significant differences inhistamine serum concentrations between mice trea-ted with anti-FceRIa antibody and those treatedwith isotype control antibody and thus it is

unlikely that the effects of this treatment are relatedto histamine

Although it is generally accepted that immunedeviation toward Th2 protects against EAE Lafailleand colleagues reported that transfer of Th2 lympho-cytes specific for MBP failed to protect against Th1cellndashmediated EAE Moreover these Th2 lympho-cytes can cause themselves EAE when transferred intoimmune-deficient RAG-1 knockout recipient micethat lack mature T and B cells3 An unusually highnumber of polymorphonuclear cells and MCs wereobserved in CNS inflammatory infiltrates of Th2cellndashinduced EAE3 In our study we have used anactive EAE model obtained in immune competentmice to evaluate the potential effects of targetingFceRI constitutively expressed on mouse MCs andbasophils We show that treatment with anti-FceRIaantibody induced an exacerbation of Th1 and Th17responses with mechanisms likely involving immunecells such as basophils and MCs both importantsources of IL-4 and other key cytokines and involvedin Th2 immunity Understanding how Th2 lympho-cytes and effector cells of Th2 immunity such asbasophils and MCs intervene in the complex patho-biology of CNS autoimmunity remains an open fieldof investigation

Of interest in the context of CNS autoimmunityit has been recently reported that subacute infectionwith intestinal parasites is associated with reduced dis-ease flares in relapsing-remitting patients with MSwith reduced disability progression and fewer MRIchanges compared with uninfected patients withMS3637 Although an increased production of IL-10from B cells and an induction of regulatory T cellswere observed in these parasite-infected patients withMS it is also possible that activation of basophilsoccurring during helminth infection could haveinduced beneficial effects and reduced encephalito-genic responses in these patients Of note increasingevidence suggests that basophil activation can con-tribute to the induction or intensity of Th2 re-sponses38 Such effects might be beneficial in thecontext of EAE and MS

The findings that we report here are associatedwith the effects of a treatment with a monoclonalantibody We confirmed that MAR-1 antibody bindsuniquely to its target the a-chain of FceRI (figure2D) and that it completely depletes basophils how-ever this antibody may also reduce the numbers andor affect the functions of other FceRI-bearing cellsAlternatively it is possible that MAR-1 antibodyresults in some activation of basophils (andor otherFceRI-bearing cells) at the time of treatmentadministration as well as depleting this populationIn that case such MAR-1ndashdependent cell activation(instead of or in addition to MAR-1ndashdependent cell

8 Neurology Neuroimmunology amp Neuroinflammation

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

Figure 3 Effects of antibody against the a-chain of the high affinity receptor for immunoglobulin E treatment onantigen-induced T-cell response and on serum concentration of histamine and IgE antibody

(A and B) Draining lymph nodes were obtained from antibody against the a-chain of the high affinity receptor for immunoglobulin E(FceRIa) or isotype control antibodyndashtreated mice (n5 5mice per group) 7 days (A) and 42 days (B) after experimental autoimmuneencephalomyelitis (EAE) induction Proliferation (mean 6 SEM from triplicate wells) and cytokine production (mean 6 SEM fromduplicate wells) were analyzed in lymph node cells stimulated in vitro with serial concentrations of myelin oligodendrocyte glyco-protein (MOG) 35ndash55 or medium alone Data are representative of 2 independent experiments that gave similar results (C and D)serumsampleswere obtained frommice treatedwith anti-FceRIa (white bars) or isotype control antibody (black bars) 7 and 42daysafter the induction of EAE Histaminewasmeasured by enzyme immunoassay in duplicatedwells (n5 3 to 6mice per group in eachtime point) (C) and total IgE was measured by ELISA in duplicated wells (n 5 8 to 16 mice per group in each time point) (D) Datarepresent mean6 SEM from 2 different experiments PI 5 postimmunization p 005 p 001 p 0005 by Studentt test 2 tailed GM-CSF 5 granulocyte-macrophage colony-stimulating factor IFN 5 interferon IL 5 interleukin

Neurology Neuroimmunology amp Neuroinflammation 7

the frequency of peritoneal MCs27 In addition to thepotential effects of anti-FceRIa antibody on baso-phils and MCs this antibody can also deplete a pop-ulation of dendritic cells namely iDC that canexpress FceRI after immunization with an allergen9

In this regard in the model of CNS autoimmunitythat we used we failed to detect by flow cytometrysuch immune cells (non-T and non-B cells expressingFceRI and lacking CD49b) in lymph nodes spleensor blood of mice during the course of EAE (data notshown) It is possible that the generation of these cellscritically depends on the type of antigen andor immu-nization protocol used and that such FceRI 1 iDCsmight play a marginal or no role in the development ofEAE Thus it is unlikely that depletion of iDCs con-tributed to the results that we observed

We found that anti-FceRIa antibodyndashtreatedmice exhibited enhanced recall responses againstMOG 35ndash55 antigen in their LNCs compared withisotype controlndashtreated mice with increased T-cellproliferation and secretion of Th1 and Th17 cyto-kines such as IFN-g IL-17 IL-6 and GM-CSFThese cytokines are known to play important rolesin the development of EAE1229 Thus immune mod-ulation might represent an important mechanism bywhich anti-FceRIa antibody treatment induces EAEexacerbation In line with our results in anothermodel of T-cell autoimmunity Gomez et al haveshown that depletion of basophils by monoclonalantibodies in a mouse model of autoimmune colitisresulted in enhanced production of Th1 and Th17cytokines and exacerbated colitis23

Among the mechanisms potentially contributingto disease exacerbation observed in anti-FceRIaantibodyndashtreated mice depletion of basophils andpossibly MCs as important sources of mediators andcytokines that can counteract Th1Th17 autoim-munity can be postulated For example IL-4 of whichthese cells represent important sources plays a key rolein counteracting CNS autoimmunity and limits theseverity of EAE2 Of interest in anti-FceRIaantibodyndashtreated mice serum concentration of IgEwhose production depends on IL-4 was significantlylower during the priming phase of EAE at a time whenbasophils were completely depleted This differencedisappeared at a later time point when basophil fre-quency had returned to normal level

Among other mediators secreted by basophilsand MCs that may potentially induce immunemodulation histamine which is synthetized andsecreted by these cells in high amounts is also con-sidered to play a role in limiting the severity of EAEHowever we did not find significant differences inhistamine serum concentrations between mice trea-ted with anti-FceRIa antibody and those treatedwith isotype control antibody and thus it is

unlikely that the effects of this treatment are relatedto histamine

Although it is generally accepted that immunedeviation toward Th2 protects against EAE Lafailleand colleagues reported that transfer of Th2 lympho-cytes specific for MBP failed to protect against Th1cellndashmediated EAE Moreover these Th2 lympho-cytes can cause themselves EAE when transferred intoimmune-deficient RAG-1 knockout recipient micethat lack mature T and B cells3 An unusually highnumber of polymorphonuclear cells and MCs wereobserved in CNS inflammatory infiltrates of Th2cellndashinduced EAE3 In our study we have used anactive EAE model obtained in immune competentmice to evaluate the potential effects of targetingFceRI constitutively expressed on mouse MCs andbasophils We show that treatment with anti-FceRIaantibody induced an exacerbation of Th1 and Th17responses with mechanisms likely involving immunecells such as basophils and MCs both importantsources of IL-4 and other key cytokines and involvedin Th2 immunity Understanding how Th2 lympho-cytes and effector cells of Th2 immunity such asbasophils and MCs intervene in the complex patho-biology of CNS autoimmunity remains an open fieldof investigation

Of interest in the context of CNS autoimmunityit has been recently reported that subacute infectionwith intestinal parasites is associated with reduced dis-ease flares in relapsing-remitting patients with MSwith reduced disability progression and fewer MRIchanges compared with uninfected patients withMS3637 Although an increased production of IL-10from B cells and an induction of regulatory T cellswere observed in these parasite-infected patients withMS it is also possible that activation of basophilsoccurring during helminth infection could haveinduced beneficial effects and reduced encephalito-genic responses in these patients Of note increasingevidence suggests that basophil activation can con-tribute to the induction or intensity of Th2 re-sponses38 Such effects might be beneficial in thecontext of EAE and MS

The findings that we report here are associatedwith the effects of a treatment with a monoclonalantibody We confirmed that MAR-1 antibody bindsuniquely to its target the a-chain of FceRI (figure2D) and that it completely depletes basophils how-ever this antibody may also reduce the numbers andor affect the functions of other FceRI-bearing cellsAlternatively it is possible that MAR-1 antibodyresults in some activation of basophils (andor otherFceRI-bearing cells) at the time of treatmentadministration as well as depleting this populationIn that case such MAR-1ndashdependent cell activation(instead of or in addition to MAR-1ndashdependent cell

8 Neurology Neuroimmunology amp Neuroinflammation

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

the frequency of peritoneal MCs27 In addition to thepotential effects of anti-FceRIa antibody on baso-phils and MCs this antibody can also deplete a pop-ulation of dendritic cells namely iDC that canexpress FceRI after immunization with an allergen9

In this regard in the model of CNS autoimmunitythat we used we failed to detect by flow cytometrysuch immune cells (non-T and non-B cells expressingFceRI and lacking CD49b) in lymph nodes spleensor blood of mice during the course of EAE (data notshown) It is possible that the generation of these cellscritically depends on the type of antigen andor immu-nization protocol used and that such FceRI 1 iDCsmight play a marginal or no role in the development ofEAE Thus it is unlikely that depletion of iDCs con-tributed to the results that we observed

We found that anti-FceRIa antibodyndashtreatedmice exhibited enhanced recall responses againstMOG 35ndash55 antigen in their LNCs compared withisotype controlndashtreated mice with increased T-cellproliferation and secretion of Th1 and Th17 cyto-kines such as IFN-g IL-17 IL-6 and GM-CSFThese cytokines are known to play important rolesin the development of EAE1229 Thus immune mod-ulation might represent an important mechanism bywhich anti-FceRIa antibody treatment induces EAEexacerbation In line with our results in anothermodel of T-cell autoimmunity Gomez et al haveshown that depletion of basophils by monoclonalantibodies in a mouse model of autoimmune colitisresulted in enhanced production of Th1 and Th17cytokines and exacerbated colitis23

Among the mechanisms potentially contributingto disease exacerbation observed in anti-FceRIaantibodyndashtreated mice depletion of basophils andpossibly MCs as important sources of mediators andcytokines that can counteract Th1Th17 autoim-munity can be postulated For example IL-4 of whichthese cells represent important sources plays a key rolein counteracting CNS autoimmunity and limits theseverity of EAE2 Of interest in anti-FceRIaantibodyndashtreated mice serum concentration of IgEwhose production depends on IL-4 was significantlylower during the priming phase of EAE at a time whenbasophils were completely depleted This differencedisappeared at a later time point when basophil fre-quency had returned to normal level

Among other mediators secreted by basophilsand MCs that may potentially induce immunemodulation histamine which is synthetized andsecreted by these cells in high amounts is also con-sidered to play a role in limiting the severity of EAEHowever we did not find significant differences inhistamine serum concentrations between mice trea-ted with anti-FceRIa antibody and those treatedwith isotype control antibody and thus it is

unlikely that the effects of this treatment are relatedto histamine

Although it is generally accepted that immunedeviation toward Th2 protects against EAE Lafailleand colleagues reported that transfer of Th2 lympho-cytes specific for MBP failed to protect against Th1cellndashmediated EAE Moreover these Th2 lympho-cytes can cause themselves EAE when transferred intoimmune-deficient RAG-1 knockout recipient micethat lack mature T and B cells3 An unusually highnumber of polymorphonuclear cells and MCs wereobserved in CNS inflammatory infiltrates of Th2cellndashinduced EAE3 In our study we have used anactive EAE model obtained in immune competentmice to evaluate the potential effects of targetingFceRI constitutively expressed on mouse MCs andbasophils We show that treatment with anti-FceRIaantibody induced an exacerbation of Th1 and Th17responses with mechanisms likely involving immunecells such as basophils and MCs both importantsources of IL-4 and other key cytokines and involvedin Th2 immunity Understanding how Th2 lympho-cytes and effector cells of Th2 immunity such asbasophils and MCs intervene in the complex patho-biology of CNS autoimmunity remains an open fieldof investigation

Of interest in the context of CNS autoimmunityit has been recently reported that subacute infectionwith intestinal parasites is associated with reduced dis-ease flares in relapsing-remitting patients with MSwith reduced disability progression and fewer MRIchanges compared with uninfected patients withMS3637 Although an increased production of IL-10from B cells and an induction of regulatory T cellswere observed in these parasite-infected patients withMS it is also possible that activation of basophilsoccurring during helminth infection could haveinduced beneficial effects and reduced encephalito-genic responses in these patients Of note increasingevidence suggests that basophil activation can con-tribute to the induction or intensity of Th2 re-sponses38 Such effects might be beneficial in thecontext of EAE and MS

The findings that we report here are associatedwith the effects of a treatment with a monoclonalantibody We confirmed that MAR-1 antibody bindsuniquely to its target the a-chain of FceRI (figure2D) and that it completely depletes basophils how-ever this antibody may also reduce the numbers andor affect the functions of other FceRI-bearing cellsAlternatively it is possible that MAR-1 antibodyresults in some activation of basophils (andor otherFceRI-bearing cells) at the time of treatmentadministration as well as depleting this populationIn that case such MAR-1ndashdependent cell activation(instead of or in addition to MAR-1ndashdependent cell

8 Neurology Neuroimmunology amp Neuroinflammation

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

depletion) might influence the course of EAE in thissetting Understanding how these cells of the Th2immunity intervene in the complex autoimmuneresponse leading to CNS inflammation and demye-lination might help defining potential new targets oftreatment for this disease

Our work demonstrates that treatment with anti-FceRIa antibody plays a detrimental role in EAE andsuggests that targeting of FceRI andor of cells express-ing this receptor can influence CNS autoimmunity

AUTHOR CONTRIBUTIONSSilvia Musio designed and performed the experiments acquired and

analyzed data performed statistical analysis and wrote the manuscript

Massimo Costanza helped performing in vitro experiments discussed results

and revised the manuscript Pietro Luigi Poliani Elena Fontana and

Manuela Cominelli performed histopathologic and immunohistochemistry

analysis and revised the manuscript Gabriella Abolafio helped with flow cy-

tometry experiments Lawrence Steinman discussed the results at all stages

and contributed to write the manuscript Rosetta Pedotti initiated the study

designed research interpreted data supervised the study and wrote the

manuscript

ACKNOWLEDGMENTThe authors thank Dr Stephen J Galli and Dr Mindy Tsai for helpful

suggestions and discussion Dr Giovanna Borsellino for precious sugges-

tions on flow cytometry experiments and Cinthia Farina for providing

reagents

STUDY FUNDINGThis research was supported by grants from Fondazione Italiana Sclerosi

Multipla and FISM2015R19 to RP and FISM 2011B6 research fel-

lowship to SM) and Italian Ministry of HealthmdashYoung research Award

(GR-2009-1607206 to RP)

DISCLOSURES Musio received research support from Fondazione Italiana Sclerosi

Multipla M Costanza PL Poliani E Fontana M Cominelli and

G Abolafio report no disclosures L Steinman served on the scientific

advisory board for Novartis Receptos Atreca Tolerion Teva and Abb-

Vie received travel funding andor speaker honoraria from Celgene and

AbbVie served on the editorial board for MS Journal and Proceedings

National Academy of Sciences holds patents on antigen specific toler-

ance has a patent pending on cytokines and type 1 interferons received

research support from Atara Celgene and Biogen and holds stock op-

tions and board membership in Tolerion Board of Directors BioAtla

R Pedotti received travel funding from Novartis has a patent pending

for CNS autoimmunity with prokineticin receptor antagonist and

received research support from Italian Ministry of Health and Fondazione

Italiana Sclerosi Multipla Go to Neurologyorgnn for full disclosure

forms

Received November 17 2016 Accepted in final form February 9 2017

REFERENCES1 Steinman L Immunology of relapse and remission in mul-

tiple sclerosis Annu Rev Immunol 201432257ndash281

2 Goverman J Autoimmune T cell responses in the central

nervous system Nat Rev Immunol 20099393ndash407

3 Lafaille JJ Keere FV Hsu AL et al Myelin basic protein-

specific T helper 2 (Th2) cells cause experimental autoimmune

encephalomyelitis in immunodeficient hosts rather than pro-

tect them from the disease J Exp Med 1997186307ndash312

4 Pedotti R De Voss JJ Steinman L Galli SJ Involvement

of both lsquoallergicrsquo and lsquoautoimmunersquo mechanisms in EAE

MS and other autoimmune diseases Trends Immunol

200324479ndash484

5 Pedotti R DeVoss JJ Youssef S et al Multiple elements

of the allergic arm of the immune response modulate

autoimmune demyelination Proc Natl Acad Sci USA

20031001867ndash1872

6 Galli SJ Metcalfe DD Arber DA Dvorak AM Chapter

63 Basophils and mast cells and their disorders In

Williams Hematology 8th ed New York McGraw-Hill

Medical 2005

7 Voehringer D Protective and pathological roles of mast

cells and basophils Nat Rev Immunol 201313362ndash375

8 Wedemeyer J Tsai M Galli SJ Roles of mast cells and

basophils in innate and acquired immunity Curr Opin

Immunol 200012624ndash631

9 Hammad H Plantinga M Deswarte K et al Inflamma-

tory dendritic cellsmdashnot basophilsmdashare necessary and suf-

ficient for induction of Th2 immunity to inhaled house

dust mite allergen J Exp Med 20102072097ndash2111

Figure 4 Treatment with antibody against thea-chain of the high affinity receptorfor immunoglobulin E inducessusceptibility to experimentalautoimmune encephalomyelitis inresistant BALBc mice

(A) Clinical experimental autoimmune encephalomyelitis(EAE) scores of myelin proteolipid protein 186-204ndashimmunized mice treated before EAE induction with anti-body against the a-chain of the high affinity receptor forimmunoglobulin E (FceRIa) (black squares) isotype controlantibody (gray squares) or saline (white circles) (n 5 5 miceper group) p 0001 by analysis of variance test (B)Basophils were analyzed by flow cytometry in the blood ofBALBc mice 7 days after treatment with anti-FceRIa orisotype control antibody at the time of EAE inductionRepresentative dot plots Numbers represent mean per-centage of total leukocytes 6 SEM (n 5 5 mice per group)

Neurology Neuroimmunology amp Neuroinflammation 9

10 Secor VH Secor WE Gutekunst CA Brown MA Mast

cells are essential for early onset and severe disease in

a murine model of multiple sclerosis J Exp Med 2000

191813ndash822

11 Gregory GD Robbie-Ryan M Secor VH Sabatino JJ

Brown MA Mast cells are required for optimal autoreac-

tive T cell responses in a murine model of multiple

sclerosis Eur J Immunol 2005353478ndash3486

12 Sayed BA Christy AL Walker ME Brown MA Menin-

geal mast cells affect early T cell central nervous system

infiltration and blood-brain barrier integrity through TNF

a role for neutrophil recruitment J Immunol 2010184

6891ndash6900

13 Feyerabend TB Weiser A Tietz A et al Cre-mediated cell

ablation contests mast cell contribution in models of anti-

body- and T cell-mediated autoimmunity Immunity

201135832ndash844

14 Li H Nourbakhsh B Safavi F et al Kit (W-sh) mice

develop earlier and more severe experimental autoimmune

encephalomyelitis due to absence of immune suppression

J Immunol 2011187274ndash282

15 Piconese S Costanza M Musio S et al Exacerbated experi-

mental autoimmune encephalomyelitis in mast-cell-deficient

Kit W-shW-sh mice Lab Invest 201191627ndash641

16 Obata K Mukai K Tsujimura Y et al Basophils are

essential initiators of a novel type of chronic allergic

inflammation Blood 2007110913ndash920

17 Ohnmacht C Schwartz C Panzer M Schiedewitz I Nau-

mann R Voehringer D Basophils orchestrate chronic

allergic dermatitis and protective immunity against hel-

minths Immunity 201033364ndash374

18 Pulendran B Artis D New paradigms in type 2 immunity

Science 2012337431ndash435

19 Charles N Hardwick D Daugas E Illei GG Rivera J

Basophils and the T helper 2 environment can promote the

development of lupus nephritis Nat Med 201016701ndash707

20 Pellefigues C Charles N The deleterious role of basophils

in systemic lupus erythematosus Curr Opin Immunol

201325704ndash711

21 Bruumlhl H Cihak J Plachyacute J et al Targeting of Gr-11

CCR21 monocytes in collagen-induced arthritis Arthritis

Rheum 2007562975ndash2985

22 Bruumlhl H Cihak J Niedermeier M et al Important role of

interleukin-3 in the early phase of collagen-induced arthri-

tis Arthritis Rheum 2009601352ndash1361

23 Gomez MR Talke Y Hofmann C et al Basophils control

T-cell responses and limit disease activity in experimental

murine colitis Mucosal Immunol 20147188ndash199

24 Dombrowicz D Flamand V Brigman KK Koller BH

Kinet JP Abolition of anaphylaxis by targeted disruption

of the high affinity immunoglobulin E receptor alpha

chain gene Cell 199375969ndash976

25 Musio S Gallo B Scabeni S et al A key regulatory role for

histamine in experimental autoimmune encephalomyelitis

disease exacerbation in histidine decarboxylase-deficient

mice J Immunol 200617617ndash26

26 Colombo E Tentorio P Musio S et al Skewed B cell

differentiation affects lymphoid organogenesis but not T

cell-mediated autoimmunity Clin Exp Immunol 2014

17658ndash65

27 Denzel A Maus UA Rodriguez Gomez M et al Basophils

enhance immunological memory responses Nat Immunol

20089733ndash742

28 Wada T Ishiwata K Koseki H et al Selective ablation of

basophils in mice reveals their nonredundant role in

acquired immunity against ticks J Clin Invest 2010120

2867ndash2875

29 Codarri L Gyuumllveacuteszi G Tosevski V et al RORgt drives

production of the cytokine GM-CSF in helper T cells

which is essential for the effector phase of autoimmune

neuroinflammation Nat Immunol 201112560ndash567

30 Ma RZ Gao J Meeker ND et al Identification of Bphs

an autoimmune disease locus as histamine receptor H1

Science 2002297620ndash623

31 Noubade R Saligrama N Spach K et al Autoimmune

disease-associated histamine receptor H1 alleles exhibit

differential protein trafficking and cell surface expression

J Immunol 20081807471ndash7479

32 Lapilla M Gallo B Martinello M et al Histamine regu-

lates autoreactive T cell activation and adhesiveness in

inflamed brain microcirculation J Leukoc Biol 201189

259ndash267

33 Saligrama N Case LK del Rio R Noubade R Teuscher

C Systemic lack of canonical histamine receptor signaling

results in increased resistance to autoimmune encephalo-

myelitis J Immunol 2013191614ndash622

34 Stone KD Prussin C Metcalfe DD IgE mast cells ba-

sophils and eosinophils J Allergy Clin Immunol 2010

125S73ndashS80

35 Trotter J Zamvil SS Steinman L Comparison of antigen

specificity class II major histocompatibility complex

restriction and in vivo behavior of myelin basic protein-

specific T cell lines and clones derived from (BALBc x

SJLJ) mice J Immunol 19871391834ndash1839

36 Correale J Farez M Association between parasite infection

and immune responses in multiple sclerosis Ann Neurol

20076197ndash108

37 Correale J Farez M Razzitte G Helminth infections asso-

ciated with multiple sclerosis induce regulatory B cells

Ann Neurol 200864187ndash199

38 Hill DA Siracusa MC Abt MC et al Commensal

bacteria-derived signals regulate basophil hematopoie-

sis and allergic inflammation Nat Med 201218

538ndash546

10 Neurology Neuroimmunology amp Neuroinflammation

DOI 101212NXI000000000000034220174 Neurol Neuroimmunol Neuroinflamm

Silvia Musio Massimo Costanza Pietro Luigi Poliani et al myelin

antibody exacerbates EAE and T-cell immunity againstαRIεTreatment with anti-Fc

This information is current as of April 14 2017

ServicesUpdated Information amp

httpnnneurologyorgcontent43e342fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent43e342fullhtmlref-list-1

This article cites 37 articles 13 of which you can access for free at

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseasesfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2017 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

DOI 101212NXI000000000000034220174 Neurol Neuroimmunol Neuroinflamm

Silvia Musio Massimo Costanza Pietro Luigi Poliani et al myelin

antibody exacerbates EAE and T-cell immunity againstαRIεTreatment with anti-Fc

This information is current as of April 14 2017

ServicesUpdated Information amp

httpnnneurologyorgcontent43e342fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent43e342fullhtmlref-list-1

This article cites 37 articles 13 of which you can access for free at

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseasesfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2017 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm