Activation of the NLRP3 inflammasome by particles from the 1

Echinococcus granulosus laminated layer 2

Cecilia Casaravilla1, Álvaro Pittini

1, Dominik Rückerl

2, Judith E. Allen

2 3

& Álvaro Díaz1*

4

1. Área Inmunología, Departamento de Biociencias (Facultad de Química) and Cátedra 5

de Inmunología, Instituto de Química Biológica (Facultad de Ciencias), Universidad de 6

la República, Montevideo, Uruguay. 7

2. Faculty of Biology, Medicine and Health, School of Biological Sciences, University 8

of Manchester, M139PT, Manchester, UK. 9

10

(*) Corresponding author. E-mail: adiaz@fq.edu.uy. Postal address: Dr. Álvaro Díaz. 11

Cátedra de Inmunología. Instituto de Higiene. Avenida Alfredo Navarro 3051. 12

Montevideo CP11600. Uruguay. Tel: + 59824874320. 13

14

Running title: NLRP3 inflammasome activation by helminth material 15

Keywords: Echinococcus; laminated layer; alum; NLRP3; PI3K; membrane affinity-16

triggered signaling 17

18

IAI Accepted Manuscript Posted Online 22 June 2020Infect. Immun. doi:10.1128/IAI.00190-20Copyright © 2020 Casaravilla et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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ABSTRACT 19

The interaction of dendritic cells and macrophages with a variety of rigid non-cellular 20

particles triggers activation of the NLRP3 inflammasome and consequent secretion of 21

IL-1β. Non-cellular particles can also be generated in the context of helminth infection, 22

as these large pathogens often shed their outermost structures during growth and/or 23

moulting. One such structure is the massive, mucin-based, soft and flexible laminated 24

layer (LL), which protects the larval stages of cestodes of the genus Echinococcus. We 25

show that particles from the E. granulosus LL (pLL) trigger NLRP3- and caspase-1-26

dependent IL-1β in LPS-primed mouse bone marrow-derived dendritic cells (BMDC). 27

This response can be elicited by pLL particles too large for phagocytosis, and 28

nonetheless requires actin dynamics, Syk and PI3K. These three requirements had 29

already been observed in our previous study on the alteration by pLL of BMDC 30

responses to LPS in terms of CD86, CD40, IL-10 and IL-12: however, we now show 31

that these alterations are independent of NLRP3 and caspase-1. In other words, an initial 32

interaction with particles requiring actin dynamics, Syk and PI3K but not phagocytosis 33

elicits both NLRP3-dependent and NLRP3-independent responses. Intraperitoneal 34

injection of pLL induced IL-1, suggesting that contact with LL materials induces IL-35

1 in the E. granulosus infection setting. Our results extend NLRP3 inflammasome 36

activation by non-cellular particulate materials both to helminth-derived and to 37

flexible/soft materials. 38

39

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INTRODUCTION 40

The NLRP3 inflammasome is an innate immune sensor-response modulus triggered by 41

an exceptionally wide range of stimuli (1, 2). Recent works suggest that it plays 42

important roles in helminth infections, antagonizing type 2 responses and potentiating 43

Th17/inflammatory responses, with impacts on parasite burden and pathology (3-8). In 44

addition and counter-intuitively, NLRP3 can promote type 2 responses, independently 45

of the inflammasome (9, 10). 46

The possible triggers for NLRP3 inflammasome activation in helminth infections 47

include endogenous signals generated by inflammation and tissue damage and helminth 48

products themselves (8). Helminth products shown to trigger the NLRP3 inflammasome 49

so far are all either soluble or exosomal (3, 4, 7, 11). Outside of the field of helminth 50

infection, rigid particulate matter, specifically of crystalline nature, is well known to 51

activate the NLRP3 inflammasome in macrophages and DCs previously stimulated 52

(primed) with TLR agonists (1, 2, 12-24). Helminths, as a result of the turnover of their 53

outermost structures, have much potential to generate insoluble matter within host 54

tissues. However, such matter is physically very different from the crystalline materials 55

known to activate the NLRP3 inflammasome; in fact, the possibility of NLRP3 56

inflammasome activation by insoluble helminth materials has not been analyzed. One 57

such outermost helminth structure is the laminated layer (LL), a mm-thick mucin-based 58

protective structure of the Echinococcus granulosus sensu lato (s.l.) larva (25-27). This 59

bladder-like larva causes cystic echinococcosis in livestock and humans (28-30). Larval 60

growth is accompanied by shedding of LL particles, observed in E. granulosus s.l. 61

experimental infections (31), and better documented for the closely related species E. 62

multilocularis (32-34). 63

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We have previously analyzed the immunological effects of a preparation of particles 64

from the E. granulosus s.l. LL (termed pLL) as a possible model of LL particles shed in 65

vivo (35-37). pLL particles are made up of an aqueous gel, and are soft and deformable 66

(35). In mouse bone marrow-derived dendritic cells (BMDC) in particular, pLL induces 67

the expression of CD86, and enhances LPS-elicited CD86 and IL-10 while blunting the 68

response to LPS in terms of CD40 and IL-12p70 (as well as its subunit IL-12/23p40) 69

(35). These changes elicited by pLL require actin dynamics, PI3K class I and probably 70

the kinase Syk but not particle phagocytosis, and appear to be receptor-independent 71

(36). These features match a mechanism termed “membrane affinity-triggered 72

signaling” (MATS), put forward by Yan Shi to explain DC and macrophage responses 73

to solid, mostly crystalline materials (38). In this proposed mechanism, solid surfaces 74

interact with polar headgroups of certain plasma membrane lipids causing the 75

coalescence of lipid rafts and/or specifically the aggregation of phosphatidylinositol 4,5-76

bisphosphate (PIP2) (38-41). The cytosolic protein moesin is then recruited to clustered 77

PIP2 and in turn causes activation of Syk and downstream signaling that does not 78

require conventional receptors (41). MATS signaling may trigger phagocytosis, but it 79

can operate from the cell surface in the absence of particle internalization (39-41). 80

Materials proposed to act on DCs via MATS include sodium urate and alum (39-41), 81

which are additionally known to activate the NLRP3 inflammasome (13, 15). 82

The mechanistic similarities between responses to pLL and those induced by MATS led 83

us to hypothesize that pLL could also activate the NLPR3 inflammasome; we also 84

wondered if such activation may underlie the changes caused by pLL on BMDC 85

responses to LPS (35, 36). In this paper we show that pLL does elicit NLRP3-dependent 86

IL-1β from BMDC, but the previously described alterations in BMDC responses to LPS 87

are NLRP3-independent. We also show that NLRP3 inflammasome activation by pLL 88

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shares the MATS-like requirements with the previously described alterations to LPS 89

responses, adding weight to the hypothesis that DC recognition of pLL involves a 90

MATS-like interaction. Our results extend the range of particulate NLRP3 91

inflammasome activators to soft/flexible materials, and suggest that additional insoluble 92

materials shed by helminths may activate the hosts’ NLRP3 inflammasome module. 93

94

Materials and Methods 95

Parasite materials. pLL, pLL treated for reduction/alkylation of disulphides, and non-96

phagocytosable pLL (pLLNP

) were generated, had their concentrations determined, and 97

were stored as described (35, 36). Preparation of pLL involves a dehydration step 98

followed by grinding into a fine powder, re-hydration and filtration of the resulting 99

suspension; of the two dehydration methods described in (35), freeze-drying was used 100

in the present work. pLL preparations tested negative for endotoxin by the Limulus 101

amebocyte lysate (LAL) method (35). 102

BMDC generation and stimulation. GMCSF-BMDC were generated as described (35, 103

36), from female C57BL/6 wild-type or NLRP3 gene-deficient mice (B6.129S6-104

Nlrp3tm1Bhk

/J; Jackson Laboratories). For inflammasome activation assays, BMDC 105

(400,000 cells per well of 96-well plates) were primed for 2 h with 10 ng/mL LPS (from 106

E. coli 0127:B8; Sigma) or incubated with medium only (final volume in priming step: 107

100 µL). Then cells were stimulated with pLL, pLLNP

, alum (AllhydrogelTM

, Invivogen) 108

or ATP (Sigma), or medium only (100 µL additional volume). Supernatants were 109

collected 3 h later. For assays determining effects on LPS-induced BMDC 110

costimulatory molecule expression and cytokine output, pLL or alum was added (final 111

volume in this step was 100 µl) followed 1 h later by LPS (10 ng/mL, in 100 µL 112

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additional volume) and cells and supernatants were harvested 18 h later (35). Cell 113

viability was measured on the basis of exclusion of the ToPro3 (Invitrogen; 50 nM)

114

viability probe in flow cytometry. 115

Generation and stimulation of bone marrow-derived macrophages (BMDM). BMDM 116

were generated from the bone marrow of female C57BL/6 mice by differentiation 117

during 7 days in the presence of L929 cell supernatant as a source of M-CSF, as 118

described in (42). Cells were stimulated for inflammasome activation as described for 119

BMDC, except that they were plated at 200,000 cells per well of 96-well plates, and the 120

doses of pLL and alum used were at 50 and 50 µg per million cells respectively. 121

Chemical inhibitors. The following chemical inhibitors were used: Z-YVAD-FMK (10 122

µM; Calbiochem/Merck), wortmannin (Sigma; 1 µM) GDC-0941 (5 µM; 123

Calbiochem/Thermo), piceatannol (25 µM; Santa Cruz Biotechnology), cytochalasin D 124

(5 µM; Sigma-Aldrich), VPS34-IN1 (1 µM) and SAR405 (1 µM) (both from the 125

Division of Signal Transduction Therapy Unit, University of Dundee). 126

Measurement of cell responses. Cytokines in supernatants were measured with 127

commercial ELISA kits from RnD Systems (DuoSet ELISA kit for IL-1β and IL-10), 128

eBioscience (IL-18 mouse ELISA kit), or with an antibody pair formed by unconjugated 129

antibody from BD Pharmingen and a biotinylated antibody from Biolegend (IL-130

12/23p40). The expression of CD40 and CD86 was measured by flow cytometry in cells 131

gated for CD11c expression, as in (35). 132

Measurement of Akt phosphorylation. BMDC were stimulated with LPS (10 ng/mL) 133

alone or together with pLL or alum during 80 min (36). Phosphorylation of Akt at Ser473

134

was measured by Western blot as described (36, 37). 135

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In vivo effects of pLL. C57BL/6 mice (female, 8-10 weeks old) were injected i.p. with 136

pLL (150 µg dry mass per mouse), LPS (15 µg per mouse), both pLL and LPS, or 137

vehicle only (200 µl of PBS). Three h later, mice were euthanized using isofluorane and 138

peritoneal lavage fluid was collected for IL-1β quantification. 139

Statistics. 140

Data were analyzed by a non-parametric method, thus avoiding having to assume 141

normality and homogeneity of variances. Specifically, the extension of the Friedman 142

test with the Conover post-test and the Bonferroni correction was applied (43). This 143

method incorporates data corresponding to internal repetitions within each of two or 144

more experiments; it allows for inter-experiment variation in the absolute values 145

obtained, and it identifies those differences between conditions that are consistent across 146

experiments. The number of independent experiments used for statistical analysis and 147

summarized in the graphs shown is indicated in each figure legend; the number of 148

internal repetitions was usually 3 and in some cases 2. For graphical presentation 149

purposes, some data were normalized over responses to alum or pLL; however 150

statistical analyses were always carried out on the crude data. Significances are given 151

represented as: (*) p<0.05, (**) p<0.01, and (***) p<0.001. 152

153

154

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RESULTS 155

pLL triggers NLRP3- and caspase 1-dependent IL-1β and IL-18 secretion in primed 156

BMDC 157

DCs and macrophages that have been primed with TLR agonists release IL-1β and IL-158

18 when subsequently exposed to alum or other crystalline materials (1, 2). To find out 159

if pLL could similarly trigger IL-1β and IL-18 release, we exposed LPS-primed BMDC 160

to pLL, or to alum for comparison purposes. pLL induced IL-1β and IL-18 secretion at 161

levels within the same order of magnitude to those elicited by alum (Figure 1a, b). 162

Either insoluble stimulus induced much less IL-1 than ATP (2 mM), a very potent, 163

soluble activator of the NLRP3 inflammasome that acts via the P2X7 receptor (1, 2). 164

Negligible amounts of IL-1β or IL-18 were produced in the absence of LPS priming 165

(Figure 1a, b), consistent with the previous conclusion that pLL does not contain TLR 166

agonists and/or activate NF-κB (35, 36). Because NLRP3 inflammasome activation is 167

often accompanied by some degree of cell death (1, 2), we measured cell viability 168

following exposure to pLL or alum. Exposure to pLL (at the highest dose used) or alum 169

under the assay conditions caused cell viability to drop from ca. 85% in cells only 170

primed with LPS to ca. 60% (Suppl. Fig 1). 171

In spite of the similar responses to pLL and alum observed in BMDC, a major 172

difference between the two materials was observed upon stimulation of primed BMDM. 173

Whereas alum robustly stimulated IL-1β also in macrophages, the impact of pLL 174

appeared to be restricted to BMDC, with only a low level response in macrophages 175

(Suppl. Fig. 2). 176

Reduction of disulfides alters the physicochemical properties of the LL and weakens the 177

effects of pLL on LPS-induced signaling, costimulatory molecule and cytokine 178

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responses (35, 36, 44). Consistent with these previous findings, pLL treated for 179

disulfide reduction elicited a diminished IL-1β response in BMDC (Suppl. Fig. 3). 180

To determine if IL-1β production by pLL in primed BMDC may reflect NLRP3 181

inflammasome activation, experiments were repeated in the presence of a caspase-1 182

inhibitor (Fig. 2a) or 45 mM extracellular K+ (Fig. 2b), both known to inhibit NLRP3-183

dependent IL-1β production (1). With both treatments, IL-1β production induced by 184

pLL was strongly inhibited (Fig. 2a, b). Moreover, no induction of IL-1β secretion by 185

pLL was observed in primed BMDC deficient in NLRP3 (Fig. 2c). The results for pLL 186

matched the results obtained for alum (Fig. 2a, b, c). 187

In sum, these data provide strong evidence that pLL activates the NLRP3 188

inflammasome in primed BMDC. 189

190

pLL triggers both NLRP3- and caspase 1-dependent and –independent responses 191

Exposure to pLL alters BMDC responses to LPS: it enhances CD86 expression and IL-192

10 secretion whereas it blunts CD40 expression and IL-12/23p40 secretion, as 193

previously reported (35, 36) and confirmed here (Fig. 3a-d). We wondered if these 194

alterations were a consequence of pLL-mediated NLRP3 inflammation activation. In the 195

18-h format of these experiments (see Materials and Methods) and consistent with 196

previous data (45), LPS by itself triggered an IL-1β response, but this response was 197

much potentiated in the addition presence of pLL (Fig. 3e). In the absence of LPS, pLL 198

induced negligible IL-1β. The response to LPS plus pLL (as well as to LPS alone) was 199

reduced by the caspase-1 inhibitor (Fig. 3e). Therefore, LPS can act as a priming 200

stimulus, and pLL as a second signal, in an assay format that is not designed to 201

elicit/measure NLRP3 inflammasome activation. In contrast to IL-1β, effects of pLL on 202

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the LPS-driven expression of CD86, CD40, IL-10 and IL-12/23p40 were completely 203

unaffected by chemical inhibition of caspase-1 (Fig. 3a-d). Moreover, the effects of pLL 204

on CD86, CD40, IL-10 and IL-12/23p40 were also independent of NLRP3, in the same 205

assay format (Fig. 4a-d). 206

Of note and in agreement with previous reports (39, 46, 47), alum also inhibited CD40 207

up-regulation and IL-12/23p40 secretion and potentiated IL-10 secretion in the context 208

of LPS stimulation (Fig. 4a, c, d); these effects were also independent of NLRP3 (Fig. 209

4a, c, d). 210

In sum, the effects of pLL on the LPS-induced changes in CD40, CD86, IL-10 and IL-211

12/23p40 are independent of NLRP3 inflammasome activation. 212

213

The IL-1β responses induced by pLL require actin dynamics, Syk and PI3K signaling 214

As previously mentioned, the effects caused by pLL on the LPS-induced BMDC 215

responses in terms of CD86, CD40, IL-10 and IL-12 are abrogated by inhibitors of actin 216

dynamics or PI3K class I, as well as affected by Syk inhibitors (35, 36). Also, NLRP3-217

dependent responses to particulate stimuli have been reported to depend on actin 218

dynamics, PI3K and/or Syk (17-21, 46). Thus, we wondered, if the NLRP3-dependent 219

responses to pLL shared these mechanistic requirements. Indeed, IL-1β production 220

elicited by pLL was strongly inhibited by blockade of actin polymerization 221

(Cytochalasin D; Fig. 5a), Syk signaling (piceatannol; Fig. 5b) or a pan-PI3K inhibitor 222

(wortmannin; Fig. 5c). Moreover, inhibition of either PI3K class I (Suppl. Fig 4) or 223

PI3K class III individually led to a similar reduction in IL-1β release (Fig. 5 c). Whereas 224

IL-1β secretion in response to alum displayed similar requirements, the corresponding 225

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response to the non-particulate stimulus ATP was unaffected or only weakly inhibited 226

(Fig 5a-c). 227

We previously reported that exposure of BMDC to pLL blunts the activation of the 228

PI3K class I effector Akt elicited by disparate agonists (LPS, GM-CSF, IL-4) (36, 37). 229

This is a somewhat paradoxical effect in view of the overarching requirement for PI3K 230

class I activity for pLL to affect BMDC responses (Fig. 5c, Suppl. Fig. 4; (36)). 231

Interestingly, alum did not share the capacity of pLL to blunt Akt phosphorylation in 232

response to LPS (Suppl. Fig. 5), suggesting that BMDC responses to pLL and alum 233

have mechanistic differences, and that the effect of pLL on Akt is not a direct 234

consequence of the participation of PI3K in the overall effects of the particles. 235

In sum, induction of NLRP3-dependent IL-1β production by pLL required several 236

elements of the phagocytic machinery, and these requirements were shared by the 237

particulate stimulus alum but not by the soluble stimulus ATP. 238

239

Laminated layer particles induce IL-1β in the absence of particle phagocytosis 240

Based on existing knowledge one would assume that the requirements of actin 241

dynamics, Syk and PI3K signaling for pLL to induce IL-1β are due to pLL particles 242

needing to be internalized to trigger NLRP3 inflammasome activation. Indeed, 243

particulate NLRP3 inflammasome activators are generally thought to act via 244

phago(lyso)somal destabilization, which requires prior internalization (1, 22, 23, 48). 245

However, we have so far been unable to detect successful phagocytosis of pLL particles 246

by BMDC (unpublished observations). Further, a pLL preparation in which all particles 247

are too large for phagocytosis (pLLNP

), which we previously showed to have effects 248

similar to pLL in terms of IL-10, IL-12, CD86 and CD40 (36), also elicited a clear IL-249

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1β response (Fig 6a). This response was quantitatively weaker than the response to pLL 250

at the same dry mass dose; this is expected if the effects of pLL originate at the cell 251

surface, since at equal masses pLLNP

has less total surface area than pLL. Moreover, the 252

response to pLLNP

was sensitive to PI3K class I, PI3K class III, Syk, and actin 253

polymerization inhibitors, similar to the response to pLL (Figs. 6b and Suppl. Fig. 6), 254

suggesting that the same mechanisms are operative in both cases. 255

Thus, interaction with LL particles at the cell-surface can trigger NLRP3-dependent 256

responses, which nonetheless require elements of the phagocytic machinery. 257

258

pLL induces IL-1β in vivo 259

To verify whether activation of the NLRP3 inflammasome by particles from the LL of 260

E. granulosus may occur in vivo, pLL was injected i.p. into C57BL/6 mice with or 261

without co-injection of LPS (Fig. 7). As expected, LPS instillation induced detectable 262

IL-1β in the lavage fluid of treated animals. In line with our in vitro data, pLL 263

drastically enhanced release of IL-1β, confirming its capacity to act as NLRP3-264

inflammasome-trigger. In the absence of LPS co-injection, pLL induced modest but 265

significant levels of IL-1β, in line with in vivo results with known particulate NLRP3 266

inflammasome activators (14). 267

268

DISCUSSION 269

In this work we report that a non-cellular insoluble helminth-derived material (pLL) 270

elicits NLRP3 inflammasome-dependent cytokine production in primed BMDC by a 271

mechanism requiring the phagocytic machinery but not particle internalization. 272

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Together with our previous results (36) the observations in this work suggest that all the 273

responses elicited by pLL in BMDC start with a MATS-like cell surface interaction. 274

This is in contrast to Schistosoma mansoni soluble egg antigen, which activates the 275

NLRP3 inflammasome by a mechanism requiring the receptor dectin-2 and is 276

insensitive to inhibition of actin dynamics (3). 277

Downstream of the proposed MATS-like cell surface interaction, pLL elicits responses 278

that are both NLRP3/caspase 1–dependent and –independent (Figs. 2 - 4). Parallel 279

NLRP3/caspase 1–dependent and –independent responses in DC or macrophages have 280

also been observed for crystalline particulate adjuvants (14, 49) (results for alum in the 281

present work; Fig. 4). This suggests that eliciting both NLRP3-dependent and 282

independent responses may be a general feature of materials that trigger MATS-like 283

signaling, independently of the type of material. However, some mechanistic features 284

must differ with the material eliciting the signaling, as suggested by the ability of pLL 285

but not alum to inhibit Akt activation (Suppl. Fig. 5). We previously proposed a 286

tentative mechanism to explain the paradoxical effect of pLL on Akt activation, based 287

on competition for the PI3K class I substrate phosphatidylinositol 4,5-bisphosphate 288

between the synapse with pLL and conventional PI3K-coupled receptors (36). If this 289

mechanism is correct, it may not be operative in the synapse with alum, due to 290

qualitative, or perhaps quantitative, differences with the synapse formed with pLL. 291

The IL-1β response by pLL was abrogated by PI3K class III inhibitors (Figs. 5 and 6). 292

An explanation for this observation is made difficult by the current major uncertainties 293

surrounding the mechanisms of NLRP3 inflammasome activation (23, 50). However, 294

since our results suggest PI3K class III is needed for NLRP3 inflammasome activation 295

in response to pLL/pLLNP

and alum but not to ATP (Figs. 5 and 6), the possibility 296

should be considered that this enzyme’s role is fulfilled at the synapse with particles. 297

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Neither pLL nor alum (39) appear to be internalized by DCs. Particles that cannot be 298

phagocytosed trigger “digestive exophagy”, in which the extracellular equivalent to a 299

lysosome is formed at the synapse with the particles (51), and PI3K class III is needed 300

for (conventional) phagolysosome biogenesis (52). Exophagy has been observed in 301

particular with the cell model used in our work (BMDC generated in the presence of 302

GM-CSF), in response to aggregated LDL, a probable NLRP3 inflammasome trigger 303

(53, 54). Therefore, we reason that elements of digestive exophagy may be required for 304

NLRP3 inflammasome activation by pLL and alum (and perhaps further particulates) 305

and this would explain the requirement for PI3K class III. In contrast, digestive 306

exophagy would not be needed for the NLRP3-independent responses to particulates 307

that also take place in parallel, as suggested by our previous observation that the effects 308

of pLL on LPS-elicited CD86, CD40, IL-10 and IL-12 do not require PI3K class III 309

(36). Integrating the ideas previously discussed, an interaction with MATS-like 310

requirements in the context of frustrated phagocytosis would trigger two lines of 311

signaling: PI3K class III-dependent (possibly exophagy-dependent) signaling leading to 312

NLRP3 inflammasome activation, and PI3K class III-independent signaling leading to 313

changes observed in other LPS-initiated responses; this is summarized in Suppl. Fig. 7. 314

In addition to the mucin-based aqueous gel, the E. granulosus LL contains nano-315

deposits of calcium inositol hexakisphosphate (InsP6) (55-57). BMDC responses to LL 316

materials in terms of CD86, CD40, IL-12 and IL-10 are not appreciably affected by the 317

presence or absence of this component (35). In vitro IL-1β production by BMDC was 318

also not appreciably affected by the presence or absence of calcium InsP6 (data not 319

shown). Thus, the LL mucin-based gel appears to be sufficient to trigger the proposed 320

MATS-like interaction with the BMDC surface. 321

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As mentioned, insoluble materials so far reported to activate the NLRP3 inflammasome 322

are crystalline or otherwise rigid. Accordingly, NLRP3 inflammasome activation by 323

these materials is generally accepted to be triggered by phago(lyso)somal disruption 324

after particle phagocytosis (1, 22, 23, 48). However, some authors have demonstrated 325

NLRP3 inflammasome activation by crystals immobilized to plastic or by particles too 326

large for phagocytosis (58, 59). These authors propose that a MATS-like cell surface 327

interaction may also trigger NLRP3 inflammasome activation. Our results support this 328

proposal and importantly extend NLRP3 inflammasome activation to a material that is 329

an aqueous gel, and hence soft. Materials-level features of soft non-cellular materials 330

probably determine their potentials to activate the NLRP3 inflammasome. This is 331

suggested by our observation that reduction of disulfides in pLL weakens its capacity to 332

elicit an IL-1β response (Suppl. Fig. 3). Disulfide reduction facilitates the solubilization 333

of the LL mucin meshwork upon sonication (44). We envisage that disulphide reduction 334

alters the physical properties of pLL so that it falls below a minimum level of stiffness 335

required for the MATS-like interaction. 336

The in vivo IL-1β response elicited by pLL was much enhanced by LPS, but it 337

nonetheless reached statistically significant levels in the absence of LPS co-injection 338

(Fig. 7). This is consistent with previous observations suggesting that priming signals 339

for myeloid cell responses to particulate adjuvants are ubiquitous in vivo (14). 340

Therefore, in the E. granulosus infection setting, DCs in contact with shed LL particles 341

probably generate IL-1 (and IL-18). In addition, and as suggested by our observations 342

with non-phagocytosable particles, cells in contact with the surface of the LL as such 343

may also respond with IL-1From precedents in other helminth infections, IL-1 (and 344

IL-18) could contribute to local inflammation, down-regulate Th2 responses, and/or 345

promote Th1 and Th17 responses, both of which are detectable in cystic echinococcosis 346

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(28, 60-62). Shed particles from the E. multilocularis LL (32-34) may also be NLRP3 347

inflammasome triggers and possibly contribute to the considerable inflammatory 348

response observed in human patients (63). 349

Since the survival strategy of larval E. granulosus is based on inflammatory control (25, 350

28, 63), we hypothesize that the parasite may have evolved means to curtail NLRP3 351

activation. A major secreted E. granulosus lipoprotein, “antigen B” (not contained in 352

pLL), inhibits IL-1β output in THP-1 macrophages (64), although it is not yet clear if 353

this effect involves inhibition of NLRP3 activation. 354

pLL is the first biological particulate material to be shown to activate the NLRP3 355

inflammasome independently of phagocytosis. Other non-cellular surface structures 356

shed by tissue-dwelling helminths may share this potential. In particular, it is 357

conceivable that nematode cuticles shed during moulting may be NLRP3 358

inflammasome triggers and thus contribute to local inflammation in response to these 359

helminths. 360

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Figure legends 361

Figure 1. pLL elicits IL-1β and IL-18 production in LPS-primed BMDC. BMDC 362

were primed with LPS (10 ng/ml) for 2 h or incubated in medium only, then incubated 363

for a further 3 h with medium only, pLL (at the indicated doses, given in terms of µg 364

dry mass per million cells), alum (50 µg per million cells), or ATP (2 mM). IL-1β (a) 365

and IL-18 (b) were measured in cell supernatants. Graphs show median and ranges of 3 366

(a) or 2 (b) independent experiments with internal triplicates. Data were normalized 367

over the corresponding responses to alum. Absolute median IL-1β responses to alum in 368

primed cells were 11 ng/mL (range 4 – 27 ng/mL). Absolute median IL-1β responses to 369

pLL in primed cells were 12 ng/mL (range 8-22 ng/mL) (25 µg dose), 6 ng/mL (range 370

2-13 ng/mL) (7.5 µg dose) and 3 ng/mL (range 1-5 ng/mL) (2.5 µg dose). Absolute 371

median IL-1β responses to pLL (25 µg) in non-primed cells were 0.4 ng/mL (range 0.2-372

0.4 ng/mL). Absolute median IL-18 responses to alum in primed cells were 120 pg/mL 373

(range 86-154 ng/mL) and the corresponding values for pLL (25 µg dose) were 54 374

pg/mL (range 34-75 pg/mL). Absolute median IL-18 responses to pLL in non-primed 375

cells were 3 pg/mL (range 0 – 5 pg/mL). Asterisks represent significant differences with 376

respect to the LPS-only (no second signal) condition. 377

378

Figure 2. IL-1β production in response to pLL depends on caspase-1 and NLRP3. 379

BMDC were primed with LPS (10 ng/ml) for 2 h, then incubated for a further 3 h with 380

medium only, pLL (25 µg dry mass per million cells) or alum (50 µg per million cells) 381

and IL-1β was measured in cell supernatants. Thirty minutes before addition of pLL or 382

alum, cells were exposed to the caspase-1 inhibitor Z-YVAD-FMK (a) or to 45 mM 383

KCl (b). Alternatively, cells from NLRP3+/+

and NLRP3-/-

mice were compared (c). 384

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Graphs show median and ranges of 2 (a), 4 (b) or 3 (c) independent experiments with 385

internal triplicates. Data normalized over the corresponding responses to alum in the 386

absence of inhibitors (a, b) and in NLRP3+/+

animals (c). The median absolute values of 387

responses to alum were 21 ng/mL (range 16-27 ng/mL) (a), 19 ng/mL (range 4-47 388

ng/mL) (b), and 14 ng/mL (range 3-34 ng/mL) (c). Median absolute values of responses 389

to pLL in the absence of inhibitors and in wild-type cells were 12 ng/mL (range 11-12 390

ng/mL) (a), 23 ng/mL (8-26 ng/mL) (b), and 14 ng/mL (range 6-33 ng/mL) (c). 391

Asterisks represent significant differences with respect to stimulation with the particles 392

in the presence of vehicle only (a, b) or with respect to NLRP3+/+

cells (c). 393

394

Figure 3. pLL induces caspase 1-independent responses in BMDC in parallel to 395

eliciting caspase-1-dependent IL-1β. BMDC were exposed to pLL (25 µg dry mass 396

per million cells) or medium only, and 1 h later stimulated with LPS (10 ng/ml) or 397

added medium only; the experiment was carried out in the presence or absence of the 398

caspase-1 inhibitor Z-YVAD-FMK. Eighteen h later, expression of CD40 (a) and CD86 399

(b) at the cell surface, and IL-10 (c) and IL-12/23p40 (d) and IL-1β (e) in supernatants 400

were measured. Graphs show median and ranges of 2 independent experiments with 401

internal duplicates. Data were normalized over the corresponding responses to LPS as 402

sole stimulus. The median absolute values of responses to LPS alone were 0.4 ng/mL 403

(range 0.2 – 0.6 ng/mL) for IL-10, 5 μg/mL (range 0.4 – 9 µg/mL) for IL-12/23p40, and 404

6 ng/mL (range 5 – 7 ng/mL) for IL-1β. The median absolute values of responses to 405

pLL + LPS in the absence of inhibitor were 1.3 ng/mL (range 0.5 – 2.1 ng/mL) for IL-406

10, 1.4 μg/mL (range 0.2 – 2.6 µg/mL) for IL-12/23p40, and 20 ng/mL (range 17– 23 407

ng/mL) for IL-1β. Asterisks not associated with connecting lines represent significant 408

differences with cells exposed to vehicle only. Note that only in terms of IL-1β output 409

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were significant differences detected between conditions in the presence vs absence of 410

the inhibitor. 411

412

Figure 4. pLL and alum both elicit NLRP3-independent cellular responses. BMDC 413

from either NLRP3+/+

or NLRP3-/-

mice were exposed to pLL (25 µg dry mass per 414

million cells), alum (25 µg per million cells) or medium only, and 1 h later stimulated 415

with LPS (10 ng/ml) or added medium only. Eighteen h later, expression of CD40 (a) 416

and CD86 (b) at the cell surface were measured by flow cytometry, and IL-10 (c) and 417

IL-12/23p40 (d) in supernatants were quantitated by ELISA. Graphs show median and 418

ranges of 3 independent experiments with internal triplicates. Data were normalized 419

over the corresponding responses of NLRP3+/+

cells to LPS as sole stimulus. The 420

median absolute values of responses to LPS alone in wild-type cells were 1.3 ng/mL 421

(range 0.9-1.7 ng/mL) for IL-10 and 0.6 μg/mL (range 0.3-0.9 g/mL) for IL-12/23p40. 422

Note that no significant differences were detected between conditions in the presence vs 423

absence of the inhibitor. 424

425

Figure 5. Induction of IL-1β production by pLL or alum requires components of 426

the phagocytic machinery. BMDC were primed with LPS (10 ng/ml) for 2 h, then 427

incubated for a further 3 h with medium only, pLL (25 µg of dry mass per million cells), 428

alum (50 µg per million cells), or ATP (2 mM), and IL-1β was measured in 429

supernatants. Thirty minutes before the second signal, cells were exposed to inhibitors 430

of actin dynamics (a), Syk (b) or PI3K enzymes (c). Graphs show median and ranges of 431

2 (a, b) or 3 (c) independent experiments with internal triplicates or duplicates. Data 432

were normalized over the corresponding responses to alum in the absence of inhibitors. 433

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The median absolute values of responses to alum in the absence of inhibitors were 39 434

ng/mL (range 32-45 ng/mL) (a), 19 ng/mL (range 17-22 ng/mL) and 8 ng/mL (range 5-435

11 ng/mL). The median absolute values of responses to pLL in the absence of inhibitors 436

were 20 ng/mL (17-23 ng/mL) (a), 23 ng/mL (range 21-24 ng/mL) (b), and 3 ng/ml 437

(range 3-7 ng/mL) (c). Asterisks represent significant differences with respect to the 438

corresponding conditions in the absence of inhibitors. 439

440

Figure 6. IL-1β production can be induced by pLL in the absence of phagocytosis. 441

BMDC were primed with LPS (10 ng/ml) for 2 h, then incubated for a further 3 h with 442

medium only, pLL, or pLLNP

(at the indicated doses, in µg dry mass per million cells), 443

and IL-1β was measured in supernatants. Different doses of pLLNP

were assayed in 444

parallel to pLL (a), or cells were exposed (30 minutes before pLLNP

) to the pan-PI3K 445

inhibitor wortmannin, the PI3K class I-specific inhibitor GDC-0941, the PI3K class III-446

specific inhibitors Vps34-IN1 and SAR405, or the Syk inhibitor piceatannol (b). Graphs 447

show median and ranges of 3 (a) or 2 (b) independent experiments with internal 448

triplicates. Data were normalized over responses to pLL (25 µg). Median absolute 449

responses to pLL (25 µg) and pLLNP

(75 µg) in the absence of inhibitors were 16 ng/mL 450

(range 8 - 22 ng/mL) and 5 ng/mL (range 2 - 8 ng/mL) respectively. Asterisks not 451

associated with connecting lines represent significant differences with cells stimulated 452

with LPS only. 453

454

Figure 7. pLL elicits IL-1β in vivo. C57BL/6 mice were injected i.p. with vehicle 455

alone (PBS), pLL (150 µg dry mass per mouse), LPS (15 µg per mouse) or pLL + LPS. 456

Three h later, IL-1β was measured in the peritoneal lavage fluid. The graph shows 457

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median and data individual mice pooled from two independent experiments (n = 3 and n 458

= 5). Statistical significances were estimated by a two-way method (specified in the 459

Materials and Methods section), in which the fact that the data arise from two separate 460

experiments is taken into account. 461

462

463

464

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ACKNOWLEDGEMENTS 465

This work was funded by Wellcome Trust Project Grant 092752 (to AD and JEA) and 466

CSIC Grupos project No 977 (to AD together with A.M. Ferreira). AP was supported by 467

studentships from ANII and CAP. The authors are very grateful to M.Sc. Carlos 468

González (Montevideo, Uruguay) for his expert statistical advice. They are also grateful 469

to Yamila Martínez for her participation in the preparation of pLLNP

. 470

471

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on October 9, 2020 by guest

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