the type 2 immune response -a manipulative exit strategy ... · 3 cryptococcus neoformansinfection....

The Type 2 immune response - a manipulative exit strategy for

Cryptococcus neoformans

Pavithra Ramakrishnan

T35 Medical Student Summer Immunology ProgramNielsen Laboratory 2019

Global burden of HIV associated cryptococcal meningitis

• Leading infectious cause of death in individuals with AIDS

• 15% of AIDS-related mortality globally• Uganda accounts for 60% of adult

meningitis

• Mortality rate is 65%, even with treatment

Rajasingham et al., 2017

CNS DiseaseDisseminationInitial LungInfection

EnvironmentalExposure

CFU

s

Time

Clearance

3

Cryptococcus neoformans infection



CFU

s

Time

LatencyClearance

4




CFU

s

Time

LatencyClearance

5




CFU

s

Time

AcuteLatencyClearance

6




CFU

s

Time

AcuteLatencyClearance

7




8


What traits allow Cryptococcus to cause disease in the host?

How does the host prevent infection?

��

�

Extracellular Pathogens

Intracellular Pathogens

ParasitesFungi

Wound healing

��

Mammalian CD4 T-cell differentiation impacts patient mortality

Modified from May et al., 2016 Nat Rev Micro

Type 1

Type 2

Low Mortality

��

�

ParasitesFungi

Wound healing

Extracellular Pathogens

Intracellular Pathogens

Type 1

Type 2

High Mortality

��

Modified from May et al., 2016 Nat Rev MicroBoulware et al., 2010 PLoS Med

Mammalian CD4 T-cell differentiation impacts patient mortality

Modulation of the immune response by Cryptococcustitan cells

May et al., 2016

?

Hypothesis

Cryptococcus survives within and disseminates using M2 macrophages produced by titan cell-induced Th2 proliferation.

• Does titan cell induced Th2 response drive M2 macrophage polarization in vivo?

• Are M2 macrophages required for dissemination and crossing of the BBB via a trojan horse mechanism?

Experimental setup and gating strategy

Day 0 Day 14

Harvest: BALIsolate Leukocytes for flow cytometry

5x104

KN99αIntranasal

Day 3

Day 9

Day 6

Day 13

• Th2 response drives M2 macrophage polarization in vivo

• CD11b+ CD11c+ CD24+ dendritic cells predominate in the lung over the course of acute infection

CD 6

4

CD 24

CD 11b

CD 11c

Arg1RELMαCD71

CD 80/86

YFP

mcherry

Macrophages

Dendritic Cells

ArgYFP mice infected with mCherry-expressing Cryptococcusshow phagocytosis by type 2 cells in the lungs

Monocytes

Dendritic Cells

ArgYFP mice infected with mCherry-expressing Cryptococcusshow phagocytosis by type 2 cells in the lungs AND BLOOD

CD 1

15

CD 24

Arg1RELMαCD71

CD 80/86

YFP

mcherry

Results

• M2 polarization of macrophages is observed in the lungs during acute Cryptococcus infection.

• A large population of dendritic cells that may also be type 2 polarized is also produced during the acute infection in the lungs and could be another population that is involved in dissemination of Cryptococcus.

• Several populations of type 2 monocytes are present in blood that contain Cryptococcus.

Future work

• Develop a gating strategy to analyze dendritic cell subsets in BAL, blood and mediastinal lymph nodes

• Develop fluorescently labelled reporter strains of latent Cryptococcus neoformans strains that do not disseminate and may produce an M1 polarized immune response

• Test the therapeutic effects of various strategies that mute the type 2 immune response

Re-programming the immune response

Potentiating Type 1• Harrison et al., 2012

• IFNγ adjunctive short course therapy reduced CSF fungal burden in HIV associated cryptococcal meningitis

• was not associated with adverse events

• BUT did not improve patient survival

May et al., 2016

Re-programming the immune response

Blocking Type 2• Anti-IL13/IL4 mAb therapy

• Already FDA approved for asthma therapy (Regeneron)

Acknowledgements

Dr. Kirsten Nielsen Jackson EdgellMarina Yoder

Dr. Sophie Altamirano

Dr. Jesse Williams Dr. Tanya Freedman

Dr. Kaylee Schwertfeger

Dr. Daniel MuellerStephanie Krischuk

Thank you!

Are M2 macrophages required for dissemination and crossing the BBB via trojan horse mechanism?

Strain Dissemination Disease Th response MØ activation statusKN99α + + Th2 M2∆gpr4∆gpr5 - - Th2 M2UGCL 552 - - Th1 M1UGCL 223 + - Th1 M1

the type 2 immune response -a manipulative exit strategy ... · 3 cryptococcus neoformansinfection....

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