Journal Club. May 1 st 2015

Transcription

1 Journal Club May 1 st 2015

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3 INTRODUCTION - What Turns a Commensal into a Pathogen? certain members of intes4nal microbiota have been linked to inflammatory responses and intes4nal pathology (e.g. Bacteroides spec. and members of Enterobaceriaceae family can promote coli4s) however, signalling pathways by which resident bacteria s4mulate host immune system to induce inflamma4on in vivo remain poorly understood cri4cal step: recogni4on of microbes or endogenous molecules in the sedng of infec4on or cellular injury by host PRRs major innate signalling pathway: inflammasome

4 INTRODUCTION The NLRP3 one of four canonical inflammasomes (ac4va4on of caspase- 1) Ac4va4on of NLRP3 inflammasome requires two signals 1. TLR4 signalling induces NFkB- mediated expression of IL- 1β and NLRP3 2. several DAMPs and PAMPs lead to deubiqui4na4on of NLRP3 Assembly of NLRP3 inflammasome promotes proximity induced autoac4va4on of caspase- 1 Caspase- 1 converts pro- IL1β and pro IL- 18 into secreted bioac4ve cytokines controversial role of NLRP3 and IL- 1β in intes4nal inflamma4on: NLRP3-, caspase- 1 and IL- 1β deficient mice protected from coli4s or more suscep4ble? differences in these studies could be caused by baseline differences in the gut microbiota some commensals, such as E. coli, can induce IL- 1β in BMDM BUT ability of resident bacteria to induce IL- 1β in vivo and the innate immune cells involved remains unclear Lamkanfi & Dixit 2014

5 Steady- state Coli:s commensal bacteria epithelial damage commensal bacteria pathobionts e.g. P. mirabilis resident mononuclear phagocytes Hyporesonsive to commensal bacteria (no NLRP3 response) recruited monocytes IL- 1β other inflammatory cytokines inflamma4on

6 Intestinal Resident Bacteria Induce IL- 1β Release in the Context of Intestinal BMDMs s4mulated with faecal contents (FC) An4bio4c cocktail i.g., daily (B) 2,5 % DSS (B) or 2 % DSS (C+D) D- 1 D0 D6 1. Treatment with an:bio:cs 2. Intes:nal Inflamma:on 3. LP cell culture D7 0 3 h Fig. 1

7 Intestinal Resident Bacteria Promote Colitis via IL- 1β An4bio4c cocktail i.g., daily (D- F) 2 % DSS (A- F) D- 1 D0 D6 WT vs. IL- 1β k.o. 1. Treatment with an:bio:cs 2. Intes:nal Inflamma:on D10 Colon length Pathology Score Fig. 2

8 Commensal Bacteria Induce IL- 1β via the NLPR3 BMDMs s4mulated with faecal contents (FC) for 18 hrs NLRP3 ASC caspase- 1 open arrows: non- specific bands Fig. 3

9 Commensal Bacteria Induce IL- 1β via the NLPR3 2,5 % DSS (B) or 2 % DSS (C+D) 2. LP cell culture D0 D7 1. Intes:nal inflamma:on 0 3 h Fig. 3

10 Steady- state Coli:s commensal bacteria epithelial damage commensal bacteria resident mononuclear phagocytes Hyporesonsive to commensal bacteria (no NLRP3 response) cellular source?? NLRP3 ac:va:on IL- 1β inflamma:on

11 Recruited Monocytes are the Major Source of IL- 1β Produced in Response to the Microbiota 2,5 % DSS 2. LP cell culture D0 D6/D7 ± 5 mm ATP 1. Intes:nal inflamma:on 0 3 h (B) 3,5 h (A) Fig. 4

12 Recruited Monocytes are the Major Source of IL- 1β Produced in Response to the Microbiota 2-2,5 % DSS 2. LP cell culture D0 D6/D7 1. Intes:nal inflamma:on 0 3 h NLRP3 ASC FACS sorted cells (CD45+) CD11b + Ly6C high = inflammatory monocytes CD11b + Ly6C int = phagocytes CD11b - Ly6C - = non- phagocytes Fig. 4

13 Recruited Monocytes are the Major Source of IL- 1β Produced in Response to the Microbiota 1. Genera:on of mixed BM chimeras WT : Ccr2 DTR/+ 2. Deple:on of CCR2+ cells 3. LP cell culture total cells 10 µg/kg DT i.p. Il1b - /- : Ccr2 DTR/+ WT lethally irradiated D0 D3 D6 D7 0 3 h Fig. 4

14 Steady- state Coli:s commensal bacteria epithelial damage commensal bacteria?? resident mononuclear phagocytes Hyporesonsive to commensal bacteria (no NLRP3 response) recruited monocytes CCR2 + Ly6C hi NLRP3 ac4va4on IL- 1β inflamma:on

15 Selective Resident Bacteria Elicit Robust Release of IL- 1β 3 hrs BMDMs s4mulated with indicated mouse commensal bacteria (isolates) in 1:1 ra4o 3 hrs cell culture Cr Ebc Ebh Ecc Ecf Ecg Ec Ko Kp Lm Lr ST Ss Sx Pm Citrobacter roden;um Enterobacter cloacae Enterobacter hormaeche Enterococcus casseliflavus Enterococcus faecalis Enterococcus gallinarum Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Lactobacillus murinius Lactobacillus reuteri Salmonella enterica serovar Typhimurium Staphylococcus sciuri Staphylococcus xylosus Proteus mirabilis Fig. 5

16 Selective Resident Bacteria Elicit Robust Release of IL- 1β 18 hrs BMDMs s4mulated with indicated mouse commensal bacteria (isolates) in 1:1 ra4o 18 hrs cell culture Cr Ebc Ebh Ecc Ecf Ecg Ec Ko Kp Lm Lr ST Ss Sx Pm Citrobacter roden6um Enterobacter cloacae Enterobacter hormaeche Enterococcus casseliflavus Enterococcus faecalis Enterococcus gallinarum Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Lactobacillus murinius Lactobacillus reuteri Salmonella enterica serovar Typhimurium Staphylococcus sciuri Staphylococcus xylosus Proteus mirabilis Bacteroides and Clostridia species were also tested but did not induce IL- 1β release (data not shown) Fig. S2

17 Selective Resident Bacteria Elicit Robust Release of IL- 1β 1. Mono- associa:on of GF mice 2. Intes:nal inflamma:on 3. LP cell culture Ba Bacteroides acidifaciens 1 % DSS Cs Ec Clostridium sporogenes Escherichia coli D0 D7 Pm Proteus mirabilis 0 3 h S3 BMDMs s4mulated with indicated bacteria for 3 hrs Fig. 5

18 Selective Resident Bacteria Elicit Robust Release of IL- 1β D0 2,5 % DSS 1. Intes:nal inflamma:on various k.o. mice 2. LP cell culture D7 s4mula4on with bacteria (1:1) 0 3 h 2 % DSS D0 1. Intes:nal inflamma:on GF or P. mirabilis colonized mice D6 D7 2. LP cell culture 0 3 h Fig. 5

19 P. mirabilis HpmA Hemolysin is Required for the Activation of the NLRP3 human P. mirabilis (WT) H14320 UM001 mouse P. mirabilis Common virulence genes chew Chemotaxis flif Flagellin hpma Hemolysin mrpa Fimbria spa47 T3SS urec Urease S:mula:on of BMDMs with P. mirabilis strains 100 ng/ml LPS WT or isogenic mutants 0 3 h 6 h 100 ng/ml LPS or ΔhpmA Pm ± 5 mm ATP 0 3 h 3,5 h IL- 1β signalling via NLRP3 inflammasome requires Signal 1 (e.g LPS): induces pro- IL- 1β) Signal 2 (e.g. ATP): ac4vates caspase- 1 Fig. 6

20 P. mirabilis HpmA Hemolysin is Required for the Activation of the NLRP3 Fig. 6 K + efflux is a common event required for NLRP3 ac4va4on

21 Fig. 7 P. mirabilis Colonization Enhances DSS- Induced Colitis via NLRP3- Mediated IL- 1 Signalling

22 Steady- state Coli:s commensal bacteria epithelial damage commensal bacteria pathobionts e.g. P. mirabilis resident mononuclear phagocytes Haemolysin A Hyporesonsive to commensal bacteria (no NLRP3 response) recruited monocytes CCR2 + Ly6C hi NLRP3 ac4va4on other inflammatory cytokines IL- 1β inflamma:on

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24 THE MOTIVATION Pathogenesis of inflammatory bowel diseases involves dysregulated inflammatory CD4 + T cell responses to commensal bacteria self- specific T cells are nega4vely selected in the thymus by presenta4on of self- an4gens on thymic epithelial cells (TECs) and DCs resul4ng in clonal dele4on commensal bacteria- specific CD4 + T cells are not nega4vely selected in the thymus they encounter their an4gen in the periphery, e.g. in the MLN, where DCs present commensal- derived an4gens that have been sampled from the intes4nal lumen Treg can in part limit dysregulated CD4 + T cell responses to commensal bacteria ILC3s were found to express MHCII and to limit commensal- specific CD4 + T cell responses in a MHCII dependent manner

25 CCR6 + T- bet - NKp46 - (Lymphoid 4ssue inducer- like) ILC3s express MHCII molecules and are able to process and present an4gen MHCII + ILC3s maintain intes4nal homeostasis by limi4ng pathological CD4 + T cell responses to commensal bacteria through MHCII- dependent mechanism

26 RORγt + CCR6+ ILC3s in MLN and clp Express MHCII Fig. 1 gated on: CD45 + LIN (x): CD3, CD5, CD8, NK1.1 LIN (y): B220, CD11c, CD11b C57BL/6 naïve Fig. S1

27 MHCII Expression by ILC3s is Controlled by a Transcriptional Pathway Previously Associated with TECs CIITA = class II transac4vator ; master transcrip4onal regulator of MHCII expression MHCII expression by cells other than APCs and TECs needs specific s4muli, par4cularly IFN- γ Reith et al MLN Fig. 1 Do TECs and ILC3s share a similar func:onal roles in the selec:on of CD4 + T cells? Fig. S3

28 Fig. S4 MHCII + ILC3s Control Commensal Bacteria- speci@ic CD4 + T Cell Responses Through Direct Presentation of Microbiota- derived Antigens Rorc cre H2- Ab1 fl/fl MHCII ΔILC 1. Purifica4on of CD4 + T cells from MLN and spleen 2. Labelling with CFSE 3. S4mula4on with 50 µg/ml of faecal or 4ssue an4gen prepara4ons (prepared from homogenized 4ssues or content)

29 ILC3 Intrinsic MHCII Selectively Controls Commensal Bacteria- CD4 + T cells in the Intestine CBir1 Vβ8.3 CBir1 pep4de OR OT- II Vβ5 OVA pep4de X MHCII ΔILC Fig. 2 Fig. S5

30 Generation of ILC3- restricted MHCII Mice and T cell Transfer Model T cell transfer ac:vated cells 1. Enrichment of T cells (MACS) 2. Ac4va4on for 18 hrs with APCs (ra4o 10:1) and 1 µg/ml αcd3/αcd28 3. Purifica4on of CD4 + T cells (FACS) 4. Transfer of 5 x 10 6 cells µg CBir1 or OVA pep4de i.p. every 2 days CBir1 T cells or OVA T cells (Cd ) MHCII pos MHCII neg MHCII ILC+ Fig. 2

31 Fig. 2 MHCII + ILC3 Control Commensal- speci@ic CD4 + Teff in the Intestine and Associated Lymph Nodes CBir1 T cells (CD ) MHCII pos MHCII neg MHCII ILC+

32 MHCII + ILC3s Directly Induce Apoptosis of Commensal Bacteria- speci@ic CD4 + T cells purified ILC3s 5x µg/ml CBir or OVA pep4de 1 hr 1:2 48 hrs purified CD4 + T cells 10x µg/ml CBir or OVA pep4de 24 hrs whole MLN & splenic lymphocytes Fig. 3

33 MHCII + ILC3s Induced Cell Death of Commensal Bacteria- speci@ic CD4 + T cells is Mediated by the Bcl- 2 Family Member Bim purified ILC3s 5x µg/ml CBir or OVA pep4de 1 hr 1:2 48 hrs purified CD4 + T cells 10x µg/ml CBir or OVA pep4de 24 hrs whole MLN & splenic lymphocytes upregula4on of Nur77 and Bim have been associated with nega:ve selec:on in the thymus Fig. 3

34 MHCII + ILC3s Directly Induce Apoptosis in Commensal Bacteria- speci@ic CD4 + T cells by Cytokine and Growth Factor Starvation Fig. 3 CA cons:tu:vely ac:ve STAT- 5

35 Fig. 4 ILC3 Intrinsic MHCII is Dysregulated in Paediatric Crohn s Disease Patients and is Associated with Increased Intestinal Th17 Cells

36 SUMMARY Intestinal Selection of Commensal- CD4 + T cells by CCR6 + MHCII + ILC3s

37 THANK YOU!

38 ILC3???? DC macrophage N BH cell?????? MRC MZ B cell TLR ligands ASC