1. Presented by Abigail Axenfeld
Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism
Kairui Mao, Antonio P. Baptista, Samira Tamoutounour, Lenan Zhuang, Nicolas Bouladoux, Andrew J. Martins, Yuefeng Huang, Michael Y. Gerner, Yasmine Belkaid, and Ronald N. Germain
Nature Volume 554. February 8th 2018
Presented by Abigail Axenfeld

2. Background
Gut of mammals colonized by microbes collectively = microbiota
Many known aspects of immunity occur sequentially – innate followed by adaptive.
NK cells previously thought to be only innate immune cells in lymphoid line
Now we know of helper-like ILCs
STAT3 is a transcription activator that mediates expression of many genes
important role in cellular processes such as cell growth and apoptosis
Phosphorylation of STAT3 = activation of STAT3
Weaning is when bacterial colonization occurs in newborns
Rag1-/- cells are cells lacking adaptive immunity

3. Abstract - summary
Microbiota matures during ontogeny to state of balanced commensalism indicated by no inflammation.
ILCs (innate cells) & CD4 T cells (adaptive cells) assumed to have same fxns in containment and clearance of pathogens,
But how these cells each contribute to shape mature microbiome was previously unknown.
Here we find extensive/persistent pSTAT3 signature in group 3 ILCs & IECs induced by IL-23 & IL-22 in mice lacking T cells (adaptive-immune-deficient).
Contrarily, pSTAT3 in immunocompetent mice induced only transiently by microbial colonization at weaning… the early signature eliminated as T cell immunity develops in response to commensal bac presence.
Persistent IL-22 prod. from ILC3s in absence of T-cells = impaired host lipid metabolism.

4. Hypothesis
ILC3 and CD4 T-cells have distinct functions and function sequentially in establishing commensalism of gut microbiota/tissue metabolic homeostasis.

5. Methods quantitative multiplex immunochemistry (histo-cytometry)
Allows for: examination of cell distribution and topography of cytokine response
Scanning Electron Microscopy
Morphologic characteristics
Whole-tissue RNA sequencing (RNA-seq)
qPCR
-Used 1 staining for cell phen & molecules indicating cytokine signaling, we can examine cell distr, cytokine production, and topography(graphic detail/structure) of cytokine responses in complex tissue.
-pSTAT3 in distal small bowel b/c many diff cytokines can induce pSTAT3
Controls: -innate/adaptive cells in situ studied only in ileum

6. Specific Aims i. What does pSTAT3 have to do with ILC3/IECs
ii. What cytokines are involved in ILC vs IEC STAT activation
iii. Is innate response to microbiota activated before adaptive immune system develops?
iv. What are exact functions of innate vs adaptive lymphocytes in regard to microbiota?
v. Which host adaptive immune cells prevent ILC3 activation/IEC signaling- T cells or B cells?
vi. CD4 or CD8 T cells responsible for preventing ILC3 activation?
vii. Does persistent ILC3 in adaptive-immune-deficient cells negatively affect homeostasis?
viii. Were these lipid abnormalities specifically due to IL-22?

7. Results: i. Fig.1 (what does pSTAT have to do with ILC3/IECs?)
-WT vs Rag1-/-
-CD3-RORyt+
& EpCAM
-cryptopatch vs villi
-SPF  abx
-GF SFB
Lots of pSTAT3 found in ILC3 & IECs of Rag-/- but not WT
Do microbes have a role in pSTAT3 presence?
Abx eliminated pSTAT3 signals
Segmented filamentous bac activates STAT3 in GF mice
Conclusion: STAT3 activation arises from signaling induced by microbiota
Cryptopatch vs villi = 2 diff areas of the ileum
Roryt indicates ILC3 EpCAM indicates IEC
Fig. 1

8. ii. Extended data Fig. 2
(What cytokines are involved in ILC vs IEC STAT activation?)
ILC3 pSTAT is dependent on IL-23
IEC pSTAT is dependent on both IL-23/22
Conclusion: since only ILC3 express IL-23 receptor, its likely activation of STAT3 in ILC3 and IEC were sequential events
Fig. 2c confirmed ILC3 is needed for pSTAT in IEC
Fig. 2d confirmed IL-22 is prod. by pSTAT+ ILC3s
ILC3- doesn’t matter if theres IL-22, only dpd on IL-23
No IL-23 no pSTAT in ILC3/IEC
No IL-22 = pSTAT in ILC3 but not IEC

9. Extended data Fig. 3 What is the source of IL-23?
Diff myeloid cell populations were isolated and dendritic cells (CD11b+) &(CCR2+) both express IL23a, but CCR2+ from Rag1-/- had more IL23a than WT
Mice depleted of CCR2+ via CCR2 antibody & in 2 weeks there was no pSTAT+ ILC3s/IECs
Conclusion: CCR2+ dendritic cells produce the IL-23 responsible for the pSTAT signature in Rag1-/- mice
-dendritic cells(CD11b+) and monocyte-derived dendritic(CCR2+)
Interesting bc we’re looking for cells responsible for IL-23 in Rag-/- deficient mice

10. Summary so far
In presence of gut microbiota (SFB) , significant pSTAT3 found prolonged in ILC3s &IECs of Rag-/- mice (lacking adaptive immunity).
ILC3 STAT3 activation & IEC STAT3 activation is sequential.
CCR2+ cells prod IL-23  pSTAT in ILC3s. pSTAT+ ILC3s prod IL-22  pSTAT in IECs
This circuit activated by commensal microbiota produces strong signaling in epithelial tissue in the absence of adaptive immunity

11. iii. Fig. 2
(Is innate response to microbiota activated before adaptive immune sys. develops?)
Progeny of SFB+ mothers
Neither WT or Rag1-/- progeny showed pSTAT3+ ILC3/IECs initially.
After weaning  substantial activation of STAT3 in ILC3 & IEC WT
As adaptive immune system matures, ILC3s no longer activated in WT but remain in Rag1-/-
Conclusion: during ontogeny, Innate Lymphoid Cells operate before adaptive sys. is developed. Adaptive sys then halts ILCs to create homeostatic state of microbiota.
Lack of IL-22-dependent IEC signaling in WT suggests innate cells activated by microbiota before adaptive immune system delevlops

12. iv. Extended data Fig 4.
(What are exact functions of innate vs adaptive lymphocytes in regard to microbiota?)
In Rag1-/-, (no adaptive) bac amt increased. In Rag1-/-IL23-/- (no pSTAT3/innate or adaptive) bac further increased (4.a)
Both innate&adaptive effector molecules regulate bac amt, but SFB morphology differed whether adaptive or innate lymphocytes present….
Activated ILC3s (in Rag-/-) prevented long length SFB, (4.c) & adaptive lymphocytes limited # SFB attached to epithelial cells (4bWT)
Conclusion: innate lymphocytes have diff mechanisms of regulating microbiota. Adaptive dominates innate.
measured amt of diff bac species in ileum of WT, Rag1-/-, and IL23-/-Rag1-/- mice

13. v. Fig. 3 & Extended data Fig 5.
(Which host adaptive immune cells prevent ILC3 activation/IEC signaling- T cells or B cells?)
No T cells = pSTAT.
When cohoused WT, Rag1−/−, Tcra−/− (T cell-deficient) and Ighm−/− (B cell-deficient) mice… pSTAT found in ILC/IEC from Tcra but not Ighm (fig. 3a.)
This is consistent with increased SFB in cohoused (ext. data 5c)
Conclusion: T cells (not B cells) critical for preventing ILC3 activation/IEC signaling induced by SFB
Fig. 3
Need SFB for Rag1-/- pSTAT signature… only Tcra cohoused with Rag1-/- showed pSTAT
Extended data 5c.
Fig. 3

14. vi. Fig. 3 contd…
(CD4 or CD8 T cells responsible for preventing ILC3 activation?)
After cohousing w/ Rag1-/-, cells lacking CD4 (h2-Ab1) & cells lacking CD4/CD8 had pSTAT3+ ILC/IECs, where cells lacking just CD8 were pSTAT3- (3c,d.)
Were CD4 sufficient to suppress ILC3 activation?
Transferred total CD4+ cells into Rag1-/- found they prevent pSTAT3 in ILC3/IEC.
YES!!!!
examined STAT3 in H2-Ab1−/− (MHCII-deficient/lack CD4), B2m−/− (MHCI-deficient/lack CD8), and H2-Ab1−/−B2m−/− (MHCI&MHCII-deficient/lack both)

15. Summary so far
In presence of gut microbiota (SFB), significant pSTAT3 found prolonged in ILC3s &IECs of Rag1-/- mice (lacking adaptive immunity)
ILC3 STAT3 activation & IEC STAT3 activation is sequential.
CCR2+ cells prod IL-23  pSTAT in ILC3s. pSTAT+ ILC3s prod IL-22  pSTAT in IECs
This circuit activated by commensal microbiota produces strong signaling in epithelial tissue in the absence of adaptive immunity
during ontogeny, Innate Lymphoid Cells operate before adaptive sys. developed. Adaptive sys then halts ILCs to create homeostatic state of microbiota.
SFB critical for STAT3 signature in Rag1-/- cells
Innate lymphocytes affect morphologic characteristics of microbiota(SFB), adaptive more so regulates amount of SFB (still contained)
T cells, specifically CD4 critical for preventing ILC3 activation/IEC signaling induced by SFB

16. vii. Fig. 5
(Does persistent ILC3 in adaptive-immune-deficient mice negatively affect homeostasis?)
Low expression of metabolic processing genes in pSTAT3+ mice (coh tcra/rag1-/-)(5a)
Using quantitative real-time PCR (qPCR), we confirmed decr. mRNA for lipid transporters (Cd36, Npc1l1, Fabp1, Fabp2) in coh tcra&rag1 mice. (5b)
Also decr. Serum levels of triglycerides&FA’s (5c)
Rag1-/- has less fat accumulation than WT (5d)
Conclusion: even tho ILC3s can constrain microbial communities, their persistant acitvation = lipid abnormalities and abnml tissue homeostasis.
Need SFB for Rag1-/- signature pSTAT… only cohoused Tcra
•Whole tissue RNA sequencing (RNA-seq) of ilea from co-housed WT, Rag1−/−, & Tcra−/− (no T cells) mice and separately housed Tcra−/− mice

17. viii. Fig. 5 & Extended data Fig. 6a
(Were these lipid abnormalities specifically due to IL-22 prod. by activated ILC3s?)
When ILC3s (and therefore IL-22) were eliminated in Rorc(γt)GFP/GFPRag1−/− mice (blue ∆), lipid transporter levels, tri, FA, & fat storage increased. {5e-g}
When adenovirus containing IL-22 {ext 7} was administered to WT and non-coh Tcra-/-, lipid transporter levels, etc. were again lowered, proving that IL-22 is indeed responsible for these abnormalities.
Ext data Fig. 7
Fig. 5e-g

18. Final summary
In presence of gut microbiota (SFB), significant pSTAT3 found prolonged in ILC3s &IECs of Rag1-/- mice (lacking adaptive immunity)
ILC3 STAT3 activation & IEC STAT3 activation is sequential.
CCR2+ cells prod IL-23  pSTAT in ILC3s. pSTAT+ ILC3s prod IL-22  pSTAT in IECs
This circuit activated by commensal microbiota produces strong signaling in epithelial tissue in the absence of adaptive immunity
during ontogeny, Innate Lymphoid Cells operate before adaptive sys. developed. Adaptive sys then halts ILCs to create homeostatic state of microbiota.
SFB critical for STAT3 signature in Rag1-/- cells
Innate lymphocytes affect morphologic characteristics of microbiota(SFB), adaptive more so regulates amount of SFB (still contained)
T cells, specifically CD4 critical for preventing ILC3 activation/IEC signaling induced by SFB
In ILC3-persistent pSTAT+ mice, decr lipid transporters, FA, tri, and fat storage.
IL-22 prod. By ILC3s is the specific cause of abnormal lipid metabolism/abnml tissue homeostasis.

19. Idea for future direction of Research
Is it possible to live a long life sparing gastrointestinal complications with lack of adaptive immunity?
What complications/future disease process arise in the gut due to over- activation of ILC3 and IL-22?
Test idea this using longitudinal study of same participants over many years.