Innate lymphoid cells

Transcription

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3 Innate lymphoid cells Cording et al. FEBS 2014 Retioic acid (RA) is a vitamine A metabolite that control early vertebrate development. RA mediates CXCL13 expression in fetal mesenchyal cells, cytokine essential to lymph nodes initiation (Van de Pavert Nature immunol 2009)

4 Fetal development of ILC in secondary lymphoids organs (SLO) (Including Ltin) ENTERIC FETAL ANALISIS E12.5 ENTERIC FETAL TRANSFER UNDER KIDNEY CAPSULE (CD w old)

5 Maternal retinoids control LTi cell differentiation within developing SLO RA treatment or RAR antagonist BMS493 from E10.5 Analyses at E13.5 Lymph nodes RAR antagonist BMS493 from E10.5 Analyses at E13.5 hcd2- GFP (T and B cell reporter mice) RAR antagonist BMS493 from E10.5 Analyses at E17.5

6 Impaired SLO development in BMS treated mice might be the consequence of RA signal ablation in LTi cells RARE: DNA RA response elements E15.5 Enteric cells E15.5 LN cultured 24h with 100nM RA

7 LTi differentiation is controlled by cell-autonomous RA signalling in developing SLO Linage targeted model to block RA signaling: Vav (protein involved in T and B cells development)- icre (Cre acuvity in fetal cells) x ROSA26- RARα403 (truncate form of RARα gene) mice E15.5 fetal liver E15.5 Enteric cells LTi 4 LTi 4neg E15.5 LTi 4neg from gut and LN are cultured for 6d with OP9 E15.5

8 RA might control LTi 4neg differentiation in LTi 4 via RORγt E15.5 intesune E15.5 LTi 4neg (LN/gut) cultured 24h with 100nM RA DIG: digoxin is a antogonist RORgt receptor acuvity E13.5 LTi 4neg (LN) cultured 24h with 100nM RA

9 RA control LTi 4neg maturation in LTi 4 via RORγt E15.5 LTi 4neg transduced with pmig.rorγt- IRES- GFP virus are cultured for 6 days

10 Can RA directly regulate RORγt expression in LTi 4neg? ChIP analyses Sorted E15.5 Embrionic ILC4neg from intesune and pln IncubaUon for 8h with RA or DMSO Cell-autonomous RA signalling provides LTi cells with critical differentiation signals via direct regulation of RORγt

11 A deficit of RA in early life results in small SLO During the pregnancy: VAC: vitamin- A control diet VAH: vitamin- A High diet VAD: vitamin- A deficient diet A`er birth: VAC Analyses 10 weeks old

12 A deficit of RA signals within haematopoieuc cells in early life results in small SLO 2 week old mice Normal SLO organisauon

13 A deficit of RA signals within haematopoieuc cells in early life results in poor capacity to control infecuon i.n p.f.u. Murid HV- 4 Intrathoracic LN

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16 Bacterial colonisation begins immediately after birth Antibiotic treatment in early life induce bacteria dysbiosis and that is associated with the development of IBD, obesity, autoimmune diseases and cancer (Kuppala et al. J Pediatric 2011) Prolonged exposure to antibiotic increase the risk of late-onset sepsis (LOS) Hypothesis: Role of microbiota in susceptibility of neonatal to late-onset sepsis (LOS)

17 Antibiotic exposure reduce total number of intestinal microbes and impact in the composition of intestinal microbiota in neonatal mice 5 antibiotics: ampicillin, gentamicin, metronidazole, vancomicyn and neomycin 3 antibiotics: ampicillin, gentamicin and vancomycin Start at E15 until the end of experiment Intestinal contents Antibiotic treatment doesn t change the gestational age, birth weight or postnatal growth

18 Microbiota could control granulopoesis in neonates Reduction of total number of circulation neutrophils Bone marrow (3d old mice) Reduction of granulocyte/macrophage restricted progenitor No antibiotic toxicity, because same result in GF mice

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20 Antibiotic treatment affect the molecules homing expression in neutrophyles but doesn t their function Bone marrow (5d) Bone marrow (5d)

21 Microbiota-stimulation postnatal granulocytosis play a key role in the susceptibility to E. coli K1 infection Neutrophyles are essential in controlling E. coli K1 infection, preventing LOS and meningitis in neonates (Pluschke et al. Microb Pathog. 1988) D5 after birth: i.p c.f.u./g of E. coli K1 4h post infection 4h post infection Blood/BM 10µg/g of body weight at d3 G-CSF: To restore neutrophils numbers

22 Microbiota control postnatal granulocytosis and modulate host resistance to infection in neonatal D3: i.g. microbiota from 3d old mice (200µg in 50µl) D5 after birth: i.p c.f.u./g of E. coli K1

23 Microbiota regulates postnatal granulocytosis via IL-17A Intestine 2d after oral microbiota transplant Blood Junjie et al. JCI 2012 Blood 3d old mice

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25 Microbiota regulates postnatal granulocytosis by inducing a population of ILC Small intestine Small intestine (3d old mice) Blood (3d old mice) Anti-CD90.2 at d0, d1,d2, d3 IL23 -/- mice have normal number of neutrophils

26 LPS is one of the microbiota-derioved signals regulating postnatal granulocytosis in neonates SI (5d old mice) TLR2 -/- and NOD2 -/- mice have normal number of neutrophils Blood (5d old mice) 10 ng at d1

27 Conclusion: ILC3 NKp46 - Junjie et al. JCI 2012