Figure 2. Intestinal commensal bacteria direct postnatal trafficking of IL-22⁺ILC3 innate lymphoid cells to murine newborn lung.

(A) Representative flow cytometry plots of distinct subsets of IL-22⁺ cells in the lungs of postnatal day 4 (P4) newborn mice. Shown are relative frequencies of IL22⁺ T cells (CD45⁺CD3⁺) or neutrophils (CD45⁺Ly6G⁺) or lineage negative (CD3⁻CD8⁻CD11b⁻CD19⁻F4/80⁻CD161⁻Ly6G⁻F4/80⁻) lymphocytes in the lungs of P4 newborn mice. (B) The relative frequencies of distinct subsets of IL-22⁺ cells in the bronchial lavage (BAL) fluid of human newborn infants.. (C) Absolute numbers of IL22⁺ILC3 in the lungs of P4 wild-type (WT) or Rorγt^iDTR newborn mice treated with diphtheria toxin (DT) or no DT. (D) Survival of P4 WT or Rorγt^iDTR newborn mice treated with DT (ILC3-depleted) that received adoptive transfer of ILC3 and then were infected with S. pneumoniae. (E) The absolute number of IL-22⁺ILC3 in the lungs of ABX-free or ABX-exposed newborn mice at different time points after birth. (F) Representative flow cytometry plots and (G) absolute numbers of IL-22⁺ILC3 in the lungs of P4 GF or ABX-free or ABX-exposed or ABX-exposed newborn mice reconstituted with intestinal commensal bacteria in early life. (H) The absolute numbers of IL-22⁺ILC3 in the BAL fluid of human newborns exposed to ABX or no ABX. (I) ILC3 from P4 ABX-free newborn mice were labeled with carboxyflourosceinsuccimidylester (CFSE). ILC3 from age-matched P4 ABX-exposed or ABX-exposed newborn mice reconstituted with commensal bacteria were labeled with chloromethylbenozylaminotetramethylrhodamine (CMMTR). An equal number of CFSE- or CMMTR-labeled ILC3 were adoptively transferred into ABX-exposed newborn mice. Representative flow cytometry plots and absolute numbers of CFSE⁺ or CMMTR⁺ ILC3 in lung, spleen or small intestine were determined 12 h following adoptive transfer. (J) Relative capability of ILC3 from ABX-free or ABX-exposed or ABX-exposed newborn mice reconstituted with intestinal commensal bacteria in early life to traffic to the lungs (Homing index). Data and plots are representative of three independent experiments. Results are shown as the mean ± s.e.m (Student’s t-test or ANOVA or Wilcoxon signed-rank test). *P ≤ 0.05; **P ≤ 0.01. Number of individual animals [n] are indicated.