Intestinal commensal bacteria mediate lung mucosal immunity and promote resistance of newborn mice to infection

Abstract

Immature mucosal defenses contribute to increased susceptibility of newborn infants to pathogens. Sparse knowledge of age-dependent changes in mucosal immunity has hampered improvements in neonatal morbidity because of infections. We report that exposure of neonatal mice to commensal bacteria immediately after birth is required for a robust host defense against bacterial pneumonia, the leading cause of death in newborn infants. This crucial window was characterized by an abrupt influx of interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (IL-22⁺ILC3) into the lungs of newborn mice. This influx was dependent on sensing of commensal bacteria by intestinal mucosal dendritic cells. Disruption of postnatal commensal colonization or selective depletion of dendritic cells interrupted the migratory program of lung IL-22⁺ILC3 and made the newborn mice more susceptible to pneumonia, which was reversed by transfer of commensal bacteria after birth. Thus, the resistance of newborn mice to pneumonia relied on commensal bacteria-directed ILC3 influx into the lungs, which mediated IL-22-dependent host resistance to pneumonia during this developmental window. These data establish that postnatal colonization by intestinal commensal bacteria is pivotal in the development of the lung defenses of newborns.

Figure 1. Intestinal commensal bacteria promote resistance to S. pneumoniae in newborn mice via IL22

(A) Study design. (B) Intestinal commensal bacteria enumerated in postnatal day 4 (P4) newborn mice exposed to antibiotics (ABX) or no antibiotics (ABX-free) quantified using real-time PCR.. (C) Survival of ABX-free or ABX-exposed P4 mice or ABX-exposed newborn mice reconstituted with intestinal commensal bacteria and infected with S. pneumonia. (D) Survival of germ-free (GF) or conventionally housed (CNV) mice or age-matched GF mice reconstituted with intestinal commensal bacteria and infected with S. pneumoniae. (E) Survival of ABX-free or ABX-exposed P14 mice or ABX-exposed newborn mice reconstituted with intestinal commensal bacteria and infected with S. pneumoniae. (F) The amount of IL-22 in the bronchial lavage (BAL) fluid of P4 ABX-free or ABX-exposed mice or GF newborn mice or ABX-exposed or GF newborn mice reconstituted with intestinal commensal bacteria in early life. None of the newborn mice in this experimental group were inoculated with S. pneumoniae. (G) The amount of IL-22 in the BAL fluid of human newborns who were exposed to ABX or no ABX. (H) Survival of P4 ABX-free or ABX-exposed newborn mice treated with IL-22 intratracheally and infected with S. pneumoniae. (I) Survival of P4 ABX-free or ABX-exposed newborn mice treated with anti-IL22 antibody or isotype control antibody before reconstitution with intestinal commensal bacteria and infection with S. pneumoniae. Data 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.

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.

Figure 3. Intestinal dendritic cells mediate cross talk between commensal bacteria and IL22⁺ILC3 innate lymphoid cells and induce ILC3 to traffic to the murine newborn lung

(A) Representative flow cytometry histograms showing expression of C-C chemokine receptor (CCR) 4, 6, 7 and 9, C-X-C chemokine receptor (CXCR) 3 and 5 and C-C chemokine ligand (CCL) 20 by IL22⁺ILC3 innate lymphoid cells in the lung of postnatal day 4 (P4) ABX-free or ABX-exposed newborn mice. (B) An equal number of ILC3 from P4 WT or Ccr4^−/− newborn mice were adoptively transferred into age-matched ABX-exposed newborn mice, and the ability of ILC3 to traffic to the lungs (homing index) was determined. (C) Survival of P4 WT or Ccr4^−/− newborn mice that received adoptive transfer of WT ILC3 after infection with S. pneumoniae. (D) Representative flow cytometry plots and relative frequencies of distinct subsets of mononuclear phagocytes in the small intestine of P4 newborn mice. (E) The absolute numbers of IL22⁺ILC3 in the lungs of P4 WT or Zbtb46^DTR newborn mice treated with DT (CD11b⁺CD103⁺ DC-depleted) that received adoptive transfer of CD11b⁺CD103⁺ DCs. (F) Survival of P4 WT or Zbtb46^DTR newborn mice treated with DT (CD11b⁺CD103⁺ DC-depleted) that then received adoptive transfer of CD11b⁺CD103⁺ DCs, after infection with S. pneumoniae . (G) ILC3 isolated from lungs of P4 ABX-exposed mice were co-cultured with CD11b⁺CD103⁺ DCs isolated from age-matched ABX-exposed or ABX-free mice and examined for surface expression of various chemokine receptors. A representative flow cytometry plot is shown and (H) relative frequencies of IL-22⁺ILC3 cells expressing CCR4. (I) ILC3 isolated from lungs of P4 ABX-exposed mice were co-cultured either alone or with CD11b⁺CD103⁺ DCs isolated from age-matched ABX-exposed or ABX-free newborn mice. The ability of these ILC3 to migrate in vitro in response to a gradient of the chemokine ligand (CCL) 17 is shown. (J) The absolute numbers of IL22⁺ILC3 in the lungs of P4 Zbtb46^DTR newborn mice either exposed to ABX or no ABX that were then treated with DT (CD11b⁺CD103⁺ DC-depleted) or no DT (no DC depletion) before they were reconstituted with commensal bacteria. 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.