Research ArticleGUT MICROBIOTA

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

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Science Translational Medicine  08 Feb 2017:
Vol. 9, Issue 376, eaaf9412
DOI: 10.1126/scitranslmed.aaf9412

Gut microbiota: A driving force in lung mucosal defense

Interactions between the host immune system and intestinal commensal bacteria shape immune system development. Gray et al. now report that host-commensal interactions extend beyond the local environment and shape the repertoires of immune cells at extraintestinal sites such as the lungs. Exposure to commensals in the developmental window of the newborn period directed lung-selective trafficking of group 3 innate lymphoid cells (ILC3), a group of sentinel cells that maintain mucosal homeostasis. This was mediated by intestinal dendritic cells, which induced expression of the lung homing signal CCR4 on the ILC3. Lung-selective trafficking of ILC3 promoted the resistance of newborn mice to pneumonia. These data explain the association between widespread use of antibiotics and an increased risk of pneumonia in newborn infants.

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.

INTRODUCTION

Development of the immune system requires a sequential series of timed and coordinated events that begin early in fetal life and continue through the early postnatal period (1). Disruption of immune development during the early neonatal period results in abnormal postnatal immune responses that are more marked and persistent than those after disruption during adult life, highlighting the importance of the neonatal period as a critical developmental window (2). Although several host genetic and environmental factors modulate the development of the immune system during fetal and early postnatal life (3), few are as important as the continued interaction with commensal bacteria, which is not only the most intimate environmental exposure (4, 5) but also represents a challenge to the developing immune system (6, 7).

Commensal colonization, which begins during birth, progresses through a choreographed succession of bacterial species and evolves rapidly during the first month of life (8). These evolving microbial signals are hypothesized to play a critical role in the functional programming of immune cells. Modern childbirth practices such as cesarean deliveries (9) and increased use of antibiotics (ABX) during early life (10) not only alter the pattern of intestinal commensal colonization in the newborn but are also associated with increased risk of sepsis and pneumonia (1014), suggesting that intestinal commensal bacteria can promote the resistance of newborn infants to pneumonia. The interaction between host and the intestinal commensal bacteria extends beyond the local enteric environment and influences immune homeostasis at peripheral sites, exemplified by intestinal complications during respiratory disease and vice versa (15, 16). Nevertheless, the mechanistic basis of cross-talk between the intestinal commensal bacteria and innate lung defense, the so-called gut-lung axis, remains poorly defined (17), and the developmental pathways underlying the association between commensal colonization during the early postnatal period and development of lung immunity in newborns remain unexplored.

Here, we show that interactions between the host immune system and the intestinal commensal bacteria shape the repertoires of immune cells in the newborn mouse lung and direct the postnatal ontogeny of IL-22–producing group 3 innate lymphoid cells (ILC3), a group of sentinel cells that maintain homeostasis at mucosal barrier sites. This postnatal influx of IL-22+ILC3 promoted the resistance of neonatal mice to pneumonia. This cross-talk was mediated by mucosal dendritic cells (DCs), which captured antigen from intestinal commensal bacteria. Disruption of commensal bacteria interrupted the migratory program of ILC3, impairing their ability to traffic to the lungs and rendering the newborn mice more susceptible to pneumonia; this was reversed by exogenous IL-22 or through adoptive transfer of ILC3. Reconstitution of intestinal commensal bacteria restored the expression of CCR4 on the ILC3, restored the ability of ILC3 to migrate to the lungs, and promoted IL-22–dependent resistance to pneumonia in newborn mice.

RESULTS

Postnatal colonization by commensal bacteria promotes resistance to pneumonia in newborn mice

Previous epidemiological studies show that human infants whose mothers received frequent ABX before birth or who were delivered by cesarean section not only had altered intestinal commensal bacteria (18, 19) but also had increased risk of developing pneumonia (20, 21). This led us to hypothesize that early-life exposure to commensal bacteria promotes resistance to pneumonia in newborns. To test this hypothesis, we exposed pregnant mouse dams to a combination of ampicillin, gentamicin, and vancomycin, three commonly used ABX in pregnant women and human newborns (Fig. 1A) (22), beginning 5 days before delivery. ABX were discontinued immediately after birth, and newborn mice were challenged intratracheally with Streptococcus pneumoniae serotype 19A, a leading cause of pneumonia in human newborns (23).

Fig. 1. Intestinal commensal bacteria promote resistance to S. pneumoniae in newborn mice via IL-22.

(A) Study design. E15, embryonic day 15. (B) Intestinal commensal bacteria enumerated in postnatal day 4 (P4) newborn mice exposed to ABX (ABX) or no ABX (ABX-free), quantified using real-time polymerase chain reaction (RT-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. pneumoniae. (D) Survival of GF or 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) Amount of IL-22 in the 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) 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. NS, not significant. (I) Survival of P4 ABX-free or ABX-exposed newborn mice treated with anti–IL-22 antibody (Ab) 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 means ± SEM [Student’s t test or analysis of variance (ANOVA) or Wilcoxon signed-rank test]. *P ≤ 0.05; **P ≤ 0.01. Numbers of individual animals (n) are indicated.

This early-life ABX exposure not only reduced the total number of commensal bacteria (Fig. 1B) but also disrupted the succession of bacterial species in the intestine of newborn mice (fig. S1, A and B, and table S1A). Six hours after infection, we observed an increased bacterial load in the lungs and the bronchial lavage (BAL) fluid (fig. S1C) and increased susceptibility in newborn mice whose dams were exposed to ABX (ABX-exposed) as compared to age-matched mice whose dams were not exposed to ABX (ABX-free) (Fig. 1C). Germ-free (GF) mice, which lack commensal bacteria, similarly were more susceptible to challenge with S. pneumoniae as compared to the age-matched conventionally raised (CNV) mice (Fig. 1D). We paralleled these observations using Escherichia coli K1 or Candida albicans, other common causes of pneumonia in newborns (fig. S1, D and E) (24). Because disruption of commensal bacteria in infancy is associated with increased susceptibility to inflammatory disorders such as allergen-induced airway hyperreactivity (25) and colitis (26) during later life, we ascertained whether disruption of postnatal commensal colonization led to durable changes in host resistance to infection. We found that increased susceptibility to pneumonia after early-life ABX exposure persisted beyond the neonatal period, until at least 4 weeks of age (Fig. 1E). This persistence in susceptibility contrasted with the transient susceptibility to infection that occurs in ABX-exposed adult mice (27, 28), highlighting the critical nature of commensal exposure during early life.

We reversed the commensal disruption in ABX-exposed newborns by transferring intestinal contents from a newborn mouse during the early postnatal period as done previously (Fig. 1A) (29). Reconstitution of intestinal commensal bacteria restored resistance to pneumonia in ABX-exposed and GF newborn mice (Fig. 1, C and D). This protection against S. pneumoniae persisted beyond the neonatal period. ABX-exposed mice that received intestinal contents during the early postnatal period likewise showed increased resistance to infection, at least, for as long as 4 weeks after birth compared to their littermates that did not receive intestinal bacterial reconstitution (Fig. 1, E and F).

Whether lung-resident commensal bacteria educate the mucosal immune system, like the intestinal commensal bacteria, remains a source of controversy (30, 31), and the effect of early-life ABX exposure to lung commensal colonization in human newborns remains unexplored. We found no difference in the composition of lung commensal bacteria in ABX-free and ABX-exposed mice (fig. S1, F and G, and table S1B), perhaps related to our experimental strategy of limiting ABX exposure to the pregnant dams and not the newborn mice. Lack of differences in lung-resident commensal bacteria in the ABX-exposed newborn mice as compared to ABX-free newborns suggests that intestinal commensal bacteria rather than lung commensals mediate the resistance to pneumonia, although this possibility cannot be completely excluded.

Postnatal colonization by commensal bacteria promotes interleukin-22–dependent mucosal defense in newborn mice

We hypothesized that disruption of commensal colonization mediated changes in the expression of genes related to various aspects of innate lung defense. We carried out RNA sequencing analysis of lung mucosal RNA isolated from newborn mice on days 0 to 4. Unsupervised analysis revealed consistent transcriptional changes in ABX-exposed newborns as compared to ABX-free murine newborns (fig. S1H and table S2). Differentially expressed genes included interleukin-22 (IL-22), a cytokine critical in lung epithelial repair (fig. S1H) (32, 33) and host defense against pathogens (32, 34, 35). We found decreased concentrations of IL-22 in the BAL fluid of ABX-exposed or GF newborn mice as compared to ABX-free newborn mice (Fig. 1F). We confirmed these observations in human newborns, finding reduced concentrations of IL-22 in the BAL fluid from human newborns exposed to prolonged duration of ABX (Fig. 1G and Table 1).

Table 1. Demographic characteristics of human newborn infants.
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Reconstitution with intestinal contents from age-matched neonatal mice restored IL-22 levels in the BAL fluid of ABX-exposed or GF newborn mice (Fig. 1F). Similarly, treatment with recombinant IL-22 intratracheally restored host resistance to pneumonia in ABX-exposed newborn mice (Fig. 1H). To interrogate the importance of IL-22 in newborn’s resistance to pneumonia, we blocked IL-22 signaling with an IL-22–neutralizing antibody (36). Treatment of newborn mice with an anti–IL-22 antibody blocked the restoration of host resistance in ABX-exposed newborn mice after reconstitution of intestinal commensal bacteria (Fig. 1I). IL-22 acts via a transmembrane receptor complex that consists of IL-22R1, a receptor subunit that is shared by related cytokine IL-20 (37). We found no difference in the concentrations of IL-20 in the BAL fluid of ABX-exposed or GF newborn mice compared to ABX-free newborn mice (fig. S1I). Blockade of IL-20 signaling by treatment with a neutralizing antibody directed against IL-20 (38) did not block restoration of host resistance in ABX-exposed newborn mice after reconstitution of intestinal commensal bacteria (fig. S1J) (36). These findings demonstrate a central and nonredundant role for IL-22 in host defense against pneumonia (32, 33) and implicate IL-22 as a critical mediator by which commensal bacteria promote resistance to pneumonia in newborn mice. IL-22 bioactivity is negatively regulated by IL-22–binding protein (IL-22BP), a secreted receptor that binds to soluble IL-22 with higher affinity than IL-22R1 and functions as an antagonist (39). We did not evaluate the role of endogenous IL-22BP in our study. Several lung-resident immune cells are known to secrete IL-22BP (40), but the role of endogenous IL-22BP in pulmonary host defense remains unclear and may represent an additional regulatory layer in the ontogeny of lung defense in the newborn.

Disruption of commensal bacteria postnatally alters the repertoire of IL-22–producing immune cells in the lungs of newborn mice, increasing susceptibility to pneumonia

The identity of the IL-22–producing cells in the newborn mouse lung is unknown. We found that neither neutrophils (CD45+Ly6G+) nor macrophages (CD45+F4/80+) nor T cells (CD45+CD4+) were a significant source of IL-22 in the lungs of newborn mice (Fig. 2A). Most of IL-22–producing cells in the murine newborn lung were lineage-negative (CD45+CD3CD8CD11bCD19MHCIIF4/80CD161Ly6G) lymphocytes. We further characterized these lineage-negative lymphocytes on the basis of the expression of the surface markers CD4, CD117, CD127, NkP46, or CCR6 and the transcription factors RORγt, T-bet, or Eomes. More than 90% of IL-22–producing cells were lineage-negative (CD3CD8CD11bCD19MHCIIF4/80CD161Ly6GF4/80) lymphocytes expressing the surface markers NKp46, CCR6, CD117 and the transcription factor RORγt, identifying them as ILC3 (Fig. 2A and fig. S2A).

Fig. 2. Intestinal commensal bacteria direct postnatal trafficking of IL-22+ILC3 to murine newborn lung.

(A) Representative flow cytometry plots of distinct subsets of IL-22+ cells in the lungs of P4 newborn mice. Relative frequencies of IL-22+ T cells (CD45+CD3+) or neutrophils (CD45+Ly6G+) or lineage-negative (CD3CD8CD11bCD19F4/80CD161Ly6GF4/80) lymphocytes in the lungs of P4 newborn mice are shown. FSC-H, forward scatter–height; NKT, natural killer T cells; LTi, lymphoid tissue inducer. (B) Relative frequencies of distinct subsets of IL-22+ cells in the BAL fluid of human newborn infants. (C) Absolute numbers of IL-22+ILC3 in the lungs of P4 wild-type (WT) or RorγtiDTR newborn mice treated with DT or no DT. (D) Survival of P4 WT or RorγtiDTR newborn mice treated with DT (ILC3-depleted) that received adoptive transfer of ILC3 and then infected with S. pneumoniae. (E) 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 during early life. (H) 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 carboxyfluorescein succimidyl ester (CFSE). ILC3 from age-matched P4 ABX-exposed or ABX-exposed newborn mice reconstituted with commensal bacteria were labeled with chloromethyl-benzoyl-amino-tetramethylrhodamine (CMTMR). An equal number of CFSE- or CMTMR-labeled ILC3 was adoptively transferred into ABX-exposed newborn mice. Representative flow cytometry plots and absolute numbers of CFSE+ or CMTMR+ ILC3 in lung, spleen, or small intestine were determined 12 hours after adoptive transfer. LP, lamina propria. (J) Relative capability (homing index) 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. Data and plots are representative of three independent experiments. Results are shown as the means ± SEM (Student’s t test or ANOVA or Wilcoxon signed-rank test). *P ≤ 0.05; **P ≤ 0.01. Numbers of individual animals (n) are indicated.

We tested these observations in human newborns. ILC3 (CD45+CD3CD8CD14CD19CD69RORγt+), but not neutrophils (CD45+CD3CD8CD19CD69+), natural killer (NK) cells (CD45+CD3CD8CD19CD56+), CD4+ T cells (CD45+CD3+CD4+), or CD8+ T cells (CD45+CD8+), were a primary source of IL-22 in the lungs of human newborns (Fig. 2B and fig. S2B). These findings illustrate an important difference in the cellular sources of IL-22 in the lungs of newborn humans compared to adult humans, as several groups have reported that NK cells (41), T helper 17 cells (42), and γδ T cells (43) are the principal sources of IL-22 in adult human lungs.

ILC3 developmentally depend on RORγt and continuously express this transcription factor (44, 45). Therefore, to interrogate the importance of ILC3 in the resistance of newborn mice to pneumonia, we bred transgenic mice expressing Cre recombinase under the control of the RORγt promoter (46) with transgenic mice expressing inducible diphtheria toxin receptor (iDTR) (47) to generate RorγtDTR mice. Treatment of newborn RorγtDTR mice with diphtheria toxin (DT) decreased the number of ILC3 in the lungs (Fig. 2C and fig. S2C), reduced IL-22 in the BAL fluid (fig. S2D), and made the DT-treated RorγtDTR newborn mice more susceptible to pneumonia (Fig. 2D). Adoptive transfer of lung ILC3 restored host resistance to pneumonia in newborn RorγtDTR mice treated with DT (Fig. 2D). Together, these data confirm that IL-22+ILC3 are necessary and sufficient for promoting host resistance to pneumonia in newborn mice (48).

We sought to determine whether disruption of commensal colonization altered the repertoire of IL-22–producing cells in the newborn mouse lung. We found significantly decreased (P < 0.01) numbers of IL-22+ILC3 (Fig. 2, E to G), but not neutrophils, T cells, or NK cells (P > 0.05 for all) (fig. S2E), in the lungs of ABX-exposed or GF newborn mice as compared to ABX-free newborn mice. The decrease in the numbers of IL-22+ILC3 persisted beyond the newborn period until at least 4 weeks of life (Fig. 2E). We confirmed these observations in human newborns and found significantly decreased numbers of lung IL-22+ILC3 in the BAL fluid of human newborns (P < 0.01) exposed to prolonged duration of ABX (Fig. 2H). We then questioned whether reversing the commensal disruption would correct the immune alterations in ABX-exposed newborn mice. We found that reconstitution with commensal bacteria restored the numbers of IL-22+ILC3 in the lungs of ABX-exposed or GF newborn mice (Fig. 2, F and G), although individual IL-22 expression did not change (fig. S2F). These data illustrate that disruption of commensal bacteria in early postnatal development alters the repertoire of IL-22–producing cells in the newborn lungs.

Commensal bacteria direct the postnatal trafficking of IL-22+ILC3 to the murine newborn lung

We tested whether reduced numbers of IL-22+ILC3 in the lungs of GF or ABX-exposed newborn mice could be explained by differences in proliferation or apoptosis of IL-22+ILC3. We assessed cell proliferation or apoptosis by quantifying the number of IL-22+ILC3 expressing Ki67 or annexin, respectively. We found that an increased number of ILC3 in the lungs was not due to changes in proliferation or apoptosis (fig. S2G). We therefore hypothesized that a decrease in the absolute numbers of IL-22+ILC3 in ABX-exposed newborn mice was due to a reduced ability of ILC3 from ABX-exposed newborns to traffic preferentially to the lungs. To test this, we used a competitive trafficking assay (49) to determine the advantage of ILC3 isolated from ABX-free newborn mice to traffic to the lungs as compared to ILC3 from ABX-exposed newborn mice. We found that ILC3 from ABX-exposed newborn mice had decreased the ability to traffic selectively into the lungs, but not the spleen or small intestine as compared to ILC3 isolated from ABX-free newborn mice (Fig. 2, I and J).

We then asked whether reversing the commensal disruption would restore the ability of ILC3 to traffic to the lungs. We similarly determined the advantage of ILC3 isolated from ABX-exposed newborn mice that had received transfer of commensal bacteria to traffic to the lungs as compared to ILC3 from ABX-exposed newborn mice that had received no such transfer. Reconstitution of commensal bacteria restored the ability of ILC3 from ABX-exposed newborns to traffic selectively to the lungs (Fig. 2, I and J). Tissue-selective ILC3 trafficking has been described for the small intestine and secondary lymphoid tissues (50, 51). Our data unveil a role for intestinal commensal bacteria in selective trafficking of ILC3 into the lungs.

Commensal bacteria modulate expression of CCR4 on ILC3 and direct their postnatal trafficking to the lungs

Chemokines control the trafficking and positioning of immune cells and are critical for development and recruitment of immune cells in disease (52). We first identified the repertoire of chemokine receptors on IL-22+ILC3 from the lungs or small intestine of newborn mice. We found that CCR4 was highly expressed by most of IL-22+ILC3 from the newborn murine lung but not from the newborn murine small intestine (fig. S3A). We found no difference in the expression of CCR6, CCR7, CCR9, or CCL20 nor CXCR3 or CXCR5 on IL-22+ILC3 from the newborn lung or small intestine (fig. S3A). Tissue-selective ILC3 trafficking has been described for the intestine (50, 51) but not for the lungs. Like the intestine, the lung has a large mucosal surface, which is in continuous contact with the environment and therefore can potentially benefit from lung-selective ILC3 trafficking.

We hypothesized that exposure to commensal bacteria during early life may modulate the expression of lung-specific homing receptors on IL-22+ILC3 and thus increase their ability to traffic to the lungs. We examined the numbers and frequencies of CCR4 expressing IL-22+ILC3 in the lungs of ABX-exposed or ABX-free newborn mice. We found that most of IL-22+ILC3 from the lungs of ABX-free newborn mice were CCR4high as compared to the lung ILC3 from ABX-exposed mice or GF mice, which were CCR4low (Fig. 3A). We found no difference in expression of CCR6, CCR7, CCR9, CCL20, CXCR3, or CXCR5 on IL-22+ILC3 from the lungs of ABX-free or ABX-exposed mice (Fig. 3A). CCR4 is critical for homeostatic trafficking of T lymphocytes (53) and regulatory T cells (54) into the lungs. CCL17, one of the ligands for CCR4, is expressed by the lung epithelium (55). We therefore hypothesized that commensal bacteria use a similar mechanism to direct trafficking of ILC3 into the newborn lungs. To test this hypothesis, we determined the capability of ILC3 isolated from newborn mice lacking CCR4 (Ccr4−/−) to traffic to the lungs as compared to ILC3 from age-matched wild-type littermates. We found that ILC3 from newborn Ccr4−/− mice had decreased ability to traffic into the lungs as compared to ILC3 isolated from wild-type littermates (Fig. 3B). Furthermore, the newborn Ccr4−/− mice were more susceptible to pneumonia compared to wild-type littermates (Fig. 3C). We then asked whether an adoptive transfer of wild-type ILC3, which express CCR4 and therefore traffic into the lungs, could improve host resistance in newborn Ccr4−/− mice. We found that adoptive transfer of ILC3 from wild-type newborn mice to age-matched Ccr4−/− mice restored host resistance to pneumonia (Fig. 3C). These data illuminate a role for CCR4 in trafficking of ILC3 into the lungs and promoting newborn’s resistance to pneumonia. Nevertheless, the origin of ILC3 that traffic to the lungs is unknown. ILC3 are concentrated within the small intestine (51), and the spatial proximity of ILC3 with commensal bacteria in the small intestine supports the notion that ILC3 in the intestinal mucosa may be directed by the commensal bacteria to traffic to the lung.

Fig. 3. Intestinal DCs mediate cross-talk between commensal bacteria and IL-22+ILC3, mediating ILC3 trafficking to murine newborn lung.

(A) Representative flow cytometry histograms showing expression of CCR4, CCR6, CCR7, and CCR9; CXCR3 and CXCR5; and CCL20 by IL-22+ILC3 in the lung of P4 ABX-free or ABX-exposed newborn mice. (B) An equal number of ILC3 from P4 WT or Ccr4−/− newborn mice was 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) Absolute numbers of IL-22+ILC3 in the lungs of P4 WT or Zbtb46DTR newborn mice treated with DT (CD11b+CD103+ DC–depleted) that received adoptive transfer of CD11b+CD103+ DCs. (F) Survival of P4 WT or Zbtb46DTR 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 cocultured 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 and (H) relative frequencies of IL-22+ILC3 expressing CCR4 are shown. (I) ILC3 isolated from lungs of P4 ABX-exposed mice were cocultured 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 CCL17 is shown. (J) Absolute numbers of IL-22+ILC3 in the lungs of P4 Zbtb46DTR 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 means ± SEM (Student’s t test or ANOVA or Wilcoxon signed-rank test,). *P ≤ 0.05; **P ≤ 0.01. Numbers of individual animals (n) are indicated.

CD103+CD11b+ DCs capture antigen from commensal bacteria and induce expression of CCR4 on ILC3

We sought to identify the mechanisms by which intestinal commensal bacteria induce the expression of CCR4 on ILC3. Murine ILC3 do not express pattern recognition receptors and therefore are unlikely to directly sense the commensal bacteria (56). Mononuclear phagocytes, such as DCs and macrophages, not only detect a range of microbial signals (57) but can also cross-talk with ILC3 in the intestine (58). We found that most of mononuclear phagocytes in the intestine of newborn mice were CD45+CD11b+CD103+F4/80 cells (CD103+CD11b+ DCs) (Fig. 3D), consistent with previous findings (59). The transcription factor ZBTB46 is selectively expressed by CD103+CD11b+ DCs and their committed progenitors but is not expressed by monocytes, macrophages, or other lymphoid or myeloid lineages (60, 61). We used mice that express DTR under the control of Zbtb46 (Zbtb46DTR), which allows for the depletion of CD103+CD11b+ DCs (60) after treatment with DT. Twenty-four hours later, there was a decreased number of intestinal CD11b+CD103+ DCs (fig. S3C). Depletion of CD103+CD11b+ DCs was associated with a decrease in IL-22 in the BAL fluid (fig. S3D), reduced numbers of ILC3 in the lungs, and increased susceptibility to pneumonia of DT-treated Zbtb46DTR newborn mice (Fig. 3, E and F). Adoptive transfer of CD103+CD11b+ DCs into age-matched newborn Zbtb46DTR mice treated with DT (CD103+CD11b+ DC–depleted newborn mice) restored the number of lung ILC3 and improved the resistance of newborn mice to pneumonia (Fig. 3, E and F).

There is increasing evidence that intestinal DCs act as conductors of ILC traffic to the intestine and secondary lymphoid tissue (50). We tested whether CD103+CD11b+ DCs induced CCR4 expression on IL-22+ILC3. We cocultured CD103+CD11b+ DCs isolated from the small intestine of ABX-free or ABX-exposed newborn mice with ILC3 isolated from the lungs of ABX-free or ABX-exposed newborn mice. CD103+CD11b+ DCs isolated from the small intestine of ABX-free newborn mice were more efficient than CD103+CD11b+ DCs isolated from the small intestine of ABX-exposed newborn mice at inducing CCR4 expression on IL-22+ILC3 (Fig. 3, G and H) and restoring the ability of lung ILC3 from ABX-exposed animals to migrate in response to the CCR4 ligand CCL17 in vitro (Fig. 3I). We found no change in the expression of CCR6, CCR7, CCR9, CCL20, CXCR3, or CXCR5 (Fig. 3G). Conversely, coculture of CD11b+CD103 DCs isolated from the small intestine of either ABX-free or ABX-exposed newborn mice did not change the CCR4 expression on IL-22+ILC3 (fig. S3E), suggesting that CD11b+CD103 DCs were not as efficient as CD11b+CD103+ DCs at modulating CCR4 expression on IL-22+ILC3. These observations are consistent with other reports (6264), which suggest that distinct DC subsets respond differentially to similar environmental cues.

Finally, to test the hypothesis that CD103+CD11b+ DCs capture antigen from commensal bacteria and direct the trafficking of ILC3 into the lungs, we treated newborn Zbtb46DTR mice with DT before reconstitution with commensal bacteria. Depletion of CD103+CD11b+ DCs abrogated the increase in the ILC3 number in ABX-exposed newborn Zbtb46DTR mice after reconstitution with intestinal commensal bacteria (Fig. 3J). Treatment of Zbtb46DTR newborn mice with DT depleted the CD103+CD11b+ DCs in extraintestinal sites including the lungs. CD103+CD11b+ DCs are exceedingly rare in the newborn murine lungs (65). Therefore, intestinal CD103+CD11b+ DCs, but not lung-resident CD103+CD11b+ DCs, are likely to play a major role in selective trafficking of ILC3 to the lung. Because previous reports (66, 67) implicated IL-1β in cross-talk between intestinal mononuclear phagocytes and small intestine ILC3, we measured IL-1β in the supernatant from cocultures of DCs and ILC3. We found no difference in the IL-1β levels in coculture supernatants of CD103+CD11b+ DCs isolated from the small intestine of ABX-free or ABX-exposed newborn mice and cocultured with ILC3 (fig. S3F), suggesting that IL-1β may not be involved in cross-talk between DCs and ILC3.

DISCUSSION

Distinct immune responses specifically adapted for fetal and early postnatal life render newborns more vulnerable to infection (1). Lack of understanding of immune development in early life contributes to our inability to reduce neonatal morbidity due to pneumonia, which unfortunately kills more newborns than any other cause. Although a series of coordinated events control the development of a newborn’s immune system (3), few are as important as the interaction with successive waves of commensal bacteria, which colonize the newborn’s intestine immediately after birth (4, 5). Disruption of commensal colonization in the critical window of the early postnatal period has enduring consequences for the developing immune system, exemplified by increased risk of inflammatory disorders such as asthma and increased risk of respiratory infections beyond infancy in those infants exposed to prolonged ABX treatment or delivered by cesarean section (1014). To develop therapeutic interventions to decrease morbidity in the newborn period and beyond, we need to better understand the role of commensal colonization in the development of the newborn immune system.

Previous studies investigating commensal bacteria–driven immune maturation have prioritized the use of GF mice (68). It is now accepted that the immune system, although not immature in early life, differs fundamentally from immune responses in adults (1). The newborn’s intestine is colonized by successive waves of diverse commensal bacteria, and the newborn’s intestinal microbiota is fundamentally distinct from that of adult mice (29). Thus, commensal reconstitution studies in adult GF mice do not fully capture the complex interaction between the developing host and evolving microbiota. We exposed pregnant mouse dams to a combination of ABX, used commonly to treat mothers and infants. This not only disrupted the sequential colonization of the newborn’s intestine by different commensal bacteria but also made the newborn mice more susceptible to pneumonia, replicating the key observations from epidemiological studies (1014). The susceptibility to pneumonia in ABX-exposed newborn mice persisted beyond the newborn period, in contrast to previous reports (27, 28), showing that ABX exposure in adult mice induced only transient susceptibility to infection. Reversing the commensal disruption restored the resistance to pneumonia in the ABX-exposed newborn mice, and this protection persisted beyond the newborn period. These results coupled with other recent reports (25, 26) highlight the importance of therapeutic interventions addressing commensal dysbiosis in early life that have lasting consequences.

T cells (32) and NK cells (41) are primary mediators of innate mucosal defense against respiratory pathogens in adults. We found that, during birth, the mouse lung is populated by a few IL-22+ T cells or NK cells but is home to a significant number of IL-22+ILC3. Although IL-22+ILC3 maintain tissue homeostasis in the small intestine, the role of IL-22+ILC3 in lung mucosal defense remains a source of controversy (69). ILC3 were dispensable for protection against influenza A pneumonia (70). Reports ascribing an important role for ILC3 in lung immune homeostasis have used adult mice deficient in recombination-activating gene 2 (Rag2) or IL-2 receptor γ (Il2rg) (48, 71), which are profoundly immunodeficient, or treated with anti-CD90.2 antibodies (35), resulting in nonspecific depletion of several immune cell types, thus confounding interpretation of results from these animals. We therefore generated RorγtiDTR mice, a genetic tool to selectively deplete ILC3. First, by showing that ILC3 depletion in early life rendered the newborn mice more susceptible to pneumonia, we were able to demonstrate that IL-22+ILC3 were necessary to promote the resistance of newborn mice to pneumonia. Second, by restoring the resistance to pneumonia in ILC3-depleted newborn mice after adoptive transfer of ILC3, we established that ILC3 were sufficient to promote newborn’s resistance to pneumonia. One limitation of RorγtiDTR mice is potential depletion of the RORγt+ T cells after DT treatment. However, both human newborns (72) and murine newborns (73) are relatively lymphopenic compared to adults. Absolute numbers of IL-22+ T cells were low in the newborn mice (Fig. 2B), suggesting that depletion of RORγt+ T cells in our RorγtiDTR mice may have had only a marginal effect on the newborn’s resistance to infections.

Having shown that ILC3 played a critical role in promoting the resistance of newborn mice to pneumonia, we investigated the role of commensal bacteria in the postnatal development of lung IL-22+ILC3. Several groups have demonstrated that colonization with commensal bacteria increased the number of IL-22+ILC3 in the small intestine of adult GF mice (74, 75). Similarly, an increase in the number of IL-22+ILC3 in the postnatal period has been described for the small intestine (76, 77) but not for the lung, raising the possibility that this phenomenon is restricted to the newborn’s intestine. Our data challenge this assumption and illuminate a role for intestinal commensal bacteria in the postnatal accumulation of IL-22+ILC3 in the lungs. We noted an abrupt increase in the number of IL-22+ILC3 in the lungs immediately after birth and observed an age-dependent increase in the number of lung IL-22+ILC3, peaking at 2 weeks of age. This postnatal increase in lung IL-22+ILC3 was absent in GF or ABX-exposed newborn mice and was reversed by transfer of commensal bacteria in the early postnatal period. Previous studies have used transcriptional activation and cytokine production to delineate a role for commensal bacteria in the ontogeny of ILC3 (75, 76). However, tissue-selective trafficking as a mechanism through which commensal bacteria direct the ontogeny of ILC3 in postnatal lungs has not been investigated. ILC3 are thought to establish residency in the developing intestine by tissue-specific migration (50, 51). We show that exposure to commensal bacteria in early life directs the lung-specific IL-22+ILC3 trafficking and thus contributes to the postnatal accumulation of IL-22+ILC3 in the newborn lung. This study did not address the potential role of commensal bacteria in long-term maintenance of lung IL-22+ILC3, which needs to be clarified in the context of recent reports that ILC homeostasis at peripheral sites is dependent primarily on self-renewal (78).

Small intestine–specific trafficking of ILC3 depends on the expression of chemokine receptors CCR9 and α4β7 by ILC3 (50). In contrast, we found that lung-specific trafficking of IL-22+ILC3 was mediated by CCR4, a homing receptor also used by other immune cells (79) for homeostatic trafficking into the lung. CCR4 is activated by chemokine CCL17 or CCL20. CCL17 is expressed by the lung epithelium (55). There is evidence that intestinal DCs act as conductors of ILC3 traffic to the small intestine and secondary lymphoid tissue, but not to the lungs (50, 51). We therefore evaluated the role of intestinal DCs in instructing IL-22+ILC3 to traffic to the lung and found that intestinal CD103+CD11b+ DCs increased the expression of the lung homing receptor CCR4 by IL-22+ILC3. The ability to up-regulate CCR4 expression was dependent on the exposure of intestinal CD103+CD11b+ DCs to commensal bacteria because CD103+CD11b+ DCs from ABX-exposed newborn mice failed to increase the expression of CCR4 on IL-22+ILC3, suggesting that the ability to induce CCR4 expression and thus direct the tissue-specific migration of IL-22+ILC3 is a cell-extrinsic property. Many factors, such as components of bacterial cell walls, bacterial metabolites, and intestinal epithelial cell–derived cytokines, are known to condition the functional properties of CD103+CD11b+ DCs (80). Identification of this signal remains an area of active investigation. The role of lung CD103+CD11b+ DCs in lung-specific trafficking of IL-22+ILC3 remains unclear. The rarity of CD103+CD11b+ DCs in the newborn murine lung (65) precluded us from evaluating whether these cells could induce IL-22+ILC3 to traffic to the lungs as efficiently as intestinal CD103+CD11b+ DCs, which is a potential limitation of our study.

In conclusion, our data demonstrate the importance of commensal exposure in a defined developmental window during the newborn period in the development of pulmonary mucosal immunity in mice. We illuminate a critical role for intestinal commensal bacteria in lung-selective trafficking of IL-22+ILC3. This was mediated by intestinal CD103+CD11b+ DCs, which induced expression of the lung homing signal CCR4 on the IL-22+ILC3. Lung-selective trafficking contributed to the postnatal accumulation of IL-22+ILC3, promoting the newborn’s resistance to pneumonia. These data also potentially explain the association between cesarean delivery or widespread use of ABX and an increased risk of infections in newborn infants. Finally, similar mechanisms could influence the development of other pulmonary inflammatory disorders such as asthma, which is also associated with cesarean delivery and ABX use during early life (81), and lead to new therapeutic agents to mitigate the risk associated with early-life ABX exposure in children.

MATERIALS AND METHODS

Study design

The Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children’s Hospital Medical Center (CCHMC) approved all the animal studies (IACUC2014-0055), which were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. We bred Rorγt-Cre mice with Rosa26-iDTR mice to generate RorγtiDTR mice. We maintained C57/BL6, Rosa26-iDTR, Rorγt-Cre, or Zbtb46DTR mice at the CCHMC animal facility. We maintained the GF C57/BL6 neonatal mice in plastic isolator cages with autoclaved feed and water at the CCHMC Germ-Free Core facility. After birth, neonatal mice from multiple litters were pooled and redistributed to control for the founder effect and to minimize in-cage variations. We used neonatal C57/BL6, Zbtb46DTR, RorγtDTR, or GF C57/BL6 mice between P1 and P14 and appropriate age-, sex-, and genetic strain–matched controls to account for any variations in data. We treated pregnant female mice (C57/BL6 or Zbtb46DTR) with sterile drinking water mixed with three different ABX (ampicillin, gentamicin, and vancomycin; all at 1 mg ml−1) starting from embryonic day 15 until the delivery of neonatal mice. We determined group sizes necessary for adequate statistical power analysis using preliminary data sets. There was no randomization designed in the experiments, and we did not exclude any samples. The investigators were not blinded to group allocation during collection and analysis of the data.

Murine neonatal pneumonia studies

We grew S. pneumoniae serotype 19A (American Type Culture Collection 700674) or E. coli serotype K1 (82) or C. albicans (37°C, 200 rpm) in tryptic soy broth to log-phase growth. To mimic S. pneumoniae or E. coli or C. albicans pneumonia, we inoculated neonatal mice (P4 or P14) with either S. pneumoniae serotype 19A [105 colony-forming units (CFUs) g−1] or E. coli (104 CFU g−1) or C. albicans (105 CFU g−1), respectively, via intratracheal route. The animals were examined every 6 hours for signs of distress and were euthanized 72 hours later or earlier if moribund. To assess bacterial burden, we homogenized the lung in sterile phosphate-buffered saline (PBS). We plated serial dilutions of lung homogenates or BAL fluid in tryptone soya broth agar plates and incubated (37°C, overnight) to count the number of CFUs.

We pooled intestinal contents from no ABX–exposed P2 newborn mice. We transferred intestinal contents (200 μg in 50 μl of PBS) or vehicle (50 μl of PBS) to ABX-exposed neonatal P2 mice by a single oral gavage via fine polyethylene tubing as described previously (29).

Isolation and characterization of IL-22+ cells in the murine neonatal lung

We pooled and cut the fresh lungs from three to four newborn mice and incubated (37°C, 30 min) the cut tissues with shaking (150 rpm) in digestion buffer [RPMI 1640 with 10% fetal bovine serum (FBS), 15 mM Hepes, 1% penicillin/streptomycin (w/v), and collagenase VIII (300 U ml−1)] and pressed through a 100-μm nylon strainer to obtain single-cell suspension. The pooled preparation constituted a single data point in our analysis. We then incubated (37°C, 5 hours, 5% CO2) the cells (1 × 107) in culture medium containing RPMI 1640 with 10% fetal calf serum (FCS), 1× nonessential amino acids, 10 mM Hepes, 2 mM l-glutamine (all from Invitrogen), and 1% penicillin/streptomycin with GolgiStop (1:1000; 554724, BD Biosciences), phorbol 12-myristate 13-acetate (PMA; 10 ng/ml), and calcium ionophore A23187 (500 ng/ml) (both from Sigma-Aldrich). We washed and incubated (4°C, 10 min) the cells (1 × 107) with anti-mouse CD16/CD32 and then reincubated (4°C, 30 min) with anti-mouse CD3 antibody (145-2C11), anti-mouse CD4 antibody (GK1.5), anti-mouse CD8 antibody (53-5.8), anti-mouse CD11b antibody (M1/70), anti-mouse CD19 antibody (6D5), anti-mouse Ly6G antibody (1A8), anti-mouse F4/80 antibody (BM8), anti-mouse CD117 antibody (2B8), anti-mouse NKp46 antibody (29A1.4), anti-mouse CCR4 antibody (2G12), anti-mouse CCR6 antibody (29-2L17), anti-mouse CCR7 antibody (4B12), anti-mouse CCR9 antibody (9B1), anti-mouse CCL20 antibody (114906), anti-mouse CXCR3 antibody (173), anti-mouse CXCR5 antibody (L138D7), and anti-mouse Ki67 antibody (16A8) (all diluted 1:100; BioLegend). For intracellular staining, we washed and fixed (4°C, 60 min) the surface-stained cells in 1× Cytofix/Cytoperm buffer (BD Biosciences) and permeabilized them (4°C, overnight) using 1× Permeabilization buffer (BD Biosciences) according to the manufacturer’s instructions. We stained the cells intracellularly with anti-mouse IL-22 antibody (5164), anti-mouse RORγt antibody (Q31-378), anti-mouse T-bet antibody (4B10), or anti-mouse Eomes antibody (WD1928) (all diluted 1:50; BioLegend) and then washed (twice) and resuspended them in flow cytometry buffer. We collected the data with LSR II (BD Biosciences) and analyzed the data with FlowJo (Tree Star).

Isolation and characterization of antigen presenting cells in the murine newborn intestine

We pooled and cut the freshly resected terminal ilea from three to four neonatal mice into 2- to 5-mm pieces and incubated (37°C, 15 min) them with extraction buffer (Hanks’ balanced salt solution, 15 mM Hepes, and 1 mM EDTA) to remove the epithelial cells. We then incubated (37°C, 30 min) the cut tissues with shaking (150 rpm) in digestion buffer [RPMI 1640 with 10% FBS, 15 mM Hepes, 1% penicillin/streptomycin (w/v), and collagenase VIII (300 U ml−1)] and pressed through a 100-μm nylon strainer to obtain single-cell suspension. The pooled preparation constituted a single data point in our analysis. We then incubated (4°C, 10 min) the cells (1 × 107) with anti-mouse CD16/CD32 and then reincubated (4°C, 30 min) with anti-mouse CD45 antibody (30-F11), anti-mouse CD103 antibody (2E7), anti-mouse CD11b antibody (M1/70), anti-mouse CD11c antibody (N418), anti-mouse MHCII antibody (M5/114.15.2), anti-mouse F4/80 antibody (BM8), and anti-mouse CX3CR1 antibody (SA011F11) (all diluted 1:100; BioLegend) and then washed (twice) and resuspended them in flow cytometry buffer. We collected the data with LSR II (BD Biosciences) and analyzed the data with FlowJo (Tree Star).

Treatment of neonatal mice with neutralizing antibodies, recombinant IL-22, or DT

We injected neonatal B6 mice with an anti–IL-22 antibody (8E11; a gift from W. Ouyang) or anti–IL-20 antibody (clone PA5-47092, Invitrogen) or anti–immunoglobulin G2A (54447, R&D Systems) (all 5 μg g−1 body weight) via intraperitoneal route on P1. For specific cell depletion, we treated neonatal RorγtiDTR or Zbtb46DTR mice with DT (1.5 ng, R&D Systems) or vehicle via intraperitoneal route on P1. We assessed ablation efficiency by flow cytometry 24 hours later. For gain-of-function studies, we treated ABX-exposed neonatal mice with recombinant IL-22 (10 μg g−1 body weight) (cat. no. 414-CS, R&D Systems) or vehicle via intratracheal route on P2.

Adoptive transfer of ILC3 or CD103+CD11b+ DCs

For adoptive transfer, we pooled lungs or small intestine from 8 to 10 newborn mice (P2). We harvested about 1 × 106 LinCCR6+ cells from the pooled specimens by positive and negative selection as done previously by other groups (71). This resulting cell population was >97% enriched for ILC3 (71). We harvested 1 × 107 intestinal CD103+CD11b+ DCs from the pooled small intestine specimens by positive and negative selection as done previously (83). We adoptively transferred LinCCR6+ cells (0.5 × 106 cells per animal) or CD103+CD11b+ DCs (0.5 × 106 cells per animal) via intraperitoneal route on P2.

In vivo ILC3 migration assay

We first isolated lung LinCCR6+ cells (>97% ILC3) by positive and negative selection as done previously (71). We then incubated (37°C, 20 min) ILC3 (1 × 107) from P3 GF or ABX-exposed newborns with CFSE (5 mM). We incubated ILC3 from control (ABX-free) neonatal mice with CMTMR (10 mM). We quenched with an equal volume of 10% FBS, diluted 10× with PBS, and resuspended the cells in RPMI 1640, supplemented with 2% FBS and 2 mM glutamine. We co-injected 106 cells of each population into ABX-exposed neonatal mice via intraperitoneal route. We euthanized host mice 24 hours later and determined the numbers of injected ILC3 migrating into the lungs, spleen, and small intestine by flow cytometry as done previously (50). We calculated the relative homing index as follows: (CFSE+ ILC3 in organ A)/(CMTMR+ ILC3 in organ A) ÷ (CFSE+ ILC3 in injected cells)/(CMTMR+ ILC3 in injected cells), as described previously (49). We performed similar experiments after reconstitution of commensal bacteria and with lung LinCCR6+ cells from Ccr4−/− or wild-type mice.

Cell coculture and chemotaxis assay

We isolated LinCCR6+ cells (>97% ILC3) from lungs (71) or CD103+CD11b+CD11c+ cells from the intestine of ABX-exposed or ABX-free newborn mice (P4). We then cocultured lung ILC3 and intestinal CD103+CD11b+ DCs or CD103CD11b+ (105 cells each) in the following combinations (ILC3 from ABX mice + DC from no-ABX mice; ILC3 from ABX mice + DC from ABX mice, ILC3 from ABX-free mice + DC from ABX-free mice, or ILC3 from ABX-free mice + DC from ABX mice; ILC3 from ABX or ABX-free mice alone, or DC from ABX and ABX-free mice alone) in round-bottom plates in RPMI 1640 supplemented with 2% FBS, 2 mM glutamine, and 50 μM β-mercaptoethanol. We harvested the supernatants, and the remaining cells were incubated with GolgiPlug and subsequently analyzed by flow cytometry. For chemotaxis assay, we loaded ILC3 (106 cells in 100 μl of RPMI 1640 supplemented with 2% FBS) into Transwell inserts with pore size of 5 μm (Corning Transwell) and placed in wells containing ±20 nM CCL17 in RPMI 1640, supplemented with 2% FBS, 2 mM glutamine, and 50 μM β-mercaptoethanol. We incubated (2 hours, 37°C) the plates and then analyzed the migrated and input cells by flow cytometry. We expressed results as percentages of ILC3 in migrated wells as compared to input wells as described previously (50).

Sample collection and analysis of commensal bacteria in the lungs and small intestine of newborn mice

Given the technical challenges in collecting adequate biospecimens for 16-s sequencing from newborn mice, we opted to pool biospecimens from 8 to 10 newborn mice from three separate litters per treatment group. An unexpected benefit of this approach is the control of variations in the commensal bacteria attributed to founder and cage effect (84). We collected the entire left lobe of the lung using one heat-treated sterile scissors per animal as described previously by other groups (85). We then cut open the terminal ileum using sterile scissors and removed intestinal contents using sterile plastic loops as done previously (29). We snap-froze (−80°C) the specimens for subsequent analysis (−80°C). We extracted the bacterial DNA from the whole lung or the intestinal contents using QIAamp DNA Stool Mini Kit (Qiagen) using a previously described protocol (85) and quantified 16S ribosomal (r) DNA by RT-PCR using degenerate primers as described previously (86). To analyze the commensal bacteria, we amplified the variable 2 region of microbial 16S rRNA by high-fidelity PCR with bar-coded 8F and 338R universal primers with A and B sequencing adaptors, respectively, and bifido primers (Roche) and sequenced them with Genome Sequencer FLX Titanium system (Roche) at the University of North Carolina Microbiome Core Facility (Chapel Hill, NC). The reagents used for DNA extraction, PCR, and sequencing reaction are a common source of contamination in microbiome sequencing studies (87). To control for variation in the reagents, we processed all the samples using a common batch of DNA extraction reagents and PCR reagents. We included appropriate controls including a negative control (no template) and positive community control (intestinal contents from age-matched mice) from each batch of harvested tissues when performing 16S rRNA PCR or 16S rRNA sequencing. We decoded and processed the sequences using the QIIME software package (version 1.7) and custom R package code (88). Analysis of the sequences from negative control indicated the presence of several bacterial species, suggesting potential contamination from the DNA extraction or sequencing reagents. The dominant bacterial species in the negative control were Rhodocyclaceae (17%), Rhizobiales (15%), Agrobacterium (14%), Micrococcus (10%), Hydrogenophilus (9%), Neisseria (5%), Lysinibacillus (4%), Micrococcus (4%), and Tenericutes (4%). The relative abundance of these contaminants in experimental samples was less than 3% (table S1) and thus unlikely to alter the conclusions. We used phylogenetic diversity to compute and visualize α diversity and unweighted and weighted UniFrac for β diversity. We tested the observed differences in UniFrac distances between ABX-treated groups and across different ages for significance using a t test, and we corrected the reported P values for multiple comparisons using a Monte Carlo permutation procedure with 10,000 iterations. We deposited all data sets in a publicly available database (figshare), which can be accessed at https://figshare.com/s/52f4aa2f8035fd505cf1.

Transcriptomics analysis

We sequenced high-quality RNA from the whole lung at CCHMC Sequencing Core Facility with an Illumina HiSeq 2500. We performed data alignment with TopHat, followed by gene quantification [fragments per kilobase million (FPKM)] using Cufflinks. We carried out differential expression analysis with both FPKM and read count–based methods. We performed pathway and network analyses with AltAnalyze as described previously (89). We deposited all data sets in a publicly available database (figshare), which can be accessed at https://figshare.com/s/52f4aa2f8035fd505cf1.

Human newborn studies

The Institutional Review Board (IRB) at CCHMC approved all the human studies. The biological samples were initially collected from infants who underwent clinical evaluation of either upper airway obstruction or tracheobronchomalacia, common anomalies of the airways in infants, after obtaining informed consent from the parents (IRB approval #2013-3309). The BAL fluid samples were centrifuged (4°C, 10 min, 400g). The resultant supernatant was frozen (−80°C), and the cells were cryopreserved (−150°C) in 90% FBS and 10% dimethyl sulfoxide. We used the frozen supernatant and cryopreserved cells in our analysis (IRB approval #2015-7983). Because pneumonia (48) and asthma (71) are associated with ILC3 activation in the lungs, we excluded infants with a history of pneumonia (defined as worsening of respiratory status, increase or change in the quality of respiratory secretions, and temperature instability with radiographic changes) or wheezing. We then selected infants who received ABX (ABX-exposed group) or no ABX (no-ABX group) and matched the respective treatment groups for gestational age, age at the time of procedure, and history of mechanical ventilation. Characteristics of the subjects are provided in Table 1. After thawing, we incubated (37°C, 5 hours, 5% CO2) the cells (0.5 × 106) in culture medium containing RPMI 1640 with 10% FCS, 1× nonessential amino acids, 10 mM Hepes, 2 mM l-glutamine (all from Invitrogen), and 1% penicillin/streptomycin with GolgiStop (1:1000; 554724, BD Biosciences), PMA (10 ng/ml), and calcium ionophore A23187 (500 ng/ml). We immunophenotyped the cells in BAL fluid as described previously (90). We stained cells with anti-human CD3 antibody (SP34-2), anti-human CD8 antibody (B9.11), anti-human CD14 antibody (RMO52), anti-human CD19 antibody (J3-119), anti-human CD45 antibody (J.33), anti-human CD56 antibody (NK901), anti-human CD69 antibody (TP1.55.3), and anti-human NKp46 antibody (9E2) (all diluted 1:1000; BioLegend). For intracellular staining, we washed and fixed (4°C, 60 min) the surface-stained cells in 1× Cytofix/Cytoperm buffer (BD Biosciences) and permeabilized them (4°C, overnight) using 1× Permeabilization buffer (BD Biosciences) according to the manufacturer’s instructions. We stained the cells intracellularly with anti-human RORγt antibody (AFKJS-9), anti-human T-bet antibody (4B10), and anti-human IL-22 antibody (BG/IL-22) (all diluted 1:50; BioLegend) and then washed (twice) and resuspended them in flow cytometry buffer. We collected the data with LSR II (BD Biosciences) and analyzed the data with FlowJo (Tree Star).

Enzyme-linked immunosorbent assay

We measured IL-22 or IL-20 in the murine (69) or human BAL fluid (91) using commercially available kits (eBioscience) as described previously. We measured IL-1β in the cell culture supernatant using commercially available kits (eBioscience) according to the manufacturer’s instructions.

Statistical analyses

Each data point represents a pool of three to four newborn mice that were pooled before the isolation of leukocytes from the indicated tissue. All data met the assumptions of the statistical tests used. We compared differences between groups by either unpaired two-tailed Student’s t test, ANOVA, or Wilcoxon signed-rank test. We used the Kaplan-Meier log-rank test to compare survival between groups (all in GraphPad Prism 6.0). P values are indicated as *P ≤ 0.05 or **P ≤ 0.01.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/9/376/eaaf9412/DC1

Fig. S1. Intestinal commensal bacteria promote resistance to S. pneumoniae in newborn mice via IL-22.

Fig. S2. Intestinal commensal bacteria direct postnatal trafficking of IL-22+ILC3 to murine newborn lung promoting resistance to pneumonia.

Fig. S3. Intestinal DCs mediate cross-talk between commensal bacteria and ILC3.

Table S1A. Relative abundance of different commensal bacteria in the intestine of ABX-free or ABX-exposed newborn mice.

Table S1B. Relative abundance of different commensal bacteria in the lungs of ABX-free or ABX-exposed newborn mice.

Table S2. Differentially expressed genes in the lungs of P4 ABX-free or ABX-exposed newborn mice.

Table S3. Tabulated raw data.

REFERENCES AND NOTES

  1. Acknowledgments: We thank W. Ouyang (Genentech) for providing IL-22–neutralizing antibody. We thank N. Butz for her assistance with microbial DNA isolation and the Children’s Hospital Research Foundation’s Flow Cytometry and Cell Sorting Core Laboratory for technical advice and support. We thank S. Way, C. Chougnet, and H. Singh for their helpful comments. We thank the physicians, nurses, and staff of the Cincinnati Children’s Hospital Medical Center Pulmonary Medicine Division and all of the participating patients and their families. Funding: H.D. is supported by 1K08HD084686 and the Francis Family Foundation. T.A. is supported by 5K08DK093784, the Burroughs Wellcome Fund, the Crohn’s and Colitis Foundation of America, and the Pew Trust. G.W. is supported by 5R01AI099479 and 5R01HL105834. J.W. is supported by 5R01HL095580, 4U01HL110964, and 5U01HL122642. Author contributions: H.D. designed the in vivo experimental studies for commensal disruption and reconstitution, innate immune cell depletion, and adoptive transfer of innate immune cells. J.G. designed in vitro coculture and cell migration studies. T.A., G.W., and J.W. provided reagents. H.D., J.G., and K.O. carried out experiments. H.D. and J.G. analyzed the data and wrote the manuscript with input from T.A., G.W., and J.W. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All the data sets are deposited in a publicly available database (figshare) and can be accessed at https://figshare.com/s/52f4aa2f8035fd505cf1. IL-22–neutralizing antibody is available from Genentech subject to a material transfer agreement.
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