Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria

Abstract

The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical containment remain unclear. Here, we show that interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses were associated with Crohn's disease and progressive hepatitis C virus infection in patients. Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases.

Figure 1. Innate lymphoid cells are resident in intestinal tissues of healthy mammals and limit bacterial dissemination and systemic immune activation in naïve mice

(A, C and E) The frequency of live IL-22⁺ cells was examined by flow cytometry from ex vivo IL-23-stimulated cells from (A) intestinal tissues from healthy humans, (C) the rectum of healthy rhesus macaques (E) or the small intestine lamina propria of naïve C57BL/6 mice. (B, D and F) The gated live IL-22⁺ populations from (B) humans, (D) rhesus macaques or (F) mice were stained with the indicated markers (open blue histogram) and compared to negative population controls (human and rhesus macaques: CD20⁺ CD56⁺, solid grey histograms; mice: CD19⁺ B cell populations, solid grey histograms). (G-N) Naïve C57BL/6 Rag1^-/- mice were administered an isotype control or anti-CD90.2 mAb starting on day 0 and sacrificed on day 3, 14 or 28. Colony forming units (CFU) present in homogenates from the (G) spleen and (H) liver of antibody treated mice. (I) LPS concentrations in homogenates from the liver of antibody treated mice. (J) H&E stained histological sections of the liver of antibody treated mice. Bar, 5 μm. Spleen (K) size and (L) weight from antibody treated mice. Bar, 5 mm. Serum concentrations of (M) IL-6 and (N) TNFα from antibody treated mice. All data are representative of 3 independent experiments of 3 individual mice per experiment, 5 total individual human donors or 2 total individual Rhesus macaques. Data shown are the mean ± SEM. Statistics compare days post depletion versus isotype using the Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001. ND, none detected.

Figure 2. ILCs regulate anatomical containment of commensal bacteria through IL-22-dependent induction of anti-microbial peptides

(A-E) Naïve C57BL/6 Rag1^-/- mice were administered an isotype control or anti-IL-22 mAb starting on day 0 and sacrificed on day 14. Colony forming units (CFU) present in homogenates from the (A) spleen and (B) liver of antibody treated mice. (C) LPS concentrations in homogenates from the liver of antibody treated mice. (D) H&E stained histological sections of the liver of antibody treated mice. Bar, 5 μm. (E) Spleen weight from antibody treated mice. (F-K) Naïve C57BL/6 Rag1^-/- mice were administered an isotype control or anti-CD90.2 mAb with PBS control or rL-22 starting on day 0 and sacrificed on day 14. (F) Colony forming units (CFU) present in homogenates from the spleen of antibody treated mice. (G) LPS concentrations in homogenates from the liver of antibody treated mice. Relative fold change of (H) Reg3b, (I) Reg3g, (J) S100a8 and (K) S100a9 transcript in terminal ileum epithelial RNA from treated mice. All data are representative of 2 or more independent experiments with a minimum of 3-4 mice per group. Data shown are the mean ± SEM. Statistics compare treatment versus isotype unless otherwise noted using the Student's t test. * p < 0.05 ** p < 0.01. ND, none detected.

Figure 3. Innate lymphoid cells regulate selective anatomical containment of Alcaligenes species to limit systemic inflammation

(A-C) Naïve C57BL/6 Rag1^-/- mice were administered an isotype control or anti-CD90.2 mAb starting on day 0 and sacrificed on day 14. Tissues from the liver and spleen were homogenized and cultured in LB broth. (A) Pyrosequencing of contents from the intestinal lumen or tissue cultures from anti-CD90.2 mAb treated Rag1^-/- mice. Top right bar represents sequence frequency. (B) Peyer's patches and (C) mesenteric lymph node (LN) were analyzed by FISH using probes to identify Alcaligenes spp. (BPA and ALBO) and epithelial cells (WGA) or DNA (DAPI). Bar, 100 μm. (D-G) Naïve germ-free (GF) or Alcaligenes monoassociated Rag1^-/- mice were administered an isotype control or anti-CD90.2 mAb starting on day 0 and sacrificed on day 5. (D) LPS concentrations in homogenates from the liver of antibody treated mice. (E) Spleen weight from antibody treated mice. Serum concentrations of (F) IL-6 and (G) TNFα from antibody treated mice. (H) Alcaligenes was cultured in the presence of PBS or recombinant (r) S100A8/S100A9 and colony forming units (CFU) were measured. Naïve Rag1^-/- mice were administered an isotype control or anti-CD90.2 mAb with PBS control or rS100A8/S100A9 on day 0 and sacrificed on day 5. Colony forming units (CFU) present in homogenates from the (I) spleen and (J) liver of antibody treated mice. All data are representative of 2 independent experiments with a minimum of 2-3 mice per group. Data shown are the mean ± SEM. Statistics compare PBS versus rS100S8/A9 treatments using Student's t test * p < 0.05. ND, none detected.

Figure 4. ILCs regulate anatomical containment of Alcaligenes in lymphocyte-replete hosts and Alcaligenes-specific responses are associated with chronic human disease

(A-H) Naïve CD90-disparate chimeric mice were administered an isotype control or anti-CD90.2 mAb starting on day 0 and sacrificed on day 3, 14 or 28. Colony forming units (CFU) present in homogenates from the (A) spleen and (B) liver of antibody treated chimeric mice. (C) LPS concentrations in homogenates from the liver of antibody treated mice. (D) H&E stained histological sections of the liver of antibody treated chimeric mice. Bar, 5 μm. (E) Spleen size from antibody treated chimeric mice. Bar, 5 mm. Serum concentrations of (F) IL-6 and (G) TNFα from antibody treated chimeric mice. (H) Relative optical density (OD) values of serum IgG specific to Alcaligenes crude antigens in treated chimeric mice. All data are representative of 3 independent experiments with a minimum of 3-5 mice per group. Statistics compare days post depletion versus isotype using the Student's t test. (I-N) Relative serum IgG (OD values per g/mL total serum IgG) specific to Alcaligenes crude antigens in (I) control (n=13) versus pediatric Crohn's disease patients (n=18) or (J) control (n=20) versus cirrhotic HCV-infected patients awaiting orthotopic liver transplantation (n=19). Statistics compare disease status using the Mann-Whitney test. Relative serum IgG specific for Alcaligenes crude antigen in chronically HCV-infected individuals (n=27) were correlated with levels of serum (K) bilirubin, (L) international normalized ratio (INR) of prothrombin time, (M) albumin and (N) platelets. The association between Alcaligenes-specific IgG levels and clinical parameters were compared by non-parametric Spearman's rank correlation coefficient.