Lymphotoxin controls the IL-22 protection pathway in gut innate lymphoid cells during mucosal pathogen challenge

Abstract

Innate lymphoid cells (ILCs) have emerged as important players, regulating the balance between protective immunity and immunopathology at mucosal surfaces. However, mechanisms that regulate ILCs' effector functions during mucosal pathogenic challenge are poorly defined. Using mice infected with the natural mouse enteric pathogen Citrobacter rodentium, we demonstrate that lymphotoxin (LT) is essential for IL-22 production by intestinal ILCs. Blocking of LTβR signaling dramatically reduced intestinal IL-22 production after C. rodentium infection. Conversely, stimulating LTβR signaling induced an IL-22 protection pathway in LT-deficient mice. Furthermore, exogenous IL-22 expression rescued LTβR-deficient mice. IL-22-producing ILCs were predominantly located in lymphoid follicles in the colon and interacted closely with dendritic cells (DCs). We find that an LT-driven positive feedback loop controls IL-22 production by RORγt(+) ILCs via LTβR signaling in DCs. Taken together, our data show that LTβR signaling in gut lymphoid follicles regulates IL-22 production by ILCs in response to mucosal pathogen challenge.

Figure 1. LTβR signaling controls innate IL-22 pathway in the gut

A. IL-22 levels in the colon of naïve and infected WT and Ltbr^-/- mice. LTbr^-/- and WT mice (n=5/group/experiment) were orally inoculated with C. rodentium. mRNA expression of IL-22 and IL-22- dependent RegIIIγ and RegIIIβ antimicrobial proteins in the colon were measured by real-time PCR on day 5 post infection. Naïve WT mice were used as control. B. WT mice were treated with 150 μg of LTβR-Ig, or control human Ig (hIg) at day 0 and 3 post infection. Expression of IL-22 and RegIIIγ and RegIIIβ antimicrobial proteins were measured in colon by real-time PCR at day 5 post infection. C. Rag1^-/- mice were treated intraperitoneally with 150 μg of LTβR-Ig at day 0 and 3 post infection, and expression of IL-22, and RegIIIγ and RegIIIβ antimicrobial proteins was measured by real-time PCR at day 5 post infection. A-C. Data represent means ± s.e.m. n=5, *p<0.05, **p<0.01. Data represent one of three independent experiments with similar results. Real-time PCR data were normalized to hprt expression.

Figure 2. IL-22 is an essential protection pathway downstream of LTβR signaling

A-C. Stimulation of LTβR signaling promotes IL-22 protection pathway. Ltb^-/- mice were injected intraperitoneally with 75μg of agonistic anti-LTβR antibody or control rat Ig (rIg) at day 0 and 3 post infection with C. rodentium. mRNA expression of IL-22 (A) and RegIIIγ (B) were measured in colon by real-time PCR at day 4 post infection. C. Bacterial titers in liver at day 4 post infection. n=4, **p<0.01, Data represent means ± s.e.m. D-G. Hydrodynamic injection of IL-22 expressing plasmid rescues Ltbr^-/- mice from lethal C. rodentium infection. IL-22 expressing plasmid or control vector were intravenously injected to WT or Ltbr^-/-mice 6h after C. rodentium infection. n=10-13/group. Survival (D) and body weight change (E) are shown. ***p<0.001 between IL-22 treated Ltbr^-/- and control untreated Ltbr^-/- mice by Mantel-Cox log-rank test. Data combined from two experiments with similar results. F. Representative hematoxylin and eosin staining of colons at day 9 post infection. Red boxes in top panel are shown at higher magnification at lower panel. Bars: 1mm for top panel, and 200μm for lower panel. G. Colitis histopathology score. n=5, *p<0.05. Data represent means ± s.e.m.

Figure 3. IL-22 is produced in lymphoid follicles by innate RORγt⁺ cells

A. IL-22 is predominantly expressed in lamina propria. Colon lamina propria (LP) and intraepithelial lymphocyte (IEL) cell populations from WT mice were purified at day 5 post infection. IL-22 expression was measured by real-time PCR. Data normalized to hprt expression. Data represent means ± s.e.m. n=5 mice, **p<0.01. B. LTi cells are predominant IL-22 producing cells in the lamina propria. Colon LP innate cell populations from RORγt-GFP^+/-/Ltbr^+/+ and RORγt-GFP^+/-/LTbr^-/- mice at day 5 post infection were purified by flow cytometry. IL-22 expression was measured by real-time PCR. Sorted cell populations are indicated as: LTi: CD45⁺CD3^-GFP⁺NK1.1^-NKp46^-, NKp46: CD45⁺CD3^-GFP⁺NKp46⁺, NK: CD45⁺CD3^-GFP^-NK1.1⁺, Th17: CD45⁺CD3⁺GFP⁺NK1.1^-NKp46^-. Data combined from two experiments, means ± s.e.m, n=4, **p<0.01. IL-22 data were normalized to hprt expression. C. IL-22 expressing cells are predominantly located in lymphoid follicles. WT mice were orally infected with C. rodentium and colon sections at day 5 post infection were stained with indicated antibodies. Nuclei were stained with DAPI. Bars: 20μm. D. IL-22 producing cells interact with DC in lymphoid follicles. Colon sections of mice at day 5 post infection were stained with indicated antibodies and analyzed by confocal microscopy. Nuclei were stained with DAPI. Arrows indicate contact of DC (green) with IL-22 producing cells (red) on top panel, and RORγt⁺CD4^- cells on bottom panel. Bars: 10μm. E. IL-22 is expressed predominantly in isolated lymphoid follicles. WT mice were orally infected with C. rodentium, and colon tissue collected at day 5 post infection. Isolated lymphoid follicles (ILF+) and surrounded tissue (ILF-) were microdissected under stereo microscope, and IL-22 expression measured by real-time PCR. IL-22 data were normalized to hprt expression. *p<0.05, n=3 mice. F. Co-culture of LTi cells with DC promotes IL-22 production by LTi cells. Lamina propria LTi cells from naïve RORγt-GFP^+/- mice were co-cultured in vitro for 18h with lamina propria DCs from WT mice at day 5 post infection. After co-culture, LTi and DC cells were separated by flow cytometry and IL-22 measured by real-time PCR. Data normalized to b-actin. One of two independent experiments with similar results is shown.

Figure 4. LT expression on RORγt⁺ cells is essential for control of IL-22 production and protection of mice against C. rodentium infection

A-B. LT expression on RORγt⁺ cells controls IL-22 protection pathway. WT and RORγt-Ltb^-/-mice were orally infected with C. rodentium. Expression of IL-22 in colon and purified lamina propria cells (A), and antimicrobial proteins RegIIIγ, and RegIIIβ (B) was measured by real-time PCR at day 5 post infection. Data represent means ± s.e.m. One out of three independent experiments with similar results is shown. n=5 mice, *p<0.05, **p<0.01. Expression data were normalized to hprt expression. C. IL-22 expression is sufficient to rescue RORγt-Ltb^-/- mice from lethal C. rodentium infection. WT and RORγt-Ltb^-/- mice were intravenously injected with IL-22 expressing plasmid or control vector at 6h after C. rodentium infection. n=10/group. Data combined from two experiments with similar results. Survival and body weight change are shown. D. Representative hematoxylin and eosin staining of colon at day 9 post infection. Red boxes in top panel are shown at higher magnification at lower panel. Bars: 1mm for top panel, and 200μm for lower panel. E. Colitis histopathology score for mice in panel A. n=5, ***p<0.001. Data represent means ± s.e.m. One of two experiments with similar results is shown.

Figure 5. LTβR signaling in DCs is required for IL-22 production

WT, CD11c-Ltbr^-/-, and Ltbr^-/- mice were orally infected with C. rodentium. Body weight change (A) and representative hematoxylin and eosin staining of colon at day 12 post infection (B). Arrows indicate bacterial lesions. Bars: 50μm (top panels), and 20 μm (lower panels). Bacterial titers were measured in feces (C) on day 14, and liver (D), and spleen (E) on day 10 post infection. F. IL-22 expression in colon at day 5 post infection. G. IL-22 levels in colon supernatants at day 5 post infection. H. Expression of RegIIIγ mRNA in colon at day 5 post infection. C-H. Data represent means ± s.e.m. n=5 mice, *p<0.05, **p<0.01. Represents one of three independent experiments with similar results. Real-time PCR data were normalized to hprt expression. I. LTβR signaling in DCs regulates IL-23 production. CD11c⁺CD11b⁺ DCs from colon lamina propria of WT and LTbr^-/- mice were purified by flow cytometry at day 5 post infection. Expression of IL23p19, IL23p40, IL-1b, IL-6 cytokines was measured by real-time PCR. Data combined from two experiments, means ± s.e.m, n=4, *p<0.05, NS-not significant. RT-PCR data normalized to hprt expression.

Figure 6. Proposed model to control IL-22 pathway against mucosal bacterial pathogen

LT expression by RORγt⁺ ILCs is necessary for IL-22 production following invasion of mucosal bacterial pathogen. LT signaling by RORγt⁺ ILC promotes the development of lymphoid follicles in the gut. Lymphoid follicles provide necessary microenvironment for interaction between innate RORγt⁺ cells and DC. Interplay between RORγt⁺ ILCs and DCs promotes IL-23 production by DCs that, in turn, activates IL-22 synthesis by RORγt⁺ cells as a positive feedback loop. IL-22 stimulates IL-22R on epithelial cells which triggers production of antimicrobial proteins RegIIIγ and RegIIIβ to eliminate mucosal bacterial pathogen.