Homeostatic Regulation of Salmonella-Induced Mucosal Inflammation and Injury by IL-23

IL-12 and IL-23 regulate innate and adaptive immunity to microbial pathogens through influencing the expression of IFN-γ, IL-17, and IL-22. Herein we define the roles of IL-12 and IL-23 in regulating host resistance and intestinal inflammation during acute Salmonella infection. We find that IL-23 alone is dispensable for protection against systemic spread of bacteria, but synergizes with IL-12 for optimal protection. IL-12 promotes the production of IFN-γ by NK cells, which is required for resistance against Salmonella and also for induction of intestinal inflammation and epithelial injury. In contrast, IL-23 controls the severity of inflammation by inhibiting IL-12A expression, reducing IFN-γ and preventing excessive mucosal injury. Our studies demonstrate that IL-23 is a homeostatic regulator of IL-12-dependent, IFN-γ-mediated intestinal inflammation

The roles of IL-12 and IL-23 in the Innate and Adaptive Immune Responses against Salmonella

Thesis (Ph.D.)--University of Washington, 2012→Non-typhoidal Salmonella is a major health concern worldwide typically causing gastroenteritis in people of all ages and severe invasive disease in immune-compromised persons in particular in individuals with genetic defects in innate immune signaling molecules Interleukin (IL)-12/IL-23. The innate immune system is pivotal for early control of Salmonella and proper induction of the adaptive immune system responsible for eradication of and future protection from this pathogen. IL-12 and IL-23 are important innate signaling molecules that typically work through the promotion of their downstream target Interferon (IFN)-γ and IL-17/-22, respectively. Our overall goal is to better define the roles of these cytokines using of the streptomycin pretreatment model of Salmonella -induced enterocolitis in order to develop/modify different modalities and therapies for this disease. →In the acute phase, we found that IL-23 alone was dispensable for protection against systemic spread of bacteria, but synergized with IL-12 for optimal protection. We also found that IL-12 promoted the production of IFN-γ by NK cells, which as mentioned above, is required for resistance against Salmonella and also for induction of intestinal inflammation and epithelial injury. In contrast, IL-23 controlled the severity of inflammation by inhibiting IL-12A expression, thereby reducing IFN-γ and prevented excessive mucosal injury. →In late phase studies our data showed that the combined loss of IL-12/-23 led to defective control of oral attenuated Salmonella infection, diminished the generation of antigen-specific CD4 T cells, but robust antigen specific antibody responses. IFN-γ -/- mice had a survival disadvantage compared to IL-12/-23 DKO mice. This differential survival may reflect the unexpected production of IFN-γ by IL-12/23 DKO T-cells. Surprisingly, antigen-specific Th1 cells were readily detected upon rechallenge of immunized IL-12/23 deficient mice, indicating that these cytokines are not required for the generation of Th1 cells. Nevertheless, the IL-12/23-independent, antigen-specific Th1 and antibody responses were insufficient for optimal control of late phase Salmonella infection. Together these data demonstrate the critical and complex relationship of IL-12 and IL-23 that are essential for early and late phase immunity against Salmonella

IL-23 inhibits <i>Salmonella</i> induced IFN-γ production by lamina propria NK cells.

<p>(A) LP populations isolated from WT, p19^−/−, p35^−/−, p40^−/− and IFN-γ^−/− mice were stained for surface expression of NK1.1 and CD3ε; numbers indicate the percentage of cells in the gated box. (B) Intracellular production of IFN-γ and TNF is shown for NK cells (the NK1.1⁺CD3⁻ population gated in A). (C) The percentage of LP IFN-γ+ NK cells were quantified for the aforementioned mice at 3 d <i>Salmonella</i> infection. Plotted are the mean ± SE for 3 mice per condition. Statistical significance was determined using the one-way ANOVA followed by Bonferroni post test. *: p<0.05, **: p<0.01, ***: p<0.001</p

IL-23 inhibits <i>Salmonella</i> induced IL-12 dependent IFN-γ mediated inflammation.

<p>(A) IFN-γ, and (B) IL-12a (IL-12p35) cecal gene expression at 3 d after oral WT <i>Salmonella</i> infection +/− mock treated mice from WT, p19^−/−, p35^−/−, p40^−/− mice was determined by qPCR. Bars show the median and the data are pooled from 3 separate infections. (C) Cecum and (D) liver were collected from WT and IFN-γ^−/− 3 days post infection. Organ homogenates were diluted and plated to determine CFU/organ. Bars represent the median bacterial load and the data are pooled from 2 separate infections. (E) Histologic changes were quantified on H&E-stained cecal sections as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037311#pone-0037311-g002" target="_blank">Fig. 2</a>. (F) TUNEL and (G) AB positive cells were quantified per high-power field in the ceca of at minimum 3 infected mice/group. Results are expressed in mean ± SE. Statistical significance was determined using one way ANOVA followed by Bonferroni post test for A and B, the Mann-Whitney test was used for C and D, and unpaired Student's t-test was used for E-G. *: p<0.05, **: p<0.01, ***: p<0.001.</p

IL-12 and IL-23 are required for resistance against S. Typhimurium (Stym).

<p>WT, p19^−/−(no IL-23), p35^−/−(no IL-12), and p40^−/− (no IL-12/-23) mice were orally infected with 1×10³ CFU of <i>S</i>. Typhimurium 1 day after 20 mg streptomycin pretreatment. (A) Cecum and (B) liver were collected from infected mice 3 days post infection. Organs were weighed, homogenized and plated to determine CFU/organ. Bars represent the median bacterial load. Pooled data from 3 separate experiments are shown in A and B. (C) Representative ceca are shown for each group at 3 d after oral inoculation with PBS (mock) or Stym. A one centimeter bar indicates the magnification. (D) 10X and (E) 40X magnification of histopathology of H&E stained cecal sections of WT, p19^−/−, p35^−/−, and p40^−/− mice 3 d after infection. Bar in (D) represents 100 micron and (E) 20 micron. Notched arrow-heads in (E) indicate cell death. (F) Blinded scoring was performed on H&E-stained cecal sections. The inflammatory score equals the sum of the separate categories (edema, PMN infiltration, lymphoplasmacytosis, and goblet cell loss). (G) H&E stained cecal sections from WT, p19^−/−, p35^−/−, and p40 mice 3 d after infection were blinded and qualitatively assessed for cell death: 0 (no significant), 1 (focal), 2 (mild), 3 (severe) in the basal epithelium. At least 3 mock treated animals and at least 13 infected mice from each group from 3 separate experiments were scored in 1F and 1G. Mean values ± SE are shown. Statistical significance was determined using the one way ANOVA test with Bonferroni post test (A, B, F and G). *: p<0.05, **: p<0.01, ***: p<0.001.</p

IL-23 limits IL-12 dependent pro-inflammatory cytokine production and the activation of gut mononuclear phagocytes.

<p>(A) TNF, (B) Nos2, (C) IL-6, (D) IL-1β, (E) IL-10 and (F) TGF-β cecal gene expression at 3 d after oral <i>S</i>. Typhimurium or mock infection of WT, p19^−/−, p35^−/−, p40^−/− mice was determined by qPCR. Data are expressed as the ratio of mRNA levels of the gene of interest divided by GAPDH expression from the same RNA. Bars show median values and the data are pooled from 3 separate infections. (G) Following mock or <i>Salmonella</i> infection, lamina propria (LP) cells were isolated from the cecum and colon of C57BL/6 mice as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037311#s4" target="_blank">Materials and Methods</a> section. LP cells were treated for 4 hrs <i>ex vivo</i> with BFA, followed by surface staining with CD11c, CD11b and class II MHC, followed by intracellular TNF. The CD11b^hi CD11c⁺ cell population (red gates) were the primary TNF producers. TNF and class II MHC expression by the CD11b^hi CD11c⁺ cell populations is shown for representative animals in the lower panels. Quantification of CD11b^hi/CD11c⁺ positive cells for (I) MHC classII^hi expression and (J) MHC classII^hi TNF⁺ populations. Statistical significance was determined using one way ANOVA followed by Bonferroni post test. *: p<0.05, **: p<0.01, ***: p<0.001.</p

Loss of IL-23 in the context of functional IL-12 results in increased epithelial cell death and loss of goblet cells.

<p>(A) 20X magnification of TUNEL-positive (brown) or (C) Alcian Blue (blue) cells of representative infected cecal sections. The bar in (A) and (C) represents 20 micron. The mean ± SE of TUNEL (B) positive cells per high-power field was quantified from cecal sections WT (n = 4), p19^−/− (n = 5), p35^−/− (n = 6) and p40^−/− (n = 3) mice. The mean ± SE Alcian Blue positive cells were quantified per high-power field from cecal sections of the same mice (n = 3 per group). Statistical significance for (B and D) was determined using the one way ANOVA followed by Bonferroni post test. *: p<0.05, **: p<0.01, ***: p<0.001.</p

Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in a murine model of PSC

Objective Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. Goal: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient ( mdr2 −/− ) mice and microbial profiles in PSC patient cohorts. Design We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2 −/− mice and targeted metagenomic analysis in PSC patients. Results GF mdr2 −/− mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2 −/− mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2 −/− mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients’ clinical severity by Mayo risk scores. Conclusions We identified novel functionally protective and detrimental resident bacterial species in mdr2 −/− mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients