Phosphatidylinositol 3-kinase-γ signaling promotes Campylobacter jejuni-induced colitis through neutrophil recruitment in mice

Abstract

Crypt abscesses caused by excessive neutrophil accumulation are prominent features of human campylobacteriosis and its associated pathology. The molecular and cellular events responsible for this pathological situation are currently unknown. We investigated the contribution of PI3K-γ signaling in Campylobacter jejuni-induced neutrophil accumulation and intestinal inflammation. Germ-free and specific pathogen-free Il10(-/-) and germ-free Il10(-/-);Rag2(-/-) mice were infected with C. jejuni (10(9) CFU/mouse). PI3K-γ signaling was manipulated using either the pharmacological PI3K-γ inhibitor AS252424 (i.p. 10 mg/kg daily) or genetically using Pi3k-γ(-/-) mice. After up to 14 d, inflammation was assessed histologically and by measuring levels of colonic Il1β, Cxcl2, and Il17a mRNA. Neutrophils were depleted using anti-Gr1 Ab (i.p. 0.5 mg/mouse/every 3 d). Using germ-free Il10(-/-);Rag2(-/-) mice, we observed that innate immune cells are the main cellular compartment responsible for campylobacteriosis. Pharmacological blockade of PI3K-γ signaling diminished C. jejuni-induced intestinal inflammation, neutrophil accumulation, and NF-κB activity, which correlated with reduced Il1β (77%), Cxcl2 (73%), and Il17a (72%) mRNA accumulation. Moreover, Pi3k-γ(-/-) mice pretreated with anti-IL-10R were resistant to C. jejuni-induced intestinal inflammation compared with Wt mice. This improvement was accompanied by a reduction of C. jejuni translocation into the colon and extraintestinal tissues and by attenuation of neutrophil migratory capacity. Furthermore, neutrophil depletion attenuated C. jejuni-induced crypt abscesses and intestinal inflammation. Our findings indicate that C. jejuni-induced PI3K-γ signaling mediates neutrophil recruitment and intestinal inflammation in Il10(-/-) mice. Selective pharmacological inhibition of PI3K-γ may represent a novel means to alleviate severe cases of campylobacteriosis, especially in antibiotic-resistant strains.

Figure 1. Innate immune cells mediate C. jejuni induced colitis in Il10^−/− mice

Cohorts of 4–7 germ-free Il10^−/− and Il10^−/−; Rag2^−/− mice were transferred to SPF conditions and immediately gavaged with a single dose of 10⁹ C. jejuni/mouse. After 12 days, colons were resected for formalin-fixation, sectioning and H&E staining, or RNA extraction for gene expression analysis. (A) Histological intestinal damage scores of C. jejuni-induced inflammation in Il10^−/− and Il10^−/−; Rag2^−/− mice. (B) Quantification of histological intestinal damage score mediated by C. jejuni infection. (C) Il1β, Cxcl2 and Il17a mRNA accumulation was quantified using an ABI 7900HT Fast Real-Time PCR System and specific primers and data were normalized to Gapdh. All graphs depict mean ± SEM. NS, P > 0.05. Scale bar is 200 μm. Results are representative of 3 independent experiments.

Figure 2. PI3K signaling pathway mediates C. jejuni-induced intestinal inflammation in Il10^−/−; NF-κB^EGFP mice

Four cohorts of 4–8 germ-free Il10^−/−; NF-κB^EGFP mice were transferred to SPF conditions and immediately gavaged with a single dose of C. jejuni (10⁹/mouse) and then i.p. injected with vehicle control (Ctl, 5% DMSO), wortmannin (wort, 1.4 mg/kg body weight) daily for 12 days. (A) H&E representation of intestinal damage. Arrow heads indicate crypt abscesses. (B) Quantification of intestinal inflammation as histological score. (C) Western blot for total and phosphorylated (S473) AKT and EGFP protein levels in pooled colonic lysates of infected mice. (D) Colonic EGFP expression levels were visualized using a CCD camera macroimaging system. (E) Il1β, Cxcl2 and Il17a mRNA accumulation was quantified using real time PCR. All graphs depict mean ± SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Scale bar is 200 μm. Results are representative of 3 independent experiments.

Figure 3. Pharmacological inhibition of PI3Kγ blocks C. jejuni-induced intestinal inflammation in Il10^−/−; NF-κB^EGFP mice

Four cohorts of 4–6 germ-free Il10^−/−; NF-κB^EGFP mice were transferred to SPF conditions and infected with C. jejuni as described in Figure1. Mice were i.p. injected with vehicle (5% DMSO) or pharmacological PI3Kγ inhibitor AS252424 (10 mg/kg body weight) daily for 6 days. (A) H&E representation of intestinal inflammation. (B) Quantitative histological score of intestinal inflammation. (C) Western blot for total and phosphorylated (S473) AKT, phosphorylated p70S6K (T389) and EGFP protein levels in pooled colonic lysates of infected mice. (D) Density of Western blot bands was quantified using ImageJ and normalized to control. (E) Colonic EGFP expression levels were visualized using a CCD camera macroimaging system. (F) Il1β, Cxcl2 and Il17a mRNA accumulation was quantified by real time PCR. All graphs depict mean ± SEM. *, P < 0.05, **, P < 0.01, NS, not significant. Scale bar is 200 μm. Results are representative of 3 independent experiments.

Figure 4. PI3Kγ deficiency attenuates C. jejuni-induced intestinal inflammation

Eight cohorts of 4–6 Wt, Il10^−/− and Pi3kγ^−/−mice were treated with antibiotic for 7 days and then gavaged with a single dose of C. jejuni (10⁹/mouse). Wt and Pi3kγ^−/−mice were injected anti-IL-10R antibody (i.p. 0.5 mg/mouse/every 3 days) to block IL-10 receptors. Mice were sacrificed after 14 days. (A–B) H&E representation of intestinal inflammation. (C) Quantitative histological score of intestinal inflammation. (D) Western blot for phosphorylated AKT (S473), phosphorylated p70S6K (T389) and total AKT protein levels in pooled colonic lysates of infected mice. (E) Il1β, Cxcl2 and Il17a mRNA accumulation was quantified using real time PCR. Data represent means ± SEM.*, P < 0.05. Scale bar is 200 μm. Results are representative of 2 independent experiments.

Figure 5. PI3Kγ signaling promotes C. jejuni invasion into colon, MLN and spleen

Cohorts of 4–6 germ-free Il10^−/−; NF-κB^EGFP mice, SPF Wt and Pi3kγ^−/− mice were treated and infected as indicated in Figure 3 and 4. (A) C. jejuni (red dots) in colonic sections of infected mice was detected using FISH. Scale bar is 10 μm. (B) C. jejuni bacterial count in the colon, MLN and spleen of vehicle- or AS252424-treated mice. (C) Presence of C. jejuni (red dots) in colonic tissue of Wt and Pi3kγ^−/− mice was determined using FISH. Data represent means ± SEM. * P < 0.05. Results are representative of 3 independent experiments.

Figure 6. PI3Kγ mediates neutrophil accumulation and crypt abscesses in C. jejuni infected mice

Cohorts of 4–6 germ-free Il10^−/−; NF-κB^EGFP mice were transferred to SPF conditions and infected/treated as indicated in Figure 3. (A) Number of crypt abscesses in C. jejuni infected mice. (B) IHC representation of MPO positive cells (brown dots) in the colon tissue of C. jejuni infected mice. (C) Peripheral blood neutrophils were isolated and plated in a transwell system and cells migration in response to CXCL-2 (250 ng/mL) at the bottom well were enumerated. Representative light images of neutrophils migrated into bottom wells. (D) Quantitative measurements of migrated neutrophils. Lower panels (scale bar 20 μm) are magnified images of area shown in the upper panels (scale bar 200 μm). Data represent means ± SEM. **, P < 0.01. Results are representative of 3 independent experiments.

Figure 7. Neutrophils enhance C. jejuni-induced colitis

Cohorts of 4–6 germ-free Il10^−/−; NF-κB^EGFP mice were transferred to SPF conditions and infected as in Figure 3. Neutrophils were depleted using anti-Gr1 antibody. (A) H&E representation of intestinal inflammation. (B) Quantitative histological score of intestinal inflammation. (C) IHC representation of MPO positive cells (brown dots) in the colon tissue of C. jejuni infected mice. (D) The colonic H&E stained sections were imaged (5 fields/mouse) and neutrophils were identified based on morphological features. Data are presented as average counts/mouse. (E) Presence of C. jejuni (red dots) in colonic tissue was determined using FISH. Lower panels (scale bar 20 μm) are magnified images of area shown in the upper panels (scale bar 200 μm). Data represent means ± SEM. *, P < 0.05. Results are representative of 3 independent experiments.