Campylobacter jejuni Induces Colitis Through Activation of Mammalian Target of Rapamycin Signaling  Xiaolun Sun, Deborah Threadgill, Christian Jobin  Gastroenterology  Volume 142, Issue 1, Pages 86-95.e5 (January 2012) DOI: 10.1053/j.gastro.2011.09.042 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Rapamycin prevents and treats C jejuni–induced colitis in Il10−/−; NF-κBEGFP mice. Cohorts of 5 to 14 germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and immediately gavaged with 109 C jejuni/mouse and then injected intraperitoneally with vehicle control (Ctl, 5% dimethyl sulfoxide) or rapamycin (rapa, 1.5 mg/kg body wt) for 12 days. For rapamycin treatment (rapa trt), C jejuni–infected mice (4 days) were injected intraperitoneally with rapamycin to day 12. Colon was resected and fixed in formaldehyde, and sections were stained with H&E or protein extracts for Western blot analysis. (A) Western blot for total and phosphorylated colonic p70S6K. (B) Histologic intestinal damage score of rapamycin prevention on C jejuni infection. (C) Histologic intestinal damage score of rapamycin treatment. (D) H&E representation of the different experimental groups with crypt abscesses (arrowheads). (E) H&E sections of rapamycin-treated mice. All graphs depict mean ± SE. **P < .01, ***P < .001. Scale bar = 200 μm. Results are representative of 4 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 C jejuni–induced intestinal inflammation is independent of CD4 T-cell activation. Three cohorts of 4 to 5 germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and immediately gavaged with 109 C jejuni/mouse and then injected intraperitoneally with vehicle control (Ctl, phosphate-buffered saline) or anti-CD4 antibody (anti-CD4, 0.5 mg/mouse every 3 days). (A) H&E representation of the different experimental groups. (B) Histologic score of intestinal inflammation. (C) Representative flow cytometry analysis of CD4+ cell population change in the spleen. (D) Percentage flow cytometry results of CD4+ cells in the spleen and MLN. All graphs depict mean ± SE. ***P < .001. Scale bar = 200 μm. Results are representative of 3 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Treatment with rapamycin reduces C jejuni–induced colonic EGFP expression in Il10−/−; NF-κBEGFP mice. Four cohorts of 5 to 14 germ-free Il10−/−; NF-κ BEGFP mice were transferred to SPF conditions and infected with C jejuni/mice as described in Figure 1. Mice were injected intraperitoneally with vehicle (5% dimethyl sulfoxide) or rapamycin (1.5 mg/kg body wt) for 12 days. (A) EGFP expression in the mouse colon was visualized using a charge-coupled device camera macro-imaging system. EGFP levels were analyzed by (B) Western blot and (C) confocal microscopy visualization of EGFP-positive cells images in 3 dimensions using Imaris. Results are representative of 3 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Rapamycin attenuates C jejuni–induced expression of proinflammatory mediators. Four cohorts of 5 to 14 germ-free Il10−/−; NF-κBEGFP mice were treated as indicated in Figure 1. Colons were resected and RNA extracted using TRIzol (Invitrogen, Carlsbad, CA). (A) Il1β, Cxcl2, and Il-17a messenger RNA accumulation was quantified using an ABI 7900HT Fast Real-Time PCR System, and specific primers and data were normalized to Gapdh. (B) Colonic tissues and MLNs were cultured in RPMI 1640 medium supplemented with 3% fetal bovine serum and 1% antibiotics for 18 hours. Supernatants were collected, and IL-1β and IL-17 secretion was measured by ELISA. Data represent means ± SE. *P < .05, **P < .01. Results are representative of 3 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Neutrophils participate in C jejuni–induced colitis. Cohorts of 5 to 7 germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and infected/treated as indicated in Figure 1. (A) Immunohistochemical representation of myeloperoxidase-positive colonic cells (brown dots) in Control (Ctl), C jejuni–infected, rapamycin (rapa)-treated mice. Lower panels (scale bar = 20 μm) are magnified images of area shown in the upper panels (scale bar = 200 μm). (B) Flow cytometry analysis for CD45+ and Gr-1+ cells in the peripheral blood of infected and rapamycin-treated mice. (C) TEM images of colonic tissues from C jejuni–infected with/without rapamycin treatment. Left panel shows accumulated neutrophils (arrowheads) in a crypt with microvilli erosion (arrows). Right panel shows absence of neutrophils with intact microvilli (arrows). Insets are magnified images of the framed area. Scale bar = 2 μm. Data represent means ± SE. **P < .01. Results are representative of 3 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 mTOR signaling promotes C jejuni invasion of the colon, MLNs, and spleen. Cohorts of 3 to 4 germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and infected/treated as indicated in Figure 1. (A) Clustered C jejuni (red dots) in colonic section of infected mice was detected using FISH. Insets are magnified images of the framed area. Scale bar = 100 μm. (B) Presence of clustered C jejuni (red dots) in whole colon tissue was determined using FISH and 3-dimensional imaging. (C) Evidence of intracellular (arrows, left panel) and luminal (right panel) C jejuni in colonic tissues of untreated or rapamycin-treated mice, respectively. Lower panels (scale bar = 200 nm) are magnified images of the area shown in the upper panels (scale bar = 2 μm). (D) C jejuni bacterial count in the stool, colon, MLNs, and spleen of untreated or rapamycin-treated mice. Data represent means ± SE. *P < .05. Results are representative of 3 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Rapamycin promotes C jejuni eradication and LC3II generation in splenocytes. Splenocytes were isolated from Il10−/− mice and infected with C jejuni (multiplicity of infection, 50) and cultured with rapamycin (100 nmol/L). A gentamycin killing assay at 0 and 4 hours and WB assay at 0.5, 1.5, and 4 hours were performed. (A) Percentage of C jejuni survival in splenocytes. (B) Western blot of colonic LC3I/II and phosphorylated p70S6K in infected splenocytes. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Rapamycin attenuates C jejuni–induced colitis in conventionally derived Il10−/−; NF-κBEGFP mice. Cohorts of 4 to 6 conventionally derived Il10−/−; NF-κBEGFP mice were treated with an antibiotic cocktail (7-day) and gavaged with 109 C jejuni/mouse. Mice were injected intraperitoneally with vehicle (5% dimethyl sulfoxide) or rapamycin (rapa, 1.5 mg/kg body wt) for 14 days. Colon was resected and fixed in formaldehyde, and sections were stained with H&E. (A) H&E representation of the different experimental groups. (B) Histologic intestinal damage scores of rapamycin prevention on C jejuni infection. All graphs depict mean ± SE. *P < .05. Scale bar = 200 μm. Results are representative of 2 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 E coli fails to induce intestinal inflammation in Il10−/−; NF-κBEGFP mice. Five germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and immediately gavaged with 107 E coli/mouse for 12 days. Colon was resected and fixed in formaldehyde, and sections were stained with H&E. Shown are H&E representations of the different experimental groups. Scale bar = 200 μm. Results are representative of 2 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Rapamycin ameliorates S typhimurium–induced intestinal inflammation in cecum and colon of Il10−/−; NF-κBEGFP mice. Cohorts of 4 to 6 germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and immediately gavaged with 107 S typhimurium/mouse. Mice were injected intraperitoneally with vehicle control (ctl, 5% dimethyl sulfoxide) or rapamycin treatment (rapa trt, 1.5 mg/kg body wt) daily to day 2. Cecum and colon were resected and fixed in formaldehyde, and sections were stained with H&E. (A) H&E representation of cecum from the different experimental groups. (B) Histologic cecal damage scores of rapamycin-treated, S typhimurium–infected mice. (C) H&E representation of colon from the different experimental groups. (D) Histologic colonic damage scores of rapamycin-treated, S typhimurium–infected mice. All graphs depict mean ± SE. *P < .05. Scale bar = 50 μm. Results are representative of 2 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 C jejuni induces early colitis in Il10−/−; NF-κBEGFP mice. Cohorts of 5 to 6 germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and immediately gavaged with 109 C jejuni/mouse. At day 4, colon was resected and fixed in formaldehyde, and sections were stained with H&E. (A) H&E representation of the different experimental groups. (B) Histologic intestinal damage scores of rapamycin-treated, C jejuni–infected mice. Scale bar = 200 μm. Results are representative of 2 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Rapamycin treatment reverses C jejuni–induced bloody diarrhea in Il10−/−; NF-κBEGFP mice. Cohorts of 5 to 14 germ-free Il10−/−; NF-κBEGFP mice were transferred to SPF conditions and immediately gavaged with 109 C jejuni/mouse. For rapamycin treatment (rapa trt), C jejuni–infected mice (4 days) were injected intraperitoneally with vehicle control (ctl, 5% dimethyl sulfoxide) or rapamycin (1.5 mg/kg body wt) daily for 12 days. (A) Rapamycin treatment prevents accumulation of blood on the anus of C jejuni–infected mice. (B) Rapamycin treatment prevents C jejuni–induced bloody colon (arrowheads indicate residual blood). Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 C jejuni–induced Il-12p40 and TNFα messenger RNA accumulation is not blocked by rapamycin. Cohorts of 5 to 14 germ-free Il10−/−; NF-κBEGFP mice were treated as indicated in Figure 4. Il-12p40 and TNFα messenger RNA accumulation was quantified using an ABI 7900HT Fast Real-Time PCR System, and specific primers and data were normalized to Gapdh. Data represent means ± SE. Results are representative of 3 independent experiments. Gastroenterology 2012 142, 86-95.e5DOI: (10.1053/j.gastro.2011.09.042) Copyright © 2012 AGA Institute Terms and Conditions