KSR1 protects from interleukin-10 deficiency-induced colitis in mice by suppressing T-lymphocyte interferon-γ production

Abstract

Background & aims: Immunological disorders of the gastrointestinal tract such as inflammatory bowel disease often result in recurrent and persistently elevated levels of proinflammatory cytokines. Kinase suppressor of Ras 1 (KSR1) is involved in tumor necrosis factor-mediated colon epithelial cell survival, yet its role in chronic inflammation has not been defined. In this study, we tested the hypothesis that KSR1 is protective against spontaneous experimental colitis.

Methods: KSR1(-/-)Interleukin-10 (Il10)(-/-) mice were generated and histolopathologic parameters of intestinal inflammation were scored. Bone marrow transplants performed on wild-type and KSR1(-/-)Il10(-/-) mice determined the contribution of KSR1 in hematopoietic lineages. Mucosal T helper (Th) 1 and Th17 cytokine were also examined. In vitro Th1 and Th17 polarization assays were conducted and interleukin (IL)-17A and interferon-γ (IFN-γ) production analyzed by flow cytometry. Neutralizing antibodies against IgG, IL-17A, or IFN-γ were administered to 3-week-old KSR1(-/-)Il10(-/-) mice for 3 weeks and scored for colitis.

Results: KSR1(-/-)Il10(-/-) mice developed accelerated and severe spontaneous colitis by 4 weeks of age. KSR1 expression in hematopoietic lineages was protective against colitis. Both IFN-γ and IL-17A transcripts were elevated in colons of KSR1(-/-) and KSR1(-/-)Il10(-/-) mice. IFN-γ production was increased in lamina propria T cells isolated from KSR1(-/-) and KSR1(-/-)Il10(-/-) mice. Additionally, in vitro Th1 polarization was increased while Th17 polarization was impaired in KSR1-deficient naïve T cells. Finally, administration of IFN-γ neutralizing antibodies attenuated colitis in KSR1(-/-)Il10(-/-) mice.

Conclusions: Mice lacking both KSR1 and IL-10 develop exacerbated colitis due to dysregulated IFN-γ production in T lymphocytes.

Figure 1. KSR1^−/−Il10^−/− mice develop accelerated spontaneous colitis

A) WT, KSR1^−/−, Il10^−/−, and KSR1^−/−Il10^−/− mice were weighed weekly from 3 weeks of age to 10 weeks. Plotted data are the mean weight for each group (n ≥ 5). Error bars represent the SEM. B) Mouse colonoscopic images taken on 10-week old mice. C) Colons were removed from 10-week old mice and flushed, weighed, and measured from the cecum to anus. Solid bars represent the mean (n ≥ 3) of the length/weight ratios. Error bars are the SEM. D) Paraffin embedded colon sections from 10-week old mice stained with Hematoxylin and Eosin (H&E). Images were taken at 20X magnification (scale bars, 50 µm). E) H&E stained 10-week old mouse colon sections were scored by a pathologist blinded to the genotype. Solid bars represent the mean injury and inflammation score for each group (n ≥ 6) and error bars are the SEM. F) KSR1^−/−Il10^−/− mice were sacrificed at each time point indicated and scored for inflammation and injury as before. * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 2. KSR1 in hematopoietic lineages suppresses colitis in Il10^−/− mice

A) 4-week old WT and KSR1^−/−Il10^−/− (DKO) recipient mice were irradiated with 9 Gy ¹³⁷Cesium. Bone marrow transplants using the indicated donor mice were performed and mice were sacrificed at 10-weeks of age. Each individual colonic injury and inflammation score is plotted with a solid line indicating the mean score for each group from three independent experiments and error bars are the SEM. B) Representative H&E stained paraffin embedded colon sections from recipient DKO mice transplanted with WT (1), Il10^−/− (2), KSR^−/− (3), or KSR1^−/−Il10^−/− (4) bone marrow as indicated. * P < 0.05, ** P < 0.01

Figure 3. Colons of KSR1^−/− and KSR1^−/−Il10^−/− mice have elevated IFN-γ

A) Total RNA was isolated from homogenized whole colon tissue from 10 week-old WT, KSR1^−/−, Il10^−/−, and KSR1^−/−Il10^−/− mice. TNF, IFN-γ, and IL-17A cytokine transcript levels were analyzed by quantitative real-time PCR. Solid bars represent the mean for each genotype and error bars are the SEM. B) Total protein from homogenized mouse colons was analyzed for the detection and quantitation of TNF, IFN-γ, and IL-17A. Solid bars represent the mean quantity of each cytokine per mg of protein. Error bars are the SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 4. Lamina propria T cells isolated from KSR1^−/− mice have increased IFN-γ production

Lamina propria T cells were isolated from WT, KSR1^−/−, Il10^−/−, and KSR1^−/−Il10^−/− mice and cultured for 5 hours in the presence of the protein transport inhibitor GolgiPlug and treated with or without PMA/Ionomycin. Cells were stained for cell surface TCRβ and intracellular IFN-γ and IL-17A. Samples were analyzed by flow cytometry gated on lymphocyte geometry and TCRβ⁺ staining. A) Representative flow cytometry contour plots of unstimulated and stimulated WT, KSR1^−/−, Il10^−/−, and KSR1^−/−Il10^−/− lamina propria TCRβ⁺ T cells stained for intracellular IFN-γ and IL-17A. B) The percent of TCRβ⁺ T cells positive for intracellular IFN-γ or IL-17A was determined and solid bars represent the mean while error bars are the SEM. * P < 0.05, ** P < 0. 01

Figure 5. In vitro Th17 polarization is impaired while in vitro Th1 polarization is increased in KSR1^−/− T cells

Splenocytes isolated from WT, KSR1^−/−, Il10^−/−, and KSR1^−/−Il10^−/− mice were cultured under Th17 or Th1 polarizing conditions. Lymphocytes were stained for cell surface CD4 and TCRβ and intracellular IFN-γ and IL-17A. Samples were analyzed by flow cytometry gated on lymphocyte geometry and CD4⁺TCRβ⁺ cell surface staining. A) Representative flow cytometry dot plots of unstimulated and stimulated T cells cultured under Th17 polarizing conditions. B) Intracellular cytokine staining on Th17 polarized cells was quantified and reported as the percent CD4⁺TCRβ⁺ cells staining positive for IL-17A or IFN-γ. Bar graphs represent the mean for each cultured T cell population from two independent experiments. Error bars are the SEM. C) Representative flow cytometry dot plots of unstimulated and stimulated T cells cultured under Th1 polarizing conditions. D) Intracellular cytokine staining on Th1 polarized cells was quantified and reported as the percent CD4⁺TCRβ⁺ cells staining positive for IL-17A or IFN-γ. Bar graphs represent the mean for each cultured T cell population. Error bars are the SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 6. Neutralizing IFN-γ attenuates colitis in KSR1^−/−Il10^−/− mice

3-week old KSR1^−/−Il10^−/− mice were administered 100 µg/mouse neutralizing antibodies against αIgG, αIFN-γ, or αIL-17A intraperitoneally twice per week for a period of 3 weeks and assessed for colitis. A) Mouse colons were removed, flushed, weighed, and measured from the cecum to anus. Solid bars represent the mean length/weight ratio. Error bars are the SEM. B) Colon sections were scored as before for inflammation and injury. Solid bars represent the mean and error bars represent the SEM. C) Representative H&E stained colon sections from KSR1^−/−Il10^−/− mice administered αIgG, αIFN-γ, or αIL-17A neutralizing antibody. * P < 0.05, ** P < 0.01