Fundamental role for the creatine kinase pathway in protection from murine colitis

Abstract

Inflammatory diseases of the digestive tract, including inflammatory bowel disease, cause metabolic stress within mucosal tissue. Creatine is a key energetic regulator. We previously reported a loss of creatine kinases (CKs) and the creatine transporter expression in inflammatory bowel disease patient intestinal biopsy samples and that creatine supplementation was protective in a dextran sulfate sodium (DSS) colitis mouse model. In the present studies, we evaluated the role of CK loss in active inflammation using the DSS colitis model. Mice lacking expression of CK brain type/CK mitochondrial form (CKdKO) showed increased susceptibility to DSS colitis (weight loss, disease activity, permeability, colon length, and histology). In a broad cytokine profiling, CKdKO mice expressed near absent interferon gamma (IFN-γ) levels. We identified losses in IFN-γ production from CD4⁺ and CD8⁺ T cells isolated from CKdKO mice. Addback of IFN-γ during DSS treatment resulted in partial protection for CKdKO mice. Extensions of these studies identified basal stabilization of the transcription factor hypoxia-inducible factor in CKdKO splenocytes and pharmacological stabilization of hypoxia-inducible factor resulted in reduced IFN-γ production by control splenocytes. Thus, the loss of IFN-γ production by CD4⁺ and CD8⁺ T cells in CKdKO mice resulted in increased colitis susceptibility and indicates that CK is protective in active mucosal inflammation.

Figure 1.

CKdKO and control mice were exposed to 1.5% DSS in their drinking water for 5 days followed by a 2-day recovery period. A Weights and B Disease Activity Index (weight change, stool consistency and bleeding score) were obtained daily. C On day 7, mice were gavaged with FITC-dextran 4 kD and plasma was obtained at 4 hours. D Colon length was measured from cecum to distal colon. E, F H & E of colonic tissue were compared and scored (see methods section). n=6-12 mice per group, scale bars 100μm. Statistical differences were analyzed by 2way ANOVA (A, B), unpaired t test (C, D) and one-way ANOVA (f). * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001

Figure 2.

A Total colonic tissue samples from DSS experiment were homogenized and protein homogenate was evaluated using IFN-γ mesoscale analysis (n=5-6). B PBL were isolated from wildtype and CKdKO mice and stimulated with PMA and ionomycin for 18 hours followed by supernatant collection and analysis by IFN-γ ELISA (n=4-6). C Splenocytes from wildtype and CKdKO mice were stimulated with PMA and ionomycin with Brefeldin A and were analyzed by flow cytometry (n=3). D PBMCs were isolated from healthy human volunteers and stimulated with PMA and ionomycin in the presence or absence of cyclocreatine (n=2-5). RNA was isolated and evaluated by qPCR relative to actin. Statistical differences were analyzed by 2way ANOVA (A, B, C) and unpaired T test (D)P<0.01, *** P<0.001

Figure 3.

CKdKO and control mice were exposed to 1.5% DSS in their drinking water for 5 days with the addition of IP injections of IFN-γ or PBS followed by a 4-day recovery period. A Weights and B Disease Activity Index (weight change, stool consistency and bleeding score) were obtained daily. C Colon length was measured from cecum to distal colon. D, E H & E of colonic tissue were compared and scored (see methods section), scale bars 100μm. n=12-14 mice per group for A, B, C and n=4 for E. Statistical differences were analyzed by 2way ANOVA (A, B), unpaired t test (C) and one-way ANOVA (E). *P<0.05, ** P<0.01

Figure 4.

A Splenocytes were isolated from wildtype and CKdKO mice. Protein was then collected from the cells and HIF-1α was assessed by western blot relative to actin. B qPCR conducted on unstimulated Control and CKdKO splenocytes for HIF target genes BNIP3L, ADM and EPO. C HIF target gene qPCR conducted on Control splenocytes treated with IOX4 from 8-12 hrs. D Isolated wildtype splenocytes were treated with HIF stabilized IOX4 in the presence or absence of PMA, ionomycin and α-CD3 activation. F Splenocytes stimulated with PMA, ionomycin or α-CD3 in the presence or absence of IOX4 were evaluated by flow cytometry for intracellular IFN-γ in CD4⁺, CD8⁺ and NK1.1⁺ populations. n=3-5. Statistical differences were analyzed by unpaired t test (B) and one-way ANOVA (C, D, E). * P<0.05, ** P<0.01, *** P<0.001.