TRPM8 activation attenuates inflammatory responses in mouse models of colitis

Abstract

Transient Receptor Potential Melastatin-8 (TRPM8), a recently identified member of the transient receptor potential (TRP) family of ion channels, is activated by mild cooling and by chemical compounds such as the supercooling agent, icilin. Since cooling, possibly involving TRPM8 stimulation, diminishes injury-induced peripheral inflammation, we hypothesized that TRPM8 activation may also attenuate systemic inflammation. We thus studied the involvement of TRPM8 in regulating colonic inflammation using two mouse models of chemically induced colitis. TRPM8 expression, localized immunohistochemically in transgenic TRPM8(GFP) mouse colon, was up-regulated in both human- and murine-inflamed colon samples, as measured by real-time PCR. Wild-type mice (but not TRPM8-nulls) treated systemically with the TRPM8 agonist, icilin showed an attenuation of chemically induced colitis, as reflected by a decrease in macroscopic and microscopic damage scores, bowel thickness, and myeloperoxidase activity compared with untreated animals. Furthermore, icilin treatment reduced the 2,4,6-trinitrobenzenesulfonic acid-induced increase in levels of inflammatory cytokines and chemokines in the colon. In comparison with wild-type mice, Dextran Sodium Sulfate (DSS)-treated TRPM8 knockout mice showed elevated colonic levels of the inflammatory neuropeptide calcitonin-gene-related peptide, although inflammatory indices were equivalent for both groups. Further, TRPM8 activation by icilin blocked capsaicin-triggered calcitonin-gene-related peptide release from colon tissue ex vivo and blocked capsaicin-triggered calcium signaling in Transient Receptor Potential Vaniloid-1 (TRPV1) and TRPM8 transfected HEK cells. Our data document an anti-inflammatory role for TRPM8 activation, in part due to an inhibiton of neuropeptide release, pointing to a novel therapeutic target for colitis and other inflammatory diseases.

Fig. 1.

TRPM8 expression in human and mouse colon. (A) Real-time PCR detection of TRPM8 expression in colonic biopsies from control (closed circle), noninflamed Crohn's disease patient colons (closed squares), or inflamed Crohn's disease patient colons (closed triangles). Data are shown as mean ± SEM. *P < 0.05. n = 5–29. (B) TRPM8 expression in the colon of mice treated with vehicle, TNBS, or DSS. A significant increase in TRPM8 mRNA levels is seen in colonic tissue from TNBS- or DSS-treated mice compared with vehicle-treated mice. Data are shown as mean ± SEM. *P < 0.05. n = 4–6.

Fig. 2.

Icilin attenuates colonic inflammation in mice. Assessment of intestinal damage scores, colonic MPO levels, and bowel thickness in mice treated with vehicle, icilin, TNBS or DSS, and TNBS or DSS plus icilin or vehicle. Mice treated with icilin exhibit reduced intestinal damage scores, colonic MPO levels, and bowel thickness compared with vehicle-treated animals in both TNBS- (A, C, and E) and DSS- (B, D, and F) induced colitis. No significant differences are observed between vehicle- and icilin-treated groups. (G) Representative pictograms of H&E-stained colon sections from mice treated with vehicle, icilin, TNBS, or TNBS plus icilin. Histogram depicts the microscopic damage scores in mice from each of the four groups. *P < 0.05, **P < 0.005, and ***P < 0.0005. n = 8 animals per group.

Fig. 3.

Icilin reduces inflammatory cytokine and chemokine levels in mice with TNBS-induced colitis. Cytokine and chemokine profiling in the colons of mice treated with vehicle, icilin, TNBS, or TNBS plus icilin. Mice treated with icilin during the course of TNBS-induced colitis show significantly reduced cytokine/chemokine levels compared with mice with TNBS-induced colitis. *P < 0.05. n = 8 animals per group.

Fig. 4.

TRPM8^−/− mice do not show enhanced DSS-induced colonic inflammation but have elevated levels of CGRP. Assessment of intestinal damage scores, colonic MPO levels, and bowel thickness in WT and TRPM8^−/− mice treated with vehicle, DSS (2.5% wt/vol), or low-dose DSS (1% wt/vol:Low DSS). TRPM8^−/− mice do not show significantly different (A) damage scores, (B) bowel thickness, or (C) MPO levels compared with WT mice. (D) Comparison of CGRP levels in colonic tissue from WT and TRPM8^−/− mice after 7 d of DSS administration. Data are shown as mean ± SEM. n = 8 animals per group. *P < 0.05.

Fig. 5.

Inhibition of TRPV1-dependent CGRP release and calcium signaling by TRPM8 activation. (A) TRPV1 activation by capsaicin triggers CGRP release from distal colon tissue. TRPM8 activation by icilin treatment does not stimulate CGRP release from the colon. Pretreatment of colonic tissue with icilin inhibits TRPV1-stimulated CGRP release. * indicates significant elevation of CGRP release compared with control. # indicates significant attenuation compared with capsaicin-stimulated CGRP release. Data are expressed as mean ± SEM. n = 3 animals per group. *P < 0.05. (B) Calcium signaling was monitored in TRPM8- and TRPV1-transfected HEK cell monolayers. Representative trace showing fluorescence intensity (0.2ΔF/F) elicited by capsaicin (1 μM) and icilin (30 μM) in human embryonic kidney-tsA-201 cells transfected with TRPV1, TRPM8, and mCherry (to visualize transfected cells). (C) Percentage of transfected cells responding to capsaicin following vehicle or icilin application. (Inset) Representative image of mCherry-transfected tsA-201cells. Cells responding to capsaicin and icilin were mCherry-positive. Capsaicin responses were considered positive when ΔF/F was >10% of the initial response before vehicle or icilin challenge.