TRPM2 cation channels modulate T cell effector functions and contribute to autoimmune CNS inflammation

Abstract

TRPM2, a highly Ca(2+)-permeable member of the transient receptor potential melastatin-related (TRPM) family of cation channels, is expressed in cells of the immune system. We demonstrate firstly that TRPM2 cation channels on T cells critically influence T cell proliferation and proinflammatory cytokine secretion following polyclonal T cell receptor stimulation. Consistently, trpm2-deficient mice exhibited an attenuated clincal phenotype of experimental autoimmune encephalomyelitis (EAE) with reduced inflammatory and demyelinating spinal cord lesions. Importantly, trmp2-deficient T cells were as susceptible as wildtype T cells to oxidative stress-induced cell death as it occurs in inflammatory CNS lesions. This supports the notion that the attenuated EAE phenotype is mainly due to reduced T cell effector functions but unaffected by potential modulation of T cell survival at the site of inflammation. Our findings suggest TRPM2 cation channels as a potential target for treating autoimmune CNS inflammation.

Figure 1. TRPM2 cation channels are upregulated in CD4⁺ T cells upon polyclonal T cell receptor stimulation and critically determine proliferation and proinflammatory cytokine secretion.

(A) RT-PCR performed with naïve (middle) and anti-CD3/CD28 bead-stimulated (right) wildtype CD4⁺ T cells using murine TRPM2-specific primers revealed upregulation of TRPM2 expression upon stimulation. As negative control water (left) instead of cDNA was used. An experiment representative of 3 independent trials is shown. (B) Immunocytochemitry using an antibody specific for the c-terminal portion of the TRPM2 protein (red) revealed positive staining in activated bead-stimulated CD4⁺ T cells. Cell nuclei were counterstained with DAPI (blue). (C, D) Proliferation as determined by ³[H]-thymidine uptake (C) and secretion of proinflammatory IL-2, IFN-γ and IL-17 determined by ELISA from the supernatants (D) were significantly reduced in CD4⁺ T cells from trpm2-deficient (TRPM2 −/−) as compared to wildtype (TRPM2 +/+) mice following anti-CD3/CD28 bead stimulation at a bead:cell ratio of 1∶4. Moreover, the TRPM2 channel blocker ACA dose-dependently reduced proliferation (filled circles) of bead-stimulated splenocytes (E, bead:cell ratio 1∶4) without overt toxic effects on cell viability as assed using flow cytometry for annexin V and propidium iodide (open circles). ACA at a concentration of 100 µM significantly reduced IFN-γ secretion of bead-stimulated splenocytes (F, bead:cell ratio 1∶4; n = 3 for all experiments) P-values ≤0.05 were considered significant (*). P-values ≤0.01 and ≤0.001 were considered highly significant (** and ***, respectively).

Figure 2. TRPM2 cation channels critically determine proliferation and proinflammatory cytokine secretion following various strengths of polyclonal T cell receptor triggering.

(A–D) Proliferation as determined by ³[H]-thymidine uptake (A; bead:cell ratio of 1∶4) and secretion of proinflammatory IL-2 (B), IFN-γ (C) and IL-17 (D) determined by ELISA from the supernatants were significantly reduced in spleenocytes from trpm2-deficient as compared to wildtype mice following anti-CD3/CD28 bead-stimulation for 3 days at various bead:cell ratios (1∶1 to 1∶16; n = 3 for all experiments). P-values ≤0.05 were considered significant (*). P-values ≤0.01 and ≤0.001 were considered highly significant (** and ***, respectively).

Figure 3. Trpm2-deficient mice exhibit an ameliorated EAE phenotype with reduced inflammatory CNS infiltrates and demyelination.

(A) Clinical MOG_35–55 peptide-induced EAE disease score in wildtype and trpm2-deficient mice was examined for 50 days post-immunization (p.i.) and revealed a significantly reduced peak- and residual score in trpm2-deficent as compared to wildtype mice (n = 3 independent trials with 10 mice each for both experimental groups). (B, C) Representative H&E (B) and LFB (C) stainig (upper panels) of spinal cord sections from wildtype (left) and trpm2-deficient (right) mice obtained at day 20 post-immunization and quantification of respective leasion sizes (lower panels; n = 2 mice from each of the 3 independent trials representing the mean clinical score of the experimental group at day 20 post-immunization, white arrows indicate lesions). P-values ≤0.05 were considered significant (*). P-values ≤0.01 and ≤0.001 were considered highly significant (** and ***, respectively).

Figure 4. TRPM2 channels do not impact survival of activated CD4⁺ T cells under altered redix conditions.

Suvival of activated bead-stimulated CD4⁺ T cells (bead:cell ratio of 1∶4) from wildtype and trpm2-deficient mice under various concentrations of H₂O₂ (0.001–10 mM) using the amount of ATP relased following cell lysis as a parameter of cell viability (n = 3; ns  =  not significant).