Inhibition of the K+ channel KCa3.1 ameliorates T cell-mediated colitis

Abstract

The calcium-activated K(+) channel KCa3.1 plays an important role in T lymphocyte Ca(2+) signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca(2+) influx. To assess the role of KCa3.1 channels in lymphocyte activation in vivo, we studied T cell function in KCa3.1(-/-) mice. CD4 T helper (i.e., Th0) cells isolated from KCa3.1(-/-) mice lacked KCa3.1 channel activity, which resulted in decreased T cell receptor-stimulated Ca(2+) influx and IL-2 production. Although loss of KCa3.1 did not interfere with CD4 T cell differentiation, both Ca(2+) influx and cytokine production were impaired in KCa3.1(-/-) Th1 and Th2 CD4 T cells, whereas T-regulatory and Th17 function were normal. We found that inhibition of KCa3.1(-/-) protected mice from developing severe colitis in two mouse models of inflammatory bowel disease, which were induced by (i) the adoptive transfer of mouse naïve CD4 T cells into rag2(-/-) recipients and (ii) trinitrobenzene sulfonic acid. Pharmacologic inhibitors of KCa3.1 have already been shown to be safe in humans. Thus, if these preclinical studies continue to show efficacy, it may be possible to rapidly test whether KCa3.1 inhibitors are efficacious in patients with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

Fig. 1.

Decreased TCR-stimulated Ca²⁺ flux and cytokine production in KCa3.1^−/− Th0 cells. (A) Whole-cell patch-clamp experiments of CD4 T cells isolated from spleens of WT or KCa3.1^−/− mice following stimulation for 48 h with anti-CD3, anti-CD28 antibodies. Bar graph summary of n = 10–15 cells. KCa3.1 current was measured as the TRAM-34 (1 μM)–inhibited current and Kv current as the ShK (1 nM)–inhibited current. (B) Cells in A were rested overnight and after loading with Fura-2 AM (5 μM), Ca²⁺ flux was determined at excitation wavelengths of 340 and 380 nm after cross-linking with anti-CD3 antibodies. (C) Cells in A were rested overnight and after stimulating with anti-CD3 and anti-CD28, IL-2 production was determined by flow cytometry following intracytoplasmic staining with anti–IL-2 antibodies. All these experiments are representative experiments that were repeated at least five times from CD4 T cells isolated from independent mice.

Fig. 2.

Impaired activation of KCa3.1^−/− Th1 and Th2, but not Th17 or Treg CD4 T cells. Whole-cell patch-clamp experiments were performed on WT and KCa3.1^−/− (A(i)) Th1 and (B(i)) Th2 cells as described in figure 1A (n = 10–12 cells). Th1 and Th2 cells were rested overnight, and anti-CD3-stimulated Ca2⁺ flux was assessed in Th1 (A(ii)) and Th2 (B(ii)) as described in figure 1B. (C) (i) Whole-cell patch-clamp data (n = 15 cells) from WT Th17 cells (for details refer to SI Materials and Methods). (ii) Cells were differentiated into Th17, and IL-17 expression was assessed by intracellular staining after stimulation with PMA and ionomycin. Expression of IL-17 in WT cells was also assessed in the presence of the KCa3.1 inhibitor TRAM-34, or the Kv inhibitor ShK. (D) (i) CD4⁺CD25^hi Treg cells isolated from WT spleens were subject to whole-cell patch-clamp experiments (n = 10 cells). (ii) Suppression Assay: CD4⁺CD25^- WT effector cells were co-cultured with either WT or KCa3.1^−/− CD4⁺CD25⁺ suppressor Tregs at an effector:suppressor ratio of 10:1, 3:1, or 1:1 and proliferation was determined by assessing uptake of [³H]thymidine.

Fig. 3.

Decreased cytokine production by KCa3.1^−/− Th1 and Th2 cells. WT and KCa3.1^−/− Th1 cells were stimulated with anti-CD3 and anti-CD28 or PMA and ionomycin, and IFN-γ (A), IL-2 (B), and TNF(C) were assessed by flow cytometry following intracytoplasmic staining with antibodies as indicated. (D) WT and KCa3.1^−/− Th2 cells were stimulated with anti-CD3 and anti-CD28, and IL-13 expression was determined 4 h after stimulation by real-time PCR. Shown is the relative amount of IL-13 standardized to β-actin control.

Fig. 4.

Decreased severity of colitis induced by KCa3.1^−/− CD4⁺CD25^-CD45RB^hi donor T cells. WT or KCa3.1^−/− CD4⁺CD25^-CD45RB^hi were transferred into rag2^−/− mice, and weight (A) and histologic score (B) on 7 pairs of animals were determined. (C) Example of histology of colon from Rag2^−/− mice adoptively transferred WT (i) and KCa3.1^−/− (ii) cells. (D) Adoptively transferred WT or KCa3.1^−/− T cells were recovered from rag2^−/− spleens or mesenteric lymph nodes and stained with antibodies to CD4 (i) or CD44) and CD45RB (ii) followed by FACS analysis. In i and ii, WT/no-injection is from a control C57BL/6 mouse, and rag2^−/−/no-injection is from a control rag2^−/− mouse.

Fig. 5.

KCa3.1^−/− cells recovered from rag2^−/− mice are defective in TCR-stimulated Ca²⁺ flux and cytokine production. CD4 T cells recovered from rag2^−/− mice underwent whole-cell patch-clamp experiment (A) or stimulation with anti-CD3 to assess Ca²⁺ flux (B) and IFN-γ production (C) as described in Fig. 1. Shown is a representative experiment from CD4 T cells isolated from three independent reconstituted rag2^−/− mice.

Fig. 6.

TRAM-34, a KCa3.1 inhibitor, inhibits TNBS-induced colitis. TNBS in 40% ethanol was administered rectally to SJL mice that were untreated or treated with TRAM-34, and histological analysis was performed at day 6 after TNBS treatment. (A) Summary of histological analysis of 6 pairs of mice. (B) Example of histological analysis of control (i) or TRAM-34–treated colon(ii).