Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and disease activity in ulcerative colitis

Abstract

Background and aims: Potassium channels, KV1.3 and KCa3.1, have been suggested to control T-cell activation, proliferation, and cytokine production and may thus constitute targets for anti-inflammatory therapy. Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by excessive T-cell infiltration and cytokine production. It is unknown if KV1.3 and KCa3.1 in the inflamed mucosa are markers of active UC. We hypothesized that KV1.3 and KCa3.1 correlate with disease activity and cytokine production in patients with UC.

Methods: Mucosal biopsies were collected from patients with active UC (n=33) and controls (n=15). Protein and mRNA expression of KV1.3 and KCa3.1, immune cell markers, and pro-inflammatory cytokines were determined by quantitative-real-time-polymerase-chain-reaction (qPCR) and immunofluorescence, and correlated with clinical parameters of inflammation. In-vitro cytokine production was measured in human CD3(+) T-cells after pharmacological blockade of KV1.3 and KCa3.1.

Results: Active UC KV1.3 mRNA expression was increased 5-fold compared to controls. Immunofluorescence analyses revealed that KV1.3 protein was present in inflamed mucosa in 57% of CD4(+) and 23% of CD8(+) T-cells. KV1.3 was virtually absent on infiltrating macrophages. KV1.3 mRNA expression correlated significantly with mRNA expression of pro-inflammatory cytokines TNF-α (R(2)=0.61) and IL-17A (R(2)=0.51), the mayo endoscopic subscore (R(2)=0.13), and histological inflammation (R(2)=0.23). In-vitro blockade of T-cell KV1.3 and KCa3.1 decreased production of IFN-γ, TNF-α, and IL-17A.

Conclusions: High levels of KV1.3 in CD4 and CD8 positive T-cells infiltrates are associated with production of pro-inflammatory IL-17A and TNF-α in active UC. KV1.3 may serve as a marker of disease activity and pharmacological blockade might constitute a novel immunosuppressive strategy.

Keywords: Colitis ulcerosa;; Interleukins;; K(Ca)3.1; KCNA3;; KCNN4;; Novel treatment strategy;.

Figure 1

mRNA expression of cell markers, pro-inflammatory cytokines, and potassium channels in mucosal biopsies of UC patients and controls. Data from individual patients are also given as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Significant and borderline significant correlations of K_v1.3 mRNA expression (in percentage of GAPDH) with clinical scores, cell markers and cytokines. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Immunohistochemical stainings visualizing the potassium channels and cell types in the normal mucosa of controls and the inflamed mucosa of patients with active ulcerative colitis. Note that the crypts in UC are more irregular and contain less mucus. K_V1.3⁺ cells are located in the interstitial tissue around crypts in UC patients. Crypts in both patient groups are positive for K_Ca3.1 and immunoreactivity is also detected in cell infiltrates in UC. Both CD4⁺ and CD8⁺ cells clearly infiltrate the tissue in UC. Infiltration of macrophages (MAC) is also evident in the inflamed mucosa of UC.

Figure 4

Immunofluorescent stainings of potassium channels, cell markers, and chemokine-receptor-7 (CCR7). In the upper part of each picture the individual fluorochromes are shown. The lower part shows the merged pictures visualizing co-localized immunofluorescence as a yellow signal. Arrows indicates co-localized immunofluorescence. The top row displays immunostainings of K_V1.3 and in the lower row K_Ca3.1.

Figure 5

The left side of the graph shows the percentage of K_V1.3 immunofluorescence that co-localize with CD8, CD4, macrophages and Chemokine Receptor 7 (CCR7) after analyses with the Cell Profiler software. The right side shows the percentage of K_Ca3.1 immunofluorescence co-localizing with CD4, CD8 and macrophages. Error bars show mean ± SEM.

Figure 6

Effect of K_V1.3 and K_Ca3.1 blockage in PMA (phorbol-12-myristate-13-acetate) + Ionomycin-stimulated human CD3⁺ T cells. First figure shows [³H]-thymidine incorporation, which is a measure of cell proliferation. Additional figures visualize production of inflammatory cytokines. PAP-1 and ShK-L5 are K_V1.3 blockers; Senicapoc is a K_Ca3.1 blocker. The blockers were tested at different concentrations, and in the right-hand side of each graph combinations of blockers are presented. Individually, the blockers decrease the level of cell proliferation and cytokine secretion and when used in combination the effect is even more prominent.