Pathophysiological significance of the two-pore domain K+ channel K2P5.1 in splenic CD4+CD25− T cell subset from a chemically-induced murine inflammatory bowel disease model

The alkaline pH-activated, two-pore domain K+ channel K2P5.1 (also known as TASK2/KCNK5) plays an important role in maintaining the resting membrane potential, and contributes to the control of Ca2+ signaling in several types of cells. Recent studies highlighted the potential role of the K2P5.1 K+ channel in the pathogenesis of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. The aim of the present study was to elucidate the pathological significance of the K2P5.1 K+ channel in inflammatory bowel disease (IBD). The degrees of colitis, colonic epithelial damage, and colonic inflammation were quantified in the dextran sulfate sodium-induced mouse IBD model by macroscopic and histological scoring systems. The expression and functional activity of K2P5.1 in splenic CD4+ T cells were measured using real-time PCR, Western blot, and fluorescence imaging assays. A significant increase was observed in the expression of K2P5.1 in the splenic CD4+ T cells of the IBD model. Concomitant with this increase, the hyperpolarization response induced by extracellular alkaline pH was significantly larger in the IBD model with the corresponding intracellular Ca2+ rises. The expression of K2P5.1 was higher in CD4+CD25− T cells than in CD4+CD25+ regulatory T cells. The knockout of K2P5.1 in mice significantly suppressed the disease responses implicated in the IBD model. Alternations in intracellular Ca2+ signaling following the dysregulated expression of K2P5.1 were associated with the disease pathogenesis of IBD. The results of the present study suggest that the K2P5.1 K+ channel in CD4+CD25− T cell subset is a potential therapeutic target and biomarker for IBD.

The alkaline pH-activated, two-pore domain K + channel K 2P 5.1 (also known as TASK2/KCNK5) plays an important role in maintaining the resting membrane potential, and contributes to the control of Ca 2+ signaling in several types of cells. Recent studies highlighted the potential role of the K + 2P 5.1 K channel in the pathogenesis of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. The aim of the present study was to elucidate the pathological significance of the K 2P 5.1 K + channel in inflammatory bowel disease (IBD). The degrees of colitis, colonic epithelial damage, and colonic inflammation were quantified in the dextran sulfate sodium-induced mouse IBD model by macroscopic and histological scoring systems. The expression and functional activity of K 2P 5.1 in splenic CD4 + T cells were measured using real-time PCR, Western blot, and fluorescence imaging assays. A significant increase was observed in the expression of K 2P 5.1 in the splenic CD4 + T cells of the IBD model. Concomitant with this increase, the hyperpolarization response induced by extracellular alkaline pH was significantly larger in the IBD model with the corresponding intracellular Ca 2+ rises. The expression of K 2P 5.1 was higher in CD4 + CD25 − T cells than in CD4 + CD25 + regulatory T cells. The knockout of K 2P 5.1 in mice significantly suppressed the disease responses implicated in the IBD model. Alternations in intracellular Ca 2+ signaling following the dysregulated expression of K 2P 5.1 were associated with the disease pathogenesis of IBD. The results of the present study suggest that the K

INTRODUCTION
Due to the overlap in the pathogeneses and pharmacological treatments, Crohn's disease (CD) and ulcerative colitis (UC) are types of inflammatory bowel diseases (IBD) (Bouma and Strober, 2003;McCole, 2014). Dextran sulfate sodium (DSS) is known to chemically induce the pathogenesis of CD and UC in rodents (Okayasu et al., 1990;Dieleman et al., 1994). A genome-wide expression profiling study showed that DSS-associated genes in mice correlated with data obtained from IBD patients (Fang et al., 2011). Therefore, the DSS-induced IBD model is a commonly used model of IBD in mice, and a number of studies have used the DSS-induced IBD model to investigate the pathogenesis of CD and UC.
An elevation in intracellular Ca 2+ by the release of Ca 2+ from intracellular Ca 2+ stores and Ca 2+ influx through store-operated Ca 2+ channels is one of the essential triggering signals for T cell activation (Cahalan and Chandy, 2009;Hogan et al., 2010). The K + channel is also one of the key molecules that modulates Ca 2+ signaling in T cells because it provides an electrochemical gradient to drive Ca 2+ influx, and two major K + channels (the voltage-gated, K V 1.3 and intermediate-conductance Ca 2+activated, K Ca 3.1) function in T and B lymphocytes (Cahalan and Chandy, 2009). Recent studies including our previous study demonstrated that the upregulation of ion channels (Orai/STIM, TRPV1, TRPA1, and K Ca 3.1) in CD4 + T cells was involved in the pathogenesis of IBD, while their pharmacological blockade elicited a significant decrease in IBD disease severity (Di Sabatino et al., 2009;Di et al., 2010;Bertin et al., 2014;Kun et al., 2014;. The background or leak two-pore domain K + (K 2P ) channel superfamily includes 16 members and plays a crucial role in diverse physiological functions through the regulation of cell excitability (Enyedi and Czirják, 2010;Es-Salah-Lamoureux et al., 2010). The K 2P 5.1 K + channel (also known as TASK2/KCNK5) is activated by extra-and intracellular alkalization, and plays an important role in maintaining the resting membrane potential and the control of Ca 2+ signaling in various types of cells. K 2P 5.1 has been shown to control diverse physiological and pathophysiological processes including innate immunity and cancer progression (Bittner et al., 2010b;Clark et al., 2011;Cid et al., 2013), and is, therefore, expected to become a potential therapeutic target for the treatment Abbreviations: K 2P , two-pore domain K + (channel); TASK, TWIK-related acid-sensitive K + (channel); TWIK, tandem of P-domains in a weakly inward rectifying K + (channel); TALK, TWIK-related alkaline pH-activated K + (channel); IBD, inflammatory bowel disease; DSS, dextran sodium sulfate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DiBAC 4 (3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol; Fura 2-AM, acetoxymethyl 2- [5-[bis[(acetoxymethoxy-oxo-methyl) of autoimmune, inflammatory, and allergic diseases as well as several types of cancers. Recent studies described the pathophysiological impact of the upregulation of K 2P 5.1 in CD4 + and CD8 + T cells on the pathogenesis of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis (Bittner et al., 2010a. K 2P 5.1 is inhibited by nonselective K + channel inhibitors such as quinidine, lidocaine, and clofilium and is activated by volatile anesthetics (Wulff and Zhorov, 2008); however, the lack of selective blockers is represents an important difficulty in experimental studies on K 2P 5.1.
In the present study, we examined the involvement of the K 2P 5.1 K + channel in CD4 + T lymphocytes in the pathogenesis of IBD using a mouse model of chemically-induced IBD. We further compared histopathologies between the DSS-induced IBD model using wild-type and K 2P 5.1-deficient mice.

Preparation of the Dextran Sulfate Sodium (DSS)-induced Mouse IBD Model
Male C57BL/6J (6-7 weeks of age) mice were purchased from Shimizu Laboratory Supplies (Kyoto, Japan), and were acclimatized for 1 week before the experiment. They were given distilled water containing 5.0% (wt/vol) dextran sulfate sodium 5000 (DSS) (Wako Pure Chemical, Osaka, Japan) ad libitum. Control mice were given drinking water only. Seven days after the administration of DSS, mice were sacrificed, tissue samples were taken, and colitis and inflammation were assessed macroscopically and scored as previously reported . All experiments were carried out in accordance with the guiding principles for the care and use of laboratory animals in Kyoto Pharmaceutical University, and the protocols were approved by the committee on the Ethics of Animal Research of Kyoto Pharmaceutical University (Permit Number: 15-12-091). K 2P 5.1 heterozygous knockout mice bred in a C57BL6 background [B6;CB-Kcnk5Gt(pU-21)81Imeg] were established by the exchangeable gene trap method (Araki et al., 2014). Homozygous (K 2P 5.1 −/− ) knockout mice were generated by crossing heterozygous (K 2P 5.1 +/− ) males with K 2P 5.1 +/− females (Cid et al., 2013). Genomic DNA was isolated from finger or tail biopsies following a 3-h digestion at 50 • C in buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 100 mM NaCl, 0.1% SDS, and 1 mg/mL proteinase K, followed by heat inactivation. PCR was performed using the primer pairs to distinguish the K 2P 5.1 wild type (forward: 5 ′ -GCTGAGAACAATAGGGACAG-3 ′ and reverse: 5 ′ -TCACCCAGCTTTGGGATTCC-3 ′ ) and gene trapped (forward: 5 ′ -GCTGAGAACAATAGGGACAG-3 ′ and reverse: 5 ′ -TACAGGCATCGTGGTGTCAC-3 ′ ). PCR conditions were as follows: 30 cycles at 94 • C for 30 s, 60 • C for 30 s, and 68 • C for 30 s. The amplified products were separated on 1.0% agarose gels, and visualized by ethidium bromide staining. DSS-induced IBD model mice were prepared using male and female K 2P 5.1 −/− (6-10 weeks of age) and K 2P 5.1 +/− (6 weeks of age) mice.

Histological Scoring
Inflammation and crypt damage in hematoxylin and eosin (H&E)-stained sections were assessed as reported previously (Dieleman et al., 1994;. Briefly, for histological assessments, 1 cm of tissue collected from the distal colon was fixed in 10% buffered formalin, embedded in a paraffin block, cut into 5-µm-thick sections, and stained with H&E. The inflammation score was determined as a multiplication of the severity grade of inflammation (grade 0, none; grade 1, slight; grade 2, moderate; grade 3, severe) as well as its extent (grade 0, none; grade 1, mucosa; grade 2, mucosa, and submucosa; grade 3, transmural). The crypt damage score was determined as a multiplication of the damage grade of the crypt (grade 0, none; grade 1, basal 1/3 damage; grade 2, basal 2/3 damage; grade 3 only the surface epithelium intact; grade 4, entire crypt, and epithelium lost) and the percent area score (grade 1, 1-25%; grade 2, 25-50%; grade 3, 51-75%; grade 4, 76-100%). Data were obtained from three sections of the colon measured at least 200 µm apart per animal from four individual mice per group.

Western Blotting
Splenic CD4 + T cells were solubilized with lysis buffer with 1% SDS. The same amount of proteins (10 µg for each) were subjected to 10% SDS-PAGE . The blots were incubated with an anti-K 2P 5.1 antibody (G-14 or H-170, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and then incubated with anti-rabbit or anti-goat horseradish peroxidaseconjugated IgG (Millipore). An enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire, England) was used to detect the bound antibody. The resulting images were analyzed by a LAS-3000mini device (Fujifilm, Tokyo, Japan). The blots were also probed with an anti-β-actin (ACTB) antibody (Medical & Biological Laboratories, Nagoya, Japan).

Measurement of the Membrane Potential and Intracellular Ca 2+ Concentrations by Fluorescent Indicators
The membrane potential was measured using the fluorescent voltage-sensitive dye DiBAC 4 (3), as previously reported (Ohya et al., 2013. Glass bottom tissue culture dishes were coated with fibronectin (Wako Pure Chemical Industries, Osaka, Japan) in Ca 2+ and Mg 2+ -free PBS at 50 µg/mL for 1 µg/cm 2 at 4 • C overnight. Cells were seeded onto fibronectin-coated dishes and cultured for 2 h at 37 • C in 5% CO 2 humidified incubator. Prior to the fluorescence measurements with DiBAC 4 (3), cells were incubated in normal HEPES buffer containing 100 nM DiBAC 4 (3) for 20 min at room temperature. The cells were continuously incubated with 100 nM DiBAC 4 (3) throughout the experiments. In membrane potential imaging, cells loaded with DiBAC 4 (3) were illuminated at a wavelength of 490 nm, and fluorescence images were recorded on the ORCA-Flash2.8 digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Additionally, intracellular Ca 2+ concentrations were measured using the fluorescent Ca 2+ indicator dye Fura 2-AM. Cells were incubated with 10 µM Fura 2-AM in normal HEPES solution for 30 min at room temperature. Cells loaded with Fura 2-AM were alternatively illuminated at wavelengths of 340 and 380 nm, and fluorescence images were recorded. The fluorescent intensity of Fura 2 was expressed as measured 340/380 nm fluorescence ratios after background subtraction. Data collection and analyses were performed using an HCImage system (Hamamatsu Photonics). Images were measured every 5 s, and the values of fluorescent intensity (F) were determined by measuring the average for 1 min (12 images).

Flow Cytometric Analysis
Cell surface markers were analyzed with BD FACSCalibur (BD Biosciences, San Jose, CA, USA), which acquired at least 10,000 events, and gated according to forward-and side-scatter (Ohya et al., 2013). Data were analyzed using CellQuest software (BD (E) splenic CD4 + cell lysates were probed by immunoblotting with an anti-K 2P 5.1 (G-14) antibody. Molecular weight standards are shown in kilodaltons (kDa) on the right. Arrowheads indicate the migrating positions of K 2P 5.1 and ACTB proteins (open: K 2P 5.1, closed: glycosylated K 2P 5.1, gray: ACTB). Lower panel shows signals after the pre-incubation of a primary antibody with excess antigens. (F) summarized data obtained from (E) as the optical density of the band signal for K 2P 5.1 (open arrowhead) in the IBD model relative to that in normal mice. (G) Expression of alkaline pH-activated K 2P K + channel subtype (K 2P 3.1, 9.1, and 16.1) transcripts in the splenic CD4 + T cells of control and IBD model mice. (H) the percentages of the CD4 + T cell population relative to CD3+ T cells were analyzed by flow cytometry, acquiring at least 10,000 events. Results were expressed as means ± SEM. Numbers used for the experiments are shown in parentheses. *, **P < 0.05, 0.01 vs. control mice (ctrl).
Biosciences). The lymphocyte gate was established by analyzing the forward angle vs. right angle light scatter. The percentage of positive-staining cells was determined by comparing the test histograms with those obtained using a fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated anti-CD4 antibody (FITC/PE-CD4), FITC-conjugated CD3 (FITC-CD3), PE-conjugated CD8 (PE-CD8), and PE-conjugated CD25 (PE-CD25) (BD Biosciences). After being incubated with the antibodies for 1 h at room temperature, excess antibodies were removed by repeated washing with PBS.

Statistical Analysis
Significant differences between two and among multiple groups were evaluated using the Student's t-and Welch's t-tests or Tukey's test after the F-test or ANOVA. Data that were nonnormal distributed were analyzed using the Mann-Whitney Utest. Significance at P < 0.05 and P < 0.01 was indicated in the figures. Data are presented as means ± SE.

RESULTS
Upregulated Expression of the K 2P 5.1 K + Channel in Splenic CD4 + T Cells from the Mouse Model of DSS-induced Inflammatory Bowel Disease Mice were sacrificed 7 days after the administration of 5.0% (wt/vol) DSS and tissue samples were collected. Control mice were given drinking water only. As previously reported , severe macroscopic symptoms (colitis and fecal blood) were detected in DSS-induced IBD model mice, and the scores of colonic inflammation and crypt damage were significantly higher in IBD model mice than in control mice (not shown). As reported by Pereira et al. (1987), enlargement of the spleen, "splenomegaly, " was detected in IBD model mice (Figure 1A), and a significant increase in the transcriptional expression of the Th1 cytokine interferon-γ (IFN-γ) and Th17 cytokine interleukin-17 (IL-17) was detected (Figures 1B,C), without any changes in the Th2 cytokine IL-4 (not shown). The flow cytometric analysis showed no significant differences of the CD4 + phenotypic ratio between isolated splenic CD3 + T cells from both groups ( Figure 1H).
The quantitative, real-time PCR assay showed that the expression level of the K 2P 5.1 transcripts was significantly higher in the splenic CD4 + T cells of IBD model mice (IBD) than in control mice (ctrl) (Figure 1D). The expression levels of K 2P 5.1 relative to ACTB (in arbitrary units) were 0.0036 ± 0.0002 and 0.0161 ± 0.0025 in the splenic CD4 + T cells of control mice (n = 8) and IBD model mice (n = 6, P < 0.01), respectively. The western blot examination showed that the amount of the K 2P 5.1 protein (∼50 kDa) in the cell lysate of splenic CD4 + T cells was higher in IBD model mice than in control mice ( Figure 1E, upper panel, open arrowhead), which was consistent with the results obtained by real-time PCR examination. The amount of the glycosylated K 2P 5.1 protein (∼65 kDa, Figure 1E, upper panel, closed arrowhead) was also higher in the IBD model. The optical density for the K 2P 5.1 protein band signal (∼50 kDa) relative to that for ACTB ( Figure 1E, middle panel) was calculated using Image J software (Ver. 1.42, NIH), and its protein expression level in control mice was expressed as 1.00. The relative optical density in the IBD model was 5.05 ± 0.48 (n = 5 for each, P < 0.01) (Figure 1F). The two band signals specific for an anti-K 2P 5.1 antibody (G-14) disappeared with a pre-incubation with excess antigens (Figure 1E, lower panel). Similar results were obtained when another anti-K 2P 5.1 antibody (H-170) was used as the primary antibody (Supplementary Figure 1). In addition, the expression levels of the other alkaline pH-activated K 2P channel subtypes, K 2P 3.1/TASK1, K 2P 9.1/TASK3, and K 2P 16.1/TALK1 transcripts, were less abundant in the IBD model: <0.004 in arbitrary units (Figure 1G).

Enhancement in Alkaline pH e -induced Hyperpolarizing Responses in Splenic CD4 + T Cells of IBD Model Mice
To perform functional analysis of K 2P 5.1 in splenic CD4 + T cells, we measured the hyperpolarizing responses induced by the alkalization of extracellular pH (pH e ) (pH e 8.0 and 8.5) using the membrane potential-sensitive dye, DiBAC 4 (3). To confirm living cells, 140 mM high K + -induced depolarization was measured at the end of protocol. As shown in Figure 2A, the fluorescence intensity of DiBAC 4 (3) decreased with the application of alkaline pH e (pH e 8.5). The peak amplitude of the relative fluorescence intensity of DiBAC 4 (3) was lower in IBD model mice than in control mice ( Figure 2B). When alkaline pH e -induced hyperpolarizing responses (pH e 8.0 and 8.5) were expressed as changes in the relative fluorescence intensity of DiBAC 4 (3) [ relative fluorescence intensity of DiBAC 4 (3)], they were significantly larger in IBD model mice than in control mice ( Figure 2C). Under alkaline pH e conditions, high K + -induced increases in the fluorescence intensity of DiBAC 4 (3) were almost the same as those under normal pH e (pH e 7.4) conditions ( Figure 2B). In the presence of the nonspecific K 2P 5.1 blocker, clofilium (5 µM), alkaline pH e (pH e 8.5)induced hyperpolarizing responses disappeared in both groups. Significantly larger depolarizing responses were observed in IBD model mice (Figure 2D), suggesting that alkaline pH e -induced hyperpolarizing responses via the activation of K 2P 5.1 in IBD model mice were underestimated. Clofilium also inhibits voltagegated K + channel K V 1.3, one of main K + channels in T cells, however, 4-aminopyridine (5 mM), a K V 1.3 blocker, did not induce any significant differences in the alkaline pH e (pH e 8.5)-induced hyperpolarizing responses [ relative fluorescence intensity of DiBAC 4 (3) = −0.17 ± 0.04 (n = 19)]. A recent study demonstrated that the Ca 2+ -release activated Ca 2+ (CRAC) channel, Orai1, which contributes to the store-operated entry of Ca 2+ into T cells, was activated by alkaline pH (Beck et al., 2014), suggesting that the function of Orai1 was upregulated in the splenic CD4 + T cells of IBD model mice. However, depolarizing responses were not suppressed by the pre-treatment with the Orai1 blocker 2-APB (10 µM) for more than 5 min (P < 0.01) (Figure 2D), and the expression levels of Orai1 transcripts were also not changed between the two groups (not shown). We subsequently observed a relationship between alkaline pH e -induced hyperpolarization responses and [Ca 2+ ] i rises via CRAC channels in the splenic CD4 + T cells of IBD model mice by simultaneously measuring DiBAC 4 (3) and Fura 2 signals ( Figure 2E). As shown in Figure 2F, alkaline pH einduced hyperpolarization responses positively correlated with [Ca 2+ ] i rises in the splenic CD4 + T cells of IBD model mice (R 2 = 0.41). The alkaline pH e -induced [Ca 2+ ] i rises were suppressed by about 40% by the pre-treatment with 2-APB (10 µM) (not shown). Higher Expression Level of K 2P 5.1 in the Inflammatory CD4 + CD25 − Subset than in the Regulatory CD4 + CD25 + Subset When K 2P 5.1 is upregulated in Th1/Th17 cells producing IFNγ/IL-17, the inhibition of K 2P 5.1 may suppress the disease progression of IBD. However, when K 2P 5.1 is upregulated in regulatory T cells producing IL-10, the inhibition of K 2P 5.1 may promote the disease progression of IBD. In order to clarify the pathophysiological significance of the K 2P 5.1 K + channel in IBD, we examined differences in the expression levels of K 2P 5.1 between the splenic CD4 + CD25 − and CD4 + CD25 + subsets in IBD model mice using a real-time PCR assay. As shown in Figure 3, the proinflammatory Th1/Th17 cytokines IFN-γ ( Figure 3B) and IL-17A ( Figure 3C) transcripts were expressed at high levels in the CD4 + CD25 − subset of IBD model mice, whereas the natural regulatory T cell (T reg ) markers CD25 (Figure 3D), IL-10 ( Figure 3E), and Foxp3 (not shown) were predominantly expressed in the CD4 + CD25 + subset. As shown in Figure 3A, K 2P 5.1 transcripts were expressed at significantly higher levels in the CD4 + CD25 − subset in IBD model mice than in control mice. Significant increases in the expression levels of the K 2P 5.1 transcripts were also found in the CD4 + CD25 + subset of IBD model mice (P<0.01 vs. control) ( Figure 3A); however, these increases were significantly lower than those observed in the CD4 + CD25 − subset of IBD model mice (P < 0.01). Correspondingly, similar levels of alkaline pH e -induced hyperpolarizing responses (pH e 8.5) were observed in the CD4 + CD25 − subset of IBD model mice but not in the CD4 + CD25 + subset (not shown). These results suggested that the pharmacological inhibition of K 2P 5.1 is an effective strategy for the treatment of IBD.
Decreased Susceptibility to the Pathogenesis of IBD in K 2P 5.1 Knockout Mice As described above, the lack of selective pharmacological agents represents the main difficulty in carrying out experimental studies on K 2P 5.1. Therefore, we examined the effects of the genetic inhibition of K 2P 5.1 on inflammatory responses during acute IBD in K 2P 5.1 homozygous knockout (K 2P 5.1 −/− ) mice. As reported in our recent study , severe clinical symptoms and enlargement of the spleen were observed in wildtype K 2P 5.1 +/+ mice (Figures 4A-C, left columns). A significant protective effect against IBD was observed in K 2P 5.1 −/− mice (Figures 4A,B, right columns). The diarrhea scores were 2.25 ± 0.16 (n = 8) and 1.41 ± 0.17 (n = 17) in K 2P 5.1 +/+ and K 2P 5.1 −/− mice, respectively (P < 0.01 vs. K 2P 5.1 +/+ ). The degrees of splenomegaly was also significantly lower in K 2P 5.1 −/− mice (P < 0.05 vs. K 2P 5.1 +/+ ). Furthermore, colonic inflammation and crypt damage were quantified by colon weight/length ratio measurements and histological scoring. As shown in Figure 4D, colonic wall thickening was significantly increased in K 2P 5.1 +/+ mice, whereas no significant differences between control and IBD were observed in K 2P 5.1 −/− mice. Histological assessments of colonic inflammation and crypt damage also revealed that both scores were significantly lower Results were expressed as means ± SEM. Numbers used for the experiments are shown in parentheses. **P < 0.01 vs. control mice (ctrl), ## P < 0.01 vs. the CD4 + CD25 − subset.

DISCUSSION
The activation of K + channels in T cells promotes Ca 2+ influx, thereby indirectly modulating Ca 2+ signaling (Vig and Kinet, 2009). Two K + channel subtypes, the voltage-gated K V 1.3 and intermediate-conductance Ca 2+ -activated K + channel K Ca 3.1, are known to mainly function in T and B lymphocytes (Di Sabatino et al., 2009). The physiological role of the two-pore domain K + channel K 2P 5.1 was recently clarified in lymphocytes (Nam et al., 2011;Cid et al., 2013;Shin et al., 2014), and several studies have shown the pathophysiological impact of the upregulation of the two-pore domain K + channel K 2P 5.1 in CD4 + T cells on the pathogenesis of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis (Bittner et al., 2010a. However, the pathophysiological impact of K 2P 5.1 in diseases associated with Th1 and Th17 cytokine profiles has not yet been examined using in vivo animal models. This is the first study to employ chemically-induced IBD model mice to characterize the pathophysiological role of K 2P 5.1 in mature CD4 + T cells. The main results of the present study were as follows: (1) Facilitation of the expression level and functional activity of the K 2P 5.1 K + channel in splenic CD4 + T cells of IBD model mice, especially in the CD4 + CD25 − subset (Figures 1, 2), (2) Decreased disease activity index (diarrhea, bloody feces, and weight loss) and histopathological scores (colonic inflammation and crypt damage) in homozygous K 2P 5.1-deficient (K 2P 5.1 −/− ) mice (Figure 4). We were unable to elucidate the mechanism underlying the transcription regulation of K 2P 5.1 in the splenic CD4 + T cells of IBD model mice. The voltage-gated K + channel K V 1.3 and Ca 2+ -activated K + channel K Ca 3.1 are the main K + conductance channels in CD4 + T cells. Although both transcripts were expressed in splenic CD4 + CD25 − cells at high levels, no significant differences were observed in their expression levels between control and IBD model mice (not shown). These results suggested that the upregulation of K 2P 5.1 played an important role in the changes observed in T cell Ca 2+ signaling in IBD pathogenesis, and may affect the facilitation of proliferation and infiltration of splenic CD4 + T cells. K 2P 5.1 also contributes to the regulation of osmotic volume, regulatory volume decreases (RVD), in several types of cells including T cells (Niemeyer et al., 2001;Bobak et al., 2011;Andronic et al., 2013), which participate in cell cycle progression. Therefore, a dysfunction in cellular volume homeostasis via the upregulation of K 2P 5.1 may also be involved in dysregulated cellular functions in the CD4 + T cells of IBD model mice.
As shown in Figure 3A, the upregulation of K 2P 5.1 was greater in splenic CD4 + CD25 − T cells of IBD model mice than in CD4 + CD25 + T cells. CD4 + CD25 − T cells include pro-inflammatory Th1 and Th17 subsets, and an increase was observed in the transcription of IFN-γ and IL-17A in the splenic CD4 + CD25 − T cells of IBD model mice (Figures 3B,C). Due to the lack of a selective K 2P 5.1 inhibitor, we were unable to examine the effects of its in vivo administration on the disease activity index and histopathological scores in IBD model mice in order to clarify the pathophysiological significance of K 2P 5.1 in IBD. However, we showed that the genetic knockdown of K 2P 5.1 significantly protected against IBD via the FIGURE 5 | Expression levels of IFN-γ and IL-17A transcripts in CD4 + CD25 − subset of IBD models from K 2P 5.1 +/+ (+/+) and K 2P 5.1 −/− (-/-) mice (A,B), and IL-10 transcripts CD4 + CD25 + subset of IBD models from K 2P 5.1 +/+ (+/+) and K 2P 5.1 −/− mice (-/-) (C). Expression levels were measured using a real-time PCR assay. Results were expressed as means ± SE. Numbers used for the experiments are shown in parentheses. *P < 0.01 vs. K 2P 5.1 +/+ mice. amelioration of severe colitis and colonic inflammation using homozygous K 2P 5.1 knockdown K 2P 5.1 −/− mice (Figure 4). Furthermore, the expression levels of the IFN-γ transcripts from the pro-inflammatory Th1 subset were significantly lower in the CD4 + CD25 − cells of the IBD model from K 2P 5.1 −/− mice than from K 2P 5.1 +/+ mice (Figure 5A), and no significant differences were noted in the expression levels of IL-17 in CD4 + CD25 − cells and IL-10 in CD4 + CD25 + cells between IBD models from K 2P 5.1 +/+ and K 2P 5.1 −/− mice (Figures 5B,C). Similar findings were obtained with the genetic and pharmacological knockdown of the Ca 2+ -activated K + channel K Ca 3.1 in an IBD model (Di et al., 2010;Ohya and Imaizumi, 2014). It has been reported that the function of Th1 was impaired in an IBD model from K Ca 3.1 −/− mice, whereas that of Th17 was normal (Di et al., 2010). We recently demonstrated the inhibition of IFN-γ transcription, but not that of IL-17A by the in vivo administration of selective K Ca 3.1 inhibitors in a DSS-induced IBD model . Therefore, the development of small molecule K 2P 5.1 modulators may represent therapeutic and/or preventative strategies for the treatment of inflammatory and autoimmune disorders and cancer, and the establishment of a novel high-throughput screening system to detect these modulators currently required (Bagriantsev et al., 2013). Furthermore, novel findings on the regulatory molecules of K 2P 5.1 will provide insights into the inhibition of K 2P 5.1.
Regulatory T (T reg ) cells play an important role in the maintenance of intestinal homeostasis, and T reg cells are considered to be beneficial for IBD therapy (Boden and Snapper, 2008;Himmel et al., 2012;Gibson et al., 2013;Mayne and Williams, 2013). As shown in Figure 3A, the expression levels of K 2P 5.1 transcripts of K 2P 5.1 were significantly increased in the splenic CD4 + CD25 + T cells of IBD model mice. This subset also included Foxp3-positive cells (not shown) and, thus, is referred to as naturally-occurring regulatory T cells. In B lymphocytes, the production of the anti-inflammatory cytokine IL-10 was shown to be stimulated by Ca 2+ influx via the CRAC Frontiers in Physiology | www.frontiersin.org channel (Matsumoto et al., 2011;Baba et al., 2014). Therefore, the upregulation of K 2P 5.1 may strengthen the production of IL-10 in the natural T reg cells of IBD model mice, and the activation of K 2P 5.1 may improve the pathogenesis of IBD. In the present study, the expression of IL-10 was significantly higher in the natural T reg cells of IBD model mice than in those of control mice (Figure 3E). No significant changes in the expression of IL-10 were found in natural T reg cells of the IBD model from K 2P 5.1 −/− mice ( Figure 5C); however, the long-lasting dysregulation of Ca 2+ signaling elicited by a K 2P 5.1 deficiency may be compensated by the upregulation of other K + channel(s), and, thus, the expression of IL-10 may be maintained within normal ranges in the T reg cells of the IBD model from K 2P 5.1 −/− mice. Further studies are needed in order to clarify the pathophysiological role of K 2P 5.1 in the natural T reg cells of IBD.
In conclusion, the results of the present study suggested that the background K + channel K 2P 5.1 in splenic CD4 + T cells was involved in the pathogenesis of IBD using a chemically-induced IBD model and provided evidence for the K 2P 5.1 K + channel as a potential therapeutic target for suppressing the progression of IBD. Dysregulated K 2P 5.1 may stimulate the Th1 imbalance in the process of intestinal inflammation. The lack of selective K 2P 5.1 blockers may facilitate future research on a novel highthroughput screening system to detect small molecule K 2P 5.1 modulators and the regulatory mechanisms underlying K 2P 5.1 transcription, translation, and protein degradation in IBD.