Expression of K_2P5.1 potassium channels on CD4⁺T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a chronic inflammatory disease which is characterized by pain, swelling and progressive destruction of multiple joints. The systemic nature of RA causes, next to loss of joint function, substantially decreased quality of life and increased mortality of patients. Current treatments are mainly based on immunosuppressive disease-modifying antirheumatic drugs, among them the rapidly expanding family of biologic agents [1]. Close monitoring of disease activity is mandatory for the evaluation of treatment efficacy as a substantial percentage of patients do not respond adequately to first-line therapy. In these cases, as well as in patients with disease exacerbations, a change in treatment strategy is required [2]. Monitoring of disease activity includes patient history, clinical examination, blood values (C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR)) and composite scores such as the widely used disease activity score (DAS28). The DAS28 score includes the number of swollen and painful joints, the ESR rate and the patient's subjective evaluation on a visual analogy scale (VAS) [3, 4].

The potassium channel K_2P5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5) belongs to the family of two-pore domain potassium channels (K_2P channels) which has recently been shown to be expressed on T lymphocytes [5, 6]. K_2P5.1 is important for T cell functions such as proliferation or cytokine production [7] as it is hypothesized that the counterbalancing efflux of potassium channels is mandatory for a longer lasting elevation of the intracellular Ca²⁺ levels during T cell stimulation [7]. Moreover, chronic repetitive stimulation leads to an upregulation of K_2P5.1 channel expression whereas pharmacological blockade or siRNA-induced gene silencing of K_2P5.1 results in a reduction of T cell effector functions. It has additionally been shown that expression levels of this channel are strongly increased on T lymphocytes from the peripheral blood from clinically active relapsing-remitting multiple sclerosis (MS) patients. Interestingly, expression and MS-specific upregulation were found predominantly on CD8⁺ T cells rather than on CD4⁺ T cells which may be due to a disease-specific pathogenic role of cytotoxic T lymphocytes. Expression of K_2P5.1 was even higher on cerebrospinal fluid (CSF)-derived T lymphocytes than in the peripheral blood and K_2P5.1-positive T lymphocytes can be found within inflammatory lesions from MS patients. So far, it was not known whether these findings are MS-specific or can be similarly found in other autoimmune disorders. CD4⁺ T helper cells play an important role in the pathogenesis of RA. This is suggested by its association with certain MHC II loci, especially HLA-DRB1, and PTPN22, which is relevant for T cell function [8]. The therapeutic effects of blockade of T cell costimulation by abatacept provides more direct evidence [9].

Therefore, we investigated the correlation of K_2P5.1 expression levels on T lymphocytes from RA with different disease activity parameters. The influence of different therapies was taken into account as they might potentially influence K_2P5.1 expression. Finally, a longitudinal study was conducted in a subset of patients who underwent therapy change due to disease exacerbation and these patients were followed up for six months.

The potassium channel K_2P5.1 has been previously shown to regulate T cell function in vitro and in vivo. In a first set of experiments, freshly isolated CD4⁺ T cells from healthy donors were either left unstimulated or stimulated with CD3/CD28 beads for two days. A clear upregulation of K_2P5.1 could be shown on protein level (one representative example is shown in Figure 1A, left panel; quantification of three independent experiments - right panel). In MS, putatively pathogenic T cells derived from the CSF - reflecting the site of inflammation - showed significantly higher levels of K_2P5.1 than T cells from the peripheral blood of the same patients [7]. Based on these findings, we next analyzed the K_2P5.1 expression on activated CD4⁺ T lymphocytes out of the synovial fluid of RA patients. Synovial fluid derived T lymphocytes showed a slight upregulation of the activation marker CD69, but not of CD25 when compared to cells from the peripheral blood (Figure 1B). In patients with RA, K_2P5.1 is upregulated both on RNA level (Figure 1C, left panel, black lines) and on protein level (Figure 1C, middle panel, black lines) as assessed by real time RT-PCR and flow cytometry, respectively. A representative flow cytometry staining can be found in Figure 1C, right panel. In contrast, no difference could be observed in one patient with reactive arthritis (Figure 1C, left and middle panel, grey lines). These findings point towards a shared pathophysiological motif of both T cell-mediated disorders - namely MS (CD8⁺ T cells) and RA (CD4⁺ T cells). Finally, TASK2 expressing T cells could be identified immunohistochemically within human synovial tissue sections. Exemplary costainings for the T cell marker CD3 and TASK2 can be found in Figure 1D.

Activity of CD4⁺ and CD8⁺ T lymphocytes critically depend on constitutively expressed K_2P5.1 potassium channels. Chronic stimulation as it may occur under autoimmune conditions leads to an upregulation of these channels on RNA and protein level. In a first set of experiments, MACS-isolated T cells from the peripheral blood of 58 RA patients were analyzed for expression of K_2P5.1 by RT-PCR. We compared the correlation of K_2P5.1 channel expression on both CD4⁺ and CD8⁺ T lymphocytes with different disease activity parameters. We found a positive correlation between K_2P5.1 expression levels measured as Δct values and DAS28 on CD4⁺ T lymphocytes (R = 0.63; Figure 2A left panel; Figure 2D). Moreover, a weaker correlation could be found for ESR (R = 0.39; Figure 2B left panel; Figure 2D) and CRP levels in the peripheral blood (R = 0.28; Figure 2C left panel; Figure 2D). In contrast, K_2P5.1 expression on CD8⁺ T lymphocytes and three disease activity parameters were found to be only weakly correlated (Figure 2A-D). In summary, K_2P5.1 expression on CD4⁺ T lymphocytes of RA patients seems to be positively correlated with the disease severity (Figure 2E). Hence, K_2P5.1 expression levels are strongly elevated in patient subgroups with high disease activity (Figure 2E). These results were corroborated on protein level in a small cohort of patients (n = 5, see Supplementary Figure S1 in Additional file 1). As a note of caution, it should be mentioned that all RA patients received disease-modifying therapies, which were divided into three classes due to their mode of action (conventional, black diamonds; TNFα inhibitors, grey diamonds; rituximab, white diamonds; Supplementary Figure S2A-B in Additional file 2). The correlation coefficients for these subgroups are shown in Supplementary Figure S2C in Additional file 2 (conventional: n = 21, R = 0.61; TNFα inhibitors: n = 27, R = 0.49; rituximab: n = 10, R = 0.91). It may be speculated that the high positive correlation which was found for rituximab-treated patients may at least partly be due to a higher disease activity in this patient subgroup (conventional: DAS28 = 2.92; TNFα inhibitors: DAS28 = 3.30; rituximab: DAS28 = 3.98). To further support these results we analyzed K2P5.1 expression in naïve and stimulated CD4⁺ T cells from healthy individuals after 24 hours in vitro treatment with methotrexate, etanercept, adalinumab, certolizumab, tocilizumab and hydroxychloroquine. No significant upregulation of K2P5.1 was observed in treated T cells compared to untreated controls (n = 5; see Table 2).

In a next set of experiments a longitudinal follow-up study was initiated including patients who underwent therapy escalation from conventional therapy to biological or from one biological to another. According to these criteria, 11 patients were recruited and followed up for three to six months. Most patients (n = 8) showed at Month 3 a reduction in the DAS28 score (Figure 3A, left panel). Mean DAS28 scores are shown on the right (t = 0 months: 4.66 ± 0.50; t = 3 months: 3.70 ± 0.42; t = 6 months: 4.18 ± 0.70). Comparable results could be found for CRP (Figure 3B) and ESR levels in the peripheral blood (Figure 3C) which decreased from 1.84 mg/dl to 0.95 mg/dl and from 30.4 mm/h to 22.9 mm/h, respectively. K_2P5.1 expression levels decreased at Month 3 in 9 out of 11 patients (Figure 3D). It should be noted that two patients who had an increase of K_2P5.1 expression at Month 3 showed a parallel increase of DAS28. In the entire group the relative expression reduction of K_2P5.1 was 28% and 47% at three and six months, respectively. In summary, this prospective study demonstrates a strong longitudinal correlation of clinical disease activity parameters with K_2P5.1 expression levels of peripheral CD4⁺ T cells in individual patients.

In an additional set of experiments, nine patients with a therapeutic switch to tocilizumab were followed up for six months as well (Figure 4). By interfering with IL-6 signaling, tocilizumab is known to have inhibitory effects on inflammatory markers such as CRP, serum amyloid A or ESR [11]. These effects of tocilizumab may be independent of its therapeutic effect in RA patients and it has been doubted whether classical surrogate markers (for example, ESR, CRP) are suitable for measuring therapy efficacy in tocilizumab-treated RA patients [12]. Indeed, we found a drastic decrease in ESR (29.3 ± 0.8 to 4.7 ± 1.5 mm/h) and CRP (1.89 ± 0.78 to 0.06 ± 0.01 mg/dl) values three months after tocilizumab initiation. At three months, all patients showed an initial decrease in DAS28, ESR and CRP values, whereas K_2P5.1 levels were clearly downregulated in four and strongly upregulated in two patients to 3.0 and 3.5, respectively (marked in red throughout Figure 4A-D). Remarkably, in both patients K_2P5.1 upregulation preceded a clinical relapse.

Disease activity in RA patients was found to correlate strongly with K_2P5.1 expression levels in CD4⁺ T lymphocytes in the peripheral blood in a cross-sectional study in 58 patients. Furthermore, longitudinal observations in individual patients showed comparable changes in all disease surrogate markers with K_2P5.1 expression. It seems plausible that K_2P5.1 expression reflects the activation status of chronically stimulated autoimmune CD4⁺ T lymphocytes.

However, a number of questions remain to be addressed which are beyond the focus of this initial study, for example: Can K_2P5.1 expression serve as a biomarker for disease activity in RA? Current serum markers (CRP and ESR levels) which are also part of composite scores like DAS28 or DAS28-CRP reflect rather the level of systemic inflammation than the specific activation status of pathogenic immune cells. Moreover, their use seems to be limited especially in the case of tocilizumab which inhibits the systemic acute phase reaction at least in part independently from its well-proven anti-rheumatic effects [13]. This problem has, for example, been addressed by Matsui et al. who evaluated neutrophil CD64 as a biomarker for otherwise masked infection under tocilizumab therapy [14]. We observed that in contrast to our cross-sectional study and our longitudinal study with other medications, K_2P5.1 expression changes behaved differently in tocilizumab-treated patients. Two out of nine patients even showed an opposite upregulation of K_2P5.1 at three months preceding a relapse about three months later. At the present, it seems too early and patient numbers are too low to state that K_2P5.1 upregulation generally precedes clinical relapses in RA, whether it reveals otherwise masked clinical developments under tolizumab therapy or whether its validity is limited under these circumstances. Therefore, in light of the current results further patient studies need to assess the prognostic value, time course and clinical validity of monitoring K_2P5.1 expression in RA patients. Furthermore, it has to be proven whether K_2P5.1 can be used for differential diagnosis.

Additionally, research efforts are needed concerning the mechanisms underlying K_2P5.1 function and regulation in the pathophysiology of RA. Especially the use of animal models for RA may help to provide insight in this context.

We show here for the first time a correlation of K_2P5.1 expression levels in CD4⁺ T lymphocytes and disease activity in patients suffering from RA. Since other studies already showed a functional role of K_2P5.1 for T cell effector function this member of the two-pore domain potassium channel family might represent an interesting molecular target for diagnostic and/or therapeutic applications. However, further studies from independent cohorts are warranted to confirm and extend the presented findings. Furthermore, the use of animal models for RA might help to shed more light on the functional role of K_2P5.1 in RA pathogenesis.