Discussion
The ENV59-GP3 peptide contains the ISD sequence from a specific human endogenous retroviral protein encoded by the envelope ENV59 gene, originally identified in SLE patients [9], and shown to be adapted by the human immune system to ameliorate autoimmunity [9, 10]. In this report, we provide evidence that treatment with the ISD ENV59-GP3 inhibits the development of colitis in dextran sulfate sodium (DSS) -induced mice, a widely recognized autoimmune model of human IBD. ENV59-GP3 showed reduction of the disease score in this model, which was comparable to effects of the KCa3.1 channel blocker NS6180, though ENV59-GP3 treatment was at five times lower doses (by weight) than that of NS6180. The very low doses of ENV59-GP3 in in vivo treatment regime are in agreement with the treatment results in other animal models [9, 10].
In our literature review for potential signaling pathways and molecular targets utilized by ENV59-GP3 peptide we came across studies by Di et al. who has reported that that KCa3.1 ( KCNN4 ) − / − mice are protected from developing severe colitis [11]. Similarly, as is the case for ENV59-GP3, NS6180 or Tram-34- small molecule inhibitors of KCa3.1 reduce the disease score in T-cell mediated colitis models [11].
This prompted us to study the effects of Env59-GP3 peptide on the calcium activated potassium channels. Here, for the first time, we show the inhibitory activity of an endogenous retroviral ISD peptide on the intermediate-conductance calcium activated potassium-ion channel KCa3.1 current.
KCa3.1 plays an important function in regulating Ca2+ influx - mediated functions such as cytokine production, proliferation and migration in the cells of the immune system [27]. In line with this mechanism, T-cell receptor engagement dramatically induces KCa3.1 expression, in parallel with increased KCa3.1 currents and enhanced [Ca2+] signaling during human T-cell activation [12, 27]. KCa3.1 has been recognized as a potent target for several autoimmune diseases indications including IBD, atherosclerosis, multiple sclerosis, rheumatoid arthritis as well as diseases such as asthma, renal fibrosis, diabetes and sickle cell anemia [15, 28, 29].
Analysis of cytokine production revealed similar inhibitory effects of the ISD ENV59-GP3 peptide and the selective KCa3.1 blocker NS6180 on the in vitro production of IL-6 and TNF-alpha in the LPS or PMA + ionomycin PBMCs (data not shown) assays reflecting a particularly important role of KCa3.1 channel when proliferation is driven by the Ca2+‐dependent NFAT and NF‐k B pathways. The identical cytokine profiles obtained with ENV59-GP3 and KCa3.1 inhibitors strongly support their identical pharmacological activity in these assays. Analysis of cytokine production from DSS-mice model treated animals revealed equipotent inhibitory effects of the ENV59-GP3 and NS6180 compounds on the production of IL-6, TNF-α, IL-1β; whereas IL-17 was not affected by the peptide and only minorly affected by the NS6180 treatment. These findings also reinforce a critical role for Th1 effector cells in murine model of colitis, and are consistent with a recent report demonstrating that IL-17 may have more of a protective function by interfering with the generation of Th1 cells [30]. Thus, by not inhibiting Treg and Th17 cell function, KCa3.1 blockers may have a unique therapeutic profile to inhibit procolitis-inducing Th1 and Th2 cells, without interfering with the beneficial function of Treg and possibly Th17 cells [10].
Env59-GP3 peptide inhibits calcium influx induced by Thapsigargin in a variety of different cell types. Thapsigargin is an inhibitor of the sarco/endoplasmic reticulum Ca2+ATPase (SERCA). Inhibition of SERCA causes a slight increase of intracellular Ca2+, which in turn activates the calcium release-activated channels (CRAC), resulting in significant influx of calcium ions into the cytoplasm. This calcium influx is inhibited by ENV59-GP3. Interestingly, ENV59-GP3 showed no direct effect on the calcium release-activated channels (CRAC) itself (data not shown), in agreement with the peptide’s inhibitory effect on the calcium activate potassium efflux through KCa3.1.
The immune suppressive effect of ENV59-GP3 on THP-1 cells described in this report can be accredited to its inhibitory effect on the ion channel, since electrophysiological recordings as well as mRNA expression levels demonstrated the presence and the activity of KCa3.1 in this human monocyte cell line.
KCa3.1 expression has been correlated with IBD in clinical settings. The impact of KCNN4 expression or activity was extensively analyzed in immune cells. T cell activation is altered by the expression level and activity of KCNN4 in T lymphocytes in pediatric patients with IBD [31]. The expression level of KCNN4 in T cells can be correlated with disease activity in UC and elevated T-cell KCNN4 expression in UC correlates with inflammatory cytokine levels and disease activity [26]. In CD a gene variant of KCNN4 has been described that is associated with ileal disease in the New Zealand and Australian population [32].
In addition to IBD, KCa3.1 channels are suggested to be a novel target for treating various diseases such as idiopathic pulmonary fibrosis [33], airway inflammation (allergic asthma) and ischemia/stroke [34]. Senicapoc, a KCa3.1 blocker, was suggested as a treatment for sickle cell disease by inhibiting red blood cell dehydration thus increasing hemoglobin. Senicapoc showed promising efficacy both in mice and humans (up to phase 3 study), with no adverse effects compared to placebo. Yet, the trial was terminated before end of study, as it seemed impossible to reach the primary endpoint [35]. Additionally, clinical studies using Clotrimazole, a small molecule inhibitor of KCa3.1, in rheumatoid arthritis were performed in the 1980’s [20]. Clotrimazole was shown to be effective in the treatment of the disease and superior to ketoprofen in certain measurements. Improvement with clotrimazole took place more slowly but was more sustained than with ketoprofen. However, further exploration of Cloritmazol’s clinical utility was hampered by its gastrointestinal side effects and liver toxicity due to inhibition of cytochrome P450 [36].
ENV-59-GP3 is isolated from an endogenous retrovirus. Endogenous retroviruses originate from ancient viral infections. Evolution of the interaction between ion channels and viral envelope proteins might originally have been a result of the optimization of the ENV structure for its primary use i.e. viral entry. It is feasible that ion channels might provide additional attachment points for the virus to target cells and thus function as co-receptors. Alternatively, changes in the membrane potential might play a role in viral entry into the target cells. It is feasible that the ISD sequences from exogenous or other families of endogenous retroviral envelopes are also inhibitors of the KCa3.1 or other similar ion channels. Indeed, it is possible that other virus groups or other pathogens have evolved similar immune modulatory activities independently. Whether this is the case remains to be investigated.
In any case, the data presented here has important implications for our understanding of endogenous retroviruses in immunology and the relationship between the ENV59 peptide and IBD disease provides further rationale for KCa3.1 constituting an attractive drug target.