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.