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\begin{document}

\title{Retroviral Glycoprotein-Mediated Immune Suppression via the potassium
Channel KCa3.1- A new strategy for treatment of Inflammatory Bowel
Diseases.}



\author[1]{Magdalena Laska}%
\author[2]{Jesper Bonnet Møller}%
\author[2]{Jonas Heilskov Graversen}%
\author[3]{Dorte Strøbæk}%
\author[3]{Linda Blomster}%
\author[3]{Palle Christophersen}%
\author[1]{Shervin Bahrami}%
\affil[1]{Aarhus University}%
\affil[2]{University of Southern Denmark}%
\affil[3]{Saniona A/S}%


\vspace{-1em}



  
  \date{June 03, 2024}


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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Peptides derived from retroviral envelope
proteins have been shown to possess a wide range of immunosuppressive
and anti-inflammatory activities. We have previously reported
identification of such a peptide derived from the envelope protein coded
by a human endogenous retrovirus (HERV). In this study we assessed
effects of this peptide treatment on inhibition of immune response in
the DSS-induced mice model of colitis. Furthermore, we identified that
\emph{in vitro} the peptide inhibits the KCa3.1 potassium channel, a
potential target for therapy of immune diseases. Experimental Approach:
We characterized an immunosuppressive peptide ENV59, from a specific
HERV envelope protein, \emph{in vivo} effects on inflammation control in
acute colitis mice model and \emph{in vitro} on the production of
pro-inflammatory cytokines. Furthermore, we described \emph{in vitro}
ENV59-GP3 effects with respect to potency of inhibition on KCa3.1
channels and calcium influx. Key Results: ENV59-GP3 peptide treatment
showed reduction of the disease score in the DSS-induced acute colitis
mice model, which was comparable to effects of the KCa3.1 channel
blocker NS6180. 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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β. \selectlanguage{english}Patch
clamp studies show that the peptide ENV59-GP3 is a blocker of the
potassium channel KCa3.1. Conclusion and Implications: Env59-GP3
represents KCa3.1 channel inhibitor underlining the implications of
using virus derived channel blockers for treatment of autoimmune
diseases. There are no drugs with a similar mechanism of action
currently on the market.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Inflammatory Bowel Disease (IBD) is a group of inflammatory conditions
of the colon and small intestine. Crohn's disease (CD) and ulcerative
colitis (UC) are the principal types of inflammatory bowel diseases. The
etiology is multifactorial, and many genetic loci have been found to
serve as susceptibility genes for different subgroups of patients with
IBD {[}1, 2{]}, likewise, environmental influences are also considered
as factors involved in IBD onset {[}3{]}. In addition, to its
complicated risk factors, the pathogenesis of IBD is not entirely clear,
making it difficult to develop new drugs with therapeutic value and
acceptable side effects {[}2{]}.

For more than two decades, animal models of intestinal inflammation have
provided a wealth of information about mucosal immunology and the
intestinal inflammation encountered in the inflammatory bowel diseases.
While no single model has proven to sufficiently represent the
complexity of the clinical characteristics of the human disease, the
combined data obtained from these various animal models have provided a
more detailed understanding of the underlying principles of human IBD
and delineate pathways that might help predict the successful outcome of
novel therapeutics in the clinic {[}4, 5{]}.

The most widely used murine model of colitis employs dextran sodium
sulfate (DSS), a chemical colitogen with anticoagulant properties, to
induce intestinal epithelial damage {[}6{]}. The DSS colitis model lends
itself to IBD research due to its rapidity, simplicity, reproducibility
and controllability. Acute, chronic and relapsing models of intestinal
inflammation can be achieved by modifying the concentration of DSS and
the frequency of administration {[}6{]}.

Numerous inflammatory mediators have been implicated in the pathogenesis
of IBD. These include cytokines, eicosanoids, reactive oxygen species,
nitric oxide, and complement activation products {[}7{]}. Similarly,
increased expression of different inflammatory mediators (TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β,
\selectlanguage{english}IFN-g, \selectlanguage{english}IL-10 and IL-12) are observed as early as the first day of DSS
treatment. The production of these inflammatory mediators increases
progressively during DSS treatment {[}8{]}.

Recently, we reported identification of an immunosuppressive peptide
(ENV59-GP3) from a specific human endogenous retroviral envelope
protein, ENV59, which has been adapted by the immune system and is
involved in ameliorating autoimmunity {[}9, 10{]}. The ENV59-GP3 peptide
showed strong immune modulating activity both \emph{in vitro}
and\emph{in vivo} in SKG mouse model for rheumatoid arthritis and EAE
model for Multiple Sclerosis {[}9, 10{]}.

In this study we follow up on our previous findings and demonstrate that
the ENV59-GP3 peptide treatment, reduces inflammation in the DSS-induced
mice model of colitis. Furthermore, we show that \emph{in vitro}
ENV59-GP3 inhibits the K\textsubscript{Ca}3.1 potassium channel, an ion
channel responsible for hyperpolarization of the plasma membrane and
regulation of calcium influx upon lymphocyte activation.

Potassium channels might be regarded as a new group of therapeutic
targets for IBD {[}11, 12{]}. The intermediate-conductance
calcium/calmodulin-gated potassium channel K\textsubscript{Ca}3.1 also
known as the IK or the Gardos channel (encoded by the \emph{KCNN4} gene)
is expressed in a variety of tissues and cell types including T cells
{[}13, 14{]}, macrophages as well as DCs {[}14{]}. In T cells, sustained
Ca\textsuperscript{2+} influx via the CRAC channel is important for
sufficient cytokine production and proliferation. Influx of
Ca\textsuperscript{2+} is dependent on a negative membrane potential.
Activation of K\textsubscript{Ca}3.1 by elevated intracellular
Ca\textsuperscript{2+} maintains the negative membrane potential through
efflux of K\textsuperscript{+}, which helps to sustain
Ca\textsuperscript{2+} entry into the cell. Upon channel opening, the
efflux of potassium is driven by its concentration gradient and results
in hyperpolarization of the membrane providing the electrochemical
gradient necessary for influx of calcium {[}15{]}.

K\textsubscript{Ca}3.1 channel is known to be involved in regulating T
cell function, proliferation, and cytokine production {[}16{]}, thus
suggesting that they could be involved in the pro-inflammatory responses
in IBD further up-stream of inflammatory cytokines such as IFN-g \selectlanguage{english}and
TNF-\selectlanguage{greek}α. \selectlanguage{english}Furthermore, studies have revealed a prominent role of KCNN4 in
Ca\textsuperscript{2+}-dependent ion transport and intestinal
restitution {[}17{]}. In a T cell-mediated mouse model of colitis,
inhibition of K\textsubscript{Ca}3.1 ameliorated the disease {[}11{]}.
Likewise, the K\textsubscript{Ca}3.1 channel blocker NS6180 prevents
T-cell activation and inflammation in a rat model of inflammatory bowel
disease {[}18{]}. Although loss of K\textsubscript{Ca}3.1 did not
interfere with CD4 T cell differentiation, both
Ca\textsuperscript{2+}~influx and cytokine production were impaired in
KCNN4\textsuperscript{-/-}~Th1 and Th2 CD4 T cells, whereas T-regulatory
and Th17 function were normal.~In animal models for other autoimmune
diseases, the K\textsubscript{Ca}3.1 inhibitor TRAM-34 or ICA-17043
(Senicapoc) have been reported to prevent experimental autoimmune
encephalomyelitis in mice {[}19{]} or to inhibit/attenuate inflammatory
responses in mice with collagen antibody induced rheumatoid arthritis.
Clinical studies with Clotrimazole (form which Tram-34 has been derived)
already provided evidence that blockers of this channel may be useful in
treatment of chronic diseases such as RA, however, non-target related
adverse effects have prevented further development in some cases
{[}20{]}.

\textbf{Materials and methods}

\emph{Preparation of a synthetic peptide}

ENV59-GP3 (AJ289711.1 envelope sequence) peptide and Flu ISU (control)
peptide were chemically synthesized and acquired from Schaefer-N,
Copenhagen, DK with. All peptides were homo-dimerized through disulfide
bond formation between cysteine residues. The dimeric forms were used in
all subsequent experiments.

\emph{Cell culture}

THP-1 human acute monocytic leukemia cells and HEK 293 cells were grown
in culture in Roswell Park Memorial Institute medium (RPMI 1640,
Invitrogen) or DMEM (Dulbecco's Modified Eagle Medium) respectively,
supplemented with 10\% fetal bovine serum (Sigma, Cat\# F7524) and 1\%
penicillin/streptomycin (P/S).

\emph{(qRT-PCR) analysis}

Quantitative reverse transcription--polymerase chain reaction (qRT-PCR)
analysis were performed as described previously using primers for
amplification of IL-6 and TNF-alpha {[}9, 21{]}.

\emph{Enzyme-linked immunosorbent assay (ELISA)}

IL-6 or TNF-alpha was assayed with a conventional sandwich ELISA
(BioLegend), using ELISA (BioLegend), using 96-well plates coated with
antibodies specific for IL-6 or TNF-alpha.

\emph{Mouse model of DSS-induced colitis}

\emph{In vivo} studies were conducted at the University of Southern
Denmark using male 8-10 weeks old C57BI/6n littermates (Taconic). The
mice were co-housed in groups of 4 and provided with water and
food\emph{ad libitum} at 20-24\textsuperscript{0}C, 55\% humidity and a
12-h light/dark cycle. All procedures were performed in accordance with
the ethical standards of the institution with approval from the Danish
Animal Experiments Inspectorate (Approval no. 2017-15-0201-01254).

\emph{DSS-induced acute intestinal inflammation}

For acute DSS-induced colitis, the mice were administrated 2.5\% DSS
(molecular weight, 36-50kD; MP Biomedicals) in their drinking water for
7 days, followed by ordinary tap water. Three treatment groups were
included in the study: animals treated with 12,5\selectlanguage{greek}µ\selectlanguage{english}g peptide (250\selectlanguage{greek}µ\selectlanguage{english}l) IP
every other day (0.5mg/kg); animals treated with saline (250\selectlanguage{greek}µ\selectlanguage{english}l) as
control; or animals treated with 62,5\selectlanguage{greek}µ\selectlanguage{english}g blocker (250\selectlanguage{greek}µ\selectlanguage{english}l) IP every other
day (2.5mg/kg). All buffers contain 1.25\% DMSO. During this time
period, the mice were weighted daily and subsequently sacrificed at day
10. At the time of sacrifice, colon specimens were collected for
histology and qPCR analyses.

\emph{Histological assessment of inflammation}

Assessment of inflammation was performed as described by {[}22{]}. In
brief, colonic sections were H\&E-stained and assessed for disease
severity by trained personnel blinded to treatment and genotype. Scoring
was based on two parameters; inflammatory cell infiltrate (0-3) and
intestinal architecture (0-3). The combined values were added to give a
histological score between 0 and 6. Images were acquired using an
inverted Nikon Eclipse Ti microscope.

\emph{Q-PCR analyses for mouse cytokines}

For transcriptional analyses, colon samples from DSS-treated mice were
homogenized in TRIzol (Thermo Fisher Scientific) and stored at -80\selectlanguage{ngerman}°C
until further processed. RNA was initially extracted with chloroform and
then further purified by lithium chloride precipitation to remove DSS a
strong inhibitor of downstream PCR applications {[}23{]}. Reverse
transcription of total RNA was performed using the High-Capacity cDNA
Reverse Transcription kit according to the manufacturer's
recommendations (Thermo Fisher Scientific). Quantification of expression
was detected on a StepOnePlus Real-Time PCR (Thermo Fisher Scientific)
using the following TaqMan Gene Expression Assays (Applied
Biosystems):\emph{Il-1b} (Mm00434228), \emph{Il-6} (Mm00446190),
\emph{IL-17a}(Mm00439618) and \emph{tnfa} (Mm00443258). Gene expression
was normalized as n-fold difference to mouse~\emph{Hprt1} ~(Mm00446968)
according to the cycling threshold.

\emph{Electrophysiology}

All experiments were performed in the whole\selectlanguage{english}-cell version of the
patch\selectlanguage{english}-clamp technique using either an EPC\selectlanguage{english}-9 or EPC\selectlanguage{english}-10 amplifier
controlled by Pulse software (HEKA Electronics, Lambrecht, Germany)
according to the previously described methodology {[}18{]}. HEK293 cells
stably expressing human wild-type K\textsubscript{Ca}3.1 channel or
THP-1 human monocyte cell line were used. IC\textsubscript{50}~values
for blocking K\textsubscript{Ca}3.1 channels were estimated either from
concentration--response curves by fitting to the Hill equation or from a
fit to the inhibition kinetics assuming that
IC\textsubscript{50}~=~\emph{K} \textsubscript{d}~as detailed previously
{[}18{]}. Selectivity experiments using other ion channels were
performed as described previously {[}24{]}.

\emph{Calcium influx assay}

Intracellular calcium measurements in cell populations were monitored by
using Fluo-8 calcium flux assay according to the manufactures protocol
(Abcam, Denmark) and fluorescence was analyzed at at Ex/Em 490/525 nm.
Depletion of intracellular stores was achieved by addition of 250nM
Thapsigargin (Tg; Sigma).

\textbf{Results}

\textbf{Effect of the ENV59-GP3 peptide and
K\textsubscript{Ca}3.1-blocker NS6180 in DSS-induced acute colitis mice
model}

The effect of blocking K\textsubscript{Ca}3.1 on the immune
response\emph{in vivo} was evaluated using DSS-induced acute colitis
model. Starting at day 0 and throughout the course of the experiment,
the mice were dosed i.p. twice weekly with a ENV59-GP3 peptide, at 0.5
mg per kg of body weight; selective K\textsubscript{Ca}3.1 blocker
NS6180 at 2.5 mg per kg of body weight, or with saline (control). The
development of IBD was monitored as daily average weight loss
development. As shown in Figure 1A, DSS administration resulted in
significant weight loss (control), whereas in mice treated with
ENV59-GP3 peptide or the channel blocker NS6180, weight loss was
significantly reduced by day 6\textasciitilde{}10. Furthermore, we found
that colon length, a macroscopic indicator of colitis severity, in
DSS-treated mice control group was significantly reduced. In contrast,
administration of ENV59-GP3 at 0.5 mg/kg or NS6180 at 2.5 mg/kg led to a
marked improvement in colon length (\emph{p} \textless{}0.0001) (Figure
1B). Additionally, we tested whether treatments with ENV59-GP3 peptide
or NS6180 blocker would have an impact on expression of proinflammatory
cytokines in colon tissues, presumably as an effect of upstream blockage
of the K\textsubscript{Ca}3.1 channel. Consistent with less severe
disease, TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-6 (Figure 1C) show decreased expression in
ENV59-GP3 and NS6180 treated animals, though only difference in IL-6
expression was statistically significant. Unfortunately, such a high
degree of variability in RT-qPCR analyses of inflammatory mediators are
often observed in similar in vivo models. Furthermore, the expression of
IL-17 mRNA was not markedly reduced by either tested treatments. At the
time of sacrifice, ENV59-GP3 and/or NS6180 treated mice displayed
significantly reduced colonic shortening and decreased intestinal
inflammation compared with control group (Figure 2).

\textbf{ENV59-GP3 Immune Suppression via Inhibition of the Intermediate
Conductance Calcium Activated Potassium Channel K\textsubscript{Ca}3.1}

Despite the extensive literature documenting a broad range of immune
suppressive activity affecting different signal transduction pathways by
retroviral derived immune suppressive peptides, no precise molecular
mechanism of action (MoA) has been described. ENV59-GP3 is derived from
an endogenous retroviral envelope protein and thus is expressed on the
surface of immune cells. Therefore, it can be expected that it will
interact with a surface receptor. We noted that K\textsubscript{Ca}3.1
is a surface protein and that~inhibition of~K\textsubscript{Ca}3.1
protected mice from developing severe~colitis~in two mouse models
of~inflammatory bowel disease {[}11{]}, furthermore
K\textsubscript{Ca}3.1 inhibitor ameliorate the disease progression in
EAE mice {[}19{]}. The overlap between the activity of
K\textsubscript{Ca}3.1 and our observations of the ENV59 activity made
it a good candidate for further investigation.

\textbf{Cytokine release and inhibiting effect of ENV59-GP3 and of
NS6180 in stimulated THP-1 cells}

The ENV59-GP3 peptide inhibits the production of pro-inflammatory
cytokines in activated THP-1 cells {[}9{]}. To elucidate whether
K\textsubscript{Ca}3.1 plays any role in this phenotypic activity of
ENV59-GP3 peptide, expression and activity of the K\textsubscript{Ca}3.1
channels in THP-1 cells were confirmed by qPCR (Figure 3B). Patch clamp
experiments confirmed existence of currents sensitive to specific
K\textsubscript{Ca}3.1 channel blocker (NS6180) and channel opener
(NS309, Figure 3A). Next we studied if the inhibitory effect of
ENV59-GP3 in cytokine production mimics that of NS6180, a potent and
highly selective small molecule inhibitor of K\textsubscript{Ca}3.1
channels {[}18{]}. Our results (Figure 3C) corroborate the notion that
K\textsubscript{Ca}3.1 is an important regulator of synthesis and
release of proinflammatory cytokines in THP-1 cells and that the
ENV59-GP3 peptide and the channel blocker NS6180 have similar effects on
cytokine production in these cells.

\textbf{Effect of ENV59-GP3 peptide on calcium influx following
Ca\textsuperscript{2+} depletion in THP-1 cells}

To measure the effects of ENV59-GP3 peptide and NS6180 blocker on
calcium flux, THP-1 cells were loaded with the calcium sensitive dye
Fluo-4 and incubated with either ENV59-GP3 peptide or NS6180 blocker,
and/or with the calcium store depleting agent Thapsigargin, an inhibitor
of the sarco/endoplasmic reticulum Ca\textsuperscript{2+} ATPase (SERCA)
{[}25{]} (Figure 4A). Similarly, both ENV59-GP3 peptide and NS6180
reduced the influx-mediated Ca\textsuperscript{2+} accumulation induced
by Thapsigargin. In comparison, an immune suppressive peptide isolated
from the influenza hemagglutinin (Flu ISU) which has a similar
immunosuppressive phenotype to ENV59-GP3 {[}21{]}, but interacts with
and inhibits dimerization of the stimulator of interferon genes (STING)
{[}21{]} did not have a significant effect in the
{[}Ca\textsuperscript{2+}{]}\textsubscript{I} in this assay. Thus, the
ENV59-GP3 peptide's biological activity is more similar to the
K\textsubscript{Ca}3.1 inhibitor than it is to an influenza
virus-derived immunosuppressive peptide.

\textbf{Selective inhibition of K\textsubscript{Ca}3.1 channels in THP-1
cells by ENV59-GP3}

We next examined the direct effect of the ENV59-GP3 peptide on the
essentially pure K\textsubscript{Ca}3.1 current (Figure 4B-C) at -40mV
in THP-1 cells (Figure 4B). ENV59-GP3 peptide was applied at
concentrations relevant for its immunosuppressive effects (Figure 3C)
and, as clearly shown, it reversibly inhibited the -40mV current
component, which was formally identified as being K\textsubscript{Ca}3.1
by the subsequent application of NS6180. Furthermore, the ENV59-GP3
peptide-like NS6180-did not exert a measurable effect on the voltage
dependent current component at positive potentials. Hence the effect
seems selective for the K\textsubscript{Ca}3.1 channel over
K\textsubscript{v} channels in THP-1 cells.

\textbf{ENV59-GP3 inhibition of recombinant human K\textsubscript{Ca}3.1
channels stably expressed in HEK293 cells}

Figure 4D-E summarizes the results for ENV59-GP3 with the curve
representing fits to the Hill equation, revealing
IC\textsubscript{50}value of 43\selectlanguage{greek}µ\selectlanguage{english}M for ENV59-GP3. To confirm that the
inhibiting effect of ENV59-GP3 peptide represent an interaction with the
K\textsubscript{Ca}3.1 channel and not an indirect effect, we evaluated
the peptide's effect on the recombinant hK\textsubscript{Ca}3.1 stably
over-expressed in HEK293 cells (Figure 5 A-D), a cell type which does
not naturally express any calcium activated potassium channels. As
observed in the previous experiment with THP-1 cells, after the initial
transient rise in activity, the K\textsubscript{Ca}3.1 current
stabilized. Initially we blocked the current by using Charybdotoxin-a
very potent K\textsubscript{Ca}3.1 blocker {[}26{]}. The ENV59-GP3
peptide exert a qualitatively similar reversible inhibition, whereas the
control peptide Flu ISU did not show any effect, in accordance with its
lack of effect in the {[}Ca\textsuperscript{2+}{]} assay (Figure 5 C-D).
Finally, the NS309 activator was applied yielding the expected specific
increase in K\textsubscript{Ca}3.1 current as also observed for the
THP-1 channel.

\textbf{Discussion}

The ENV59-GP3 peptide contains the ISD sequence from a specific human
endogenous retroviral protein encoded by the envelope \emph{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 K\textsubscript{Ca}3.1 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 \emph{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 K\textsubscript{Ca}3.1 ( 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 K\textsubscript{Ca}3.1 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
K\textsubscript{Ca}3.1 current.

K\textsubscript{Ca}3.1 plays an important function in regulating
Ca\textsuperscript{2+} 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 K\textsubscript{Ca}3.1 expression, in parallel with
increased K\textsubscript{Ca}3.1 currents and enhanced
{[}Ca\textsuperscript{2+}{]} signaling during human T-cell activation
{[}12, 27{]}. K\textsubscript{Ca}3.1 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 K\textsubscript{Ca}3.1
blocker NS6180 on the \emph{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 K\textsubscript{Ca}3.1 channel when
proliferation is driven by the Ca\textsuperscript{2+}-dependent NFAT and
NF-\emph{k} B pathways. The identical cytokine profiles obtained with
ENV59-GP3 and K\textsubscript{Ca}3.1 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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β; \selectlanguage{english}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, K\textsubscript{Ca}3.1 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 Ca\textsuperscript{2+}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
K\textsubscript{Ca}3.1.

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
K\textsubscript{Ca}3.1 in this human monocyte cell line.

K\textsubscript{Ca}3.1 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 \emph{KCNN4} has been
described that is associated with ileal disease in the New Zealand and
Australian population {[}32{]}.

In addition to IBD, K\textsubscript{Ca}3.1 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 K\textsubscript{Ca}3.1 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 K\textsubscript{Ca}3.1, 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
K\textsubscript{Ca}3.1 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 K\textsubscript{Ca}3.1 constituting an attractive drug
target.

\textbf{Figure legends}

\textbf{Figure 1 Effect of the ENV59-GP3 peptide and
K\textsubscript{Ca}3.1-blocker NS6180 in DSS-induced acute colitis mice
model} .

Either saline (control) or ENV59-GP3 peptide (0.5mg/mg) or NS6180
blocker (2.5mg/kg) were administrated intraperitoneally every other day
of DSS treatment. Weight development was monitored from day 0 to day 10.
(A) daily weight development is shown for each of the treatment groups
(pool of 2 independent experiments, 9-10 mice in each group). (B) The
comparison of colon length. Data are expressed as means \selectlanguage{ngerman}± SEM (n = 10
per group). * p \textless{} 0.05, * * * p \textless{} 0.001, and * * * *
p \textless{} 0.0001, one-way ANOVA with post hoc Tukey's test. (C)
Evaluation of inflammatory cytokines. Total distal colon tissue was
isolated from mice 10 days after the induction of DSS colitis and
relevant treatments and equivalent biopsy sections were taken for
inflammatory mRNA analysis (RT-qPCR). All gene values were normalized to
the expression of Hprt1. Data are presented as mean ± SEM (n = 10 per
group).

\textbf{Figure 2 Histomorphology of colon tissue and inflammation
scores} according to the scale described in the Materials and Methods.
(A) Inflammation score for each treatment group. The histological H\&E
stains were scored by 2 individuals blinded to treatment and
experiments. Right panel, Representative H\&E staining from mouse colon.
Bar~=~100~\selectlanguage{greek}μ\selectlanguage{english}m. Differences were calculated using a one-way analysis of
variance (ANOVA) followed by post hoc Tukey's test test. Values are
presented as mean~\selectlanguage{ngerman}±~SD.~\textsuperscript{**}\emph{P}
~\textless{}~0.01,~\textsuperscript{***}\emph{P} ~\textless{}~0.001.

\textbf{Figure 3} A) \textbf{Functional expression of
K\textsubscript{Ca}3.1 and Kv channels in THP-1 cells.} Application of
NS309 or 1 \selectlanguage{greek}μ\selectlanguage{english}M NS6180 occurred by switching to the appropriate tube
feeding the inflow-line. From left to the right: The IV curve recorded
after stabilization of the current after the initial run up (Control)
and \textasciitilde{}1 min after application of 500 nM NS309.: After
NS309 wash-out and establishment of new stable baseline (NS309 wash-out)
1 \selectlanguage{greek}μ\selectlanguage{english}M NS6180 was applied (NS6180) which completely blocked the KCa3.1
component. Note the distinct depolarizing shift in reversal potential. :
The difference curve obtained by subtracting the curves in the middle
panel revealing the NS6180 sensitive component. B) \textbf{Expression
of\emph{KCNN4} mRNA in THP-1 cells} . Melting curve plot showing Q-PCR
product specific for \emph{KCNN4} mRNA. C) \textbf{Cytokine release and
inhibiting effect of Env59-GP3 and of NS6180 in stimulated THP-1
cells.}mRNA and protein levels for IL-6 and TNF-alpha.

\textbf{Figure 4} A) \textbf{Calcium influx following Ca 2+ depletion in
THP-1 cells.} Calcium flux to the cytosol following cell activation is
reduced in the presence of Env59-GP3 or NS6180. B-C) \textbf{Selective
inhibition of THP-1 K\textsubscript{Ca}3.1 channels by Env59-GP3} .
Whole cell current recording from THP-1 cell. B shows the IV curves
after current stabilization for about 15 min (a) and after perfusion
with Env59-GP3 peptide at a concentration of 20\selectlanguage{greek}µ\selectlanguage{english}M for 5 min (b) (left
figure). The IV curve after Env59-GP3 peptide wash-out (c ) (right
figure) and after perfusion with 1\selectlanguage{greek}µ\selectlanguage{english}M NS6180 for 5 min (d). The arrows
indicate -40mV, the membrane potential used for putative ``pure''
K\textsubscript{Ca}3.1 current recording. The -40mM current recordings
were used for construction of the experimental time course relation
shown in C, where the letters mark the times for the trace recordings.
D-E) \textbf{IC\textsubscript{50} value for ENV59-GP3 in recombinant
human K\textsubscript{Ca}3.1 channels stably expressed in HEK293
cells.}The fraction of unblocked current at the end of compound
application as a function of the test concentration. The lines are the
curves resulting from fits of the data to the Hill equation described by
IC50 values of 43\selectlanguage{greek}µ\selectlanguage{english}M for Env59-GP3 peptide.

\textbf{Figure 5} A-D. \textbf{ENV59-GP3 inhibition of recombinant human
K\textsubscript{Ca}3.1 channels stably expressed in HEK293 cells} .
A,B,C show the K\textsubscript{Ca}3.1 currents in the absence (a,c.e)
and presence (b,d,f) of compounds, with the letters also showing the
times of recording at the time course in D. After establishment of the
whole cell configuration K\textsubscript{Ca}3.1 currents increased as
expected due to equilibration with the buffered Ca2+ solution in the
pipette (compare also to Fig. 4) and then ran down to a stable current
(D). The reversible K\textsubscript{Ca}3.1 blocking scorpion toxin
peptide Charybdotoxin (10 nM) (A,D) and the K\textsubscript{Ca}3.1
positive modulator NS309 (500 nM) (D) framed two additions of ENV59-GP3
(50 \selectlanguage{greek}μ\selectlanguage{english}M) (B,D) and the control peptide Flu ISU F\#2 (50 \selectlanguage{greek}μ\selectlanguage{english}M) (C,D),
respectively.

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