BACKGROUND
several inflammatory diseases are characterized by an unbalanced inflammation and an increased expression of inflammatory cytokines such as tnf-α, il- <dig>  il-17a, il-17c, il- <dig>  il- <dig>  il- <dig>  ifnγ and cxcl <dig>  especially tnf-α, il-17a and il-12/il- <dig> are believed to play a key role in the pathogenesis of psoriasis, which is confirmed by the successful use of antibodies directed against these cytokines in the treatment of psoriasis
<cit> .
a subset of these cytokines such as tnf-α, il-17c, il- <dig> and cxcl2) have also been implicated in sepsis
<cit> .
it has been known for many years that retroviruses are capable of inhibiting proliferation of immune cells upon stimulation
<cit> .
these effects have been localized to the viral fusion protein.
expression of such fusion proteins is sufficient to enable allogenic cells to grow into a tumor in immune competent mice
<cit> .
immunosuppressive  fusion proteins have been found in a variety of different viruses with type i fusion mechanism such as retroviruses like mpmv and hiv and in filoviruses such as ebola and marburg
<cit> .
this isu-activity was in all these cases located to a very well-defined structure in the transmembrane protein  the isu effects range from significant inhibition of lymphocyte proliferation
<cit>  cytokine skewing
<cit> , inhibition of monocytic burst
<cit> , polyclonal b cell activation and cytotoxic t cell activity
<cit> .
peptides spanning the isu domain are as active as the whole protein in these assays but only when presented in a dimeric form or coupled to a carrier protein .
such multimeric peptides are capable of reducing or abolishing immune responses like cytokine secretion or proliferation of activated t-cells.
the isu-domain has been investigated extensively using a 17-meric peptide  derived from murine leukemia virus  which shows significant immunosuppressive activity in vitro as described above
<cit> .
the ability of cks- <dig> to repress t-cell mediated immune responses makes this peptide an interesting drug candidate for the treatment of inflammatory diseases.
the aim of this study was to verify the in vivo effects of a retrovirally derived cks- <dig> peptide on the immune system.
we used two known mouse models for skin inflammation: acute allergic contact dermatitis model and tpa toxic eczema model
<cit> .
in both models, the application of the peptide reduced the inflammation induced by the irritants, thereby confirming the viability of utilization of this virus-derived peptide for treatment of inflammatory diseases.
RESULTS
in order to retain the isu-peptide on the ears of the mice for longer periods the dissolved peptide was mixed with natusan®, first touch protection cream at a 1: <dig> ratio to create a homogenous solution.
all peptides were freshly dissolved at the start of treatments and stored for 4°c during the animal experiments.
acute toxic contact eczema model
two variants of the tpa-induced acute toxic contact eczema model were used to evaluate the anti-inflammatory potential of topical applied cks- <dig> peptide.
initially, mice were pretreated for 3 days with peptide-containing cream followed by induction of ear inflammation by tpa.
figure
2a depicts the time-course.
evidently, mice treated with cks- <dig> peptide have significantly reduced swelling of the ears upon inflammation induction.
the direct comparison with a scrambled peptide in figure
2b  points to a specific effect of cks- <dig> dimer, which is significantly more potent than the scrambled peptide control to reduce ear inflammation .
in the same model an experiment was performed in which inflammation was induced five days prior to treatment with peptide cream .
in this setup a monomer peptide vs a dimer was tested since the isu-activity of the peptides are consistently correlated with di- or multimeric forms of the peptides
<cit> .
this was confirmed in a direct comparison experiment, where cks- <dig> dimer-containing cream significantly reduced ear inflammation in contrast to the monomeric peptide.
figure
3b clearly shows a significant reduction of inflammation two days after cks- <dig> dimer treatment in contrast to the monomeric peptide .
monomeric peptide was not different from the pure cream vehicle.
furthermore, in the same experiment we performed a direct comparison to a potent anti-inflammatory topical glucocorticoid based cream .
clobetasol propionate treatment was very potent in this model and reduced inflammation close to the basal level with minimal inter-animal variation.
oxazolone-induced murine allergic contact dermatitis
this model is representative of allergic contact dermatitis induced inflammation by sensitizing the mice 7 days prior to challenge.
three days before induction of inflammation the mice were treated with peptide-containing cream.
evidently, as in the tpa-induced eczema model treatment with cks- <dig> containing cream lowered swelling of the ears  following inflammation induction by oxazolone .
however, the effect was less pronounced at day two post oxazolone challenge  as shown in the box-plot in figure
4b.
in general, this contact dermatitis mouse model induces much less inflammation of the ears in comparison to the tpa mouse model.
the results of a total of four independent experiments  each with  <dig> mice pr treatment group are compiled in figure
<dig>  the figure depicts the relative reduction in ear thickness of the treatment groups  compared with tpa or oxazolone alone.
across the experiments the reduction in the cks- <dig> treatment group ranged from 20-43% with no effect of scrambled/monomer.
clobetasol propionate reduced the swelling of the ears by 80% in the single study where it was applied.
the consistent reduction of treatment with cks-17-containing cream independent of the model and scheme of application was significantly different from treatment with a scrambled peptide .
the impact of cks- <dig> treatment on cytokines implicated in inflammatory diseases
rna from ear biopsies of the mice treated with tpa  was harvested and subjected to mrna relative quantification of il-1α, il- <dig>  il- <dig>  il-17c, tnf-α and cxcl <dig>  all expression levels were adjusted for input mrna by gapdh gene expression and the results normalized to levels for vehicle only treated mice.
there was a clear and significant reduction of mrna levels of il-17c, tnf-α, il- <dig> and cxcl <dig> in mice treated with cks- <dig> containing cream .
when correlating the relative mrna expression of these cytokines/chemokines to the ear thickness of individual animals, a statistically significant correlation was observed .
in turn, expression levels of neither il-1α nor il- <dig> were affected by treatment with cks-17-containing cream compared to cream with scrambled peptide.
notably, plotting the expression levels of il-1α and il- <dig> against the ear thickness revealed no linear correlations where observed .
the results are summarized in table
<dig>
interestingly, treating mouse ears with cream containing cks- <dig> peptide results in a potent blockage of tnf-α mrna expression   <dig> ) to levels below the background in untreated animals in the vehicle control group  1).
discussion
in this report we have tested the hypothesis that a retrovirus-derived immunosuppressive peptide may reduce inflammation.
we found that a topically delivered retroviral derived peptide  reduced ear inflammation in two different mouse models.
neither a dimeric version of a scrambled isu-peptide nor a monomer of the isu-peptide had any effect, similar to what was previously reported in cell-culture-based assays
<cit> .
the lowering of inflammation in the mouse models can be coupled to significantly reduced expression of key inflammatory cytokines like il- <dig>  tnf-α and il-17c.
these results complement those of cianciolo et al.
who recently reported that the cks- <dig> isu-peptide or shorter versions thereof had an immunosuppressive effect in mouse models of inflammatory peritonitis and disseminated intravascular coagulation
<cit> .
parallel work on hydrophobic regions of hiv- <dig> gp <dig>  both the fusion peptide and the isu domain, has suggested control of t cell activation
<cit> .
a different applicability of modifying the isu-peptide from hiv- <dig> was reported by morozov and colleagues who were able to enhance the vaccine induced igg titer towards recombinant gp41
<cit> .
previously, tpa-induced skin inflammation in mice has been shown to involve potent upregulation of tnf-α
<cit> .
here we report that tnf-α expression levels were the most severely affected cytokine level by peptide treatment.
despite a four-fold increase in mrna by tpa treatment the application of cks- <dig> containing cream reduced tnf-α expression to below baseline levels .
in addition, levels of another important regulator in autoimmune diseases, il-17c was significantly reduced upon treatment of inflamed ears with cream containing retroviral peptide.
recently, anti-il-17a receptor antibodies have proven successful in early clinical trials
<cit> .
interestingly, we find that the level of il-1α expression is not regulated by cks- <dig> peptide application.
considering the effect of il-1α in the contact acute eczema model this was somewhat surprising although in agreement with earlier studies of the intrinsic effects of retrovirus-derived envelope peptides highlighting that cks- <dig> does not block production of il-1α but reduce the immunobiological effect
<cit> .
the varied effect on several key cytokines suggests that the local application of peptide containing cream pursued in this study could be of greater relevance than systemic use of monoclonal antibodies mainly because of the plethora of targets and the fact that for example anti-tnf-α blockers carry increased infection risks
<cit> .
in the model of oxazolone induced inflammation a similar trend was observed in that ear inflammation was reduced in peptide treated mice.
the degree of inflammation induced in this model is of a smaller magnitude.
in turn, the relative reduction by peptide treatment was comparable to what was obtained in the tpa-induced model suggesting that the peptide did indeed impact this model as well, although the inhibitory effect failed to reach statistical significance.
in this model we have previously shown mitogen-activated protein kinase ap  kinase  <dig> regulated tnf-α as important for ear inflammation
<cit> .
CONCLUSIONS
we have shown that the immunosuppressive retroviral peptide cks- <dig> is capable of reducing proinflammatory cytokines as well as the level of inflammation as measured by ear thickness in two mouse models of inflammatory skin disease.
together with the work of cianciolo et al.
<cit>  our results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines such as tnf-α, il- <dig>  and il-17c may represent a novel class of drugs for the treatment of excess inflammation.

 several inflammatory diseases are characterized by an unbalanced inflammation and an increased expression of inflammatory cytokines such as tnf-α, il- il-17a, il-17c, il- il- il- ifnγ and cxcl especially tnf-α, il-17a and il-12/il- are believed to play a key role in the pathogenesis of psoriasis, which is confirmed by the successful use of antibodies directed against these cytokines in the treatment of psoriasis. a subset of these cytokines such as tnf-α, il-17c, il- and cxcl2) have also been implicated in sepsis. it has been known for many years that retroviruses are capable of inhibiting proliferation of immune cells upon stimulation. these effects have been localized to the viral fusion protein. expression of such fusion proteins is sufficient to enable allogenic cells to grow into a tumor in immune competent mice. immunosuppressive fusion proteins have been found in a variety of different viruses with type i fusion mechanism such as retroviruses like mpmv and hiv and in filoviruses such as ebola and marburg. this isu-activity was in all these cases located to a very well-defined structure in the transmembrane protein the isu effects range from significant inhibition of lymphocyte proliferation cytokine skewing, inhibition of monocytic burst, polyclonal b cell activation and cytotoxic t cell activity. peptides spanning the isu domain are as active as the whole protein in these assays but only when presented in a dimeric form or coupled to a carrier protein. such multimeric peptides are capable of reducing or abolishing immune responses like cytokine secretion or proliferation of activated t-cells. the isu-domain has been investigated extensively using a 17-meric peptide derived from murine leukemia virus which shows significant immunosuppressive activity in vitro as described above. the ability of cks- to repress t-cell mediated immune responses makes this peptide an interesting drug candidate for the treatment of inflammatory diseases. the aim of this study was to verify the in vivo effects of a retrovirally derived cks- peptide on the immune system. we used two known mouse models for skin inflammation: acute allergic contact dermatitis model and tpa toxic eczema model. in both models, the application of the peptide reduced the inflammation induced by the irritants, thereby confirming the viability of utilization of this virus-derived peptide for treatment of inflammatory diseases.  in order to retain the isu-peptide on the ears of the mice for longer periods the dissolved peptide was mixed with natusan®, first touch protection cream at a 1: ratio to create a homogenous solution. all peptides were freshly dissolved at the start of treatments and stored for 4°c during the animal experiments. acute toxic contact eczema model two variants of the tpa-induced acute toxic contact eczema model were used to evaluate the anti-inflammatory potential of topical applied cks- peptide. initially, mice were pretreated for 3 days with peptide-containing cream followed by induction of ear inflammation by tpa. figure  2a depicts the time-course. evidently, mice treated with cks- peptide have significantly reduced swelling of the ears upon inflammation induction. the direct comparison with a scrambled peptide in figure  2b points to a specific effect of cks- dimer, which is significantly more potent than the scrambled peptide control to reduce ear inflammation. in the same model an experiment was performed in which inflammation was induced five days prior to treatment with peptide cream. in this setup a monomer peptide vs a dimer was tested since the isu-activity of the peptides are consistently correlated with di- or multimeric forms of the peptides. this was confirmed in a direct comparison experiment, where cks- dimer-containing cream significantly reduced ear inflammation in contrast to the monomeric peptide. figure  3b clearly shows a significant reduction of inflammation two days after cks- dimer treatment in contrast to the monomeric peptide. monomeric peptide was not different from the pure cream vehicle. furthermore, in the same experiment we performed a direct comparison to a potent anti-inflammatory topical glucocorticoid based cream. clobetasol propionate treatment was very potent in this model and reduced inflammation close to the basal level with minimal inter-animal variation. oxazolone-induced murine allergic contact dermatitis this model is representative of allergic contact dermatitis induced inflammation by sensitizing the mice 7 days prior to challenge. three days before induction of inflammation the mice were treated with peptide-containing cream. evidently, as in the tpa-induced eczema model treatment with cks- containing cream lowered swelling of the ears following inflammation induction by oxazolone. however, the effect was less pronounced at day two post oxazolone challenge as shown in the box-plot in figure  4b. in general, this contact dermatitis mouse model induces much less inflammation of the ears in comparison to the tpa mouse model. the results of a total of four independent experiments each with mice pr treatment group are compiled in figure  the figure depicts the relative reduction in ear thickness of the treatment groups compared with tpa or oxazolone alone. across the experiments the reduction in the cks- treatment group ranged from 20-43% with no effect of scrambled/monomer. clobetasol propionate reduced the swelling of the ears by 80% in the single study where it was applied. the consistent reduction of treatment with cks-17-containing cream independent of the model and scheme of application was significantly different from treatment with a scrambled peptide. the impact of cks- treatment on cytokines implicated in inflammatory diseases rna from ear biopsies of the mice treated with tpa was harvested and subjected to mrna relative quantification of il-1α, il- il- il-17c, tnf-α and cxcl all expression levels were adjusted for input mrna by gapdh gene expression and the results normalized to levels for vehicle only treated mice. there was a clear and significant reduction of mrna levels of il-17c, tnf-α, il- and cxcl in mice treated with cks- containing cream. when correlating the relative mrna expression of these cytokines/chemokines to the ear thickness of individual animals, a statistically significant correlation was observed. in turn, expression levels of neither il-1α nor il- were affected by treatment with cks-17-containing cream compared to cream with scrambled peptide. notably, plotting the expression levels of il-1α and il- against the ear thickness revealed no linear correlations where observed. the results are summarized in table  interestingly, treating mouse ears with cream containing cks- peptide results in a potent blockage of tnf-α mrna expression ) to levels below the background in untreated animals in the vehicle control group 1). discussion in this report we have tested the hypothesis that a retrovirus-derived immunosuppressive peptide may reduce inflammation. we found that a topically delivered retroviral derived peptide reduced ear inflammation in two different mouse models. neither a dimeric version of a scrambled isu-peptide nor a monomer of the isu-peptide had any effect, similar to what was previously reported in cell-culture-based assays. the lowering of inflammation in the mouse models can be coupled to significantly reduced expression of key inflammatory cytokines like il- tnf-α and il-17c. these results complement those of cianciolo et al. who recently reported that the cks- isu-peptide or shorter versions thereof had an immunosuppressive effect in mouse models of inflammatory peritonitis and disseminated intravascular coagulation. parallel work on hydrophobic regions of hiv- gp both the fusion peptide and the isu domain, has suggested control of t cell activation. a different applicability of modifying the isu-peptide from hiv- was reported by morozov and colleagues who were able to enhance the vaccine induced igg titer towards recombinant gp41. previously, tpa-induced skin inflammation in mice has been shown to involve potent upregulation of tnf-α. here we report that tnf-α expression levels were the most severely affected cytokine level by peptide treatment. despite a four-fold increase in mrna by tpa treatment the application of cks- containing cream reduced tnf-α expression to below baseline levels. in addition, levels of another important regulator in autoimmune diseases, il-17c was significantly reduced upon treatment of inflamed ears with cream containing retroviral peptide. recently, anti-il-17a receptor antibodies have proven successful in early clinical trials. interestingly, we find that the level of il-1α expression is not regulated by cks- peptide application. considering the effect of il-1α in the contact acute eczema model this was somewhat surprising although in agreement with earlier studies of the intrinsic effects of retrovirus-derived envelope peptides highlighting that cks- does not block production of il-1α but reduce the immunobiological effect. the varied effect on several key cytokines suggests that the local application of peptide containing cream pursued in this study could be of greater relevance than systemic use of monoclonal antibodies mainly because of the plethora of targets and the fact that for example anti-tnf-α blockers carry increased infection risks. in the model of oxazolone induced inflammation a similar trend was observed in that ear inflammation was reduced in peptide treated mice. the degree of inflammation induced in this model is of a smaller magnitude. in turn, the relative reduction by peptide treatment was comparable to what was obtained in the tpa-induced model suggesting that the peptide did indeed impact this model as well, although the inhibitory effect failed to reach statistical significance. in this model we have previously shown mitogen-activated protein kinase ap kinase regulated tnf-α as important for ear inflammation.  we have shown that the immunosuppressive retroviral peptide cks- is capable of reducing proinflammatory cytokines as well as the level of inflammation as measured by ear thickness in two mouse models of inflammatory skin disease. together with the work of cianciolo et al. our results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines such as tnf-α, il- and il-17c may represent a novel class of drugs for the treatment of excess inflammation. 
furthermore, ear biopsies from mice treated with the dimeric immunosuppressive peptide showed a significant reduction in mrna of the pro-inflammatory cytokines tnf-α, il-17c, and il- as well as the chemokine cxcl compared to mice treated with control peptides. the anti-inflammatory effect of a synthetic retrovirus-derived immunosuppressive peptide of amino acids was tested in two murine skin inflammation models, a tpa-induced acute toxic contact eczema model and an oxazolone-induced allergic contact dermatitis. the results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines may represent a novel class of drugs for the treatment of excess inflammation. here we test the hypothesis that such immunosuppressive peptides may serve as immunomodulatory reagents for treatment of inflammatory disorders. overall, mice treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of % in ear thickness, whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect.
 short dimeric or mulitmeric peptides derived from a highly conserved stretch of amino acids from gammaretroviral envelope proteins has been found to have immunosuppressive properties in vitro. here we test the hypothesis that such immunosuppressive peptides may serve as immunomodulatory reagents for treatment of inflammatory disorders.  the anti-inflammatory effect of a synthetic retrovirus-derived immunosuppressive peptide of amino acids was tested in two murine skin inflammation models, a tpa-induced acute toxic contact eczema model and an oxazolone-induced allergic contact dermatitis. overall, mice treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of % in ear thickness, whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect. furthermore, ear biopsies from mice treated with the dimeric immunosuppressive peptide showed a significant reduction in mrna of the pro-inflammatory cytokines tnf-α, il-17c, and il- as well as the chemokine cxcl compared to mice treated with control peptides.  using two murine skin inflammation models, we show that an immunosuppressive retroviral peptide is capable of reducing inflammatory disorders. the results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines may represent a novel class of drugs for the treatment of excess inflammation. 