Anti-inflammatory effect of a retrovirus-derived immunosuppressive peptide in mouse models
- Martin Tolstrup†1Email author,
- Claus Johansen†2,
- Lars Toft†1,
- Finn S Pedersen3,
- Anne Funding2,
- Shervin Bahrami4,
- Lars Iversen2,
- Lars Østergaard1 and
- Mogens Duch3
© Tolstrup et al.; licensee BioMed Central Ltd. 2013
Received: 9 August 2013
Accepted: 12 November 2013
Published: 18 November 2013
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 17 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 (n = 24) treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of 28.8% in ear thickness (range 20.1-42.5), whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect (p = 0.028). 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-6 as well as the chemokine CXCL2 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.
Several inflammatory diseases are characterized by an unbalanced inflammation and an increased expression of inflammatory cytokines such as TNF-α, IL-6, IL-17A, IL-17C, IL-20, IL-22, IL-23, IFNγ and CXCL2. Especially TNF-α, IL-17A and IL-12/IL-23 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[1–3]. A subset of these cytokines such as TNF-α, IL-17C, IL-6 and CXCL2) have also been implicated in sepsis[4–8].
The ISU-domain has been investigated extensively using a 17-meric peptide (CKS-17) derived from murine leukemia virus (MLV) which shows significant immunosuppressive activity in vitro as described above[4, 5, 21–23].
The ability of CKS-17 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-17 peptide on the immune system. We used two known mouse models for skin inflammation: Acute allergic contact dermatitis model and TPA toxic eczema model[24, 25].
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 1:1 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
Oxazolone-induced murine allergic contact dermatitis
The impact of CKS-17 treatment on cytokines implicated in inflammatory diseases
Cytokine mRNA expression index from ear biopsies
TPA w. scrambled
TPA w. CKS-17
Interestingly, treating mouse ears with cream containing CKS-17 peptide results in a potent blockage of TNF-α mRNA expression (mean index (range) 0.3(0.17-0.52)) to levels below the background in untreated animals in the vehicle control group (mean index (range) 1(0.49-1.51)).
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 (CKS-17) 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[9, 10, 14, 15, 18–20, 26]. The lowering of inflammation in the mouse models can be coupled to significantly reduced expression of key inflammatory cytokines like IL-6, TNF-α and IL-17C.
These results complement those of Cianciolo et al. who recently reported that the CKS-17 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-1 gp41, both the fusion peptide and the ISU domain, has suggested control of T cell activation[17, 28]. A different applicability of modifying the ISU-peptide from HIV-1 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[30, 31]. 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-17 containing cream reduced TNF-α expression to below baseline levels (average of 70% reduction compared to baseline). 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[2, 30].
Interestingly, we find that the level of IL-1α expression is not regulated by CKS-17 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-17 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 (MAPKAP) kinase 2 regulated TNF-α as important for ear inflammation.
We have shown that the immunosuppressive retroviral peptide CKS-17 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-6, and IL-17C may represent a novel class of drugs for the treatment of excess inflammation.
CKS-17 peptide (LQNRRGLDLLFLKEGGLC) or scrambled CKS-17 (LGGEKLFLLDLGRRNQLC) both linked into dimers via the C-terminal cysteine were purchased as lyophilized powder from Anaspec (California, USA). CKS-17 monomer was purchased without the C-terminal disulphide linkage. All peptides were dissolved in sterile water.
BALB/cJ and C57BL/6 mice were purchased from Taconic Europe (Ry, Denmark). All mice used in this study were female and 6–8 weeks of age when experiments were initiated. The animals were kept in animal facilities that maintained a temperature of 19–25°C, and a 12-hour day/night cycle. They were given access to food and water ad libitum. All experiments were approved by the Committee for Animal Experiments in Denmark.
Acute toxic contact eczema model
Two different dosings were pursued in this model. Firstly, C57BL/6 mice were treated once daily from day −3 until day 3 on the ears with cream (commercially available baby lotion; Natusan®, First Touch Protection cream) + 50% H2O) alone, cream + scrambled peptide or cream + CKS-17 peptide. Before the first application of cream at day −3 the ear thickness was measured. At day 0 and day 2 (3 hours before treatment with cream) the mice were treated with phorbol 12-myristate 13-acetate (TPA)(Sigma-Aldrich, St Louis, MO, cat no. P8139) on the ears (2 μg/ear). TPA was dissolved in acetone. Before the first challenge of TPA the ear thickness was measured (day 0) and again post-challenge at the times indicated, using a Mitutyo digimatic indicator. In the second experiment, BALB/cJ were treated at day 0, 2, 5 and 7 with phorbol 12-myristate 13-acetate (TPA)(Sigma-Aldrich, St Louis, MO, cat no. P8139) on the ears (2 μg/ear). TPA was dissolved in acetone. Before the first challenge of TPA the ear thickness was measured (day 0) and again post-challenge at the times indicated, using a Mitutyo digimatic indicator. At day 5 and 7 the mice were treated one time with either cream alone, cream + CKS-17 monomér, cream + CKS-dimér or Clobetasolpropionat 0.05% cream formulation applied neat (GSK, UK). The mice were anaesthetised during the ear measurement.
Oxazolone-induced murine allergic contact dermatitis
4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) was purchased from Sigma-Aldrich (cat no. E0753). Oxazolone was dissolved in acetone. At day −7 mice were sensitized with 1.5% oxazolone by application to the clipped abdomen (100 μl). Once daily from day −3 until day 1 the ears of the mice were treated with cream (commercial available baby lotion; Natusan®, First Touch Protection cream + 50% H2O) alone, cream + scrambled peptide or cream + CKS-17 peptide. Before the first application of cream at day −3 the ear thickness was measured. At day 0 the mice were challenged with 0.5% oxazolone at the ears (20 μl per ear). This was done 3 hours prior to treatment with the different cream formulations. Before challenge (day 0), the ear thickness was measured and again post-challenge at the times indicated, using a Mitutyo digimatic indicator.
At termination of one study of acute toxic contact eczema punch biopsies (4 mm) from mice ears were transferred to 1 ml of −80°C cold RNAlater-ICE (Ambion inc., Austin, TX). Samples were kept at −80°C until 24 hours before RNA purification at which time they were transferred to −20°C. On RNA purification, biopsies were removed from RNAlater-ICE and transferred to 175 μl of SV RNA lysis buffer added β-mercaptoethanol (SV Total RNA Isolation System; Promega, Madison, WI) and homogenized. RNA purification was completed according to the manufacturer’s instructions (SV Total RNA Isolation System; Promega). RNA was stored until further use at −80°C.
Quantitative polymerase chain reaction (qPCR)
For reverse transcription, Taqman Reverse Transcription reagents (Applied Biosystems, Foster City, CA, U.S.A.) were used. For qPCR we used Platinum® qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA, U.S.A.) with primers and probes were Taqman 20× Assays-On-Demand (FAM-labeled MGB-probes) gene expression assay mix (Applied Biosystems). Each sample was loaded as triplets and analyzed on a Rotorgene-3000 real-time PCR machine (Corbett Research, Cambridge, U.K.). Relative gene expression levels were determined by using the relative standard curve method as outlined in User Bulletin 2 (ABI PRISM 7700 sequencing detection system, Applied Biosystems). Briefly, a standard curve for each gene was made of fourfold serial dilutions of total RNA from a punch biopsy from the ears of mice. The curve was then used to calculate relative amounts of target mRNA in the samples. As housekeeping GAPDH was used. Assay ID for the primers and probes used in this study were as follows: IL-17c (Mm00439619_m1); TNF-α (Mm00443258_m1); IL-1α (Mm00439620_m1); IL-6 (Mm00446190_m1); IL-10 (Mm00439616_m1); CXCL2 (Mm00436450_m1); GAPDH (Mm99999915_g1).
Comparisons of ear thickness and relative mRNA levels between treatment groups were performed using a student’s t-test. Correlations between ear thickness and mRNA levels were determined using spearman correlations. Lastly, the percentage effect on reduction in ear thickness between scrambled and CKS-17 treated groups across four independent mouse experiments were compared using a Mann–Whitney test. All p-values reported are two-tailed and we set the significance level at 5%.
This study was supported by a grant from the Aarhus University Hospital Research Fund and a proof-of-concept grant from Aarhus University.
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