SummaryAlpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide exhibiting anti-inflammatory activity in experimental models of autoimmune diseases. However, no studies thus far have examined the effects of α-MSH on systemic lupus erythematosus (SLE). This study aimed to determine the effects of an α-MSH agonist in induced murine lupus. Here we employed female Balb/cAn mice in which lupus was induced by pristane. Groups of lupus animals were treated daily with the α-MSH analogue [Nle4, DPhe7]-α-MSH (NDP-MSH) (1·25 mg/kg) injected intraperitoneally or saline for 180 days. Normal animals comprised the control group. Arthritis incidence, plasma immunoglobulin (Ig)G isotypes, anti-nuclear antibodies (ANA) and plasma cytokines were evaluated. Renal function was assessed by proteinuria and histopathological lesion. Glomerular levels of IgG, α-smooth muscle actin (α-SMA), inducible nitric oxide synthase (iNOS), C3, CD3, melanocortin receptors (MCR)1, corticotrophin-releasing factor (CRF) and α-MSH was estimated by immunohistochemistry. When compared with normal controls, lupus animals exhibited increased arthritis, IgG levels, ANA, interleukin (IL)-6, IL-10, proteinuria and mesangial cell proliferation together with glomerular expression of α-SMA and iNOS. Glomerular expression of MCR1 was reduced in lupus animals. NDP-MSH treatment reduced arthritis scores by 70% and also diminished IgG1 and IgG2a levels and ANA incidence. In the glomerulus, NDP-MSH treatment reduced cellularity by 50% together with reducing IgG deposits, and expression levels of α-SMA, iNOS and CRF were also all decreased. Taken together, our results suggest for the first time that α-MSH treatment improves several parameters of SLE disease activity in mice, and indicate that this hormone is an interesting potential future treatment option.

Background: Adaptive immune cells, including CD4 + CD69 + and CD4 + CD25 + FoxP3 + regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4 + CD25 + FoxP3 + Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4 + CD69 + and CD4 + CD25 + FoxP3 + T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4 + CD69 + T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4 + CD69 + T cells and reduced numbers of CD4 + CD25 + FoxP3 + Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

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Backgroundα-MSH is a neuropeptide with several biological functions including down regulation of pro-inflammatory pathways and inhibition of the development of some experimental rheumatic diseases. Although pristane-induced SLE murine has clinical and serological similarities to human disease, cellular characteristics that contributes to autoimmunity and the effect of α-MSH in this model remains unknown.ObjectivesTo analyze, in pristane-induced SLE balb/c mice, the expressions of CD69 activation receptor on CD4 Tcells and of CD4+CD25+FoxP3+ peripheral Treg cells; and to evaluate the potential therapeutic effects of an α-MSH analogue (NDP-α-MSH) this model.MethodsTwenty balb/c mice were studied: 15 were injected with a single intraperitoneal (IP) dose of 0.5ml pristane for SLE-like model induction, whereas 5 age/gender matched control mice were given saline. Among the 15 pristane-induced mice (PIM), 5 received daily IP saline and 10 were treated with 1,25mg/kg/d NDP-α-MSH for 90 days. Samples of 200ul heparinized peripheral blood were collected from submandibular area from all 20 animals initially (time 0, T0) and following 90 days (T90). PBMC were isolated by erythrocyte lysis and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD69 and FoxP3 molecules (BD Pharmingen™). Cell surface expressions were analyzed by flow cytometry using Guava EasyCyte™ HT (Millipore) and all results are shown as mean ± SD. Kruskal-Wallis test was used for statistical analysis, and P<0.05 considered significant.ResultsUntreated PIM compared to control mice had significantly increased expression of CD69 on CD4 Tcells (22.1±2.1% vs 5.3±1.9%, P<0.005) even though the percentage of Treg cells was significantly lower (2.3±0.7% vs 6.9±1.4%, P<0.005). The comparison of NDP-α-MSH treated PIM and untreated PIM revealed similar expression of CD69 on CD4 Tcells (16.5±8.4% vs 22.1±2.1%, P>0.005), whereas treated PIM had significantly higher percentage of peripheral Treg cells (10.2±3.9% vs 2.3±0.7%, P<0.005).ConclusionsPristane-induced SLE model develops similar expression patterns of activated CD4+CD69+ Tcells and CD4+CD25+FoxP3+ Treg cells to human lupus, which were in part reverted by α-MSH treatment. These murine findings generate new knowledge concerning immunological abnormalities of human SLE contributing to the understanding of this disease, and may indicate a potential therapeutical role for α-MSH.ReferencesCatania, A. et al. The melanocortin system in control of inflammation, 2010.Abdel-Malek, Z.A. Melanocortin receptors: their functions and regulation by physiological agonists and antagonists,2001.P mATEL, H.B. et al. Melanocortin control of cell trafficking in vascular inflammation, 2010.Capsoni, F. et al. Melanocortin peptides inhibit urate Crystal-induced activation of phagocytic cells, 2009.Zhou, L.L. et al. Regulatory effect of melatonina on cytokine disturbances in the pristane-induced lupus mice, 2010.Satoh, M. et al. Induction of lupus-associated autoantibodies in Balb/c mice by intraperitoneal injection of prista...

BackgroundProliferation of auto reactive T cells and loss of immunological tolerance are features of SLE. In human lupus, enhanced expression of CD69 in peripheral CD4 T cells indicates incremented activation and lower numbers of CD4+CD25+FoxP3+Treg cells contributes to autoimmunity. Pristane-induced lupus murine model has clinical similarities to human disease, nonetheless whether cellular characteristics of this model resemble those of lupus patients remains unknown.ObjectivesTo analyse the expressions of CD69 activation receptor on CD4 Tcells and of CD4+CD25+FoxP3+Treg cells in peripheral blood and spleen of pristane-induced SLE balb/c mice.MethodsTweenty one female Balb/c mice were analyzed: 13 received a single intraperitoneal 0,5 ml dose of pristane and 8 the same dose of saline. Euthanized mice samples of blood and spleen were collected 120 (T120) days after inoculation of pristane or saline. Mononuclear cells from peripheral blood (PBMC) and splenocytes were obtained by lysis of erythrocytes followed by washings with RPMI medium 1640 and centrifugation, subsequently criopreserved until evaluation by flow cytometry using the appliance GuavaEasyCyteTM HT (Millipore). For this step, cells were unfrozen, washed with RPMI medium 1640 and incubated with monoclonal antibodies against CD3, CD4, CD25, CD28, CD69 and FoxP3 (BD PharmingenTM). The results are expressed as mean ± SD and Mann-Whitney test was used for statistical analysis, being considered significant p<0,05.ResultsCompared to control, SLE pristane-induced animals presented increase of CD4+CD69+ T cells in blood and in spleen (p=0.008 and p=0.049, respectively), while the porcentage of Treg CD4+CD25+FoxP3+ was lower in blood and in spleen (p=0.012 and p=0.018, respectively).ConclusionsIncrease of CD4+CD69+ T cell and reduction of CD4+CD25+FoxP3+ Treg expression suggests activated T CD4 cells and loss of peripheral autotolerance in pristane-induced SLE mice. These alterations are similar to observations in human lupus, in indicating that this model may be useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in LES.ReferencesLahita, R. G. Systemic Lupus Erythematosus. Fourth Edition. Toronto: Academic Press; 2004.Mak, A.; Kow, N.Y. Journal of Immunology Research. 2014;Moulton, V.R.; Tsokos, G.C. The Journal of Clinical Investigation. 2015.McGaha, T.L.; Sorrentino, B.; Ravetch, J.V. Science. 2005.Nagarkatti, P. Medical Microbiology. Lecture. 16–17.Abbas, A.K.; Lichtman, A.H.; Pillai, S. Imunologia Básica. 4ª edição. 2013.Doyle, H.A.; Yan, J.; Liang, B.; Mamula, M.J.Immunology Research. 2001.Kuhn, A.; Beissert, S.; Crammer, P.H. Archives of Dermatological Research. 2009.Bonelli, M.; Von Dalwigk, K.; Savitskaya, A.; Smolen, J.S.; Scheinecker, C. Annals of the Rheumatic Diseases. 2008.Bonelli, M.; Von Dalwigk, K.; Savitskaya, A.; Smolen, J.S.; Scheinecker, C. Annals of the Rheumatic Diseases. 2008.AcknowledgementWe thank the Division of Rheumatology and the Clinical Emergency, University of São...