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Abstract / Cytokine 63 (2013) 243–314

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92 STAT5 activates the C-Myc proximal promoter: Elevated STAT5 or C-Myc expression relative to STAT1 is linked with reduced survival of melanoma patients given IFNc therapy

kine levels could explain the natural immunity of baboons to SIV infection. Further work is underway to identify genes associated with this natural immunity. Identification of these genes ultimately may help inform the development of new therapeutic or preventive approaches to AIDS.

Ibtisam Ghazawi a, Albert S. Mellick a, Samuel J. Cutler a, James Doecke b, Jeanette Raleigh c, Grant McArthur c,d,e, Stephen J. Ralph a, a School of Medical Science and Griffith Health Institute, Griffith University, Southport, Qld 4222, Australia, b CMIS, Royal Brisbane Women’s Hospital, Herston, Brisbane, Australia, c Division of Cancer Medicine and Research, Peter MacCallum Cancer Centre, East Melbourne, Vic., Australia, d Department of Pathology, University of Melbourne, Parkville, Vic., Australia, e Department of Medicine, St Vincent’s Hospital, University of Melbourne, Fitzroy, Vic., Australia

http://dx.doi.org/10.1016/j.cyto.2013.06.096

Recently, a role for IFNc in the genesis of melanoma was reported. Here, we define the mechanism whereby IFNc becomes a growth stimulator of melanoma cells. In this study, IFNc stimulated STAT5 to activate the expression of c-myc, an oncogene linked with reduced melanoma patient survival. In a preliminary retrospective study of 27 stage III melanoma patients who received IFNa2 therapy, STAT5A and c-myc expression levels in metastatic lymph node biopsies were highly correlated (Pearson’s r = 0.914; P < 0.0001). Kaplan–Meier survival analyses revealed high expression levels of c-myc or STAT5 relative to STAT1 were prognostic markers indicating reduced patient survival. In vitro studies showed that STAT5 activated the c-myc proximal promoter via a functional STAT5A binding site, conferring significant inducibility by IFNc or EGF (but not IFNa) in luciferase reporter assays of melanoma cells overexpressing STAT5A. IFNc caused markedly different responses in wild-type A375 cells compared with A375-STAT5A cells, supporting a key role for STAT5A in IFNc-mediated regulation of c-myc expression in melanoma. ChIP assays confirmed direct STAT5A binding during IFNc-mediated activation of the c-myc proximal SBE regulatory region. 50 RACE and Q-PCR showed that increased levels of c-myc in A375-STAT5A cells induced by IFNc were associated with a shift in the initiation of transcription near to or at the proximal STAT5 binding element. These results indicate that overexpression of STAT5 in cancer cells is responsible for growth-stimulation by IFNc. http://dx.doi.org/10.1016/j.cyto.2013.06.095

93 Role of cytokines in the natural resistance of baboons to SIV infection Luis D. Giavedoni a,b, Laura M. Parodi a,b, Veronica Obregon-Perko a, Lisa M. Smith a, Jessica Callery a, Vida L. Hodara a,b, a Department of Virology & Immunology, Texas Biomedical Research Institute, San Antonio, TX, USA, b Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA The establishment of human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections in humans is the consequence of multiple cross-species transmissions of simian immunodeficiency viruses (SIVs) from chimpanzees (SIVcpz) and sooty mangabeys (SIVsmm), respectively. While over 40 African nonhuman primate (NHP) species carry their unique strain of SIV, many of which resulted from simian-to-simian transmissions, these natural SIV infections are chronic and productive but not associated with immunodeficiency. Baboons (Papio hamadryas ssp.), one of the most widely distributed African NHP species, do not seem to be naturally infected with SIV, even though their home ranges overlap those of many SIV-infected NHP species. Despite some serological evidence for SIV infection in very few wild-living baboons, isolation of a replicating virus from these animals has not been reported. The reasons for this apparent natural resistance to SIV infection are not known. Baboons have been shown to be susceptible to experimental infections with some HIV-1, HIV-2 or SHIVs strains. Further, in our hands, baboon lymphocytes support productive infection with SIVmac and SIVagm, indicating that intracellular antiviral factors like APOBEC3 proteins, Trim-5a, and Tetherin probably do not contribute to this resistance. Our long-term goal is to identify the factors responsible for natural resistance to SIV infection in baboons. We hypothesized that cytokines and cytotoxic factors from cells from the innate immune system are involved in this natural resistance. As a first step towards testing this hypothesis, we performed in vitro infections with SIV of lymphocytes from baboons and Indian rhesus macaques. We determined kinetics of viral replication, and measured cytokine production and cell activation in order to identify differences between these NHP species. While all the rhesus PBMC preparations supported SIV replication, there were PBMC from some baboons that were refractory to SIV infection; this difference was more pronounced for infections with SIVagm than with SIVmac. We also observed that in vitro stimulation of rhesus cells resulted in higher levels of CD4 T cell activation in comparison to stimulated cells from baboons; however, the concentration of cytokines that compete with SIV for binding to CCR5 (such as RANTES, MIP-1a, and MIP-1b) was higher for stimulated baboon cells than for macaque cells. Taken together, these differences in T cell activation status and cyto-

94 Interleukin-22 mediates protection during a murine model of cutaneous leishmaniasis Ciara Gimblet a, David Artis b, Phillip Scott a, a Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, USA, b Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA Cutaneous leishmaniasis is a disease characterized by ulcerating skin lesions that are normally self-healing. However some patients develop more severe disease that fails to resolve. While parasite control by the immune response has been well studied, the mechanisms of how lesions resolve, or in some cases fail to resolve, is not well understood. IL-22, a member in the IL-10 family of cytokines, has been shown to contribute to the wound healing process through maintenance of the epithelial barrier. In this study, we investigated whether IL-22 contributed to lesion resolution during Leishmania infection. We infected IL-22 deficient mice in the ear with L. major and found that these mice developed more severe disease compared with C57BL/6 control mice. The lesions of the IL-22 deficient mice had significantly more inflammation, which resulted in tissue loss at the site of infection. However, we found that parasite burdens were similar in the wild-type and IL-22 deficient mice, suggesting that the increased inflammation was not due to an increased parasite burden. Instead, the lesions from the IL-22 deficient mice expressed significantly higher levels of IL-1a, IL-1b, and S100A8/S100A9, molecules associated with tissue damage, increased epithelial cell death, and delayed wound healing. These studies suggest that during L. major infection, IL-22 plays a previously unappreciated role in controlling Leishmania-induced inflammation, presumably by protecting epithelial cells from cell death and maintaining the epithelial barrier for an efficient wound healing response.

http://dx.doi.org/10.1016/j.cyto.2013.06.097

95 Type I interferon regulates acute IL-5 and IL-13 expression in human memory CD4+ T cells Sarah R. Gonzales-van Horn, Jonathan P. Huber, J. David Farrar, Department of Immunology, UT Southwestern Medical Center, Dallas, TX, USA Type I Interferon (IFN-a/b) is a critical anti-viral cytokine and is currently being used to treat Hepatitis C viral infections and multiple sclerosis, among other diseases. Although the positive regulatory effects of this cytokine have been studied, less is known about its role as a negative regulator of gene expression. Previously, we demonstrated that IFN-a/b suppresses Th2 development in human naïve CD4+ T cells by negatively regulating GATA3 expression, leading to the suppression of IL-4, IL-5 and IL-13. Based on this data, we were interested in how IFN-a/b might affect other T cell types. In the current study, we interrogated the effects of IFN-a/b on human memory CD4+ T cells. We hypothesized that IFN-a/b has similar negative regulatory effects on memory CD4+ T cells, leading to decreased Th2 cytokine expression. Human memory CD4+ T cells were acutely stimulated in the presence of IFN-a. Interestingly, IL-5 and IL-13 gene expression was suppressed, however, IL-4 expression was unaffected. Further, IFN-a/b signaling, but not IFN-c or IFN-k, suppressed IL-5 and IL-13 expression. Nuclear run-on studies demonstrated that IFN-a/b signaling suppressed de novo transcription of IL-5 and IL-13, and this suppression did not require the translation of interferon-sensitive genes. Our data indicate that this negative regulatory pathway acted upstream of gene expression, and suggested that IFN-a/b was able to acutely suppress specific gene expression. Currently, we are assessing direct STAT2 binding by ChIP-Seq, which will identify potential cis-regulatory regions necessary for IL-5 and L-13 gene modulation. Together, these results suggest that IFN-a/b could have many benefits in suppressing multiple aspects of Th2 development and effector function in atopic asthma. http://dx.doi.org/10.1016/j.cyto.2013.06.098