br Funding M I is supported by European Research Council
Funding M.I. is supported by European Research Council (ERC) Consolidator Grant 725038, Italian Association for Cancer Research (AIRC) Grant 19891, Italian Ministry of Health (MoH) Grant GR-2011-02347925, Lombardy Foundation for Biomedical Research (FRRB) Grant 2015-0010, the European Molecular Biology Organization (EMBO) Young Investigator Program, and a Career Development from the Giovanni Armenise-Harvard Foundation; M.K. is the recipient of the Italian Ministry of Education (MIUR) grant SIR-RBSI14BAO5.
Introduction Oclacitinib (OCL; APOQUEL®) is a novel immunomodulatory agent that has been registered for the treatment of pruritus in allergic dermatitis and atopic dermatitis (AD) in dogs. OCL acts as an inhibitor of the function of important pro-inflammatory, pro-allergic and pruritogenic cytokines via inhibition of the Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling. JAKs are a group of 4 enzymes (JAK1, JAK2, JAK3, TYK2) that function by facilitating the transmission of signals from cell membrane receptors to the nucleus (Gonzales et al., 2014). Although first described as the signal-transducing pathway of interferons, the JAK/STAT appeared to have a role in regulating cytokine signaling and is engaged in signal transduction of several pivotal cytokines (Igaz et al., 2001). These cytokines are involved in the pathogenesis of inflammation, allergy and pruritus. Therefore, all the evidence indicates that the main mechanism responsible for anti-inflammatory and anti-allergic effects of OCL is related to the inhibition of the production of these cytokines (Gonzales et al., 2014). OCL is recognised as a selective inhibitor of JAK1, but additionally, it inhibits the activity of JAK2 and, to a lesser degree, JAK3 (Gonzales et al., 2014; Roskoski Jr, 2016). The JAK3/STAT5 signaling pathway is important for the maintenance of the transcription factor Foxp3 (Forkhead Box P3 protein) in CD25+CD4+ regulatory T SU11274 receptor (hereinafter referred to as CD4+ Treg cells) (Goldstein et al., 2016), which is essential for development and function of these cells. Thus, Foxp3 is not only a marker of Treg cells, but it also confers their suppressive properties (Fontenot et al., 2003). Treg cells are responsible for the regulation of the immune response and play a leading role in inducing immune tolerance through suppression of various effector T cells. Functions of Treg cells include prevention of autoimmune disease, suppression of allergy and pathogen-induced immunopathology and induction of oral tolerance (Workman et al., 2009). Recently, Hauck et al. (2016) have found increased numbers of CD4+ Treg cells in peripheral blood from dogs with AD and their correlation with disease severity. In turn, Jassies-Van Der Lee et al. (2014) demonstrated that the percentage of Foxp3+CD25+CD8+ T regulatory cells (hereinafter referred to as CD8+ Treg cells) was reduced in the skin of dogs with AD. These results (Jassies-Van Der Lee et al., 2014; Hauck et al., 2016) strongly suggest that CD4+ Treg cells and CD8+ Treg cells may participate in the pathogenesis of canine AD. Considering the fact that OCL can inhibit JAK3, we put forward the hypothesis that inhibition of JAK3 induced by OCL may down-regulate Foxp3 expression in canine Treg cells, thereby depleting them. This effect should be seen as an unfavorable one, especially in patients with allergic disorders, because Treg cells counteract allergic and autoimmune diseases. Thus, the first aim of this study has been to determine the effect of OCL on Foxp3 expression in CD25+CD4+ and CD25+CD8+ T cells, and on counts of CD4+ and CD8+ Treg cells. It should be added that in contrast to research on humans and mice, the available literature contains a single report (Jassies-Van Der Lee et al., 2014) concerning the presence of CD8+ Treg cells in dogs. Hence, this study will provide new data on canine Foxp3-expressing CD8+ T cells. The crucial event in the induction of an immune response is the activation of CD4+ T cells that needs binding of IL-2 to its high-affinity receptor (i.e. CD25) for optimal signaling. CD25 is extensively induced after T cell activation, which enables the effective binding of IL-2 to effector T cells. Inappropriate activation of effector CD4+ T cells could favor and promote the allergic response, including canine AD (Beccati et al., 2016). It has been demonstrated that ruxolitinib, a potent JAK1/JAK2 inhibitor, reduces activation and differentiation of human CD4+ T cells both in vitro and in vivo (Parampalli Yajnanarayana et al., 2015). Therefore, our hypothesis was that one of the mechanisms responsible for the anti-allergic action of OCL might be the prevention/reduction of T cell activation. To determine this effect, induction of the early T cell activation marker, i.e. CD25, was assessed on mitogen-activated CD4+ T cells.