Duchenne Muscular Dystrophy DMD is

Duchenne Muscular Dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity (Mercuri and Muntoni, 2013; Muntoni, 2003; Muntoni et al., 2003). Indeed, dystrophin is an essential component of the dystrophin glycoprotein complex (DGC) which connects the extracellular matrix with the intracellular SC 144 filaments (Muntoni et al., 2003). Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle (Kumar et al., 2004). However, several altered mechanisms are involved in DMD onset and progression, resulting in muscle weakness and progressive muscle wasting (Evans et al., 2009a). Because of muscle damage, cytokines expression is stimulated and immune cells infiltrate in dystrophic muscle, contributing to worsen the dystrophic phenotype (Evans et al., 2009b; Rosenberg et al., 2015; Villalta et al., 2015). Indeed, dystrophic muscles are characterized by up-regulation of inflammatory gene expression, such as NF-kB, and by an increase in inflammatory cell infiltration, mostly composed by macrophages, neutrophils, and T cells. Macrophages, neutrophils, and T cells are the primary cells that invade the dystrophic muscle. It is currently believed that neutrophils and macrophages infiltrate the skeletal muscle around age 2weeks in mdx mice, while CD8+ and CD4+ T cells infiltrate the muscle between age 4–8weeks (Evans et al., 2009a, 2010; Rosenberg et al., 2015). NF-kB pathway results persistently activated in dystrophic muscle and it has been proposed as one of the major mediators of muscle wasting: it is activated prior the onset of the disease in response of increased Calcium influx and mechanical stretch and it is responsible for the increased cytokine expression and the immune response activation (Acharyya et al., 2007; Kumar and Boriek, 2003; Messina et al., 2006, 2009). The repeated cycles of myofibers degeneration and regeneration, due to lack of dystrophin, lead to the establishment of a chronic inflammatory environment (Madaro and Bouche, 2014). Thus, the functional muscle tissue is replaced by a fibrotic and adipose non-functional connective tissue (Kharraz et al., 2014; Pessina et al., 2015). Therapeutic strategies based on the restoration of dystrophin expression or the administration of dystrophin stem cells are promising, but are often limited in efficacy, and long-lasting efficacy has still to be reached (Bengtsson et al., 2016; Cossu et al., 2015; Odom et al., 2011; Sampaolesi et al., 2003, 2006; Seto et al., 2012). The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a “cure” for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology, which may eventually converge, may be successful. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit (Angelini, 2007; Angelini and Peterle, 2012; Schacke et al., 2002; Schoepe et al., 2006). Numerous other anti-inflammatory therapies, aimed to target more specific mediators of inflammation, have been proposed to improve healing (Heier et al., 2013; Pelosi et al., 2015). In this context, we previously showed that lack of PKCθ in mdx, the mouse model of DMD, in the mdx/PKCθ−/− mouse model we generated, was associated with reduced muscle wasting, improved muscle regeneration and maintenance of performance compared to mdx mice (Madaro et al., 2012). We further demonstrated, by bone marrow transplantation experiments, that PKCθ expression in immune cells is required to mount a robust inflammatory response in mdx, which, in turn, exacerbates the muscle pathology (Madaro et al., 2012). PKCθ belongs to the novel class of PKCs and is expressed in both hematopoietic cells and in striated muscle (Marrocco et al., 2014; Nath and Isakov, 2014). In T cells, it is the key member of the PKC family to play a critical role in the Ca2+/NFAT, AP-1 and NF-kB pathways to activate the IL-2 and IL-4 promoters, and it appears to be required for the development of a robust inflammatory response in vivo (Pfeifhofer et al., 2003; Thuille et al., 2013; Wachowicz et al., 2014; Wachowicz and Baier, 2014; Kwon et al., 2012). Previous studies showed that PKCθ−/− mice fail to develop experimental allergic encephalomyelitis, display drastically reduced lung inflammation after induction of allergic asthma, and have a significantly diminished response in experimental colitis and a type II collagen induced arthritis model (Anderson et al., 2006; Fang et al., 2012; Tan et al., 2006; Wang et al., 2009). Of note, PKCθ−/− mice may still mount a normal protective immune response to clear viral infections (Giannoni et al., 2005), and maintain Treg function (Gupta et al., 2008; Ma et al., 2012; Sun, 2012; Zanin-Zhorov et al., 2010). Taken together, these evidences validate PKCθ as a particularly attractive target to selectively manipulate Teff cell functions that are relevant to pathogenesis of different diseases, including, as our results suggested, muscular dystrophy. Noteworthy, a large effort is now devoted to the synthesis of pharmacological PKCθ inhibitors, to be used for anti-inflammatory therapies, and several PKCθ inhibitors have been identified and reported in recent literature (Boschelli et al., 2010; Chand et al., 2012; Curnock et al., 2014; Cywin et al., 2007; George et al., 2015; Hage-Sleiman et al., 2015; Jimenez et al., 2013; Katoh et al., 2016). Some of them bind selectively PKCθ while others are non-selective in nature. One of the highly selective inhibitor for PKCθ reported was C20, (compound 20 from Boheringer-Inglheim Pharmaceuticals, Inc), belonging to the amino pyrimidine class of inhibitors (Cywin et al., 2007; Sims et al., 2007; Zanin-Zhorov et al., 2010). In any case, to propose a pharmacological protocol to inhibit PKCθ, eventual possible effects on other tissues should be considered. Indeed, PKCθ is expressed also in other tissue, including skeletal and cardiac muscle. In skeletal muscle, we and others showed that PKCθ is mostly involved in mediating signaling pathways regulating fetal and early post-natal growth and maturation (Madaro et al., 2011, 2013; Marrocco et al., 2014; Messina et al., 2010; Zappelli et al., 1996). Employing PKCθ−/− mice, we also showed that PKCθ maintains the correct structure and function of the heart by preventing cardiomyocyte cell death in response to work demand and to neuro-hormonal signals, to which heart cells are continuously exposed (Paoletti et al., 2010).