**2. Natural killer cells**

Natural killer (NK) cells are capable of distinguishing between infected/transformed cells and uninfected/non-transformed cells and are able to kill the former using perforin or granzyme dependent mechanism (Topham & Hewitt, 2009). The best recognized mechanism for NK cell activation is through Type 1 interferons (IFNα/β). Type 1 interferons upregulate multiple interferon response factors (IRFs) and two of these IRFs are strongly implicated in NK cell proliferation and activation (Taniguchi et al., 2001). NK cell numbers are dramatically reduced in IRF2-/- mice (Lohoff et al., 2000). NK cell numbers are also reduced in IRF1-/- mice but in this case, the defect is not inherent in the NK cell progenitor since adoptive transfer of IRF1-/- bone marrow into wild-type mice results in NK cell proliferation (Ogasawara et al., 1998). Rather, IRF-1 appears to control IL-15 expression in bone marrow stromal cells and IL-15 promotes NK cell generation. Similarly, other cytokines including IL-2, IL-12 and IL-18 promote NK cell responses (Agaugue et al., 2008). In contrast to IRF1, IRF2 is inherently important in the NK cell progenitor since adoptive transfer of IRF2-/- bone marrow into wild-type recipients fails to generate NK cells. How NK cells recognize aberrant cells has received substantial study since these effectors are non-T cells, lack the T cell receptor and CD3, and do not undergo genetic recombination of recognition receptors (Biron et al., 1999; Orange et al., 2002). NK cells express substantial numbers of both activating and inhibiting receptors (reviewed in (Lanier, 2008)), and despite lacking classical T cell receptors, NK cells can recognize microbial molecules. Examples include NKp46 recognition of the influenza hemagglutinin protein (Mandelboim et al., 2001), Ly49H recognition of m157 (mCMV) (Arase et al., 2002), NKp44/NKp46 recognition of NDV hemagglutinin-neuraminidase (Jarahian et al., 2009), and Ly49P recognition of m04 (mCMV) (Kielczewska et al., 2009). NK activation receptors pair with ITAM-bearing DAP12, FcεRI-γ and CD3-ζ signaling molecules. Stimulation of the NK activating receptors leads to phosphorylation of the ITAM components and recruitment of Syk and ZAP-70. This results in actin cytoskeletal reorganization, which promotes secretion of preformed cytokines. The cytokines primarily produced by NK cells are IFNγ/TNFα, or perforin/granzyme. Activation also increases transcription of cytokine genes. In contrast, inhibitory NK receptors are either monomeric type 1 glycoproteins of the immunoglobulin superfamily [examples include: killer cell immunoglobulin-like receptors (KIRs) and leukocyte immunoglobulin-like receptors (LILRs)] or type II glycoproteins containing a C-type lectin like scaffold [examples include: Ly49 and CD94-NKG2A]. Both types of receptor contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their intracellular domains which, when activated, recruit tyrosine phosphatases that block the phosphorylation steps initiated by the NK activating receptors and thus inhibit NK cell functions (Lanier, 2008).

take 7-10 days after virus inoculation. The best known innate immunity results from microbial products binding to and activating Toll-Like Receptors (TLR) or RNA helicases (RIG-I and MDA-5) which activate transcription factors (NFkB) leading to expression of cytokines (TNFα, IL-1β and IL-6) and nitric oxide (Hosoi et al., 2004; Michelsen et al., 2004); or interferon response factors (IRF3/7) leading to expression of type 1 interferons (IFNα/β) RANTES and IP-10 (O'Neill, 2004; Vogel et al., 2003). These roles for TLR are discussed elsewhere. This review will concentrate on lymphocytes belonging to the innate immune response and discuss their role in myocarditis. These lymphocytes include natural killer

Natural killer (NK) cells are capable of distinguishing between infected/transformed cells and uninfected/non-transformed cells and are able to kill the former using perforin or granzyme dependent mechanism (Topham & Hewitt, 2009). The best recognized mechanism for NK cell activation is through Type 1 interferons (IFNα/β). Type 1 interferons upregulate multiple interferon response factors (IRFs) and two of these IRFs are strongly implicated in NK cell proliferation and activation (Taniguchi et al., 2001). NK cell numbers are dramatically reduced in IRF2-/- mice (Lohoff et al., 2000). NK cell numbers are also reduced in IRF1-/- mice but in this case, the defect is not inherent in the NK cell progenitor since adoptive transfer of IRF1-/- bone marrow into wild-type mice results in NK cell proliferation (Ogasawara et al., 1998). Rather, IRF-1 appears to control IL-15 expression in bone marrow stromal cells and IL-15 promotes NK cell generation. Similarly, other cytokines including IL-2, IL-12 and IL-18 promote NK cell responses (Agaugue et al., 2008). In contrast to IRF1, IRF2 is inherently important in the NK cell progenitor since adoptive transfer of IRF2-/- bone marrow into wild-type recipients fails to generate NK cells. How NK cells recognize aberrant cells has received substantial study since these effectors are non-T cells, lack the T cell receptor and CD3, and do not undergo genetic recombination of recognition receptors (Biron et al., 1999; Orange et al., 2002). NK cells express substantial numbers of both activating and inhibiting receptors (reviewed in (Lanier, 2008)), and despite lacking classical T cell receptors, NK cells can recognize microbial molecules. Examples include NKp46 recognition of the influenza hemagglutinin protein (Mandelboim et al., 2001), Ly49H recognition of m157 (mCMV) (Arase et al., 2002), NKp44/NKp46 recognition of NDV hemagglutinin-neuraminidase (Jarahian et al., 2009), and Ly49P recognition of m04 (mCMV) (Kielczewska et al., 2009). NK activation receptors pair with ITAM-bearing DAP12, FcεRI-γ and CD3-ζ signaling molecules. Stimulation of the NK activating receptors leads to phosphorylation of the ITAM components and recruitment of Syk and ZAP-70. This results in actin cytoskeletal reorganization, which promotes secretion of preformed cytokines. The cytokines primarily produced by NK cells are IFNγ/TNFα, or perforin/granzyme. Activation also increases transcription of cytokine genes. In contrast, inhibitory NK receptors are either monomeric type 1 glycoproteins of the immunoglobulin superfamily [examples include: killer cell immunoglobulin-like receptors (KIRs) and leukocyte immunoglobulin-like receptors (LILRs)] or type II glycoproteins containing a C-type lectin like scaffold [examples include: Ly49 and CD94-NKG2A]. Both types of receptor contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their intracellular domains which, when activated, recruit tyrosine phosphatases that block the phosphorylation steps initiated by the NK activating receptors and thus inhibit NK cell functions (Lanier, 2008).

(NK), natural killer T (NKT) and γδ T cells.

**2. Natural killer cells** 

NK inhibitory receptors recognize major histocompatibility complex class I (MHC I) molecules which represents one mechanism by which NK cells distinguish between infected and normal cells, as many viruses attempt to evade the immune system through downregulation of MHC molecules (Hewitt, 2003).

Evidence for a major role of NK cells in clinical myocarditis is rather weak. NK cells were not observed in heart tissue from 18 cases of biopsy proven myocarditis (Chow et al., 1989). Other studies have found either increased numbers of NK like cells in peripheral blood of dilated cardiomyopathy patients (Yokoyama, 1988) or diminution of NK cell activity in such patients (Maisch et al., 1985). Studies in patients with Chagas' disease find no alteration in NK cells early in the disease but an increase in these innate effectors occurs at later stages (Sathler-Avelar et al., 2003). One potential problem with clinical studies is that diagnosis of myocarditis or dilated cardiomyopathy is a relatively late event in the disease process and may be quite removed from the initiating acute infection (Woodruff, 1980). In fact, while viral genomic sequences can be detected in clinical heart biopsies for months and possibly for years, it is rare for infectious virus to be isolated from the hearts of myocarditis patients. Any role for NK cells may be over by the time human tissue is studied. The best evidence that NK cells might participate in viral myocarditis comes from mouse models. These studies indicate that NK cells are important in controlling coxsackievirus B infections in vivo (Gauntt et al., 1988; Gauntt et al., 1989; Vella & Festenstein, 1992) as depletion of these cells substantially increases virus titers in the heart or pancreas. The ability of NK cells to suppress virus infection may relate to their cytolytic activity to infected cardiocytes. Rapid elimination of infected cells before virus replication is complete would restrict the number of progeny virions produced and therefore limit the next cycle of infection. The second mechanism by which NK cells may help control virus infection is through either augmenting or accelerating the adaptive immune response to the virus. NK cells directly interact with both dendritic cells and activated T cells causing maturation of the dendritic cells and increased activation of the T cells (Zingoni et al., 2005). Interactions occur through up-regulation of OX40L on the NK cells and OX40 on activate CD4+ lymphocytes. Also, NK cells contain high concentration of pre-formed cytokines which can be rapidly released upon NK receptor engagement and these cytokines provide the environment necessary for optimal adaptive immunity development. As with the mouse model of CVB3 myocarditis, NK cells also control spread of Trypanosoma cruzi in the mouse model of Chagas' disease (Brener & Gazzinelli, 1997).
