**1.2.3 IFNγ signalling**

IFNγ is a homodimeric cytokine. It binds to two IFNγ receptor α (IFNγRα) chains (Fig. 3). A third unit of IFNγRα and two molecules of IFNγ receptor β (IFNγRβ, also termed accessory factor 1, AF-1) bind to the IFNγRα (Thiel et al., 2000). This leads to the activation and transphosphorylation of Janus tyrosine kinase 1 and 2 (JAK1 and JAK2) which are associated with IFNγRα and IFNγRβ, respectively, and are brought together upon receptor oligomerisation (Igarashi et al., 1994, Kotenko et al., 1995). JAK1 and JAK2 phosphorylate IFNγR leading to the recruitment of two molecules of the transcription factor, signal transducer and activator of transcription-1 (STAT-1). After phosphorylation and activation by JAK2, STAT-1 homodimerises and translocates to the nucleus where it stimulates the expression of target genes (Takeda & Akira, 2000). Islet cells isolated from STAT-1-/- nonobese diabetic (NOD) mice were resistant to apoptosis induced by combined treatment with IFNγ and TNFα or IFNγ and IL-1β (S. Kim et al., 2007). In support of this, blockade of STAT-1 protected against diabetes induced by injection of multiple low doses of streptozotocin in mice (Callewaert et al., 2007, C.A. Gysemans et al., 2005). A recent gene expression analysis showed that nearly two thousand genes are regulated by STAT-1 in response to cytokine exposure (IL-1β and IFNγ) in β-cells (Moore et al., 2011). STAT-1 was found to regulate the IL-1β/IFNγ-mediated induction of chemokines, including CXCL9, CXCL10, CXCL11 and CCL20 (Moore et al., 2011) and islets from STAT-1-/- mice have decreased production of CXCL10 upon cytokine exposure both *in vitro* and *in vivo* (C.A. Gysemans et al., 2005).

STAT-1 also down-regulates several genes specific to β-cell functions, such as insulin, glucokinase, Glut2, prohormone convertases, as well as many transcription factors involved in the differentiation and maintenance of β-cell phenotype (e.g. Pdx1, MafA, Nkx2.2) (Moore et al., 2011, Perez-Arana et al., 2010).

Finally, STAT-1 is an important regulator of genes mediating intracellular stress and apoptotic pathways. Several apoptosis-related genes such as Puma, CHOP, Bax, Bid, caspase-3, -4, -7, DP5/Hrk and endoplasmic reticulum stress-transducing genes (XBP1, ATF4) are regulated by STAT-1 (Eizirik & Darville, 2001, Moore et al., 2011, Anastasis Stephanou et al., 2000). IFNγ has been found to profoundly accelerate IL-1β-mediated iNOS induction and thus cause oxidative stress. We have demonstrated that treatment of a rat insulinoma cell line (RIN-r) with a combination of IL-1β and IFNγ induces the mitochondrial apoptotic pathway in an iNOS-dependent manner (Holohan et al., 2008). This is in line with reports from other groups (Gurzov et al., 2009).

The inflammatory effects of IFNγ are controlled by negative feedback regulation, exerted by interferon regulated factor-1 (IRF-1) (Moore et al., 2011) and SOCS-1 and -3 (Alexander, 2002). IRF-1 is likely to exert its STAT-1 regulatory role by up-regulation of SOCS-1 (Moore et al., 2011). IRF-1 expression reduces chemokine expression in β-cells and resulting T cell infiltration in Langerhans islets (C. Gysemans et al., 2008, Moore et al., 2011), however the effect of IRF-1 on STAT-1-mediated β-cell de-differentiation (loss of β-cell function) and cell stress is minor (Moore et al., 2011). In line with this, transgenic expression of SOCS-1 in β-cells reduced diabetes development in non-obese diabetic (NOD) mice (FlodströmTullberg et al., 2003) and protected β-cells against infiltrating autoreactive T cells (Chong et al., 2004). In summary, the effect of IFNγ in β-cells is primarily mediated by STAT-1 through which IFNγ controls key processes culminating in loss of β-cell function, stress and finally death. IFNγ regulates a number of genes that increase the sensitivity of β-cells to apoptotic stimuli and intracellular stress.
