**4. Therapeutic strategies**

The number of people affected by T1DM is approximately 20 million worldwide and is rapidly rising (Chabot, 2002). While exogenous administration of insulin is an effective treatment for acute hyperglycaemia in T1DM, it does not prevent secondary complications (White et al., 2008) and can in some cases lead to hypoglycaemia (Kort et al., 2011). Alternative therapeutic strategies include pancreas transplantation and islet transplantation. While whole pancreas transplantation is an invasive surgical method associated with major complications, islet transplantation is less invasive and associated with significantly lower morbidity and mortality. Successful islet transplantation would result in insulin independence, protection from hypoglycaemia, improvement of microvascular complications, improved patient survival and enhanced quality of life (Kort et al., 2011). The method is currently in clinical trials and has been used to treat around 1,000 individuals worldwide (Kort et al., 2011). Islet transplantation has many limitations, including limited availability of suitable islet graft donors, high cost and high rate of partial or total graft failure. Islet graft failure can be caused by allorejection, toxicity of immunosuppressive drugs that are required to reduce immune rejection, glucotoxicity, and recurrence of autoimmunity (Kort et al., 2011).

An approach to reduce β-cell death in islet grafts is the transfer of therapeutically useful genes into islet cells prior to transplantation (McCabe et al., 2006). The development of gene therapy techniques that can protect β-cells from autoimmune destruction may not only improve outcomes after islet transplantation but may also lead to preventive therapies for patients at high risk of developing T1DM (McCabe et al., 2006).

Cytokine-Induced -Cell Stress and Death in Type 1 Diabetes Mellitus

**4.3 Anti-oxidant gene transfer** 

cytokine exposure and oxidative stress.

**5. Conclusion** 

**6. Acknowledgement** 

JNK is another candidate target for anti-cytokine gene therapy. Inhibition of JNK has been shown to protect pig islet cells from apoptosis and loss of JNK function after isolation and also after transplantation suppresses IL-1β induced apoptosis in insulin-secreting rodent cell lines (Nikulina et al., 2003, Noguchi et al., 2005). Other potential transgenes interfering with cytokine signalling include feedback inhibitors, e.g. SOCS (Yasukawa et al., 2000). It is thought that a compromised ability to up-regulate SOCS in response to cytokine exposure makes βcells particularly susceptible to cytokine-induced damage (Karlsen et al., 2001, Yasukawa et al., 2000). The overexpression of SOCS-3 in response to IL-1β was shown to be slower in β-cells compared to other cell lines (Karlsen et al., 2001). It was also demonstrated that SOCS-3 overexpression can protect rodent β-cells from cytokine-induced death (Karlsen et al., 2001).

The protective effects of several antioxidant enzymes including catalase, glutathione peroxidase and the superoxide dismutases (SODs) MnSOD and CuZnSOD have been investigated. While results have not been entirely consistent, many studies have demonstrated that activation or overexpression of these enzymes can protect β-cells against oxidative stress or cytokine-induced destruction at least to some extent (Benhamou et al., 1998, Bertera et al., 2003, Hohmeier et al., 1998, Lortz & Tiedge, 2003, Lortz et al., 2000). These studies have shown that antioxidant gene transfer is a promising strategy in prolonging islet graft longevity. However, it has also been observed that transfer of antioxidant genes alone could not protect β-cells long term against toxicity caused by

In recent years basic biomedical research has delivered a wealth of knowledge about the pathways by which inflammatory cytokines sensitise β-cells to cell death during the course of T1DM pathogenesis. Although the picture is still incomplete, we have learned about the major stresses to which β-cells are exposed. Some of the molecular players mediating these stresses have been identified. In particular, pro-inflammatory cytokines IL-1β, TNFα and IFNγ have been implicated as main mediators of β-cell stress and death during T1DM. It emerges that these cytokines synergistically activate transcriptional programs that lead to NO signalling, oxidative stress, ER stress, as well as modulation of Bcl-2 family protein expression. How these pathways precisely intersect has not yet been fully clarified. Studies elucidating these mechanisms may provide the knowledge to improve therapy. Islet transplantation, a therapeutic approach that would overcome the need of continuous insulin administration, is still in its infancy. Modern gene transfer techniques offer a huge potential for improvement to islet transplantation as it can help overcoming the cellular and autoimmune-mediated stress transplanted islets are exposed to. The experiments mentioned at the end of this chapter are encouraging that the accumulating knowledge of the molecules and pathways mediating β-cell stress will help to develop gene therapeutic approaches

We are grateful to Dr. Sandra Healy for critical reading of this manuscript and Anna McCormick for providing the microscopy images of mouse pancreatic tissue sections. LV is

alleviating these stresses, thus improving survival of transplanted islets.

227

Various candidate transgenes are being examined for their potential in protecting β-cells under various stresses including cytokine-exposure and oxidative stress. The rational choice of therapeutic genes is helped by understanding the mechanism of -cell destruction which has been the subject of this chapter. Potential targets will be reviewed in this section. Target genes studied to date encode regulators of the cytokine signal transduction pathways, molecules that inhibit -cell apoptosis, antioxidant enzymes, immunoregulatory proteins and pro-survival cytokines (McCabe et al., 2006).

#### **4.1 Anti-apoptotic gene transfer**

Apoptosis plays a major role in β-cell death in T1DM (see section 2.). The transfer of antiapoptotic genes as a strategy to counteract islet destruction has been explored. Candidate genes include those expressing cytoprotective and anti-apoptotic heat shock proteins (Hsps) and anti-apoptotic Bcl-2 family proteins. Hsp70 is one of the major heat shock proteins in mammals and is thought to be responsible for the relative resistance of human β-cells to cytokine-induced stress and death (Burkart et al., 2000). Hsp70 can protect cells under conditions of stress by directly inhibiting the transduction of the apoptotic signal, by decreasing the amount of oxidative stress and also by reducing ER stress via its chaperone activity. It has been shown that pre-conditioning by heat shock could protect pancreatic islet cells from insults by NO, ROS and the cytotoxic drug streptozocin and this increased resistance correlated with induced expression of Hsp70 (Bellmann et al., 1995). Another Hsp that is potentially capable of protecting β-cells is heme oxygenase (HO-1), also known as Hsp32. HO-1 exerts its cytoprotective effects mainly by reduction of oxidative stress (McCabe et al., 2006) and overexpression of HO-1 could protect cytokine-exposed islet cells from apoptosis (Pileggi et al., 2001, Ye & Laychock, 1998). Bcl-2 family proteins, such as the anti-apoptotic Bcl-2, are major regulators of the apoptotic signalling cascade. It has been suggested that an impaired induction of anti-apoptotic Bcl-2 plays a role in cytokineinduced dysfunction and cell death of human islet cells relative to porcine islets (Piro et al., 2001). Moreover, overexpression of Bcl-2 was shown to protect β-cells from cytokineinduced apoptosis (Y. Liu et al., 1996) and increase the longevity of islet grafts after transplantation (Contreras et al., 2001). Several mechanisms by which Bcl-2 might exert βcell protection have been suggested (McCabe et al., 2006). These include inhibition of cytochrome c release from mitochondria, inhibition of ER stress-induced apoptosis and blocking of Ca2+ release from the ER. It was shown that Bcl-2 overexpression can reduce ER stress-induced apoptosis in islet cells (Contreras et al., 2003). Both of these mechanisms have been associated with cytokine-induced β-cell death. Another candidate transgene may be the gene encoding the cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (cFlip) as its overexpression has been shown to inhibit the activation of caspase-8 in β-cells exposed to TNFα (Cottet et al., 2002).

#### **4.2 Anti-cytokine gene transfer**

Inhibition of NF-κB, a main effector of cytokine-signalling, was shown to reduce cytokineinduced apoptosis in rodent β-cells *in vitro* (Baker et al., 2001, Heimberg et al., 2001) and *in vivo* (Eldor et al., 2006) and Fas-induced apoptosis in human islet cells (Giannoukakis et al., 2000). It should be noted that active NF-κB has been shown to be an essential factor in mediating glucose-stimulated insulin secretion (Norlin et al., 2005) and while NF-κB inhibition may protect β-cells from apoptosis it may also interfere with insulin secretion.

JNK is another candidate target for anti-cytokine gene therapy. Inhibition of JNK has been shown to protect pig islet cells from apoptosis and loss of JNK function after isolation and also after transplantation suppresses IL-1β induced apoptosis in insulin-secreting rodent cell lines (Nikulina et al., 2003, Noguchi et al., 2005). Other potential transgenes interfering with cytokine signalling include feedback inhibitors, e.g. SOCS (Yasukawa et al., 2000). It is thought that a compromised ability to up-regulate SOCS in response to cytokine exposure makes βcells particularly susceptible to cytokine-induced damage (Karlsen et al., 2001, Yasukawa et al., 2000). The overexpression of SOCS-3 in response to IL-1β was shown to be slower in β-cells compared to other cell lines (Karlsen et al., 2001). It was also demonstrated that SOCS-3 overexpression can protect rodent β-cells from cytokine-induced death (Karlsen et al., 2001).

### **4.3 Anti-oxidant gene transfer**

The protective effects of several antioxidant enzymes including catalase, glutathione peroxidase and the superoxide dismutases (SODs) MnSOD and CuZnSOD have been investigated. While results have not been entirely consistent, many studies have demonstrated that activation or overexpression of these enzymes can protect β-cells against oxidative stress or cytokine-induced destruction at least to some extent (Benhamou et al., 1998, Bertera et al., 2003, Hohmeier et al., 1998, Lortz & Tiedge, 2003, Lortz et al., 2000). These studies have shown that antioxidant gene transfer is a promising strategy in prolonging islet graft longevity. However, it has also been observed that transfer of antioxidant genes alone could not protect β-cells long term against toxicity caused by cytokine exposure and oxidative stress.
