Sex Differences in the Efficacy of Combined IL-10 and IL-10R1 Gene Therapy for Neuropathic Pain in Mice

Neuropathic pain is a debilitating condition that affects millions of Americans. Despite decades of research, the disease is refractory to current treatments in a substantial population of patients. Recent advances in the understanding of neuropathic pain have led to the targeting of glial cells to better control the condition. Current clinical therapies do not manage the inflammatory response that originates with glial cells and contributes to this affliction. IL-10 is an anti-inflammatory cytokine that attenuates the inflammatory response that occurs at the neuroimmune interface in neuropathic pain models. IL-10 gene therapy has alleviated neuropathic pain in a number of animal studies. We tested whether gene therapy increasing the expression of IL-10 and IL-10R1 could attenuate allodynia in a chronic constriction injury (CCI) model of neuropathic pain. Our experiments did not produce reversal of neuropathic pain using the combined gene therapy. However, the CCI surgeries did not produce neuropathic pain in the male mice. While the female mice developed neuropathic pain, the expression of plasmid genes could not be definitively confirmed. Therefore, while the present study did not provide convincing evidence that the combined gene therapy could alleviate neuropathic pain, additional experiments should be performed to further investigate the potential of combined IL-10/IL-10R1 gene therapy for combating neuropathic pain and to investigate the sex differences in the biochemical pathways underlying neuropathic pain. Introduction Neuropathic pain is a debilitating illness that affects a large portion of the world population. It arises as a direct consequence of a lesion or disease affecting the somatosensory system (Treede et al., 2008). Studies have reported variable numbers for the prevalence of neuropathic pain, ranging from 1.5% to 8% (Taylor, 2006; Torrance et al., 2006; Bouhassira et al., 2008). Regardless of where the prevalence falls within this range, millions of Americans and millions more worldwide are affected. Neuropathic pain is expensive to manage and significantly decreases the quality of life of afflicted patients (Taylor, 2006). Intrathecal delivery of IL-10 gene therapy has shown promise in alleviating neuropathic pain in an animal model of neuropathic pain in several studies (Milligan et al., 2005a, 2005b, 2006a, 2006b; Ledeboer et al., 2007; Sloane et al., 2009a, 2009b; Soderquist et al., 2010). We hypothesized that overexpressing IL-10R1, part of the IL-10 receptor, could enhance the effectiveness of IL-10 gene therapy. To test this hypothesis, we acquired an AAV9 viral vector that leads to overexpression of the IL-10 receptor (IL-10R1). This technique extends the effective duration of the pain relief (Figure 1). In addition, a recent report suggests that chronic pain develops by different cellular mechanisms in males and females (Sorge et al., 2015). This is of particular concern because the studies of IL-10 gene therapy have been conducted almost exclusively in male animals (Kwilasz et al., 2014), but the majority of chronic pain patients are female (Torrance et al., 2006). My work compared the efficacy of combined IL-10 and IL-10R1 gene therapy in both male and female mice to determine whether over-expression of the IL-10 receptor could create a longer-lasting reversal of allodynia than treatment with IL-10 gene therapy alone. Aims 1. Compare the effects of combined IL-10 and IL-10R1 gene therapy, as compared to each gene therapy technique in isolation, to determine if the combined therapy creates a longer duration anti-allodynia. 2. Compare the anti-allodynic effects of IL-10 gene therapy techniques in male and female mice. 3. Quantify expression of IL-10, IL-10R1, P2X4, and neuroinflammatory markers at different time points after surgery to determine the effects of gene therapy treatments on mRNA expression. Background Neuropathic Pain Neuropathic pain is a debilitating condition that affects a large portion of the world population. It arises as a direct consequence of a lesion or disease affecting the somatosensory system (Treede et al., 2008). A conservative estimate by Taylor (Taylor, 2006) predicts that the prevalence of neuropathic pain is 1.5%; studies in France and Britain have arrived at higher estimates of 7-8% of the population (Torrance et al., 2006; Bouhassira et al., 2008). Regardless of the exact number, millions of Americans suffer from neuropathic pain, presenting a significant clinical challenge. Despite years of research, current therapeutics do not effectively manage neuropathic pain in a substantial population of patients. Notably, all currently approved therapeutics focus on regulating neuronal processes. However, recent evidence has implicated glial cells, specifically microglia and astrocytes, as the major players in the induction and maintenance of neuropathic pain (Watkins et al., 2001; Watkins and Maier, 2003). Specifically, inflammation has been implicated as a key mechanism underlying the condition, and glial cells play an important role in generating the inflammatory response (Grace et al., 2014). Taken together, these findings make the inhibiting the inflammatory response produced by glial cells a key target for drug discovery. Neuroimmune Signaling The neuroimmune signaling generated by activated glial cells plays a key role in initiating and maintaining neuropathic pain (Grace et al., 2014). Three key cytokines among the myriad of pro-inflammatory substances that are released are tumor necrosis factor (TNF), interleukin-1 (IL-1), and interleukin 6 (IL-6). The neuroimmune interface is extremely complex, with a number of parallel and redundant mechanisms that can enhance pain signals. Two general mechanisms contribute to the neuroimmune enhancement of pain: the enhancement of excitatory synaptic transmissions, and the attenuation of inhibitory synaptic messages (Grace et al., 2014). Within each of these broad categories, multiple distinct pathways exist that can each enhance pain signaling and contribute to neuropathic pain (Grace et al., 2014). Pharmacological substances exist which can inhibit the actions of IL-1 and TNF (Watkins and Maier, 2003, 2004). However, blocking the actions of single pro-inflammatory cytokines is not a viable clinical approach. One issue is that these cytokines perform redundant functions. For example, if IL-1 is pharmacologically inhibited, TNF can perform the same biochemical actions as IL-1 (Watkins and Maier, 2003; Grace et al., 2014). Additionally, the potential effectiveness of inhibiting these molecules is limited by the fact that they perform important protective functions in the neuroimmune interface (Grace et al., 2014). Specifically, both TNF and IL-1 promote neuronal regeneration in different parts of the nervous system (Fontaine et al., 2002; Nadeau et al., 2011). Taken in combination, the presence of redundant mechanisms and the beneficial effects of proinflammatory cytokines in certain situations disqualifies the inhibition of any specific substance as a viable therapeutic method for neuropathic pain. Instead of inhibiting one or more proinflammatory cytokines, introducing a cytokine with broad-spectrum anti-inflammatory properties presents an alternate approach (Watkins and Maier, 2003; Grace et al., 2014). IL-10 A promising candidate for attenuating neuropathic pain is the anti-inflammatory cytokine IL-10 (Watkins and Maier, 2003, 2004). IL-10 performs potent anti-inflammatory actions. Neurons, astrocytes, and microglial cells in the CNS express IL-10. Activation of the IL-10 receptor has been shown to inhibit the release of numerous pro-inflammatory cytokines, including TNF, IL-1β, and IL-6 (Kwilasz et al., 2014). Additionally, IL-10 receptor activation stimulates the release of factors which mediate pro-inflammatory cytokines, including IL-1 receptor antagonist (Kwilasz et al., 2014). IL-10 exhibits neuroprotective properties through multiple mechanisms. Its inhibition of microglial secretion of pro-inflammatory cytokines increases excitatory amino acid transporter-2 (EAAT2) expression. This, in turn, reduces the buildup of excitatory amino acids and consequently prevents the neurotoxic effects of glutamate buildup (Bachis et al., 2001; Kim et al., 2011). IL-10 also appears to directly promote neuronal survival. Specific subsets of neurons display IL-10 receptors, and the activation of these receptors can promote neuron survival (Boyd et al., 2003). Taken together, its neuroprotective properties and broad-spectrum anti-inflammatory effects make IL-10 a promising candidate for treating neuropathic pain.