Samuel S. Duffy

The Contribution of Immune and Glial Cell Types in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterised by widespread areas of focal demyelination. Its aetiology and pathogenesis remain unclear despite substantial insights gained through studies of animal models, most notably experimental autoimmune encephalomyelitis (EAE). MS is widely believed to be immune-mediated and pathologically attributable to myelin-specific autoreactive CD4+ T cells. In recent years, MS research has expanded beyond its focus on CD4+ T cells to recognise the contributions of multiple immune and glial cell types to the development, progression, and amelioration of the disease. This review summarises evidence of T and B lymphocyte, natural killer cell, macrophage/microglial, astrocytic, and oligodendroglial involvement in both EAE and MS and the intercommunication and influence of each cell subset in the inflammatory process. Despite important advances in the understanding of the involvement of these cell types in MS, many questions still remain regarding the various subsets within each cell population and their exact contribution to different stages of the disease.

Characterisation of Immune and Neuroinflammatory Changes Associated with Chemotherapy-Induced Peripheral Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) and associated neuropathic pain is a debilitating adverse effect of cancer treatment. Current understanding of the mechanisms underpinning CIPN is limited and there are no effective treatment strategies. In this study, we treated male C57BL/6J mice with 4 cycles of either Paclitaxel (PTX) or Oxaliplatin (OXA) over a week and tested pain hypersensitivity and changes in peripheral immune responses and neuroinflammation on days 7 and 13 post 1st injection. We found that both PTX and OXA caused significant mechanical allodynia. In the periphery, PTX and OXA significantly increased circulating CD4+ and CD8+ T-cell populations. OXA caused a significant increase in the percentage of interleukin-4+ lymphocytes in the spleen and significant down-regulation of regulatory T (T-reg) cells in the inguinal lymph nodes. However, conditional depletion of T-reg cells in OXA-treated transgenic DEREG mice had no additional effect on pain sensitivity. Furthermore, there was no leukocyte infiltration into the nervous system of OXA- or PTX-treated mice. In the peripheral nervous system, PTX induced expression of the neuronal injury marker activating transcription factor-3 in IB4+ and NF200+ sensory neurons as well as an increase in the chemokines CCL2 and CCL3 in the lumbar dorsal root ganglion. In the central nervous system, PTX induced significant astrocyte activation in the spinal cord dorsal horn, and both PTX and OXA caused reduction of P2ry12+ homeostatic microglia, with no measurable changes in IBA-1+ microglia/macrophages in the dorsal and ventral horns. We also found that PTX induced up-regulation of several inflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL11, CCL4, CCL3, IL-12p70 and GM-CSF) in the spinal cord. Overall, these findings suggest that PTX and OXA cause distinct pathological changes in the periphery and nervous system, which may contribute to chemotherapy-induced neuropathic pain.

Immunotherapy targeting cytokines in neuropathic pain.

Pain is a complex warning system activated in response to potential or apparent danger and the absence of pain is detrimental. Nociceptive pain is high-threshold pain activated in the presence of intense stimuli, such as contact with a burning object, and is a protective system essential for detection of noxious stimuli. Inflammatory pain, caused by immune system activation in response to tissue injury or infection, is also protective, as it discourages physical contact with the damaged area and assists healing (Woolf, 2010). In contrast, neuropathic pain emanating from disease or damage to the somatosensory system, is somewhat unique in that it is not protective, but rather pathological. Neuropathic pain encompasses a series of heterogeneous conditions with some similar clinical manifestations. Peripheral examples include traumatic nerve injury, diabetic peripheral neuropathy and chemotherapy-induced peripheral neuropathy, whilst multiple sclerosis is an example of a disease which can result in centrally derived neuropathic pain. These conditions are characterized by a low-threshold chronic pain emanating from aberrant peripheral and central neuronal sensitization. Symptoms include paraesthesia, spontaneous ongoing pain, and evoked pain (e.g., hyperalgesia and allodynia). Recent studies investigating neuropathic pain have demonstrated significant associated immune system activation and a fundamental role for cytokine signaling (Austin and Moalem-Taylor, 2010). In this Opinion Article, we briefly summarize the progress made on research of cytokine involvement in neuropathic pain states and suggest that targeting key cytokines may prove useful in the development of new immune-therapeutics. However, further studies are required to determine which cytokine is the appropriate target for specific neuropathic pain conditions.

Depletion of Foxp3+ regulatory T cells increases severity of mechanical allodynia and significantly alters systemic cytokine levels following peripheral nerve injury

Neuropathic pain is a debilitating condition caused by damage to the somatosensory nervous system, such as peripheral nerve injury. The immune system, and in particular the adaptive T cell response, plays a key role in mediating such pain. Regulatory T (Treg) cells are a small subpopulation of inhibitory T cells that prevent autoimmunity, limit immunopathology and maintain immune homeostasis. Here, we investigated the effects of conditional depletion of Treg cells on mechanical allodynia and serum cytokines in mice with chronic constriction injury (CCI) of the sciatic nerve, an animal model of neuropathic pain. We demonstrate that CCI induced the infiltration of small numbers of Treg cells within effected neuronal tissue. Utilising the transgenic DEREG (DEpletion of REGulatory T cells) mice, we confirmed effective depletion of Foxp3+ Treg cells by diphtheria toxin injections. Following CCI we observed a transient, though significant, increase in pain hypersensitivity for Treg-depleted DEREG mice compared to non-Treg-depleted mice. Analysis of systemic cytokine levels demonstrated significant changes in serum cytokine expression profiles. In particular, we observed significant increases in systemic concentration of RANTES, IL-2 and IL-5, and significant decreases in IL-12 and IFN-γ in nerve-injured Treg-depleted DEREG mice. Further analysis indicated a substantial increase in the serum concentration of IL-12p40 as a direct result of Treg cell depletion. These results suggest that depletion of Foxp3+ Treg cells promote nerve injury-induced pain hypersensitivity, partially by inducing altered systemic concentrations of cytokines, which may act to regulate neuropathic pain.

Active immunization with myelin-derived altered peptide ligand reduces mechanical pain hypersensitivity following peripheral nerve injury

BackgroundT cells have been implicated in neuropathic pain that is caused by peripheral nerve injury. Immunogenic myelin basic protein (MBP) peptides have been shown to initiate mechanical allodynia in a T cell-dependent manner. Antagonistic altered peptide ligands (APLs) are peptides with substitutions in amino acid residues at T cell receptor contact sites and can inhibit T cell function and modulate inflammatory responses. In the present study, we studied the effects of immunization with MBP-derived APL on pain behavior and neuroinflammation in an animal model of peripheral nerve injury.MethodsLewis rats were immunized subcutaneously at the base of the tail with either a weakly encephalitogenic peptide of MBP (cyclo-MBP87-99) or APL (cyclo-(87-99)[A91,A96]MBP87-99) in complete Freund’s adjuvant (CFA) or CFA only (control), following chronic constriction injury (CCI) of the left sciatic nerve. Pain hypersensitivity was tested by measurements of paw withdrawal threshold to mechanical stimuli, regulatory T cells in spleen and lymph nodes were analyzed by flow cytometry, and immune cell infiltration into the nervous system was assessed by immunohistochemistry (days 10 and 30 post-CCI). Cytokines were measured in serum and nervous tissue of nerve-injured rats (day 10 post-CCI).ResultsRats immunized with the APL cyclo-(87-99)[A91,A96]MBP87-99 had significantly reduced mechanical pain hypersensitivity in the ipsilateral hindpaw compared to cyclo-MBP87-99-treated and control rats. This was associated with significantly decreased infiltration of T cells and ED1+ macrophages in the injured nerve of APL-treated animals. The percentage of anti-inflammatory (M2) macrophages was significantly upregulated in the APL-treated rats on day 30 post-CCI. Compared to the control rats, microglial activation in the ipsilateral lumbar spinal cord was significantly increased in the MBP-treated rats, but was not altered in the rats immunized with the MBP-derived APL. In addition, immunization with the APL significantly increased splenic regulatory T cells. Several cytokines were significantly altered after CCI, but no significant difference was observed between the APL-treated and control rats.ConclusionsThese results suggest that immune deviation by active immunization with a non-encephalitogenic MBP-derived APL mediates an analgesic effect in animals with peripheral nerve injury. Thus, T cell immunomodulation warrants further investigation as a possible therapeutic strategy for the treatment of peripheral neuropathic pain.

Effects of active immunisation with myelin basic protein and myelin-derived altered peptide ligand on pain hypersensitivity and neuroinflammation

Neuropathic pain is a debilitating condition in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Specific myelin basic protein (MBP) peptides are encephalitogenic, and myelin-derived altered peptide ligands (APLs) are capable of preventing and ameliorating EAE. We investigated the effects of active immunisation with a weakly encephalitogenic epitope of MBP (MBP87-99) and its mutant APL (Cyclo-87-99[A(91),A(96)]MBP87-99) on pain hypersensitivity and neuroinflammation in Lewis rats. MBP-treated rats exhibited significant mechanical and thermal pain hypersensitivity associated with infiltration of T cells, MHC class II expression and microglia activation in the spinal cord, without developing clinical signs of paralysis. Co-immunisation with APL significantly decreased pain hypersensitivity and neuroinflammation emphasising the important role of neuroimmune crosstalk in neuropathic pain.

Peripheral and Central Neuroinflammatory Changes and Pain Behaviors in an Animal Model of Multiple Sclerosis

Pain is a widespread and debilitating symptom of multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system. Although central neuroinflammation and demyelination have been implicated in MS-related pain, the contribution of peripheral and central mechanisms during different phases of the disease remains unclear. In this study, we used the animal model experimental autoimmune encephalomyelitis (EAE) to examine both stimulus-evoked and spontaneous pain behaviors, and neuroinflammatory changes, over the course of chronic disease. We found that mechanical allodynia of the hind paw preceded the onset of clinical EAE but was unmeasurable at clinical peak. This mechanical hypersensitivity coincided with increased microglial activation confined to the dorsal horn of the spinal cord. The development of facial mechanical allodynia also emerged in preclinical EAE, persisted at the clinical peak, and corresponded with pathology of the peripheral trigeminal afferent pathway. This included T cell infiltration, which arose prior to overt central lesion formation and specific damage to myelinated neurons during the clinical peak. Measurement of spontaneous pain using the mouse grimace scale, a facial expression-based coding system, showed increased facial grimacing in mice with EAE during clinical disease. This was associated with multiple peripheral and central neuroinflammatory changes including a decrease in myelinating oligodendrocytes, increased T cell infiltration, and macrophage/microglia and astrocyte activation. Overall, these findings suggest that different pathological mechanisms may underlie stimulus-evoked and spontaneous pain in EAE, and that these behaviors predominate in unique stages of the disease.

Attenuation of mechanical pain hypersensitivity by treatment with Peptide5, a connexin-43 mimetic peptide, involves inhibition of NLRP3 inflammasome in nerve-injured mice

ABSTRACT Connexin43 (Cx43) hemichannels in spinal cord astrocytes are implicated in the maintenance of neuropathic pain following peripheral nerve injury. Peptide5 is a Cx43 mimetic peptide that blocks hemichannels. In this study, we investigated the effects of spinal delivery of Peptide5 on mechanical pain hypersensitivity in two mouse models of neuropathic pain, peripheral nerve injury and chemotherapy‐induced peripheral neuropathy (CIPN). We demonstrated that 10 days following a chronic constriction injury (CCI) of the sciatic nerve, Cx43 expression, co‐localised predominantly with astrocytes, was increased in the ipsilateral L3–L5 lumbar spinal cord. An intrathecal injection of Peptide5 into nerve‐injured mice, on day 10 when pain was well‐established, caused significant improvement in mechanical pain hypersensitivity 8 h after injection. Peptide5 treatment resulted in significantly reduced Cx43, and microglial and astrocyte activity in the dorsal horn of the spinal cord, as compared to control saline‐treated CCI mice. Further in vitro investigations on primary astrocyte cultures showed that 1 h pre‐treatment with Peptide5 significantly reduced adenosine triphosphate (ATP) release in response to extracellular calcium depletion. Since ATP is a known activator of the NOD‐like receptor protein 3 (NLRP3) inflammasome complex, a key mediator of neuroinflammation, we examined the effects of Peptide5 treatment on NLRP3 inflammasome expression. We found that NLRP3, its adaptor apoptosis‐associated spec‐like protein (ASC) and caspase‐1 protein were increased in the ipsilateral spinal cord of CCI mice and reduced to naïve levels following Peptide5 treatment. In the models of oxaliplatin‐ and paclitaxel‐induced peripheral neuropathy, treatment with Peptide5 had no effect on mechanical pain hypersensitivity. Interestingly, in these CIPN models, although spinal Cx43 expression was significantly increased at day 13 following chemotherapy, NLRP3 expression was not altered. These results suggest that the analgesic effect of Peptide5 is specifically achieved by reducing NLRP3 expression. Together, our findings demonstrate that blocking Cx43 hemichannels with Peptide5 after nerve injury attenuates mechanical pain hypersensitivity by specifically targeting the NLRP3 inflammasome in the spinal cord. HighlightsCx43 is increased in the spinal cord following peripheral nerve injury.Blocking Cx43 with Peptide5 reduces mechanical pain hypersensitivity.Peptide5 treatment reduces spinal Cx43 expression and glial activationPeptide5 treatment reduces the expression of spinal NLRP3 inflammasome.The analgesic effect of Peptide5 depends on the assembly of the NLRP3 inflammasome.

The role of regulatory T cells in nervous system pathologies

Regulatory T (Treg) cells are a special subpopulation of immunosuppressive T cells that are essential for sustaining immune homeostasis. They maintain self‐tolerance, inhibit autoimmunity, and act as critical negative regulators of inflammation in various pathological states including autoimmunity, injury, and degeneration of the nervous system. Treg cells are known to convey both beneficial and detrimental influences in certain disease contexts, and accumulating research suggests that their action may be altered in a range of peripheral and central nervous system pathologies. In this review, we discuss emerging evidence for the dichotomous role of Treg cells in various neurological pathologies including multiple sclerosis, Guillain‐Barré syndrome, neuropathic pain, traumatic central nervous system injury, stroke, and neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimers disease, and Parkinsons disease. We are in the early stages of uncovering the role of Treg cells in these conditions, and a better understanding of the ways in which these cells operate in the nervous system will enable us to develop novel therapeutic interventions.

Managing Neuropathic Pain in Multiple Sclerosis: Pharmacological Interventions

BACKGROUND Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Of the plethora of motor and sensory disturbances experienced by sufferers, neuropathic pain is a highly prevalent and debilitating symptom, and at present remains extremely difficult to treat. Common forms of neuropathic pain seen in MS patients include central neuropathic pain, Lhermittes phenomenon and trigeminal neuralgia, which are all speculated to arise from specific patterns of lesion formation. OBJECTIVE Efficacious pharmacological interventions for the treatment of neuropathic pain associated with MS are lacking, and have been largely informed by drug trials in peripheral neuropathies and spinal cord injury. METHOD/RESULTS Neuropathic pain in MS is inadequately relieved by conventional analgesics, and first-line therapies are generally comprised of anti-depressive and anti-convulsive drugs. A range of alternatives have been proposed and tested with variable success, including cannabinoids and certain opioid analgesics. Animals with experimental autoimmune encephalomyelitis (EAE), an autoimmune model of MS, also exhibit neuropathic pain symptoms. CONCLUSION Studies aimed at understanding the mechanisms underlying EAE-induced neuropathic pain and investigating the efficacy of novel pharmacological interventions at the animal level offer an exciting area of future research, and may inform future therapeutic options for MS-associated neuropathic pain.