Veronica I. Shubayev

Immunotherapy holds promise as a non-addictive treatment of refractory chronic pain states. Increasingly, sex is recognized to impact immune regulation of pain states, including mechanical allodynia (pain from non-painful stimulation) that follows peripheral nerve trauma. This study aims to assess the role of B cells in sex-specific responses to peripheral nerve trauma. Using a rat model of sciatic nerve chronic constriction injury (CCI), we analyzed sex differences in (i) the release of the immunodominant neural epitopes of myelin basic protein (MBP); (ii) the levels of serum immunoglobulin M (IgM)/immunoglobulin G (IgG) autoantibodies against the MBP epitopes; (iii) endoneurial B cell/CD20 levels; and (iv) mechanical sensitivity behavior after B cell/CD20 targeting with intravenous (IV) Rituximab (RTX) and control, IV immunoglobulin (IVIG), therapy. The persistent MBP epitope release in CCI nerves of both sexes was accompanied by the serum anti-MBP IgM autoantibody in female CCI rats alone. IV RTX therapy during CD20-reactive cell infiltration of nerves of both sexes reduced mechanical allodynia in females but not in males. IVIG and vehicle treatments had no effect in either sex. These findings provide strong evidence for sexual dimorphism in B-cell function after peripheral nervous system (PNS) trauma and autoimmune pathogenesis of neuropathic pain, potentially amenable to immunotherapeutic intervention, particularly in females. A myelin-targeted serum autoantibody may serve as a biomarker of such painful states. This insight into the biological basis of sex-specific response to neuraxial injury will help personalize regenerative and analgesic therapies.

Background: Autoantibodies, a hallmark of autoimmune disorders, are thought to contribute to the pathogenesis of chronic pain. Mechanical hypersensitivity is a severe complication of peripheral nerve injury mediated through sex-specific activity of immune and glial cells. Whether B cell contribution to nerve injury and pain is sexually dimorphic remains unknown. Methods: CD20 immunoblotting and immunostaining was conducted in female and male sciatic nerve after chronic constriction injury (CCI). B cell/CD20 targeting by bolus intravenous (IV) Rituximab or control immunoglobulin (IVIG) therapy or vehicle was administered in female and male rats post-CCI, followed by von Frey mechanical sensitivity testing. The cryptic myelin basic protein (MBP) epitopes were identified by immunostaining (EMD-AB5864 Millipore antibody) in female and male CCI nerves. ELISA was used to detect serum autoantibodies against the immunodominant MBP(84-104) epitope was conducted in female and male rats post-CCI. Results: Infiltration of CD20-positive cells, presumably B cell, was observed in female and male nerve at one week post-CCI. A single bolus IV anti-CD20/Rituximab therapy at one week post-CCI reduced the degree of mechanical hypersensitivity in female, but not in male, rats. IVIG therapy and vehicle treatment had no effect in either sex. Sustained release of the cryptic MBP epitopes from myelin was observed in nerves post-CCI in both sexes. Remarkably, serum anti-MBP(84-104) autoantibody was detected in female, but not in male, rats post-CCI. Conclusions: B cell activity and autoantibody production after peripheral nerve injury are sexually dimorphic. In females, mechanical hypersensitivity arising due to autoimmune remodeling of myelin on sensory neuraxis is amenable to immunotherapeutic intervention.

In the peripheral nerve, pro-inflammatory matrix metalloproteinase (MMP)-9 performs essential functions in the acute response to injury. Whether MMP-9 activity contributes to late-phase injury or whether MMP-9 expression or activity after nerve injury is sexually dimorphic remains unknown. Patterns of MMP-9 expression, activity and excretion were assessed in a model of painful peripheral neuropathy, sciatic nerve chronic constriction injury (CCI), in female and male rats. Real-time Taqman RT-PCR for MMP-9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) of nerve samples over a 2-month time course of CCI was followed by gelatin zymography of crude nerve extracts and purified MMP-9 from the extracts using gelatin Sepharose-beads. MMP excretion was determined using protease activity assay of urine in female and male rats with CCI. The initial upsurge in nerve MMP-9 expression at day 1 post-CCI was superseded more than 100-fold at day 28 post-CCI. The high level of MMP-9 expression in late-phase nerve injury was accompanied by the reduction in TIMP-1 level. The absence of MMP-9 in the normal nerve and the presence of multiple MMP-9 species (the proenzyme, mature enzyme, homodimers, and heterodimers) was observed at day 1 and day 28 post-CCI. The MMP-9 proenzyme and mature enzyme species dominated in the early- and late-phase nerve injury, consistent with the high and low level of TIMP-1 expression, respectively. The elevated nerve MMP-9 levels corresponded to the elevated urinary MMP excretion post-CCI. All of these findings were comparable in female and male rodents. The present study offers the first evidence for the excessive, uninhibited proteolytic MMP-9 activity during late-phase painful peripheral neuropathy and suggests that the pattern of MMP-9 expression, activity, and excretion after peripheral nerve injury is universal in both sexes.

Complex regional pain syndrome is an extremely painful condition that develops after trauma to a limb. Complex regional pain syndrome exhibits autoimmune features in part mediated by autoantibodies against muscarinic‐2 acetylcholine (M2) receptor. The mechanisms underlying the M2 receptor involvement in complex regional pain syndrome remain obscure. Based on our recent work demonstrating that limb nerve trauma releases a potent proalgesic, immunodominant myelin basic protein fragment, our present sequence database analyses reveal an unexpected and previously undescribed structural homology of the proalgesic myelin basic protein fragment with the M2 receptor. As both complex regional pain syndrome and the proalgesic myelin basic protein activity are prevalent in females, this myelin basic protein/M2 homology presents an inviting hypothesis explaining the mechanisms of autoimmune pathogenesis and sexual dimorphism that underlies vulnerability toward developing complex regional pain syndrome and other pain states with neuropathic features. This hypothesis may aid in the development of novel diagnostic and therapeutic strategies to chronic pain.

Neural Regen Res, Official publication of Publishing House of Neural Regeneration Research China Association of Rehabilitation Medicine,China