Cystatin C and sex dimorphism in experimental autoimmune encephalomyelitis

This dissertation characterizes the role of a lysosomal enzyme inhibitor, Cystatin C (CysC), in an animal model of multiple sclerosis (MS), called experimental allergic encephalomyelitis (EAE). MS is an autoimmune disease characterized by infiltration of myelin reactive immune cells into the central nervous system (CNS) that is more prevalent in genetically susceptible women compared to men. Alteration in the level of CysC in the CNS of MS patients is known, but its role has not been determined. Towards clarifying the role of CysC in EAE, I initially hypothesized that CysC would have a beneficial function in EAE due to its protective role in another autoimmune disease, collagen-induced arthritis. Unexpectedly, using C57BL/6 and CysC null (CysCKO) mice, I discovered that CysC plays a detrimental role in female but not male EAE mice. This sex dimorphism in the function of CysC could also be observed in immune cell infiltration and demyelination in EAE. Towards elucidating the cellular mechanism(s) underlying the sex difference driven by CysC in EAE, I discovered that the protease inhibitor promotes the activation of antigen presenting cells such as macrophages in females that subsequently mediates stimulation of CD4+ T cells. The T cell activation-promoting ability of CysC in female macrophages appears to be related to antigen presentation in macrophages since molecular mediators of antigen processing and loading (LC3II) and presentation (CD80, CD86, MHC II) were disrupted in female, but not male, macrophages that lacked CysC. Cumulatively, these observations indicate that CysC drives sex dimorphism in macrophages. Lastly, to understand why CysC would induce sex differences in macrophage function and thus EAE susceptibility, I investigated if sex hormones were involved. I found that CysC plays a detrimental role in EAE in the presence of estrogen. This dissertation thus provides novel evidence that CysC has a role in mediating sex dimorphism in EAE

Presence and activation of pro-inflammatory macrophages are associated with CRYAB expression in vitro and after peripheral nerve injury

Abstract Background Inflammation constitutes both positive and negative aspects to recovery following peripheral nerve injury. Following damage to the peripheral nervous system (PNS), immune cells such as macrophages play a beneficial role in creating a supportive environment for regrowing axons by phagocytosing myelin and axonal debris. However, a prolonged inflammatory response after peripheral nerve injury has been implicated in the pathogenesis of negative symptoms like neuropathic pain. Therefore, the post-injury inflammation must be carefully controlled to prevent secondary damage while allowing for regeneration. CRYAB (also known as alphaB-crystallin/HSPB5) is a small heat shock protein that has many protective functions including an immunomodulatory role in mouse models of multiple sclerosis, spinal cord injury, and stroke. Because its expression wanes and rebounds in the early and late periods respectively after PNS damage, and CRYAB null mice with sciatic nerve crush injury display symptoms of pain, we investigated whether CRYAB is involved in the immune response following PNS injury. Methods Sciatic nerve crush injuries were performed in age-matched Cryab knockout (Cryab−/−) and wildtype (WT) female mice. Nerve segments distal to the injury site were processed by immunohistochemistry for macrophages and myelin while protein lysates of the nerves were analyzed for cytokines and chemokines using Luminex and enzyme-linked immunosorbent assay (ELISA). Peritoneal macrophages from the two genotypes were also cultured and polarized into pro-inflammatory or anti-inflammatory phenotypes where their supernatants were analyzed for cytokines and chemokines by ELISA and protein lysates for macrophage antigen presenting markers using western blotting. Results We report that (1) more pro-inflammatory CD16/32+ macrophages are present in the nerves of Cryab−/− mice at days 14 and 21 after sciatic nerve crush-injury compared to WT counterparts, and (2) CRYAB has an immunosuppressive effect on cytokine secretion [interleukin (IL)-β, IL-6, IL-12p40, tumor necrosis factor (TNF)-α] from pro-inflammatory macrophages in vitro. Conclusions CRYAB may play a role in curbing the potentially detrimental pro-inflammatory macrophage response during the late stages of peripheral nerve regeneration