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

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)-ß, 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.

Biochemically altered myelin triggers autoimmune demyelination.

Although immune attack against central nervous system (CNS) myelin is a central feature of multiple sclerosis (MS), its root cause is unresolved. In this report, we provide direct evidence that subtle biochemical modifications to brain myelin elicit pathological immune responses with radiological and histological properties similar to MS lesions. A subtle myelinopathy induced by abbreviated cuprizone treatment, coupled with subsequent immune stimulation, resulted in lesions of inflammatory demyelination. The degree of myelin injury dictated the resulting immune response; biochemical damage that was too limited or too extensive failed to trigger overt pathology. An inhibitor of peptidyl arginine deiminases (PADs), enzymes that alter myelin structure and correlate with MS lesion severity, mitigated pathology even when administered only during the myelin-altering phase. Moreover, cultured splenocytes were reactive against donor myelin isolates, a response that was substantially muted when splenocytes were exposed to myelin from donors treated with PAD inhibitors. By showing that a primary biochemical myelinopathy can trigger secondary pathological inflammation, "cuprizone autoimmune encephalitis" potentially reconciles conflicting theories about MS pathogenesis and provides a strong rationale for investigating myelin as a primary target for early, preventative therapy.

AlphaB-crystallin regulates remyelination after peripheral nerve injury.

AlphaB-crystallin (αBC) is a small heat shock protein that is constitutively expressed by peripheral nervous system (PNS) axons and Schwann cells. To determine what role this crystallin plays after peripheral nerve damage, we found that loss of αBC impaired remyelination, which correlated with a reduced presence of myelinating Schwann cells and increased numbers of nonmyelinating Schwann cells. The heat shock protein also seems to regulate the cross-talk between Schwann cells and axons, because expected changes in neuregulin levels and ErbB2 receptor expression after PNS injury were disrupted in the absence of αBC. Such dysregulations led to defects in conduction velocity and motor and sensory functions that could be rescued with therapeutic application of the heat shock protein in vivo. Altogether, these findings show that αBC plays an important role in regulating Wallerian degeneration and remyelination after PNS injury.

Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis.

The cysteine protease inhibitor Cystatin C (CST3) is highly expressed in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental autoimmune encephalomyelitis (EAE; a model of MS), but its roles in the diseases are unknown. Here, we show that CST3 plays a detrimental function in myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE but only in female animals. Female Cst3 null mice display significantly lower clinical signs of disease compared to wild-type (WT) littermates. This difference is associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, major histocompatibility complex [MHC] II, LC3A/B) involved in antigen processing, presentation, and co-stimulation in antigen-presenting cells (APCs). In contrast, male WT and Cst3-/- mice and cells show no differences in EAE signs or APC function. Further, the sex-dependent effect of CST3 in EAE is sensitive to gonadal hormones. Altogether, we have shown that CST3 has a sex-dependent role in MOG35-55-induced EAE.

Microglia response following acute demyelination is heterogeneous and limits infiltrating macrophage dispersion.

Microglia and infiltrating macrophages are thought to orchestrate the central nervous system (CNS) response to injury; however, the similarities between these cells make it challenging to distinguish their relative contributions. We genetically labeled microglia and CNS-associated macrophages to distinguish them from infiltrating macrophages. Using single-cell RNA sequencing, we describe multiple microglia activation states, one of which was enriched for interferon associated signaling. Although blood-derived macrophages acutely infiltrated the demyelinated lesion, microglia progressively monopolized the lesion environment where they surrounded infiltrating macrophages. In the microglia-devoid sciatic nerve, the infiltrating macrophage response was sustained. In the CNS, the preferential proliferation of microglia and sparse microglia death contributed to microglia dominating the lesion. Microglia ablation reversed the spatial restriction of macrophages with the demyelinated spinal cord, highlighting an unrealized macrophages-microglia interaction. The restriction of peripheral inflammation by microglia may be a previously unidentified mechanism by which the CNS maintains its "immune privileged" status.

Intradorsal hippocampal microinjection of lithium reverses morphine-induced impairment of memory in mice: interactions with dopamine receptor mechanism(s).

In this study, the influence of lithium administration on morphine-induced memory impairment and possible interactions with dopamine receptor mechanism have been investigated. Considering that the dorsal hippocampus is involved in memory, lithium and dopamine agents were injected into the CA1 regions of this site (intra-CA1). For memory assessment, a one-trial step-down inhibitory avoidance task was used in adult male mice. The results showed that posttraining subcutaneous administration of morphine (5 mg/kg) decreased the step-down latency on the test day, and this effect was reversed by pretest administration of the drug, which may be a form of morphine state-dependent learning. Interestingly, pretest intra-CA1 microinjection of different doses of lithium (2 and 4 microg/mouse) reversed memory impairment induced by posttraining morphine (5 mg/kg, subcutaneously). In contrast, pretest intra-CA1 administration of either D1 receptor antagonist SCH23390 (0.01-1 microg/mouse) or D2 receptor antagonist sulpiride (5-10 microg/mouse) inhibited the improvement of memory retrieval by lithium (4 microg/mouse, intra-CA1). Single pretest administration of SCH23390 or sulpiride neither elicited any response, nor reversed morphine-induced amnesia. In conclusion, it can be suggested that the dorsal hippocampal dopaminergic system is involved in the improving response of lithium on morphine-induced amnesia.

Inhibition of morphine-induced amnesia in morphine-sensitized mice: involvement of dorsal hippocampal GABAergic receptors.

In the present study, the effects of bilateral injections of the GABAergic receptor agents into the dorsal hippocampal CA1 regions (intra-CA1) on morphine-induced amnesia were examined in morphine sensitized-mice. Pre-training subcutaneous (s.c.) administration of morphine (5 mg/kg) suppressed the learning of a one-trial passive avoidance task. Amnesia induced by pre-training morphine was significantly reversed in mice which had previously received once daily injections of morphine (20 and 30 mg/kg, s.c.) for 3 days, which may be due to behavioral sensitization. Intra-CA1 injections of GABAA receptor agonist, muscimol (0.125, 0.25, 0.5 and 1 microg/mouse) or the GABAB receptor agonist, baclofen (1, 1.5 and 2 microg/mouse) prior to injection of morphine (20 mg/kg per dayx3 days) decreased the reversion of morphine-induced amnesia in morphine sensitized-mice. Daily intra-CA1 injections of muscimol or baclofen plus saline for 3 days did not alter memory formation. Furthermore, during development of sensitization, the combination of GABAA receptor antagonist, bicuculline (0.25, 0.5, 1 and 2 microg/mouse) with an ineffective dose of morphine (5 mg/kg, s.c.) reversed the amnesia induced by pre-training morphine. However, the same treatment with GABAB receptor antagonist, CGP35348 (2.5-40 microg/mouse) had no effect on the morphine response. On the other hand, daily intra-CA1 injections of bicuculline or CGP35348 alone for 3 days did not alter the amnesia induced by pre-training injection of morphine. The results suggest that morphine sensitization reverses the impairment of memory induced by morphine and that GABAergic receptors of the dorsal hippocampus may play an important role in this effect.

Characterization of migration parameters on peripheral and central nervous system T cells following treatment of experimental allergic encephalomyelitis with CRYAB.

CRYAB, a small heat shock protein, was previously shown to decrease neuroinflammation in experimental allergic encephalomyelitis (EAE). We investigated whether the expression of cell adhesion molecules and chemokine receptors on peripheral and spinal cord T cells, that could possibly affect their migration to the central nervous system, was altered following EAE CRYAB treatment. Less LFA-1+ lymphocytes and lower levels of iTAC, MCP-5 and MIG were observed in spinal cords of CRYAB-injected EAE animals. In addition, fewer blood T cells expressed CCR6, CXCR4 and CCR7 and in vivo-derived CRYAB EAE CD4+ lymphocytes were less migratory towards a MIP-3alpha gradient in vitro.