Anti-CLEC7A monoclonal antibody, clone 2A11 [R-PE]

We have previously identified activation of microglia and induction of the early growth response gene 1 (Egr1) in the retina of retinoschisin-deficient (Rs1h(-/Y)) mice. We hypothesized that microglial expression of Egr1 might support retinal microgliosis. To test this, Egr1 transcript levels were determined in RNAs isolated from early postnatal retinas and primary microglia from Rs1h(-/Y) mice and wild-type controls. Egr1 mRNA expression was strongly induced in retinoschisin-deficient retinas as well as in ex vivo isolated microglia. Increased microglial Egr1 protein expression was concordantly detected in retinal sections of Rs1h(-/Y) mice using immunohistochemistry. Prominent activation-dependent Egr1 mRNA and protein expression was also confirmed in murine BV-2 microglia. Using binding site prediction and chromatin immunoprecipitation, we identified that the Egr1 promoter itself and the microglial marker genes Clec7a and Caspase11 are direct transcriptional targets of Egr1. Over-expression of Egr1 in BV-2 cells by adenoviral infection promoted Clec7a and Caspase11 mRNA synthesis, whereas expression of the Egr1 repressor NAB2 blocked the transcription of these genes. To analyze whether Egr1 was absolutely required for microglial marker expression in vivo, transcript levels were quantified in Rs1h(-/Y)/Egr1(-/-) retinas. No significant differences in activation marker expression could be measured in retinal tissue from Rs1h(-/Y)/Egr1(-/-) mice compared to Rs1h(-/Y) mice, suggesting that lack of Egr1 does not impair transcription of microglia genes in vivo. Taken together, our findings suggest that increased Egr1 expression is present in activated retinal microglia and contributes to their activation. However, up-regulation of Egr1 is not absolutely required for retinal microglia activation in vivo.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of various autoantibodies. This disease causes disabling neuropsychiatric symptoms even in the absence of apparent inflammation in the central nervous system (CNS), but the mechanisms involved remain unknown. Innate immune-mediated inflammation has attracted attention as a pathogenic mechanism in neuropsychiatric diseases. Methods: We investigated the CNS of lupus-prone mice focusing on innate immunity. Three strains of lupus-prone mice, Fc gamma RIIB(-/-)Yaa, an F1 hybrid of NZB and NZW (NZB/NZW) mice, and MRL/Fas(lpr) (MRL/lpr) mice were used to analyze CNS immunopathology. Results: Flow cytometry analysis demonstrated the numbers of brain CD45(+) cells were increased compared with controls in lupus-prone mice. Upregulation of MHC class I and PDCA1 was observed in microglia and CD11b(+) myeloid cells of lupus-prone mice, indicating they were activated in response to interferons (IFN). Microglial gene expression analysis of Fc gamma RIIB(-/-)Yaa mice revealed the upregulation of IFN-responsive genes and inflammationrelated genes including Axl, Clec7a, and Itgax, which were previously reported in neurodegenerative conditions and primed conditions. Upregulated chemokine gene expressions including Ccl5 and Cxcl10 were concurrent with increased numbers of T cells and monocytes, especially Ly6C(lo) monocytes in the CNS. Upregulation of Axl, Clec7a, Itgax, Ccl5, and Cxcl10 was also observed in NZB/NZW mice, indicating common lupus pathology. The primed status of microglia in Fc gamma RIIB(-/-)Yaa mice was also demonstrated by morphological changes such as enlarged cell bodies with hypertrophic processes, and hyperreactivity to lipopolysaccharide. Immunohistochemistry of Fc gamma RIIB(-/-)Yaa mice indicated reactive responses of astrocytes and vascular endothelium. Behavioral studies of Fc gamma RIIB(-/-)Yaa mice revealed depressive-like behavior and heat hyperalgesia in the forced swim test and the tail-flick test, respectively. Conclusions: Our data indicated that microglia in lupus exhibit a unique primed phenotype characterized by the upregulated expressions of neurodegeneration-related genes and IFN-responsive genes. Interaction with peripheral cells and brain resident cells was presumed to orchestrate neuroinflammation. Targeting innate immune cells, such as microglia and monocytes, may be a promising therapeutic approach for neuropsychiatric SLE.