Decreased miR-4512 Levels in Monocytes and Macrophages of Individuals With Systemic Lupus Erythematosus Contribute to Innate Immune Activation and Neutrsophil NETosis by Targeting TLR4 and CXCL2.

Objective: Systemic lupus erythematosus (SLE) is an autoimmune disease with complex etiology that is not yet entirely understood. We aimed to elucidate the mechanisms and therapeutic potential of microRNAs (miRNAs) in SLE in a Tibetan population. Methods: Peripheral blood mononuclear cells from SLE patients (n = 5) and healthy controls (n = 5) were used for miRNA-mRNA co-sequencing to detect miRNAs related to immune abnormalities associated with SLE. Luciferase reporter assay was used to identify potential targets of candidate miRNA. The target genes were verified in miRNA-agomir/antagomir transfection assays with multiple cells lines and by expression analysis. The effects of candidate miRNA on monocyte and macrophage activation were evaluated by multiple cytokine profiling. Neutrophil extracellular traps (NETs) formation was analyzed in vitro by cell stimulation with supernatants of monocytes and macrophages transfected with candidate miRNA. The rodent MRL/lpr lupus model was used to evaluate the therapeutic effect of CXCL2Ab on SLE and the regulation effect of immune disorders. Results: Integrated miRNA and mRNA expression profiling identified miRNA-4512 as a candidate miRNA involved in the regulation of neutrophil activation and chemokine-related pathways. MiR-4512 expression was significantly reduced in monocytes and macrophages from SLE patients. MiR-4512 suppressed the TLR4 pathway by targeting TLR4 and CXCL2. Decreased monocyte and macrophage miR-4512 levels led to the expression of multiple proinflammatory cytokines in vitro. Supernatants of miR-4512 antagomir-transfected monocytes and macrophages significantly promoted NETs formation (P < 0.05). Blocking of CXCL2 alleviated various pathogenic manifestations in MRL/lpr mice, including kidney damage and expression of immunological markers of SLE. Conclusions: We here demonstrated the role of miR-4512 in innate immunity regulation in SLE. The effect of miR-4512 involves the regulation of monocytes, macrophages, and NETs formation by direct targeting of TLR4 and CXCL2, indicating the miR-4512-TLR4-CXCL2 axis as a potential novel therapeutic target in SLE.

The immune function of prophenoloxidase from red swamp crayfish (Procambarus clarkii) in response to bacterial infection.

Prophenoloxidase (proPO) is the zymogen form of phenoloxidase (PO), a key enzyme in melanization cascade that has been co-opted in invertebrate immune reactions. There have been reported that proPO plays many essential roles in the crustacean immune system. However, little is known about the function of proPO from red swamp crayfish (Procambarus clarkii) which is an important cultured species worldwide. Here, we cloned and expressed proPO gene from red swamp crayfish (PcproPO). Subsequently, specific antibody against PcproPO was generated. The immune function of PcproPO was further characterized in vitro and in vivo. The results showed that the expression of PcproPO mRNA could be significantly up-regulated during the challenge of Gram-positive-negative (Vibrio parahaemolyticus) and Gram-positive-positive bacterial (Staphylococcus aureus). Furthermore, the purified recombinant PcproPO protein had a strong affinity binding to both bacteria and polysaccharides. In vivo knockdown of PcproPO could significantly reduce the crayfish bacterial clearance ability, resulting in the higher mortality of the crayfish during V. parahaemolyticus infection. In addition, in vitro knockdown of PcproPO in the hemocytes significantly reduced the phenoloxidase (PO) activity and the bacterial clearance ability, indicating that PcproPO might involve in hemocyte-mediated melanization. Our results will shed a new light on the immune function of PcproPO in the crayfish.

PcToll3 was involved in anti-Vibrio response by regulating the expression of antimicrobial peptides in red swamp crayfish, Procambarus clarkii.

Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus.