Nuclear Carbonic Anhydrase 6B Associates With Prmt5 To Epigenetically Promote IL-12 Expression In Innate Response

Innate immune response is critical for host defense against invading pathogens.Microbial and dangerous signals induce pattern-recognition receptors(PRRs)-dependent activation of inflammatory signaling in innate immune cells such as macrophages and dendritic cells,leading to production of a plethora of proinflammatory cytokines which display diversified immunological and inflammatory effects.Immune response is comprised of immune recognition,immune activation and immune effect.This process is mediated by a number of immune organs,immune cells and immune molecules.Among these molecules,interleukin 12(IL-12)is a critical pro-inflammatory cytokine produced by macrophages and dendritic cells(DCs)in response to pathogens infection.IL-12 can induce the production of interferon-γ(IFN-γ)by Thl cells and Natural Killer cells(NK cells),contributing to the innate and adaptive immunity against infection.Therefore,IL-12 forms a link between innate immunity and adaptive immunity.IL-12 belongs to IL-12 family and is composed of IL-12p35 and IL-12p40 subunit.IL-12 family has three members.One is interleukin 23(IL-23),composed of IL-23p19 and IL-12p40.The other is IL-27,composed of EBIB and IL-27p28.IL-12 exerts important function in immune response.However,the regulation mechanisms of the expression of IL-12 are incompletely understood.Here we describe a new mechanism for regulation of the IL-12 expression by B type of carbonic anhydrase 6(Car6-b,encoding CA-VI B).E-selectin is a key adhesion molecule encoded by Sele mediating adhension and migration of leukocytes.In our previous study,we found that E-selectin not only mediated the leucocytes rolling on endothelial cells in the vessels,but also promoted IFN-γ receptor β(IFNyR2)expression on the membrane of macrophages to mediate macrophages activation.To study LM-triggered innate response,we compared mRNA expression in macrophages from Sele+/+ and Sele-/-mice infected with LM in vivo.Interestingly,Car6,which encoded CA-VI,was significantly decreased in Sele--macrophages.Carbonic anhydrases(CAs)are ubiquitous in all mammals,belonging to a-CA.These enzymes are metalloenzymes including Zn2+.Physiologically,they catalyze the interconversion between carbon dioxide and bicarbonate.This reaction is critical for pH regulation and homeostasis.So far,13 acti-ve isoenzymes in the mammalian a-CA family have been identified.Among these 13 CAs,only CA-VI is secreted in saliva and milk.Car6 has two transcripts,one is secreted A type of CA-VI A(encoded by Car6-a)and the other is intracellular CA-VI B.We firstly screened Car6 expression profile in immune cells and found that only Car6-b expressed in immune cells and preferially expressed in macrophages.In order to identify the function of Car6-b in innate immune response,we downregulated Car6-b expression in primary peritoneal macrophages through siRNA interference and found that knocking down Car6-b reduced the expression of IL-12 in macrophages stimulated by Toll-like receptors(TLRs)ligands.Also,deficiency of Car6-b significantly reduced the production of IL-12 upon LM infection both in vitro and in vivo.Car6-/-mice impaired the clearance of LM,which caused much lower survival rate of Car6 KO mice compared to wild type mice after LM infection.We further investigated the mechanisms of IL-12 regulation by Car6-b and discovered that the nuclear localized CA-VI B selectively promotes IL-12 expression by interaction with protein arginine N-methyltransferase 5(PRMT5),which reduces symmetric dimethylation of histone H3 arginine 8 modification(H3R8me2s)at 1112 promoter to facilitate chromatin accessibility,enhancing c-Rel binding to Il12b promoter.Our findings demonstrate a novel function of Car6 in innate immunity by regulating IL-12,which might provide the insight of potential treatment for the future.