Epigenetic Regulatory Mechanisms Of Histone Demethylase In Mastitis

Mastitis,characterized by inflammation of parenchyma of mammary glands,is one of the most common inflammatory diseases affecting dairy cattle worldwide.Mastitis is an economically important pathology associated with loss of milk production,deterioration of the milk quality,increased treatment cost and premature culling of infective dairy cattle,being considered one of the most costly to dairy industry.Currently,antibiotic treatment is a common component of mastitis control programs.On the contrary,emergence and spread of bacterial resistance caused by antibiotic therapy and antibiotic residue in milk are urgent matter of particular public interest.Therefore,the continuous search for new therapeutic alternatives,effective in the prevention and treatment of bovine mastitis,is urgent.As an extension of the human genome,epigenetics of inflammation has attracted the attention of researchers in recent years.Our previous studies have confirmed that histone acetylation is involved in regulating the inflammatory response of mastitis.However,the epigenetic regulatory mechanisms of histone methylation in the innate immune response have not been reported.Using in vivo models of lipopolysaccharide(LPS)-induced inflammation and in vitro assays in mammary epithelial cells,we identified the novel regulatory roles and underlying mechanisms of histone demethylase(HDMs)on LPS-induced mastitis.The expressions of histone demethylases were observed,and specific inhibitors were given to explore the correlation between HDMs and inflammation.Small interfering RNA(siRNA)and chromatin immunoprecipitation(ChIP)were measured to explore the novel regulatory roles and underlying mechanisms of HDMs on inflammatory gene expression,revealing the pathogenesis of bovine mastitis.Together,these findings possibly provide a novel insight for treatment,prevention and disease resistance breeding of bovine mastitis.Firstly,in LPS-induced inflammatory model in vivo,the correlation between HDMs and inflammation was explored.Mammary gland were collected for the following experiments after treatment.Our results showed that LPS treatment resulted in a significant increase in LSD1 and JMJD3 gene and protein expression.GSK-LSD1 2HCl and GSK-J1 are selective inhibitor of LSD1 and JMJD3 enzyme activity,respectively.Treatment of mice with GSK-LSD1 2HCl and GSK-J1 inhibited LSD1 and JMJD3 protein expression,inhibited infiltration of inflammatory cells and myeloperoxidase(MPO)activity as well as decreased the production of TNF-?,IL-6 and IL-1? in mammary gland.Mechanistic investigations suggested that LSD1 and JMJD3 inhibition led to the increase of histone H3K4me2 and H3K27me3.Here,we investigated that HDMs expression are positively correlated with the inflammatory response in mastitis.Mechanismly,pharmacological inhibition of HDMs expression reduce the inflammatory response of mammary gland.The results showed that histone methylation is involved in epigenetic regulation of inflammatory response.Secondly,an in vitro inflammatory model of mouse mammary epithelial cells induced by LPS was established,to explore the mechanism of HDMs involved in the regulation of inflammatory gene expression.The correlation between HDMs and inflammatory response was revivified in vitro,and the results were consistent with those obtained in vivo.Given the role of HDMs and inflammatory response,further work is needed to identify potential point for crosstalk betwwen LSD1,JMJD3 and inflammatory gene expression.In LPS-stimulated mouse mammary epithelial cells,LSD1 and JMJD3 were silenced or inhibited,respectively.The mRNA expression levels of inflammatory cytokines(TNF-?,IL-1? and IL-6)were detected by qRT-PCR.The results showed that the mRNA expression levels of TNF-?,IL-1? and IL-6 were significantly decreased by silencing or inhibiting LSD1 and JMJD3.The recruitment of LSD1,JMJD3 and related modification sites H3K4me2 and H3K27me3 at TNF-?,IL-1? and IL-6 promoters was detected by ChIP-q PCR.Compared with the blank control group,the recruitment of histone demethylase LSD1 and JMJD3 in TNF-?,IL-1? and IL-6 promoter regions was significantly decreased,while the recruitment of H3K4me2 and H3K27me3 were increased.These results suggest that HDMs may regulate the expression of inflammatory genes by affecting histone methylation of H3K4me2 and H3K27me3.TLR4 is known to be a member of the TLRs,a pattern recognition receptor that plays a crucial role in innate immunity and inflammation.The results further showed that inhibition or knockdown of LSD1 and JMJD3 could significantly inhibit the expression of TLR4 in LPS-induced MMECs.We further investigated the the level of LSD1,JMJD3,H3K4me2 and H3K27me3 in the promotor of TLR4 with a ChIP assay.The results of ChIP experiment showed that inhibited LSD1 and JMJD3 could decrease the recruitment of LPS-stimulated LSD1 and JMJD3 at TLR4 promoter.Moreover,enrichment of H3K4me2 and H3K27me3 was observed in the promotor of TLR4.Subsequently,the phosphorylation levels of I?B? and p65 of NF-?B signaling pathway were significantly decreased after HDMs inhibitors treatment.Finally,the mechanism of histone demethylase LSD1 and JMJD3 involved in the regulation of inflammation was further confirmed in bovine mammary epithelial cells.The bovine mammary epithelial cells were isolated from the mammary tissue of lactating cows.CCK8,qRT-PCR,western blotting and other experimental methods were used to verify the mechanism of HDMs regulating the expression of inflammatory genes,and the results we obtained were consistent with those obtained in the inflammatory model of MMECs.In summary,we investigated the novel regulatory roles and underlying mechanisms of HDMs on LPS-induced mastitis.On the one hand,histone demethylases LSD1 and JMJD3 were involved in inflammatory gene expression by the demethylation of H3K4me2 and H3K27me3 on the promoter region of inflammatory genes and regulate by changing the modification.In addition,LSD1 and JMJD3 directly participate in TLR4 expression by affecting the methylation of H3K4me2 and H3K27me3 at the promoter,thereby affecting the expression of key signaling protein of NF-?B signaling pathway and regulating the expression of inflammatory genes.Together,these findings possibly provide a novel insight into the epigenetic control on inflammation in mastitis.