The Regulatory Mechanism Of CD36 On Immune Repsonse Of Mammary Epithelial Cells Of Dairy Goats

The scavenger receptor CD36 is involved in danger particles recognition with other receptors such as TLR4, acts as a fatty acid translocase(FAT/CD36) during long-chain fatty acid transport, and activates downstream transcription factors, howver, the immunity of CD36 in mammary gland has not been reported. The aim of this study was to illuminate CD36 as a scavenger receptor in ruminants. Additionally, this study indicates that CD36 plays a vital role in the LPS-induced activation of downstream signaling cascades and mediates E. coli phagocytosis via TLR4 in pGMECs, which offers a novel treatment strategy for mastitis. As well as the function of long-chain polyunsaturated fatty acids(e.g. γ-linolenic acid) plays in LPS induced inflammation on primary goat mammary gland epithelial cells(pGMECs). In this study, dairy goat CD36 and TLR4 sequence were cloned. We manipulated CD36 expression in LPS induced pGMECs to explore the connection between CD36 and TLR4 in which could activate downstream cascades. Bimolecular fluorescence complementation(BiFC) and immunoprecipitation(IP) were used for detection CD36 and TLR4 interaction in E.coli internalization. In this study, we deleted CD36 fatty acid-binding domain to understand whether or not long-chain polyunsaturated fatty acids(LC-PUFA) via CD36 receptor to play anti-inflammation role. The main conclusions are shown below:1. Activation of TLR4 and CD36 in E. coli-induced mastitisMicroscopic examination showed that the interstitium was infiltrated with inflammatory cells following infection. Compared with the control group, the acinar lumina and acinar structure of the infection group were disrupted by E. coli invasion, and the bacteria destroyed the epithelial tight junctions. TLR4 and CD36 mRNA levels were higher in infected goats than in healthy goats(P < 0.01). Surprisingly, MyD88 mRNA levels were also significantly increased in E. coli-induced mastitis compared with healthy goats(P < 0.01). Downstream signal changes revealed that NF-kB-p65, c-JNK, p38-MAPK, and TRAF6 were activated in E. coli-induced mastitis tissue samples compared with normal tissue samples. The results indicated that E. coli-induced mastitis could trigger TLR4 and CD36 mRNA expression and activate the downstream signaling pathways in Xinong Saanen dairy goats.2. Cloning Xinong Saanen goat CD36 and TLR4 gene and construction of vectorssXinong Saanen dairy goat CD36 and TLR4 gene were successfully cloned. Recombinant adenovirus vector(Ad-CD36) containing CD36 cDNA sequence was constructed. Infection pGMECs with Ad-CD36 for 24 h found that CD36 mRNA and protein levels were increased dramatically in contrast to control groups(p<0.001). No changes were observed between AdGFP groups and blank control gourp(p>0.05). CD36 and TLR4 bimolecular fluorescence complementation vector: pBiFC-VN155-CD36 and pBiFC-VC155-TLR4 were successfully constructed. CD36 and TLR4 overexpression vector pef-NEO-Flag-CD36 and pef-NEO-MycTLR4 were also constructed. These two types of vectors are used to understand proteinprotein intereaction between CD36 and TLR4.3. CD36 participates in LPS-induced inflammation and regulates NF-κB and c-JNK activation but not p38-MAPK kinase pathways following LPS stimulation in pGMECsFinding out a suitable concentration of LPS did not induce cell apoptosis or necrosis but did trigger CD36 and TLR4 expression for bluiding LPS-induced inflammation cell model. CD36 works with TLR4 to activate adaptor proteins(MyD88 and TRAF6) and regulates the transcriptional activation of NF-κB and AP-1 downstream signaling pathway in LPS-induced inflammation in pGMECs. Specifically, AP-1 was primarily activated through c-JNK signaling, not p38 MAPK signaling, in LPS-stimulated pGMECs. And the downstream of inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α, except TGF-b, were influenced by manipulation CD36 expression in LPS-indcued pGMECs.4. CD36 cooperates with TLR4 during E. coli internalization and phagocytosisFrom the BiFC results of this study, CD36 and TLR4 might interact during the phagocytic process of cells. Then, we used immunoprecipitation for detection E. coli-induced infection in pGMECs might stimulate the interaction between TLR4 and CD36. Antibiotic protection assay was used to demonstrate CD36 played an important role in E. coli phagocytosis in pGMECs.5. Gamma-linolenic acid via CD36 suppresses the LPS-induced inflammatory response in pGMECsCD36127-279 was successfully cloned and over-expression Ad-CD36127-279 and pef-NEOFlag-CD36127-279 vectors were constructed. Nclear factor kappa B(NF-kB) levels were significantly decreased by CD36 receptor signaling following treatment with GLA but not LA. GLA inhibited NF-κB activation in LPS-induced pGMECs. Silencing CD36 or deleting its fatty acid-binding domain blocked the anti-inflammatory effects of GLA, resulting in an increase in NF-κB activation and disrupting its localization, as well as influence downstream inflammatory cytokines expression during LPS-induced inflammation.In conclusion, our study demonstrated that CD36 could mediate E.coli-induced inflammation and activate the downstream signaling pathways as well as interplay with TLR4 in E. coli phagocytosis in pGMECs. Long-chian polyunsaturated fatty acids exert it therapeutic effects at least in part via CD36 to inhibit LPS-induced inflammation. This result would help people to understand goat mammary gland immunity for improvmennt the quality of milk.