Methamphetamine Inhibits Toll Like Receptor-9-Mediated Activation Anti-HIV Activity In Macrophages

Part one:The Impact of METH on TLR-9-mediate Anti-HIV Activity in Human MacrophagesObsjective:To investigate the impact of Meth inhibition TLR-9 ligand (CpG-ODN) blocking HIV replication in macrophages and explore the mechanisms underlying the Meth impair TLR-9 mediated activation anti-HIV activity.Method:Peripheral blood samples from healthy adult donors were freshly isolated, and then refered to 7-day-cultured monocytes in vitro.7-day-cultured macrophages were treated with Meth and/or CpG-ODN and/or D1R antagonist. Macrophages were infected with equal amounts of cell-free HIV Bal. Culture supernatants were collected for HIV RT activity assay at days 4,8, and 12 after infection. Determined HIV RT activity and hotographed cell macrophages under a light microscopyat day 8 postinfection.Results:1. Increased levels of HIV RT activity were found in Meth group when compared with control group.2. Reduced levels of HIV RT activity were found in ODN group when compared with control group.3. Increased levels of HIV RT activity were found in Meth/ODN group when compared with ODN group.4. Reduced levels of HIV RT activity were found in Meth/ODN/SCH group when compared with Meth/ODN group.Conclusion:1. Meth compromise TLR-9-mediate anti-HIV activity in macrophages.2. D1R is involved in Meth-mediated down-regulation of TLR-9 anti-HIV in macrophages.Part two:Investigate the Mechanisms of Meth Compromising the Activation of TLR-9-mediate anti-HIV in Human MacrophagesObjective:Demonstrates whether Meth can modulate the activation of TLR-9. Investigates the potential mechanism whether due to the compromise of TLR-9-MyD88-IRF7-IFN-a signaling pathway by Meth.Method:Peripheral blood samples from healthy adult donors were freshly isolated, and then refered to 7-day-cultured monocytes in vitro.7-day-cultured macrophages were treated with Meth and/or CpG-ODN and/or D1R Antagonist. Detect the quantities of targets mRNA. Then perfonned IFN-a enzyme-linked immunosorbent assay and examine expression of TLR-9 by immunofluorescence assay. Meanwhile, cellular RNA extracted from cells was subjected to the real time RT-PCR for IRF-7,MyD88,MxA,ISG56.Results:1. A trend toward lower expression of TLR-9 was observed in Meth group compared with control. In addition, the decrease was does-and time-dependent.2. A trend toward lower expression of IFN-a was observed in Meth group compared with control. In addition, the decrease was does-and time-dependent.3. TLR-9 gene expression levels in Meth/ODN group were lower than in ODN group. Higher expression of TLR-9 was observed in Meth/ODN/SCH group compared with Meth/ODN group.4. IFN-a expression levels in Meth/ODN group were lower than in ODN group. Higher expression of IFN-a was observed in Meth/ODN/SCH group compared with Meth/ODN group.5. IRF-7, MyD88 and MxA gene expression in ODN group were higher than in control group, but not ISG56 gene. IRF-7, MyD88, MxA and ISG56 gene expression in Meth/ODN group were lower than in ODN group. Meanwhile, MyD88 and ISG56 gene expression in Meth/ODN/SCH group were higher than in Meth/ODN group, but not IRF-7 and MxA.Conclusion:1. Meth suppresses the expression of the endogenous TLR-9.2. Meth suppresses the expression of the endogenous IFN-α.3. CpG-ODN-induced TLR-9 and IFN-a expression were compromised by Meth.4. Meth inhibits IRF-7,MyD88,MxA,ISG56 expression in macrophagesea.5. D1R can block the Meth-mediated inhibition of TLR-9 and IFN-αexpression in macrophages...