| Objective This study aimed to explore the in vitro impact of TLR9 signaling pathway on HIV-1 replication mediated by morphine in macrophages,based on a macrophages-HIV-1 Bal model,and to further illustrate the underlying biological mechanism(s)of high prevalence of HIV-1 among opioid abusers.Methods Fresh peripheral blood was obtained from healthy volunteers,and peripheral blood mononuclear Cells(PBMCs)were isolated from the blood samples and then used to purify monocytes by utilizing adherence method.The human macrophages were differentiated from one-week-cultured mononuclear cells under the condition of 37?,5%C02.Macrophages were treated with or withour morphine,ODN2216(TLR9 receptor agonist),ODN2243(TLR gradeTM,ODN 2216 Control),then infected by HIV-1 Bal strain.After 4,8,12 days postinfection,the cell samples and culture supernatants were collected.The mRNA expression levels of TLR9,MyD88,IRF7,IFN-?/?,ISG56 and MxA of monocyte-derived macrophage were determined by RT-PCR.In addition,HIV-1 RNA expression was also detected by RT-PCR.Protein expression of TLR9,MyD88,IRF7,ISG56 and MxA were determined by Western blot.IFN-a/p,HIV p24 protein levels in the supernatants were measured by ELISA.The analysis of variance(ANOVA)and LSD-t test were used to analyse data.Results1.Morphine could enhance HIV-1 infection in macrophages.After 4,8,12 days postinfection,the relative expression levels(fold)of HIV-1 RNA in morphine treated group were 1.45±0.31,1.36±0.24,1.42±0.27,compared to that in control group,respectively.Consistently,the levels of HIV-p24 antigen were 3.79±2.03,37.13±17.82,51.83±24.75(ng/ml)at different time points,respectively.Both the levels of HIV RNA and HIV-p24 antigen in morphine treated group were significantly higher than that in control group(without morphine and ODN2216 treatment,P<0.05).After 8,12 days postinfection,the relative expression levels(fold)of HIV-1 RNA in morphine+ODN2243 treated group were 1.44±0.40,1.40±0.26,compared to that in control group.And the levels of HIV-p24 antigen were 37.03±18.53,48.17±18.51(ng/ml)in morphine+ODN2243 treated group.2.Morphine could down-regulate the expression of TLR9 and its downstream factors MyD88 and IRF7 in macrophages.After 4,8,12 days postinfection,the mRNA relative expression(fold)of TLR9,MyD88,IRF7 in morphine treated group were(0.671±0.17,0.55±0.29,0.63±0.24),(0.74±0.15,0.71 ±0.18,0.76±0.16),(0.61±0.23,0.63±0.17,0.65±0.13),compared to those in control group,respectively.The mRNA expression of TLR9,MyD88,IRF7 in morphine treated group were significantly lower than those in the control group(P<0.05).The results indicate that morphine could down-regulate the mRNA expression of several key factors in TLR9 signaling pathway in human macrophages,including TLR9,MyD88 and IRF7.Furthermore,the down-regulations of TLR9,MyD88 and IRF7 by morphine at protein level were confirmed by Western blot.3.Morphine could down-regulate the expression of antiviral molecules IFN-a,IFN-?,MxA and ISG56 in macrophages.After 4,8,12 days postinfection,the mRNA relative expression(fold)of IFN-a,IFN-P,MxA in morphine treated group were(0.72±0.17,0.70±0.14,0.71±0.14),(0.721±0.18,0.62±0.16,0.73±0.11),(0.58±0.16,0.62±0.18,0.70±0.11),compared to those in control group,respectively.Significance differences were observed between morphine treated group and control group on the mRNA expression of above factors(P<0.05).The results indicate that morphine could down-regulate the mRNA expression of antiviral molecules IFN-?,IFN-? and MxA.Further,the results was confirmed by Western blot,showing that morphine did down-regulate the protein expression of IFN-?,IFN-?,MxA and ISG56.4.Morphine could antagonize the induced expression of TLR9,MyD88 and IRF7 by activation of TLR9 signaling pathway,which was achieved via ODN2216 treatment.After the activating TLR9 signaling pathway through ODN2216 treatment,the mRNA relative expression levels(fold)of TLR9,MyD88 and IRF7 were detetected after 4,8,12 days postinfection in ODN2216 treated group.The results were(1.79±0.30,1.9910.34,1.57±0.43),(1.53±0.28,1.64±0.26,1.54±0.22),(1.56±0.41,1.67±0.45,1.83±0.36),compared to those in ODN2243 and morphine+ODN2216 groups,respectively.They were significantly higher in ODN2216 treated group than those in ODN2243 and morphine+ODN2216 treated groups(P<0.05).The results indicate that morphine could block the ODN2216 activation of TLR9,MyD88 and IRF7.Furthermore,the results were confirmed by Western blot,showing that morphine could block the activation of MyD88 and IRF7 proteins.5.Morphine could block the inhibition of antiviral responses mediated by TLR9 signaling pathway activation.After 4,8,12 days postinfection,the relative expression levels(fold)of HIV-1 RNA in ODN2216 treated group were 0.61 ±0.29,0.65±0.23,0.54±0.19,the levels of HIV-p24 antigen were 0.81±0.76,6.64±6.12,10.11±4.59(ng/ml),compared to those in ODN2243 and morphine+ODN2216 groups,respectively.There was a significant decrease compared with those in ODN2243 and morphine+ ODN2216 treated groups(P<0.05).The relative expression levels(fold)of antiviral molecules IFN-?,IFN-?,MxA mRNA in ODN2216 treated group were(1.73±0.14,2.09±0.22,1.69±0.18),(1.64±0.16,2.11±0.27,1.63±0.22),(1.59±0.37,1.74±0.30,1.61±0.23),compared to those in ODN2243 and morphine+ODN2216 groups,respectively.They were significantly higher than those of ODN2243 and morphine+ODN2216 treated groups(P<0.05).Although the blockade of antiviral responses at mRNA level due to morphine was not observed,blockade of morphine to antiviral responses at protein level was observed,as evidenced by the results that HIV-p24 antigen levels in morphine+ODN2216 treated group was significantly higher than that in ODN2216 treated group.In addition,morphine could block the induction of IFN-?,IFN-?,MxA,ISG56 by ODN2216.Conclusion Under the culture condition in vitro,morphine could inhibit the expression of several key factors in TLR9 signaling pathway in human macrophages,including TLR9,MyD88 and IRF7,which resulted in down-expression of antiviral molecules,including IFN-?,IFN-?,MxA,ISG56,thus enhancing HIV-1 replication in macrophages.In addition,morphine could block the antiviral responses induced by activation of TLR9 signaling pathway.The results illustrate a novel mechanism that morphine,through inhibiting TLR9 signaling pathway,disrupts the innate immunity and promotes HIV-1 infection/replication in human macrophages. |
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