PMC:7025468 / 25977-33279
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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7025468","sourcedb":"PMC","sourceid":"7025468","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7025468","text":"Meth Enhances HIV-1 Replication via IL-1 Signaling in CD4+ T-Cells\nSince Meth was found to induce IL-1β and miR-146a expression, and these play important roles in immune regulation, we explored their effects on HIV-1 infection of CD4+ T-cells.\nWe first confirmed that Meth increases HIV-1 replication in CD4+ T-cells, as shown in previous studies (Figure 5A) (5, 54). Next, we assessed the effects of Meth on release of IL-1β under various conditions. Cells were pretreated for 24 h with Meth before exposure to HIV-1; Meth was then administered daily. At 1 day post infection, we observed significantly increased release of IL-1β in cells either exposed to HIV-1 or HIV+Meth (Figure 5B). However, at 2 days post infection, we observed decreased release of IL-1β.\nFigure 5 Methamphetamine enhances HIV-1 replication via IL-1 signaling in CD4+ T-cells. CD4+ T-cells were uninfected, infected with HIV-1, treated with Meth alone, or treated with Meth and infected with HIV-1 concomitantly. Cells were harvested at 2 days post infection and supernatants were harvested at the time points indicated on graphs. (A) Culture supernatants were analyzed for HIV-1 replication by p24 ELISA, and p-values were calculated relative to untreated controls (**p \u003c 0.01). (B) IL-1β ELISA was used to determine the concentration of IL-1β from culture supernatants harvested on days 1 and 2 P.I. Relative expression was calculated by normalizing HIV and HIV+Meth samples to untreated controls. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to untreated controls (**p \u003c 0.01, ***p \u003c 0.001). (C) miR-146a and IL-1β mRNA expression was determined by RT-qPCR. Fold change was calculated by normalizing Meth, HIV, or HIV+Meth samples to untreated controls. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to untreated controls (*p \u003c 0.05, **p \u003c 0.01). (D) RT-qPCR was performed to assess changes in TRAF6 and IRAK1 mRNA expression on day 2 P.I. Fold change was calculated by normalizing Meth, HIV or HIV+Meth samples to untreated control cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to untreated controls (*p \u003c 0.05, **p \u003c 0.01). (E) Cells were harvested and lysed on day 2 P.I. Protein extracts were analyzed for IRAK1 and TRAF6 by Western Blot. GAPDH was used as a loading control. Relative band intensity was calculated using ImageJ software, and p-values were calculated relative to untreated controls (*p \u003c 0.05, **p \u003c 0.01). (F) CD4+ T-cells were infected with HIV-1 alone, infected with HIV-1 and treated with Meth daily, or infected with HIV-1 and treated with Meth and IL-1RA (200 or 400 ng/mL) daily. Culture supernatants were analyzed for HIV-1 replication by p24 ELISA, and p-values were calculated relative to HIV+ for HIV+Meth samples, or relative to HIV+Meth for HIV+Meth+Il-1RA samples (*p \u003c 0.05, **p \u003c 0.01, ***p \u003c 0.001). (G) Cells were harvested at 2 days P.I. IL-1β mRNA expression was analyzed by RT-qPCR. Fold change was calculated by normalizing HIV+Meth or HIV+IL-1RA to HIV+ samples. Data represent the mean ± SD of 3 independent experiments, and p values were calculated relative to HIV+ for HIV+Meth samples, and relative to HIV+Meth for HIV+Meth+IL-1RA samples (*p \u003c 0.05, **p \u003c 0.01). We then analyzed the expression of IL-1β mRNA and miR-146a in Meth, HIV+, and HIV+Meth treated cells. We observed significantly increased IL-1β mRNA levels as well as increased miR-146a expression across all treatments 2 days post infection (Figure 5C).\nNext, we assessed the expression of TRAF6 and IRAK1. Interestingly, TRAF6 mRNA levels were unchanged in Meth treated cells, but showed significantly decreased expression in HIV+ and HIV+Meth samples (Figure 5D). In contrast, IRAK1 mRNA showed significantly increased expression in the presence of Meth alone, but significantly decreased expression in HIV+ samples (Figure 5D). When cells were treated with Meth and HIV-1 in combination, IRAK1 mRNA displayed baseline expression (Figure 5D). By Western Blot, we observed decreased TRAF6 protein levels in Meth, HIV+, and HIV+Meth samples (Figure 5E). IRAK1 protein levels showed no change in Meth treated vs. untreated cells, but decreased expression in HIV+ and HIV+Meth treated cells 2 days post infection (Figure 5E).\nThese data demonstrate that both HIV-1 and Meth increased IL-1β and miR-146a expression. We also observed that HIV-1 inhibited TRAF6 and IRAK1 expression. Unlike Meth treatment, HIV-1 inhibited TRAF6 at the RNA level. Moreover, IRAK1 was inhibited only in HIV-1 infected samples, consistent with previous reports (55). These findings suggest that HIV-1 inhibits expression of TRAF6 and IRAK1 independent of miR-146a expression.\nInterestingly, HIV+ and HIV+Meth samples displayed increased extracellular IL-1β levels on day 1 post infection, while IL-1β mRNA levels were significantly increased at day 2 post infection. These data suggest an important role for a Meth mediated IL-1β auto-regulatory feedback loop, which may augment the inflammatory state triggered during HIV-1 infection.\nTo assess the involvement of Meth induced IL-1β in enhanced HIV-1 replication, we blocked IL-1 signaling using exogenous IL-1RA. Cells were pretreated with IL-1RA and/or Meth, for 24 h before exposure to HIV-1; IL-1RA and Meth were then administered daily. When HIV-1 infected CD4+ T-cells were co-treated with IL-1RA and Meth, the effect of Meth on enhancing HIV-1 replication was significantly attenuated in a dose dependent manner. We observed that while 200 ng/mL IL-1RA was sufficient to reduce the effect of Meth on HIV-1 replication, virus replication in these cells was still significantly higher than in HIV+ cells (Figure 5F). However, when cells were treated with higher concentrations of IL-1RA (400 ng/mL) prior to Meth treatment, HIV-1 replication was significantly inhibited (Figure 5F). Notably, when HIV-1 infected CD4+ T-cells were treated with IL-1RA alone, there was no change in HIV-1 replication (Figure S1A).\nNext, we analyzed IL-1β mRNA expression in HIV-1 infected CD4+ T-cells that were untreated, or treated with Meth, or treated with Meth and IL-1RA together. We observed that HIV+Meth+IL-1RA200ng/mL samples showed only slightly increased IL-1β mRNA expression, while IL-1β mRNA expression remained unchanged in HIV+Meth+IL-1RA400ng/mL samples relative to HIV+ controls (Figure 5G). These results are consistent with the pattern we observed during HIV-1 replication, wherein Meth augments IL-1β mRNA and HIV-1 replication, but these effects are slightly reduced in the presence of 200 ng/mL IL-1RA, and completely abrogated in the presence of 400 ng/mL IL-1RA.\nTaken together, these results demonstrate that IL-1 signaling is important for Meth mediated effects on HIV-1 replication. Specifically, our results showed that increased IL-1β expression during Meth treatment plays a role in enhanced HIV-1 replication. Further, we observed relatively unchanged expression of miR-146a, TRAF6, and IRAK1 among HIV+, HIV+Meth, HIV+Meth+IL1RA200ng/mL, and HIV+Meth+IL-1RA400ng/mL samples (Figures S1B,C), suggesting that an IL-1β positive feedback loop is central to enhanced HIV-1 replication in the presence of Meth.","divisions":[{"label":"title","span":{"begin":0,"end":66}},{"label":"p","span":{"begin":67,"end":243}},{"label":"p","span":{"begin":244,"end":763}},{"label":"figure","span":{"begin":764,"end":3350}},{"label":"label","span":{"begin":764,"end":772}},{"label":"caption","span":{"begin":774,"end":3350}},{"label":"p","span":{"begin":774,"end":3350}},{"label":"p","span":{"begin":3351,"end":3604}},{"label":"p","span":{"begin":3605,"end":4374}},{"label":"p","span":{"begin":4375,"end":4802}},{"label":"p","span":{"begin":4803,"end":5162}},{"label":"p","span":{"begin":5163,"end":6094}},{"label":"p","span":{"begin":6095,"end":6752}}],"tracks":[{"project":"TEST0","denotations":[{"id":"32117283-116-121-3683925","span":{"begin":360,"end":361},"obj":"[\"18458093\"]"},{"id":"32117283-119-125-3683926","span":{"begin":363,"end":365},"obj":"[\"30700725\"]"},{"id":"32117283-96-102-3683927","span":{"begin":4689,"end":4691},"obj":"[\"25472883\"]"}],"attributes":[{"subj":"32117283-116-121-3683925","pred":"source","obj":"TEST0"},{"subj":"32117283-119-125-3683926","pred":"source","obj":"TEST0"},{"subj":"32117283-96-102-3683927","pred":"source","obj":"TEST0"}]},{"project":"2_test","denotations":[{"id":"32117283-18458093-35221731","span":{"begin":360,"end":361},"obj":"18458093"},{"id":"32117283-30700725-35221732","span":{"begin":363,"end":365},"obj":"30700725"},{"id":"32117283-25472883-35221733","span":{"begin":4689,"end":4691},"obj":"25472883"}],"attributes":[{"subj":"32117283-18458093-35221731","pred":"source","obj":"2_test"},{"subj":"32117283-30700725-35221732","pred":"source","obj":"2_test"},{"subj":"32117283-25472883-35221733","pred":"source","obj":"2_test"}]},{"project":"MyTest","denotations":[{"id":"32117283-18458093-35221731","span":{"begin":360,"end":361},"obj":"18458093"},{"id":"32117283-30700725-35221732","span":{"begin":363,"end":365},"obj":"30700725"},{"id":"32117283-25472883-35221733","span":{"begin":4689,"end":4691},"obj":"25472883"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"attributes":[{"subj":"32117283-18458093-35221731","pred":"source","obj":"MyTest"},{"subj":"32117283-30700725-35221732","pred":"source","obj":"MyTest"},{"subj":"32117283-25472883-35221733","pred":"source","obj":"MyTest"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"TEST0","color":"#939cec","default":true},{"id":"2_test","color":"#b6ec93"},{"id":"MyTest","color":"#ec93d0"}]}]}}