Zed the neuropeptide dependence of these receptors to inhibit HIV-1 production using two distinct assays. Initially, we added specific antagonists of PAC1 or VPAC1/2 to HIV-1-infected cells before treating them with VIP or PACAP. As shown in Fig 3A, VIP-induced HIV-1 inhibition is largely dependent on VPAC1/2, since blockade of both receptors abrogated the VIP-mediated inhibition of HIV-1 production with no significant changes following PAC1 blockade. We also observed that PACAP could inhibit HIV-1 replication via activation of all three receptors, as its ability to decrease viral growth wasVIP and PACAP increase IL-10 production by macrophagesThe immunomodulatory activities of VIP and PACAP are at least partially dependent of IL-10 [9], an anti-inflammatory cytokine able to inhibit the HIV-1 replication [35,36]. We also detected that VIP and PACAP increase macrophage production of IL-10 (Fig. S1C), which peaked 48 h after the stimuli. Based onVIP and PACAP Inhibit HIV-1 InfectionFigure 2. Effects of combined VIP and PACAP treatment on HIV replication. HIV-1-infected macrophages were simultaneously treated with 1 nM, 5 nM or 10 nM of VIP and PACAP (A, B and C, respectively), and virus replication was measured as above. Data represent means 6 SEM of three independent experiments. (D) Equation used to calculate the interaction coefficient of VIP and PACAP at the indicated concentrations, based on the levels of HIV-1 inhibition shown in A, B and C. *p#.05; **p#.01; ***p#.001. doi:10.1371/journal.pone.0067701.gAUC analyses (Fig. 6C), VIP treatment doubled IL-10 production, whereas PACAP treatment tripled macrophage release of this cytokine.that CCL3, CCL4 and CCL5 and IL-10 are implicated in neuropeptide-mediated inhibition of HIV-1 growth.Discussion Contribution of b-chemokines and IL-10 to the VIP- and PACAP- mediated inhibition of HIV-1 replicationWe next evaluated whether these mediators were implicated in the ability of VIP and PACAP to reduce HIV-1 growth by adding the neuropeptides to infected macrophages together with neutralizing antibodies to CCL3, CCL4 and CCL5, or to the IL-10 receptor. This experiment was conducted five days after Title Loaded From File infection because this is the approximate time when the infection becomes productive, thus allowing the antibodies to neutralize the antiHIV-1 effector molecules when new rounds of infections were occurring. Indeed, neutralization of those three b-chemokines and blocking of the IL-10 receptor significantly reduced the inhibitory effects of VIP and PACAP on HIV-1 replication (Fig. 7), showing VIP and PACAP are pleiotropic factors associated with a number of physiological FCCP supplier processes, such as endocrine, metabolic and gastrointestinal effects, also including modulatory effects on the immune system. The receptors for VIP and PACAP, the G coupled-receptors VPAC1, VPAC2 and PAC1, are widely distributed, a feature that allows VIP and PACAP to exert their large range of effects. Here, we show that VIP and PACAP treatment increased resistance to HIV-1 replication in primary macrophages by inducing the production of the b-chemokines CCL3 and CCL5 and the cytokine IL-10, molecules that are 23977191 able to reduce HIV-1 growth in vitro. Our study focused, for the first time, on the ability of the natural peptides VIP and PACAP to modulate HIV-1 infection in a primary target cell, in addition to defining the relative contribution of each of their receptors to thisVIP and PACAP Inhibit HIV-1 InfectionFigure 4. Specific activi.Zed the neuropeptide dependence of these receptors to inhibit HIV-1 production using two distinct assays. Initially, we added specific antagonists of PAC1 or VPAC1/2 to HIV-1-infected cells before treating them with VIP or PACAP. As shown in Fig 3A, VIP-induced HIV-1 inhibition is largely dependent on VPAC1/2, since blockade of both receptors abrogated the VIP-mediated inhibition of HIV-1 production with no significant changes following PAC1 blockade. We also observed that PACAP could inhibit HIV-1 replication via activation of all three receptors, as its ability to decrease viral growth wasVIP and PACAP increase IL-10 production by macrophagesThe immunomodulatory activities of VIP and PACAP are at least partially dependent of IL-10 [9], an anti-inflammatory cytokine able to inhibit the HIV-1 replication [35,36]. We also detected that VIP and PACAP increase macrophage production of IL-10 (Fig. S1C), which peaked 48 h after the stimuli. Based onVIP and PACAP Inhibit HIV-1 InfectionFigure 2. Effects of combined VIP and PACAP treatment on HIV replication. HIV-1-infected macrophages were simultaneously treated with 1 nM, 5 nM or 10 nM of VIP and PACAP (A, B and C, respectively), and virus replication was measured as above. Data represent means 6 SEM of three independent experiments. (D) Equation used to calculate the interaction coefficient of VIP and PACAP at the indicated concentrations, based on the levels of HIV-1 inhibition shown in A, B and C. *p#.05; **p#.01; ***p#.001. doi:10.1371/journal.pone.0067701.gAUC analyses (Fig. 6C), VIP treatment doubled IL-10 production, whereas PACAP treatment tripled macrophage release of this cytokine.that CCL3, CCL4 and CCL5 and IL-10 are implicated in neuropeptide-mediated inhibition of HIV-1 growth.Discussion Contribution of b-chemokines and IL-10 to the VIP- and PACAP- mediated inhibition of HIV-1 replicationWe next evaluated whether these mediators were implicated in the ability of VIP and PACAP to reduce HIV-1 growth by adding the neuropeptides to infected macrophages together with neutralizing antibodies to CCL3, CCL4 and CCL5, or to the IL-10 receptor. This experiment was conducted five days after infection because this is the approximate time when the infection becomes productive, thus allowing the antibodies to neutralize the antiHIV-1 effector molecules when new rounds of infections were occurring. Indeed, neutralization of those three b-chemokines and blocking of the IL-10 receptor significantly reduced the inhibitory effects of VIP and PACAP on HIV-1 replication (Fig. 7), showing VIP and PACAP are pleiotropic factors associated with a number of physiological processes, such as endocrine, metabolic and gastrointestinal effects, also including modulatory effects on the immune system. The receptors for VIP and PACAP, the G coupled-receptors VPAC1, VPAC2 and PAC1, are widely distributed, a feature that allows VIP and PACAP to exert their large range of effects. Here, we show that VIP and PACAP treatment increased resistance to HIV-1 replication in primary macrophages by inducing the production of the b-chemokines CCL3 and CCL5 and the cytokine IL-10, molecules that are 23977191 able to reduce HIV-1 growth in vitro. Our study focused, for the first time, on the ability of the natural peptides VIP and PACAP to modulate HIV-1 infection in a primary target cell, in addition to defining the relative contribution of each of their receptors to thisVIP and PACAP Inhibit HIV-1 InfectionFigure 4. Specific activi.
