Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Macrophages are key targets of HIV-1 infection. We have previously described that the expressionof CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is furtherup-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication ininfected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibitsHIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restrictionfactors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoproteinB mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.Results:CCL2 neutralization potently reduced the number of p24 Gag+cells during the course of either productive orsingle cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thusdemonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viralDNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates ofHIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of themodulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression,to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replicationmediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was typeI IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expressionrevealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in thedefence response to viruses.Conclusions:Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primaryMDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which mightcontribute to regulate the expression of innate intracellular viral antagonistsin vivo. Thus, our study may potentially leadto the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection

Study of the role of the CCL2/CCR2 axis in HIV-1 infection: molecular mechanisms and potential therapeutic approaches

The chemokine CCL2 plays a key role in chronic inflammation and tissue damage in HIV-1 infected patients. We recently demonstrated that CCL2 neutralization by specific antibodies strongly reduces HIV-1 replication in primary macrophages by inhibiting viral DNA accumulation. In this study, we performed a global gene expression analysis by RNAseq to identify cellular factors modulated by CCL2 blocking and potentially involved in the regulation of HIV-1 replication. Our results show that 1915 and 311 genes are differentially expressed following 4 and 20 hours of treatment with anti-CCL2 antibodies, respectively. Upregulated genes include restriction factors (APOBEC3A), component of the interferon signaling, cytokines/chemokines, genes involved in vitamin D response . Downregulated genes comprise CXCR4 and some TLR family members. Furthermore, we investigated the effect of CCL2/CCR2 blocking in vivo on APOBEC3A expression in HIV-1 infected patients treated with, Cenicriviroc, a novel CCR5/CCR2 antagonist,. The longitudinal analysis performed on samples derived from the same patient prior to treatment initiation and after 4,12,24, 48 weeks suggest an increase of APOBEC3A expression following treatment with Cenicriviroc from baseline compared to control patients treated with conventional therapy. Overall, these data suggest that the CCL2/CCR2 axis may represent a new therapeutic target to strengthen host innate immunity thus limiting HIV-1 infection

The CCL2/CCR2 axis in the pathogenesis of HIV-1 infection: a new cellular target for therapy?

The identification of chemokine receptors as necessary co-receptors for HIV entry into target cells represented a breakthrough in the understanding of the pathogenesis of this viral infection. Since this initial discovery, it was unraveled that chemokines, in addition to their role in blocking viral entry by binding to co-receptors, have other functions in HIV pathogenesis. Indeed, chemokines can either inhibit or enhance HIV replication, and these effects may involve both entry and post-entry events of the viral life cycle. Depending on the balance of their negative versus positive effects on HIV replication and spreading, chemokines contribute to different outcomes of HIV pathogenesis. CCL2 is unique among the chemokines in that mostly enhancing effects on viral replication and pathogenesis have been reported. Either HIV infection itself or exposure to viral products can induce the expression of this chemokine and of its receptor CCR2, and high levels of CCL2/CCR2 are indeed found in HIV-infected subjects. The CCL2/CCR2 axis is tightly linked to the high level of immune activation and inflammation that is the hallmark of HIV infection even in patients undergoing antiretroviral therapy. In addition, more direct effects of CCL2 on HIV replication are becoming apparent. Thus, modulation of CCL2/CCR2-driven effects may have significant impact on HIV disease progression. In this review, we will discuss the complex interaction between CCL2/CCR2 and HIV and the emerging therapeutic approaches based on the inhibition of this axis

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation.

BackgroundMacrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.ResultsCCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses.ConclusionsNeutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection

SAMHD1-independent inhibition of HIV-1 DNA accumulation by endogenous CCL2 neutralization: possible role of APOBEC3A

Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Furthermore, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. We found that CCL2 neutralization potently reduced viral DNA accumulation and the number of p24 Gag+ cells during the course of either a productive or a single cycle infection with HIV-1BaL or (VSV-G) HIV-1, respectively. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that SAMHD1 expression was not affected by CCL2 neutralization. Furthermore, CCL2 blocking reduced the percentage of p24 Gag+ cells both in the absence and in the presence of exogenous dNTPs, and restricted the transduction of macrophages with Vpx+ lentiviral particles, which determined a strong down-modulation of SAMHD1 expression. These results demonstrated that altered SAMHD1 expression or function cannot account for the CCL2 neutralization-mediated restriction of HIV-1 replication in macrophages. Conversely, a strong and selective induction of APOBEC3A transcript and protein expression was associated with the inhibition of HIV-1 replication mediated by CCL2 neutralization. Interestingly, the level of CCL2 blocking induced APOBEC3A expression was comparable to those of freshly isolated monocytes. Finally, induction of A3A was type I IFN independent, since neutralizing Abs to IFN-α or IFN-β did not abolish CCL2 blocking-mediated A3A expression. Overall, these data demonstrate that neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle likely involving the up-regulated expression of APOBEC3A. These results highlight a novel mechanism which may contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defenses against HIV-1 and protecting macrophages from infection