HIV-1 infection of macrophages is dependent on evasion of innate immune cellular activation

Abstract

Objective: The cellular innate immune response to HIV-1 is poorly characterized. In view of HIV-1 tropism for macrophages, which can be activated via pattern recognition receptors to trigger antimicrobial defences, we investigated innate immune responses to HIV-1 by monocyte-derived macrophages.

Design: In a model of productive HIV-1 infection, cellular innate immune responses to HIV-1 were investigated, at the level of transcription factor activation, specific gene expression and genome-wide transcriptional profiling. In addition, the viral determinants of macrophage responses and the physiological effect of innate immune cellular activation on HIV-1 replication were assessed.

Results: Productive HIV-1 infection did not activate nuclear factor-kappaB and interferon regulatory factor 3 transcription factors or interferon gene expression (IFN) and caused remarkably small changes to the host-cell transcriptome, with no evidence of inflammatory or IFN signatures. Evasion of IFN induction was not dependent on HIV-1 envelope-mediated cellular entry, inhibition by accessory proteins or reverse transcription of ssRNA that may reduce innate immune cellular activation by viral RNA. Furthermore, IFNbeta priming did not sensitize responses to HIV-1. Importantly, exogenous IFNbeta or stimulation with the RNA analogue poly I:C to simulate innate immune activation invoked HIV-1 restriction.

Conclusion: We conclude that macrophages lack functional pattern recognition receptors for this virus and that HIV-1 tropism for macrophages helps to establish a foothold in the host without triggering innate immune cellular activation, which would otherwise block viral infection effectively.

Figure 1. HIV-1 does not induce NF-κB nuclear translocation or type 1 IFN responses in MDM

MDM stimulation with LPS (100 ng/mL) or CL075 (1 μg/mL) induces significant (p<0.0001, ANOVA) nuclear translocation of NF-κB RelA that is maximal at 1 h (A). In contrast, HIV-1 Ba-L derived from MDM or PBL and additional macrophage tropic strains ADA-M and JR-CSF propgated in PBL do not induce any detectable NF-κB activation (B) Median, interquartile and full ranges derived from approximately 500 cells are shown. Data are representative of 3 replicate experiments.

Figure 2. HIV-1 does not induce type 1 IFN responses in MDM

Stimulation of M-CSF differentiated MDM with poly I:C (10 μg/mL) or Infuenza A (MOI 3) induces siginificantly increased gene expression of IFNβ (A) and IP10 (B) quantified by qPCR (p<0.01, Wicoxon sign-ranked test of paired samples). Neither macrophage tropic HIV-1 strains induced detectable expression of either of these genes. Comparable observations are evident in alternatively differentiated MDM after 4 h stimulation (C-D) and after 24 h stimulation (E-F). Individual data paints are shown for each experiment.

Figure 3. HIV-1 induces modest transcriptional changes in MDM

(A) Significant gene expression changes (p<0.05, t-test) detected by whole genome microarray transcriptional profiling in HIV-1 Ba-L infected M-CSF differentiated MDM (at 24 h and 7 d after infection) appear quantitatively modest compared to 4 h stimulation with IFNβ (2 ng/mL), IFNγ (10 ng/mL) or LPS (10 ng/mL). Data are derived from 3 separate microarray experiments for each stimulus compared to 8 unstimulated MDM microarrays. (B) Functional annotation clustering analysis for significant gene expression differences identified by microarray transcriptional profiling in MDM infected with HIV-1 or stimulated for 4 hours with LPS (10 ng/mL), IFNβ (2 ng/ml) or IFNγ (10 ng/ml). Multidimensional scaling of the top 1000 (C) IFN- or (D) LPS-regulated genes shows that transcriptional profiles of 24 h and 7 d HIV-1 Ba-L infected M-CSF differentiated MDM cluster together with uninfected /unstimulated control MDM. Each node is derived from separate experiments indexed in the left-hand panel.

Figure 4. IFNβ and poly I:C restrict productive HIV-1 infection in MDM

(A) Titration of HIV-1 Ba-L infection in M-CSF differentiated MDM assessed by the proportion of p24 positive cells at 7 d, shows a significant (p<0.001, ANOVA) reduction in MDM stimulated with IFNβ (2 ng/mL) 24 h before or 24 h after viral infection. Mean±SD of 3 separate experiments are shown. (B) Similar significant (p<0.001, ANOVA) reduction of HIV-1 infection (for each of the strains indicated) is induced by stimulation of MDM with poly I:C (10 μg/mL) at the time of inoculation with HIV-1 (mean of 2 experimental replicates).

Figure 5. MDM lack constitutive or IFN-inducible expression of a functional PRR for HIV-1

IFNβ (A) and IP10 (B) gene expression levels, measured by qPCR and normalized to GAPDH, are shown for M-CSF differentiated MDM at 4 h and 24 h and at 4 h for IFNβ-primed (2ng/mL 24 h before infection) and unprimed control MDM (D) after infection or stimulation with the panel of reagents indicated. In contrast to the significant response to Infuenza (p<0.01, Wicoxon sign-ranked test of paired samples), no significant IFNβ or IP10 response was evident to any of the lentiviruses. IFNβ priming consistently enhanced the response to poly I:C stimulation but not HIV-1 or VSV-G pseudotyped lentiviral vector. Values for at least 3 separate experiments shown. (C) IFNβ-induced upregulation of IP10 and selected viral PRRs is shown in an expression matrix, derived from microarray transcriptional profiling of 4 h IFNβ-stimulated and control M-CSF differentiated MDM. * denotes genes that are significantly upregulated by IFNβ (p<0.05, t-test).