Reshaping of the Dendritic Cell Chromatin Landscape and Interferon Pathways during HIV Infection

Abstract

Myeloid dendritic cells (DCs) have the innate capacity to sense pathogens and orchestrate immune responses. However, DCs do not mount efficient immune responses to HIV-1, primarily due to restriction of virus reverse transcription, which prevents accumulation of viral cDNA and limits its detection through the cGAS-STING pathway. By allowing reverse transcription to proceed, we find that DCs detect HIV-1 in distinct phases, before and after virus integration. Blocking integration suppresses, but does not abolish, activation of the transcription factor IRF3, downstream interferon (IFN) responses, and DC maturation. Consistent with two stages of detection, HIV-1 "primes" chromatin accessibility of innate immune genes before and after integration. Once primed, robust IFN responses can be unmasked by agonists of the innate adaptor protein, MyD88, through a process that requires cGAS, STING, IRF3, and nuclear factor κB. Thus, HIV-1 replication increases material available for sensing, and discrete inflammatory inputs tune cGAS signaling to drive DC maturation.

A comprehensive map of the dendritic cell transcriptional network engaged upon innate sensing of HIV

“…Among other potential TF candidates, we additionally tested KLF13, based on its relatively high expression and known role in recruiting coactivators p300/CBP and PCAF to drive expression of CCL5 (Ahn et al, 2007). We confirmed knockdown of these targets either by immunoblot (( Figure 5C) or qPCR ( Figure S5A), and as expected, knockdown of cGAS and NONO potently inhibited DC maturation during HIV-1-GFP infection (Johnson et al, 2018;Lahaye et al, 2018;Lahaye et al, 2013) (Figures 5D-F; S5B-E). The most effective shRNA clones for HIVEP1, KLF13, and PRDM1 had no effect on viral infection.…”

“…Towards this end we performed hypergeometric tests to assess TF enrichment, and found high enrichment for IRF, STAT, and NF-κB family members during stimulation with HIV-1-GFP, LPS, and pIC ( Figure S4D; Table S6), with kinetics closely matching what was observed for IFN production and DC maturation (Figures S1A, S1D). We also found mild enrichment of these TFs during LKO-GFP infection, supporting the concept that non-replicating lentiviral vectors are partially, if inefficiently, sensed by the innate immune system ( Figure S4D) (Johnson et al, 2018). By highlighting network topology where genes are differentially expressed during treatment with HIV-1-GFP, LKO-GFP, LPS, and pIC, we determined that the majority of the transcriptional response is concentrated in Clusters 2, 5, and 8 (Regulation of Cell Activation, IRFs & Interferon, and NF-κB & Inflammation) ( Figure S4E).…”

“…Gene expression profiles were consistent with the timing of DC maturation as scored by flow cytometry (Figure S1A), with LPS and pIC stimulation leading to early and robust induction of CD86. DCs infected with HIV-1-GFP did not mature until 48 h, and only minimally responded to LKO-GFP ( Figure S1A), as we previously described (Johnson et al, 2018;Manel et al, 2010).…”

“…Similarly, infection with HIV-1-GFP led to induction of innate immune genes ( Figure 1B; clusters 5 & 8), but did so with delayed kinetics compared to LPS and pIC, likely due to time-dependent accumulation of reverse transcription products, integration, and virus replication progressing over the first 24 h (Gao et al, 2013;Johnson et al, 2018). Gene expression profiles were consistent with the timing of DC maturation as scored by flow cytometry (Figure S1A), with LPS and pIC stimulation leading to early and robust induction of CD86.…”

“…Analyzing genome-wide chromatin accessibility represents a powerful way to assess the presence of regulatory elements such as promoters and enhancers in mammalian cells (Buenrostro et al, 2013;Johnson et al, 2018). To match the experimental conditions used for RNA-seq, we profiled changes in open chromatin by performing ATAC-seq on DCs that were mock treated or infected with HIV-1-GFP for 2, 8, 24, and 48 h, and sorted by flow cytometry (Figure 2A-B).…”