Rapid Inflammasome Activation following Mucosal SIV Infection of Rhesus Monkeys

Abstract

The earliest events following mucosal HIV-1 infection, prior to measurable viremia, remain poorly understood. Here, by detailed necropsy studies, we show that the virus can rapidly disseminate following mucosal SIV infection of rhesus monkeys and trigger components of the inflammasome, both at the site of inoculation and at early sites of distal virus spread. By 24 hr following inoculation, a proinflammatory signature that lacked antiviral restriction factors was observed in viral RNA-positive tissues. The early innate response included expression of NLRX1, which inhibits antiviral responses, and activation of the TGF-β pathway, which negatively regulates adaptive immune responses. These data suggest a model in which the virus triggers specific host mechanisms that suppress the generation of antiviral innate and adaptive immune responses in the first few days of infection, thus facilitating its own replication. These findings have important implications for the development of vaccines and other strategies to prevent infection.

Figure 1. Viral RNA in tissues

Tissue viral RNA (log RNA copies / 10⁸ cell equivalents) across multiple tissues at necropsy in animals on days 1, 3, 7, or 10 following inoculation with SIVmac251. Colors on each plot reflect individual animals. Values plotted below the horizontal line indicate samples in which viral RNA was not measured above the threshold of detection, which varied depending on the amount of each tissue analyzed. See also Supplementary Figure S1 and Supplementary Data S1.

Figure 2. Viral dissemination to distal tissues

(A) Mean log RNA copies / 10⁸ cell equivalents and probability of each tissue positive for virus is plotted versus the day of necropsy. Decline in levels of viral RNA in female reproductive tract tissues from day 1 to day 7 likely reflects clearance of residual inoculum challenge virus, with the subsequent increase from day 7 to day 10 likely reflecting productive infection. (B) Statistical comparisons of the tissue viral loads and the percent of each tissue positive for virus in animals necropsied on days 1, 3, 7, and 10 following SIVmac251 inoculation as compared with uninoculated controls (Naïve) by linear mixed models and Fisher’s exact tests.

Figure 3. SIV Gag-specific T lymphocyte responses in tissues

SIV Gag-specific CD8+ and CD4+ T lymphocyte responses analyzed by IFN-γ intracellular cytokine staining assay across multiple tissues at necropsy in animals on days 7 and 10 following inoculation with SIVmac251. See also Supplementary Data S1.

Figure 4. Proinflammatory transcriptomic signature in viral RNA positive tissues

(A) Classic antiviral restriction factors (ISG15, IRF7, APOBEC, MX2, TRIM5) are not observed in gastrointestinal (GI) tissues until day 10. F-test heatmap depicts viral RNA positive GI tissues over time by ANOVA with an antiviral gene filter (51 samples from 5 monkeys). (B) Heatmaps reveal an inflammasome signature in female reproductive tract (FRT) on day 1 as compared with uninoculated controls (17 samples from 5 monkeys) as well as in viral RNA positive as compared with viral RNA negative GI tissues (20 samples from 4 monkeys) on day 1. Gene expression is represented as a gene-wise standardized expression (Z-score) with P < 0.05. Red and blue correspond to up- and down-regulated genes respectively. (C) Gene Mania network representing co-expression of upregulated genes, including NLRX1 (arrow), on day 1 in viral RNA positive tissues reveals a proinflammatory signature. (D) Correlation between expression of the day 1 common gene signature and expression of the inflammasome signature (red) and antiviral signature (blue). (E) Correlation between expression of NLRX1 and expression of IRF7. P-values reflect Spearman correlation tests. See also Supplementary Figures S2-S5 and and Supplementary Data S1.

Figure 5. NLRX1 expression is correlated inversely with antiviral gene expression and is correlated directly with viral RNA

(A) Early NLRX1 expression is inversely correlated with antiviral restriction factors in FRT. F-test heatmap depicts viral RNA positive FRT tissues for NLRX1 or with an antiviral gene filter (20 samples from 8 monkeys). (B) Linear regression analysis of viral RNA levels and gene expression in viral RNA positive gastrointestinal tissues. Heatmap represents the top positively correlated proinflammatory genes, including NLRX1, which were part of the full regression signature. (C) Correlation analysis between NLRX1 expression and viral RNA levels in viral RNA positive gastrointestinal tissues. (D) Bee-swarm plot shows that samples with the highest NLRX1 expression had the highest viral RNA levels. P-values reflect Wilcoxon rank sum tests. See also Supplementary Figures S4 and S6.

Figure 6. Subset analysis reveals activation of NK cells in all SIV infected tissues on day 1 and day 3 following inoculation

(A) Gene Set Enrichment Analysis (GSEA) using Nakaya modules and pathway heatmap representing enrichment of genes characteristic of specific cell subsets in each tissue type on day 1 and day 3. Red and blue squares represent a positive or negative enrichment of genes characteristic of a subset, respectively. (B) Checkerboard figures reflecting NK, monocyte, and T cell subset leading edge analysis on day 1. Gene members (5% FDR) contributing to enrichment are shown, with magnitude represented as log₂ FC. Linear regression analysis of positive viral RNA with (C) NK, (D) monocyte, and (E) T cell specific gene expression in viral RNA positive gastrointestinal tissues. Gene expression is represented as a gene-wise standardized expression (Z-score) with P < 0.05. Red and blue correspond to up- and down-regulated genes respectively. See also Supplementary Figure S7.

Figure 7. Transcriptomic signatures in tissues with cellular immune responses

(A) Checkerboard figure shows gene expression pathways positively (red) and negatively (blue) associated with Gag-specific CD8+ T lymphocyte responses in gastrointestinal tissues. Pathways (MSigDB Hallmark database) are plotted on the y-axis and leading edge analysis (gene members contributing most to enrichment) are plotted along the x-axis. (B) TGF-β signature genes upregulated on Day 1-7 and Day 7-10. F-test heatmap depicts viral RNA positive gastrointestinal tissue over time by ANOVA with a TGF-β filter (51 samples from 5 monkeys). (C) Heatmap reveals a TGF-β signature in viral RNA positive gastrointestinal tissue on day 1 as compared with day 10. Linear regression analyses of viral RNA positive (D) gastrointestinal and (E) lymph node tissues reveal TGF-β specific genes that are correlated directly with the magnitude of the Gag-specific CD8+ and CD4+ T lymphocyte responses in these tissues. CD8+ T lymphocyte responses in tissues are (F) correlated inversely with TGIF1 expression and (G) correlated directly with SMAD7 expression. P values reflect Spearman correlation tests. See also Supplementary Figure S7.