Opposing roles of Toll-like receptor and cytosolic DNA-STING signaling pathways for <i>Staphylococcus aureus</i> cutaneous host defense

<div><p>Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs) to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways contributing to the early transcriptional response to <i>Staphylococcus aureus</i>, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, two PRR pathways—the Toll-like receptor (TLR) and Stimulator of Interferon Gene (STING) pathways—were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live <i>S</i>. <i>aureus</i>. TLR signaling predominantly activated a pro-inflammatory program while STING signaling activated an antiviral/type I interferon response with live but not killed <i>S</i>. <i>aureus</i>. This STING response was largely dependent on the cytosolic DNA sensor cyclic guanosine-adenosine synthase (cGAS). Using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to <i>S</i>. <i>aureus</i>. TLR signaling was required for host defense, with its absence reducing interleukin (IL)-1β production and neutrophil recruitment, resulting in increased bacterial growth. In contrast, absence of STING signaling had the opposite effect, enhancing the ability to restrict the infection. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered by <i>S</i>. <i>aureus</i> and uncover opposing roles of TLR and STING in cutaneous host defense to <i>S</i>. <i>aureus</i>.</p></div

STING signaling suppresses neutrophil recruitment and IL-1β production following cutaneous infection with <i>S</i>. <i>aureus</i>.

<p>(A) 18 hours after infection, skin was harvested and histology and immunohistochemistry was performed for Ly6G and IL-1β. Representative example of high power (200X) images of skin from a WT, <i>Sting</i>^<i>Gt/Gt</i> and <i>Myd88</i>^-/- mouse are shown. (B) Skin homogenates from WT and <i>Sting</i>^<i>Gt/Gt</i> mice at 8 and 18 hours (n = 6 per group) and <i>Myd88</i>^<i>-/-</i> mice at 8 hours (n = 5) * p<0.05 vs baseline; ** p<0.05 vs WT stimulated at same time point sample by one way ANOVA with Tukey test. (C) IL-1β expression at 8 and 18 hours after <i>S</i>. <i>aureus</i> infection in WT (n = 6 per time point), <i>Sting</i>^<i>Gt/Gt</i> mice (n = 6 per time point), and <i>Myd88</i>^-/- mice (n = 5, 8 hour time point only). * p<0.05 vs baseline; ** p<0.05 vs WT stimulated at same time point by one way ANOVA with Tukey test. All error bars denote standard error of the mean.</p

Kinetic properties of the transcriptional cascade to live and HK <i>S</i>. <i>aureus</i>.

<p>(A) Activation kinetics are shown for inducible genes from BMDMs stimulated with an MOI of 10 of <i>S</i>. <i>aureus</i> at indicated time points. Shades of blue to red indicate percentile values from low to high expression. Shown are all genes meeting RPKM >1, Fold change >5, and p<0.05 by DESeq. The average of at least 4 separate experiments is shown for each time point (B) The average fold of transcript levels of inducible genes separated within representative clusters by time point the genes reach their maximal expression level are shown. (C) Known, highly significant Jaspar 2016 motif enrichment of genes which reach maximal expression levels at the indicated time points following <i>S</i>. <i>aureus</i> infection. (D) Activation kinetics are shown for inducible genes from BMDMs stimulated with the equivalent of an MOI of 10 HK <i>S</i>. <i>aureus</i> at indicated time points. Shades of blue to red indicate percentile values from low to high expression. Induction and expression criteria are as in A. Shown are all genes meeting RPKM >1, Fold change >5, and p<0.05 by DESeq. The average of at least 2 separate experiments is shown for each time point (E) The average fold of transcript levels of inducible genes separated within representative clusters by time point the genes reach their maximal expression level are shown. (F) Known, highly significant Jaspar 2016 motif enrichment of genes which reach maximal expression levels at the indicated time points following <i>S</i>. <i>aureus</i> infection. (G) Venn diagram showing degree of overlap between all genes which meet expression and induction threshold criteria in macrophages in response to live or HK <i>S</i>. <i>aureus</i> treatment.</p

Modes of regulation of a gene signature induced by live <i>S</i>. <i>aureus</i> but not HK <i>S</i>. <i>aureus</i>.

<p>(A) Heat map demonstrating 68 genes were induced by live <i>S</i>. <i>aureus</i> that were not significantly induced by HK <i>S</i>. <i>aureus</i> (failed RPKM, fold change, or significance criteria) in BMDMs. TLR signaling was required for the induction of the majority of these genes induced by live <i>S</i>. <i>aureus</i> but not HK <i>S</i>. <i>aureus</i>, with only 2 requiring STING, and 2 genes requiring both TLRs and STING. 11 genes were induced independently of MyD88/TRIF or STING signaling in response to live <i>S</i>. <i>aureus</i> that failed to be induced by HK <i>S</i>. <i>aureus</i>. (B) GO Biologic Process of the 20 genes separated by whether they were (10 genes) or were not (10 genes) also induced by HK <i>S</i>. <i>aureus</i>, reveals genes induced by live bacteria possess the hypoxia signature. The number of genes listed next to the GO Process number refers to the number of <i>S</i>. <i>aureus</i>-induced genes in our dataset that contribute to the significant enrichment of the GO term dataset. (C) Comparison of the 10 “MyD88/TRIF or STING Independent” cluster of 20 genes that are not induced by HKSA with known monocyte/macrophage datasets stimulated with hypoxia reveals significant overlap of signatures (p-value by Fisher Exact Test).</p

Integrated framework of transcriptional activation of genes induced in BMDMs by live <i>S</i>. <i>aureus</i>.

<p>(A) Heat map of percentile induction of genes induced by treatment of BMDMs with live <i>S</i>. <i>aureus</i> (MOI 10) in wild type (B6), <i>Myd88</i>^<i>-/-</i><i>Trif</i>^<i>-/-</i>, or <i>Sting</i>^<i>Gt/Gt</i> mice reveals four distinct clusters of genes. Genes are separated into 4 clusters (I, II, III, and IV) based on mode of induction. (B) Venn diagram demonstrating the breakdown of genes in the four clusters of genes based on >50% dependence on the two main pathways induced. (C) Enriched Jaspar 2016 motifs within promoters of genes within the 4 clusters of genes defined by dependence on TLR and/or STING pathways. (D) GO Biologic Process reveals functions of genes within each cluster. The number of genes listed next to the GO Biologic Process number refers to the number of <i>S</i>. <i>aureus</i>-induced genes in our dataset that contribute to the significant enrichment of the GO term dataset. (E) Comparison of “MyD88/TRIF or STING Independent” cluster of 20 genes with known monocyte/macrophage datasets stimulated with hypoxia reveals significant overlap of signatures (p-value by Fisher's Exact Test).</p

Toll-like receptor and STING signaling dominate the transcriptional response to <i>S</i>. <i>aureus</i> in murine macrophages.

<p>(A) RNA-Seq of <i>Myd88/Trif</i>^<i>-/-</i> macrophages was compared to that of WT macrophages. Shown is the % expression value of the 369 genes induced by live <i>S</i>. <i>aureus</i> in <i>Myd88/Trif</i>^<i>-/-</i> macrophages compared to WT macrophages. The average expression from two separate experiments of WT and <i>Myd88/Trif</i>^<i>-/-</i> macrophages treated at the same time with the same dose of <i>S</i>. <i>aureus</i> at 5 different time points in each experiment is shown. (B) Known, highly significant Jaspar 2016 motif enrichment of genes separated by their degree of dependence on <i>Myd88/Trif</i>^<i>-/-</i> following <i>S</i>. <i>aureus</i> infection. (C) RNA-Seq of <i>Sting</i>^<i>Gt/Gt</i> macrophages was compared to that of WT macrophages. Shown is the % expression value of the 369 genes induced by live <i>S</i>. <i>aureus</i> in <i>Sting</i>^<i>Gt/Gt</i> macrophages compared to WT macrophages. The average expression from two separate experiments of WT and <i>Sting</i>^<i>Gt/Gt</i> macrophages treated at the same time with the same dose of <i>S</i>. <i>aureus</i> at 5 different time points in each experiment is shown. (D) Known, highly significant Jaspar 2016 motif enrichment of genes separated by their degree of dependence on STING following <i>S</i>. <i>aureus</i> infection.</p