Upon recognition of a microbial pathogen, the innate and adaptive immune systems are linked to generate a cell-mediated immune response against the foreign invader. The culture filtrate of Mycobacterium tuberculosis contains ligands, such as M. tuberculosis tRNA, that activate the innate immune response and secreted Ags recognized by T cells to drive adaptive immune responses. In this study, bioinformatics analysis of gene-expression profiles derived from human PBMCs treated with distinct microbial ligands identified a mycobacterial tRNA-induced innate immune network resulting in the robust production of IL-12p70, a cytokine required to instruct an adaptive Th1 response for host defense against intracellular bacteria. As validated by functional studies, this pathway contained a feed-forward loop, whereby the early production of IL-18, type I IFNs, and IL-12p70 primed NK cells to respond to IL-18 and produce IFN-γ, enhancing further production of IL-12p70. Mechanistically, tRNA activates TLR3 and TLR8, and this synergistic induction of IL-12p70 was recapitulated by the addition of a specific TLR8 agonist with a TLR3 ligand to PBMCs. These data indicate that M. tuberculosis tRNA activates a gene network involving the integration of multiple innate signals, including types I and II IFNs, as well as distinct cell types to induce IL-12p70.

The identification of the components of Mycobacterium tuberculosis that trigger host immune responses has long been a goal of scientists studying the disease. In the 19th century, R. Koch, after identifying M. tuberculosis as the cause of tuberculosis, went on to show that the bacilli induced protective immunity, which he ascribed to a culture filtrate preparation called “tuberculin” (1). Although tuberculin was later disproved to be a cure for tuberculosis, Koch had discovered what would become a standard diagnostic test for exposure to M. tuberculosis. The protocol for purifying tuberculin from the bacterial culture filtrate was refined by F. Seibert and colleagues (27), who demonstrated that the preparations confer antigenicity when injected into skin. This work led to the identification of the protein Ags from M. tuberculosis culture filtrate that are recognized by, and can elicit adaptive responses by, T cells (810). The proteins purified from the culture filtrate elicit robust delayed-type hypersensitivity responses in patients previously exposed to M. tuberculosis and individuals vaccinated with bacillus Calmette–Guérin (BCG). Yet protein purification largely hinders their ability to generate adaptive immune responses by themselves, suggesting that an immune adjuvant is present in the culture filtrate.

To combat the intracellular pathogen M. tuberculosis, the innate and adaptive immune responses interact, with the innate response responsible for instructing the type of the adaptive T cell response. The innate immune system uses pattern recognition receptors (PRRs) to recognize a diverse array of microbial ligands known as pathogen associated molecular patterns (PAMPs). The host has deployed PRRs in a variety of subcellular locations recognizing distinct microbial ligands, resulting in the activation of common and specific innate immune responses. For example, distinct dendritic cell (DC) differentiation programs are induced by muramyl dipeptide of mycobacteria via the cytoplasmic receptor NOD2, compared to triacylated lipopeptide, a ligand for the cell surface receptor TLR2/1 (11). Furthermore, mycobacterial DNA, released from bacteria that reside in the host phagosome, gains access to the cytoplasm to trigger nucleotide sensor pathways, such as STING, leading to a type I IFN response (1215). In chronic bacterial infections, such as tuberculosis, the induction of type I IFNs opposes the action of the type II IFN, IFN-γ, which is required for an effective antimicrobial response against the causative pathogen (16, 17).

Although the M. tuberculosis culture filtrate contains several protein Ags that elicit T cell responses, it also contains one or more microbial ligands (i.e., PAMPs) that trigger innate instruction of the adaptive T cell response, which are largely removed during the purification of the purified protein derivative. In addition to proteins, M. tuberculosis culture filtrate contains nucleic acids (7), with tRNA being an abundant form of RNA (18). Treatment of human monocytes with tRNA purified from M. tuberculosis culture filtrate induced their apoptosis, which is thought to contribute to the pathogenesis of tuberculosis (18); however, the extent and specificity of the immune response triggered by M. tuberculosis tRNA remain unknown. Therefore, we investigated whether M. tuberculosis tRNA triggers a distinct innate immune response for instruction of the adaptive T cell response.

Whole blood was obtained from healthy donors who provided written informed consent (University of California, Los Angeles Institutional Review Board). PBMCs were isolated by Ficoll-Hypaque (GE Healthcare) density gradient centrifugation and cultured in RPMI 1640 (Life Technologies) supplemented with 10% FCS (HyClone) and 1% Pen/Strep glutamine (Life Technologies), at a density of 2 × 106 cells per milliliter in 96-well flat-bottom plates (Corning) at 37°C with 4% CO2.

TLR2/1L (10 μg/ml), a synthetic lipopeptide derived from the 19-kDa mycobacterial lipoprotein, was obtained from EMC Microcollections. Polyinosinic-polycytidylic acid (poly I:C; HMW; 2 μg/ml) and TLR-506 (500 nM) were from InvivoGen. ssRNA40 (phosphothioate backbone, HPLC purified; 500 ng/ml) was synthesized by IDT.

Total RNA was isolated from M. tuberculosis H37Rv that was grown to OD 0.8–1 (2 × 108 cells per milliliter) and lysed using TRIzol Reagent in bead-beating tubes, in the presence of antioxidants (19). The cells were agitated with four cycles of beating, each followed by a 5-min rest period on ice. Chloroform (0.2 ml/ml TRIzol Reagent) was added, followed by incubation at ambient temperature for 5 min. The samples were shaken and then centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase was removed for further tRNA purifications using a PureLink miRNA Isolation Kit (Invitrogen), according to the manufacturer’s instructions. Ethanol (100%) was added to the lysate to give a 35% concentration. The mixture was loaded onto a PureLink column and centrifuged at 12,000 × g for 1 min. The flow-through was mixed with 100% ethanol to give a final concentration of 70%, and the mixture was loaded onto a PureLink column and centrifuged at 12,000 × g for 1 min to yield small RNAs (<200 nt). The columns were washed twice using wash buffer (Invitrogen). The small RNAs were eluted by adding RNase-free water and centrifuging again. tRNA was finally purified by size-exclusion chromatography with an Agilent SEC-3 column (3 μm, 300 A, 7.8 × 300 mm) and eluted with 100% 8 mM ammonium acetate at 65°C to remove contaminating microRNA and other size-resolvable RNA fragments (19). tRNA was eluted for 11–13 min for each sample. Fractions containing tRNA were collected, and tRNA quantity and quality were checked using an Agilent Bioanalyzer (19). RNA purity and quality were assessed using an Agilent Bioanalyzer (19). Using endotoxin-free reagents, endotoxin levels were <2 pg/μg tRNA.

Nucleic acid ligands (1 μg/ml) were complexed with DOTAP (Roche), according to the manufacturer’s instructions, to facilitate delivery to the endosome. DOTAP alone did not induce cytokine release. Reagents were determined to be endotoxin-free by a Limulus amebocyte lysate assay (Lonza).

Primary cells were stimulated on the same day as isolation. Cell supernatants were harvested at 24 h, unless otherwise noted. The following cytokines were measured by sandwich ELISA using Ab pairs: IL-18 (MBL International), IFN-γ (BD Biosciences), IL-6, and IL-12p40 (Invitrogen). IFN-α, IL-1β, IL-10, IL-12p70, and TNF-α were measured using BD CBA Flex Sets (BD Biosciences).

PBMCs were treated with the following monoclonal neutralizing Ab compounds for 30 min before stimulation: IL-18 (10 μg/ml; MBL International) and IFN-γ (10 μg/ml; BD Biosciences). IgG1 (10 μg/ml) from the corresponding manufacturer was used as a control. The specific TLR8-inhibitor VTX-3119 and related control compound VTX-764 were gifts from VentiRx Pharmaceuticals and were used at a concentration of 100 nM, which was determined by dose titration to provide optimal inhibition without off-target effects.

PBMCs were depleted of CD56+ cells using CD56 MicroBeads (Miltenyi Biotec), as directed by the manufacturer’s protocol. Depletion was confirmed at >99% purity by flow cytometry. CD56-depleted PBMCs were cultured at 1.8 × 106 cells per milliliter to reflect the loss of CD56+ cells, which are estimated to account for 10% of PBMCs.

PBMCs were labeled with Abs to CD3 (CD3-FITC; Invitrogen) and CD56 (CD56-PE; eBioscience) or isotype control. For intracellular detection of IFN-γ, PBMCs were treated with GolgiPlug (BD Biosciences) 4 h prior to harvest. Following surface staining and fixation, cells were treated with Perm/Wash Buffer (BD Biosciences) and stained with IFN-γ–allophycocyanin (Invitrogen) or isotype control. Flow cytometry was performed on an LSR II (BD Biosciences) in the University of California, Los Angeles Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility, which is supported by National Institutes of Health awards P30 CA016042 and 5P30 AI028697. Analysis was performed using FlowJo (TreeStar).

PBMCs were stimulated as described. RNA was harvested at 1, 6, and 24 h and isolated with an RNeasy Micro Kit (QIAGEN), according to the manufacturer’s directions, including on-column DNase digestion. RNA was quantified by NanoDrop, and quality was assessed using an Agilent 2100 Bioanalyzer. Libraries were created from high-quality RNA using a TruSeq RNA Library Prep Kit v2 (Illumina). Libraries were quantified by Qubit, pooled by donor in groups of 12, and sequenced in duplicate on a HiSeq 2500 Sequencing System (Illumina).

Sequenced reads were aligned to the human reference genome (build hg19 UCSC) using TopHat and Bowtie 2. Raw counts were calculated with HTSeq using the hg19 Ensembl annotation. Normalization and differential expression analysis were performed using the DESeq2 package for R, which used the data for all three donors in all cases. The false discovery rate (FDR) was controlled by applying the Benjamin–Hochberg correction to the p values. Differentially expressed genes were identified using these cutoffs: fold-change (FC) > 2 versus media and adjusted p value < 0.05. Hierarchical clustering was performed with “hclust,” and principle component analysis was performed via “prcomp” in R (version 3.2.4). Weighted Gene Correlation Network Analysis (WGCNA) was performed using the WGCNA package, as described (20). A network of relevant gene relationships, as calculated by WGCNA, was visualized using VisANT.

Functional analysis of differentially expressed genes was performed using Ingenuity Pathway Analysis (IPA; QIAGEN). Gene Ontology (GO) term analysis was performed using the ClueGO plugin (version 2.2.5) for Cytoscape (version 3.3.0) and the GO term database files from August 6, 2016. Significantly enriched terms were identified by a right-sided hypergeometric test with a Bonferroni p value correction, using a cutoff of p < 0.05. A network connecting canonical pathways and biological functions relevant to the induction of Th1 responses derived from IPA, GO terms, and differentially expressed genes was visualized using Gephi (β version 0.9.1). This was accomplished by the following strategy: pathways chosen from ClueGO analysis of WGCNA turquoise module genes, pathways chosen from IPA analysis of turquoise module genes and similar pathways identified in a parallel analysis of genes induced by activation of PBMCs by tRNA, select IPA biological functions from tRNA relevant to cell types, select genes of interest from tRNA genes differentially expressed versus TLR2/1L, Pearson correlations for IL18/IL12/IFNG calculated on the regularized logarithm (rLog) DESeq2 results matrix, and Gephi used to illustrate connections between pathways and genes connected to IL18, IL12, and IFNG, as well as significantly correlated genes.

Results are shown as mean ± SEM. Cytokine data were transformed using log10(x+1), and the Shapiro–Wilk test was used to test for normality. One-way ANOVA was performed for comparisons among three or more groups. Two-way repeated-measures ANOVA was used to assess significance in experiments with multiple factors. Individual details of statistical analyses for individual experiments, such as a post test for multiple comparisons, are provided in the figure legends. Statistical analyses were performed using GraphPad Prism 7 and R 3.4.1 (r-project.org).

To investigate the innate immune response to M. tuberculosis tRNA, we measured the gene-expression profiles induced by M. tuberculosis tRNA in human PBMCs to include many of the immune cell types that are likely to be involved. M. tuberculosis tRNA was prepared from strain H37Rv, as described (19) An ssRNA 30-mer, derived from the HIV-1 long terminal repeat and known to activate TLR7 and TLR8 (21), served as a positive control for detecting innate immune responses to RNA. In addition, we compared the response to a mycobacterial 19-kDa triacylated lipopeptide, which activates cell surface TLR2/1 (TLR2/1 ligand, TLR2/1L) and is known to be a major activator of transcriptional pathways in response to M. tuberculosis. The optimal concentrations of these ligands were determined by dose titration or used as previously established in our laboratory (11).

PBMCs from three donors were stimulated with the various ligands, and the cells were collected after 1, 6, and 24 h. mRNAs were isolated, libraries were prepared, and gene-expression profiles were obtained by RNA sequencing, which, after filtering out background expression, yielded a dataset of 14,637 genes. The microarray data presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE110325 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110325). Principal component analysis (PCA) of the DESeq2-normalized counts was used to identify samples displaying similar trends in gene expression (Fig. 1A) (5). At the 6- and 24-h time points, PCA indicated that gene expression for PBMCs treated with M. tuberculosis tRNA and control ssRNA was similar, whereas gene expression for TLR2/1L-treated and untreated PBMCs formed distinct groups. PCA of the gene-expression data at 1 h formed a single group, with all three ligands inducing similar profiles as the control media.

FIGURE 1.

Network analysis of M. tuberculosis tRNA-, ssRNA-, and TLR2/1L-induced gene-expression profiles in PBMCs. (A) PCA of correlation of gene expression from RNA sequencing on rLog matrix output from DESeq2. Ellipse denotes 95% confidence interval from k-means clustering of PC1 and PC2 variance. (B) Hierarchical clustering of rLog-transformed counts. Euclidean distance, complete clustering. (C) Overlap of significantly induced genes by M. tuberculosis tRNA, ssRNA, and TLR2/1L, defined as FC > 2 over media and FDR < 0.05. Hypergeometric p value was calculated for overlaps excluding the 396 common genes. (D) Top Bio Functions identified by IPA of significantly induced genes for M. tuberculosis tRNA, ssRNA, and TLR2/1L. (E) Comparison of significantly induced functional pathways induced by M. tuberculosis tRNA to ssRNA or TLR2/1L.

FIGURE 1.

Network analysis of M. tuberculosis tRNA-, ssRNA-, and TLR2/1L-induced gene-expression profiles in PBMCs. (A) PCA of correlation of gene expression from RNA sequencing on rLog matrix output from DESeq2. Ellipse denotes 95% confidence interval from k-means clustering of PC1 and PC2 variance. (B) Hierarchical clustering of rLog-transformed counts. Euclidean distance, complete clustering. (C) Overlap of significantly induced genes by M. tuberculosis tRNA, ssRNA, and TLR2/1L, defined as FC > 2 over media and FDR < 0.05. Hypergeometric p value was calculated for overlaps excluding the 396 common genes. (D) Top Bio Functions identified by IPA of significantly induced genes for M. tuberculosis tRNA, ssRNA, and TLR2/1L. (E) Comparison of significantly induced functional pathways induced by M. tuberculosis tRNA to ssRNA or TLR2/1L.

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A second unsupervised analysis, hierarchical clustering, was also performed to characterize the relationships between samples (Fig. 1B). Consistent with the PCA, gene-expression profiles for tRNA and ssRNA at the 6- and 24-h time points clustered together. The samples for TLR2/1L at 6 and 24 h formed their own group, as did the samples for the untreated controls at 6 and 24 h. All of the 1-h samples, regardless of the stimulus and including the media control, clustered on a separate branch from the 6- and 24-h samples, indicating that there was little difference in the transcriptional response at this early time point.

To determine the gene signatures induced in PBMCs by each ligand versus the media control, differential gene expression was calculated with DESeq2 (22). For each ligand, we compared the two groups of samples (ligand versus media), with each group containing data from three donors. Differentially expressed genes for each time point were those that had FC > 2 between the groups and an FDR < 0.05. We calculated a gene signature of significantly induced genes for each ligand by taking the union of the differentially expressed genes at each time point for a given ligand. Comparison of gene signatures overall showed a striking overlap (p < 10−10) of the tRNA and ssRNA signatures, with 1465 common genes out of a total of 1692 genes for tRNA and 1791 for ssRNA (Fig. 1C). Of these RNA-induced genes, 396 were also induced by TLR2/1L, leaving 1069 induced by only tRNA and ssRNA. In contrast, TLR2/1L induced a distinct signature, with 720 significantly induced genes, of which 215 were unique to TLR2/1 activation.

To investigate the immune pathways induced by each ligand, functional analysis of the gene signatures was performed using IPA. Analysis using the biologic functions tool showed common pathways between the tRNA and ssRNA gene signatures, identifying roles for NK cells, DC, and monocytes and differentiation of Th1 cells (Fig. 1D). In contrast, the biologic functions of the TLR2/1L-induced gene profile indicated monocyte, macrophage, and neutrophil-associated pathways. IPA using the canonical pathways tool identified “DC maturation,” “NK–DC cross-talk,” “IFN signaling,” “PRR recognition of bacteria and viruses,” and “communication between innate and adaptive immune cells” as being more significantly induced by tRNA compared with TLR2/1L (>10−5 difference in p value, Fig. 1E). In comparison, the significance of canonical pathways identified for the tRNA and ssRNA signatures was similar. Together, these data suggest that tRNA and ssRNA, but not TLR2/1L, induced common gene signatures in PBMCs indicative of an innate pathway for the induction of Th1 cells.

To further define the potential interaction between genes associated with tRNA/ssRNA induction and those associated with a Th1 response, we used WGCNA, an unbiased approach that defines modules of highly interconnected genes based on pairwise correlations (20). We tried to identify modules that were specifically induced by one of the ligands. This was accomplished by first encoding the ligands in a binary vector that was 1 for a specific ligand, as well as all of its time points, and 0 for all other ligands. The module-expression levels were then correlated with these binary vectors to identify specific module/ligand associations.

WGCNA identified 31 gene modules, of which 9 were positively correlated with at least one microbial ligand (Fig. 2A). In addition to the modules associated with tRNA and ssRNA activation of PBMC, TLR2/1L activation was associated with MEantiquewhite4, indicating significant enrichment of terms including “regulation of macroautophagy” and “IL-1 signaling pathway” (Fig. 2B). The MElightyellow module was also associated with TLR2/1L activation and enriched for genes associated with “chemotaxis” and “chemokine signaling pathway.” Several modules were associated with the media-treated cells. Also, there were modules that were negatively associated with a given stimulus, but they were not studied further.

FIGURE 2.

Identification of RNA-correlated module and associated functional analysis. (A) WGCNA eigengene modules correlated to at least one treatment condition (p < 0.05). Red indicates positive correlation, and green indicates inverse correlation. (B) Top hits for functional term annotation of WGCNA modules positively correlated with M. tuberculosis tRNA and ssRNA or TLR2/1L signatures. Padj is calculated by ClueGO as the p value, with the Bonferroni correction for the association of the functional term with the gene-expression data. Ratio represents the genes for a given functional term that are present in the module/total number of genes for the term. (C) Visualization of the gene network derived from the WGCNA turquoise module. The module was filtered for genes exhibiting significant differential expression during tRNA treatment and was annotated with the GO term “immune pathway” to select genes associated with response to M. tuberculosis infection.

FIGURE 2.

Identification of RNA-correlated module and associated functional analysis. (A) WGCNA eigengene modules correlated to at least one treatment condition (p < 0.05). Red indicates positive correlation, and green indicates inverse correlation. (B) Top hits for functional term annotation of WGCNA modules positively correlated with M. tuberculosis tRNA and ssRNA or TLR2/1L signatures. Padj is calculated by ClueGO as the p value, with the Bonferroni correction for the association of the functional term with the gene-expression data. Ratio represents the genes for a given functional term that are present in the module/total number of genes for the term. (C) Visualization of the gene network derived from the WGCNA turquoise module. The module was filtered for genes exhibiting significant differential expression during tRNA treatment and was annotated with the GO term “immune pathway” to select genes associated with response to M. tuberculosis infection.

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MEturquoise was the only module that correlated significantly with tRNA and ssRNA treatment of PBMCs. GO analysis (23) of MEturquoise revealed association with the terms “IFN-γ production” and “IFN-γ signaling” (Fig. 2B). This module contained IL18, IFNG, and IL12A, key cytokines involved in induction of a Th1 response, thereby identifying a correlated gene network for Th1 cell differentiation, as revealed by the biologic function analysis of the tRNA-induced genes. The other modules that were significantly associated with TLR2/1L or ssRNA stimulation of PBMCs were not associated with immune GO terms.

To map the gene network involved in Th1 differentiation, we filtered the tRNA-associated MEturquoise module using the “immune system” GO term, identifying 339 genes, which included the key genes involved in induction of Th1 cells. This gene set was further filtered by differential expression in tRNA versus media control (FC > 2, FDR < 0.05, calculated by DESeq2 using data from all three donors), identifying 241 genes. The correlation among these individual genes, as calculated by WGCNA, was visualized by a connectivity map (24) (Fig. 2C), centered on IL18, IFNG, and IL12A, with connections of edge weight indicated (and requiring a pairwise correlation between genes ≥0.2). Highlighting the genes connected to IL18, IFNG, and IL12A revealed a module of 120 genes, in which IFNG and IL12A were connected to each other and had multiple connections to other genes in the module. These included IL18R1, IL18RAP, and IL12RB2, which encode relevant cytokine receptors, as well as STAT1, STAT2, STAT3, and STAT5A, which encode proteins involved in signaling pathways related to the induction or downstream effects of IL-18, IFNG, and IL12A. Strikingly, the type II IFN, IFNG, and several type I IFN genes, as well as IFN-induced downstream genes, were identified in the top 250 genes induced by tRNA (Supplemental Table IA, IB). In fact, of the top 25 genes, 16 were type I IFN or downstream genes, including ISG15, CASP4, and TLR3. These genes were strongly induced by tRNA and ssRNA but not by TLR2/1L. These data implicated type I IFN and type II IFN (IFNG) in the induction of a Th1 response, which was surprising to us, given that the induction of type I IFN in tuberculosis is known to downregulate IFN-γ responses.

Integration of the bioinformatics analyses of the gene-expression profiles for the tRNA treatment of PBMCs and the WGCNA turquoise module was performed to link cell type–associated pathways with specific genes related to Th1 functional response pathways (Fig. 3). This analysis identified that M. tuberculosis tRNA directly or indirectly triggered pathways associated with monocytes, NK cells, DCs, and T cells, including Th1 cells, to induce a set of genes, including IL18, IL18R1, IFNG, JAK2, STAT1, IL12A, IL12B, IL12RB2, and STAT4. These genes are contained within functional pathways, including “role of PRR recognition of bacteria and viruses,” “TLR signaling,” “IFN-γ signaling,” “DC maturation,” “T helper differentiation,” and “defense response.” In contrast, the top four TLR2/1L-induced pathways were “granulocyte adhesion and diapedesis,” “agranulocyte adhesion and diapedesis,” “differential regulation of cytokine production in macrophages and T helper cells by IL-17A and IL-17F,” and “differential regulation of cytokine production in intestinal epithelial cells by IL-17A and IL-17F.” Together, these data suggest a model in which tRNA activates specific cell types that interact to establish a host-defense gene network involved in the innate instruction of an adaptive Th1 response.

FIGURE 3.

Integrated network of gene expression and functional analysis terms. Gephi was used to create a functional annotation network, showing connections among significant GO terms, IPA canonical pathways, WGCNA modules, and significantly expressed genes.

FIGURE 3.

Integrated network of gene expression and functional analysis terms. Gephi was used to create a functional annotation network, showing connections among significant GO terms, IPA canonical pathways, WGCNA modules, and significantly expressed genes.

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Examination of the log2-normalized counts of key genes identified by the informatics analysis, as part of the M. tuberculosis tRNA–induced Th1-differentiation pathway, confirmed that tRNA and ssRNA, but not TLR2/1L, significantly induced expression of IL18 mRNA (Fig. 4A) and its receptor heterodimer, IL18R1 and IL18RAP (Supplemental Fig. 1A). IFNG and IL12A mRNAs were also upregulated by tRNA and ssRNA, but not by TLR2/1L. IL12B was upregulated by all stimuli, whereas IL23A was not significantly induced by any of the treatments. IL6 mRNA was strongly induced by all three ligands. This analysis revealed that additional genes connected to the central genes in the Th1-differentiation pathway, including type I IFN and downstream genes, as well as genes encoding TLR3, CASP4, and ISG15, were upregulated; again, this was in response to tRNA and ssRNA but not TLR2/1L (Supplemental Fig. 1B).

FIGURE 4.

Analysis of genes characteristic of Th1 differentiation and validation in PBMCs. (A) Log-transformed normalized counts for select genes at 6 and 24 h. Data are mean ± SEM. (B) PBMCs were stimulated with M. tuberculosis tRNA, ssRNA, or TLR2/1L, and cytokine secretion was measured at 24 h (n = 20). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus media, Benjamini-Hochberg adjusted p value (A), one-way repeated-measures ANOVA, followed by the Dunnett multiple-comparisons test (B).

FIGURE 4.

Analysis of genes characteristic of Th1 differentiation and validation in PBMCs. (A) Log-transformed normalized counts for select genes at 6 and 24 h. Data are mean ± SEM. (B) PBMCs were stimulated with M. tuberculosis tRNA, ssRNA, or TLR2/1L, and cytokine secretion was measured at 24 h (n = 20). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus media, Benjamini-Hochberg adjusted p value (A), one-way repeated-measures ANOVA, followed by the Dunnett multiple-comparisons test (B).

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To validate the gene-expression profile, we determined whether induction of the cytokine mRNAs leads to secretion of the encoded protein. PBMCs were stimulated with M. tuberculosis tRNA, ssRNA40, and TLR2/1L, supernatants were collected at 24 h, and cytokine production was measured. Consistent with the gene-expression data, PBMCs released IL-18, IFN-γ, and IL-12p70 in response to tRNA (76 ± 10, 16,586 ± 2,932, and 211 ± 33 pg/ml, respectively) and ssRNA, but not media or TLR2/1L (Fig. 4B). IL-12p40 was secreted in response to all treatments, although tRNA and ssRNA induced approximately 7-fold more IL-12p40 protein than did TLR2/1L (data not shown). IL-6 secretion served as a control to demonstrate cell activation by all treatments (Fig. 4B). Low levels of IL-23 were detected in supernatants of all three treatments (Supplemental Fig. 1C). The differential production of IL-12p70 and IL-23 is consistent with the informatics analysis, indicating a gene network for instruction of a Th1 immune response. In addition, IFN-α was induced by tRNA and ssRNA, but not by TLR2/1L, consistent with the RNA sequencing data.

Given that M. tuberculosis tRNA induced the robust production of IL-12p70, we next investigated the immune networks that led to production of this cytokine. The kinetic sequence of cytokine induction in response to M. tuberculosis tRNA was determined by measuring the time course of IL-18, IFN-γ, and IL-12p70 protein production (Fig. 5A). The earliest detected response was the production of IL-18 at 6 h in response to tRNA and ssRNA (tRNA: 45 ± 5 pg/ml, p = 0.0001). IL-18 protein increased 2-fold by 24 h (tRNA: 76 ± 2 pg/ml, p = 0.0001). In contrast, little IFN-α, IFN-γ, or IL-12p70 was detected at 1 or 6 h, but there was significant induction by 24 h (tRNA: 2,351 ± 481 pg/ml, p = 0.0001; 15,751 ± 4,287 pg/ml, p = 0.0001; 139 ± 22 pg/ml, p = 0.0001, respectively). The temporal pattern of IL-6 secretion was similar to that of IL-18: protein was detected by 6 h, and the concentration doubled by 24 h (Supplemental Fig. 1D). These data indicate that IL-18 secretion is triggered early, followed by secretion of IFN-α, IFN-γ, and IL-12p70.

FIGURE 5.

Roles for IL-18 and type I IFN. (A) PBMCs were stimulated with M. tuberculosis tRNA, ssRNA or TLR2/1L, and cytokine secretion was measured at 1, 6, and 24 h (n = 7). PBMCs were treated with monoclonal anti–IL-18 neutralizing Ab or IgG1 isotype control (n = 4) (B) or with anti-IFNAR neutralizing Ab or IgG2a isotype control (n = 3) (C) for 30 min before stimulation with TLR2/1L, ssRNA, or M. tuberculosis tRNA. Supernatants were collected at 24 h, and cytokine secretion was measured. Data are mean ± SEM. The data within each subpanel were derived from independent donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus media control, two-way repeated-measures ANOVA, followed by the Dunnett multiple-comparisons test (A), one-way ANOVA, followed by the Tukey post hoc test (B and C).

FIGURE 5.

Roles for IL-18 and type I IFN. (A) PBMCs were stimulated with M. tuberculosis tRNA, ssRNA or TLR2/1L, and cytokine secretion was measured at 1, 6, and 24 h (n = 7). PBMCs were treated with monoclonal anti–IL-18 neutralizing Ab or IgG1 isotype control (n = 4) (B) or with anti-IFNAR neutralizing Ab or IgG2a isotype control (n = 3) (C) for 30 min before stimulation with TLR2/1L, ssRNA, or M. tuberculosis tRNA. Supernatants were collected at 24 h, and cytokine secretion was measured. Data are mean ± SEM. The data within each subpanel were derived from independent donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus media control, two-way repeated-measures ANOVA, followed by the Dunnett multiple-comparisons test (A), one-way ANOVA, followed by the Tukey post hoc test (B and C).

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Because IL-18 was detected early (i.e., at 6 h), we determined whether its secretion was required for downstream cytokine production. Pretreatment of PBMCs with anti–IL-18 neutralizing Abs dramatically reduced tRNA-induced secretion of IFN-γ (93% reduction, p = 0.0001) and IL-12p70 (70% reduction, p = 0.0008) (Fig. 5B). As a control, we measured induction of IL-6, which was not affected by anti–IL-18 treatment.

It has been suggested that the early production of type I IFN can contribute to optimal induction of IL-12p70 (25). To determine the role of type I IFN production in M. tuberculosis tRNA induction of IL-12p70, we performed experiments using a neutralizing Ab to IFN-α/β receptor A (IFNAR). Addition of anti-IFNAR mAbs had no effect on tRNA induction of IL-18, but it blocked the induction of IFN-γ by ∼80% (Fig. 5C). In addition, anti-IFNAR mAbs blocked tRNA induction of IFN-α, consistent with the presence of a type I IFN–driven positive-feedback loop (26, 27). Anti-IFNAR Ab significantly blocked tRNA induction of IL-12p70, whereas the difference compared with the isotype control was not significant. There was no effect of anti-IFNAR treatment on tRNA induction of IL-10.

Previous studies have shown that, in PBMCs, TLR7/8 agonists induce CD56+ (NK and NKT) cells to secrete IFN-γ, dependent on the production of IL-18, even with the concomitant induction of type I IFNs (28). To determine whether CD56+ cells were required for M. tuberculosis tRNA induction of IFN-γ, PBMCs were depleted of this subset prior to stimulation, and induction of IFN-γ release was assessed. In PBMCs depleted of CD56+ cells, M. tuberculosis tRNA induction of IFN-γ was almost entirely inhibited (141 ± 118 pg/ml CD56 depleted versus 4557 ± 4157 pg/ml PBMCs, 97% reduction, p = 0.0215) (Fig. 6A). Although TLR3 ligands have been reported to directly activate NK cells (29), we did not find such activation (data not shown).

FIGURE 6.

Roles for IFN-γ and NK cells. (A) PBMCs were depleted of CD56+ cells and stimulated as shown, and cytokine secretion was measured at 24 h (n = 3). (B) PBMCs were stimulated, and brefeldin A was added at 20 h to arrest cytokine secretion. Cells were collected at 24 h, stained with CD3-FITC, CD56-PE, and IFN-γ–allophycocyanin and measured by flow cytometry. IFN-γ+ lymphocytes were divided into subpopulations determined by CD3/CD56 staining and shown as the percentage of the parent (IFN-γ+) population (n = 3). (C) PBMCs were treated with monoclonal anti–IFN-γ neutralizing Ab for 30 min before stimulation. Cytokine secretion was measured at 24 h (n = 6). (D) PBMCs were treated with monoclonal anti–IFN-γ neutralizing Ab for 30 min before stimulation. Cytokine secretion was measured at 24 h (n = 3). Data are mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, one-way repeated-measures ANOVA and Sidak correction (A), two-way ANOVA, followed by the Tukey post hoc test (B–D).

FIGURE 6.

Roles for IFN-γ and NK cells. (A) PBMCs were depleted of CD56+ cells and stimulated as shown, and cytokine secretion was measured at 24 h (n = 3). (B) PBMCs were stimulated, and brefeldin A was added at 20 h to arrest cytokine secretion. Cells were collected at 24 h, stained with CD3-FITC, CD56-PE, and IFN-γ–allophycocyanin and measured by flow cytometry. IFN-γ+ lymphocytes were divided into subpopulations determined by CD3/CD56 staining and shown as the percentage of the parent (IFN-γ+) population (n = 3). (C) PBMCs were treated with monoclonal anti–IFN-γ neutralizing Ab for 30 min before stimulation. Cytokine secretion was measured at 24 h (n = 6). (D) PBMCs were treated with monoclonal anti–IFN-γ neutralizing Ab for 30 min before stimulation. Cytokine secretion was measured at 24 h (n = 3). Data are mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, one-way repeated-measures ANOVA and Sidak correction (A), two-way ANOVA, followed by the Tukey post hoc test (B–D).

Close modal

We further demonstrated that CD56+ cells were the major source of IFN-γ by intracellular cytokine labeling and flow cytometry. PBMCs were stimulated as above and then stained with Abs for IFN-γ, CD3, and CD56 to measure intracellular IFN-γ, with the goal to differentiate NK, NKT, and T cell populations. IFN-γ+ cells were detectable above background in tRNA- and ssRNA-treated PBMCs (Supplemental Fig. 2). Of IFN-γ+ lymphocytes, the majority were CD56+CD3 NK cells (tRNA 62 ± 9, ssRNA 69 ± 9%), followed by CD56CD3+ T cells (tRNA 28 ± 10, ssRNA 22 ± 8%), and a small number of CD56+CD3+ T cells (tRNA 2.8 ± 1.5, ssRNA 2.4 ± 1%) (Fig. 6B). Because the frequency of NK cells in PBMCs is much lower than that of T cells (∼10 versus ∼75%), this indicated that the populations were differentially predisposed to produce IFN-γ in response to the RNA stimuli. Although T cells are major producers of IFN-γ during the adaptive immune response, this early time point measures the innate immune response, in which NK cells produce the majority of IFN-γ.

Because IL-18 is known to trigger secretion of IFN-γ (3032), and IFN-γ primes IL12A transcription (33), we hypothesized that IFN-γ contributed to the production of IL-12p70. The pretreatment of PBMCs with neutralizing anti–IFN-γ Abs reduced the M. tuberculosis tRNA–induced secretion of IL-12p70 (62% reduction, p < 0.0001) (Fig. 6C). Secretion of IL-18 was unaffected by anti–IFN-γ treatment, consistent with the detection of IL-18 protein prior to the induction of IFN-γ. IL-6 production, which is IFN-γ independent, served a control.

The ability of TLR ligands to activate NK cells to produce IFN-γ involved the production of IL-18 and was dependent on secretion of IL-12p70 (28). Purified NK cells did not produce IFN-γ in response to the same TLR ligands. We had detected production of IL-12p70 as early as 6 h after stimulation with tRNA or ssRNA (Fig. 5A). tRNA and ssRNA induction of IFN-γ was blocked by anti–IL-12p70 neutralizing Abs, with little effect on IL-18 or IL-6 secretion (Fig. 6D). Thus, we conclude that IL-12p70 and IL-18 are essential for the IFN-γ response to microbial RNA, and IFN-γ contributes to robust production of IL-12p70. In addition, a multicellular response to M. tuberculosis ligands involving monocytes and NK cells contributes to induction of the Th1 pathway.

The secondary structure of tRNA includes ssRNA and dsRNA regions. Given that we detected upregulation of TLR8, a PRR for ssRNA, and TLR3, a PRR for dsRNA, mRNAs by M. tuberculosis tRNA, we explored the role of these PRRs in triggering the Th1 cytokine network, starting with TLR8.

To test the requirement of TLR8 signaling for tRNA induction of the Th1-differentiation pathway, we used a specific TLR8 antagonist, VTX-3119 (34). A dose titration was performed to determine the optimal concentration for the antagonist and preclude off-target effects (Supplemental Fig. 3). PBMCs were treated with the TLR8 antagonist VTX-3119 or control molecule VTX-764 and stimulated with TL8-506, a synthetic TLR8-specific agonist (35). Pretreatment with the TLR8 antagonist suppressed cytokine responses to the TLR8 agonist compared with pretreatment with the control compound (IL-18: 60% reduction, p < 0.0001; IFN-γ: 67% reduction, p = 0.024; IL-12p70: 47% reduction, p = 0.0004). The induction of IL-6 by all three TLR ligands was not affected by the TLR8 inhibitor.

Next, PBMCs were incubated with the TLR8 antagonist or control compound and then stimulated with M. tuberculosis tRNA, ssRNA, or TLR2/1L (Fig. 7A). Treatment with the TLR8 antagonist reduced secretion of IL-18 by 50% for activation by M. tuberculosis tRNA (12 ± 6 versus 24 ± 10 pg/ml, p = 0.0106) and 67% for ssRNA. IFN-γ secretion was 90% lower for M. tuberculosis tRNA (695 ± 224 versus 6778 ± 2433 pg/ml, p < 0.0001) and 58% reduced for ssRNA. Secretion of IL-12p70 was diminished by 84% for M. tuberculosis tRNA (42 ± 15 versus 264 ± 108 pg/ml, p < 0.0001) and 73% for ssRNA. IL-6 secretion was not significantly affected by any treatment (data not shown).

FIGURE 7.

Role of TLR8 and synergy with TLR3. (A) PBMCs were treated with TLR8 antagonist or control for 30 min before treatment with TLR2/1L, ssRNA, and M. tuberculosis tRNA. Cytokine secretion was measured at 24 h (IL-18 and IL-6, n = 3; IL-12p70 and IFN-γ, n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, two-way repeated-measures ANOVA and multiple comparisons by the Tukey post hoc test. (B) PBMCs were stimulated with nucleotide oligomers complexed with DOTAP (endosomal PRR), Lipofectamine 2000 (cytosolic PRR), or synthetic agonists for 24 h (n ≥ 4). Statistical significance of ligand-stimulated PBMC versus media calculated by one-way repeated-measures ANOVA, followed by the Dunnett multiple-comparisons test. **p ≤ 0.01, ***p ≤ 0.001. (C) PBMCs were stimulated with poly I:C, TL8-506, or a combination, and cytokine secretion was measured at 24 h (n = 3). Data were summarized using a mixed-effects model with a fixed effect for ligand and a random effect for subject. A two-way interaction term was fit to each ligand to test synergy. *p ≤ 0.05, ***p ≤ 0.001, likelihood ratio test. Data are mean ± SEM.

FIGURE 7.

Role of TLR8 and synergy with TLR3. (A) PBMCs were treated with TLR8 antagonist or control for 30 min before treatment with TLR2/1L, ssRNA, and M. tuberculosis tRNA. Cytokine secretion was measured at 24 h (IL-18 and IL-6, n = 3; IL-12p70 and IFN-γ, n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, two-way repeated-measures ANOVA and multiple comparisons by the Tukey post hoc test. (B) PBMCs were stimulated with nucleotide oligomers complexed with DOTAP (endosomal PRR), Lipofectamine 2000 (cytosolic PRR), or synthetic agonists for 24 h (n ≥ 4). Statistical significance of ligand-stimulated PBMC versus media calculated by one-way repeated-measures ANOVA, followed by the Dunnett multiple-comparisons test. **p ≤ 0.01, ***p ≤ 0.001. (C) PBMCs were stimulated with poly I:C, TL8-506, or a combination, and cytokine secretion was measured at 24 h (n = 3). Data were summarized using a mixed-effects model with a fixed effect for ligand and a random effect for subject. A two-way interaction term was fit to each ligand to test synergy. *p ≤ 0.05, ***p ≤ 0.001, likelihood ratio test. Data are mean ± SEM.

Close modal

To define further the role of TLR8 in the induction of IL-12p70, we surveyed the ability of various oligomers and base analogs targeting endosomal and cytosolic nucleic acid PRRs to induce secretion of this cytokine in PBMCs (Fig. 7B). As before, M. tuberculosis tRNA and ssRNA induced IL-18, with induction also observed for synthetic TLR8 agonist. IFN-γ was strongly induced by ssRNA, as well as, to a lesser extent, M. tuberculosis tRNA and the TLR8-specific ligand. High levels of IL-12p70 were secreted in response to ssRNA and M. tuberculosis tRNA. Despite their induction of IL-18 and IFN-γ, only low amounts of IL-12p70 were produced in response to synthetic agonists for R848, 3M-002 (TLR7/8), and TL8-506 (TLR8). By itself, poly I:C (dsRNA, TLR3) was a weak inducer of IL-18, IFN-γ, and IL-12p70. Even lower levels of these cytokines were observed in response to imiquimod (TLR7), CpG DNA (TLR9), cyclic-di-GMP (STING), and poly(deoxyadenylic-deoxythymidylic) acid (STING/CDS) (3638). These data suggest that TLR8 activation alone was not sufficient to explain the strong induction of IL-12p70 by M. tuberculosis tRNA.

We next addressed whether a combination of a specific TLR8 ligand plus a second ligand activating a different PRR could lead to IL-12p70 secretion. Given that tRNA has single- and double-stranded regions and it has previously been shown to induce type I IFN via activation of TLR3 (39), it was logical to ask whether activation of TLR8 and TLR3 could induce IL-12p70. We found that the TLR3 agonist weakly induced IL-18 to a greater extent than the TLR8 agonist; when combined, the induction of IL-18 by the TLR3 and TLR8 ligands was not synergistic. In contrast, by themselves, the TLR3 and TLR8 ligands weakly induced IFN-γ and IL-12p70, but the combination of the two ligands resulted in the synergistic induction of these cytokines (Fig. 7C). TLR3 and TLR7, but not TLR8, ligands induced IFN-α, but the combination was not synergistic. These data suggest that the mechanism by which TLR3 synergizes with TLR8 in the induction of the Th1 pathway likely includes, but is not limited to, the production of type I IFN. Together, these data identify an innate pathway, which requires recognition of the pathogen by distinct PRRs and distinct cell types and involves the induction of types I and II IFNs and downstream genes, that leads to the production of IL-12p70, a cytokine required for the induction of a Th1 response as part of a major host defense pathway (Fig. 8).

FIGURE 8.

M. tuberculosis tRNA triggers the induction of IL-12p70. This model, based on the experimental data in this article, as well as the literature, shows that M. tuberculosis tRNA induces secretion of IL-18 via TLR8. Type I IFNs may contribute to enhanced induction of IL-12p35 via upregulation of TLR3, which, in combination with IL-12p40 induction, leads to secretion of bioactive IL-12p70. IL-12p70 upregulates IL-18R on NK cells, facilitating the ability of IL-18 and IL-12p70 to synergize to induce secretion of IFN-γ. IFN-γ, in turn, enhances IL-12p70 secretion. The key cell types in this process are monocytes (Mo)/macrophages (MΦ), NK cells, and myeloid DCs (mDC).

FIGURE 8.

M. tuberculosis tRNA triggers the induction of IL-12p70. This model, based on the experimental data in this article, as well as the literature, shows that M. tuberculosis tRNA induces secretion of IL-18 via TLR8. Type I IFNs may contribute to enhanced induction of IL-12p35 via upregulation of TLR3, which, in combination with IL-12p40 induction, leads to secretion of bioactive IL-12p70. IL-12p70 upregulates IL-18R on NK cells, facilitating the ability of IL-18 and IL-12p70 to synergize to induce secretion of IFN-γ. IFN-γ, in turn, enhances IL-12p70 secretion. The key cell types in this process are monocytes (Mo)/macrophages (MΦ), NK cells, and myeloid DCs (mDC).

Close modal

The ability of the immune system to mount a cell-mediated immune response involving Th1 cells is critical for host defense against many intracellular bacteria, including M. tuberculosis. To generate cell-mediated immunity, the innate immune system must recognize the microbial invader and instruct the adaptive T cell response. Yet the classic PAMPs found in mycobacteria, lipoproteins that activate TLR2/1 and muramyl dipeptide that activates NOD2, only weakly induce IL-12p70, a cytokine that is required for induction of a Th1 response. In this study, we found that M. tuberculosis tRNA, detected in the bacterial culture filtrate, is a potent inducer of IL-12p70. Exploring tRNA-induced gene-expression profiles in human PBMCs, we identified an integrated cellular and molecular pathway, beginning with the production of IL-18, type I IFNs, and IL-12p70, that results in NK cell secretion of IFN-γ and the subsequent robust induction of IL-12p70. The ability of M. tuberculosis tRNA to trigger this gene network was dependent on TLR8, yet TLR8 activation by itself was not sufficient for induction. Instead, activation of TLR3 and TLR8 synergized for the robust induction of IFN-γ and IL-12p70, suggesting a role for the concurrent activation of nucleotide receptors in mounting a cell-mediated immune response against intracellular mycobacteria.

Initial studies revealed that the culture filtrate of M. tuberculosis was sufficient by itself to induce T cells responses in a naive animal, suggesting the presence of an adjuvant to activate the innate immune response, along with the protein/Ag signals required to stimulate an adaptive immune response (27). Subsequently, a purified protein derivative fraction of the culture filtrate was isolated and shown to be useful as a diagnostic test for tuberculosis exposure, identifying individuals who had developed a delayed-type hypersensitivity response because of exposure to M. tuberculosis or vaccination with BCG. The component(s) of the culture filtrate that can act as an adjuvant to stimulate the innate immune response has not been evaluated extensively, but tRNA was previously found to be a major component of the nucleotide fraction of the filtrate that was shown to induce apoptosis in monocytes (18). Our data demonstrate a mechanism by which mycobacterial tRNA can robustly induce IL-12p70, a key cytokine in the induction of a Th1 response and, hence, could serve as an adjuvant for the adaptive T cell response.

It was unexpected that a single mycobacterial ligand simultaneously induced the type II IFN, IFN-γ, and the type I IFNs because, in chronic mycobacterial infections, including tuberculosis and leprosy, the type I IFNs inhibit the production and action of IFN-γ (40, 41). Yet, as part of the pathway to induce IL-12p70, tRNA and ssRNA were found to be potent inducers of both types of IFN, as well as IFN-downstream genes. IFN-α, at low levels, has been shown to augment the ability of TLR ligands to trigger IL-12p70 production in DCs (25, 42, 43), consistent with our data that type I IFNs, as part of the acute immune response to M. tuberculosis tRNA, are involved in the production of IL-12p70. The findings presented in this article also demonstrate that type I IFN was required for M. tuberculosis tRNA induction of IFN-γ by NK cells, thereby linking the production of type I IFN to the production of type II IFN. The induction of type I IFNs, together with the early production of IFN-γ following tRNA activation of PBMCs, is also likely to contribute to IL-12p70 production, because IFN-γ can augment the ability of other TLR ligands to induce IL-12p40 and IL-12p35 (44, 45). In contrast, in chronic bacterial infection, types I and II IFN have opposing functional roles, resulting in the inhibition of IFN-γ–induced antimicrobial responses (41). The ability of type I IFN to directly interfere with transcription of IL12B (46), leading to inhibition of IL-12p70 (47, 48) and blockade of Th1 responses (49, 50), can be overcome by the production of ISG15, which downregulates the type I IFN response (51, 52). We note that tRNA and ssRNA strongly induced ISG15. In viral infection, the initial induction of type I IFN is required to clear the infection but, in chronic infections, it contributes to pathogenesis.

In determining the source of IFN-γ production, our data underscore the importance of NK cell activation in the robust induction of IL-12p70. We identified that M. tuberculosis tRNA, by triggering TLR8 to induce the production of IL-18, induces NK cell production of IFN-γ. The activation of NK cells to produce IFN-γ was dependent on the early production of IL-12p70, presumably through its ability to upregulate both subunits of IL-18R on NK cells (53, 54), thereby enhancing responsiveness to IL-18. Consistent with this mechanism, tRNA upregulated IL18R1 and IL18RAP mRNA in PBMCs, and the addition of neutralizing Abs to IL-12p70 inhibited tRNA induction of IFN-γ. This early production of IFN-γ by NK cells was required for the later, more robust, induction of IL-12p70, because depletion of NK cells or the addition of anti–IFN-γ blocking Abs reduced tRNA induction of IL-12p70. A role for NK cells in tuberculosis has been demonstrated in mouse models by their recruitment to the lung as early as 7 d postinfection (55), with a role in preventing tissue damage (56). In human tuberculosis, NK cells have been identified at the site of infection in patients: in the pleural fluid (57) and in granulomas in pulmonary lesions (58). In addition, when used to revaccinate individuals, BCG boosted IFN-γ–producing CD56+ cells in vivo; when added to whole blood, it upregulated IFN-γ–producing CD56+ cells in vitro via an IL-12 and IL-18–dependent mechanism (59).

The ability of M. tuberculosis tRNA to induce IL-12p70 is likely related to its natural location when released by M. tuberculosis in specific subcellular compartments. M. tuberculosis resides primarily in endosomes that contain the nucleotide-sensing TLRs, including TLR3 (dsRNA), TLR7 (ssRNA), TLR8 (ssRNA), and TLR9 (dsDNA). TLR8 is primarily expressed in human monocytes and myeloid DCs (60) and is localized to endosomes/phagosomes, allowing the innate immune system to distinguish self-RNA (nucleus, cytoplasm) from non-self RNA (endosome/phagosome). Our data indicate that the ability of M. tuberculosis tRNA to induce IL-18, IFN-γ, and IL-12p70 was blocked by a specific TLR8 antagonist, consistent with previous studies (61) The mechanism by which TLR8 recognizes tRNA likely involves recognition of the ssRNA regions present in the tRNA stem loop sequences (e.g., in the anticodon sequence) (62). Of potential clinical relevance, a TLR8 gain-of-function polymorphism (TLR8 A1G) correlates with increased resistance to tuberculosis in humans (6369). Presumably, the abundance of M. tuberculosis tRNA in the bacterial culture filtrate is due to a combination of accumulation through bacterial lysis and resistance to degradation (18). Although TLR8 has been shown to recognize Borrelia burgdorferi RNA in infected human macrophages (70), experiments to demonstrate a role for TLR8 during an in vitro infection of monocytes/macrophages with M. tuberculosis have not been successful, perhaps because of the slow turnover of bacteria in the short term in in vitro cultures.

Although M. tuberculosis tRNA was a potent inducer of IL-12p70 and did so in a TLR8-dependent fashion, a TLR8 agonist alone weakly induced IL-12p70. In addition to ssRNA regions, tRNA contains dsRNA regions and has been shown to trigger TLR3 (39), prompting us to measure the response to a combination of ligands. The combination of dsRNA that activates TLR3 and a synthetic ligand that activates TLR8 induced IL-18 in an additive manner, but they were synergistic in the induction of IFN-γ and IL-12p70. Therefore, we propose a model for the ability of tRNA to potently induce IL-12p70 (Fig. 8) that involves TLR8 induction of IL-18. TLR3 activation leads to production of type I IFN, which can enhance induction of IL-18 (71) feedback to enhance TLR3 expression (72, 73), leading to induction of IL-12p35 via an IRF3-dependent pathway (74). This, in combination with early TLR8 induction of IL-12p40, leads to the formation of IL-12p70, which upregulates both subunits of IL-18R on NK cells (53, 54), thus synergizing with IL-18 to induce secretion of IFN-γ from NK cells (75). We infer that, because IL-12p70 is assembled by a single cell, myeloid DCs are involved, given that they express TLR3 and TLR8 (60) and are key producers of this cytokine. The production of IFN-γ subsequently enhances the production of IL-12p70, thereby amplifying its effects.

The innate pathway that we describe for induction of IL-12p70 is relevant, as there is evidence from both murine models and from genetic studies in humans that the induction of IFN-γ is required for protective immunity against tuberculosis. Mice lacking the gene for IFN-γ died of M. tuberculosis challenge 2–3 wk after i.v. challenge and within a month after aerosol challenge (76). Activation of mouse macrophages by IFN-γ and TNF-α induced killing of intracellular bacteria through the induction of NO (77, 78). Individuals with genetic disorders leading to the decreased production of, or response to, IFN-γ are highly susceptible to tuberculosis and other mycobacterial diseases (7981). One mechanism by which IFN-γ contributes to host defense against M. tuberculosis in humans is through activation of macrophage antimicrobial activity via the vitamin D–dependent induction of the antimicrobial peptides cathelicidin and DEFB4 (82).

Our data suggest that a single microbial ligand may trigger multiple PRRs, leading to upregulation of IL-12p35 and IL-12p40 to synergize in the production of IL-12p70, thereby inducing a distinct innate immune response critical to host defense. Combinatorial analysis of how multiple innate signals trigger gene expression identified synergistic and antagonistic interactions, suggesting a functional role in the adaptation to complex stimuli (83). As such, tRNA induced a gene network that was associated with innate instruction of an adaptive T cell response. In addition to IL18, IL12A, IL12B, and IFNG, which are known to polarize toward a Th1 adaptive response, we found upregulation of multiple T cell costimulatory molecules, including CD40 and CD80 (42, 84). Also of note were chemoattractants, such as CXCL10, which recruit T cells, NK cells, DCs, and monocytes to the site of infection (85). Several of the genes in this network have been shown to be critical for host defense against mycobacterial infection in humans based on the enhanced susceptibility of individuals with genetic mutations, including IFNG, IFNGR1, and IL12RB1 (7981). ISG15 is required to maintain IFN-γ production during mycobacterial infection (51, 52), such that its genetic alteration increased the susceptibility of individuals to mycobacteria infection and abrogated the IFN-γ response to mycobacterial infection in vitro (86). As stated, the roles of IL-12, IL-18, and type I IFN in driving NK cell activation, as well as the ability of IFN-γ to amplify IL-12, have been established. In this study, we have used molecular and cellular approaches to identify a network by which a single microbial ligand triggers multiple PRRs, leading to the production of types I and II IFNs and resulting in types I and II IFN–dependent production of IL-12p70, as part of the innate immune response against many intracellular pathogens.

This work was supported by National Institutes of Health Grants R01HL119068, R01AI022553, R01HL129887, R01AR040312, and P50AR063020 (to R.L.M.) and by the Singapore–MIT Alliance for Research and Technology under a grant from the National Research Foundation of Singapore (to P.C.D.).

The microarray data presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110325) under accession number GSE110325.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BCG

bacillus Calmette–Guérin

DC

dendritic cell

FC

fold change

FDR

false discovery rate

GO

Gene Ontology

IFNAR

IFN-α/β receptor A

IPA

Ingenuity Pathway Analysis

PAMP

pathogen associated molecular pattern

PCA

principal component analysis

poly I:C

polyinosinic-polycytidylic acid

PRR

pattern recognition receptor

rLog

regularized logarithm

WGCNA

Weighted Gene Correlation Network Analysis.

1
Bloom
,
B. R.
,
C. J. L.
Murray
.
1992
.
Tuberculosis: commentary on a reemergent killer.
Science
257
:
1055
1064
.
2
Seibert
,
F. B.
1926
.
The isolation of a crystalline protein with tuberculin activity.
Science
63
:
619c
620c
.
3
Long
,
E. R.
,
F. B.
Seibert
.
1926
.
The chemical composition of the active principle of tuberculin. VII. The evidence that the active principle is a protein.
Am. Rev. Tuberc.
13
:
448
.
4
Seibert
,
F. B.
1928
.
X. The isolation in crystalline form and identification of the active principle of tuberculin.
Am. Rev. Tuberc.
17
:
402
421
.
5
Seibert
,
F. B.
1934
.
The isolation and properties of the purified protein derivative of tuberculin.
Am. Rev. Tuberc.
30
:
713
720
.
6
Seibert
,
F. B.
,
J. T.
Glenn
.
1941
.
Tuberculin purified protein derivative. Preparation and analyses of a large quantity for standard.
Am. Rev. Tuberc. Pulm. Dis.
44
:
9
25
.
7
Seibert
,
F. B.
1944
.
The chemistry of tuberculin.
Chem. Rev.
34
:
107
127
.
8
Collins
,
F. M.
,
J. R.
Lamb
,
D. B.
Young
.
1988
.
Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate.
Infect. Immun.
56
:
1260
1266
.
9
Pal
,
P. G.
,
M. A.
Horwitz
.
1992
.
Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis.
Infect. Immun.
60
:
4781
4792
.
10
Hubbard
,
R. D.
,
C. M.
Flory
,
F. M.
Collins
.
1992
.
Immunization of mice with mycobacterial culture filtrate proteins.
Clin. Exp. Immunol.
87
:
94
98
.
11
Schenk
,
M.
,
S. R.
Krutzik
,
P. A.
Sieling
,
D. J.
Lee
,
R. M.
Teles
,
M. T.
Ochoa
,
E.
Komisopoulou
,
E. N.
Sarno
,
T. H.
Rea
,
T. G.
Graeber
, et al
.
2012
.
NOD2 triggers an interleukin-32-dependent human dendritic cell program in leprosy.
Nat. Med.
18
:
555
563
.
12
Watson
,
R. O.
,
P. S.
Manzanillo
,
J. S.
Cox
.
2012
.
Extracellular M. tuberculosis DNA targets bacteria for autophagy by activating the host DNA-sensing pathway.
Cell
150
:
803
815
.
13
Collins
,
A. C.
,
H.
Cai
,
T.
Li
,
L. H.
Franco
,
X. D.
Li
,
V. R.
Nair
,
C. R.
Scharn
,
C. E.
Stamm
,
B.
Levine
,
Z. J.
Chen
,
M. U.
Shiloh
.
2015
.
Cyclic GMP-AMP synthase is an innate immune DNA sensor for Mycobacterium tuberculosis.
Cell Host Microbe
17
:
820
828
.
14
Manzanillo
,
P. S.
,
M. U.
Shiloh
,
D. A.
Portnoy
,
J. S.
Cox
.
2012
.
Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages.
Cell Host Microbe
11
:
469
480
.
15
Wassermann
,
R.
,
M. F.
Gulen
,
C.
Sala
,
S. G.
Perin
,
Y.
Lou
,
J.
Rybniker
,
J. L.
Schmid-Burgk
,
T.
Schmidt
,
V.
Hornung
,
S. T.
Cole
,
A.
Ablasser
.
2015
.
Mycobacterium tuberculosis differentially activates cGAS- and inflammasome-dependent intracellular immune responses through ESX-1.
Cell Host Microbe
17
:
799
810
.
16
Manca
,
C.
,
L.
Tsenova
,
A.
Bergtold
,
S.
Freeman
,
M.
Tovey
,
J. M.
Musser
,
C. E.
Barry
III.
,
V. H.
Freedman
,
G.
Kaplan
.
2001
.
Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta.
Proc. Natl. Acad. Sci. USA
98
:
5752
5757
.
17
Ottenhoff
,
T. H.
,
R. H.
Dass
,
N.
Yang
,
M. M.
Zhang
,
H. E.
Wong
,
E.
Sahiratmadja
,
C. C.
Khor
,
B.
Alisjahbana
,
R.
van Crevel
,
S.
Marzuki
, et al
.
2012
.
Genome-wide expression profiling identifies type 1 interferon response pathways in active tuberculosis.
PLoS One
7
:
e45839
.
18
Obregón-Henao
,
A.
,
M. A.
Duque-Correa
,
M.
Rojas
,
L. F.
García
,
P. J.
Brennan
,
B. L.
Ortiz
,
J. T.
Belisle
.
2012
.
Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism.
PLoS One
7
:
e29970
.
19
Cai
,
W. M.
,
Y. H.
Chionh
,
F.
Hia
,
C.
Gu
,
S.
Kellner
,
M. E.
McBee
,
C. S.
Ng
,
Y. L.
Pang
,
E. G.
Prestwich
,
K. S.
Lim
, et al
.
2015
.
A platform for discovery and quantification of modified ribonucleosides in RNA: application to stress-induced reprogramming of tRNA modifications.
Methods Enzymol.
560
:
29
71
.
20
Langfelder
,
P.
,
S.
Horvath
.
2008
.
WGCNA: an R package for weighted correlation network analysis.
BMC Bioinformatics
9
:
559
.
21
Heil
,
F.
,
H.
Hemmi
,
H.
Hochrein
,
F.
Ampenberger
,
C.
Kirschning
,
S.
Akira
,
G.
Lipford
,
H.
Wagner
,
S.
Bauer
.
2004
.
Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.
Science
303
:
1526
1529
.
22
Love
,
M. I.
,
W.
Huber
,
S.
Anders
.
2014
.
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
Genome Biol.
15
:
550
.
23
Bindea
,
G.
,
B.
Mlecnik
,
H.
Hackl
,
P.
Charoentong
,
M.
Tosolini
,
A.
Kirilovsky
,
W. H.
Fridman
,
F.
Pagès
,
Z.
Trajanoski
,
J.
Galon
.
2009
.
ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks.
Bioinformatics
25
:
1091
1093
.
24
Hu
,
Z.
,
J.
Mellor
,
J.
Wu
,
C.
DeLisi
.
2004
.
VisANT: an online visualization and analysis tool for biological interaction data.
BMC Bioinformatics
5
:
17
.
25
Gautier
,
G.
,
M.
Humbert
,
F.
Deauvieau
,
M.
Scuiller
,
J.
Hiscott
,
E. E.
Bates
,
G.
Trinchieri
,
C.
Caux
,
P.
Garrone
.
2005
.
A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells.
J. Exp. Med.
201
:
1435
1446
.
26
Marié
,
I.
,
J. E.
Durbin
,
D. E.
Levy
.
1998
.
Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7.
EMBO J.
17
:
6660
6669
.
27
Ma
,
F.
,
B.
Li
,
Y.
Yu
,
S. S.
Iyer
,
M.
Sun
,
G.
Cheng
.
2015
.
Positive feedback regulation of type I interferon by the interferon-stimulated gene STING.
EMBO Rep.
16
:
202
212
.
28
Gorski
,
K. S.
,
E. L.
Waller
,
J.
Bjornton-Severson
,
J. A.
Hanten
,
C. L.
Riter
,
W. C.
Kieper
,
K. B.
Gorden
,
J. S.
Miller
,
J. P.
Vasilakos
,
M. A.
Tomai
,
S. S.
Alkan
.
2006
.
Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists.
Int. Immunol.
18
:
1115
1126
.
29
Schmidt
,
K. N.
,
B.
Leung
,
M.
Kwong
,
K. A.
Zarember
,
S.
Satyal
,
T. A.
Navas
,
F.
Wang
,
P. J.
Godowski
.
2004
.
APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA.
J. Immunol.
172
:
138
143
.
30
Ushio
,
S.
,
M.
Namba
,
T.
Okura
,
K.
Hattori
,
Y.
Nukada
,
K.
Akita
,
F.
Tanabe
,
K.
Konishi
,
M.
Micallef
,
M.
Fujii
, et al
.
1996
.
Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.
J. Immunol.
156
:
4274
4279
.
31
García
,
V. E.
,
K.
Uyemura
,
P. A.
Sieling
,
M. T.
Ochoa
,
C. T.
Morita
,
H.
Okamura
,
M.
Kurimoto
,
T. H.
Rea
,
R. L.
Modlin
.
1999
.
IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection.
J. Immunol.
162
:
6114
6121
.
32
Lee
,
H. R.
,
S. Y.
Yoon
,
S. B.
Song
,
Y.
Park
,
T. S.
Kim
,
S.
Kim
,
D. Y.
Hur
,
H. K.
Song
,
H.
Park
,
D.
Cho
.
2011
.
Interleukin-18-mediated interferon-gamma secretion is regulated by thymosin beta 4 in human NK cells.
Immunobiology
216
:
1155
1162
.
33
Liu
,
J.
,
X.
Guan
,
T.
Tamura
,
K.
Ozato
,
X.
Ma
.
2004
.
Synergistic activation of interleukin-12 p35 gene transcription by interferon regulatory factor-1 and interferon consensus sequence-binding protein.
J. Biol. Chem.
279
:
55609
55617
.
34
Howbert, J. J., G. Dietsch, R. Hershberg, L. E. Burgess, J. P. Lyssikatos, B. Newhouse, and H. W. Yang, inventors; Ventirx Pharmaceuticals Inc., Array Biopharma Inc., assignees. Substituted benzoazepines as toll-like receptor modulators. United States patent application PCT/US2010/045934, Publication No. WO2011022508 A2. 2011 Feb 24
.
35
Lu
,
H.
,
G. N.
Dietsch
,
M. A.
Matthews
,
Y.
Yang
,
S.
Ghanekar
,
M.
Inokuma
,
M.
Suni
,
V. C.
Maino
,
K. E.
Henderson
,
J. J.
Howbert
, et al
.
2012
.
VTX-2337 is a novel TLR8 agonist that activates NK cells and augments ADCC.
Clin. Cancer Res.
18
:
499
509
.
36
Ablasser
,
A.
,
F.
Bauernfeind
,
G.
Hartmann
,
E.
Latz
,
K. A.
Fitzgerald
,
V.
Hornung
.
2009
.
RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate.
Nat. Immunol.
10
:
1065
1072
.
37
Zhang
,
Z.
,
B.
Yuan
,
M.
Bao
,
N.
Lu
,
T.
Kim
,
Y. J.
Liu
.
2011
.
The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells.
Nat. Immunol.
12
:
959
965
.
38
Ma
,
F.
,
B.
Li
,
S. Y.
Liu
,
S. S.
Iyer
,
Y.
Yu
,
A.
Wu
,
G.
Cheng
.
2015
.
Positive feedback regulation of type I IFN production by the IFN-inducible DNA sensor cGAS.
J. Immunol.
194
:
1545
1554
.
39
Wang
,
Z.
,
L.
Xiang
,
J.
Shao
,
Z.
Yuan
.
2006
.
The 3′ CCACCA sequence of tRNAAla(UGC) is the motif that is important in inducing Th1-like immune response, and this motif can be recognized by Toll-like receptor 3.
Clin. Vaccine Immunol.
13
:
733
739
.
40
Berry
,
M. P.
,
C. M.
Graham
,
F. W.
McNab
,
Z.
Xu
,
S. A.
Bloch
,
T.
Oni
,
K. A.
Wilkinson
,
R.
Banchereau
,
J.
Skinner
,
R. J.
Wilkinson
, et al
.
2010
.
An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis.
Nature
466
:
973
977
.
41
Teles
,
R. M.
,
T. G.
Graeber
,
S. R.
Krutzik
,
D.
Montoya
,
M.
Schenk
,
D. J.
Lee
,
E.
Komisopoulou
,
K.
Kelly-Scumpia
,
R.
Chun
,
S. S.
Iyer
, et al
.
2013
.
Type I interferon suppresses type II interferon-triggered human anti-mycobacterial responses.
Science
339
:
1448
1453
.
42
Hermann
,
P.
,
M.
Rubio
,
T.
Nakajima
,
G.
Delespesse
,
M.
Sarfati
.
1998
.
IFN-alpha priming of human monocytes differentially regulates gram-positive and gram-negative bacteria-induced IL-10 release and selectively enhances IL-12p70, CD80, and MHC class I expression.
J. Immunol.
161
:
2011
2018
.
43
Heystek
,
H. C.
,
B.
den Drijver
,
M. L.
Kapsenberg
,
R. A.
van Lier
,
E. C.
de Jong
.
2003
.
Type I IFNs differentially modulate IL-12p70 production by human dendritic cells depending on the maturation status of the cells and counteract IFN-gamma-mediated signaling.
Clin. Immunol.
107
:
170
177
.
44
Ma
,
X.
,
J. M.
Chow
,
G.
Gri
,
G.
Carra
,
F.
Gerosa
,
S. F.
Wolf
,
R.
Dzialo
,
G.
Trinchieri
.
1996
.
The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells.
J. Exp. Med.
183
:
147
157
.
45
Hayes
,
M. P.
,
F. J.
Murphy
,
P. R.
Burd
.
1998
.
Interferon-gamma-dependent inducible expression of the human interleukin-12 p35 gene in monocytes initiates from a TATA-containing promoter distinct from the CpG-rich promoter active in Epstein-Barr virus-transformed lymphoblastoid cells.
Blood
91
:
4645
4651
.
46
Byrnes
,
A. A.
,
X.
Ma
,
P.
Cuomo
,
K.
Park
,
L.
Wahl
,
S. F.
Wolf
,
H.
Zhou
,
G.
Trinchieri
,
C. L.
Karp
.
2001
.
Type I interferons and IL-12: convergence and cross-regulation among mediators of cellular immunity.
Eur. J. Immunol.
31
:
2026
2034
.
47
Manca
,
C.
,
L.
Tsenova
,
S.
Freeman
,
A. K.
Barczak
,
M.
Tovey
,
P. J.
Murray
,
C.
Barry
,
G.
Kaplan
.
2005
.
Hypervirulent M. tuberculosis W/Beijing strains upregulate type I IFNs and increase expression of negative regulators of the Jak-Stat pathway.
J. Interferon Cytokine Res.
25
:
694
701
.
48
Guarda
,
G.
,
M.
Braun
,
F.
Staehli
,
A.
Tardivel
,
C.
Mattmann
,
I.
Förster
,
M.
Farlik
,
T.
Decker
,
R. A.
Du Pasquier
,
P.
Romero
,
J.
Tschopp
.
2011
.
Type I interferon inhibits interleukin-1 production and inflammasome activation.
Immunity
34
:
213
223
.
49
de Paus
,
R. A.
,
A.
van Wengen
,
I.
Schmidt
,
M.
Visser
,
E. M.
Verdegaal
,
J. T.
van Dissel
,
E.
van de Vosse
.
2013
.
Inhibition of the type I immune responses of human monocytes by IFN-α and IFN-β.
Cytokine
61
:
645
655
.
50
Orme
,
I. M.
,
R. T.
Robinson
,
A. M.
Cooper
.
2015
.
The balance between protective and pathogenic immune responses in the TB-infected lung.
Nat. Immunol.
16
:
57
63
.
51
Bogunovic
,
D.
,
M.
Byun
,
L. A.
Durfee
,
A.
Abhyankar
,
O.
Sanal
,
D.
Mansouri
,
S.
Salem
,
I.
Radovanovic
,
A. V.
Grant
,
P.
Adimi
, et al
.
2012
.
Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency.
Science
337
:
1684
1688
.
52
Zhang
,
X.
,
D.
Bogunovic
,
B.
Payelle-Brogard
,
V.
Francois-Newton
,
S. D.
Speer
,
C.
Yuan
,
S.
Volpi
,
Z.
Li
,
O.
Sanal
,
D.
Mansouri
, et al
.
2015
.
Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation.
Nature
517
:
89
93
.
53
Yoshimoto
,
T.
,
K.
Takeda
,
T.
Tanaka
,
K.
Ohkusu
,
S.
Kashiwamura
,
H.
Okamura
,
S.
Akira
,
K.
Nakanishi
.
1998
.
IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-gamma production.
J. Immunol.
161
:
3400
3407
.
54
Fehniger
,
T. A.
,
M. H.
Shah
,
M. J.
Turner
,
J. B.
VanDeusen
,
S. P.
Whitman
,
M. A.
Cooper
,
K.
Suzuki
,
M.
Wechser
,
F.
Goodsaid
,
M. A.
Caligiuri
.
1999
.
Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response.
J. Immunol.
162
:
4511
4520
.
55
Junqueira-Kipnis
,
A. P.
,
A.
Kipnis
,
A.
Jamieson
,
M. G.
Juarrero
,
A.
Diefenbach
,
D. H.
Raulet
,
J.
Turner
,
I. M.
Orme
.
2003
.
NK cells respond to pulmonary infection with Mycobacterium tuberculosis, but play a minimal role in protection.
J. Immunol.
171
:
6039
6045
.
56
Feng
,
C. G.
,
M.
Kaviratne
,
A. G.
Rothfuchs
,
A.
Cheever
,
S.
Hieny
,
H. A.
Young
,
T. A.
Wynn
,
A.
Sher
.
2006
.
NK cell-derived IFN-gamma differentially regulates innate resistance and neutrophil response in T cell-deficient hosts infected with Mycobacterium tuberculosis.
J. Immunol.
177
:
7086
7093
.
57
Schierloh
,
P.
,
M.
Alemán
,
N.
Yokobori
,
L.
Alves
,
N.
Roldán
,
E.
Abbate
,
M.
del C Sasiain
,
S.
de la Barrera
.
2005
.
NK cell activity in tuberculosis is associated with impaired CD11a and ICAM-1 expression: a regulatory role of monocytes in NK activation.
Immunology
116
:
541
552
.
58
Portevin
,
D.
,
L. E.
Via
,
S.
Eum
,
D.
Young
.
2012
.
Natural killer cells are recruited during pulmonary tuberculosis and their ex vivo responses to mycobacteria vary between healthy human donors in association with KIR haplotype.
Cell. Microbiol.
14
:
1734
1744
.
59
Suliman
,
S.
,
H.
Geldenhuys
,
J. L.
Johnson
,
J. E.
Hughes
,
E.
Smit
,
M.
Murphy
,
A.
Toefy
,
L.
Lerumo
,
C.
Hopley
,
B.
Pienaar
, et al
.
2016
.
Bacillus calmette-guérin (BCG) revaccination of adults with latent Mycobacterium tuberculosis infection induces long-lived BCG-reactive NK cell responses.
J. Immunol.
197
:
1100
1110
.
60
Kadowaki
,
N.
,
S.
Ho
,
S.
Antonenko
,
R. W.
Malefyt
,
R. A.
Kastelein
,
F.
Bazan
,
Y. J.
Liu
.
2001
.
Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.
J. Exp. Med.
194
:
863
869
.
61
Sha
,
W.
,
H.
Mitoma
,
S.
Hanabuchi
,
M.
Bao
,
L.
Weng
,
N.
Sugimoto
,
Y.
Liu
,
Z.
Zhang
,
J.
Zhong
,
B.
Sun
,
Y. J.
Liu
.
2014
.
Human NLRP3 inflammasome senses multiple types of bacterial RNAs.
Proc. Natl. Acad. Sci. USA
111
:
16059
16064
.
62
Chan
,
P. P.
,
T. M.
Lowe
.
2009
.
GtRNAdb: a database of transfer RNA genes detected in genomic sequence.
Nucleic Acids Res.
37
(
Database
):
D93
D97
.
63
Davila
,
S.
,
M. L.
Hibberd
,
R.
Hari Dass
,
H. E.
Wong
,
E.
Sahiratmadja
,
C.
Bonnard
,
B.
Alisjahbana
,
J. S.
Szeszko
,
Y.
Balabanova
,
F.
Drobniewski
, et al
.
2008
.
Genetic association and expression studies indicate a role of toll-like receptor 8 in pulmonary tuberculosis.
PLoS Genet.
4
:
e1000218
.
64
Bukhari
,
M.
,
M. A.
Aslam
,
A.
Khan
,
Q.
Iram
,
A.
Akbar
,
A. G.
Naz
,
S.
Ahmad
,
M. M.
Ahmad
,
U. A.
Ashfaq
,
H.
Aziz
,
M.
Ali
.
2015
.
TLR8 gene polymorphism and association in bacterial load in southern Punjab of Pakistan: an association study with pulmonary tuberculosis.
Int. J. Immunogenet.
42
:
46
51
.
65
Daya
,
M.
,
L.
van der Merwe
,
P. D.
van Helden
,
M.
Möller
,
E. G.
Hoal
.
2015
.
Investigating the role of gene-gene interactions in TB susceptibility.
PLoS One
10
:
e0123970
.
66
Lai
,
Y. F.
,
T. M.
Lin
,
C. H.
Wang
,
P. Y.
Su
,
J. T.
Wu
,
M. C.
Lin
,
H. L.
Eng
.
2016
.
Functional polymorphisms of the TLR7 and TLR8 genes contribute to Mycobacterium tuberculosis infection.
Tuberculosis
98
:
125
131
.
67
Salie
,
M.
,
M.
Daya
,
L. A.
Lucas
,
R. M.
Warren
,
G. D.
van der Spuy
,
P. D.
van Helden
,
E. G.
Hoal
,
M.
Möller
.
2015
.
Association of toll-like receptors with susceptibility to tuberculosis suggests sex-specific effects of TLR8 polymorphisms.
Infect. Genet. Evol.
34
:
221
229
.
68
Sun
,
Q.
,
Q.
Zhang
,
H. P.
Xiao
,
C.
Bai
.
2015
.
Toll-like receptor polymorphisms and tuberculosis susceptibility: a comprehensive meta-analysis.
J. Huazhong Univ. Sci. Technolog. Med. Sci.
35
:
157
168
.
69
Wu
,
L.
,
Y.
Hu
,
D.
Li
,
W.
Jiang
,
B.
Xu
.
2015
.
Screening toll-like receptor markers to predict latent tuberculosis infection and subsequent tuberculosis disease in a Chinese population.
BMC Med. Genet.
16
:
19
.
70
Cervantes
,
J. L.
,
C. J.
La Vake
,
B.
Weinerman
,
S.
Luu
,
C.
O’Connell
,
P. H.
Verardi
,
J. C.
Salazar
.
2013
.
Human TLR8 is activated upon recognition of Borrelia burgdorferi RNA in the phagosome of human monocytes.
J. Leukoc. Biol.
94
:
1231
1241
.
71
Fang
,
R.
,
H.
Hara
,
S.
Sakai
,
E.
Hernandez-Cuellar
,
M.
Mitsuyama
,
I.
Kawamura
,
K.
Tsuchiya
.
2014
.
Type I interferon signaling regulates activation of the absent in melanoma 2 inflammasome during Streptococcus pneumoniae infection.
Infect. Immun.
82
:
2310
2317
.
72
Miettinen
,
M.
,
T.
Sareneva
,
I.
Julkunen
,
S.
Matikainen
.
2001
.
IFNs activate toll-like receptor gene expression in viral infections.
Genes Immun.
2
:
349
355
.
73
Sirén
,
J.
,
J.
Pirhonen
,
I.
Julkunen
,
S.
Matikainen
.
2005
.
IFN-alpha regulates TLR-dependent gene expression of IFN-alpha, IFN-beta, IL-28, and IL-29.
J. Immunol.
174
:
1932
1937
.
74
Goriely
,
S.
,
C.
Molle
,
M.
Nguyen
,
V.
Albarani
,
N. O.
Haddou
,
R.
Lin
,
D.
De Wit
,
V.
Flamand
,
F.
Willems
,
M.
Goldman
.
2006
.
Interferon regulatory factor 3 is involved in Toll-like receptor 4 (TLR4)- and TLR3-induced IL-12p35 gene activation.
Blood
107
:
1078
1084
.
75
Matikainen
,
S.
,
A.
Paananen
,
M.
Miettinen
,
M.
Kurimoto
,
T.
Timonen
,
I.
Julkunen
,
T.
Sareneva
.
2001
.
IFN-alpha and IL-18 synergistically enhance IFN-gamma production in human NK cells: differential regulation of Stat4 activation and IFN-gamma gene expression by IFN-alpha and IL-12.
Eur. J. Immunol.
31
:
2236
2245
.
76
Flynn
,
J. L.
,
J.
Chan
,
K. J.
Triebold
,
D. K.
Dalton
,
T. A.
Stewart
,
B. R.
Bloom
.
1993
.
An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection.
J. Exp. Med.
178
:
2249
2254
.
77
Chan
,
J.
,
Y.
Xing
,
R. S.
Magliozzo
,
B. R.
Bloom
.
1992
.
Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages.
J. Exp. Med.
175
:
1111
1122
.
78
MacMicking
,
J. D.
,
R. J.
North
,
R.
LaCourse
,
J. S.
Mudgett
,
S. K.
Shah
,
C. F.
Nathan
.
1997
.
Identification of nitric oxide synthase as a protective locus against tuberculosis.
Proc. Natl. Acad. Sci. USA
94
:
5243
5248
.
79
Newport
,
M. J.
,
C. M.
Huxley
,
S.
Huston
,
C. M.
Hawrylowicz
,
B. A.
Oostra
,
R.
Williamson
,
M.
Levin
.
1996
.
A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection.
N. Engl. J. Med.
335
:
1941
1949
.
80
Jouanguy
,
E.
,
S.
Lamhamedi-Cherradi
,
D.
Lammas
,
S. E.
Dorman
,
M. C.
Fondanèche
,
S.
Dupuis
,
R.
Döffinger
,
F.
Altare
,
J.
Girdlestone
,
J. F.
Emile
, et al
.
1999
.
A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.
Nat. Genet.
21
:
370
378
.
81
Altare
,
F.
,
A.
Durandy
,
D.
Lammas
,
J. F.
Emile
,
S.
Lamhamedi
,
F.
Le Deist
,
P.
Drysdale
,
E.
Jouanguy
,
R.
Döffinger
,
F.
Bernaudin
, et al
.
1998
.
Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.
Science
280
:
1432
1435
.
82
Fabri
,
M.
,
S.
Stenger
,
D. M.
Shin
,
J. M.
Yuk
,
P. T.
Liu
,
S.
Realegeno
,
H. M.
Lee
,
S. R.
Krutzik
,
M.
Schenk
,
P. A.
Sieling
, et al
.
2011
.
Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages.
Sci. Transl. Med.
3
:
104ra102
.
83
Cappuccio
,
A.
,
R.
Zollinger
,
M.
Schenk
,
A.
Walczak
,
N.
Servant
,
E.
Barillot
,
P.
Hupé
,
R. L.
Modlin
,
V.
Soumelis
.
2015
.
Combinatorial code governing cellular responses to complex stimuli.
Nat. Commun.
6
:
6847
.
84
Klug-Micu
,
G. M.
,
S.
Stenger
,
A.
Sommer
,
P. T.
Liu
,
S. R.
Krutzik
,
R. L.
Modlin
,
M.
Fabri
.
2013
.
CD40 ligand and interferon-γ induce an antimicrobial response against Mycobacterium tuberculosis in human monocytes.
Immunology
139
:
121
128
.
85
Lande
,
R.
,
E.
Giacomini
,
T.
Grassi
,
M. E.
Remoli
,
E.
Iona
,
M.
Miettinen
,
I.
Julkunen
,
E. M.
Coccia
.
2003
.
IFN-alpha beta released by Mycobacterium tuberculosis-infected human dendritic cells induces the expression of CXCL10: selective recruitment of NK and activated T cells.
J. Immunol.
170
:
1174
1182
.
86
Fan
,
J. B.
,
D. E.
Zhang
.
2013
.
ISG15 regulates IFN-γ immunity in human mycobacterial disease.
Cell Res.
23
:
173
175
.
87
Waddell
,
S. J.
,
S. J.
Popper
,
K. H.
Rubins
,
M. J.
Griffiths
,
P. O.
Brown
,
M.
Levin
,
D. A.
Relman
.
2010
.
Dissecting interferon-induced transcriptional programs in human peripheral blood cells.
PLoS One
5
:
e9753
.

The authors have no financial conflicts of interest.

Supplementary data