Mouse transcriptome reveals potential signatures of protection and pathogenesis in human tuberculosis.

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection.

Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.

CARD9+ microglia promote antifungal immunity via IL-1ß- and CXCL1-mediated neutrophil recruitment.

The C-type lectin receptor-Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clearance. In the present study we investigated host and pathogen factors that promote protective neutrophil recruitment during invasion of the CNS by Candida albicans. The cytokine IL-1ß served an essential function in CNS antifungal immunity by driving production of the chemokine CXCL1, which recruited neutrophils expressing the chemokine receptor CXCR2. Neutrophil-recruiting production of IL-1ß and CXCL1 was induced in microglia by the fungus-secreted toxin Candidalysin, in a manner dependent on the kinase p38 and the transcription factor c-Fos. Notably, microglia relied on CARD9 for production of IL-1ß, via both transcriptional regulation of Il1b and inflammasome activation, and of CXCL1 in the fungus-infected CNS. Microglia-specific Card9 deletion impaired the production of IL-1ß and CXCL1 and neutrophil recruitment, and increased fungal proliferation in the CNS. Thus, an intricate network of host-pathogen interactions promotes antifungal immunity in the CNS; this is impaired in human deficiency in CARD9, which leads to fungal disease of the CNS.

Routemaps for Highly Effective Tuberculosis Vaccination.

There is no highly effective tuberculosis vaccine. Darrah et al. (2020) and Tait et al. (2019) are setting new benchmarks for protection against infection and pulmonary disease by changing the route of vaccine delivery and by using a protein subunit vaccine with a potent adjuvant.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Endogenous and Therapeutic 25-Hydroxycholesterols May Worsen Early SARS-CoV-2 Pathogenesis in Mice.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-Hydroxycholesterol (25HC), a product of the activity of cholesterol-25-hydroxylase (CH25H) on cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, 25HC can also amplify inflammation and be converted by CYP7B1 (cytochrome P450 family 7 subfamily B member 1) to 7α,25-dihydroxycholesterol, a lipid with chemoattractant activity, via the G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2)/GPR183 (G protein-coupled receptor 183). Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that although 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 (angiotensin-converting enzyme 2) mouse model in vivo. Treatment with 25HC also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma proinflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points but no change in weight loss. Consistent with these findings, although Ch25h and 25HC were upregulated in the lungs of SARS-CoV-2-infected wild-type mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the ß variant were similar to those in control animals. Taken together, endogenous 25HCs do not significantly regulate early SARS-CoV-2 replication or pathogenesis, and supplemental 25HC may have proinjury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Mycobacterium tuberculosis inhibits the NLRP3 inflammasome activation via its phosphokinase PknF.

Mycobacterium tuberculosis (Mtb) has evolved to evade host innate immunity by interfering with macrophage functions. Interleukin-1ß (IL-1ß) is secreted by macrophages after the activation of the inflammasome complex and is crucial for host defense against Mtb infections. We have previously shown that Mtb is able to inhibit activation of the AIM2 inflammasome and subsequent pyroptosis. Here we show that Mtb is also able to inhibit host cell NLRP3 inflammasome activation and pyroptosis. We identified the serine/threonine kinase PknF as one protein of Mtb involved in the NLRP3 inflammasome inhibition, since the pknF deletion mutant of Mtb induces increased production of IL-1ß in bone marrow-derived macrophages (BMDMs). The increased production of IL-1ß was dependent on NLRP3, the adaptor protein ASC and the protease caspase-1, as revealed by studies performed in gene-deficient BMDMs. Additionally, infection of BMDMs with the pknF deletion mutant resulted in increased pyroptosis, while the IL-6 production remained unchanged compared to Mtb-infected cells, suggesting that the mutant did not affect the priming step of inflammasome activation. In contrast, the activation step was affected since potassium efflux, chloride efflux and the generation of reactive oxygen species played a significant role in inflammasome activation and subsequent pyroptosis mediated by the Mtb pknF mutant strain. In conclusion, we reveal here that the serine/threonine kinase PknF of Mtb plays an important role in innate immune evasion through inhibition of the NLRP3 inflammasome.

Incidence of Nontuberculous Mycobacterial Pulmonary Infection, by Ethnic Group, Hawaii, USA, 2005-2019.

To further clarify differences in the risk for nontuberculous mycobacterial pulmonary infection (NTM-PI) among ethnic populations in Hawaii, USA, we conducted a retrospective cohort study among beneficiaries of Kaiser Permanente Hawaii (KPH). We abstracted demographic, socioeconomic, clinical, and microbiological data from KPH electronic health records for 2005-2019. An NTM-PI case-patient was defined as a person from whom >1 NTM pulmonary isolate was obtained. We performed Cox proportional hazards regression to estimate incidence of NTM-PI while controlling for confounders. Across ethnic groups, risk for NTM-PI was higher among persons who were underweight (body mass index [BMI] <18.5 kg/m2). Among beneficiaries who self-identified as any Asian ethnicity, risk for incident NTM-PI was increased by 30%. Low BMI may increase susceptibility to NTM-PI, and risk may be higher for persons who self-identify as Asian, independent of BMI.

Mycobacterium tuberculosis Inhibits Autocrine Type I IFN Signaling to Increase Intracellular Survival.

The type I IFNs (IFN-α and -ß) are important for host defense against viral infections. In contrast, their role in defense against nonviral pathogens is more ambiguous. In this article, we report that IFN-ß signaling in murine bone marrow-derived macrophages has a cell-intrinsic protective capacity against Mycobacterium tuberculosis via the increased production of NO. The antimycobacterial effects of type I IFNs were mediated by direct signaling through the IFN-α/ß-receptor (IFNAR), as Ab-mediated blocking of IFNAR1 prevented the production of NO. Furthermore, M. tuberculosis is able to inhibit IFNAR-mediated cell signaling and the subsequent transcription of 309 IFN-ß-stimulated genes in a dose-dependent way. The molecular mechanism of inhibition by M. tuberculosis involves reduced phosphorylation of the IFNAR-associated protein kinases JAK1 and TYK2, leading to reduced phosphorylation of the downstream targets STAT1 and STAT2. Transwell experiments demonstrated that the M. tuberculosis-mediated inhibition of type I IFN signaling was restricted to infected cells. Overall, our study supports the novel concept that M. tuberculosis evolved to inhibit autocrine type I IFN signaling to evade host defense mechanisms.

Innate and adaptive interferons suppress IL-1α and IL-1ß production by distinct pulmonary myeloid subsets during Mycobacterium tuberculosis infection.

Interleukin-1 (IL-1) receptor signaling is necessary for control of Mycobacterium tuberculosis (Mtb) infection, yet the role of its two ligands, IL-1α and IL-1ß, and their regulation in vivo are poorly understood. Here, we showed that both IL-1α and IL-1ß are critically required for host resistance and identified two multifunctional inflammatory monocyte-macrophage and DC populations that coexpressed both IL-1 species at the single-cell level in lungs of Mtb-infected mice. Moreover, we demonstrated that interferons (IFNs) played important roles in regulating IL-1 production by these cells in vivo. Type I interferons inhibited IL-1 production by both subsets whereas CD4(+) T cell-derived IFN-γ selectively suppressed monocyte-macrophages. These data provide a cellular basis for both the anti-inflammatory effects of IFNs and probacterial functions of type I IFNs during Mtb infection and reveal differential regulation of IL-1 production by distinct cell populations as an additional layer of complexity in the activity of IL-1 in vivo.

Cutting Edge: IL-1R1 Mediates Host Resistance to Mycobacterium tuberculosis by Trans-Protection of Infected Cells.

IL-1R1 deficiency in mice causes severe susceptibility to Mycobacterium tuberculosis Mice and macrophage cultures lacking IL-1R1 display increased bacterial growth, suggesting that phagocytes may require IL-1R1-dependent antimicrobial signals to limit intracellular M. tuberculosis replication directly. However, the myeloid-cell-intrinsic versus -extrinsic requirements for IL-1R1 to control M. tuberculosis infection in mice have not been directly addressed. Using single-cell analysis of infected cells, competitive mixed bone marrow chimeras, and IL-1R1 conditional mutant mice, we show in this article that IL-1R1 expression by pulmonary phagocytes is uncoupled from their ability to control intracellular M. tuberculosis growth. Importantly, IL-1R1-dependent control was provided to infected cells in trans by both nonhematopoietic and hematopoietic cells. Thus, IL-1R1-mediated host resistance to M. tuberculosis infection does not involve mechanisms of cell-autonomous antimicrobicidal effector functions in phagocytes but requires the cooperation between infected cells and other cells of hematopoietic or nonhematopoietic origin to promote bacterial containment and control of infection.

Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk.

Tuberculosis remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent. Despite chemotherapy, the global tuberculosis epidemic has intensified because of HIV co-infection, the lack of an effective vaccine and the emergence of multi-drug-resistant bacteria. Alternative host-directed strategies could be exploited to improve treatment efficacy and outcome, contain drug-resistant strains and reduce disease severity and mortality. The innate inflammatory response elicited by Mycobacterium tuberculosis (Mtb) represents a logical host target. Here we demonstrate that interleukin-1 (IL-1) confers host resistance through the induction of eicosanoids that limit excessive type I interferon (IFN) production and foster bacterial containment. We further show that, in infected mice and patients, reduced IL-1 responses and/or excessive type I IFN induction are linked to an eicosanoid imbalance associated with disease exacerbation. Host-directed immunotherapy with clinically approved drugs that augment prostaglandin E2 levels in these settings prevented acute mortality of Mtb-infected mice. Thus, IL-1 and type I IFNs represent two major counter-regulatory classes of inflammatory cytokines that control the outcome of Mtb infection and are functionally linked via eicosanoids. Our findings establish proof of concept for host-directed treatment strategies that manipulate the host eicosanoid network and represent feasible alternatives to conventional chemotherapy.

Changes in inflammatory protein and lipid mediator profiles persist after antitubercular treatment of pulmonary and extrapulmonary tuberculosis: A prospective cohort study.

BACKGROUND: The identification of meaningful biomarkers of tuberculosis (TB) has potential to improve diagnosis, disease staging and prediction of treatment outcomes. It has been shown that active pulmonary TB (PTB) is associated with qualitative and quantitative changes in systemic immune profile, suggesting a chronic inflammatory imbalance. Here we characterized the profile of PTB and extrapulmonary TB (EPTB) in a prospective cohort study. METHODS: We measured a panel of 27 inflammatory cytokines, soluble receptors, and lipid mediators in peripheral blood from patients with PTB or EPTB from a prospective clinical study in China. Multidimensional analyses were performed to describe associations between plasma levels of biomarkers and different TB disease presentation profiles. RESULTS: Mycobacterium tuberculosis infection induced changes in both the expression and correlation profiles of plasma mediators of inflammation in patients with PTB compared to those with EPTB. Increases in mycobacterial loads in sputum smears were associated with rises in concentrations of several molecules involved in TB pathogenesis, such as IL-1ß, IFN-α, IL-10 and PGF2α. Moreover, PTB patients presenting with severe disease exhibited a distinct inflammatory profile hallmarked by heightened levels of TNF-α, IL-1ß, IL17, IL-18 and IL-27. Interestingly, while antitubercular treatment (ATT) resulted in early changes of plasma concentrations of markers in PTB, changes were delayed in EPTB patients. Exploratory analyses of the molecular degree of perturbation (MDP) of the inflammatory mediators before and during ATT suggested the occurrence of infection and/or treatment-induced long lasting "inflammatory imprinting" of biomarker profiles in TB. At 24 weeks post ATT commencement, markers underlying the observed increases in MDP scores were IL-27 in PTB and IL-1ß in EPTB patients. CONCLUSION: Our findings describe systemic and durable changes in the concentrations of inflammatory cytokines and lipid mediators in both PTB and EPTB and emphasize the role of M. tuberculosis bacterial burden and site of disease in modulating patient immune biomarkers.

Cytokine and lipid mediator networks in tuberculosis.

A major approach for immunologic intervention in tuberculosis involves redirecting the outcome of the host immune response from the induction of disease to pathogen control. Cytokines and lipid mediators known as eicosanoids play key roles in regulating this balance and as such represent important targets for immunologic intervention. While the evidence for cytokine/eicosanoid function derives largely from the investigation of murine and zebrafish experimental infection models, clinical studies have confirmed the existence of many of the same pathways in tuberculosis patients. Here, we summarize new data that reveal important intersections between the cytokine and eicosanoid networks in the host response to mycobacteria and discuss how targeting this crosstalk can promote resistance to lethal Mycobacterium tuberculosis infection. This approach could lead to new host-directed therapies to be used either as an adjunct for improving the efficacy of standard antibiotic treatment or for the management of drug-resistant infections.

The Nlrp3 inflammasome, IL-1ß, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL/6 mice.

Infection of C57BL/6 mice with most Leishmania major strains results in a healing lesion and clearance of parasites from the skin. Infection of C57BL/6 mice with the L. major Seidman strain (LmSd), isolated from a patient with chronic lesions, despite eliciting a strong Th1 response, results in a nonhealing lesion, poor parasite clearance, and complete destruction of the ear dermis. We show here that in comparison to a healing strain, LmSd elicited early upregulation of IL-1ß mRNA and IL-1ß-producing dermal cells and prominent neutrophil recruitment to the infected skin. Mice deficient in Nlrp3, apoptosis-associated speck-like protein containing a caspase recruitment domain, or caspase-1/11, or lacking IL-1ß or IL-1 receptor signaling, developed healing lesions and cleared LmSd from the infection site. Mice resistant to LmSd had a stronger antigen-specific Th1 response. The possibility that IL-1ß might act through neutrophil recruitment to locally suppress immunity was supported by the healing observed in neutropenic Genista mice. Secretion of mature IL-1ß by LmSd-infected macrophages in vitro was dependent on activation of the Nlrp3 inflammasome and caspase-1. These data reveal that Nlrp3 inflammasome-dependent IL-1ß, associated with localized neutrophil recruitment, plays a crucial role in the development of a nonhealing form of cutaneous leishmaniasis in conventionally resistant mice.

Type I IFN Induction via Poly-ICLC Protects Mice against Cryptococcosis.

Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.

Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis.

Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.

Cutting edge: control of Mycobacterium tuberculosis infection by a subset of lung parenchyma-homing CD4 T cells.

Th1 cells are critical for containment of Mycobacterium tuberculosis infection, but little else is known about the properties of protective CD4 T cell responses. In this study, we show that the pulmonary Th1 response against M. tuberculosis is composed of two populations that are either CXCR3(hi) and localize to lung parenchyma or are CX3CR1(hi)KLRG1(hi) and are retained within lung blood vasculature. M. tuberculosis-specific parenchymal CD4 T cells migrate rapidly back into the lung parenchyma upon adoptive transfer, whereas the intravascular effectors produce the highest levels of IFN-γ in vivo. Importantly, parenchymal T cells displayed greater control of infection compared with the intravascular counterparts upon transfer into susceptible T cell-deficient hosts. Thus, we identified a subset of naturally generated M. tuberculosis-specific CD4 T cells with enhanced protective capacity and showed that control of M. tuberculosis correlates with the ability of CD4 T cells to efficiently enter the lung parenchyma rather than produce high levels of IFN-γ.

Cutting edge: Endoplasmic reticulum stress licenses macrophages to produce mature IL-1ß in response to TLR4 stimulation through a caspase-8- and TRIF-dependent pathway.

The accumulation of improperly folded proteins within the endoplasmic reticulum (ER) generates perturbations known as ER stress that engage the unfolded protein response. ER stress is involved in many inflammatory pathologies that are also associated with the production of the proinflammatory cytokine IL-1ß. In this study, we demonstrate that macrophages undergoing ER stress are able to drive the production and processing of pro-IL-1ß in response to LPS stimulation in vitro. Interestingly, the classical NLRP3 inflammasome is dispensable, because maturation of pro-IL-1ß occurs normally in the absence of the adaptor protein ASC. In contrast, processing of pro-IL-1ß is fully dependent on caspase-8. Intriguingly, we found that neither the unfolded protein response transcription factors XBP1 and CHOP nor the TLR4 adaptor molecule MyD88 is necessary for caspase-8 activation. Instead, both caspase activation and IL-1ß production require the alternative TLR4 adaptor TRIF. This pathway may contribute to IL-1-driven tissue pathology in certain disease settings.

Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome.

Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1ß/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1ß processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1ß expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.