Mycobacterial Cord Factor Reprograms the Macrophage Response to IFN-γ towards Enhanced Inflammation yet Impaired Antigen Presentation and Expression of GBP1.

Mycobacteria survive in macrophages despite triggering pattern recognition receptors and T cell-derived IFN-γ production. Mycobacterial cord factor trehalose-6,6-dimycolate (TDM) binds the C-type lectin receptor MINCLE and induces inflammatory gene expression. However, the impact of TDM on IFN-γ-induced macrophage activation is not known. In this study, we have investigated the cross-regulation of the mouse macrophage transcriptome by IFN-γ and by TDM or its synthetic analogue trehalose-6,6-dibehenate (TDB). As expected, IFN-γ induced genes involved in Ag presentation and antimicrobial defense. Transcriptional programs induced by TDM and TDB were highly similar but clearly distinct from the response to IFN-γ. The glycolipids enhanced expression of a subset of IFN-γ-induced genes associated with inflammation. In contrast, TDM/TDB exerted delayed inhibition of IFN-γ-induced genes, including pattern recognition receptors, MHC class II genes, and IFN-γ-induced GTPases, with antimicrobial function. TDM downregulated MHC class II cell surface expression and impaired T cell activation by peptide-pulsed macrophages. Inhibition of the IFN-γ-induced GTPase GBP1 occurred at the level of transcription by a partially MINCLE-dependent mechanism that may target IRF1 activity. Although activation of STAT1 was unaltered, deletion of Socs1 relieved inhibition of GBP1 expression by TDM. Nonnuclear Socs1 was sufficient for inhibition, suggesting a noncanonical, cytoplasmic mechanism. Taken together, unbiased analysis of transcriptional reprogramming revealed a significant degree of negative regulation of IFN-γ-induced Ag presentation and antimicrobial gene expression by the mycobacterial cord factor that may contribute to mycobacterial persistence.

Macrophage Phosphoproteome Analysis Reveals MINCLE-dependent and -independent Mycobacterial Cord Factor Signaling.

Immune sensing of Mycobacterium tuberculosis relies on recognition by macrophages. Mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM), is the most abundant cell wall glycolipid and binds to the C-type lectin receptor (CLR) MINCLE. To explore the kinase signaling linking the TDM-MINCLE interaction to gene expression, we employed quantitative phosphoproteome analysis. TDM caused upregulation of 6.7% and suppressed 3.8% of the 14,000 phospho-sites identified on 3727 proteins. MINCLE-dependent phosphorylation was observed for canonical players of CLR signaling (e.g. PLCγ, PKCδ), and was enriched for PKCδ and GSK3 kinase motifs. MINCLE-dependent activation of the PI3K-AKT-GSK3 pathway contributed to inflammatory gene expression and required the PI3K regulatory subunit p85α. Unexpectedly, a substantial fraction of TDM-induced phosphorylation was MINCLE-independent, a finding paralleled by transcriptome data. Bioinformatics analysis of both data sets concurred in the requirement for MINCLE for innate immune response pathways and processes. In contrast, MINCLE-independent phosphorylation and transcriptome responses were linked to cell cycle regulation. Collectively, our global analyses show substantial reprogramming of macrophages by TDM and reveal a dichotomy of MINCLE-dependent and -independent signaling linked to distinct biological responses.

Gene trap mice reveal an essential function of dual specificity phosphatase Dusp16/MKP-7 in perinatal survival and regulation of Toll-like receptor (TLR)-induced cytokine production.

MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16.

TNF promotes DECTIN2 family C-type lectin receptor expression in human macrophages.

TNF blockade constitutes an effective therapy for inflammatory bowel disease, yet increases the risk for infection, including active tuberculosis. The DECTIN2 family C-type lectin receptors MINCLE, MCL, and DECTIN2 sense mycobacterial ligands and activate myeloid cells. In mice, upregulation of DECTIN2 family C-type lectin receptor after stimulation with Mycobacterium bovis Bacille Calmette-Guérin requires TNF. Here, we investigated whether TNF controls inducible C-type lectin receptor expression in human myeloid cells. Monocyte-derived macrophages were stimulated with Bacille Calmette-Guérin and the TLR4 ligand lipopolysaccharide, and expression of C-type lectin receptor was analyzed. Bacille Calmette-Guérin and lipopolysaccharide strongly upregulated messenger RNA expression of DECTIN2 family C-type lectin receptor but not of DECTIN1. Bacille Calmette-Guérin and lipopolysaccharide also induced robust production of TNF. Recombinant TNF was sufficient to upregulate expression of DECTIN2 family C-type lectin receptor. Blocking TNF with the TNFR2-Fc fusion protein etanercept abrogated, as expected, the effect of recombinant TNF and impaired induction of DECTIN2 family C-type lectin receptor by Bacille Calmette-Guérin and lipopolysaccharide. Flow cytometry confirmed upregulation of MCL at the protein level by recombinant TNF and showed inhibition of Bacille Calmette-Guérin-induced MCL by etanercept. To investigate the impact of TNF on C-type lectin receptor expression in vivo, we analyzed peripheral blood mononuclear cells of patients with inflammatory bowel disease and observed downregulation of MINCLE and MCL expression after therapeutic TNF blockade. Together, TNF is sufficient to upregulate DECTIN2 family C-type lectin receptor in human myeloid cells and contributes to this process after encounter with Bacille Calmette-Guérin or lipopolysaccharide. Impaired C-type lectin receptor expression in patients receiving TNF blockade may dampen the sensing of microbes and defense against infection.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity.

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.

Cutting edge: Mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate.

The mycobacterial cord factor trehalose-6,6-dimycolate (TDM) and its synthetic analog trehalose-6,6-dibehenate (TDB) are potent adjuvants for Th1/Th17 vaccination that activate Syk-Card9 signaling in APCs. In this study, we have further investigated the molecular mechanism of innate immune activation by TDM and TDB. The Syk-coupling adapter protein FcRgamma was essential for macrophage activation and Th17 adjuvanticity. The FcRgamma-associated C-type lectin receptor Mincle was expressed in macrophages and upregulated by TDM and TDB. Recombinant Mincle-Fc fusion protein specifically bound to the glycolipids. Genetic ablation of Mincle abolished TDM/TDB-induced macrophage activation and induction of T cell immune responses to a tuberculosis subunit vaccine. Macrophages lacking Mincle or FcRgamma were impaired in the inflammatory response to Mycobacterium bovis bacillus Calmette-Guérin. These results establish that Mincle is a key receptor for the mycobacterial cord factor and controls the Th1/Th17 adjuvanticity of TDM and TDB.

DGCR8 deficiency impairs macrophage growth and unleashes the interferon response to mycobacteria.

The mycobacterial cell wall glycolipid trehalose-6,6-dimycolate (TDM) activates macrophages through the C-type lectin receptor MINCLE. Regulation of innate immune cells relies on miRNAs, which may be exploited by mycobacteria to survive and replicate in macrophages. Here, we have used macrophages deficient in the microprocessor component DGCR8 to investigate the impact of miRNA on the response to TDM. Deletion of DGCR8 in bone marrow progenitors reduced macrophage yield, but did not block macrophage differentiation. DGCR8-deficient macrophages showed reduced constitutive and TDM-inducible miRNA expression. RNAseq analysis revealed that they accumulated primary miRNA transcripts and displayed a modest type I IFN signature at baseline. Stimulation with TDM in the absence of DGCR8 induced overshooting expression of IFNß and IFN-induced genes, which was blocked by antibodies to type I IFN. In contrast, signaling and transcriptional responses to recombinant IFNß were unaltered. Infection with live Mycobacterium bovis Bacille Calmette-Guerin replicated the enhanced IFN response. Together, our results reveal an essential role for DGCR8 in curbing IFNß expression macrophage reprogramming by mycobacteria.

MyD88 Is Required for Efficient Control of Coxiella burnetii Infection and Dissemination.

The intracellular pathogen Coxiella (C.) burnetii causes Q fever, a usually self-limiting respiratory infection that becomes chronic and severe in some patients. Innate immune recognition of C. burnetii and its role in the decision between resolution and chronicity is not understood well. However, TLR2 is important for the response to C. burnetii in mice, and genetic polymorphisms in Myd88 have been associated with chronic Q fever in humans. Here, we have employed MyD88-deficient mice in infection models with the attenuated C. burnetii Nine Mile phase II strain (NMII). Myd88-/- macrophages failed to restrict the growth of NMII in vitro, and to upregulate production of the cytokines TNF, IL-6, and IL-10. Following intraperitoneal infection, NMII bacterial burden was significantly higher on day 5 and 20 in organs of Myd88-/- mice. After infection via the natural route by intratracheal injection, a higher bacterial load in the lung and increased dissemination of NMII to other organs was observed in MyD88-deficient mice. While wild-type mice essentially cleared NMII on day 27 after intratracheal infection, it was still readily detectable on day 42 in multiple organs in the absence of MyD88. Despite the elevated bacterial load, Myd88-/- mice had less granulomatous inflammation and expressed significantly lower levels of chemoattractants, inflammatory cytokines, and of several IFNγ-induced genes relevant for control of intracellular pathogens. Together, our results show that MyD88-dependent signaling is essential for early control of C. burnetii replication and to prevent systemic spreading. The continued presence of NMII in the organs of Myd88-/- mice constitutes a new mouse model to study determinants of chronicity and resolution in Q fever.

Calcineurin is essential for virulence in Candida albicans.

Calcineurin is a conserved Ca(2+)-calmodulin-activated, serine/threonine-specific protein phosphatase that regulates a variety of physiological processes, e.g., cell cycle progression, polarized growth, and adaptation to salt and alkaline pH stresses. In the pathogenic yeast Cryptococcus neoformans, calcineurin is also essential for growth at 37 degrees C and virulence. To investigate whether calcineurin plays a role in the virulence of Candida albicans, the major fungal pathogen of humans, we constructed C. albicans mutants in which both alleles of the CMP1 gene, encoding the calcineurin catalytic subunit, were deleted. The C. albicans Delta cmp1 mutants displayed hypersensitivity to elevated Na(+), Li(+), and Mn(2+) concentrations and to alkaline pH, phenotypes that have been described after calcineurin inactivation in the related yeast Saccharomyces cerevisiae. Unlike S. cerevisiae calcineurin mutants, which exhibit reduced susceptibility to high Ca(2+) concentrations, growth of C. albicans was inhibited in the presence of 300 mM CaCl(2) after the deletion of CMP1, demonstrating that there are also differences in calcineurin-mediated cellular responses between these two yeast species. In contrast to C. neoformans, inactivation of calcineurin did not cause temperature sensitivity in C. albicans. In addition, hyphal growth, an important virulence attribute of C. albicans, was not impaired in the Delta cmp1 mutants under a variety of inducing conditions. Nevertheless, the virulence of the mutants was strongly attenuated in a mouse model of systemic candidiasis, demonstrating that calcineurin signaling is essential for virulence in C. albicans.