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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.

Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4+ T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection. Fil: Velasquez, Lis Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Milillo, María Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Delpino, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Trotta, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernandez, Pablo Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Pozner, Roberto Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Lang, Roland. Freidrich Alexander Universität Erlangen‐Nürnberg; Alemania Fil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Barrionuevo, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

HIF1A is a transcription factor that plays a central role for the adaptation to tissue hypoxia and for the inflammatory response of myeloid cells, including DCs. HIF1A is stabilized by hypoxia but also by TLR ligands under normoxic conditions. The underlying signaling events leading to the accumulation of HIF1A in the presence of oxygen are still poorly understood. Here, we show that in contrast to hypoxic stabilization of HIF1A, normoxic, TLR-mediated HIF1A accumulation in DCs follows a different pathway that predominantly requires MYD88-dependent NF-κB activity. The TLR-induced HIF1A controls a subset of proinflammatory genes that are insufficiently induced following hypoxia-mediated HIF1A induction. Thus, TLR activation and hypoxia stabilize HIF1A via distinct signaling pathways, resulting in differential HIF1A-dependent gene expression.

Dendritic cells interact with T cells in intestinal mucosa in an MHCII-dependent manner, suggesting presentation outside organized lymphoid tissue may be important in determining T cell phenotype. The MAPK phosphatase DUSP1 is an essential negative regulator of TLR-triggered innate immune activation. Here, we have investigated the impact of DUSP1 on inflammatory and antimicrobial host responses to the intracellular pathogen Chlamydophila pneumoniae. Following nasal infection, DUSP1-deficient mice mounted an enhanced pulmonary cytokine (IL-1β, IL-6) and chemokine response (CCL3, CCL4, CXCL1, CXCL2), leading to increased leukocyte infiltration. Of interest, the increased inflammatory response, in the absence of DUSP1, was associated with higher bacterial numbers in the lungs, although the expression of IFN-γ and critical antichlamydial effector molecules, such as iNOS, was intact. Blockade of IL-6 trans-signaling by injection of a soluble gp130-Fc fusion protein corrected the overshooting chemokine production as well as the increased chlamydial load in Dusp1−/− mice. Furthermore, IL-6 enhanced the replication of C. pneumoniae in embryonic fibroblasts in vitro. These data show that DUSP1 is required to achieve a balanced response to chlamydial infection and identify IL-6 as critical for amplifying inflammation and benefiting chlamydial growth through direct effects on infected cells.

Innate immune cells produce NO via inducible NO synthase (iNOS) in response to certain infections or upon stimulation with cytokines such as IFN-γ and TNF. NO plays an important role in host defense against intracellular bacteria including Chlamydophila pneumoniae as a result of its microbicidal activity. In MyD88-deficient mice, which succumb to C. pneumoniae infection, iNOS induction is impaired 6 days postinfection, although pulmonary levels of IFN-γ and TNF are elevated as in wild-type mice at this time-point. Here, we demonstrate that induction of iNOS in macrophages upon C. pneumoniae infection is controlled by MyD88 via two pathways: NF-κB activation and phosphorylation of the MAPK JNK, which leads to the nuclear translocation of c-Jun, one of the two components of the AP-1 complex. In addition, phosphorylation of STAT1 and expression of IFN regulatory factor 1 (IRF-1) were delayed in the absence of MyD88 after C. pneumoniae infection but not after IFN-γ stimulation. Taken together, our data show that for optimal induction of iNOS during C. pneumoniae infection, the concerted action of the MyD88-dependent transcription factors NF-κB and AP-1 and of the MyD88-independent transcription factors phosphorylated STAT1 and IRF-1 is required.

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen-associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up-regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1- or TLR6-dependent manner. There are also di- and triacylated LP, which stimulate TLR1-deficient cells and TLR6-deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)2C-(VPGVG)4VPGKG, fibroblast-stimulating LP-1, and Pam2C-SK4. Dominant-negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram-positive and Gram-negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.