Search

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.

Drugs are a frequent cause of severe anaphylactic reactions. Here we analyze a large data set on drug induced anaphylaxis regarding elicitors, risk factors, symptoms and treatment. Data from the European Anaphylaxis Registry (2007-2019) with 1,815 reported cases of drug-induced anaphylaxis were studied accordingly. Drugs are the third most frequent cause of anaphylaxis reported in the Anaphylaxis Registry. Among the eliciting groups of drugs analgesics and antibiotics were far most often reported. Female and senior patients were more frequently affected, while the number of children with DIA was low. DIA patients had symptoms affecting the skin and mucous membranes (n = 1,525, 84.02%), the respiratory (n = 1,300, 71.63%), the cardiovascular (n = 1,251, 68.93%) and the gastrointestinal system (n = 549, 30.25%). Drugs caused significant more severe reactions, occurred more often in medical facilities and led to increased hospitalisation rates in comparison to food and insect venom induced anaphylaxis. Adrenaline was used more often in patients with DIA than in anaphylaxis due to other causes. Patients with skin symptoms received more antihistamines and corticosteroids in the acute treatment, while gastrointestinal symptoms led to less adrenaline use. The study contributes to a better understanding of DIA, with a large number of cases from Europe supporting previous data, e.g., analgesics and antibiotics being the most frequent culprits for DIA. Female gender and higher age are relevant risk factors and despite clear recommendations, the emergency treatment of DIA is not administered according to the guidelines.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant lipoproteins within the lung alveoli. Alveolar macrophages (AMs) are crucial for surfactant clearance, and their differentiation depends on colony-stimulating factor 2 (CSF2), which regulates the establishment of an AM-characteristic gene regulatory network. Here, we report that the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is essential for the development of the AM identity, as demonstrated by transcriptome and chromatin accessibility analysis. Furthermore, C/EBPβ-deficient AMs showed severe defects in proliferation, phagocytosis, and lipid metabolism, collectively resulting in a PAP-like syndrome. Mechanistically, the long C/EBPβ protein variants LAP* and LAP together with CSF2 signaling induced the expression of Pparg isoform 2 but not Pparg isoform 1, a molecular regulatory mechanism that was also observed in other CSF2-primed macrophages. These results uncover C/EBPβ as a key regulator of AM cell fate and shed light on the molecular networks controlling lipid metabolism in macrophages.

Successful subunit vaccination with recombinant proteins requires adjuvants. The glycolipid trehalose-dibehenate (TDB), a synthetic analog of the mycobacterial cord factor, potently induces Th1 and Th17 immune responses and is a candidate adjuvant for human immunization. TDB binds to the C-type lectin receptor Mincle and triggers Syk-Card9-dependent APC activation. In addition, interleukin (IL)-1 receptor/MyD88-dependent signaling is required for TDB adjuvanticity. The role of different innate immune cell types in adjuvant-stimulated Th1/Th17 responses is not well characterized. We investigated cell recruitment to the site of injection (SOI) and to the draining lymph nodes (dLNs) after immunization with the TDB containing adjuvant CAF01 in a protein-based vaccine. Recruitment of monocytes and neutrophils to the SOI and the dramatic increase in lymph node cellularity was partially dependent on both Mincle and MyD88. Despite their large numbers at the SOI, neutrophils were dispensable for the induction of Th1/Th17 responses. In contrast, CCR2-dependent monocyte recruitment was essential for the induction of Th1/Th17 cells. Transport of adjuvant to the dLN did not require Mincle, MyD88, or CCR2. Together, adjuvanticity conferred by monocytes can be separated at the cellular level from potential tissue damage by neutrophils.

Airway infection is a major cause of mortality worldwide. The identification of new mechanisms aiding in effective host immune response is therefore required. Here, we show that the specific depletion of the pleural immune cell compartment during bacterial pneumonia resulted in a reduced pulmonary immune response and increased mortality in mice. Bacterial airway infection provoked early pleural space (PS) inflammation characterized by innate response activator (IRA) B cell development and pleural large resident macrophage (LRM) necroptosis, the repopulation of LRMs being driven by cellular proliferationin situ. Necroptotic LRMs amplified PS inflammation by stimulating pleural Mincle-expressing macrophages whereas IRA B cells contributed partially to GM-CSF-induced PS inflammation. Upon pulmonary infection, the induction of PS inflammation resulted in reduced bacterial burden whereas the specific depletion of pleural resident macrophages led to increased mortality and bacterial burden and reduced pulmonary immunity. Moreover, mice in which B cells were unable to produce GM-CSF exhibited reduced CD103+dendritic cells and reduced CD4+T cell numbers in the draining lymph node. Altogether, our results describe a previously unrecognized mechanism of pleural space inflammation necessary for effective protection against bacterial airway infection.

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.

TNF blockade is a successful treatment for human autoimmune disorders like rheumatoid arthritis and inflammatory bowel disease yet increases susceptibility to tuberculosis and other infections. The C-type lectin receptors (CLR) MINCLE, MCL, and DECTIN-2 are expressed on myeloid cells and sense mycobacterial cell wall glycolipids. In this study, we show that TNF is sufficient to upregulate MINCLE, MCL, and DECTIN-2 in macrophages. TNF signaling through TNFR1 p55 was required for upregulation of these CLR and for cytokine secretion in macrophages stimulated with the MINCLE ligand trehalose-6,6-dibehenate or infected with Mycobacterium bovis bacillus Calmette–Guérin. The Th17 response to immunization with the MINCLE-dependent adjuvant trehalose-6,6-dibehenate was specifically abrogated in TNF-deficient mice and strongly attenuated by TNF blockade with etanercept. Together, interference with production or signaling of TNF antagonized the expression of DECTIN-2 family CLR, thwarting vaccine responses and possibly increasing infection risk.

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 down-regulates 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 withMycobacterium bovisBacille 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 nematodeNippostrongylus brasiliensisin 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 withN. brasiliensisor withSchistosoma mansonipromoted 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. brasiliensisinfection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ-specific manner. Taken together, our results demonstrate down-regulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

Background There is controversy whether taking beta-blockers or ACE inhibitors (ACEI) is a risk factor for more severe systemic insect sting reactions (SSR) and whether it increases the number or severity of adverse events (AE) during venom immunotherapy (VIT). Methods in this open, prospective, observational, multicenter trial, we recruited patients with a history of a SSR and indication for VIT. The primary objective of this study was to evaluate whether patients taking beta-blockers or ACEI show more systemic AE during VIT compared to patients without such treatment. Results in total, 1,425 patients were enrolled and VIT was performed in 1,342 patients. of all patients included, 388 (27.2%) took antihypertensive (AHT) drugs (10.4% took beta-blockers, 11.9% ACEI, 5.0% beta-blockers and ACEI). Only 5.6% of patients under AHT treatment experienced systemic AE during VIT as compared with 7.4% of patients without these drugs (OR: 0.74, 95% CI: 0.43-1.22, p = 0.25). The severity of the initial sting reaction was not affected by the intake of beta-blockers or ACEI (OR: 1.14, 95% CI: 0.89-1.46, p = 0.29). in total, 210 (17.7%) patients were re-stung during VIT and 191 (91.0%) tolerated the sting without systemic symptoms. of the 19 patients with VIT treatment failure, 4 took beta-blockers, none an ACEI. Conclusions This trial provides robust evidence that taking beta-blockers or ACEI does neither increase the frequency of systemic AE during VIT nor aggravate SSR. Moreover, results suggest that these drugs do not impair effectiveness of VIT. (Funded by Medical University of Graz, Austria; Clinicaltrials.gov number, NCT04269629)., Medizinische Universitat Graz Funding Source: Medline

BackgroundCheckpoint inhibitors revolutionized the treatment of metastatic melanoma patients. Although tumor burden and lactate dehydrogenase (LDH) are associated with overall survival (OS), the impact of tumor growth kinetics remains elusive and in part contradictory. The aims of this study were to develop a novel simple and rapid method that estimates pretreatment metastatic growth rate (MGR) and to investigate its prognostic impact in melanoma patients treated with antiprogrammed death receptor-1 (PD-1) antibodies.MethodsMGR was assessed in three independent cohorts of a total of 337 unselected consecutive metastasized stage IIIB–IV melanoma patients (discovery cohort: n=53, confirmation cohort: n=126, independent multicenter validation cohort: n=158). MGR was computed during the pretreatment period before initiation of therapy with anti-PD-1 antibodies nivolumab or pembrolizumab by measuring the increase of the longest diameter of the largest target lesion. Tumor doubling time served as quality control. Kaplan-Meier analysis and univariable as well as multivariable Cox regression were used to examine the prognostic impact of MGR.ResultsPretreatment MGR >3.9 mm/month was associated with impaired OS in the discovery cohort (HR 6.19, 95% CI 2.92 to 13.10, pConclusionsHigh pretreatment MGR is an independent strong prognostic biomarker associated with unfavorable survival of melanoma patients receiving anti-PD-1 antibodies. Further investigations are warranted to assess the predictive impact of MGR in distinct systemic therapeutic regimens.

BACKGROUND Patients with a history of anaphylaxis are at risk of future anaphylactic reactions. Thus, secondary prevention measures are recommended for these patients to prevent or attenuate the next reaction. METHODS Data from the Anaphylaxis Registry were analyzed to identify secondary prevention measures offered to patients who experienced anaphylaxis. Our analysis included 7788 cases from 10 European countries and Brazil. RESULTS The secondary prevention measures offered varied across the elicitors. A remarkable discrepancy was observed between prevention measures offered in specialized allergy centers (84% of patients were prescribed adrenaline autoinjectors following EAACI guidelines) and outside the centers: Here, EAACI guideline adherence was only 37%. In the multivariate analysis, the elicitor of the reaction, age of the patient, mastocytosis as comorbidity, severity of the reaction, and reimbursement/availability of the autoinjector influence physician's decision to prescribe one. CONCLUSIONS Based on the low implementation of guidelines concerning secondary prevention measures outside of specialized allergy centers, our findings highlight the importance of these specialized centers and the requirement of better education for primary healthcare and emergency physicians.

In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understoo Fil: Bénard, Alan. Centre National de la Recherche Scientifique; Francia Fil: Sakwa, Imme. Leibniz - Institute Of Freshwater Ecology And Inland Fisheries; Alemania Fil: Schierloh, Luis Pablo. Instituto National de Recherches Agronomiques. Centre de Recherches de Toulouse; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Colom, André. Centre National de la Recherche Scientifique; Francia Fil: Mercier, Ingrid. Instituto National de Recherches Agronomiques. Centre de Recherches de Toulouse; Francia Fil: Tailleux, Ludovic. Instituto Pasteur; Francia Fil: Jouneau, Luc. Institut National de la Recherche Agronomique; Francia Fil: Boudinot, Pierre. Institut National de la Recherche Agronomique; Francia Fil: Al Saati, Talal. Université Paul Sabatier; Francia Fil: Lang, Roland. Universitat Erlangen-Nuremberg; Alemania Fil: Rehwinkel, Jan. University of Oxford; Reino Unido Fil: Loxton, Andre G.. Stellenbosch University; Sudáfrica Fil: Kaufmann, Stefan H. E.. Max Planck Institut für Infektionsbiologie; Alemania Fil: Anton Leberre, Veronique. Institut National de la Recherche Agronomique; Francia Fil: O'Garra, Anne. National Institute for Medical Research; Reino Unido Fil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Gicquel, Brigitte. Institut National de la Recherche Agronomique; Francia Fil: Fillatreau, Simon. Centre National de la Recherche Scientifique; Francia Fil: Neyrolles, Olivier. Centre National de la Recherche Scientifique; Francia Fil: Hudrisier, Denis. Centre National de la Recherche Scientifique; Francia

Significance Many patients with B cell lymphoma carry alterations in the gene coding for the transcription factor Foxp1. High Foxp1 expression has been linked to poor prognosis in those malignancies; however, the physiological functions of Foxp1 in mature B cells remain unknown. By employing genetic mouse models, we show that Foxp1 deletion results in reduced B cell numbers and impaired antibody production upon T cell-independent immunization. Foxp1-deficient mature B cells are impaired in survival and exhibit an increased proliferation capacity, and transcriptional analysis identified defective expression of the prosurvival Bcl-xl gene. Our results provide insight into the regulation of mature B cell survival by Foxp1 and have implications for understanding the role of Foxp1 in the development of B cell malignancies., The transcription factor Foxp1 is critical for early B cell development. Despite frequent deregulation of Foxp1 in B cell lymphoma, the physiological functions of Foxp1 in mature B cells remain unknown. Here, we used conditional gene targeting in the B cell lineage and report that Foxp1 disruption in developing and mature B cells results in reduced numbers and frequencies of follicular and B-1 B cells and in impaired antibody production upon T cell-independent immunization in vivo. Moreover, Foxp1-deficient B cells are impaired in survival even though they exhibit an increased capacity to proliferate. Transcriptional analysis identified defective expression of the prosurvival Bcl-2 family gene Bcl2l1 encoding Bcl-xl in Foxp1-deficient B cells, and we identified Foxp1 binding in the regulatory region of Bcl2l1. Transgenic overexpression of Bcl2 rescued the survival defect in Foxp1-deficient mature B cells in vivo and restored peripheral B cell numbers. Thus, our results identify Foxp1 as a physiological regulator of mature B cell survival mediated in part via the control of Bcl-xl expression and imply that this pathway might contribute to the pathogenic function of aberrant Foxp1 expression in lymphoma.

The neuropeptide galanin is distributed in the central and peripheral nervous systems and in non-neuronal peripheral organs, including the skin. Galanin acts via three G protein-coupled receptors which, except galanin receptor 1, are expressed in various skin structures. The galanin system has been associated with inflammatory processes of the skin and of several other organs. Psoriasis is an inflammatory skin disease with increased neovascularization, keratinocyte hyperproliferation, a proinflammatory cytokine milieu, and immune cell infiltration. In this study, we showed that galanin receptor 3 is present in endothelial cells in human and murine dermal vessels and is co-expressed with nestin in neo-vessels of psoriatic patients. Moreover, in a murine psoriasis model, we showed that C57/BL6 mice lacking galanin receptor 3 display a milder course of psoriasis upon imiquimod treatment, leading to decreased disease severity, delayed neo-vascularization, reduced infiltration of neutrophils, and significantly lower levels of proinflammatory cytokines compared with wild-type mice. In contrast, galanin receptor 2-knockout animals did not differ significantly from wild type mice at both the macroscopic and molecular levels in their inflammatory response to imiquimod treatment. Our data indicate that galanin receptor 3, but not galanin receptor 2, plays an important role in psoriasis-like skin inflammation.

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.

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

The T-cell adjuvanticity of mycobacterial cord factor trehalose 6,6'-dimycolate (TDM) is well established. The identification of the C-type lectin Mincle on innate immune cells as the receptor for TDM and its synthetic analogue trehalose 6,6'-dibehenate (TDB) has raised interest in development of synthetic Mincle ligands as novel adjuvants. Trehalose mono- (TMXs) and diesters (TDXs) with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), and myristate (M)] were tested. Upon stimulation of murine macrophages, G-CSF secretion and NO production were strongly augmented by all TDXs tested, in a wide concentration range. In contrast, the TMXs triggered macrophage activation only at high concentrations. Macrophage activation by all TDXs required Mincle, but was independent of MyD88. The superior capacity of TDXs for activating macrophages was paralleled by direct binding of TDXs, but not of TMXs, to a Mincle-Fc fusion protein. Insertion of a short polyethylene glycol between the sugar and acyl chain in TDS reduced Mincle-binding and macrophage activation. Immunization of mice with cationic liposomes containing the analogues demonstrated the superior adjuvant activity of trehalose diesters. Overall, immune activation in vitro and in vivo by trehalose esters of simple fatty acids requires two acyl chains of length and involves Mincle.

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.

Aims Polymorphonuclear neutrophils are key players in innate immunity. The innate immune system needs to be tightly controlled to ensure proper activation but also no overactivation. Galanin has been shown to regulate inflammatory reactions, and therefore, we aimed to elucidate the expression of galanin and its three receptors (GAL1-GAL3) in polymorphonuclear neutrophils and to evaluate whether galanin exerts direct or indirect effects on human and murine polymorphonuclear neutrophils. Methods Human peripheral polymorphonuclear neutrophils were isolated from fresh blood of healthy donors, and murine polymorphonuclear neutrophils were isolated from bone marrow of C57BL/6N mice. Gene expression was evaluated by qRT-PCR. As a marker for polymorphonuclear neutrophil activation, CD11b integrin surface expression was measured by FACS analysis. Furthermore, a label-free technology measuring ligand-induced dynamic mass redistribution was used to evaluate the response of polymorphonuclear neutrophils to galanin. Results GAL2 receptor expression was found in both human and murine polymorphonuclear neutrophils, galanin and GAL3 receptor were exclusively expressed in murine bone marrow polymorphonuclear neutrophils, and GAL1 receptor was not detectable in polymorphonuclear neutrophils of either species. Galanin treatment was not able to induce CD11b integrin surface expression or dynamic mass redistribution in human polymorphonuclear neutrophils and murine bone marrow polymorphonuclear neutrophils. However, galanin treatment significantly enhanced the response of polymorphonuclear neutrophils of both species to interleukin-8. Conclusion Galanin can be regarded as an immunomodulatory peptide as it can sensitize polymorphonuclear neutrophils towards pro-inflammatory cytokines in humans and mice.

Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220− bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF–induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-β, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9flox/flox; CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-β and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-β expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-β production by these cells.

Suppressor of cytokine signaling 3 (SOCS3) is a feedback inhibitor of interleukin (IL)-6 signaling in macrophages. In the absence of this molecule, macrophages become extremely prone to an IL-6-dependent expression of arginase-1 (Arg1) and nitric oxide synthase (NOS)2, the prototype markers for alternative or classical macrophage activation, respectively. Because both enzymes are antipodean macrophage effector molecules in Mycobacterium tuberculosis (Mtb) infection, we assessed the relevance of SOCS3 for macrophage activation during experimental tuberculosis using macrophage-specific SOCS3-deficient (LysMcreSOCS3loxP/loxP) mice. Aerosol infection of LysMcreSOCS3loxP/loxP mice resulted in remarkably higher bacterial loads in infected lungs and exacerbated pulmonary inflammation. This increased susceptibility to Mtb infection was accompanied by enhanced levels of both classical and alternative macrophage activation. However, high Arg1 expression preceded the increased induction of NOS2 and at early time points of infection mycobacteria were mostly found in cells positive for Arg1. This sequential activation of Arg1 and NOS2 expression in LysMcreSOCS3loxP/loxP mice appears to favor the initial replication of Mtb particularly in Arg1-positive cells. Neutralization of IL-6 in Mtb-infected LysMcreSOCS3loxP/loxP mice reduced arginase activity and restored control of mycobacterial replication in LysMcreSOCS3loxP/loxP mice. Our data reveal an unexpected role of SOCS3 during experimental TB: macrophage SOCS3 restrains early expression of Arg1 and helps limit Mtb replication in resident lung macrophages, thereby limiting the growth of mycobacteria. Together, SOCS3 keeps IL-6-dependent divergent macrophage responses such as Nos2 and Arg1 expression under control and safeguard protective macrophage effector mechanisms.

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications., Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

Immunoglobulin G (IgG) glycosylation can modulate antibody effector functions. Depending on the precise composition of the sugar moiety attached to individual IgG glycovariants either pro- or anti-inflammatory effector pathways can be initiated via differential binding to type I or type II Fc-receptors. However, an in depth understanding of how individual IgG subclasses are glycosylated during the steady state and how their glycosylation pattern changes during vaccination is missing. To monitor IgG subclass glycosylation during the steady state and upon vaccination of mice with different T-cell dependent and independent antigens, tryptic digests of serum, and antigen-specific IgG preparations were analyzed by reversed phase-liquid chromatography-mass spectrometry. We show that there is a remarkable difference with respect to how individual IgG subclasses are glycosylated during the steady state. More importantly, upon T-cell dependent and independent vaccinations, individual antigen-specific IgG subclasses reacted differently with respect to changes in individual glycoforms, suggesting that the IgG subclass itself is a major determinant of restricting or allowing alterations in specific IgG glycovariants.

Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro . Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcRγ) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors.

Corynebacterium diphtheriae is the causative agent of diphtheria, a toxin mediated disease of upper respiratory tract, which can be fatal. As a member of the CMNR group, C. diphtheriae is closely related to members of the genera Mycobacterium, Nocardia and Rhodococcus. Almost all members of these genera comprise an outer membrane layer of mycolic acids, which is assumed to influence host-pathogen interactions. In this study, three different C. diphtheriae strains were investigated in respect to their interaction with phagocytic murine and human cells and the invertebrate infection model Caenorhabditis elegans. Our results indicate that C. diphtheriae is able to delay phagolysosome maturation after internalization in murine and human cell lines. This effect is independent of the presence of mycolic acids, as one of the strains lacked corynomycolates. In addition, analyses of NF-κB induction revealed a mycolate-independent mechanism and hint to detrimental effects of the different strains tested on the phagocytic cells. Bioinformatics analyses carried out to elucidate the reason for the lack of mycolates in one of the strains led to the identification of a new gene involved in mycomembrane formation in C. diphtheriae.

TLR-mediated recognition of microbial danger induces substantial changes in macrophage migration, adherence, and phagocytosis. Recently, we described the LPS-regulated phosphorylation of many cytoskeleton-associated proteins by phosphoproteomics. The functional role of these cytoskeletal and motor proteins in innate immune cell responses is largely unexplored. Here, we first identified both long-tailed class I myosins Myo1e and Myo1f as important contributors to LPS-triggered macrophage spreading. Mouse bone marrow-derived macrophages and DCs deficient in Myo1e selectively secreted increased amounts of the chemokine CCL2. In addition, the cell surface expression of MHC class II (MHC-II) on both cell types was reduced in the absence of Myo1e. However, transcriptional changes in CCL2 and MHC-II were not observed in the absence of Myo1e, indicating that Myo1e regulates specific intracellular transport processes. The capacity of macrophages and DCs lacking Myo1e to stimulate antigen-specific CD4(+) T-cell proliferation was impaired, consistent with the reduced MHC-II surface protein levels. Surprisingly, in Myo1e-deficient DCs, the proteolytic cleavage of endocytosed antigen was also increased. Together, our results provide evidence for a non-redundant function of the motor protein Myo1e in the regulation of TLR4-controlled, cytoskeleton-associated functional properties of macrophages and DCs, and in induction of a full MHC-II-restricted adaptive immune response.

The DC-derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady-state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α⁻CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α-galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α⁻ and CD8α⁺ splenic DC subsets and enhances cross-presentation of exogenous antigens. Based on genome-wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN-γ-mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN-γR and are much less responsive to IFN-γ. Under inflammatory conditions, particularly in the absence of IFN-γ signaling in IFN-γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM-CSF in concert with IL-4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression.

Individually and combined, Toll-like receptors (TLR)-2, -4, -9, nucleotide oligomerization domain (NOD) 2 and NALP3 contribute to the Mycobacterium tuberculosis (Mtb)-induced innate immune response only to a limited extent, particularly in terms of inducing antibacterial protection and granuloma formation in vivo. A singular essential sensory component of this initial response has not been discovered yet. Trehalose-6,6'-dimycolate (TDM), a well known mycobacterial cell wall glycolipid, is believed to be involved in these early inflammatory processes after Mtb infection. Only recently the macrophage inducible C-type lectin (Mincle) was demonstrated as an essential receptor for TDM. However, not much is known about the sensing capacity of Mincle during infection with live mycobacteria. To determine the significance of Mincle during tuberculosis (TB), we analyzed the outcome of Mtb infection in Mincle-deficient mice. Whereas in the absence of Mincle macrophages did not respond to TDM, Mincle-deficient mice were capable of mounting an efficient granulomatous and protective immune response after low and high dose infections with Mtb. Mutant mice generated a normal T helper (TH) 1 and TH17 immune response followed by the induction of efficient macrophage effector mechanisms and control of mycobacterial growth identical to wildtype mice. From our results we conclude that absence of the innate receptor Mincle can be fully compensated for in vivo in terms of sensing Mtb and mounting a protective inflammatory immune response.

SummaryCell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB’s function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.PaperFlick

Background Allergen-specific IgE antibodies are a hallmark of type I allergy. The aim of this cross-sectional study was to analyze the sensitization profiles of an Austrian adolescent population utilizing molecule-based IgE diagnosis. Methods Serum samples of 501 nonselected pupils from Salzburg, Austria, were tested in ImmunoCAP ISAC® for IgE reactivity to 112 single allergens. Sensitization profiles were assessed and statistically coordinated with reported allergies. Results In the population aged 12–21 years, 53.5% showed IgE reactivity to at least one allergen tested. The highest prevalence was found for Phl p 1 from grass pollen (26.5%), group 2 mite allergens (18.2%), Bet v 1 from birch pollen (16.3%) and Fel d 1 from cat (14.4%). The majority of participants showed a complex sensitization profile and reacted on average to 9 allergens. Pollen sensitization was highly prevalent (41.7%) and mainly driven by group I grass and PR-10 allergens of the Betulaceae family, while Pla l 1 represented the most relevant weed. Diagnosed and self-reported allergies were noted in 21.9% and 45.5% of participants, respectively, and correlated well with in vitro results. Among atopic individuals, 71.4% reported to suffer from at least one allergy; concordance was found for grass and cat sensitization, while venom- and weed pollen-positive individuals were frequently asymptomatic. Conclusions More than half of the tested adolescent population had already established an atopic status presenting a complex IgE reactivity profile dominated by pollen sensitization. Detailed molecule-based analysis allows determining relevant biomarkers and monitoring of the atopic status in populations.

The C-type lectin receptors (CLRs) Mincle, Mcl, and Dectin-2 bind mycobacterial and fungal cell wall glycolipids and carbohydrates. Recently, we described that expression of these CLR is downregulated during differentiation of human monocytes to dendritic cells (DC) in the presence of GM-CSF and IL-4. Here, we demonstrate that the Th2 cytokine IL-4 specifically inhibits expression of Mincle, Mcl, and Dectin-2 in human antigen-presenting cells (APC). This inhibitory effect of IL-4 was observed across species, as murine macrophages and DC treated with IL-4 also downregulated these receptors. IL-4 blocked upregulation of Mincle and Mcl mRNA expression and cell surface protein by murine macrophages in response to the Mincle ligand Trehalose-6,6-dibehenate (TDB), whereas the TLR4 ligand LPS overcame inhibition by IL-4. Functionally, downregulation of Mincle expression by IL-4 was accompanied by reduced cytokine production upon stimulation with TDB. These inhibitory effects of IL-4 were dependent on the transcription factor Stat6. Together, our results show that the key Th2 cytokine IL-4 exerts a negative effect on the expression of Mincle and other Dectin-2 cluster CLR in mouse and human macrophages and DC, which may render these sentinel cells less vigilant for sensing mycobacterial and fungal ligands. © 2016 Hupfer, Schick, Jozefowski, Voehringer, Ostrop and Lang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).

Several spleen tyrosine kinase–coupled C-type lectin receptors (CLRs) have emerged as important pattern recognition receptors for infectious danger. Because encounter with microbial pathogens leads to the simultaneous ligation of several CLRs and TLRs, the signals emanating from different pattern recognition receptors have to be integrated to achieve appropriate biological responses. In this review, we briefly summarize current knowledge about ligand recognition and core signaling by Syk-coupled CLRs. We then address mechanisms of synergistic and antagonistic crosstalk between different CLRs and with TLRs. Emerging evidence suggests that signal integration occurs through 1) direct interaction between receptors, 2) regulation of expression levels and localization, and 3) collaborative or conflicting signaling interference. Accordingly, we aim to provide a conceptual framework for the complex and sometimes unexpected outcome of CLR ligation in bacterial and fungal infection. Copyright © 2017 by The American Association of Immunologists, Inc. This is the pre-peer reviewed version of the following article: [Contact, Collaboration, Conflict: Signal Integration of Syk-coupled C-type Lectin Receptors], which has been published in final form at https://doi.org/10.4049/jimmunol.1601665

CD16-positive (CD14++CD16+ and CD14+CD16++) monocytes have unique features with respect to phenotype and function. We have used transcriptional profiling for comparison of CD16-positive monocytes and classical monocytes. We show herein that 187 genes are greater than fivefold differentially expressed, including 90 genes relevant to immune response and inflammation. Hierarchical clustering of data for monocyte subsets and CD1c+ myeloid blood dendritic cells (DCs) demonstrate that CD16-positive cells are more closely related to classical monocytes than to DCs. Reverse transcriptase polymerase chain reaction for ten genes with the strongest differential expression confirmed the pattern including a lower messenger RNA level for CD14, CD163, and versican in CD16-positive monocytes. The pattern was similar for CD16-positive monocytes at rest and after exercise mobilization from the marginal pool. By contrast, alveolar macrophages, small sputum macrophages, breast milk macrophages, and synovial macrophages all showed a different pattern. When monocyte-derived macrophages (MDMs) were generated from CD16-positive monocytes by culture with macrophage colony-stimulating factor in vitro, then the MDMs maintained properties of their progeny with lower expression of CD14, CD163, and versican compared with CD14++CD16−MDMs. Furthermore, CD16-positive MDMs showed a higher phagocytosis for opsonized Escherichia coli. The data demonstrate that CD16-positive monocytes form a distinct type of cell, which gives rise to a distinct macrophage phenotype.

Background Anaphylaxis is the most severe manifestation of an IgE-dependent allergy. Standardized acquired clinical data from large cohorts of well-defined cases are not available. The aim of this study was to analyse the symptom profile and risk factors of anaphylaxis in a large Central European cohort. Methods We acquired data from patients in Germany, Austria and Switzerland who experienced a severe allergic reaction defined by the onset of severe pulmonary and/or severe cardiovascular symptoms. The data were gained via an online questionnaire from 83 medical centres specialized in allergy. Data were collected from 2006 to 2010 and analysed by using a multinomial regression model. Results A total of 2012 paediatric and adult patients were included into the present analysis. The skin (84%) was the most frequently affected organ followed by the cardiovascular (72%) and the respiratory (68%) system. The regression model analysing the onset of cardiovascular versus respiratory symptoms revealed a strong impact of age (adjusted OR = 6.08; 95% CI, 3.35–11.01; P

Background: Knowledge of allergen-specific T cell epitopes is a prerequisite not only for therapeutic approaches but also for elucidating immunological mechanisms of type I allergy. Ex vivo detection of allergen-specific T cells using class II tetramer technology has become an important tool for investigating immune responses in atopic and healthy individuals. Methods: Using 3H-thymidine incorporation assays, T cell epitopes specific for the major timothy grass pollen allergen Phl p 5.0101 were mapped in 11 allergic donors and two different mouse strains. Different protocols for expansion/restimulation of T cells from the blood of allergic donors and detection of allergen-specific T cells by Class II Ultimer staining were evaluated. Results: We identified several new Phl p 5.0101 class II T cell epitopes in allergic patients and confirmed previously published ones. Additionally, we discovered the major T cell epitopes in BALB/c and C57BL/6 mice. Using a novel Class II Ultimer, we detected epitope-specific T cells expanded from the blood of an allergic donor. Conclusions: Epitope mapping of Phl p 5.0101 revealed an immunodominant epitope in BALB/c and C57BL/6 mice and an immunodominant region in humans (amino acids 259–282), which was recognized by 8 out of 11 allergic donors. Detection of Phl p 5-specific T cells was demonstrated using a Class II Ultimer specific for epitope 196–210. Successful detection of ultimer-positive T cells was strongly dependent on a resting phase after in vitro expansion.

Pathogen recognition by the innate immune system is essential for the induction of adaptive T cell responses. A diverse range of pathogen-associated molecular patterns (PAMPs) are recognized by a variety of pathogen recognition receptors (PRRs). Among these are the well known Toll-like receptors (TLR) and the more recently described C-type lectin receptors (CLR) which utilize distinct signaling pathways leading to a diverse repertoire of effector molecules produced. The composition of the inflammatory juice released from activated innate immune cells has a major impact on the polarization of Th cell responses. Defined PAMPs may therefore be used as adjuvants to direct adaptive immune responses to subunit vaccines. Targeting CLR is an alternative or complementary strategy to TLR-triggering adjuvants that will benefit the development of more efficient subunit vaccines for prevention of major human infectious diseases. In this short review, we discuss the potential of CLRs activating APC via the Syk-Card9 pathway as receptors for adjuvants that direct the development of robust Th17 and Th1 responses to subunit vaccines.

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.

The immune system defends the organism against invading pathogens. In recent decades it became evident that elimination of such pathogens, termination of inflammation, and restoration of host homeostasis all depend on bidirectional crosstalk between the immune system and the neuroendocrine system. This crosstalk is mediated by a complex network of interacting molecules that modulates inflammation and cell growth. Among these mediators are neuropeptides released from neuronal and non-neuronal components of the central and peripheral nervous systems, endocrine tissues, and cells of the immune system. Neuropeptide circuitry controls tissue inflammation and maintenance, and an imbalance of pro- and anti-inflammatory neuropeptides results in loss of host homeostasis and triggers inflammatory diseases. The galanin peptide family is undoubtedly involved in the regulation of inflammatory processes, and the aim of this review is to provide up-to-date knowledge from the literature concerning the regulation of galanin and its receptors in the nervous system and peripheral tissues in experimental models of inflammation. We also highlight the effects of galanin and other members of the galanin peptide family on experimentally induced inflammation and discuss these data in light of an anti-inflammatory role for this family of peptides.

The mitogen-activated protein kinase phosphatase Dusp1 (also known as MKP-1) is essential for control of the inflammatory response to systemic challenge with the lipopolysaccharide of Gram-negative bacteria. Here, we have investigated the consequences of Dusp1-deficiency in colon ascendens stent peritonitis (CASP) and caecal ligation and puncture (CLP), two mouse models of septic peritonitis. Following CASP, Dusp1(-/-) mice had increased serum levels of CCL4, interleukin-10 (IL-10) and IL-6, with differences from wild-type mice being dependent on severity of sepsis. These cytokines, along with inducible nitric oxide synthase messenger RNA, were also expressed at higher levels in spleen and liver. Similar over-production of these cytokines was detected in the CLP model, with even larger differences from wild-type mice. Despite the increased inflammatory response, bacterial clearance was impaired in Dusp1(-/-) mice subjected to CASP and CLP. Dusp1(-/-) mice suffered increased lethality in both peritonitis models. Together our data indicate that exaggerated inflammatory responses to gut bacteria introduced into the peritoneum in the absence of Dusp1 do not help to control bacterial replication but are detrimental for the host.

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.

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 FcRγ was essential for macrophage activation and Th17 adjuvanticity. The FcRγ-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 FcRγ 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.

Elevated IL-10 has been implicated in reactivation tuberculosis (TB). Since macrophages rather than T cells were reported to be the major source of IL-10 in TB, we analyzed the consequences of a macrophage-specific overexpression of IL-10 in transgenic mice (macIL-10-transgenic) after aerosol infection with Mycobacterium tuberculosis (Mtb). MacIL-10 transgenic mice were more susceptible to chronic Mtb infection than nontransgenic littermates, exhibiting higher bacterial loads in the lung after 12 wk of infection and dying significantly earlier than controls. The differentiation, recruitment, and activation of Th1 cells as well as the induction of IFN-γ-dependent effector genes against Mtb were not affected by macrophage-derived IL-10. However, microarray analysis of pulmonary gene expression revealed patterns characteristic of alternative macrophage activation that were overrepresented in Mtb-infected macIL-10 transgenic mice. Importantly, arginase-1 gene expression and activity were strikingly enhanced in transgenic mice accompanied by a reduced production of reactive nitrogen intermediates. Moreover, IL-10-dependent arginase-1 induction diminished antimycobacterial effector mechanisms in macrophages. Taken together, macrophage-derived IL-10 triggers aspects of alternative macrophage activation and promotes Mtb recrudescence independent of overt effects on anti-TB T cell immunity.

IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls proinflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is TNF-α, a potent proinflammatory mediator that is the target for multiple anti-TNF-α clinical strategies in Crohn’s disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. In this study, we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNF-α and IL-1α in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3′ untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38 MAPK activity. The reduction of p38 MAPK activity releases TTP from the p38 MAPK-mediated inhibition, thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.

Novel vaccination strategies against Mycobacterium tuberculosis (MTB) are urgently needed. The use of recombinant MTB antigens as subunit vaccines is a promising approach, but requires adjuvants that activate antigen-presenting cells (APCs) for elicitation of protective immunity. The mycobacterial cord factor Trehalose-6,6-dimycolate (TDM) and its synthetic analogue Trehalose-6,6-dibehenate (TDB) are effective adjuvants in combination with MTB subunit vaccine candidates in mice. However, it is unknown which signaling pathways they engage in APCs and how these pathways are coupled to the adaptive immune response. Here, we demonstrate that these glycolipids activate macrophages and dendritic cells (DCs) via Syk–Card9–Bcl10–Malt1 signaling to induce a specific innate activation program distinct from the response to Toll-like receptor (TLR) ligands. APC activation by TDB and TDM was independent of the C-type lectin receptor Dectin-1, but required the immunoreceptor tyrosine-based activation motif–bearing adaptor protein Fc receptor γ chain (FcRγ). In vivo, TDB and TDM adjuvant activity induced robust combined T helper (Th)-1 and Th-17 T cell responses to a MTB subunit vaccine and partial protection against MTB challenge in a Card9-dependent manner. These data provide a molecular basis for the immunostimulatory activity of TDB and TDM and identify the Syk–Card9 pathway as a rational target for vaccine development against tuberculosis.

Background: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons. Objective: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA). Methods: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays. Results: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods. Conclusions: Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

SummaryThe action of type I interferons in the central nervous system (CNS) during autoimmunity is largely unknown. Here, we demonstrate elevated interferon beta concentrations in the CNS, but not blood, of mice with experimental autoimmune encephalomyelitis (EAE), a model for CNS autoimmunity. Furthermore, mice devoid of the broadly expressed type I IFN receptor (IFNAR) developed exacerbated clinical disease accompanied by a markedly higher inflammation, demyelination, and lethality without shifting the T helper 17 (Th17) or Th1 cell immune response. Whereas adoptive transfer of encephalitogenic T cells led to enhanced disease in Ifnar1−/− mice, newly created conditional mice with B or T lymphocyte-specific IFNAR ablation showed normal EAE. The engagement of IFNAR on neuroectodermal CNS cells had no protective effect. In contrast, absence of IFNAR on myeloid cells led to severe disease with an enhanced effector phase and increased lethality, indicating a distinct protective function of type I IFNs during autoimmune inflammation of the CNS.