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1.
Front Immunol ; 11: 598636, 2020.
Article in English | MEDLINE | ID: mdl-33250899

ABSTRACT

Breathing allows a multitude of airborne microbes and microbial compounds to access the lung. Constant exposure of the pulmonary microenvironment to immunogenic particles illustrates the need for proper control mechanisms ensuring the differentiation between threatening and harmless encounters. Discrimination between live and dead bacteria has been suggested to be such a mechanism. In this study, we performed infection studies of murine precision cut lung slices (PCLS) with live or heat-killed P. aeruginosa, in order to investigate the role of viability for induction of an innate immune response. We demonstrate that PCLS induce a robust transcriptomic rewiring upon infection with live but not heat-killed P. aeruginosa. Using mutants of the P. aeruginosa clinical isolate CHA, we show that the viability status of P. aeruginosa is assessed in PCLS by TLR5-independent sensing of flagellin and recognition of the type three secretion system. We further demonstrate that enhanced cytokine expression towards live P. aeruginosa is mediated by uptake of viable but not heat-killed bacteria. Finally, by using a combined approach of receptor blockage and genetically modified PCLS we report a redundant involvement of MARCO and CD200R1 in the uptake of live P. aeruginosa in PCLS. Altogether, our results show that PCLS adapt the extent of cytokine expression to the viability status of P. aeruginosa by specifically internalizing live bacteria.


Subject(s)
Cytokines/metabolism , Host-Pathogen Interactions , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/microbiology , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/physiology , Animals , Biopsy , Computational Biology/methods , Disease Models, Animal , Flagellin/metabolism , Gene Expression Profiling , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Immunohistochemistry , Mice , Mice, Knockout , Microbial Viability , Pneumonia, Bacterial/genetics , Pneumonia, Bacterial/immunology , Pseudomonas Infections/genetics , Pseudomonas Infections/immunology , Transcriptome , Type III Secretion Systems/metabolism
2.
Redox Biol ; 28: 101325, 2020 01.
Article in English | MEDLINE | ID: mdl-31606550

ABSTRACT

Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with 'internal tandem duplications (FLT3ITD)' drive production of reactive oxygen in leukemic cells. FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions. The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein.


Subject(s)
Cyclohexanones/pharmacology , Cysteine/metabolism , Mutation , fms-Like Tyrosine Kinase 3/chemistry , fms-Like Tyrosine Kinase 3/metabolism , Cell Line , Cytoplasm/metabolism , HEK293 Cells , Humans , Models, Molecular , Oxidation-Reduction , Protein Conformation , Reactive Oxygen Species/metabolism , Signal Transduction , fms-Like Tyrosine Kinase 3/genetics
3.
Front Immunol ; 10: 198, 2019.
Article in English | MEDLINE | ID: mdl-30846984

ABSTRACT

Streptococcus pyogenes is a major human pathogen causing a variety of diseases ranging from common pharyngitis to life-threatening soft tissue infections and sepsis. Microbial nucleic acids, especially bacterial RNA, have recently been recognized as a major group of pathogen-associated molecular patterns (PAMPs) involved in the detection of Streptococcus pyogenes via endosomal Toll-like receptors (TLRs) in vitro. However, the individual contribution and cooperation between TLRs as well as cell-type and strain specific differences in dependency on nucleic acid detection during S. pyogenes infection in vitro have not been clarified in detail. Moreover, the role of particularly bacterial RNA for the defense of S. pyogenes infection in vivo remains poorly defined. In this study, we report that in all investigated innate immune cells involved in the resolution of bacterial infections, including murine macrophages, dendritic cells and neutrophils, recognition of S. pyogenes strain ATCC12344 is almost completely dependent on nucleic acid sensing via endosomal TLRs at lower MOIs, whereas at higher MOIs, detection via TLR2 plays an additional, yet redundant role. We further demonstrate that different S. pyogenes strains display a considerable inter-strain variability with respect to their nucleic acid dependent recognition. Moreover, TLR13-dependent recognition of S. pyogenes RNA is largely non-redundant in bone marrow-derived macrophages (BMDMs), but less relevant in neutrophils and bone marrow-derived myeloid dendritic cells (BMDCs) for the induction of an innate immune response in vitro. In vivo, we show that a loss of nucleic acid sensing blunts early recognition of S. pyogenes, leading to a reduced local containment of the bacterial infection with subsequent pronounced systemic inflammation at later time points. Thus, our results argue for a crucial role of nucleic acid sensing via endosomal TLRs in defense of S. pyogenes infection both in vitro and in vivo.


Subject(s)
Endosomes/metabolism , Host-Pathogen Interactions/immunology , Streptococcal Infections/immunology , Streptococcal Infections/metabolism , Streptococcus pyogenes/physiology , Toll-Like Receptors/metabolism , Biomarkers , Cytokines/metabolism , Humans , Immunity, Cellular , Immunity, Innate , Nitric Oxide/metabolism , Nucleic Acids/immunology , RNA, Bacterial/immunology , Reactive Oxygen Species/metabolism , Streptococcal Infections/microbiology
4.
Int J Mol Sci ; 19(7)2018 06 21.
Article in English | MEDLINE | ID: mdl-29933628

ABSTRACT

High expression levels of the calcium-binding proteins S100A8 and S100A9 in myeloid cells in kidney transplant rejections are associated with a favorable outcome. Here we investigated the myeloid cell subset expressing these molecules, and their function in inflammatory reactions. Different monocyte subsets were sorted from buffy coats of healthy donors and investigated for S100A8 and S100A9 expression. To characterize S100A9high and S100A9low subsets within the CD14+ classical monocyte subset, intracellular S100A9 staining was combined with flow cytometry (FACS) and qPCR profiling. Furthermore, S100A8 and S100A9 were overexpressed by transfection in primary monocyte-derived macrophages and the THP-1 macrophage cell line to investigate the functional relevance. Expression of S100A8 and S100A9 was primarily found in classical monocytes and to a much lower extent in intermediate and non-classical monocytes. All S100A9+ cells expressed human leukocyte antigen—antigen D related (HLA-DR) on their surface. A small population (<3%) of CD14+ CD11b+ CD33+ HLA-DR− cells, characterized as myeloid derived suppressor cells (MDSCs), also expressed S100A9 to high extent. Overexpression of S100A8 and S00A9 in macrophages led to enhanced extracellular reactive oxygen species (ROS) production, as well as elevated mRNA expression of anti-inflammatory IL-10. The results suggest that the calcium-binding proteins S100A8 and S100A9 in myeloid cells have an immune regulatory effect.


Subject(s)
Calgranulin A/immunology , Calgranulin B/immunology , Interleukin-10/immunology , Macrophages/immunology , Myeloid-Derived Suppressor Cells/immunology , Calcium/immunology , Calcium/metabolism , Calgranulin A/genetics , Calgranulin B/genetics , Cell Lineage/genetics , Cell Lineage/immunology , Flow Cytometry , Gene Expression Regulation , HLA-DR Antigens/genetics , HLA-DR Antigens/immunology , Healthy Volunteers , Humans , Immunophenotyping , Interleukin-10/genetics , Macrophages/cytology , Myeloid-Derived Suppressor Cells/cytology , Primary Cell Culture , RNA, Messenger/genetics , RNA, Messenger/immunology , Reactive Oxygen Species/immunology , Reactive Oxygen Species/metabolism , THP-1 Cells
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