Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated ...receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.
The calcium- and arachidonic acid (AA)-binding proteins S100A8 and S100A9 are involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in phagocytes. They are specifically ...expressed in myeloid cells, and are also found in epithelial cells in various (patho)physiological conditions. We have investigated the consequences of S100A8/A9 overexpression in epithelial cell lines on reactive oxygen species (ROS) generation and downstream signaling. Epithelial carcinoma HeLa cells, which exclusively express Nox2, showed dramatically increased activation of NADPH oxidase by phorbol 12-myristate 13-acetate after S100A8/A9 gene transfection. HaCaT keratinocytes overexpressing S100A8/A9 showed enhanced, transient ROS generation in response to the calcium ionophore A23187 compared to mock-transfected cells. Polymerase chain reaction analysis revealed mRNA transcripts for Nox1, Nox2, and Nox5 in HaCaT keratinocytes. Detailed transfection studies confirmed that NADPH oxidase activities in Nox1- and Nox5-transfected HeLa cells were enhanced after S100A8/A9 gene complementation. Furthermore, mutational analysis revealed that AA binding and Thr113 phosphorylation are important for S100A8/A9-enhanced activation of NADPH oxidase. Nuclear factor-κB (NF-κB) activation and interleukin-8 mRNA levels were increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-κB is a redox-sensitive transcription factor. Because they are expressed in epithelia under specific conditions, S100A8 and S100A9 might be involved in skin pathogenesis by modulating aspects of downstream signaling.
S100A8/A9 promotes NADPH oxidase in HaCaT keratinocytes and subsequently increases NFκB activation, which plays important roles in the balance between epidermal growth and differentiation. ...S100A8/A9-positive HaCaT cells present with a significantly reduced rate of cell division and greater expression of two keratinocyte differentiation markers, involucrin and filaggrin, than control cells. S100A8/A9 mutants fail to enhance NFκB activation, TNFα-induced IL-8 gene expression and NFκB p65 phosphorylation, and S100A8/A9-positive cells demonstrate better cell survival in forced suspension culture than mutant cells. S100A8/A9 is induced in epithelial cells in response to stress. Therefore, S100A8/A9-mediated growth arrest could have implications for tissue remodeling and repair.
Several reports have recently demonstrated a detrimental role of Toll-like receptors (TLR) in cerebral ischemia, while there is little information about the endogenous ligands which activate ...TLR-signaling. The myeloid related proteins-8 and-14 (Mrp8/S100A8; Mrp14/S100A9) have recently been characterized as endogenous TLR4-agonists, and thus may mediate TLR-activation in cerebral ischemia. Interestingly, not only TLR-mRNAs, but also Mrp8 and Mrp14 mRNA were found to be induced in mouse brain between 3 and 48 h after transient 1 h focal cerebral ischemia/reperfusion. Mrp-protein was expressed in the ischemic hemisphere, and co-labeled with CD11b-positive cells. To test the hypothesis that Mrp-signaling contributes to the postischemic brain damage, we subjected Mrp14-deficient mice, which also lack Mrp8 protein expression, to focal cerebral ischemia. Mrp14-deficient mice had significantly smaller lesion volumes when compared to wild-type littermates (130±16 mm3 vs. 105±28 mm3) at 2 days after transient focal cerebral ischemia (1 h), less brain swelling, and a reduced macrophage/microglia cell count in the ischemic hemisphere. We conclude that upregulation and signaling of Mrp-8 and-14 contribute to neuroinflammation and the progression of ischemic damage.
Here the nucleotide sequence of three cDNAs from
Xenopus laevis are reported. The corresponding genes belong to the AAA-family of genes showing the strongest sequence homology to the human HIV-1 Tat ...binding protein 1 (TBP-1) and to the yeast SUG1 gene, respectively. Analysis of their expression pattern indicate that they are maternally stored in oocytes and involved in very early stages of vertebrate embryogenesis.
Myeloid-related proteins 8 and 14 (MRP8 and MRP14) are two Ca2+-binding proteins of the S-100 family highly abundant in myelomonocytic cells. The expression is not only dependent on the developmental ...status of the cell but also on the inflammatory situation in the tissue. In order to identify regulatory elements responsible for the high expression of MRP14 in myeloid cells, reporter gene constructs have been transfected into HL-60 cells, Mono Mac 6 cells, and L132 cells. We demonstrated that a DNA element in the first intron (positions 153–361) enhances the transcriptional activity of the homologous promoter and of the heterologous herpes simplex virus thymidine kinase promoter up to 37-fold. To further identify the functional site, the region between positions 153 and 192 was analyzed functionally using the thymidine kinase promoter. The region increased the expression in the same magnitude as the complete intron. This enhancer is highly conserved in the human and murine MRP genes, indicative of its involvement in the transcription of MRPs. Protein binding to the region is demonstrated using EMSA, DNA cross-linking, Southwestern blotting, and affinity purification. Affinity purification confirms that four proteins bind to the enhancer element.
► S100A8/A9 gene induction in keratinocytes by TLR3 ligand polyI:C. ► Confirmation in organotypic epithelial “raft” culture after herpes simplex virus infection. ► S100A8/A9 gene induction is not ...dependent on IL-10. ► Involvement of NF-κB, p38 MAPK, PI-3K and Jak-Stat signal transduction pathways in polyI:C-induced S100A8/A9 gene expression. ► S100A8/A9 gene induction is abrogated by treatment with either cycloheximide or bleomycin.
Viral double-stranded RNA (dsRNA) and its synthetic analog polyI:C are recognized via multiple pathways and induce the expression of genes related to inflammation. In the present study, we demonstrated the polyI:C-induced gene expression of the damage associated molecular pattern (DAMP) molecules S100A8 and S100A9, while other S100 genes were not affected. Cycloheximide and Brefeldin A treatment revealed both the expression of S100A8 and S100A9 as secondary response genes and the involvement of polyI:C-induced cytokines herein. Several type I and type III interferons such as IFNβ, IL-20, IL-24, and IFNλ/IL-29 were expressed in response to polyI:C, however, they failed to induce S100A8 and S100A9 gene expression. These data indicate the involvement of the danger molecule S100A8/A9 in the resistance against viruses.
Recently, we showed that S100A8/A9 were secreted from phorbol ester-stimulated neutrophil-like HL-60 cells, thereby carrying arachidonic acid Kerkhoff et al. (1999) J. Biol. Chem. 274, 32672−32679. ...The present study was undertaken to evaluate whether the secreted S100A8/A9−AA complex might be involved in transcellular eicosanoid metabolism. In the presence of S100A8/A9, arachidonic acid was rapidly taken up by human umbilical vein endothelial cells in a saturable and energy-dependent fashion. Protein-facilitated arachidonate uptake was confirmed by its sensitivity toward the protein modifiers bromobimane and phloretin. Both potassium and sodium depletion did not affect the arachidonate transport, indicating that arachidonate influx was independent of endocytosis. The uptake of exogenous arachidonic acid by HUVEC was predominantly mediated by FAT/CD36. This conclusion was drawn by the findings that (i) arachidonate uptake was drastically inhibited by sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36; (ii) the maximal inhibition of arachidonate uptake induced by SSO was similar to that effected by ATP depletion; and (iii) the arachidonate transport was 2-fold higher in COS-7 cells transfected with the pEF.BOS-CD36 expression vector than in the empty vector-transfected COS-7 cells. Kinetic studies of arachidonate uptake were indicative for an interaction between fatty acid transporter and S100A8/A9−AA complex that was confirmed by an in vitro protein−protein interaction assay. FAT/CD36 has been suggested to be involved in inflammatory responses, and S100A8/A9 are released from activated leukocytes at inflammatory loci. Therefore, it can be envisioned that their interaction might propagate host response by perpetuating recruitment and activation of cellular effectors.
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