SUMMARYHistamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-jB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.
Although histamine plays an essential role in inflammation, its influence on cyclooxygenases (COX) and prostanoid homeostasis is not well understood. In this study, we investigated the effects of histamine on the expression of COX-1 and COX-2 and determined their contribution to the production of PGE2, prostacyclin (PGI2), and thromboxane A2 in human coronary artery endothelial cells (HCAEC). Incubation of HCAEC monolayers with histamine resulted in marked increases in the expression of COX-2 and production of PGI2 and PGE2 with no significant change in the expression of COX-1. Histamine-induced increases in PGI2 and PGE2 production were due to increased expression and function of COX-2 because gene silencing by small interfering RNA or inhibition of the catalytic activity by a COX-2 inhibitor blocked prostanoid production. The effects of histamine on COX-2 expression and prostanoid production were mediated through H1 receptors. In addition to the direct effect, histamine was found to amplify LPS-stimulated COX-2 expression and PGE2 and PGI2 production. In contrast, histamine did not stimulate thromboxane A2 production in resting or LPS-activated HCAEC. Histamine-induced increases in the production of PGE2 and PGI2 were associated with increased expression of mRNA encoding PGE2 and PGI2 synthases. The physiological role of histamine on the regulation of COX-2 expression in the vasculature is indicated by the findings that the expression of COX-2 mRNA, but not COX-1 mRNA, was markedly reduced in the aortic tissues of histidine decarboxylase null mice. Thus, histamine plays an important role in the regulation of COX-2 expression and prostanoid homeostasis in vascular endothelium.
Sarcoidosis is a complex systemic granulomatous disease of unknown etiology characterized by the presence of activated macrophages and Th1/Th17 effector cells. Data mining of our RNA-Seq analysis of CD14+monocytes showed enrichment for metabolic and hypoxia inducible factor (HIF) pathways in sarcoidosis. Further investigation revealed that sarcoidosis macrophages and monocytes exhibit higher protein levels for HIF-α isoforms, HIF-1β, and their transcriptional co-activator p300 as well as glucose transporter 1 (Glut1). In situ hybridization of sarcoidosis granulomatous lung tissues showed abundance of HIF-1α in the center of granulomas. The abundance of HIF isoforms was mechanistically linked to elevated IL-1β and IL-17 since targeted down regulation of HIF-1α via short interfering RNA or a HIF-1α inhibitor decreased their production. Pharmacological intervention using chloroquine, a lysosomal inhibitor, decreased lysosomal associated protein 2 (LAMP2) and HIF-1α levels and modified cytokine production. These data suggest that increased activity of HIF-α isoforms regulate Th1/Th17 mediated inflammation in sarcoidosis.
Sarcoidosis is a multisystem granulomatous disease of unknown etiology that primarily affects the lungs. Our previous work indicates that activation of p38 plays a pivotal role in sarcoidosis inflammatory response. Therefore, we investigated the upstream kinase responsible for activation of p38 in sarcoidosis alveolar macrophages (AMs) and peripheral mononuclear cells (PBMCs). We identified that sustained p38 phosphorylation in sarcoidosis AMs and PBMCs is associated with active MKK4 but not with MKK3/6. Additionally, we found that sarcoidosis AMs exhibit a higher expression of IRAK1, IRAK-M and Rip2. Surprisingly, ex vivo treatment of sarcoidosis AMs or PBMCs with IRAK1/4 inhibitor led to a significant increase in IL-1β mRNA expression both spontaneously and in response to TLR2 ligand. However, a combination of Rip2 and IRAK-1/4 inhibitors significantly decreased both IL-1β and IL-6 production in sarcoidosis PBMCs and moderately in AMs. Importantly, a combination of Rip2 and IRAK-1/4 inhibitors led to decreased IFN-γ and IL-6, and decreased percentage of activated CD4+CD25+ cells in PBMCs. These data suggest that in sarcoidosis both pathways, namely IRAK and Rip2 are deregulated. Targeted modulation of Rip2 and IRAK pathways may prove to be a novel treatment for sarcoidosis.
Sarcoidosis is a complex systemic granulomatous disorder of unknown etiology. Genome-wide association studies have not been able to explain a causative role for nucleotide variation in its pathogenesis. The goal of the present study was to identify the gene expression profile and the cellular pathways altered in sarcoidosis monocytes via RNA-sequencing. Peripheral blood monocytes play a role in sarcoidosis inflammation. Therefore, we determined and compared the transcriptional signature of monocytes from peripheral blood from sarcoidosis patients and healthy controls via RNA-sequencing. We found 2,446 differentially expressed (DE) genes between sarcoidosis and healthy control monocytes. Analysis of these DE genes showed enrichment for ribosome, phagocytosis, lysosome, proteasome, oxidative phosphorylation and metabolic pathways. RNA-sequencing identified upregulation of genes involved in phagocytosis and lysosomal pathway in sarcoidosis monocytes, whereas genes involved in proteasome degradation and ribosomal pathways were downregulated. Further studies are needed to investigate the role of specific genes involved in the identified pathways and their possible interaction leading to sarcoidosis pathology.
Inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components are initiated by Toll-like receptor 2 (TLR2) and TLR4, respectively. Therefore, the existence of functionally active TLR2 and TLR4 in human conjunctival epithelial cells (HCEC) are critical for the effective host defense against bacterial infections in the eye. We examined the ability of HCEC to respond to TLR4 ligand, lipopolysaccharide (LPS), or TLR2 ligands, lipoteichoic acid (LTA) and peptidoglycan (PGN) using the Chang conjunctival epithelial cell line and the primary conjunctival epithelial cell line (IOBA-NHC) as in vitro models. Incubation of Chang cells with LPS (1 to 1,000 ng/ml) failed to stimulate IL-6 production where as stimulation with LTA or PGN resulted in marked increases in IL-6 production. Semi-quantitative RT-PCR and immunofluorescence analyses showed that Chang cells express TLR2 and TLR4 mRNA and proteins. However, these cells expressed little or no mRNA encoding MD2, an accessory molecule required for TLR4 signaling. Incubation of Chang epithelial cells with interferon-gamma (IFNgamma), but not TNF-alpha, stimulated MD2 mRNA expression and restored LPS responsiveness. In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production. The lack of LPS response, deficient expression of MD2, and induction of MD2 expression and LPS response after IFNgamma priming, were also evident in IOBA-NHC cells. These results demonstrate that HCEC lack LPS responsiveness due to deficient expression of MD2 and that the response can be restored by IFN-gamma priming or MD2 supplementation.
SUMMARYHost defence against tuberculosis infection involves T-lymphocyte mediated cellular immune responses. In this study we assessed T-cell activation by studying the early signal transduction events and production of cytokines by human CD4 + T-cells. The study constituted of five groups of subjects: ]i were seen in TB patients as compared to T + ve healthy controls. TB patients showed significantly lower levels of IL-2 and IFN g and higher levels of IL-4 as compared to normal healthy controls, suggesting a diminished Th1 response. Thus, the reciprocal changes in cytokines, reduced [Ca 2 + ] i levels, and CD54 expression in patients imply phenotype shifting of Th precursors to Th2 type in TB patients.
Post viral infection bacterial pneumonia is a major cause of morbidity and mortality associated with both seasonal and pandemic influenza virus illness. Despite much efforts put into the discovery of mechanisms of post viral–bacterial infections and their complications in recent years, the molecular mechanisms underlying the increased susceptibility to bacterial infection remain poorly understood. In this study, we focused on the pathways regulating immune responses in murine macrophages and modeled post viral–bacterial infections through pretreatment of bone marrow-derived macrophages (BMDMs) with a toll-like receptor (TLR) 7/8 ligand (R848) and subsequent challenge with TLR2/4 agonists to mimic bacterial infection. We found R848-primed BMDMs upon subsequent exposure to TLR2/4 ligands respond with enhanced inflammatory cytokine production, especially IL-6 and TNF-α. The enhanced cytokine production in R848-primed BMDMs in response to TLR2/4 was due to increased TGF-β-activated kinase (TAK) 1 phosphorylation with subsequent activation of ERK and p38 MAPKs. Furthermore, we identified that R848 priming leads to increased K63-linked polyubiquitination on TRAF6. K63-linked polyubiquitination on TRAF6 is a signal leading to enhanced activation of downstream pathways including TAK1. Importantly, R848-primed BMDMs infected with live bacteria exhibited decreased bacterial clearance. Small-molecule enhancer of rapamycin 3, an ubiquitin ligase inhibitor reversed the K63-linked polyubiquitination on TRAF6 in R848-primed BMDMs and subsequently decreased TAK1 and MAPK phosphorylation, and cytokine production as well as reversed the decreased bacterial clearance capacity of BMDMs. Our study may provide a novel molecular target to alleviate post viral–bacterial infections.
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