Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA) oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1) metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE), can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE), is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC) to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis

Distinct Signature of Oxylipid Mediators of Inflammation during Infection and Asymptomatic Colonization by E. coli

Urinary tract infection (UTI) is an extremely common infectious disease. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Asymptomatic bacteriuric E. coli (ABEC) strains successfully colonize the urinary tract resulting in asymptomatic bacteriuria (ABU) and do not induce symptoms associated with UTI. Oxylipids are key signaling molecules involved in inflammation. Based on the distinct clinical outcomes of E. coli colonization, we hypothesized that UPEC triggers the production of predominantly proinflammatory oxylipids and ABEC leads to production of primarily anti-inflammatory or proresolving oxylipids in the urinary tract. We performed quantitative detection of 39 oxylipid mediators with proinflammatory, anti-inflammatory, and proresolving properties, during UTI and ABU caused by genetically distinct E. coli strains in the murine urinary bladder. Our results reveal that infection with UPEC causes an increased accumulation of proinflammatory oxylipids as early as 6 h postinoculation, compared to controls. To the contrary, ABEC colonization leads to decreased accumulation of proinflammatory oxylipids at the early time point compared to UPEC infection but does not affect the level of proresolving oxylipids. This report represents the first comprehensive investigation on the oxylipidome during benign ABEC colonization observed in ABU and acute inflammation triggered by UPEC leading to UTI

A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses

IntroductionMetastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM.MethodsWe developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-β) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNβ-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNβ-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective.Results12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses.DiscussionOur study found that VSV-IFNβ -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNβ-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen

Critical involvement of GPCR kinase-5 in E.coli induced pneumonia (INC7P.427)

Abstract G-protein coupled receptor kinases are serine/threonine kinases involved in a range of pathophysiological processes including inflammation. We recently demonstrated, deficiency of G-protein coupled receptor kinase-5 (GRK5) improves sepsis outcome in a polymicrobial septic. In this study we examined the role of GRK5 in various immunopathological features associated with E.coli induced pulmonary infection using wild type (WT) and GRK5 knockout (KO) mice. Interestingly, 12-hours post-infection, GRK5KO exhibited significantly reduced inflammation and bacterial load as well as decreased neutrophils in the lung. Ex vivo assays demonstrated, GRK5KO and WT neutrophils have similar capacity to kill bacteria suggesting an early increase in neutrophils in the KO mice might decrease bacterial load. Consistent with this prediction, 4 hours post-infection, GRK5KO mice had markedly elevated neutrophil numbers in the lungs, compared to the WT mice. Interestingly, at later time points after infection (12 hours) even though the neutrophil (Ly6G+) numbers were reduced in the KO, the number of efferocytosing macrophages (Ly6g+ F4/80+) were markedly higher in the GRK5KO animals, suggesting that GRK5 deficiency advances the inflammatory resolution phase (increased efferocytosing macrophages). Taken together, our results show that GRK5 deficiency improves pulmonary infection and inflammation likely via early-enhanced neutrophil recruitment as well as swifter clearance of neutrophils in the lung.</jats:p

Overlapping and distinct roles of GRK5 in TLR2-, and TLR3-induced inflammatory response in vivo

G-protein coupled receptor kinase-5 (GRK5) is a recently described NFκB regulator in TLR4 signaling pathway. To determine whether the role of GRK5 is MyD88- or TRIF-dependent, we injected wild type and GRK5 knockout mice with Pam3CSK4 (MyD88-dependent TLR1/2 ligand) and Poly(I:C) (TRIF-dependent TLR3 ligand) and examined the in vivo systemic inflammatory response. Our results demonstrate that GRK5 regulates IL-12p40 and G-CSF via a mechanism that is common to both MyD88 and TRIF. However, GRK5 regulates IL-5 and MCP-1 in a MyD88-dependent but TNFα in a TRIF-dependent manner. Together, our results demonstrate multiple roles of GRK5 in TLR signaling

Nonhematopoietic β-Arrestin-1 Inhibits Inflammation in a Murine Model of Polymicrobial Sepsis

β-Arrestin-1 (βArr1), a scaffolding protein critical in G-protein coupled receptor desensitization has more recently been found to be important in the pathogenesis of various inflammatory diseases. We sought to understand the role of βArr1 in sepsis pathogenesis using a mouse model of polymicrobial sepsis. Although in previous studies we established that βArr1 deficiency protects mice from endotoxemia, here we demonstrate that the absence of βArr1 remarkably renders mice more susceptible to mortality in polymicrobial sepsis. In accordance with the mortality pattern, early production of inflammatory mediators was markedly enhanced in βArr1 knockout mice systemically and locally in various organs. In addition, enhanced inflammation in the heart was associated with increased NFκB activation. Compared to these effects, immune cell infiltration, thymic apoptosis, and immune suppression during polymicrobial sepsis were unaffected by a deficiency of βArr1. Additionally, enhanced inflammation and consequent higher mortality were not observed in heterozygous mice, suggesting that one allele of βArr1 was sufficient for this protective negative regulatory role. We further demonstrate that, unexpectedly, βArr1 in nonhematopoietic cells is critical and sufficient for inhibiting sepsis-induced inflammation, whereas hematopoietic βArr1 is likely redundant. Taken together, our results reveal a novel and previously unrecognized negative regulatory role of the nonhematopoietic βArr1 in sepsis-induced inflammation

β-arrestin-1 negatively regulates inflammatory response to polymicrobial sepsis in mice (110.11)

Abstract β-arrestins are scaffolding proteins that regulate a number of receptor signaling pathways including Toll-like receptors. We recently demonstrated that mice lacking either β-arrestin-1 or β-arrestin-2 are protected from lipopolysaccharide-induced lethality and have a markedly reduced inflammatory response. To assess the role of β-arrestin-1 in a clinically relevant model of sepsis, we subjected wild type and β-arrestin-1 knockout mice to cecal-ligation and puncture (CLP) to mimick septic peritonitis and polymicrobial sepsis. Surprisingly, we found that, mortality of β-arrestin-1 knockout mice was significantly enhanced compared to the wild type mice after CLP. Consistent with lethality, β-arrestin-1 knockout mice had markedly elevated inflammatory cytokine levels in the plasma, peritoneal cavity, and bronchoalveolar fluid. Enhanced systemic inflammatory response of β-arrestin-1 knockout mice was associated with significantly enhanced infiltration of immune cells into the peritoneal cavity after induction of septic peritonitis. Together, these results demonstrate that, contrary to its role in lipolysaccharide-TLR4 signaling in vivo, β-arrestin-1 is a negative regulator of inflammation induced by polymicrobial sepsis and that the phenotype of the mice may be related to a potentially aberrant immune response from excess infiltration of immune cells. These results also suggest that the role of β-arrestin-1 in this model is likely independent of its role in TLR4 signaling in vivo.</jats:p