Nandakumar Packiriswamy

G-protein coupled receptor kinase 5 mediates lipopolysaccharide-induced NFκB activation in primary macrophages and modulates inflammation in vivo in mice.

G‐protein coupled receptor kinase‐5 (GRK5) is a serine/threonine kinase discovered for its role in the regulation of G‐protein coupled receptor signaling. Recent studies have shown that GRK5 is also an important regulator of signaling pathways stimulated by non‐GPCRs. This study was undertaken to determine the physiological role of GRK5 in Toll‐like receptor‐4‐induced inflammatory signaling pathways in vivo and in vitro. Using mice genetically deficient in GRK5 (GRK5−/−) we demonstrate here that GRK5 is an important positive regulator of lipopolysaccharide (LPS, a TLR4 agonist)‐induced inflammatory cytokine and chemokine production in vivo. Consistent with this role, LPS‐induced neutrophil infiltration in the lungs (assessed by myeloperoxidase activity) was markedly attenuated in the GRK5−/− mice compared to the GRK5+/+ mice. Similar to the in vivo studies, primary macrophages from GRK5−/− mice showed attenuated cytokine production in response to LPS. Our results also identify TLR4‐induced NFκB pathway in macrophages to be selectively regulated by GRK5. LPS‐induced IκBα phosphorylation, NFκB p65 nuclear translocation, and NFκB binding were markedly attenuated in GRK5−/− macrophages. Together, our findings demonstrate that GRK5 is a positive regulator of TLR4‐induced IκBα–NFκB pathway as well as a key modulator of LPS‐induced inflammatory response. J. Cell. Physiol. 226: 1323–1333, 2011.

G-protein-coupled receptor kinases in inflammation and disease

G-protein-coupled receptor kinases (GRKs) are serine/threonine protein kinases originally discovered for their role in G-protein-coupled receptor (GPCR) phosphorylation. Recent studies have demonstrated a much broader function for this kinase family including phosphorylation of cytosolic substrates involved in cell signaling pathways stimulated by GPCRs, as well as by non-GPCRs. In addition, GRKs modulate signaling via phosphorylation-independent functions. Because of these various biochemical functions, GRKs have been shown to affect critical physiological and pathophysiological processes, and thus are considered as drug targets in diseases such as heart failure. Role of GRKs in inflammation and inflammatory diseases is an evolving area of research and several studies including work from our lab in the recent years have demonstrated critical role of GRKs in the immune system. In this review, we discuss the classical and the newly emerging functions of GRKs in the immune system and their role in inflammation and disease processes.

G-Protein-Coupled Receptor Kinase-5 Mediates Inflammation but Does Not Regulate Cellular Infiltration or Bacterial Load in a Polymicrobial Sepsis Model in Mice

NFκB-dependent signaling is an important modulator of inflammation in several diseases including sepsis. G-protein-coupled receptor kinase-5 (GRK5) is an evolutionarily conserved regulator of the NFκB pathway. We hypothesized that GRK5 via NFκB regulation plays an important role in the pathogenesis of sepsis. To test this we utilized a clinically relevant polymicrobial sepsis model in mice that were deficient in GRK5. We subjected wild-type (WT) and GRK5 knockout (KO) mice to cecal ligation and puncture (CLP)-induced polymicrobial sepsis and assessed the various events in sepsis pathogenesis. CLP induced a significant inflammatory response in the WT and this was markedly attenuated in the KO mice. To determine the signaling mechanisms and the role of NFκB activation in sepsis-induced inflammation, we assessed the levels of IκBa phosphorylation and expression of NFκB-dependent genes in the liver in the two genotypes. Both IκBa phosphorylation and gene expression were significantly inhibited in the GRK5 KO compared to the WT mice. Interestingly, however, GRK5 did not modulate either immune cell infiltration (to the primary site of infection) or local/systemic bacterial load subsequent to sepsis induction. In contrast GRK5 deficiency significantly inhibited sepsis-induced plasma corticosterone levels and the consequent thymocyte apoptosis in vivo. Associated with these outcomes, CLP-induced mortality was significantly prevented in the GRK5 KO mice in the presence of antibiotics. Together, our studies demonstrate that GRK5 is an important regulator of inflammation and thymic apoptosis in polymicrobial sepsis and implicate GRK5 as a potential molecular target in sepsis.

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.

Role of endothelial cells in bovine mammary gland health and disease.

Abstract The bovine mammary gland is a dynamic and complex organ composed of various cell types that work together for the purpose of milk synthesis and secretion. A layer of endothelial cells establishes the blood–milk barrier, which exists to facilitate the exchange of solutes and macromolecules necessary for optimal milk production. During bacterial challenge, however, endothelial cells divert some of their lactation function to protect the underlying tissue from damage by initiating inflammation. At the onset of inflammation, endothelial cells tightly regulate the movement of plasma components and leukocytes into affected tissue. Unfortunately, endothelial dysfunction as a result of exacerbated or sustained inflammation can negatively affect both barrier integrity and the health of surrounding extravascular tissue. The objective of this review is to highlight the role of endothelial cells in supporting milk production and regulating optimal inflammatory responses. The consequences of endothelial dysfunction and sustained inflammation on milk synthesis and secretion are discussed. Given the important role of endothelial cells in orchestrating the inflammatory response, a better understanding of endothelial function during mastitis may support development of targeted therapies to protect bovine mammary tissue and mammary endothelium.

Supplementation of linoleic acid (C18:2n-6) or α-linolenic acid (C18:3n-3) changes microbial agonist-induced oxylipid biosynthesis

Oxylipids are derived from polyunsaturated fatty acids (PUFA) in cellular membranes and the relative abundance or balance may contribute to disease pathogenesis. Previous studies documented unique oxylipid profiles from cows with either coliform or Streptococcus uberis mastitis, suggesting that lipid mediator biosynthesis may be dependent on the type of microbial-derived agonist. Changing the fatty acid content of peripheral blood leukocytes also may be critical to the relative expression of oxylipid profiles and the outcome of bacterial infection. No information is available in dairy cows describing how changing cellular PUFA content will modify oxylipids in the context of a microbial agonist challenge. Therefore, the hypothesis for the current study was that PUFA supplementation would change bovine leukocyte fatty acid content and respective oxylipid profiles from ex vivo microbial agonist-challenged leukocytes. Fatty acid content of leukocytes and plasma was quantified in (1) samples from cows not supplemented with PUFA, (2) cows supplemented with linoleic acid (LnA), and (3) cows supplemented with α-linolenic acid (ALA). Plasma oxylipids were assessed after S. uberis or lipopolysaccharide exposure and was compared with unstimulated oxylipid profiles. Fatty acid supplementation with ALA significantly increased ALA content of blood leukocytes and plasma relative to LnA. Fatty acid supplementation affected several S. uberis-induced oxylipids, but only S. uberis-induced 15-oxoETE was greater with ALA supplementation compared with LnA. Notably, only LPS-induced 5,6 LXA4 was altered with fatty acid supplementation, but no significant effect of LnA vs. ALA treatment was identified. Future studies are needed to understand how leukocyte activation and membrane PUFA availability collectively contribute to differential oxylipid profiles.

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.

Bacterial Dose-Dependent Role of G Protein-Coupled Receptor Kinase 5 in Escherichia coli-Induced Pneumonia

ABSTRACT G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose.

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.

Poly(I:C) Priming Exacerbates Cecal Ligation and Puncture-Induced Polymicrobial Sepsis in Mice

Sepsis continues to be a major healthcare issue with one of the highest mortality rates in intensive care units. Toll-like receptors are pattern recognition receptors that are intricately involved in the pathogenesis of sepsis. TLR3 is a major receptor for double-stranded RNA and is largely associated with immunity to viral infection. In this study, we examined the role of TLR3 priming in the immunopathology of sepsis using cecal-ligation and puncture (CLP) model of sepsis in mice. Mice injected with vehicle or poly(I:C) were subjected to sham or CLP surgery and various parameters of sepsis, including mortality, inflammation, and bacterial clearance were assessed. Poly(I:C) pre-treatment significantly enhanced mortality in mice subjected to CLP. Consistent with this, inflammatory cytokines including TNFα, IL-12p40, IFNγ, and MCP-1 were enhanced both systemically and locally in the poly(I:C)-treated group compared to the vehicle control. In addition, bacterial load was significantly higher in the poly(I:C)-treated septic mice. These changes were associated with reduced macrophage activation (but not neutrophils) in the peritoneal cavity of poly(I:C) pre-treated mice compared to vehicle pre-treatment. Together our results demonstrate that poly(I:C) priming in sepsis is likely to be detrimental to the host due to effects on systemic inflammatory cytokines and bacterial clearance.