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1.
Matrix Biol ; 121: 149-166, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37391162

RESUMO

In response to tissue injury, changes in the extracellular matrix (ECM) can directly affect the inflammatory response and contribute to disease progression or resolution. During inflammation, the glycosaminoglycan hyaluronan (HA) becomes modified by tumor necrosis factor stimulated gene-6 (TSG6). TSG6 covalently transfers heavy chain (HC) proteins from inter-α-trypsin inhibitor (IαI) to HA in a transesterification reaction and is to date is the only known HC-transferase. By modifying the HA matrix, TSG6 generates HC:HA complexes that are implicated in mediating both protective and pathological responses. Inflammatory bowel disease (IBD) is a lifelong chronic disorder with well-described remodeling of the ECM and increased mononuclear leukocyte influx into the intestinal mucosa. Deposition of HC:HA matrices is an early event in inflamed gut tissue that precedes and promotes leukocyte infiltration. However, the mechanisms by which TSG6 contributes to intestinal inflammation are not well understood. The aim of our study was to understand how the TSG6 and its enzymatic activity contributes to the inflammatory response in colitis. Our findings indicate that inflamed tissues of IBD patients show an elevated level of TSG6 and increased HC deposition and that levels of HA strongly associate with TSG6 levels in patient colon tissue specimens. Additionally, we observed that mice lacking TSG6 are more vulnerable to acute colitis and exhibit an aggravated macrophage-associated mucosal immune response characterized by elevated pro-inflammatory cytokines and chemokines and diminished anti-inflammatory mediators including IL-10. Surprisingly, along with significantly increased levels of inflammation in the absence of TSG6, tissue HA levels in mice were found to be significantly reduced and disorganized, absent of typical "HA-cable" structures. Inhibition of TSG6 HC-transferase activity leads to a loss of cell surface HA and leukocyte adhesion, indicating that the enzymatic functions of TSG6 are a major contributor to stability of the HA ECM during inflammation. Finally, using biochemically generated HC:HA matrices derived by TSG6, we show that HC:HA complexes can attenuate the inflammatory response of activated monocytes. In conclusion, our data suggests that TSG6 exerts a tissue-protective, anti-inflammatory effect via the generation of HC:HA complexes that become dysregulated in IBD.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Adesão Celular , Colite/induzido quimicamente , Colite/genética , Ácido Hialurônico/metabolismo , Inflamação/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo
3.
Front Immunol ; 13: 1046574, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36733389

RESUMO

Introduction: Neutrophil extracellular traps (NETs) clear pathogens but may contribute Q8 pathogenically to host inflammatory tissue damage during sepsis. Innovative therapeutic agents targeting NET formation and their potentially harmful collateral effects remain understudied. Methods: We investigated a novel therapeutic agent, neonatal NET-Inhibitory Factor (nNIF), in a mouse model of experimental sepsis - cecal ligation and puncture (CLP). We administered 2 doses of nNIF (1 mg/ kg) or its scrambled peptide control intravenously 4 and 10 hours after CLP treatment and assessed survival, peritoneal fluid and plasma NET formation using the MPO-DNA ELISA, aerobic bacterial colony forming units (CFU) using serial dilution and culture, peritoneal fluid and stool microbiomes using 16S rRNA gene sequencing, and inflammatory cytokine levels using a multiplexed cytokine array. Meropenem (25 mg/kg) treatment served as a clinically relevant treatment for infection. Results: We observed increased 6-day survival rates in nNIF (73%) and meropenem (80%) treated mice compared to controls (0%). nNIF decreased NET formation compared to controls, while meropenem did not impact NET formation. nNIF treatment led to increased peritoneal fluid and plasma bacterial CFUs consistent with loss of NET-mediated extracellular microbial killing, while nNIF treatment alone did not alter the peritoneal fluid and stool microbiomes compared to vehicle-treated CLP mice. nNIF treatment also decreased peritoneal TNF-a inflammatory cytokine levels compared to scrambled peptide control. Furthermore, adjunctive nNIF increased survival in a model of sub-optimal meropenem treatment (90% v 40%) in CLP-treated mice. Discussion: Thus, our data demonstrate that nNIF inhibits NET formation in a translationally relevant mouse model of sepsis, improves survival when given as monotherapy or as an adjuvant with antibiotics, and may play an important protective role in sepsis.


Assuntos
Armadilhas Extracelulares , Sepse , Camundongos , Animais , Neutrófilos/patologia , Meropeném/farmacologia , RNA Ribossômico 16S/genética , Sepse/patologia , Citocinas/farmacologia , Receptores Proteína Tirosina Quinases , Punções
4.
JCI Insight ; 6(17)2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314391

RESUMO

Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness, as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbating disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in patients with COVID-19 and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, 2 signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in patients with COVID-19. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation, leading to production of pathological HA fragments that are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in a ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.


Assuntos
COVID-19/metabolismo , Endotélio Vascular/metabolismo , Ácido Hialurônico/metabolismo , Idoso , COVID-19/sangue , COVID-19/patologia , Citocinas/sangue , Endotélio Vascular/patologia , Feminino , Glicocálix/metabolismo , Glicocálix/patologia , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/sangue , Hialuronoglucosaminidase/sangue , Hialuronoglucosaminidase/metabolismo , Masculino , Pessoa de Meia-Idade , Quinases Associadas a rho/metabolismo
5.
J Histochem Cytochem ; 69(1): 25-34, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32870756

RESUMO

On the surface of endothelial cells (ECs) lies the glycocalyx, a barrier of polysaccharides that isolates the ECs from the blood. The role of the glycocalyx is dynamic and complex, thanks to not only its structure, but its vast number of components, one being hyaluronan (HA). HA is a critical component of the glycocalyx, having been found to have a wide variety of functions depending on its molecular weight, its modification, and receptor-ligand interactions. As HA and viscous blood are in constant contact, HA can transmit mechanosensory information directly to the cytoskeleton of the ECs. The degradation and synthesis of HA directly alters the permeability of the EC barrier; HA modulation not only alters the physical barrier but also can signal the initiation of other pathways. EC proliferation and angiogenesis are in part regulated by HA fragmentation, HA-dependent receptor binding, and downstream signals. The interaction between the CD44 receptor and HA is a driving force behind leukocyte recruitment, but each class of leukocyte still interacts with HA in unique ways during inflammation. HA regulates a diverse repertoire of EC functions.


Assuntos
Células Endoteliais/metabolismo , Glicocálix/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Animais , Humanos , Inflamação/metabolismo , Neovascularização Fisiológica
6.
JCI Insight ; 1(21): e90240, 2016 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-28018974

RESUMO

Life-sustaining responses to low oxygen, or hypoxia, depend on signal transduction by HIFs, but the underlying mechanisms by which cells sense hypoxia are not completely understood. Based on prior studies suggesting a link between the ß-adrenergic receptor (ß-AR) and hypoxia responses, we hypothesized that the ß-AR mediates hypoxia sensing and is necessary for HIF-1α accumulation. Beta blocker treatment of mice suppressed hypoxia induction of renal HIF-1α accumulation, erythropoietin production, and erythropoiesis in vivo. Likewise, beta blocker treatment of primary human endothelial cells in vitro decreased hypoxia-mediated HIF-1α accumulation and binding to target genes and the downstream hypoxia-inducible gene expression. In mechanistic studies, cAMP-activated PKA and/or GPCR kinases (GRK), which both participate in ß-AR signal transduction, were investigated. Direct activation of cAMP/PKA pathways did not induce HIF-1α accumulation, and inhibition of PKA did not blunt HIF-1α induction by hypoxia. In contrast, pharmacological inhibition of GRK, or expression of a GRK phosphorylation-deficient ß-AR mutant in cells, blocked hypoxia-mediated HIF-1α accumulation. Mass spectrometry-based quantitative analyses revealed a hypoxia-mediated ß-AR phosphorylation barcode that was different from the classical agonist phosphorylation barcode. These findings indicate that the ß-AR is fundamental to the molecular and physiological responses to hypoxia.

7.
J Clin Invest ; 126(7): 2465-81, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27214549

RESUMO

High levels of arginine metabolizing enzymes, including inducible nitric oxide synthase (iNOS) and arginase (ARG), are typical in asthmatic airway epithelium; however, little is known about the metabolic effects of enhanced arginine flux in asthma. Here, we demonstrated that increased metabolism sustains arginine availability in asthmatic airway epithelium with consequences for bioenergetics and inflammation. Expression of iNOS, ARG2, arginine synthetic enzymes, and mitochondrial respiratory complexes III and IV was elevated in asthmatic lung samples compared with healthy controls. ARG2 overexpression in a human bronchial epithelial cell line accelerated oxidative bioenergetic pathways and suppressed hypoxia-inducible factors (HIFs) and phosphorylation of the signal transducer for atopic Th2 inflammation STAT6 (pSTAT6), both of which are implicated in asthma etiology. Arg2-deficient mice had lower mitochondrial membrane potential and greater HIF-2α than WT animals. In an allergen-induced asthma model, mice lacking Arg2 had greater Th2 inflammation than WT mice, as indicated by higher levels of pSTAT6, IL-13, IL-17, eotaxin, and eosinophils and more mucus metaplasia. Bone marrow transplants from Arg2-deficient mice did not affect airway inflammation in recipient mice, supporting resident lung cells as the drivers of elevated Th2 inflammation. These data demonstrate that arginine flux preserves cellular respiration and suppresses pathological signaling events that promote inflammation in asthma.


Assuntos
Arginina/metabolismo , Asma/imunologia , Asma/metabolismo , Mitocôndrias/metabolismo , Adulto , Animais , Hiper-Reatividade Brônquica , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Feminino , Humanos , Inflamação , Interleucina-13/metabolismo , Interleucina-17/metabolismo , Masculino , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Fator de Transcrição STAT6/metabolismo , Células Th2
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