TSG6 hyaluronan matrix remodeling dampens the inflammatory response during colitis.

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.

Corrigendum: Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial sepsis by inhibiting neutrophil extracellular traps.

[This corrects the article DOI: 10.3389/fimmu.2022.1046574.].

Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial sepsis by inhibiting neutrophil extracellular traps.

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.

COVID-19 generates hyaluronan fragments that directly induce endothelial barrier dysfunction.

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.

Barrier Housing and Gender Effects on Allergic Airway Disease in a Murine House Dust Mite Model.

Allergic airway disease models use laboratory mice housed in highly controlled and hygienic environments, which provide a barrier between the mice and a predetermined list of specific pathogens excluded from the facility. In this study, we hypothesized that differences in facility barrier level and, consequently, the hygienic quality of the environment that mice inhabit impact the severity of pulmonary inflammation and lung function. Allergen-naive animals housed in the cleaner, high barrier (HB) specific pathogen-free facility had increased levels of inflammatory cytokines and higher infiltration of immune cells in the lung tissue but not in the bronchoalveolar lavage compared with mice housed in the less hygienic, low barrier specific pathogen-free facility. In both genders, house dust mite-induced airway disease was more severe in the HB than the low barrier facility. Within each barrier facility, female mice developed the most severe inflammation. However, allergen-naive male mice had worse lung function, regardless of the housing environment, and in the HB, the lung function in female mice was higher in the house dust mite model. Severe disease in the HB was associated with reduced lung microbiome diversity. The lung microbiome was altered across housing barriers, gender, and allergen-exposed groups. Thus, the housing barrier level impacts microbial-driven disease and gender phenotypes in allergic asthma. The housing of laboratory mice in more clean HB facilities aggravates lung immunity and causes a more severe allergic lung disease.

Hyaluronan and Its Receptors as Regulatory Molecules of the Endothelial Interface.

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.

Endothelial cells in the innate response to allergens and initiation of atopic asthma.

Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

Quantification of airway fibrosis in asthma by flow cytometry.

Airway fibrosis is a prominent feature of asthma, contributing to the detrimental consequences of the disease. Fibrosis in the airway is the result of collagen deposition in the reticular lamina layer of the subepithelial tissue. Myofibroblasts are the leading cell type involved with this collagen deposition. Established methods of collagen deposition quantification present various issues, most importantly their inability to quantify current collagen biosynthesis occurring in airway myofibroblasts. Here, a novel method to quantify myofibroblast collagen expression in asthmatic lungs is described. Single cell suspensions of lungs harvested from C57BL/6 mice in a standard house dust mite model of asthma were employed to establish a flow cytometric method and compare collagen production in asthmatic and non-asthmatic lungs. Cells found to be CD45- αSMA+ , indicative of myofibroblasts, were gated, and median fluorescence intensity of the anti-collagen-I antibody labeling the cells was calculated. Lung myofibroblasts with no, medium, or high levels of collagen-I expression were distinguished. In asthmatic animals, collagen-I levels were increased in both medium and high expressers, and the number of myofibroblasts with high collagen-I content was elevated. Our findings determined that quantification of collagen-I deposition in myofibroblastic lung cells by flow cytometry is feasible in mouse models of asthma and indicative of increased collagen-I expression by asthmatic myofibroblasts. © 2018 International Society for Advancement of Cytometry.

Bone marrow transplantation prevents right ventricle disease in the caveolin-1-deficient mouse model of pulmonary hypertension.

Accumulating evidence shows a causative role for the bone marrow (BM) in the genesis and progression of pulmonary hypertension (PH). Engraftment of BM hematopoietic stem cells from PH patients to mice reproduces the cardiopulmonary pathology of PH. However, it is unknown whether healthy BM can prevent the development of right heart disease. Caveolin-1-deficient (CAV-1 KO) mice develop cardiopulmonary disease with manifestations resembling PH, including elevated right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary endothelial proliferative disease. Here, we hypothesize that engraftment of healthy BM to CAV-1 KO mice will prevent pulmonary vascular remodeling and development of the cardiopulmonary disease. CAV-1 KO mice and wild-type (WT) mice underwent transplantation with WT or CAV-1 KO BM. Hematopoietic differentiation was analyzed by flow cytometry. Pulmonary endothelial remodeling was quantified by CD31 image analysis. RVSP and RV cardiomyocyte area or Fulton's index were used to analyze RV hypertrophy. Maladaptive RV hypertrophy was determined by quantification of RV fibrosis. Transplantation of CAV-1 KO BM into healthy recipient WT mice led to elevation of RVSP, RV hypertrophy, and pulmonary endothelial remodeling. Reconstitution of CAV-1 KO with WT BM prevented spontaneous development of PH, including elevation of RVSP and maladaptive RV hypertrophy, but not pulmonary endothelial remodeling. Healthy BM has a protective role in the right ventricle independent of pulmonary vascular disease.

Hypoxia sensing through ß-adrenergic receptors.

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.

Flow Cytometric Quantification of Peripheral Blood Cell ß-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although ß-adrenergic receptor (ßAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of ßAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated ß-blocker alprenolol was synthesized and validated as a ßAR specific probe that was combined with immunophenotyping to quantify ßAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that ßAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell ßAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

Increased mitochondrial arginine metabolism supports bioenergetics in asthma.

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.

Eotaxin-Rich Proangiogenic Hematopoietic Progenitor Cells and CCR3+ Endothelium in the Atopic Asthmatic Response.

Angiogenesis is closely linked to and precedes eosinophilic infiltration in asthma. Eosinophils are recruited into the airway by chemoattractant eotaxins, which are expressed by endothelial cells, smooth muscles cells, epithelial cells, and hematopoietic cells. We hypothesized that bone marrow-derived proangiogenic progenitor cells that contain eotaxins contribute to the initiation of angiogenesis and inflammation in asthma. Whole-lung allergen challenge of atopic asthma patients revealed vascular activation occurs within hours of challenge and before airway inflammation. The eotaxin receptor CCR3 was expressed at high levels on submucosal endothelial cells in patients and a murine model of asthma. Ex vivo exposure of murine endothelial cells to eotaxins induced migration and angiogenesis. In mechanistic studies, wild-type mice transplanted with eotaxin-1/2-deficient bone marrow had markedly less angiogenesis and inflammation in an atopic asthma model, whereas adoptive transfer of proangiogenic progenitor cells from wild-type mice in an atopic asthma model into the eotaxin-1/2-deficient mice led to angiogenesis and airway inflammation. The findings indicate that Th2-promoting hematopoietic progenitor cells are rapidly recruited to the lung upon allergen exposure and release eotaxins that coordinately activate endothelial cells, angiogenesis, and airway inflammation.