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Background and Purpose: Fibrotic lung remodelling after a respiratory viral infection represents a debilitating clinical sequela. Studying or managing viral-fibrotic sequela remains challenging, due to limited therapeutic options and lack of understanding of mechanisms. This study determined whether protein disulfide isomerase A3 (PDIA3) and secreted phosphoprotein 1 (SPP1), which are associated with pulmonary fibrosis, can promote influenza-induced lung fibrotic remodelling and whether inhibition of PDIA3 or SPP1 can resolve viral-mediated fibrotic remodelling., Experimental Approach: A retrospective analysis of TriNetX data sets was conducted. Serum from healthy controls and influenza A virus (IAV)-infected patients was analysed. An inhibitor of PDIA3, punicalagin, and a neutralizing antibody for SPP1 were administered in mice. Macrophage cells treated with macrophage colony-stimulating factor (M-CSF) were used as a cell culture model., Key Results: The TriNetX data set showed an increase in lung fibrosis and decline in lung function in flu-infected acute respiratory distress syndrome (ARDS) patients compared with non-ARDS patients. Serum samples revealed a significant increase in SPP1 and PDIA3 in influenza-infected patients. Lung PDIA3 and SPP1 expression increased following viral infection in mouse models. Punicalagin administration 2 weeks after IAV infection in mice caused a significant decrease in lung fibrosis and improved oxygen saturation. Administration of neutralizing SPP1 antibody decreased lung fibrosis. Inhibition of PDIA3 decreased SPP1secretion from macrophages, in association with diminished disulfide bonds in SPP1., Conclusion and Implications: The PDIA3-SPP1 axis promotes post-influenza lung fibrosis in mice and that pharmacological inhibition of PDIA3 or SPP1 can treat virus-induced lung fibrotic sequela., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Obesity is often accompanied by increased adipose tissue inflammation, a process that is partially driven by adipose tissue-resident macrophages. In this study, we explored the potential for plant-derived dietary compounds to exert anti-inflammatory effects in macrophages that alleviate obesity-associated adipocyte dysfunction. Capsaicin (CAP), schisandrin A (SA), enterodiol (END), and enterolactone (ENL) treatment polarized J774 macrophages to an "M2" or anti-inflammatory phenotype and inhibited responses to stimulation with lipopolysaccharide (LPS). Furthermore, these compounds blocked inflammasome activation when administered just before ATP-induced NLRP3 activation, as evidenced by the abrogation of IL-1β release in mouse macrophages and human peripheral blood monocytes. The addition of CAP, SA, or ENL during the differentiation of bone marrow-derived macrophages was also sufficient to inhibit LPS-induced IL-6 and TNFα production. Finally, CAP, END, and ENL treatment during differentiation of 3T3-L1 adipocytes induced an adiponectin-high phenotype accompanied by increases in thermogenic gene expression, and conditioned media from these adipocytes inhibited LPS-induced production of IL-1β, IL-6, and TNFα from J774 macrophages. These polarizing effects were partially mediated by the elevated adiponectin and decreased syndecan-4 in the adipocyte-conditioned media. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity. NEW & NOTEWORTHY The utility of food-based products to prevent or alleviate chronic conditions such as obesity and its associated comorbidities is an attractive approach. Capsaicin, schisandrin A, enterodiol, and enterolactone, phytochemicals present in traditional medicinal food, decreased proinflammatory cytokine production from macrophages that, in turn, reduced obesity-associated adipocyte dysfunction. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.

Obesity is a global health problem characterized by excessive fat accumulation, driven by adipogenesis and lipid accumulation. Long non-coding RNAs (lncRNAs) have recently been implicated in regulating adipogenesis and adipose tissue function. Mouse lncRNA U90926 was previously identified as a repressor of in vitro adipogenesis in 3T3-L1 preadipocytes. Consequently, we hypothesized that, in vivo, U90926 may repress adipogenesis, and hence its deletion would increase weight gain and adiposity. We tested the hypothesis by applying U90926-deficient (U9-KO) mice to a high-throughput phenotyping pipeline. Compared with WT, U9-KO mice showed no major differences across a wide range of behavioral, neurological, and other physiological parameters. In mice fed a standard diet, we have found no differences in obesity-related phenotypes, including weight gain, fat mass, and plasma concentrations of glucose, insulin, triglycerides, and free fatty acids, in U9-KO mice compared to WT. U90926 deficiency lacked a major effect on white adipose tissue morphology and gene expression profile. Furthermore, in mice fed a high-fat diet, we found increased expression of U90926 in adipose tissue stromal vascular cell fraction, yet observed no effect of U90926 deficiency on weight gain, fat mass, adipogenesis marker expression, and immune cell infiltration into the adipose tissue. These data suggest that the U90926 lacks an essential role in obesity-related phenotypes and adipose tissue biology in vivo., (© 2024 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Obesity is a risk factor for asthma. Individuals with asthma and obesity often have poor asthma control and do not respond as well to therapies such as inhaled corticosteroids and long-acting bronchodilators. Weight loss improves asthma control, with a 5%-10% loss in body mass necessary and sufficient to lead to clinically relevant improvements. Preclinical studies have demonstrated the pathogenic contribution of adipocytes from obese mice to the augmented production of proinflammatory cytokines from airway epithelial cells and the salutary effects of diet-induced weight loss to decrease these consequences. However, the effects of adipocyte-derived products on airway epithelial function in human obesity remain incompletely understood. We utilized samples collected from a 12-mo longitudinal study of subjects with obesity undergoing weight loss (bariatric) surgery including controls without asthma and subjects with allergic and nonallergic obese asthma. Visceral adipose tissue (VAT) samples were collected during bariatric surgery and from recruited normal weight controls without asthma undergoing elective abdominal surgery. Human bronchial epithelial (HBEC3-KT) cells were exposed to plasma or conditioned media from cultured VAT adipocytes with or without agonists. Human bronchial smooth muscle (HBSM) cells were similarly exposed to adipocyte-conditioned media. Proinflammatory cytokines were augmented in supernatants from HBEC3-KT cells exposed to plasma as compared with subsequent visits. Whereas exposure to obese adipocyte-conditioned media induced proinflammatory responses, there were no differences between groups in both HBEC3-KT and HBSM cells. These data show that bariatric surgery and subsequent weight loss beneficially change the circulating factors that augment human airway epithelial and bronchial smooth muscle cell proinflammatory responses. NEW & NOTEWORTHY This longitudinal study following subjects with asthma and obesity reveals that weight loss following bariatric surgery decreases the capacity for plasma to augment proinflammatory cytokine secretion by human bronchial epithelial cells, implicating that circulating but not adipocyte-derived factors are important modulators in obese asthma.

Background: Cancer and its treatment can adversely affect skeletal muscle, impacting physical function, treatment response and survival. No studies, however, have comprehensively characterized these muscle adaptations longitudinally in human patients at the cellular level., Methods: We examined skeletal muscle size and function from the whole body to the sub-cellular level in 11 patients with non-small cell lung cancer (NSCLC; 6 male/5 female, mean age 58 ± 3 years) studied over a 2-month observation period starting during their first cycle of standard of care cancer treatment and in 11 age- and sex-matched healthy controls (HC) without a current or past history of cancer. Biopsies of the vastus lateralis were performed to assess muscle fibre size, contractility and mitochondrial content, along with assessments of physical function, whole muscle size and function, and circulating cytokines., Results: Body weight, composition and thigh muscle area and density were unaltered over time in patients with NSCLC, while muscle density was lower in patients with NSCLC versus HC (P = 0.03). Skeletal muscle fibre size decreased by 18% over time in patients (all P = 0.02) and was lower than HC (P = 0.02). Mitochondrial fractional area and density did not change over time in patients, but fractional area was lower in patients with NSCLC compared with HC (subsarcolemmal, P = 0.04; intermyofibrillar, P = 0.03). Patients with NSCLC had higher plasma concentrations of IL-6 (HC 1.40 ± 0.50; NSCLC 4.71 ± 4.22; P < 0.01), GDF-15 (HC 569 ± 166; NSCLC 2071 ± 1168; P < 0.01) and IL-8/CXCL8 (HC 4.9 ± 1.8; NSCLC 10.1 ± 6.0; P = 0.02) compared with HC, but there were no changes in inflammatory markers in patients with NSCLC over time. No changes were observed in markers of satellite cell activation or DNA damage in patients and no group differences were noted with HC. Whole-muscle strength was preserved over time in patients with NSCLC coincident with improved single fibre contractility., Conclusions: This study is the first to comprehensively examine longitudinal alterations in skeletal muscle fibre size and function in patients with NSCLC and suggests that muscle fibre atrophy occurs during cancer treatment despite weight stability and no changes in conventional clinical measurements of whole body or thigh muscle size over this period., (© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

Obesity is a risk factor for severe influenza, and asthma exacerbations caused by respiratory viral infections. We investigated mechanisms that increase the severity of airway disease related to influenza in obesity using cells derived from obese and lean individuals, and in vitro and in vivo models. Primary human nasal epithelial cells (pHNECs) derived from obese compared with lean individuals developed increased inflammation and injury in response to influenza A virus (IAV). Obese mice infected with influenza developed increased airway inflammation, lung injury and elastance, but had a decreased interferon response, compared with lean mice. Lung arachidonic acid (AA) levels increased in obese mice infected with IAV; arachidonic acid increased inflammatory cytokines and injury markers in response to IAV in human bronchial epithelial (HBE) cells. Obesity in mice, and AA in HBE cells, increased activation of p38 MAPK signaling following IAV infection; inhibiting this pathway attenuated inflammation, injury and tissue elastance responses, and improved survival. In summary, obesity increases disease severity in response to influenza infection through activation of the p38 MAPK pathway in response to altered arachidonic acid signaling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chandrasekaran, Morris, Butzirus, Mark, Kumar, Souza De Lima, Daphtary, Aliyeva, Poynter, Anathy and Dixon.)

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but challenges with dosing or side effects such as bleeding limit clinical application. In this study, we demonstrate that suramin, a widely available polyanionic drug, completely neutralizes the toxic effects of individual histones, but not citrullinated histones from neutrophil extracellular traps. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones in vivo. Suramin also prevented histone-induced lung endothelial cell cytotoxicity in vitro and lung edema, intra-alveolar hemorrhage, and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Mitochondria are multifaceted organelles necessary for numerous cellular signaling and regulatory processes. Mitochondria are dynamic organelles, trafficked and anchored to subcellular sites depending upon the cellular and tissue requirements. Precise localization of mitochondria to apical and basolateral membranes in lung epithelial cells is important for key mitochondrial processes. Miro1 is an outer mitochondrial membrane GTPase that associates with adapter proteins and microtubule motors to promote intracellular movement of mitochondria. We show that deletion of Miro1 in lung epithelial cells leads to perinuclear clustering of mitochondria. However, the role of Miro1 in epithelial cell response to allergic insults remains unknown. We generated a conditional mouse model to delete Miro1 in Club Cell Secretory Protein (CCSP) positive lung epithelial cells to examine the potential roles of Miro1 and mitochondrial trafficking in the lung epithelial response to the allergen, house dust mite (HDM). Our data show that Miro1 suppresses epithelial induction and maintenance of the inflammatory response to allergen, as Miro1 deletion modestly induces increases in pro-inflammatory signaling, specifically IL-6, IL-33, CCL20 and eotaxin levels, tissue reorganization, and airway hyperresponsiveness. Furthermore, loss of Miro1 in CCSP + lung epithelial cells blocks resolution of the asthmatic insult. This study further demonstrates the important contribution of mitochondrial dynamic processes to the airway epithelial allergen response and the pathophysiology of allergic asthma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Bruno, Lamberty, McCoy, Mark, Daphtary, Aliyeva, Butnor, Poynter, Anathy and Cunniff.)

Background: The purpose of this study was to investigate physiological phenotypes of asthma in obesity., Research Question: Do physiological responses during bronchoconstriction distinguish different groups of asthma in people with obesity, and also differentiate from responses simply related to obesity?, Study Design and Methods: Cross-sectional study of people with obesity (31 with asthma and 22 without lung disease). Participants underwent methacholine challenge testing with measurement of spirometry and respiratory system impedance by oscillometry., Results: Participants had class III obesity (BMI, 46.7 ± 6.6 kg/m 2 in control subjects and 47.2 ± 8.2 kg/m 2 in people with asthma). Most participants had significant changes in peripheral airway impedance in response to methacholine: in control subjects, resistance at 5 Hz measured by oscillometry increased by 45% ± 27% and area under the reactance curve (AX) by 268% ± 236% in response to 16 mg/mL methacholine; in people with asthma, resistance at 5 Hz measured by oscillometry increased by 52% ± 38% and AX by 361% ± 295% in response to provocation concentration producing a 20% fall in FEV 1 dose of methacholine. These responses suggest that obesity predisposes to peripheral airway reactivity. Two distinct groups of asthma emerged based on respiratory system impedance: one with lower reactance (baseline AX, 11.8; interquartile range, 9.9-23.4 cm H 2 O/L) and more concordant bronchoconstriction in central and peripheral airways; the other with high reactance (baseline AX, 46.7; interquartile range, 23.2-53.7 cm H 2 O/L) and discordant bronchoconstriction responses in central and peripheral airways. The high reactance asthma group included only women, and reported significantly more gastroesophageal reflux disease, worse chest tightness, more wheeze, and more asthma exacerbations than the low reactance group., Interpretation: Peripheral airway reactivity detected by oscillometry is common in obese control subjects and obese people with asthma. There is a subgroup of obese asthma characterized by significant peripheral airway dysfunction by oscillometry out of proportion to spirometric airway dysfunction. This peripheral dysfunction represents clinically significant respiratory disease not readily assessed by spirometry., (Copyright © 2023 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Thousands of long noncoding RNAs are encoded in mammalian genomes, yet most remain uncharacterized. In this study, we functionally characterized a mouse long noncoding RNA named U90926. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene was highly induced in macrophages and dendritic cells by TLR activation, in a p38 MAPK- and MyD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. An in vivo model of endotoxic shock revealed that, in comparison with wild type mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Prior studies in younger adults showed that reducing the normally high intake of the saturated fatty acid, palmitic acid (PA), in the North American diet by replacing it with the monounsaturated fatty acid, oleic acid (OA), decreased blood concentrations and secretion by peripheral blood mononuclear cells (PBMCs) of interleukin (IL)-1β and IL-6 and changed brain activation in regions of the working memory network. We examined the effects of these fatty acid manipulations in the diet of older adults. Ten subjects, aged 65-75 years, participated in a randomized, cross-over trial comparing 1-week high PA versus low PA/high OA diets. We evaluated functional magnetic resonance imaging (fMRI) using an N-back test of working memory and a resting state scan, cytokine secretion by lipopolysaccharide (LPS)-stimulated PBMCs, and plasma cytokine concentrations. During the low PA compared to the high PA diet, we observed increased activation for the 2-back minus 0-back conditions in the right dorsolateral prefrontal cortex (Broadman Area (BA) 9; p < 0.005), but the effect of diet on working memory performance was not significant (p = 0.09). We observed increased connectivity between anterior regions of the salience network during the low PA/high OA diet (p < 0.001). The concentrations of IL-1β (p = 0.026), IL-8 (p = 0.013), and IL-6 (p = 0.009) in conditioned media from LPS-stimulated PBMCs were lower during the low PA/high OA diet. This study suggests that lowering the dietary intake of PA down-regulated pro-inflammatory cytokine secretion and altered working memory, task-based activation and resting state functional connectivity in older adults., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Obesity is associated with severe, difficult-to-control asthma, and increased airway oxidative stress. Mitochondrial reactive oxygen species (mROS) are an important source of oxidative stress in asthma, leading us to hypothesize that targeting mROS in obese allergic asthma might be an effective treatment. Using a mouse model of house dust mite (HDM)-induced allergic airway disease in mice fed a low- (LFD) or high-fat diet (HFD), and the mitochondrial antioxidant MitoQuinone (MitoQ), we investigated the effects of obesity and ROS on HDM-induced airway inflammation, remodeling, and airway hyperresponsiveness (AHR). Obese allergic mice showed increased lung tissue eotaxin, airway tissue eosinophilia, and AHR compared with lean allergic mice. MitoQ reduced airway inflammation, remodeling, and hyperreactivity in both lean and obese allergic mice, and tissue eosinophilia in obese-allergic mice. Similar effects were observed with decyl triphosphonium (dTPP + ), the hydrophobic cationic moiety of MitoQ lacking ubiquinone. HDM-induced oxidative sulfenylation of proteins was increased particularly in HFD mice. Although only MitoQ reduced sulfenylation of proteins involved in protein folding in the endoplasmic reticulum (ER), ER stress was attenuated by both MitoQ and dTPP + suggesting the anti-allergic effects of MitoQ are mediated in part by effects of its hydrophobic dTPP + moiety reducing ER stress. In summary, oxidative signaling is an important mediator of allergic airway disease. MitoQ, likely through reducing protein oxidation and affecting the UPR pathway, might be effective for the treatment of asthma and specific features of obese asthma.

More than 50% of people with asthma in the United States are obese, and obesity often worsens symptoms of allergic asthma and impairs response to treatment. Based on previously established roles of the epithelial NADPH oxidase DUOX1 in allergic airway inflammation, we addressed the potential involvement of DUOX1 in altered allergic inflammation in the context of obesity. Intranasal house dust mite (HDM) allergen challenge of subjects with allergic asthma induced rapid secretion of IL-33, then IL-13, into the nasal lumen, responses that were significantly enhanced in obese asthmatic subjects (BMI >30). Induction of diet-induced obesity (DIO) in mice by high-fat diet (HFD) feeding similarly enhanced acute airway responses to intranasal HDM challenge, particularly with respect to secretion of IL-33 and type 2/type 3 cytokines, and this was associated with enhanced epithelial DUOX1 expression and was avoided in DUOX1-deficient mice. DIO also enhanced DUOX1-dependent features of chronic HDM-induced allergic inflammation. Although DUOX1 did not affect overall weight gain by HFD feeding, it contributed to glucose intolerance, suggesting a role in glucose metabolism. However, glucose intolerance induced by short-term HFD feeding, in the absence of adiposity, was not sufficient to alter HDM-induced acute airway responses. DIO was associated with enhanced presence of the adipokine leptin in the airways, and leptin enhanced DUOX1-dependent IL-13 and mucin production in airway epithelial cells. In conclusion, augmented inflammatory airway responses to HDM in obesity are associated with increases in airway epithelial DUOX1, and by increased airway epithelial leptin signaling.

Background: Individuals with allergic asthma exhibit lung inflammation and remodeling accompanied by methacholine hyperresponsiveness manifesting in proximal airway narrowing and distal lung tissue collapsibility, and they can present with a range of mild-to-severe disease amenable or resistant to therapeutic intervention, respectively. There remains a need for alternatives or complements to existing treatments that could control the physiologic manifestations of allergic asthma., Objectives: Our aim was to examine the hypothesis that because ketone bodies elicit anti-inflammatory activity and are effective in mitigating the methacholine hyperresponsiveness associated with obese asthma, increasing systemic concentrations of ketone bodies would diminish pathologic outcomes in asthma-relevant cell types and in mouse models of allergic asthma., Methods: We explored the effects of ketone bodies on allergic asthma-relevant cell types (macrophages, airway epithelial cells, CD4 T cells, and bronchial smooth muscle cells) in vitro as well as in vivo by using preclinical models representative of several endotypes of allergic asthma to determine whether promotion of ketosis through feeding a ketogenic diet or providing a ketone precursor or a ketone ester dietary supplement could affect immune and inflammatory parameters as well as methacholine hyperresponsiveness., Results: In a dose-dependent manner, the ketone bodies acetoacetate and β-hydroxybutyrate (BHB) decreased proinflammatory cytokine secretion from mouse macrophages and airway epithelial cells, decreased house dust mite (HDM) extract-induced IL-8 secretion from human airway epithelial cells, and decreased cytokine production from polyclonally and HDM-activated T cells. Feeding a ketogenic diet, providing a ketone body precursor, or supplementing the diet with a ketone ester increased serum BHB concentrations and decreased methacholine hyperresponsiveness in several acute HDM sensitization and challenge models of allergic asthma. A ketogenic diet or ketone ester supplementation decreased methacholine hyperresponsiveness in an HDM rechallenge model of chronic allergic asthma. Ketone ester supplementation synergized with corticosteroid treatment to decrease methacholine hyperresponsiveness in an HDM-driven model of mixed-granulocytic severe asthma. HDM-induced morphologic changes in bronchial smooth muscle cells were inhibited in a dose-dependent manner by BHB, as was HDM protease activity., Conclusions: Increasing systemic BHB concentrations through dietary interventions could provide symptom relief for several endotypes of allergic asthmatic individuals through effects on multiple asthma-relevant cells., Competing Interests: Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

Macrophages are important mediators of skeletal muscle function in both healthy and diseased states. In vivo specific depletion of macrophages provides an experimental method to understand physiological and pathophysiological effects of macrophages. Systemic depletion of macrophages can deplete skeletal muscle macrophages but also alters systemic inflammatory responses and metabolism, which confounds the muscle specific effects of macrophage depletion. The primary aim of this manuscript is to evaluate two methods of murine intramuscular macrophage depletion in an acute lung injury-associated indirect skeletal muscle wasting mouse model. Adult C57BL/6 (WT) and Macrophage Fas-Induced Apoptosis (MaFIA, C57BL/6-Tg) mice received clodronate liposomes or the dimerization drug AP20187 through intramuscular injection of the tibialis anterior muscle compartment, respectively. Vehicle control was injected in the contralateral muscle. We demonstrate intramuscular AP20187 in the MaFIA mouse depletes macrophages but causes an infiltration of CD45 intermediate neutrophils. In contrast, intramuscular clodronate liposomes successfully depletes macrophages without an associated increase in CD45 intermediate cells. In conclusion, intramuscular clodronate is effective for selective depletion of muscle macrophages without eliciting acute inflammation seen with AP20187 in MaFIA mice. This technique is an important tool to study the functional roles of macrophages in skeletal muscle., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest in this work., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The abundance, anatomical distribution, and vascularity of skeletal muscle make it a potentially important contributor to local cytokine production and systemic cytokine abundance during inflammatory events. An orchestrated balance between the production of pro- and anti-inflammatory mediators is necessary for proper immune function, yet the contribution of the body's largest organ system, comprised primarily of skeletal muscle myocytes that fuse to form myofibers, to this process is largely unknown. Endotoxin (lipopolysaccharide, LPS) stimulates toll-like receptor 4 (TLR4) to induce the production of several pro-inflammatory cytokines, including interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2), by a of myriad cell types. We sought to quantify the influence of myofibers on systemic cytokine concentrations following an acute endotoxemia challenge. To accomplish this, we generated muscle specific conditional knockouts for TLR4 (TLR4SMKO), IL-6 (IL6SMKO), and CCL2 (CCL2SMKO). We administered low concentrations of intravenous LPS (IV LPS) to these receptor and effector knockout mice and collected samples after 3 h. Using gene expression analysis of gastrocnemius muscle and serum cytokine measurements after IV LPS, we determined that deletion of myofiber IL-6 or CCL2 led to a 93% and 57% reduction of these specific cytokines in the systemic circulation, respectively. Myofiber specific TLR4 deletion decreased the expression of IL-6, CCL2, and C-X-C motif chemokine ligand 1 (CXCL1) in the gastrocnemius muscle. These data indicate the critical involvement and direct contribution of myofibers during the early systemic inflammatory cytokine response to endotoxin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bivona III, Mank, Stapleton, Files, Toth and Poynter.)

The NADPH oxidase DUOX1 contributes to epithelial production of alarmins, including interleukin (IL)-33, in response to injurious triggers such as airborne protease allergens, and mediates development of mucus metaplasia and airway remodeling in chronic allergic airways diseases. DUOX1 is also expressed in non-epithelial lung cell types, including macrophages that play an important role in airway remodeling during chronic lung disease. We therefore conditionally deleted DUOX1 in either lung epithelial or monocyte/macrophage lineages to address its cell-specific actions in innate airway responses to acute airway challenge with house dust mite (HDM) allergen, and in chronic HDM-driven allergic airway inflammation. As expected, acute responses to airway challenge with HDM, as well as type 2 inflammation and related features of airway remodeling during chronic HDM-induced allergic inflammation, were largely driven by DUOX1 with the respiratory epithelium. However, in the context of chronic HDM-driven inflammation, DUOX1 deletion in macrophages also significantly impaired type 2 cytokine production and indices of mucus metaplasia. Further studies revealed a contribution of macrophage-intrinsic DUOX1 in macrophage recruitment upon chronic HDM challenge, as well as features of macrophage activation that impact on type 2 inflammation and remodeling., (© 2022. The Author(s), under exclusive licence to Society for Mucosal Immunology.)

Obese asthmatics tend to have severe, poorly controlled disease and exhibit methacholine hyperresponsiveness manifesting in proximal airway narrowing and distal lung tissue collapsibility. Substantial weight loss in obese asthmatics or in mouse models of the condition decreases methacholine hyperresponsiveness. Ketone bodies are rapidly elevated during weight loss, coinciding with or preceding relief from asthma-related comorbidities. As ketone bodies may exert numerous potentially therapeutic effects, augmenting their systemic concentrations is being targeted for the treatment of several conditions. Circulating ketone body levels can be increased by feeding a ketogenic diet or by providing a ketone ester dietary supplement, which we hypothesized would exert protective effects in mouse models of inherent obese asthma. Weight loss induced by feeding a low-fat diet to mice previously fed a high-fat diet was preceded by increased urine and blood levels of the ketone body β-hydroxybutyrate (BHB). Feeding a ketogenic diet for 3 wk to high-fat diet-fed obese mice or genetically obese db/db mice increased BHB concentrations and decreased methacholine hyperresponsiveness without substantially decreasing body weight. Acute ketone ester administration decreased methacholine responsiveness of normal mice, and dietary ketone ester supplementation of high-fat diet-fed mice decreased methacholine hyperresponsiveness. Ketone ester supplementation also transiently induced an "antiobesogenic" gut microbiome with a decreased Fermicutes/Bacteroidetes ratio. Dietary interventions to increase systemic BHB concentrations could provide symptom relief for obese asthmatics without the need for the substantial weight loss required of patients to elicit benefits to their asthma through bariatric surgery or other diet or lifestyle alterations.

Current methods of small animal exercise involve either voluntary (wheel running) or forced (treadmill running) protocols. Although commonly used, each have several drawbacks which cause hesitancy to adopt these methods. While mice will instinctively run on a wheel, the distance and time spent running can vary widely. Forced exercise, while controllable, puts animals in stressful environments in which they are confined and often shocked for "encouragement." Additionally, both methods require expensive equipment and software, which limit these experiments to well-funded laboratories. To counter these issues, we developed a non-invasive mouse running device aimed to reduce handler-induced stress, provide time- and distance-based stopping conditions, and enable investigators with limited resources to easily produce and use the device. The Lockable Open-Source Training-Wheel (LOST-Wheel) was designed to be 3D printed on any standard entry-level printer and assembled using a few common tools for around 20 USD. It features an on-board screen and is capable of tracking distances, running time, and velocities of mice. The LOST-Wheel overcomes the largest drawback to voluntary exercise, which is the inability to control when and how long mice run, using a servo driven mechanism that locks and unlocks the running surface according to the protocol of the investigator. While the LOST-Wheel can be used without a computer connection, we designed an accompanying application to provide scientists with additional analyses. The LOST-Wheel Logger, an R-based application, displays milestones and plots on a user-friendly dashboard. Using the LOST-Wheel, we implemented a timed running experiment that showed distance-dependent decreases in serum myostatin as well as IL-6 gene upregulation in muscle. To make this device accessible, we are releasing the designs, application, and manual in an open-source format. The implementation of the LOST-Wheel and future iterations will improve upon existing murine exercise equipment and research., Competing Interests: The authors have declared that no competing interests exist.

Background: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2., Methods: We used house dust mite (HDM) or interleukin-1β in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1β and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database., Results: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1β-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1β-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13 C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma., Conclusions: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Obesity alters the risks and outcomes of inflammatory lung diseases. It is important to accurately recapitulate the obese state in animal models to understand these effects on the pathogenesis of disease. Diet-induced obesity is a commonly used model of obesity, but when applied to other disease models like acute respiratory distress syndrome, pneumonia, and asthma, it yields widely divergent. We hypothesized high-fat chow storage conditions would affect lipid oxidation and inflammatory response in the lungs of lipopolysaccharide (LPS)-challenged mice. For 6 weeks, C57BL/6crl mice were fed either a 10% (low-fat diet, LFD) or 60% (high-fat diet, HFD) stored at room temperature (RT, 23°C) for up to 7, 14, 21, or 42 days. Mice were treated with nebulized LPS to induce lung inflammation, and neutrophil levels in bronchoalveolar lavage were determined 24 h later. Lipid oxidation (malondialdehyde, MDA) was assayed by thiobarbituric acid reactive substances in chow and mouse plasma. Concentrations of MDA in chow and plasma rose in proportion to the duration of RT chow storage. Mice fed a HFD stored <2 weeks at RT had an attenuated response 24 h after LPS compared with mice fed an LFD. This effect was reversed after 2 weeks of chow storage at RT. Chow stored above freezing underwent lipid oxidation associated with significant alterations in the LPS-induced pulmonary inflammatory response. Our data show that storage conditions affect lipid peroxidation, which in turn affects pulmonary inflammatory responses in a mouse model of disease. It also suggests changes in the microbiome, although not significantly different suggests decreased variety and richness of bacteria in the gut, a large aspect of the immune system. Dietary composition and storage of chow may also affect pulmonary inflammation and the gut microbiome in humans., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: The purpose of this study was to investigate how 8-isoprostanes, used as a marker of airway oxidative stress, were related to sinus disease and asthma., Methods: We analyzed samples and data from two separate studies, one investigating sinonasal disease in asthma, the other investigating the effect of BMI on airway disease. We measured airway (nasal lavage) 8-isoprostanes and investigated the relationship with measures of sinus and asthma symptoms, asthma control and lung function., Results: The study of people with sinonasal disease and poorly controlled asthma included 48 obese, 31 overweight and 23 lean participants. In multivariate analysis, nasal lavage 8-isoprostane levels increased with increasing BMI (p < 0.01), and were higher in Caucasian than African American participants (p = 0.01). Sinus symptoms were inversely related to nasal 8-isoprostanes (p = 0.02) independent of BMI and Race. In the study investigating the effect of BMI on airway disease, we enrolled 13 controls with obesity and 21 people with obesity and asthma: 8-isoprostane levels were higher in obese controls than in obese people with asthma (p < 0.01), and levels were inversely related to sinus symptoms (p = 0.02) and asthma control (p < 0.01)., Interpretation: 8-isoprostanes in nasal lavage are increased in obesity, and increased in Caucasians compared with African Americans. However, levels are higher in obese controls than obese people with asthma, and appear inversely related to symptoms of airway disease., Clinical Implication: Airway 8-isoprostanes likely reflect complex oxidative signaling pathways, which are altered in obesity and those of different race, rather than being a simple marker of airway oxidative injury., Capsule Summary: Increased airway oxidative signaling (8-isoprostanes), may reflect normal physiology in the setting of obesity, as decreased levels are associated with disease activity in people with chronic sinonasal disease and asthma., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Although recognized as an important endocrine organ, little is known about the mechanisms through which adipose tissue can regulate inflammatory responses in distant tissues, such as lung that are affected by obesity. To explore potential mechanisms, male C57BL/6J mice were provided either high-fat diet, low-fat diet, or were provided a high-fat diet then switched to the low-fat diet to promote weight loss. Visceral adipocytes were then cultured in vitro to generate conditioned media (CM) that was used to treat both primary (mouse tracheal epithelial cells; MTECs) and immortalized (mouse-transformed club cells; MTCCs) airway epithelial cells. Adiponectin levels were greatly depressed in the CM from both obese and diet-switched adipocytes relative to mice continually fed the low-fat diet. MTECs from mice with obesity secreted higher baseline levels of inflammatory cytokines than MTECs from lean or diet-switched mice. MTECs treated with obese adipocyte CM increased their secretion of these cytokines compared with MTECs treated with lean CM. Diet-switched CM modestly decreased the production of cytokines compared with obese CM, and these effects were recapitulated when the CM was used to treat MTCCs. Adipose stromal vascular cells from mice with obesity expressed genes consistent with an M1 macrophage phenotype and decreased eosinophil abundance compared with lean stromal vascular fraction, a profile that persisted in the lean diet-switched mice despite substantial weight loss. Soluble factors secreted from obese adipocytes exert a proinflammatory effect on airway epithelial cells, and these alterations are attenuated by diet-induced weight loss, which could have implications for the airway dysfunction related to obese asthma and its mitigation by weight loss.

Obesity is a risk factor for the development of asthma and represents a difficult-to-treat disease phenotype. Aerobic glycolysis is emerging as a key feature of asthma, and changes in glucose metabolism are linked to leukocyte activation and adaptation to oxidative stress. Dysregulation of PKM2 (pyruvate kinase M2), the enzyme that catalyzes the last step of glycolysis, contributes to house dust mite (HDM)-induced airway inflammation and remodeling in lean mice. It remains unclear whether glycolytic reprogramming and dysregulation of PKM2 also contribute to obese asthma. The goal of the present study was to elucidate the functional role of PKM2 in a murine model of obese allergic asthma. We evaluated the small molecule activator of PKM2, TEPP46, and assessed the role of PKM2 using conditional ablation of the Pkm2 allele from airway epithelial cells. In obese C57BL/6NJ mice, parameters indicative of glycolytic reprogramming remained unchanged in the absence of stimulation with HDM. Obese mice that were subjected to HDM showed evidence of glycolytic reprogramming, and treatment with TEPP46 diminished airway inflammation, whereas parameters of airway remodeling were unaffected. Epithelial ablation of Pkm2 decreased central airway resistance in both lean and obese allergic mice in addition to decreasing inflammatory cytokines in the lung tissue. Lastly, we highlight a novel role for PKM2 in the regulation of glutathione-dependent protein oxidation in the lung tissue of obese allergic mice via a putative IFN-γ-glutaredoxin1 pathway. Overall, targeting metabolism and protein oxidation may be a novel treatment strategy for obese allergic asthma.

Muscle may contribute to the systemic inflammatory environment during critical illness, but leukocyte interaction and cytokine influence on muscle and its response has not been fully explored in this context. Using an in vivo model of intratracheal lipopolysaccharide (IT LPS)-induced acute lung injury, we show that skeletal muscle rapidly responds with expression of proinflammatory genes, which may be explained by migration of LPS into the circulation. Treatment of mature C2C12 myotubes with LPS at a level achieved in the circulation following IT LPS elicited a proinflammatory cytokine expression profile similar to that of in vivo murine muscle following IT LPS. Stimulation with toll-like receptor (TLR) 2 and 3 agonists provoked comparable responses in C2C12 myotubes. Additionally, co-cultures of C2C12 myotubes and bone marrow-derived macrophages (BMDM) identified the capacity of macrophages to increase myotube proinflammatory gene expression, with tumor necrosis factor-α (TNFα) gene and protein expression largely attributable to BMDM. To investigate the contribution of TNFα in the synergy of the co-culture environment, C2C12 myotubes were treated with recombinant TNFα, co-cultures were established using TNF-deficient BMDM, and co-cultures were also depleted of TNFα using antibodies. To determine whether the in vitro observations were relevant in vivo, mice received intramuscular administration of LPS ± TNFα or TNFα-neutralizing antibodies and showed that TNFα is both sufficient and necessary to induce synergistic cytokine release from muscle. Taken together, these data demonstrate how skeletal muscle tissue may contribute proinflammatory cytokines following acute endotoxin injury and the potential of leukocytes to augment this response via TNFα secretion., (© 2021 Federation of American Societies for Experimental Biology.)

Objectives: There is an incomplete understanding of the host humoral immune response to severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2, which underlies COVID-19, during acute infection. Host factors such as age and sex as well as the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor-binding domain of the CoV spike (RBD-S) protein mediates host cell binding and infection and is a major target for vaccine design to elicit neutralising antibodies., Methods: We assessed serum anti-SARS-CoV-2 RBD-S IgG, IgM and IgA antibodies by a two-step ELISA and neutralising antibodies in a cross-sectional study of hospitalised COVID-19 patients of varying disease severities. Anti-RBD-S IgG levels were also determined in asymptomatic seropositives., Results: We found equivalent levels of anti-RBD-S antibodies in male and female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti-RBD-S IgG, IgM and IgA responses were simultaneously induced within 10 days after onset, with anti-RBD-S IgG sustained over a 5-week period. Anti-RBD-S antibodies strongly correlated with neutralising activity. Lastly, anti-RBD-S IgG responses were higher in symptomatic COVID-19 patients during acute infection compared with asymptomatic seropositive donors., Conclusion: Our results suggest that anti-RBD-S IgG reflect functional immune responses to SARS-CoV-2, but do not completely explain age- and sex-related disparities in COVID-19 fatalities., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Many mouse models of allergic asthma exhibit eosinophil-predominant cellularity rather than the mixed-granulocytic cytology in steroid-unresponsive severe disease. Therefore, we sought to implement a novel mouse model of antigen-driven, mixed-granulocytic, severe allergic asthma to determine biomarkers of the disease process and potential therapeutic targets. C57BL/6J wild-type, interleukin-6 knockout (IL-6-/-), and IL-6 receptor knockout (IL-6R-/-), mice were injected with an emulsion of complete Freund's adjuvant and house dust mite antigen (CFA/HDM) on day 1 . Dexamethasone, a lymphocyte-depleting biological, or anti-IL-17A was administered during the intranasal HDM challenge on days 19-22 . On day 23 , the CFA/HDM model elicited mixed bronchoalveolar lavage (BAL) cellularity (typically 80% neutrophils and 10% eosinophils), airway hyperresponsiveness (AHR) to methacholine, diffusion impairment, lung damage, body weight loss, corticosteroid resistance, and elevated levels of serum amyloid A (SAA), pro-inflammatory cytokines, and T helper type 1/ T helper type 17 (Th1/Th17) cytokines compared with eosinophilic models of HDM-driven allergic airway disease. BAL cells in IL-6- or IL-6R-deficient mice were predominantly eosinophilic and associated with elevated T helper type 2 (Th2) and reduced Th1/Th17 cytokine production, along with an absence of SAA. Nevertheless, AHR remained in IL-6-deficient mice even when dexamethasone was administered. However, combined administration of anti-IL-17A and systemic corticosteroid significantly attenuated both overall and neutrophilic airway inflammation and also reduced AHR and body weight loss. Inhibition of IL-17A combined with systemic corticosteroid treatment during antigen-driven exacerbations may provide a novel therapeutic approach to prevent the pathological pulmonary and constitutional changes that greatly impact patients with the mixed-granulocytic endotype of severe asthma.

S-glutathionylation of reactive protein cysteines is a post-translational event that plays a critical role in transducing signals from oxidants into biological responses. S-glutathionylation can be reversed by the deglutathionylating enzyme glutaredoxin (GLRX). We have previously demonstrated that ablation of Glrx sensitizes mice to the development of parenchymal lung fibrosis(1). It remains unclear whether GLRX also controls airway fibrosis, a clinical feature relevant to asthma and chronic obstructive pulmonary disease, and whether GLRX controls the biology of airway epithelial cells, which have been implicated in the pathophysiology of these diseases. In the present study we utilized a house dust mite (HDM) model of allergic airway disease in wild type (WT) and Glrx -/- mice on a C57BL/6 background prone to develop airway fibrosis, and tracheal basal stem cells derived from WT mice, global Glrx -/- mice, or bi-transgenic mice allowing conditional ablation of the Glrx gene. Herein we show that absence of Glrx led to enhanced HDM-induced collagen deposition, elevated levels of transforming growth factor beta 1 (TGFB1) in the bronchoalveolar lavage, and resulted in increases in airway hyperresponsiveness. Airway epithelial cells isolated from Glrx -/- mice or following conditional ablation of Glrx showed spontaneous increases in secretion of TGFB1. Glrx -/- basal cells also showed spontaneous TGFB pathway activation, in association with increased expression of mesenchymal genes, including collagen 1a1 and fibronectin. Overall, these findings suggest that GLRX regulates airway fibrosis via a mechanism(s) that involve the plasticity of basal cells, the stem cells of the airways., (Published by Elsevier B.V.)

Background: Pharmacokinetics (PK) of pharmaceuticals and pharmaconutrients are poorly understood in critically ill patients, and dosing is often based on healthy subject data. This might be particularly problematic with enteral medications due to metabolic abnormalities and impaired gastrointestinal tract absorption common in critically ill patients. Utilizing enteral fish oil, this study was undertaken to better understand and define PK of enteral omega-3 fatty acids (eicospentaenoic acid [EPA] and docosahexaenoic acid [DHA]) in critically ill patients with severe sepsis., Materials and Methods: Healthy volunteers (n = 15) and mechanically ventilated (MV) adults with severe sepsis (n = 10) were recruited and received 9.75 g EPA and 6.75 g DHA daily in two divided enteral doses of fish oil for 7 days. Volunteers continued their normal diet without other sources of fish oil, and sepsis patients received standard enteral feeding. Blood was collected at frequent intervals during the 14-day study period. Peripheral blood mononuclear cells (PMBCs) and neutrophils were isolated and analyzed for membrane fatty acid (FA) content. Mixed linear models and t-tests were used to analyze changes in FA levels over time and FA levels at individual time points, respectively. PK parameters were obtained based on single compartment models of EPA and DHA kinetics., Results: Healthy volunteers were 41.1 ± 10.3 years; 67% were women. In patients with severe sepsis (55.6 ± 13.4 years, 50% women), acute physiologic and chronic health evaluation (APACHE) II score was 27.2 ± 8.8 at ICU admission and median MV duration was 10.5 days. Serum EPA and DHA were significantly lower in sepsis vs. healthy subjects over time. PBMC EPA concentrations were generally not different between groups over time, while PBMC DHA was higher in sepsis patients. Neutrophil EPA and DHA concentrations were similar between groups. The half-life of EPA in serum and neutrophils was significantly shorter in sepsis patients, whereas other half-life parameters did not vary significantly between healthy volunteers and sepsis patients., Conclusions: While incorporation of n-3 FAs into PBMC and neutrophil membranes was relatively similar between healthy volunteers and sepsis patients receiving identical high doses of fish oil for one week, serum EPA and DHA were significantly lower in sepsis patients. These findings imply that serum concentrations and EPA and DHA may not be the dominant driver of leukocyte membrane incorporation of EPA and DHA. Furthermore, lower serum EPA and DHA concentrations suggest that either these n-3 FAs were being metabolized rapidly in sepsis patients or that absorption of enteral medications and pharmaconutrients, including fish oil, may be impaired in sepsis patients. If enteral absorption is impaired, doses of enteral medications administered to critically ill patients may be suboptimal., (Copyright © 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Asthma is a chronic disorder characterized by inflammation, mucus metaplasia, airway remodeling, and hyperresponsiveness. We recently showed that IL-1-induced glycolytic reprogramming contributes to allergic airway disease using a murine house dust mite model. Moreover, levels of pyruvate kinase M2 (PKM2) were increased in this model as well as in nasal epithelial cells from asthmatics as compared with healthy controls. Although the tetramer form of PKM2 converts phosphoenolpyruvate to pyruvate, the dimeric form of PKM2 has alternative, nonglycolysis functions as a transcriptional coactivator to enhance the transcription of several proinflammatory cytokines. In the current study, we examined the impact of PKM2 on the pathogenesis of house dust mite-induced allergic airways disease in C57BL/6NJ mice. We report, in this study, that activation of PKM2, using the small molecule activator, TEPP46, augmented PKM activity in lung tissues and attenuated airway eosinophils, mucus metaplasia, and subepithelial collagen. TEPP46 attenuated IL-1β-mediated airway inflammation and expression of proinflammatory mediators. Exposure to TEPP46 strongly decreased the IL-1β-mediated increases in thymic stromal lymphopoietin (TSLP) and GM-CSF in primary tracheal epithelial cells isolated from C57BL/6NJ mice. We also demonstrate that IL-1β-mediated increases in nuclear phospho-STAT3 were decreased by TEPP46. Finally, STAT3 inhibition attenuated the IL-1β-induced release of TSLP and GM-CSF, suggesting that the ability of PKM2 to phosphorylate STAT3 contributes to its proinflammatory function. Collectively, these results demonstrate that the glycolysis-inactive form of PKM2 plays a crucial role in the pathogenesis of allergic airways disease by increasing IL-1β-induced proinflammatory signaling, in part, through phosphorylation of STAT3., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Dendritic cells (DCs) activated via TLR ligation experience metabolic reprogramming, in which the cells are heavily dependent on glucose and glycolysis for the synthesis of molecular building blocks essential for maturation, cytokine production, and the ability to stimulate T cells. Although the TLR-driven metabolic reprogramming events are well documented, fungal-mediated metabolic regulation via C-type lectin receptors such as Dectin-1 and Dectin-2 is not clearly understood. Here, we show that activation of DCs with fungal-associated β-glucan ligands induces acute glycolytic reprogramming that supports the production of IL-1β and its secretion subsequent to NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. This acute glycolytic induction in response to β-glucan ligands requires spleen tyrosine kinase signaling in a TLR-independent manner, suggesting now that different classes of innate immune receptors functionally induce conserved metabolic responses to support immune cell activation. These studies provide new insight into the complexities of metabolic regulation of DCs immune effector function regarding cellular activation associated with protection against fungal microbes., (©2019 The Authors. Society for Leukocyte Biology Published by Wiley Periodicals, Inc.)

Conjugated bile acids (CBAs), such as tauroursodeoxycholic acid (TUDCA), are known to resolve the inflammatory and unfolded protein response (UPR) in inflammatory diseases, such as asthma. Whether CBAs exert their beneficial effects on allergic airway responses via 1 arm or several arms of the UPR, or alternatively through the signaling pathways for conserved bile acid receptor, remains largely unknown. We used a house dust mite-induced (HDM-induced) murine model of asthma to evaluate and compare the effects of 5 CBAs and 1 unconjugated bile acid in attenuating allergen-induced UPR and airway responses. Expression of UPR-associated transcripts was assessed in airway brushings from human patients with asthma and healthy subjects. Here we show that CBAs, such as alanyl β-muricholic acid (AβM) and TUDCA, significantly decreased inflammatory, immune, and cytokine responses; mucus metaplasia; and airway hyperresponsiveness, as compared with other CBAs in a model of allergic airway disease. CBAs predominantly bind to activating transcription factor 6α (ATF6α) compared with the other canonical transducers of the UPR, subsequently decreasing allergen-induced UPR activation and resolving allergic airway disease, without significant activation of the bile acid receptors. TUDCA and AβM also attenuated other HDM-induced ER stress markers in the lungs of allergic mice. Quantitative mRNA analysis of airway epithelial brushings from human subjects demonstrated that several ATF6α-related transcripts were significantly upregulated in patients with asthma compared with healthy subjects. Collectively, these results demonstrate that CBA-based therapy potently inhibits the allergen-induced UPR and allergic airway disease in mice via preferential binding of the canonical transducer of the UPR, ATF6α. These results potentially suggest a novel avenue to treat allergic asthma using select CBAs.

Invariant NKT (iNKT) cells are tissue-resident innate-like T cells critical to the host immune response. We previously identified a 6.6 Mbp region on chromosome 1 as a major regulator of iNKT cell number and function in C57BL/6 and 129X1/SvJ mice. Here, we fine-mapped this locus by assessing the iNKT cell response to alpha-galactosylceramide (αGalCer) in a series of B6.129 congenic lines. This analysis revealed the presence of at least two genetic elements that regulate iNKT cell cytokine production in response to αGalCer. While one of these genetic elements mapped to the B6.129c6 interval containing Slam genes, the dominant regulator in this region mapped to the 0.14 Mbp B6.129c3 interval. In addition, we found that numbers of thymic iNKT cells and DP thymocytes were significantly lower in B6.129c3 mice, indicating that this interval also regulates iNKT cell development. Candidate gene analysis revealed a fivefold increase in Fcgr3 expression in B6.129c3 iNKT cells, and we observed increased expression of FcγR3 protein on B6.129c3 iNKT cells, NK cells, and neutrophils. These data identify the B6.129c3 interval as a novel locus regulating the response of iNKT cells to glycosphingolipid, revealing a link between this phenotype and a polymorphism that regulates Fcgr3 expression.

Serum amyloid A (SAA) proteins are a family of acute phase apolipoproteins implicated to directly modulate innate and adaptive immune responses. However, new studies comparing endogenous SAAs and recombinant forms of these proteins have questioned the function of SAA in inflammation and immunity. We generated SAA3 knockout mice to evaluate the contribution of SAA3 to lung development and immune-mediated lung disease. While SAA3 deficiency does not affect the generation of house dust mite-induced allergic asthma, mice lacking SAA3 develop adult-onset obesity, intrinsic airway hyperresponsiveness, increased inflammatory and fibrotic gene expression in the lung, and elevated levels of lung citrullinated proteins. Polyclonally stimulated CD4 + T cells from SAA3-/- mice exhibit impaired glycolytic activity, decreased T H 2 and T H 1 cytokine secretion, and elevated IL-17A production compared to wild type cells. Polyclonally stimulated CD8 + T cells from SAA3-/- mice also exhibit impaired glycolytic activity as well as a diminished capacity to produce IL-2 and IFNγ. Finally, SAA3-/- mice demonstrate increased mortality in response to H1N1 influenza infection, along with higher copy number of viral RNAs in the lung, a lack of CD8 + T cell IFNγ secretion, and decreased flu-specific antibodies. Our findings indicate that endogenous SAA3 regulates lung development and homeostasis, and is required for protection against H1N1 influenza infection.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli , hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli -derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell-derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma., Objectives: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma., Methods: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis., Results: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1β into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1β or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1β- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1β on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1β and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1β levels, and lactate content correlated negatively with lung function., Conclusions: Collectively, these findings demonstrate that IL-1β/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Asthma is a chronic respiratory condition, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. The hypothesis that the substantially increased expression of arginase 1 in activated macrophages limits the availability of L-arginine for nitric oxide synthesis, and thus increases AHR in lungs of mice with experimentally induced allergic asthma was recently refuted by several studies. In the present study, we tested the hypothesis that, instead, a low circulating concentration of arginine aggravates AHR in the same murine asthma model. Female FVB F/A2 tg/tg transgenic mice, which overexpress rat arginase 1 in their enterocytes, exhibit a ~ 50% decrease of their plasma L-arginine concentration., Methods: Adult female F/A2 tg/tg mice and their wild-type littermates (F/A2 wt/wt ) were sensitized and challenged with ovalbumin (OVA/OVA). Lung function was assessed with the flexiVent™ system. Adaptive changes in the expression of arginine-metabolizing or -transporting enzymes, chemokines and cytokines, and lung histology were quantified with qPCR, ELISA, and immunohistochemistry, respectively., Results: Reduction of circulating L-arginine concentration significantly increased AHR in OVA/OVA-treated mice and, to a lesser extent, even in PBS/OVA-treated mice. The pulmonary inflammatory response in OVA/OVA-treated F/A2 tg/tg and F/A2 wt/wt mice was comparable. OVA/OVA-treated F/A2 tg/tg mice differed from similarly treated female mice, in which arginase 1 expression in lung macrophages was eliminated, by a complete absence of an adaptive increase in the expression of arginine-metabolizing or -transporting enzymes., Conclusion: A reduction of the circulating L-arginine concentration rather than the macrophage-mediated increase of arginine catabolism worsens AHR.

Serum amyloid A (SAA) is an apolipoprotein that is robustly upregulated in numerous inflammatory diseases and has been implicated as a candidate pro-inflammatory mediator. However, studies comparing endogenous SAAs and recombinant forms of the acute phase protein have generated conflicting data on the function of SAA in immunity. We generated SAA3 knockout mice to evaluate the contribution of SAA3 to immune-mediated disease, and found that mice lacking SAA3 develop adult-onset obesity and metabolic dysfunction along with defects in innate immune development. Mice that lack SAA3 gain more weight, exhibit increased visceral adipose deposition, and develop hepatic steatosis compared to wild-type littermates. Leukocytes from the adipose tissue of SAA3-/- mice express a pro-inflammatory phenotype, and bone marrow derived dendritic cells from mice lacking SAA3 secrete increased levels of IL-1β, IL-6, IL-23, and TNFα in response to LPS compared to cells from wild-type mice. Finally, BMDC lacking SAA3 demonstrate an impaired endotoxin tolerance response and inhibited responses to retinoic acid. Our findings indicate that endogenous SAA3 modulates metabolic and immune homeostasis.

Background: (Over-)expression of arginase may limit local availability of arginine for nitric oxide synthesis. We investigated the significance of arginase1 (ARG1) for the development of airway hyperresponsiveness (AHR) and lung inflammation in female mice with ovalbumin (OVA)-induced allergic asthma., Methods: Arg1 was ablated in the lung by crossing Arg1 fl/fl and Tie2Cre tg/- mice. OVA sensitization and challenge were conducted, and AHR to methacholine was determined using the Flexivent system. Changes in gene expression, chemokine and cytokine secretion, plasma IgE, and lung histology were quantified using RT-qPCR, ELISA, and immunohistochemistry, respectively., Results: Arg1 ablation had no influence on the development of OVA-induced AHR, but attenuated OVA-induced increases in expression of Arg2 and Nos2, Slc7a1, Slc7a2, and Slc7a7 (arginine transporters), Il4, Il5 and Il13 (T H 2-type cytokines), Ccl2 and Ccl11 (chemokines), Ifng (T H 1-type cytokine), Clca3 and Muc5ac (goblet cell markers), and OVA-specific IgE. Pulmonary IL-10 protein content increased, but IL-4, IL-5, IL-13, TNFα and IFNγ content, and lung histopathology, were not affected. Arg1 elimination also decreased number and tightness of correlations between adaptive changes in lung function and inflammatory parameters in OVA/OVA-treated female mice. OVA/OVA-treated female mice mounted a higher OVA-IgE response than males, but the correlation between lung function and inflammation was lower. Arg1-deficient OVA/OVA-treated females differed from males in a more pronounced decline of arginine-metabolizing and -transporting genes, higher plasma arginine levels, a smaller OVA-specific IgE response, and no improvement of peripheral lung function., Conclusion: Complete ablation of Arg1 in the lung affects mRNA abundance of arginine-transporting and -metabolizing genes, and pro-inflammatory genes, but not methacholine responsiveness or accumulation of inflammatory cells.

Obesity affects numerous diseases, including asthma, for reasons that remain incompletely understood. Recent research suggests that the asthma of obesity is not a single disease, and that it breaks out into at least two distinct phenotypes. One phenotype is conventional allergic asthma modulated by obesity, whereas another arises solely due to the presence of obesity. The latter is postulated to be a consequence of the chronic lung compression caused by the obese chest wall in individuals with particularly collapsible lungs. Allergic obese asthma, on the other hand, appears to result from the way that obesity affects the immune system, which we hypothesize can be understood in terms of effects on the dynamic regulation of the inflammatory response.

iNKT cells and mast cells have both been implicated in the syndrome of allergic asthma through their activation-induced release of Th2 type cytokines and secretion of histamine and other mediators, respectively, which can promote airways hyperresponsiveness (AHR) to agents such as methacholine. However, a mechanistic link between iNKT cells and mast cell recruitment or activation has never been explored. Our objective was to determine whether iNKT cells are necessary for the recruitment of mast cells and if iNKT cells can influence the acute allergen induced bronchoconstriction (AIB) caused by mast cell mediator release. To do so, we pharmacologically eliminated iNKT cells using a specific antibody (NKT-14) and examined its impact on airway inflammation and physiological phenotype. In mice treated with NKT-14, the elimination of iNKT cells was sufficient to prevent AHR and pulmonary eosinophilic inflammation elicited by administration of the iNKT cell agonist αGalCer. In mice treated with NKT-14 and then sensitized and challenged with house dust mite extract (HDM), eliminating the iNKT cells significantly reduced both AHR and AIB but did not affect pulmonary inflammation, the mast cell population, nor the release of the mast cell mediators mast cell protease-1 and prostaglandin D2. We conclude that while iNKT cells contribute to the phenotype of allergic airways disease through the manifestation of AIB and AHR, their presence is not required for mast cell recruitment and activation, or to generate the characteristic inflammatory response subsequent to allergen challenge., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Alcohol use disorders are common both in the United States and globally, and are associated with a variety of co-morbid, inflammation-linked diseases. The pathogenesis of many of these ailments are driven by the activation of the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18. We hypothesized that protracted exposure of leukocytes to ethanol would amplify inflammasome activation, which would help to implicate mechanisms involved in diseases associated with both alcoholism and aberrant NLRP3 inflammasome activation. Here we show that long-term ethanol exposure of human peripheral blood mononuclear cells and a mouse macrophage cell line (J774) amplifies IL-1β secretion following stimulation with NLRP3 agonists, but not with AIM2 or NLRP1b agonists. The augmented NRLP3 activation was mediated by increases in iNOS expression and NO production, in conjunction with increases in mitochondrial membrane depolarization, oxygen consumption rate, and ROS generation in J774 cells chronically exposed to ethanol (CE cells), effects that could be inhibited by the iNOS inhibitor SEITU, the NO scavenger carboxy-PTIO, and the mitochondrial ROS scavenger MitoQ. Chronic ethanol exposure did not alter K + efflux or Zn 2+ homeostasis in CE cells, although it did result in a lower intracellular concentration of NAD + . Prolonged administration of acetaldehyde, the product of alcohol dehydrogenase (ADH) mediated metabolism of ethanol, mimicked chronic ethanol exposure, whereas ADH inhibition prevented ethanol-induced IL-1β hypersecretion. Together, these results indicate that increases in iNOS and mitochondrial ROS production are critical for chronic ethanol-induced IL-1β hypersecretion, and that protracted exposure to the products of ethanol metabolism are probable mediators of NLRP3 inflammasome hyperactivation., (Copyright © 2017. Published by Elsevier B.V.)

Males of many species, ranging from humans to insects, are more susceptible than females to parasitic, fungal, bacterial, and viral infections. One mechanism that has been proposed to account for this difference is the immunocompetence handicap model, which posits that the greater infectious disease burden in males is due to testosterone, which drives the development of secondary male sex characteristics at the expense of suppressing immunity. However, emerging data suggest that cell-intrinsic (chromosome X and Y) sex-specific factors also may contribute to the sex differences in infectious disease burden. Using a murine model of influenza A virus (IAV) infection and a panel of chromosome Y (ChrY) consomic strains on the C57BL/6J background, we present data showing that genetic variation in ChrY influences IAV pathogenesis in males. Specific ChrY variants increase susceptibility to IAV in males and augment pathogenic immune responses in the lung, including activation of proinflammatory IL-17-producing γδ T cells, without affecting viral replication. In addition, susceptibility to IAV segregates independent of copy number variation in multicopy ChrY gene families that influence susceptibility to other immunopathological phenotypes, including survival after infection with coxsackievirus B3. These results demonstrate a critical role for genetic variation in ChrY in regulating susceptibility to infectious disease.

Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite-induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management.

Background: Previous literature suggests that a higher ratio of palmitic acid (PA)/oleic acid (OA) in the diet induces inflammation, which may result in deficient brain insulin signaling, and, secondarily, impaired physical activity, sleep efficiency, and cognitive functioning., Objective: We hypothesized that lowering the typical dietary PA/OA would affect the activation of relevant brain networks during a working memory task and would also lower secretion of pro-inflammatory cytokines., Design: In 12 female subjects participating in a randomized, cross-over trial comparing 3-week high PA diet (HPA) and low PA and a high OA diet (HOA), we evaluated functional magnetic resonance imaging (fMRI) using an N-back test of working memory, cytokine secretion by lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC), and plasma cytokine concentrations., Results: Brain activation during the HPA diet compared to the HOA diet was increased in regions of the basal ganglia including the caudate and putamen (p<0.005). In addition, compared to the HOA diet, during the HPA diet, the plasma concentrations of IL-6 (p=0.04) and IL-1β (p=0.05) were higher, and there was a higher secretion of IL-18 (p=0.015) and a trend for higher IL-1β secretion (p=0.066) from LPS-stimulated PBMCs., Conclusions: The HPA diet resulted in increased brain activation in the basal ganglia compared to the HOA diet as well as increased secretion of pro-inflammatory cytokines. These data provide evidence that short-term (2week) diet interventions impact brain network activation during a working memory task and that these effects are reversible since the order of the study diets was randomized. These data are consistent with the hypothesis that lowering the dietary PA content via substitution with OA also could affect cognition., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sepsis is characterized by systemic inflammation, edema formation and hypo-perfusion leading to organ dysfunction and ultimately death. Activation of the transient receptor potential vanilloid type 4 (TRPV4) channel is associated with edema formation and circulatory collapse. Here, we show that TRPV4 channels are involved in the hyper-inflammatory response and mortality associated with sepsis. Pharmacological inhibition of TRPV4 channels in mice reduced mortality in lipopolysaccharide and cecal-ligation-and-puncture models of sepsis, but not in a tumor necrosis factor-α (TNFα)-induced sepsis model. These protective effects of TRPV4 channel inhibition were attributable to prevention of the sepsis-induced surge of a broad spectrum of pro-inflammatory cytokines, including TNFα, interleukin (IL)-1 and IL-6, and subsequent preservation of endothelial cell function, including Ca 2+ signaling, integrity and endothelium-dependent vasodilation. These results suggest that TRPV4 antagonists may be of therapeutic utility in the management of sepsis.