Lung Inflammation Research Articles

Downregulation of Mirlet7 miRNA family promotes Tc17 differentiation and emphysema via de-repression of RORγt

Environmental air irritants including nanosized carbon black (nCB) can drive systemic inflammation, promoting chronic obstructive pulmonary disease (COPD) and emphysema development. The let-7 microRNA (Mirlet7 miRNA) family is associated with IL-17-driven T cell inflammation, a canonical signature of lung inflammation. Recent evidence suggests the Mirlet7 family is downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated. Here, we show that overall expression of the Mirlet7 clusters, Mirlet7b/Mirlet7c2 and Mirlet7a1/Mirlet7f1/Mirlet7d, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)- or nCB-elicited emphysema. We demonstrate that loss of the Mirlet7b/Mirlet7c2 cluster in T cells predisposed mice to exaggerated CS- or nCB-elicited emphysema. Furthermore, ablation of the Mirlet7b/Mirlet7c2 cluster enhanced CD8+IL17a+ T cells (Tc17) formation in emphysema development in mice. Additionally, transgenic mice overexpressing Mirlet7g in T cells are resistant to Tc17 and CD4+IL17a+ T cells (Th17) development when exposed to nCB. Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of Mirlet7 in T cells. Overall, our findings shed light on the Mirlet7/RORγt axis with Mirlet7 acting as a molecular brake in the generation of Tc17 cells and suggest a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.

Systematic investigation of the material basis, effectiveness and safety of Thesium chinense Turcz. and its preparation Bairui Granules against lung inflammation

BackgroundThesium chinense Turcz. (Named as Bai Rui Cao in Chinese) and its preparations (e.g., Bairui Granules) have been used to treat inflammatory diseases, such as acute mastitis, lobar pneumonia, tonsillitis, coronavirus disease 2019 (COVID-19), and upper respiratory tract infection. However, the material basis, pharmacological efficiency, and safety have not been illustrated.MethodsAnti-inflammatory activity-guided isolation of constituents has been performed using multiple column chromatography, and their structures were elucidated by NMR spectroscopy and ECD calculations. The inhibitory effects on lung inflammation and safety of the crude ethanol extract (CE), Bairui Granules (BG), and the purified active constituents were evaluated using lipopolysaccharide (LPS)-stimulated acute lung inflammation (ALI) mice model or normal mice.ResultsSeven new compounds (1–7) and fifty-six known compounds (8–63) were isolated from T. chinense, and fifty-four were reported from this plant for the first time. The new flavonoid glycosides 1–2, new fatty acids 4–5, new alkaloid 7 as well as the known constituents including flavonoid aglycones 8–11, lignans 46–54, alkaloids 34 and 45, coumarins 57, phenylpropionic acids 27, and simple aromatic compounds 39, 44 and 58 exhibited anti-inflammatory activity. Network pharmacology analysis indicated that anti-inflammation of T. chinense was attributed to flavonoids and alkaloids by regulating inflammation-related proteins (e.g., TNF, NF-κB, TGF-β). Furthermore, constituents of T. chinense including kaempferol-3-O-glucorhamnoside (KN, also named as Bairuisu I, 19), astragalin (AG, Bairuisu II, 12), and kaempferol (KF, Bairuisu III, 8), as well as CE and BG could alleviate lung inflammation caused by LPS in mice by preventing neutrophils infiltration and the expression of the genes for pro-inflammatory cytokines NLRP3, caspase-1, IL-1β, and COX-2. After a 28-day subacute toxicity test, BG at doses of 4.875 g/kg and 9.750 g/kg (equivalent to onefold and twofold the clinically recommended dose) and CE at a dose of 11.138 g/kg (equivalent to fourfold the clinical dose of BG) were found to be safe and non-toxic.ConclusionsThe discovery of sixty-three constituents comprehensively illustrated the material basis of T. chinense. T. chinense and Bairui Granules could alleviate lung inflammation by regulating inflammation-related proteins and no toxicity was observed under the twofold of clinically used doses.

Prophylactic use of standardized extract of propolis of Apis mellifera (EPP-AF®) reduces lung inflammation and improves survival in experimental lethal sepsis

Ethnopharmacological relevanceSepsis poses one of the biggest public health problems, necessitating the search for new therapeutic alternatives. For centuries, propolis has been widely used in folk medicine to treat various inflammatory and infectious diseases. Given its extensive use, it has excellent potential as an adjuvant treatment for patients with sepsis. ObjectiveThis study evaluated prophylactic treatment with standardized propolis extract (EPP-AF®) and followed the prognosis of sepsis induced by ligation and cecal ligation and puncture (CLP). MethodsInitially, for survival assessment, Swiss mice were separated into five groups: Sham (false operated), control (PBS), ATB (received antibiotic, 8 mg/kg), P10 (received EPP-AF®, 10 mg/kg), and P100 (received EPP-AF®, 100 mg/kg). The animals received PBS, antibiotic, or EPP-AF® by the subcutaneous route 6 h before the CLP procedure. Animal survival was assessed every 12 h for five days when all of them were euthanized. ResultsWe show that the treatment with EPP-AF® significantly increased the life expectancy of animals with sepsis compared to the control group. Interestingly, prophylactic treatment with EPP-AF® showed no effect on the number of colony-forming units in the peritoneum, blood, or lung. However, there was a decrease in cellular influx in the peritoneum. This alteration was unrelated to the number of bone marrow cells or the differential counting of peripheral blood cells. The coagulogram remained unchanged, including the number of platelets and prothrombin time-activated partial thromboplastin time. However, the inflammatory infiltrate and bleeding in the lung tissue were lower in the animals that received EPP-AF®. ConclusionThus, it was possible to conclude that prophylactic treatment with EPP-AF® preserved the lung parenchyma, resulting in an increased lifespan of mice with sepsis. It can be a helpful adjuvant in prophylactic treatment with antibiotics in presurgical conditions.

Polyhexamethylene guanidine phosphate induces pyroptosis via reactive oxygen species-regulated mitochondrial dysfunction in bronchial epithelial cells

Pyroptosis is a form of programmed cell death characterized by gasdermin (GSDM)-mediated pore formation in the cell membrane, resulting in the release of pro-inflammatory cytokines and cellular lysis. Increasing evidence has shown that pyroptosis is responsible for the progression of various pulmonary disorders. The inhalation of polyhexamethylene guanidine (PHMG) causes severe lung inflammation and pulmonary toxicity; however, the underlying mechanisms are unknown. Therefore, in this study, we investigate the role of pyroptosis in PHMG-induced pulmonary toxicity. We exposed bronchial epithelial cells, BEAS-2B, to PHMG phosphate (PHMG-p) and evaluated cell death type, reactive oxygen species (ROS) levels, and relative expression levels of pyroptosis-related proteins. Our data revealed that PHMG-p reduced viability and induced morphological alterations in BEAS-2B cells. Exposure to PHMG-p induced excessive accumulation of mitochondrial ROS (mtROS) in BEAS-2B cells. PHMG-p activated caspase-dependent apoptosis as well as NLRP3/caspase-1/GSDMD-mediated- and caspase-3/GSDME-mediated pyroptosis through mitochondrial oxidative stress in BEAS-2B cells. Notably, PHMG-p reduced mitochondrial respiratory function and induced the translocation of Bax and cleaved GSDM into the mitochondria, leading to mitochondrial dysfunction. Our results enhanced our understanding of PHMG-p-induced lung toxicity by demonstrating that PHMG-p induces pyroptosis via mtROS-induced mitochondrial dysfunction in bronchial epithelial cells.

Antenatal creatine supplementation reduces persistent fetal lung inflammation and oxidative stress in an ovine model of chorioamnionitis.

Chorioamnionitis is a common antecedent of preterm birth and induces inflammation and oxidative stress in the fetal lungs. Reducing inflammation and oxidative stress in the fetal lungs may improve respiratory outcomes in preterm infants. Creatine is an organic acid with known anti-inflammatory and antioxidant properties. The objective of the study was to evaluate the efficacy of direct fetal creatine supplementation to reduce inflammation and oxidative stress in fetal lungs arising from an in utero proinflammatory stimulus. Fetal lambs (n = 51) were instrumented at 90 days gestation to receive a continuous infusion of creatine monohydrate (6 mg·kg-1·h-1) or saline for 17 days. Maternal chorioamnionitis was induced with intra-amniotic lipopolysaccharide (LPS; 1 mg, O55:H6) or saline 7 days before delivery at 110 days gestation. Tissue creatine content was assessed with capillary electrophoresis, and inflammatory markers were analyzed with Luminex Magpix and immunohistochemistry. Oxidative stress was measured as the level of protein thiol oxidation. The effects of LPS and creatine were analyzed using a two-way ANOVA. Fetal creatine supplementation increased lung creatine content by 149% (PCr<0.0001) and had no adverse effects on lung morphology. LPS-exposed groups showed increased levels of interleukin-8 in the bronchoalveolar lavage (PLPS<0.0001) and increased levels of CD45+ leukocytes (PLPS<0.0001) and MPO+ (PLPS<0.0001) cells in the lung parenchyma. Creatine supplementation significantly reduced the levels of CD45+ (PCr=0.045) and MPO+ cells (PCr=0.012) in the lungs and reduced thiol oxidation in plasma (PCr<0.01) and lung tissue (PCr=0.02). In conclusion, fetal creatine supplementation reduced markers of inflammation and oxidative stress in the fetal lungs arising from chorioamnionitis.NEW & NOTEWORTHY We evaluated the effect of antenatal creatine supplementation to reduce pulmonary inflammation and oxidative stress in the fetal lamb lungs arising from lipopolysaccharide (LPS)-induced chorioamnionitis. Fetal creatine supplementation increased lung creatine content and had no adverse effects on systemic fetal physiology and overall lung architecture. Importantly, fetuses that received creatine had significantly lower levels of inflammation and oxidative stress in the lungs, suggesting an anti-inflammatory and antioxidant benefit of creatine.

IRG1/ACOD1 Promotes Neutrophil Reverse Migration and Alleviates Local Inflammation.

Polymorphonuclear neutrophil (PMN) infiltration at inflammatory site plays a critical role in inflammation. PMN reverse migration (rM) describes the phenomenon that PMNs migrate away from inflammatory site back into the vasculature, and its role within inflammatory scenarios remains to be fully determined. This study aimed to investigate the mechanism underlying PMN rM and its role in inflammation. First, we demonstrated PMN rM in a mouse model of LPS-induced acute lung inflammation. By single-cell RNA sequencing (scRNA-seq), we demonstrated that reverse migrated (rM-ed) PMNs in blood expressed high level of immuneresponsive gene 1 (Irg1), the encoding gene of cis-aconitate decarboxylase (ACOD1). Using a mouse air pouch model, which enables us to directly track rM-ed PMNs in vivo, we detected higher expression of ACOD1 in the rM-ed PMNs in circulation. Furthermore, mice with Irg1 knockout exhibited decreased PMN rM and higher levels of inflammatory cytokine in inflammatory site. Mechanistically, we found that itaconate, the product of ACOD1 catalyzation, decreased PMN ICAM-1 expression at the inflammation site. Furthermore, inflammatory site showed a high level of shed CD11a, the ligand of ICAM-1. Neutralization of either ICAM-1 or CD11a leading to increased PMN rM. These findings suggest that the binding of ICAM-1 and shed CD11a serves as a retaining force to hold PMNs in the site of inflammation, and ACOD1-decreased PMN surface expression of ICAM-1 weakens the retaining force, so promoting PMNs to leave the inflammatory site. These results indicate a regulatory role of IRG1 in PMN rM and subsequent contributions to inflammation resolution.

Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling

Acute lung injury (ALI) is characterized by respiratory failure resulting from the disruption of the epithelial and endothelial barriers as well as immune system. In this study, we evaluated the therapeutic potential of airway epithelial cell-derived extracellular vesicles (EVs) in maintaining lung homeostasis. We isolated human bronchial epithelial cell-derived EVs (HBEC-EVs), which endogenously express various immune-related surface markers and investigated their immunomodulatory potential in ALI. In ALI cellular models, HBEC-EVs demonstrated immunosuppressive effects by reducing the secretion of proinflammatory cytokines in both THP-1 macrophages and HBECs. Mechanistically, these effects were partially ascribed to nine of the top 10 miRNAs enriched in HBEC-EVs, governing toll-like receptor-NF-κB signaling pathways. Proteomic analysis revealed the presence of proteins in HBEC-EVs involved in WNT and NF-κB signaling pathways, pivotal in inflammation regulation. ANXA1, a constituent of HBEC-EVs, interacts with formyl peptide receptor (FPR)2, eliciting anti-inflammatory responses by suppressing NF-κB signaling in inflamed epithelium, including type II alveolar epithelial cells. In a mouse model of ALI, intratracheal administration of HBEC-EVs reduced lung injury, inflammatory cell infiltration, and cytokine levels. Collectively, these findings suggest the therapeutic potential of HBEC-EVs, through their miRNAs and ANXA1 cargo, in mitigating lung injury and inflammation in ALI patients.

Ethanol-induced lung and cardiac right ventricular inflammation and remodeling underlie progression to pulmonary arterial hypertension.

Current research on ethanol-induced cardiovascular anomalies has focused on left ventricular (LV) function and blood pressure. To extend this area of research, we sought to determine whether ethanol-induced alterations in the structure and function of the right cardiac ventricle (RV) and pulmonary artery (PA) lead to pulmonary arterial hypertension (PAH). Two groups of male Sprague-Dawley rats received a balanced liquid diet containing 5% ethanol (w/v) or a pair-fed isocaloric liquid diet for 8 weeks. Weekly echocardiography was conducted to evaluate cardiopulmonary function, and lung and RV tissues were collected for exvivo histological and molecular studies. The ethanol-treated rats exhibited: (1) Elevated mean pulmonary arterial pressure and decreased pulmonary artery acceleration time/ejection time; (2) Pulmonary vascular remodeling comprising intrapulmonary artery medial layer thickening; and (3) RV hypertrophy along with increased RV/LV + septum, RV diameter, RV cardiomyocyte cross-sectional area, and LV mass/body weight ratio. These responses were associated with increased lung and RV pro-inflammatory markers, endothelin-1 (ET-1), TNF-α, and IL-6 levels and higher ET-1,ET-1 type A/B receptor ratio, and downregulation of the cytoprotective protein, bone morphogenetic protein receptor 2 (BMPR2), in the lungs. These findings show that moderate ethanol-induced cardiopulmonary changes underlie progression to PAH via an upregulated proinflammatory ET1-TNFα-IL6 pathway and suppression of the anti-inflammatory BMPR2.

Non-allergic eosinophilic inflammation and airway hyperresponsiveness induced by diesel engine exhaust through activating ILCs

Diesel engine exhaust (DEE) is associated with the development and exacerbation of asthma. Studies have shown that DEE can aggravate allergen-induced eosinophilic inflammation in lung. However, it remains not clear that whether DEE alone could initiate non-allergic eosinophilic inflammation and airway hyperresponsiveness (AHR) through innate lymphoid cells (ILCs) pathway. This study aims to investigate the airway inflammation and hyperresponsiveness and its relationship with ILC after DEE exposure. Non-sensitized BALB/c mice were exposed in the chamber of diesel exhaust or filtered air for 2, 4, and 6 weeks (4 h/day, 6 days/week). Anti-CD4 mAb or anti-Thy1.2 mAb was administered by intraperitoneal injection to inhibit CD4+T or ILCs respectively. AHR、airway inflammation and ILCs were assessed. DEE exposure induced significantly elevated level of neutrophils, eosinophils, collagen content at 4, 6 weeks. Importantly, the airway AHR was only significant in the 4weeks-DEE exposure group. No difference of the functional proportions of Th2 cells was found between exposure group and control group. The proportions of IL-5+ILC2, IL-17+ILC significantly increased in 2, 4weeks-DEE exposure group. After depletion of CD4+T cells, both the proportion of IL-5+ILC2 and IL-17A ILCs was higher in the 4weeks-DEE exposure group which induced AHR, neutrophilic and eosinophilic inflammation accompanied by the IL-5, IL-17A levels. Diesel engine exhaust alone can imitate asthmatic characteristics in mice model. Lung-resident ILCs are one of the major effectors cells responsible for a mixed Th2/Th17 response and AHR.

17β-estradiol and methylprednisolone association as a therapeutic option to modulate lung inflammation in brain-dead female rats.

Brain death (BD) is known to compromise graft quality by causing hemodynamic, metabolic, and hormonal changes. The abrupt reduction of female sex hormones after BD was associated with increased lung inflammation. The use of both corticoids and estradiol independently has presented positive results in modulating BD-induced inflammatory response. However, studies have shown that for females the presence of both estrogen and corticoids is necessary to ensure adequate immune response. In that sense, this study aims to investigate how the association of methylprednisolone (MP) and estradiol (E2) could modulate the lung inflammation triggered by BD in female rats. Female Wistar rats (8 weeks) were divided into four groups: sham (animals submitted to the surgical process, without induction of BD), BD (animals submitted to BD), MP/E2 (animals submitted to BD that received MP and E2 treatment 3h after BD induction) and MP (animals submitted to BD that received MP treatment 3h after BD induction). Hemodynamics, systemic and local quantification of IL-6, IL-1β, VEGF, and TNF-α, leukocyte infiltration to the lung parenchyma and airways, and adhesion molecule expression were analyzed. After treatment, MP/E2 association was able to reinstate mean arterial pressure to levels close to Sham animals (p<0.05). BD increased leukocyte infiltration to the airways and MP/E2 was able to reduce the number of cells (p=0.0139). Also, the associated treatment modulated the vasculature by reducing the expression of VEGF (p=0.0616) and maintaining eNOS levels (p=0.004) in lung tissue. Data presented in this study show that the association between corticoids and estradiol could represent a better treatment strategy for lung inflammation in the female BD donor by presenting a positive effect in the hemodynamic management of the donor, as well as by reducing infiltrated leukocyte to the airways and release of inflammatory markers in the short and long term.

Regnase-1 D141N mutation induces CD4+ T cell-mediated lung granuloma formation via upregulation of Pim2.

Regnase-1 is an RNase that plays a critical role in negatively regulating immune responses by destabilizing inflammatory mRNAs. Dysfunction of Regnase-1 can be a major cause of various inflammatory diseases with tissue injury and immune cell infiltration into organs. This study focuses on the role of RNase activity of Regnase-1 in developing inflammatory diseases. We have constructed mice with a single point mutation at the catalytic center of Regnase-1 RNase domain, which lacks endonuclease activity. D141N mutant mice demonstrated systemic inflammation, immune cell infiltration into various organs and progressive development of lung granuloma. CD4+ T cells, mainly affected by this mutation, upregulated mTORC1 pathway and facilitated the autoimmune trait in D141N mutation. Moreover, serine/threonine kinase Pim2 contributed to lung inflammation in this mutation. Inhibition of Pim2 kinase activity ameliorated granulomatous inflammation, immune cell infiltration and proliferation in the lungs. Additionally, Pim2 inhibition reduced the expression of adhesion molecules on CD4+ T cells, suggesting a role for Pim2 in facilitating leukocyte adhesion and migration to inflamed tissues. Our findings provide new insights into the role of Regnase-1 RNase activity in controlling immune function and underscore the therapeutic relevance of targeting Pim2 to modulate abnormal immune responses.

CD8 T cells exert a critical role in the transition from left heart failure to lung remodeling and right ventricular hypertrophy

Chronic left ventricular (LV) failure or heart failure (HF) often progress to pulmonary remodeling and right ventricular (RV) hypertrophy even with optimal medical care. Cytotoxic CD8+ T cells play an important role in modulating virus infection and tissue injury by producing proinflammatory cytokines and cytolysis of the infected or injured cells. However, the role of CD8+ T cells in heart failure (HF)-induced lung remodeling and RV hypertrophy is unknown. To determine the role of CD8 T cells in modulating HF progression under preexisting HF conditions, wild type mice with existing LV failure produced by transverse aortic constriction (TAC) were randomized to depletion of cytotoxic CD8+ T cells, regulatory T cells (Tregs), or both by using specific blocking antibodies. The cardiac function, lung inflammation, fibrosis, vascular remodeling, and right ventricular remodeling were determined. As anticipated, LV failure caused lung inflammation and activation of pulmonary CD4+ T cells and CD8+ T cells. Interestingly, depletion of CD8+ T cells dramatically attenuated the increase of lung weight, lung inflammation, lung vascular remodeling, and RV hypertrophy in mice with existing LV failure. LV failure was associated with an increased ratio of activated T cells to Tregs, and Treg depletion exacerbated lung inflammation and the progression of LV failure. Treg depletion also exacerbated the lung CD4+ and CD8+ T cell infiltration, and the percentage of effector memory T cells (Tem) in HF mice. Depletion of CD8+ T cells was suffcient to rescued HF mice from the exacerbated lung inflammation and the progression of LV failure that was caused by Treg depletion. These findings demonstrate that CD8+ T cells play a critical role in promoting the transition from LV failure to lung remodeling and RV hypertrophy. NIH funding. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Generation of a lethal mouse model expressing human ACE2 and TMPRSS2 for SARS-CoV-2 infection and pathogenesis

Mouse models expressing human ACE2 for coronavirus disease 2019 have been frequently used to understand its pathogenesis and develop therapeutic strategies against SARS-CoV-2. Given that human TMPRSS2 supports viral entry, replication, and pathogenesis, we established a double-transgenic mouse model expressing both human ACE2 and TMPRSS2 for SARS-CoV-2 infection. Co-overexpression of both genes increased viral infectivity in vitro and in vivo. Double-transgenic mice showed significant body weight loss, clinical disease symptoms, acute lung injury, lung inflammation, and lethality in response to viral infection, indicating that they were highly susceptible to SARS-CoV-2. Pretreatment with the TMPRSS2 inhibitor, nafamostat, effectively reduced virus-induced weight loss, viral replication, and mortality in the double-transgenic mice. Moreover, the susceptibility and differential pathogenesis of SARS-CoV-2 variants were demonstrated in this animal model. Together, our results demonstrate that double-transgenic mice could provide a highly susceptible mouse model for viral infection to understand SARS-CoV-2 pathogenesis and evaluate antiviral therapeutics against coronavirus disease 2019.

GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment

Background aimsExtracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. MethodshUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. ResultshUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. ConclusionshUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.

L-citrulline Mitigates Systemic Inflammation and Associated Pulmonary Vascular Remodeling in a Neonatal Rat Model of Postnatal Undernutrition

Background &amp; Aim: Preterm infants with postnatal growth restriction and undernutrition are at increased risk of developing bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), metabolic syndrome, and sepsis. Preclinical and clinical studies report increased systemic inflammation, and lung and pancreatic histopathology in growth-restricted neonates. Safe therapies to mitigate these adverse outcomes associated with postnatal growth restriction and undernutrition in preterm infants are needed. L-citrulline is a non-essential amino acid, involved in the urea and nitric oxide cycles, that exhibits anti-inflammatory properties. This study aims to investigate whether L-citrulline may decrease systemic inflammation and improve histopathology in the lung, heart, and pancreas in a neonatal model of postnatal undernutrition. Methods: Neonatal Sprague Dawley rat pups were divided into 2 groups, normal nutrition (litter size n=12) and undernutrition (litter size n=18) by altering the litter size of the nursing dam. Body weight was measured daily and both groups received daily intraperitoneal injections of saline or L-citrulline (2.5g/kg) for 21 days. On postnatal day 21, heart, lung, and pancreatic tissue were collected for histology and mRNA analyses. Lung and pancreatic tissue were processed for qPCR to detect mRNA expression of pro-inflammatory mediators (MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α). Right ventricular hypertrophy was calculated using Fulton’s index (weight of the right ventricle divided by the combined weight of the left ventricle and the intraventricular septum). Lung sections were stained with Movat Pentachrome and examined for the presence of immune cells and medial wall thickening of pulmonary arteries. In a separate group of animals, bronchoalveolar lavage fluid (BALF) and blood obtained by cardiac puncture were cytocentrifuged and stained with hematoxylin &amp; eosin for immune cell counts. Results: Undernourished rats had a lower body weight than normally nourished rats at postnatal day 21 (n=11-17; p&lt;0.0001). Undernourished animals had an increased number of immune cells in BALF and lung tissue and an elevated neutrophil count in the blood which were all decreased with concurrent L-citrulline treatment (n=3-4; p&lt;0.001). L-citrulline treatment also prevented the increased mRNA expression of pro-inflammatory mediators in the lungs [MCP-1/CCL2, TNF-α, and IL-1β] (n=8; p&lt;0.05) and pancreas [MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α] (n=3; p&lt;0.05) of undernourished animals. These animals had a greater pulmonary artery medial wall area and Fulton’s index when compared to normally nourished animals treated with saline or L-citrulline (n=3-4; p&lt;0.001). Concurrent L-citrulline treatment in undernourished animals prevented pulmonary artery vascular remodeling and heart tissue histopathology such that the medial wall area and Fulton’s index did not differ from that of animals in control groups (n=4; p&lt;0.01). Conclusion: L-citrulline effectively mitigates the adverse effects of postnatal undernutrition, reducing lung, pancreatic, and systemic inflammation and histopathology in the lung and heart associated with PH. As a safe, well-tolerated, and inexpensive treatment, L-citrulline presents promising therapeutic potential that could prevent short and long-term morbidities associated with growth restriction and undernutrition in preterm infants. This work was funded by the Women's Auxiliary of The Hospital for Sick Children. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Research hotspots and evolving trends of barrier dysfunction in acute lung injury and acute respiratory distress syndrome

Endothelial and epithelial barrier dysfunction due to increased permeability and heightened inflammatory reactions influences the emergence of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Nevertheless, bibliometric research comparing endothelial and epithelial barriers is limited. Therefore, this bibliometric study analyzed the Web of Science Core Collection (WoSCC) of the Science Citation Index Expanded literature to explore present research priorities and development tendencies within this field. We conducted a comprehensive search (October 18, 2023) on WoSCC from January 1, 2010, to October 18, 2023, focusing on articles related to endothelial and epithelial barriers in ALI and ARDS. Retrieved data were visualized and analyzed using R-bibliometrix, VOS viewer 1.6.19, and CiteSpace 6.2. R4. Functional enrichment analysis of gene targets identified in the keyword list using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene ontology databases, and based on the STRING database to construct a PPI network to predict core genes. A total of 941 original articles and reviews were identified. The United States had the highest number of publications and citations and the highest H-index and G-index. According to the Collaboration Network Analysis graph, the United States and China had the strongest collaboration. Birukova AA had the most publications and citations among all authors, while eight of the top ten institutions with mediator centrality were located in the United States. The American Journal of Physiology-Lung Cellular and Molecular Physiology was the leading journal and had the most well-established publication on endothelial and epithelial barriers in ALI and ARDS. Bibliometric analysis revealed that the most frequently used keywords were acute lung injury, ARDS, activation, expression, and inflammation. RHOA appeared most frequently among gene-related keywords, and the PI3K-AKT signaling pathway had the highest count in KEGG pathway enrichment. Research on endothelial versus epithelial barriers in ALI and ARDS remains preliminary. This bibliometric study examined cooperative network connections among countries, authors, journals, and network associations in the cited references. Investigation of the functions of the endothelial and epithelial barriers in ALI/ARDS associated with COVID-19 has recently gained significant attention.

Homeobox B4 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on endotoxin-associated acute lung injury in rats

BackgroundAlveolar capillary endothelial cell (EC) injury has a pivotal role in driving acute respiratory distress syndrome (ARDS) progression and maintaining endothelial homeostasis. A previous ex vivo study revealed that overexpression of homeobox B4 (HOXB4) in bone marrow mesenchymal stem cells (BMSCs) enhanced protection against lipopolysaccharide (LPS)-induced EC injury by activating the Wnt/β-catenin pathway. This in vivo study was performed to verify whether BMSCs overexpressing HOXB4 exert similar protective effects on LPS-induced acute lung injury (ALI) in an animal model. MethodsThe ALI rat model was established by intraperitoneal injection of LPS. Wildtype BMSCs or BMSCs overexpressing HOXB4 were then injected via the tail vein. The lung characteristics of rats were visualized by computed tomography. Lung histopathological characteristics and collagen deposition were assessed by hematoxylin-eosin and Masson's staining, respectively, which were combined with the lung wet/dry ratio and proinflammatory factor levels in bronchoalveolar lavage fluid to further evaluate therapeutic effects. Expression of β-catenin and VE-cadherin was assessed by western blotting and immunofluorescence. ResultsCompared with wildtype BMSCs, overexpression of HOXB4 optimized the therapeutic effects of BMSCs, which manifested as improvements in lung exudation and histopathological features, reduced lung collagen deposition, amelioration of lung permeability, attenuation of lung inflammation, and enhanced expression of β-catenin and VE-cadherin proteins. ConclusionsHOXB4-overexpressing BMSCs optimized the protective effect against LPS-induced ALI by partially activating Wnt/β-catenin signaling.

Cardiac safety of ozanimod, a novel sphingosine-1-phosphate receptor ligand, in COVID-19 patients requiring oxygen

BackgroundOzanimod is a novel immune modulator that could be useful in viral pulmonary infections by reducing lung inflammation. It’s an S1P receptor ligand known to induce bradycardia and more serious adverse cardiac effects such as atrioventricular block (AVB) and QT prolongation. We present a sub-study of the COZI trial where ozanimod was administered in acute pulmonary infection patients, to assess cardiac safety. MethodsIn this pilot randomized open label trial, COVID-19 patients requiring oxygen support were randomized in 2 groups: standard of care + ozanimod (OZA) versus standard of care alone (SOC). All patients were monitored with a 14-day ECG monitor (CardioSTAT®) during their hospitalization. We evaluated the cardiac effects of ozanimod on heart rate (HR), PR length, and QT duration. ResultsA total of 42 patients were analyzed: 23 in SOC and 19 in OZA. Mean hourly HR over the first 10 days of treatment decrease in OZA compared with SOC (p<0.0001). The maximum decrease in HR occurred on day 3, The maximum decrease in heart rate occurred on day 3, without significant difference between groups: 49 bpm (42-59) in OZA and 54 bpm (48-60) in SOC, p=0.45. No high degree atrioventricular block was recorded. QT and PR median values were within normal in both groups without significant difference. ConclusionThe maximal reduction in HR occurred 3 days after the onset of ozanimod treatment in patients hospitalized for COVID-19 but it did not remain significant over the 10-day treatment. No relevant cardiac adverse event was observed.

Inflammatory lung injury is associated with endothelial cell mitochondrial fission and requires the nitration of RhoA and cytoskeletal remodeling

Higher levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a TLR4 agonist, are associated with poor clinical outcomes in sepsis-induced acute lung injury (ALI). Little is known regarding the mechanisms by which eNAMPT is involved in ALI. Our recent work has identified a crucial role for mitochondrial dysfunction in ALI. Thus, this study aimed to determine if eNAMPT-mediated inflammatory injury is associated with the loss of mitochondrial function. Our data show that eNAMPT disrupted mitochondrial bioenergetics. This was associated with cytoskeleton remodeling and the loss of endothelial barrier integrity. These changes were associated with enhanced mitochondrial fission and blocked when Rho-kinase (ROCK) was inhibited. The increases in mitochondrial fission were also associated with the nitration-mediated activation of the small GTPase activator of ROCK, RhoA. Blocking RhoA nitration decreased eNAMPT-mediated mitochondrial fission and endothelial barrier dysfunction. The increase in fission was linked to a RhoA-ROCK mediated increase in Drp1 (dynamin-related protein 1) at serine(S)616. Another TLR4 agonist, lipopolysaccharide (LPS), also increased mitochondrial fission in a Drp1 and RhoA-ROCK-dependent manner. To validate our findings in vivo, we challenged C57BL/6 mice with eNAMPT in the presence and absence of the Drp1 inhibitor, Mdivi-1. Mdivi-1 treatment protected against eNAMPT-induced lung inflammation, edema, and lung injury. These studies demonstrate that mitochondrial fission-dependent disruption of mitochondrial function is essential in TLR4-mediated inflammatory lung injury and identify a key role for RhoA-ROCK signaling. Reducing mitochondrial fission could be a potential therapeutic strategy to improve ARDS outcomes.

Conditional epithelial sodium channel (ENaC) over-expression in C57Bl6 mouse lung triggers innate immune responses via activation of high mobility group box 1 (HMGB1) signaling

Background: One of the more sinister examples of a lung disease borne by chronic inflammation is cystic fibrosis (CF). Inherited mutations in the CFTR protein hinders the secretion of Cl−, depolarizing the membrane potential and enabling excessive Na+ flux across epithelial sodium channels (ENaCs). The mechanism by which these electrochemical imbalances contribute to chronic inflammation are unclear. Based on our preliminary observations, we hypothesize that hyperactive ENaC dysfunction plays a pivotal role in initiating sterile inflammation via activation of HMGB1 cytokine activity. Our research group has shown that high HMGB1 levels in CF sputum predicts incidence and recurrence of future acute pulmonary exacerbation. Methods: In support of this new line of investigation, we generated a conditional lung-ENaC overexpression model ( Dox-Scnn1b), which uniquely allows us to control the timing (and thus severity) of ENaC hyperactive dysfunction in C57Bl6 mice. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected from WT and Dox-Scnn1b mice (± 625 mg/kg dox chow) in order to evaluate changes in transcript, protein, immune cell differentials, and lung injury following conditional lung ENaC overexpression. Student’s t-tests; ANOVA followed by post-hoc testing; and multivariate linear regression analysis (SigmaPlot 14.0 Systat Software) were used to evaluate statistical significance and determine whether ENaC feedforward signaling to HMGB1 effects pro-inflammatory signaling in lung epithelia. Results: qRT-PCR indicates that high dox chow (625 mg/kg) increased Scnn1b gene expression (encoding for β-ENaC subunit responsible for hyperactive channel dysfunction) by three-log fold over control fed mice, (n=6; p=0.01). Indeed, increase in Scnn1b transcript corresponded with significant increase in β-ENaC protein as detected by western blot analysis and amiloride sensitive current measurements. Importantly, Scnn1b overexpression correlated positively with increased HMGB1 protein in the lung (by 1.5 fold, p=.01 vs control; n= 6 from 2 independent studies) and % neutrophil migration. Dox induction of ENaC and HMGB1 also resulted in increased lung injury scores (from 0.24±0.1 to 0.45±0.06, n=3; p&lt;.01). Conclusions: Conditional lung ENaC overexpression mediates HMGB1 cytokine activity and closely recapitulates the sterile lung injury phenotype observed in CF patients. Significance: The mechanisms identified through this study can be targeted for new drug therapy, which is highly significant, since there are no effective therapies currently available for reversing lung inflammation. Funding: T35DK103596 (MZ) and R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.