Lung Neutrophil Infiltration Research Articles

Progranulin derivative attenuates lung neutrophilic infiltration from diesel exhaust particle exposure.

Air pollutants, such as diesel exhaust particles (DEPs), induce respiratory disease exacerbation with neutrophilic infiltration. Progranulin (PGRN), an epithelial cell and macrophage-derived secretory protein, is associated with neutrophilic inflammation. PGRN is digested into various derivatives at inflammatory sites and is involved in several inflammatory processes. PGRN and its derivatives likely regulate responses to DEP exposure in allergic airway inflammation. To investigate the role of PGRN and its derivatives in the regulation of responses to DEP exposure in allergic airway inflammation. A murine model of allergic airway inflammation was generated in PGRN-deficient mice, and they were simultaneously exposed to DEP followed by intranasal administration of full-length recombinant PGRN (PGRN-FL) and a PGRN-derived fragment (FBAC). Inflammatory status was evaluated by bronchoalveolar lavage fluid and histopathologic analyses. Human bronchial epithelial cells were stimulated with DEPs and house dust mites (HDMs), and the effect of FBAC treatment was evaluated by assessing various intracellular signaling molecules, autophagy markers, inflammatory cytokines, and intracellular oxidative stress. DEP exposure exaggerated neutrophilic inflammation, enhanced IL-6 and CXCL15 secretions, and increased oxidative stress in the murine model; this effect was greater in PGRN-deficient mice than in wild-type mice. The DEP-exposed mice with PGRN-FL treatment revealed no change in neutrophil infiltration and higher oxidative stress status in the lungs. On the contrary, FBAC administration inhibited neutrophilic infiltration and reduced oxidative stress. In human bronchial epithelial cells, DEP and HDM exposure increased intracellular oxidative stress and IL-6 and IL-8 secretion. Decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and increased phosphor-p62 and LC3B expression were also observed. FBAC treatment attenuated oxidative stress from DEP and HDM exposure. FBAC reduced neutrophilic inflammation exaggerated by DEP exposure in a mouse model of allergic airway inflammation by reducing oxidative stress. PGRN and PGRN-derived proteins may be novel therapeutic agents in attenuating asthma exacerbation induced by air pollutant exposure.

The STAT3 inhibitor B9 alleviates lipopolysaccharide-induced acute lung injury through its anti-inflammatory effects

The development of acute lung injury (ALI), a common respiratory condition with multiple causes, is significantly influenced by the pro-inflammatory environment of signal transducer and activator of transcription 3 (STAT3) in macrophages. Our study aimed to evaluate the anti-inflammatory effects of B9 (N-(4-hydroxyphenyl)-9, 10-dioxo-9, 10-dihydroanthracene-2-sulfonamide), a novel inhibitor targeting the STAT3 SH2 domain, in macrophages and to assess its therapeutic potential for ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI. We found that B9 (30 mg/kg) significantly reduced lung pathological damage and neutrophil infiltration caused by the intratracheal administration of LPS. Additionally, the high expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in alveolar lavage fluid was also inhibited by B9 treatment. The decreased expression of CD86 and increased CD206 in lung tissue demonstrated the anti-inflammatory effect of B9, which was due to its inhibition of the STAT3 signaling pathway in macrophages of ALI mice. Furthermore, B9 suppressed the activation of RAW264.7 cells induced by LPS, characterized by its ability to inhibit the activation of iNOS and STAT3 in a dose-dependent manner, as well as reduce the secretion of IL-6 and IL-1β. The in vivo preliminary safety evaluation indicated that B9 had a favorable safety profile at the administered doses. These results suggest that B9 exerts a therapeutic effect on LPS-induced ALI, potentially by preventing the phosphorylation of STAT3 Y705 and S727 without affecting the STAT3 protein level. Taken together, these findings provide a foundation for developing B9 as a novel anti-inflammatory agent for ameliorating LPS-induced ALI.

Overexpression of PIK3CG in Cancer Cells Promotes Lung Cancer Cell Migration and Metastasis Through Enhanced MMPs Expression and Neutrophil Recruitment and Activation.

Metastasis is a major cause of death in lung cancer. The aim of this study is to analyze the role and mechanism of PI3K catalytic subunit gamma (PIK3CG, also known as p110γ) in lung cancer cell migration and metastasis. Knockdown (KD) and overexpression (OE) of PIK3CG expression in lung cancer cell lines A549 and H1299 in vitro cultured was achieved. Two PIK3CG-specific inhibitors, Eganelisib and CAY10505, were used to treat A549 and H1299 cells. An experimental lung metastasis mouse model was constructed using tail vein injection of LLC cells. Finally, a co-culture system was established using Transwell chambers. Compared with the NC group, the number of cells that completed migration and the expression levels of matrix metalloproteinases (MMPs) were significantly reduced in the KD group and Eganelisib and CAY10505 treatment groups, while the number of cells that migrated successfully and the expression levels of MMPs were significantly increased in the OE group. Lung tissues of mice injected with PIK3CG-stabilized overexpressed LLC cells showed more pronounced lung cancer growth, lung metastatic nodules, neutrophil infiltration and MMPs expression. Co-culture with neutrophils, soluble extracts of neutrophils and cathepsin G all promoted the migration of lung cancer cells. PIK3CG overexpression in tumor cells significantly promoted the migration and metastasis of lung cancer cell.

UNVEILING THE PROTECTIVE MECHANISMS OF PUERARIN AGAINST ACUTE LUNG INJURY: A COMPREHENSIVE EXPLORATION OF THE ROLES AND MECHANISMS OF MST1/ERS SIGNALING.

Objectives: Puerarin, the principal active constituent extracted from Pueraria, is believed to confer protection against sepsis-induced lung injury. The study aimed to elucidate the role and mechanism of Mst1/ERS in puerarin-mediated protection against acute lung injury (ALI). Methods: Monolayer vascular endothelial cell permeability was assessed by gauging the paracellular flow of FITC-dextran 40,000 (FD40). ELISA was employed for the quantification of inflammatory cytokines. Identification of target proteins was conducted through western blotting. Histological alterations and apoptosis were scrutinized using hematoxylin-eosin staining and TUNEL staining, respectively. The ultrastructure of the endoplasmic reticulum was observed via transmission electron microscopy. Results: Puerarin significantly protected mice from LPS-induced ALI, reducing lung interstitial width, neutrophil and lymphocyte infiltration, pulmonary interstitial and alveolar edema, and lung apoptosis. Puerarin treatment also markedly attenuated levels of TNF-α and IL-1β in both alveolar lavage fluid and serum. Furthermore, puerarin significantly attenuated LPS-induced increases in Mst1, GRP78, CHOP, and Caspase12 protein expression and blunted LPS-induced decrease in ZO-1 protein expression in lung tissues. Puerarin obviously reduced endoplasmic reticulum expansion and vesiculation. Similarly, puerarin significantly mitigated the LPS-induced reduction in HUVEC cell viability and ZO-1 expression. Puerarin also attenuated LPS-induced increase in apoptosis, TNF-α and IL-1β, FD40 flux, and Mst1, GRP78, CHOP, and Caspase12 expression in HUVEC cells. Nevertheless, the inhibitory impact of puerarin on vascular endothelial cell injury, lung injury, and endoplasmic reticulum stress (ERS) was diminished by Mst1 overexpression. Conclusion: These findings demonstrated that the Mst1/ERS signaling pathway played a pivotal role in the development of LPS-induced vascular endothelial cell dysfunction and ALI. Puerarin exhibited the ability to attenuate LPS-induced vascular endothelial cell dysfunction and ALI by inhibiting the Mst1/ERS signaling pathway.

A NOVEL OLIGONUCLEOTIDE MRNA MIMIC ATTENUATES HEMORRHAGE-INDUCED ACUTE LUNG INJURY.

Hemorrhagic shock (HS) is accompanied by a pronounced activation of the inflammatory response in which acute lung injury (ALI) is one of the most frequent consequences. Among the pivotal orchestrators of this inflammatory cascade, extracellular cold-inducible RNA-binding protein (eCIRP) emerges as a noteworthy focal point, rendering it as a promising target for the management of inflammation and tissue injury. Recently, we have reported that oligonucleotide poly(A) mRNA mimic termed A 12 selectively binds to the RNA binding region of eCIRP and inhibits eCIRP binding to its receptor TLR4. Furthermore, in vivo administration of eCIRP induces lung injury in healthy mice and that mouse deficient in CIRP showed protection from inflammation-associated lung injury. We hypothesize that A 12 inhibits systemic inflammation and ALI in HS. To test the impacts of A 12 on systemic and lung inflammation, extent of inflammatory cellular infiltration and resultant lung damage were evaluated in a mouse model of HS. Male mice were subjected to controlled hemorrhage with a mean arterial pressure of 30 mm Hg for 90 min and then resuscitated with Ringer's lactate solution containing phosphate-buffered saline (vehicle) or A 12 at a dose of 4 nmol/g body weight (treatment). The infusion volume was twice that of the shed blood. At 4 h after resuscitation, mice were euthanized, and blood and lung tissues were harvested. Blood and tissue markers of inflammation and injury were evaluated. Serum markers of injury (lactate dehydrogenase, alanine transaminase, and blood urea nitrogen) and inflammation (TNF-α, IL-6) were increased after HS and A 12 treatment significantly decreased their levels. A 12 treatment also decreased lung levels of TNF-α, MIP-2, and KC mRNA expressions. Lung histological injury score, neutrophil infiltration (Ly6G staining and myeloperoxidase activity), and lung apoptosis were significantly attenuated after A 12 treatment. Our study suggests that the capacity of A 12 in attenuating HS-induced ALI and may provide novel perspectives in developing efficacious pharmaceutics for improving hemorrhage prognosis.

Resveratrol modulates the Nrf2/NF-κB pathway and inhibits TSLP-mediated atopic march.

Resveratrol has been found to have anti-inflammatory and anti-allergic properties. The effects of resveratrol on thymic stromal lymphopoietin (TSLP)-mediated atopic march remain unclear. To explore the potential role of resveratrol in TSLP-mediated atopic march. The atopic march mouse model was established by topical application of MC903 (a vitamin D3 analog). Following the treatment with resveratrol, airway resistance in mice was discovered by pulmonary function apparatus, and the number of total cells, neutrophils, and eosinophils in bronchoalveolar lavage fluid was counted. The histopathological features of pulmonary and ear skin tissues, inflammation, and cell infiltration were determined by hematoxylin and eosin staining. The messenger RNA (mRNA) levels of TSLP, immunoglobulin E, interleukin (IL)-4, IL-5, and IL-13 were measured by real-time quantitative polymerase chain reaction. The protein expression of nuclear factor kappa B (NF-κB)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling-associated molecules (p-p65, p65, p-I kappa B kinase alpha (IκBα), IκBα, Nrf2, and TSLP) in lung and ear skin tissues were assessed by Western blot analysis. Resveratrol attenuated airway resistance and infiltration of total cells, eosinophils, and neutrophils in both lung and ear skin tissues. Resveratrol ameliorates serum inflammatory markers in allergic mice. Moreover, the phosphorylation levels of NF-κB pathway-related proteins were significantly reduced by administration of resveratrol in allergic lung and ear skin tissues. Similarly, the protein expression of TSLP in both lung and ear skin tissues was reduced by resveratrol, and Nrf2, a protector molecule, was increased with resveratrol treatment. Resveratrol attenuates TSLP-reduced atopic march through ameliorating inflammation and cell infiltration in pulmonary and ear skin tissues by inhibiting the abnormal activation of NF-κB signaling pathway.

Cichorium intybus L. significantly alleviates cigarette smoke-induced acute lung injury by lowering NF-κB pathway activation and inflammatory mediators

BackgroundCigarette smoke (CS) is one of the primary causes of acute lung injury (ALI) via provoking pulmonary inflammation and oxidative stress. Despite substantial studies, no effective treatment for ALI is presently available. PurposeNew prospective treatment options for ALI are required. Thus, this project was designed to investigate the in vivo and in vitro protective effects of 70 % methanolic-aqueous crude extract of whole plant of Cichorium intybus (Ci.Mce) against CS-induced ALI. Study design/methods: Initially, male Swiss albino mice were subjected to whole-body CS exposure for 10 continuous days to prepare CS-induced ALI models. Normal saline (10 mL/kg), Ci.Mce (100, 200, 300 mg/kg), and Dexamethasone (1 mg/kg) were orally administered to respective animal groups 1 h prior to CS-exposure. 24 hrs after the last CS-exposure, BALF and lungs were harvested to study the key characteristics of ALI. Next, HPLC analysis was done to explore the phytoconstituents. ResultsCi.Mce exhibited significant reductions in lung macrophage and neutrophil infiltration, lung weight coefficient, and albumin exudation. Additionally, it effectively ameliorated lung histopathological alterations and hypoxemia. Notably, Ci.Mce exerted inhibitory effects on the excessive generation of IL-6, IL-1β, and KC in both CS-induced ALI murine models and CSE-stimulated RAW 264.7 macrophages. Noteworthy benefits included the attenuation of oxidative stress induced by CS, evidenced by decreased levels of MDA, TOS, and MPO, alongside enhanced TAC production. Furthermore, Ci.Mce demonstrated a marked reduction in CS-induced NF-κB expression, both in vivo and in vitro. ConclusionConsequently, Cichorium intybus could be a therapeutic option for CS-induced ALI due to its ability to suppress inflammatory reactions, mitigate oxidative stress, and quell NF-κB p65 activation.

Protective effect of olopatadine hydrochloride against LPS-induced acute lung injury: via targeting NF-κB signaling pathway.

Morbidity and mortality rates associated with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are high (30-40%). Nuclear factor-kappa B (NF-κB) is a transcription factor, associated with transcription of numerous cytokines leading to cytokine storm, and thereby, plays a major role in ALI/ARDS and in advanced COVID-19 syndrome. Considering the role of NF-κB in ALI, cost-effective in silico approaches were utilized in the study to identify potential NF-κB inhibitor based on the docking and pharmacokinetic results. The identified compound was then pharmacologically validated in lipopolysaccharide (LPS) rodent model of acute lung injury. LPS induces ALI by altering alveolar membrane permeability, recruiting activated neutrophils and macrophages to the lungs, and compromising the alveolar membrane integrity and ultimately impairs the gaseous exchange. Furthermore, LPS exposure is associated with exaggerated production of various proinflammatory cytokines in lungs. Based on in silico studies Olopatadine Hydrochloride (Olo), an FDA-approved drug was found as a potential NF-κB inhibitor which has been reported for the first time, and consideredfurther for the pharmacological validation. Intraperitoneal LPS administration resulted in ALI/ARDS by fulfilling 3 out of the 4 criteria described by ATS committee (2011) published workshop report. However, treatment with Olo attenuated LPS-induced elevation of proinflammatory markers (IL-6 and NF-κB), oxidative stress, neutrophil infiltration, edema, and damage in lungs. Histopathological studies also revealed that Olo treatment significantly ameliorated LPS-induced lung injury, thus conferring improvement in survival. Especially, the effects produced by Olo medium dose (1mg/kg) were comparable to dexamethasone standard. In nutshell, inhibition of NF-κB pathway by Olo resulted in protection and reduced mortality in LPS- induced ALI and thus has potential to be used clinically to arrest disease progression in ALI/ARDS, since the drug is already in the market. However, the findings warrant further extensive studies, and also future studies can be planned to elucidate its role in COVID-19-associated ARDS or cytokine storm.

Modeling Ventilator-Induced Lung Injury and Neutrophil Infiltration to Infer Injury Interdependence.

Acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI) are heterogeneous conditions. The spatiotemporal evolution of these heterogeneities is complex, and it is difficult to elucidate the mechanisms driving its progression. Through previous quantitative analyses, we explored the distributions of cellular injury and neutrophil infiltration in experimental VILI and discovered that VILI progression is characterized by both the formation of new injury in quasi-random locations and the expansion of existing injury clusters. Distributions of neutrophil infiltration do not correlate with cell injury progression and suggest a systemic response. To further examine the dynamics of VILI, we have developed a novel computational model that simulates damage (cellular injury progression and neutrophil infiltration) using a stochastic approach. Optimization of the model parameters to fit experimental data reveals that the range and strength of interdependence between existing and new damaged regions both increase as mechanical ventilation patterns become more injurious. The interdependence of cellular injury can be attributed to mechanical tethering forces, while the interdependence of neutrophils is likely due to longer-range cell signaling pathways.

Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Emphysema.

The activity of PP2A (protein phosphatase 2A), a serine-threonine phosphatase, is reduced by chronic cigarette smoke (SM) exposure and α-1 antitrypsin (AAT) deficiency, and chemical activation of PP2A reduces the loss of lung function in SM-exposed mice. However, the previously studied PP2A-activator tricyclic sulfonamide compound DBK-1154 has low stability to oxidative metabolism, resulting in fast clearance and low systemic exposure. Here we compare the utility of a new more stable PP2A activator, ATUX-792, versus DBK-1154 for the treatment of SM-induced emphysema. ATUX-792 was also tested in human bronchial epithelial cells and a mouse model of AAT deficiency, Serpina1a-e-knockout mice. Human bronchial epithelial cells were treated with ATUX-792 or DBK-1154, and cell viability, PP2A activity, and MAP (mitogen-activated protein) kinase phosphorylation status were examined. Wild-type mice received vehicle, DBK-1154, or ATUX-792 orally in the last 2 months of 4 months of SM exposure, and 8-month-old Serpina1a-e-knockout mice received ATUX-792 daily for 4 months. Forced oscillation and expiratory measurements and histology analysis were performed. Treatment with ATUX-792 or DBK-1154 resulted in PP2A activation, reduced MAP kinase phosphorylation, immune cell infiltration, reduced airspace enlargements, and preserved lung function. Using protein arrays and multiplex assays, PP2A activation was observed to reduce AAT-deficient and SM-induced release of CXCL5, CCL17, and CXCL16 into the airways, which coincided with reduced neutrophil lung infiltration. Our study indicates that suppression of the PP2A activity in two models of emphysema could be restored by next-generation PP2A activators to impact lung function.

Sinomenine alleviates lipopolysaccharide-induced acute lung injury via a PPARβ/δ-dependent mechanism

Evidence is mounting that sinomenine and peroxisome proliferator-activated receptor β/δ (PPARβ/δ) are effective against lipopolysaccharide (LPS)-induced acute lung injury (ALI) via anti-inflammatory properties. However, it is unknown whether PPARβ/δ plays a role in the protective effect of sinomenine on ALI. Here, we initially observed that preemptive administration of sinomenine markedly alleviated lung pathological changes, pulmonary edema and neutrophil infiltration, accompanied by inhibition of the expression of the pro-inflammatory cytokines Tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6), which were largely reversed following the addition of a PPARβ/δ antagonist. Subsequently, we also noticed that sinomenine upregulated adenosine A2A receptor expression in a PPARβ/δ-dependent manner in LPS-stimulated bone marrow-derived macrophages (BMDMs). Further investigation indicated that PPARβ/δ directly bound to the functional peroxisome proliferator responsive element (PPRE) in the adenosine A2A receptor gene promoter region to enhance the expression of the adenosine A2A receptor. Sinomenine was identified as a PPARβ/δ agonist. It could bind with PPARβ/δ, and promote the nuclear translocation and transcriptional activity of PPARβ/δ. In addition, combined treatment with sinomenine and an adenosine A2A receptor agonist exhibited synergistic effects and better protective roles than their single use against ALI. Taken together, our results reveal that sinomenine exerts advantageous effects on ALI by activating of PPARβ/δ, with the subsequent upregulation of adenosine A2A receptor expression, and provide a novel and potential therapeutic application for ALI.

Epicutaneous Staphylococcus aureusexposure triggers lung inflammation via epithelia-intrinsic IL-36R signaling

Abstract Allergic diseases have been increasing recently and affect nearly 20% of the human population, including 200 million people with atopic dermatitis (AD), 250 million people with food allergies, and 600 million people with allergic rhinitis or asthma. The atopic march is the concept whereby having early-in-life AD and a dysfunctional skin barrier during initial allergen sensitization increases the predisposition of allergic diseases at distant epithelial sites (e.g., asthma). We have previously shown that IL-36R signaling was required to exacerbate the progression from AD-like skin inflammation to subsequent allergic lung inflammation in mice. However, whether IL-36R acts directly on the skin and/or lung epithelia to trigger the progression towards lung inflammation is not entirely clear. To address this gap in knowledge, we performed our allergic lung inflammation model in WT, IL-36R−/−, keratinocyte-specific IL-36R deficient (K14-cre×IL-36R fl/fl), and lung epithelial-specific IL-36R deficient (Nkx2-1-cre×IL-36R fl/fl) mice, whereby first epicutaneous Staphylococcus aureus and cockroach antigen (CrA) exposure initiates AD-like skin inflammation, which is followed by intratracheal CrA exposure to trigger subsequent allergic lung inflammation. Evaluation of skin and lung inflammation by image, histologic and flow cytometric analyses revealed that both keratinocyte- and lung epithelial-intrinsic IL-36R signaling were required for the development of lung inflammation, weight loss, and neutrophil lung infiltration despite no differences in circulating neutrophils. Collectively, our findings suggest that IL-36R signaling in skin and lung epithelia is a critical mechanism for the progression of the atopic march. NIAMS: R01AR073665 and LEO Foundation: LF-OC-22-000953; SJN is funded by NIAMS 1T32AR074920

A 3-O-sulfated heparan sulfate dodecasaccharide (12-mer) suppresses thromboinflammation and attenuates early organ injury following trauma and hemorrhagic shock.

Dysregulated inflammation and coagulation are underlying mechanisms driving organ injury after trauma and hemorrhagic shock. Heparan sulfates, cell surface glycosaminoglycans abundantly expressed on the endothelial surface, regulate a variety of cellular processes. Endothelial heparan sulfate containing a rare 3-O-sulfate modification on a glucosamine residue is anticoagulant and anti-inflammatory through high-affinity antithrombin binding and sequestering of circulating damage-associated molecular pattern molecules. Our goal was to evaluate therapeutic potential of a synthetic 3-O-sulfated heparan sulfate dodecasaccharide (12-mer, or dekaparin) to attenuate thromboinflammation and prevent organ injury. Male Sprague-Dawley rats were pre-treated subcutaneously with vehicle (saline) or dekaparin (2 mg/kg) and subjected to a trauma/hemorrhagic shock model through laparotomy, gut distention, and fixed-pressure hemorrhage. Vehicle and dekaparin-treated rats were resuscitated with Lactated Ringer's solution (LR) and compared to vehicle-treated fresh-frozen-plasma-(FFP)-resuscitated rats. Serial blood samples were collected at baseline, after induction of shock, and 3 hours after fluid resuscitation to measure hemodynamic and metabolic shock indicators, inflammatory mediators, and thrombin-antithrombin complex formation. Lungs and kidneys were processed for organ injury scoring and immunohistochemical analysis to quantify presence of neutrophils. Induction of trauma and hemorrhagic shock resulted in significant increases in thrombin-antithrombin complex, inflammatory markers, and lung and kidney injury scores. Compared to vehicle, dekaparin treatment did not affect induction, severity, or recovery of shock as indicated by hemodynamics, metabolic indicators of shock (lactate and base excess), or metrics of bleeding, including overall blood loss, resuscitation volume, or hematocrit. While LR-vehicle-resuscitated rodents exhibited increased lung and kidney injury, administration of dekaparin significantly reduced organ injury scores and was similar to organ protection conferred by FFP resuscitation. This was associated with a significant reduction in neutrophil infiltration in lungs and kidneys and reduced lung fibrin deposition among dekaparin-treated rats compared to vehicle. No differences in organ injury, neutrophil infiltrates, or fibrin staining between dekaparin and FFP groups were observed. Finally, dekaparin treatment attenuated induction of thrombin-antithrombin complex and inflammatory mediators in plasma following trauma and hemorrhagic shock. Anti-thromboinflammatory properties of a synthetic 3-O-sulfated heparan sulfate 12-mer, dekaparin, could provide therapeutic benefit for mitigating organ injury following major trauma and hemorrhagic shock.

NADPH oxidase 2 limits amplification of IL-1β-G-CSF axis and animmature neutrophil subset in murine lung inflammation.

The leukocyte NADPH oxidase 2 (NOX2) regulates inflammation independent of its antimicrobial activity. Inherited defects in NOX2 lead to chronic granulomatous disease (CGD), associated with recurrent bacterial and fungal infections, often with excessive neutrophilic inflammation that results in significant inflammatory burden and tissue damage. We previously showed that excessive leukotriene B4 (LTB4) production by NOX2-deficient mouse neutrophils was a key driver of elevated lung neutrophil infiltration in the initial response to pulmonary challenge with the model fungal particle zymosan. We now identify interleukin-1β (IL-1β) and downstream granulocyte colony-stimulating factor (G-CSF) as critical amplifying signals that augment and sustain neutrophil accrual in CGD mice. Neutrophils, delivered into the lung via LTB4, were the primary source of IL-1β within the airways, and their increased numbers in CGD lungs led to significantly elevated local and plasma G-CSF. Elevated G-CSF simultaneously promoted increased granulopoiesis and mobilized the release of higher numbers of an immature CD101- neutrophil subset from the marrow, which trafficked to the lung and acquired a significantly more proinflammatory transcriptome in CGD mice compared with wild-type mice. Thus, neutrophil-produced IL-1β and downstream G-CSF act sequentially but nonredundantly with LTB4 to deploy neutrophils and amplify inflammation in CGD mice after inhalation of zymosan. NOX2 plays a critical role in dampening multiple components of a feed-forward pipeline for neutrophil recruitment, and these findings highlight NOX2 as a key regulator of neutrophil number, subsets, and function at inflamed sites.

Characteristics of inflammatory response and repair after experimental blast lung injury in rats.

Blast-induced lung injury is associated with inflammatory, which are characterised by disruption of the alveolar-capillary barrier, haemorrhage, pulmonary infiltrateration causing oedema formation, pro-inflammatory cytokine and chemokine release, and anti-inflammatory counter-regulation. The objective of the current study was to define sequence of such alterations in with establishing blast-induced lung injury in rats using an advanced blast generator. Rats underwent a standardized blast wave trauma and were euthanised at defined time points. Non-traumatised animals served as sham controls. Obtained samples from bronchoalveolar lavage fluid (BALF) at each time-point were assessed for histology, leukocyte infiltration and cytokine/chemokine profile. After blast lung injury, significant haemorrhage and neutrophil infiltration were observed. Similarly, protein accumulation, lactate dehydrogenase activity (LDH), alveolar eicosanoid release, matrix metalloproteinase (MMP)-2 and -9, pro-Inflammatory cytokines, including tumour necrosis factor (TNF) and interleukin (IL) -6 raised up. While declining in the level of anti-inflammatory cytokine IL-10 occurred. Ultimately, pulmonary oedema developed that increased to its maximum level within the first 1.5 h, then recovered within 24 h. Using a stablished model, can facilitate the study of inflammatory response to blast lung injury. Following the blast injury, alteration in cytokine/chemokine profile and activity of cells in the alveolar space occurs, which eventuates in alveolar epithelial barrier dysfunction and oedema formation. Most of these parameters exhibit time-dependent return to their basal status that is an indication to resilience of lungs to blast-induced lung injury.

Isoegomaketone alleviates inflammatory response and oxidative stress in sepsis lung injury.

Sepsis is a life-threatening condition characterized by acute organ dysfunction, which frequently leads to acute lung injury (ALI) in approximately 40% of cases. Isoegomaketone (IK) is a constituent of essential oil found in P. frutescens, known for its diverse biological properties, including anti-inflammatory and antitumor effects. However, the regulatory impact of IK on ALI in the context of sepsis remains poorly understood. Pathological alterations in lung tissues were assessed using hematoxylin and eosin staining. Enumeration of total leukocytes and neutrophils in bronchoalveolar lavage fluid (BALF) was performed using a hematocytometer, while the levels of interleukin (IL)-6, IL-1β, IL-10, and IL-17 in BALF were quantified using enzyme-linked immunosorbent serological assay. In addition, the levels of malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), and glutathione (GSH) in lung tissues were assessed using respective commercial kits; cell apoptosis was evaluated using the terminal deoxynucleotide transferase--mediated dUTP nick end-labeling assay, and protein expressions were determined through Western blot analysis. Our findings revealed that cecal ligation and puncture (CLP) treatment in mice induced severe lung injury, characterized by increased lung injury scores, significant bleeding, neutrophil infiltration, and alveolar edema. However, treatment with IK at a dose of 10 mg/kg ameliorated CLP-induced lung injury, while IK dose of 5 mg/kg showed no significant effect. Additionally, IK treatment at 10 mg/kg reduced CLP-induced inflammation by decreasing levels of IL-6, IL-1β, IL-10, and IL-17. Furthermore, IK at 10 mg/kg attenuated CLP-induced oxidative stress by modulating levels of MDA, MPO, SOD, and GSH. Moreover, IK treatment with a dose of 10 mg/kg activated the nuclear factor erythroid 2-related factor 2-heme oxygenase-1 (Nrf2-HO-1) pathway by enhancing the protein expressions of Nrf2 and HO-1. This study demonstrates that IK could mitigate the inflammatory response and oxidative stress associated with sepsis-induced ALI, supporting IK as a promising therapeutic agent for the treatment of sepsis-associated ALI.

SEX-DEPENDENT EFFECTS OF ADIPOCYTE STAT3 INHIBITION ON THE INFLAMMATORY RESPONSE DURING SEVERE SEPSIS.

Introduction: Sepsis is a dysregulated host response to infection that can lead to life-threatening organ dysfunction. Clinical and animal studies consistently demonstrate that female subjects are less susceptible to the adverse effects of sepsis, demonstrating the importance of understanding how sex influences sepsis outcomes. The signal transducer and activator of transcription 3 (STAT3) pathway are a major signaling pathway that facilitates inflammation during sepsis. STAT3 is abundantly expressed in white adipose tissue; however, little is known about the contribution of white adipose tissue STAT3 activation during sepsis. We hypothesize that adipocyte STAT3 inhibition during severe sepsis will exaggerate the inflammatory response and impact organ injury, in a sex-dependent manner. Methods: We generated STAT3 flox/flox (wild-type [WT]) and adipocyte STAT3 knock out (A-STAT3 KO) mice using Cre-lox technology. Studies were done in 12- to 16-week-old male and female mice. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Control nonseptic mice did not undergo CLP (0 h CLP). Tissues were harvested 18 h after CLP. Body composition was determined by echo magnetic resonance imaging. Energy metabolism was determined by indirect calorimetry. White adipose tissue morphology was determined by hematoxylin and eosin staining, while STAT3 activation in the white adipose tissue was determined by western blot analysis and immunohistochemistry staining of STAT3 activation/phosphorylation at tyrosine 705. Plasma cytokines (TNF-α, IL-6, and leptin) were determined by luminex assay. Neutrophil infiltration of the lung and liver was assessed by myeloperoxidase activity assay. Histological signs of organ injury on lung and liver tissue were assessed by hematoxylin and eosin staining. Liver injury was further assessed by measuring plasma alanine and aspartate aminotransferase. In a separate cohort of mice, sepsis was induced by CLP and mice were monitored every 6-12 h over a 7-day period to assess survival rate. Results: We demonstrate that neither body composition nor energy metabolism is altered with adipocyte STAT3 inhibition in male or female mice, under nonseptic conditions. Sepsis was associated with reduced adipocyte size in female WT and A-STAT3 KO mice, suggesting that this event is STAT3 independent. Sepsis did not alter adipocyte size in male WT and A-STAT3 KO mice, suggesting that this event is also sex dependent. Although STAT3 phosphorylation at tyrosine 705 expression is negligible in male and female A-STAT3 KO mice, septic female WT and A-STAT3 KO mice have higher white adipose tissue STAT3 activation than male WT and A-STAT3 KO mice. Adipocyte STAT3 inhibition did not alter the proinflammatory cytokine response during sepsis in male or female mice, as measured by plasma TNF-α, IL-6, and leptin levels. Adipocyte STAT3 inhibition reduced lung neutrophil infiltration and histological signs of lung injury during sepsis in male mice. On the contrary, adipocyte STAT3 inhibition had no effect on lung neutrophil infiltration or lung injury in female mice. We further demonstrate that neither liver neutrophil infiltration nor histological signs of liver injury are altered by adipocyte STAT3 inhibition during sepsis, in male or female mice. Lastly, adipocyte STAT3 inhibition did not affect survival rate of male or female mice during sepsis. Conclusions: Our study demonstrates that sex influences white adipose tissue STAT3 activation and morphology during sepsis, which is not dependent on the presence of functional STAT3 in mature adipocytes. Furthermore, genetic inhibition of adipocyte STAT3 activation in male, but not female mice, results in reduced lung neutrophil infiltration and lung injury during sepsis. The results from our study demonstrate the importance of considering biological sex and the white adipose tissue as potential sources and targets of inflammation during sepsis.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

Neutrophils drive pulmonary vascular leakage in MHV-1 infection of susceptible A/J mice.

Lung inflammation, neutrophil infiltration, and pulmonary vascular leakage are pathological hallmarks of acute respiratory distress syndrome (ARDS) which can lethally complicate respiratory viral infections. Despite similar comorbidities, however, infections in some patients may be asymptomatic while others develop ARDS as seen with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections for example. In this study, we infected resistant C57BL/6 and susceptible A/J strains of mice with pulmonary administration of murine hepatitis virus strain 1 (MHV-1) to determine mechanisms underlying susceptibility to pulmonary vascular leakage in a respiratory coronavirus infection model. A/J animals displayed increased lung injury parameters, pulmonary neutrophil influx, and deficient recruitment of other leukocytes early in the infection. Moreover, under basal conditions, A/J neutrophils overexpressed primary granule protein genes for myeloperoxidase and multiple serine proteases. During infection, myeloperoxidase and elastase protein were released in the bronchoalveolar spaces at higher concentrations compared to C57BL/6 mice. In contrast, genes from other granule types were not differentially expressed between these 2 strains. We found that depletion of neutrophils led to mitigation of lung injury in infected A/J mice while having no effect in the C57BL/6 mice, demonstrating that an altered neutrophil phenotype and recruitment profile is a major driver of lung immunopathology in susceptible mice. These results suggest that host susceptibility to pulmonary coronaviral infections may be governed in part by underlying differences in neutrophil phenotypes, which can vary between mice strains, through mechanisms involving primary granule proteins as mediators of neutrophil-driven lung injury.

OBESE MICE WITH PNEUMONIA HAVE HYPERLEPTINEMIA AND INCREASED PULMONARY SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 ACTIVATION.

Obesity is an ongoing epidemic that influences pathobiology in numerous disease states. Obesity is associated with increased plasma leptin levels, a hormone that activates the signal transducer and activator of transcription 3 (STAT3) pathway. Pneumonia is a significant cause of morbidity and mortality. During pneumonia, inflammatory pathways including STAT3 are activated. Outcomes in obese patients with pneumonia are mixed, with some studies showing obesity increases harm and others showing benefit. It is unclear whether obesity alters STAT3 activation during bacterial pneumonia and how this might impact outcomes from pneumonia. We used a murine model of obesity and pneumonia challenge with Pseudomonas aeruginosa in obese and nonobese mice to investigate the effect of obesity on STAT3 activation. We found obese mice with bacterial pneumonia had increased mortality compared with nonobese mice. Inflammatory markers, IL-6 and TNF-α, and lung neutrophil infiltration were elevated at 6 h after pneumonia in both nonobese and obese mice. Obese mice had greater lung injury compared with nonobese mice at 6 h after pneumonia. Leptin and insulin levels were higher in obese mice compared with nonobese mice, and obese mice with pneumonia had higher pulmonary STAT3 activation compared with nonobese mice.