Neutrophils In Bronchoalveolar Lavage Fluid Research Articles

478. Lung Hybrid Neutrophils and Subsequent Extracellular Traps Are Protective In COVID-19-Associated Pulmonary Aspergillosis

Abstract Background COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus frequently affecting critically ill COVID-19 patients. Pathophysiological insight, key to improve diagnostic and immunomodulatory therapeutic options, is lacking. Methods We performed single-cell RNA sequencing (scRNA-seq) on 37 bronchoalveolar lavage (BAL) samples from 37 critically ill COVID-19 patients. Three groups were defined: patients who did not develop aspergillosis (COVID-19-only, n=22), CAPA patients with sampling < 5 days after CAPA diagnosis (early CAPA, n=6) and CAPA patients with sampling 5-11 days after CAPA diagnosis (late CAPA, n=9). All CAPA patients had probable/proven CAPA according to the 2020 ECMM/ISHAM consensus criteria. Additionally, we assessed neutrophil extracellular trap (NET) levels in a separate cohort of 33 biobanked COVID-19-only BAL samples and 24 early CAPA samples. Results A total of 69008 cells passed quality filtering. CAPA patients had significantly lower BAL neutrophil proportions than COVID-19-only patients, particularly in early CAPA (Fig. 1A). Pseudotime inference revealed two neutrophil trajectories: a regular maturation trajectory, and a trajectory giving rise to “hybrid” neutrophils which express genes encoding proteins with antigen-presenting functions (Fig. 1B). The latter trajectory was dominant in CAPA patients (Fig. 1C). NETosis analyses revealed significantly higher levels of citrullinated histone H3 DNA complexes (H3Cit-DNA) in CAPA patients (Fig. 2A). This explains the low CAPA BAL neutrophil proportions, as neutrophils that underwent NETosis are no longer detected via scRNA-seq. CAPA patients with the lowest H3Cit-DNA levels had significantly decreased survival rates (Fig. 2B). Figure 1:Bronchoalveolar lavage fluid (BALF) neutrophil proportions are significantly decreased in CAPA patients, accompanied by a shift to hybrid neutrophil formation. Panel (A): BALF neutrophil proportions as analyzed by single-cell RNA sequencing using the Seurat R package are significantly lower in CAPA patients compared to COVID-19-only patients. Patients with early CAPA have significantly lower BALF neutrophil proportions than patients with late CAPA. Macrophage/monocyte and epithelial cell proportions are reciprocally increased in CAPA patients compared to COVID-19-only patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Panel (B): Two trajectories are defined using pseudotime inference calculated using the Slingshot R package: a trajectory dominant in COVID-19-only patients with regular maturation of progenitor neutrophils, and a trajectory dominant in CAPA patients with maturation towards a ‘hybrid neutrophil’ state, with neutrophils expression genes encoding proteins with functions in antigen presentation. Subsequently, the hybrid neutrophil proportion is significantly higher in CAPA patients compared to COVID-19-only patients, and is significantly higher in patients with early CAPA than those with late CAPA. The mature neutrophil proportion is reciprocally reduced in CAPA patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Figure 2:Neutrophil extracellular trap (NET) levels are increased in early CAPA and are associated with increased survival in CAPA patients specifically. Panel (A): Myeloperoxidase (MPO) DNA levels were analyzed as measure for general NET-formation, while citrullinated histone H3 bound DNA (H3Cit-DNA) levels were analyzed as more specific PAD4-dependent NET-formation, in BALF samples from early CAPA and COVID-19-only patients. A trend towards higher MPO-DNA levels was found in early CAPA patients, while H3Cit-DNA levels were significantly higher in early CAPA compared to COVID-19-only patients. P-values calculated using Mann-Whitney U test. Panel (B): Kaplan-Meier analysis of patients with NETosis analyses, divided in early CAPA and COVID-19-only patients and subdivided according to H3Cit-DNA levels (cut-off at 20000 ng/mL for early CAPA and at 8000 ng/mL for COVID-19-only). Log-rank test was used to compare survival distributions. For the comparison early CAPA (low H3Cit-DNA) versus early CAPA (high H3Cit-DNA), the log-rank p-value was 0.033. Conclusion CAPA patients display extremely high levels of released NETs in the lower respiratory tract, associated with a shift from the normal neutrophil maturation process towards “hybrid neutrophil” formation, probably upon encountering the fungus. In contrast to high NETosis contributing to mortality in severe COVID-19, CAPA patients likely require these NETs to survive aspergillosis. BAL NET levels hold promise as a tool to guide diagnosis, prognosis and treatment in these patients. Disclosures Simon Feys, MD, Pfizer: Travel support Katrien Lagrou, PharmD, PhD, FUJIFILM Wako: Speaker fee|Gilead: Advisor/Consultant|Gilead: Speaker fee|MRM Health: Advisor/Consultant|MSD: Advisor/Consultant|Pfizer: Speaker fee|Thermo fisher Scientific: Service fee Joost Wauters, MD, PhD, Gilead: Grant/Research Support|Gilead: Speaker's fee, travel support, advisory board|MSD: Grant/Research Support|MSD: Speaker's fee, travel support, posaconazole for interventional trial|Pfizer: Grant/Research Support|Pfizer: Speaker's fee, travel support, advisory board.

Effects of low-dust forages on dust exposure, airway cytology, and plasma omega-3 concentrations in Thoroughbred racehorses: A randomized clinical trial.

Racehorses commonly develop evidence of mild asthma in response to dust exposure. Diets deficient in omega-3 polyunsaturated fatty acids (Ω-3) might exacerbate this response. To compare dust exposure, bronchoalveolar lavage fluid (BALF) cytology, and plasma Ω-3 and specialized pro-resolving mediators (SPM) concentrations amongst racehorses fed dry hay, steamed hay, and haylage. Forty-three Thoroughbred racehorses. Prospective clinical trial. Horses were randomly assigned to be fed dry hay, steamed hay, or haylage for 6 weeks. Measures of exposure to dust in the breathing zone were obtained twice. At baseline, week-3, and week-6, BALF cytology was examined. Plasma lipid profiles and plasma SPM concentrations were examined at baseline and week 6. Generalized linear mixed models examined the effect of forage upon dust exposure, BALF cytology, Ω-3, and SPM concentrations. Respirable dust was significantly higher for horses fed hay (least-square mean ± s.e.m. 0.081 ± 0.007 mg/m3 ) when compared with steamed hay (0.056 ± 0.005 mg/m3 , P=.01) or haylage (0.053 ± 0.005 mg/m3 , P < .01). At week 6, BALF neutrophil proportions in horses eating haylage (3.0% ± 0.6%) were significantly lower compared with baseline (5.1 ± 0.7, P=.04) and horses eating hay (6.3% ± 0.8%, P < .01). Plasma eicosapentaenoic acid to arachidonic acid ratios were higher in horses eating haylage for 6 weeks (0.51 ± 0.07) when compared with baseline (0.34 ± 0.05, P < .01) and horses eating steamed (0.24 ± 0.02, P < .01) or dry hay (0.25 ± 0.03, P < .01). Steamed hay and haylage reduce dust exposure compared with dry hay, but only haylage increased the ratio of anti-inflammatory to pro-inflammatory lipids while reducing BAL neutrophil proportions within 6 weeks.

Acinetobacter baumannii reinforces the pathogenesis by promoting IL-17 production in a mouse pneumonia model.

Interleukin-17 (IL-17) is involved in host defense against bacterial infection.Little is known about the role of IL-17 in A. baumannii-infected pneumonia. Our objective was to investigate the role of IL-17 in pulmonary A. baumannii infection in a mouse model. We infected C57BL/6 mice intra-tracheally (i.t.) with A. baumannii to establish pneumonia model and found A. baumannii infection elevated IL-17 expression in lungs. IL-17-deficient (Il17-/-) mice were resistant to pulmonary A. baumannii infection, showing improved mice survival, reduced bacteria burdens, and alleviated lung inflammation.Further, treatment of A. baumannii-infected Il17-/- mice with IL-17 exacerbated the severity of pneumonia. These data suggest a pathogenic role of IL-17 in pulmonary A. baumannii infection. Further, the infiltration and phagocytic function of neutrophils in broncho-alveolar lavage fluid were detected by flow cytometry. The results showed that Il17-/- mice had increased neutrophil infiltration and enhanced phagocytosis in neutrophils at the early time of infection. Treatment of mice with IL-17 suppressed phagocytic function of neutrophils. All data suggest that IL-17 promotes susceptibility of mice to pulmonary A. baumannii infection by suppressing neutrophil phagocytosis at early time of infection. Targeting IL-17 might be a potential therapeutic strategy in controlling the outcome of A. baumanniipneumonia.

Ketamine Attenuates Airway Inflammation via Inducing Inflammatory Cells Apoptosis and Activating Nrf2 Pathway in a Mixed-Granulocytic Murine Asthma Model.

The use of ketamine, an anesthetic, as a treatment for asthma has been investigated in numerous studies. However, how ketamine affects asthma is unclear. The present study examined the effects of ketamine on a murine model of mixed-granulocytic asthma, and the role of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. The murine model of mixed-granulocytic asthma was established using ovalbumin (OVA) for sensitization and the combination of OVA and lipopolysaccharides (LPS) for challenge. The main characteristics of asthma, oxidative stress biomarkers, and the expression of the Nrf2 pathway were examined. ML385 was administered to verify the role of the Nrf2 pathway. Mice in the OVA +LPS group developed asthmatic characteristics, including airway hyperresponsiveness, mixed-granulocytic airway inflammation, mucus overproduction, as well as increased levels of oxidative stress and impaired apoptosis of inflammatory cells. Among the three concentrations, ketamine at 75mg/kg effectively attenuated these asthmatic symptoms, activated the Nrf2 pathway, decreased oxidative stress, and induced apoptosis of eosinophils and neutrophils in bronchoalveolar lavage fluid (BALF) with a reducing level of myeloid cell leukemia 1(Mcl-1). ML385 (an Nrf2 inhibitor) eliminated the protective effects of ketamine on the mixed-granulocytic asthma model. The study concluded that ketamine reduced oxidative stress and attenuated asthmatic symptoms (neutrophilic airway inflammation) by activating the Nrf2-Keap1 pathway, with 75 mg/kg ketamine showing the best results. Ketamine administration also increased neutrophil and eosinophil apoptosis in BALF, which may contribute to the resolution of inflammation. The use of ketamine as a treatment for asthma may therefore be beneficial.

Nicotinamide Mononucleotide Attenuates LPS-Induced Acute Lung Injury With Anti-Inflammatory, Anti-Oxidative and Anti-Apoptotic Effects

IntroductionNicotinamide mononucleotide (NMN) is a nucleotide that is commonly recognized for its role as an intermediate of nicotinamide adenine dinucleotide (NAD+) biosynthesis with multiple pharmacological effects. The purpose of this study was to evaluate the protective effect of nicotinamide mononucleotide (NMN) against lipopolysaccharide (LPS)–induced acute lung injury (ALI). MethodsWe investigated the effect of NMN on ALI-induced inflammatory response, oxidative stress, and cell apoptosis. The ALI mouse model was performed by injecting LPS intratracheally at a dose of 10 mg/kg in 50 μL saline. Flow cytometry was used to detect neutrophil infiltration in bronchoalveolar lavage fluid (BALF), and ELISA was used to detect the contents of inflammatory cytokines TNF-α, IL-1β and IL-6 in BALF. Oxidative stress was evaluated by determining the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in lung tissue. ROS formation was analyzed by immunofluorescence. Western blotting was performed to detect apoptotic levels and p38MAPK/NF-κB phosphorylation levels in lung tissue. ResultsIn the ALI mouse model, NMN showed a significant therapeutic effect compared to the LPS group. NMN attenuated the pathological damage and cell apoptosis in lung tissue, decreased the levels of TNF-α, IL-1β, and IL-6 in BALF, and reduced the number of total cells and neutrophils in BALF. In addition, NMN attenuated the LPS-induced elevation of dry-to-wet ratio, MDA content, p38 MAPK and p65 NF-κB phosphorylation levels, and the SOD activity was increased by NMN treatment. ConclusionsIn conclusion, the present study showed that NMN exerted a protective effect on LPS-induced ALI with anti-inflammatory, antioxidative, and antiapoptotic effects.

Gastrointestinal consequences of lipopolysaccharide-induced lung inflammation

BackgroundRespiratory inflammation is the body’s response to lung infection, trauma or hypersensitivity and is often accompanied by comorbidities, including gastrointestinal (GI) symptoms. Why respiratory inflammation is accompanied by GI dysfunction remains unclear. Here, we investigate the effect of lipopolysaccharide (LPS)-induced lung inflammation on intestinal barrier integrity, tight-junctions, enteric neurons and inflammatory marker expression.MethodsFemale C57bl/6 mice (6–8 weeks) were intratracheally administered LPS (5 µg) or sterile saline, and assessed after either 24 or 72 h. Total and differential cell counts in bronchoalveolar lavage fluid (BALF) were used to evaluate lung inflammation. Intestinal barrier integrity was assessed via cross sectional immunohistochemistry of tight junction markers claudin-1, claudin-4 and EpCAM. Changes in the enteric nervous system (ENS) and inflammation in the intestine were quantified immunohistochemically using neuronal markers Hu + and nNOS, glial markers GFAP and S100β and pan leukocyte marker CD45.ResultsIntratracheal LPS significantly increased the number of neutrophils in BALF at 24 and 72 h. These changes were associated with an increase in CD45 + cells in the ileal mucosa at 24 and 72 h, increased goblet cell expression at 24 h, and increased expression of EpCAM at 72 h. LPS had no effect on the expression of GFAP, S100β, nor the number of Hu + neurons or proportion of nNOS neurons in the myenteric plexus.ConclusionsIntratracheal LPS administration induces inflammation in the ileum that is associated with enhanced expression of EpCAM, decreased claudin-4 expression and increased goblet cell density, these changes may contribute to systemic inflammation that is known to accompany many inflammatory diseases of the lung.

Effect of Maxing Shigan Decoction and dissembled prescriptions against airway inflammation in RSV-aggravated asthma and mechanism of regulating TRPV1

This study investigated the effect of Maxing Shigan Decoction(MXSGD) and its disassembled prescriptions against the airway inflammation in respiratory syncytial virus(RSV)-aggravated asthma and the regulation of transient receptor potential vanilloid-1(TRPV1). To be specific, ovalbumin(OVA) and RSV were used to induce aggravated asthma in mice(female, C57BL/6). Then the model mice were intervened by MXSGD and the disassembled prescriptions. The eosinophil(EOS) in peripheral blood, inflammatory cells in bronchoalveolar lavage fluid(BALF), enhanced pause(Penh) variation, and lung pathological damage in each group were observed, and the changes of interleukin(IL)-4, IL-13, substance P(SP), and prostaglandin E2(PGE2) in BALF were mea-sured by enzyme-linked immunosorbent assay(ELISA). Quantitative real time polymerase chain reaction(qPCR) and Western blot were used to detect mRNA and protein of TRPV1 in mouse lung tissue. In the in vitro experiment, 16 HBE cells were stimulated with IL-4 and RSV. Then the changes of TRPV1 expression after the intervention with the serum containing MXSGD and its disassembled prescriptions were observed. Besides, the intracellular Ca~(2+) level after the stimulation with TRPV1 agonist was evaluated. The results showed that the mice in the model group had obvious asthma phenotype, the levels of various inflammatory cells in the peripheral blood and BALF and Penh were significantly increased(P&lt;0.05, P&lt;0.01), and the lung tissue was severely damaged compared with the control group. Compared with the model group, the levels of EOS in the peripheral blood and BALF were significantly decreased in the MXSGD group, the SG group and the MXC group(P&lt;0.05, P&lt;0.01). The levels of WBC and neutrophils in BALF were significantly decreased in the MXSGD group and SG group(P&lt;0.01), the levels of neutrophils in BALF were decreased in the MXC group(P&lt;0.05). The improvement effect of the MXGSD on the level of inflammatory cells in peripheral blood and BALF was better than that of two disassembled groups(P&lt;0.05, P&lt;0.01). After 50 mg·mL~(-1) acetylcholine chloride stimulation, the Penh values of the MXSGD group and the MXC group significantly decreased(P&lt;0.01), and the Penh value of the SG group decreased(P&lt;0.05). The levels of IL-4, IL-13, PGE2 and SP in BALF could be significantly decreased in the MXSGD group(P&lt;0.05, P&lt;0.01), the levels of IL-13 and PGE2 in BALF could be decreased in the MXC group(P&lt;0.05, P&lt;0.01), and the levels of IL-13, PGE2 and SP in BALF could be decreased in the SG group(P&lt;0.05, P&lt;0.01). MXSGD could down-regulate the protein and mRNA expression of TRPV1 in lung tissue(P&lt;0.05, P&lt;0.01). The serum containing MXSGD and its disassembled prescriptions could down-regulate TRPV1 expression in 16 HBE cells stimulated by IL-4 combined with RSV and inhibit the inward flow of Ca~(2+) induced by TRPV1 agonist, especially the serum containing MXSGD which showed better effect than the serum containing disassembled ones(P&lt;0.05). In vivo and in vitro experiments verified the protective effect of MXSGD and its disassembled prescriptions against airway inflammation in RSV-exacerbated asthma, the whole decoction thus possessed synergy in treating asthma, with better performance than the dissembled prescriptions. Different groups of prescription had made contributions in improving airway hyperresponsiveness, anti-allergy and anti-inflammation. The mechanism is the likelihood that it regulates TRPV1 channel and levels of related inflammatory mediators.

CTRP6 suppresses neutrophil extracellular traps formation to ameliorate sepsis-induced lung injury through inactivation of ERK pathway

Septic lung injury is associated with excessive neutrophil activation, while neutrophil extracellular traps formation contributes to inflammatory lung injury in sepsis. C1q/tumor necrosis factor-related protein-6 (CTRP6) is a paralog of adiponectin and exerts anti- inflammatory and antioxidant properties. The role of CTRP6 in sepsis-associated inflammatory lung injury was investigated in this study. Mice were injected with lipopolysaccharides (LPS) intraperitoneally to establish the mouse sepsis model. They were first tail-vein injected with adenovirus-mediated overexpression CTRP6 (Ad-CTRP6) and then subjected to the LPS injection. Pathological changes in lungs were detected by hematoxylin and eosin staining. Inflammation cytokine levels in bronchoalveolar lavage fluid were assessed by qRT-PCR and ELISA. Flow cytometry was used to detect the number of neutrophils in bronchoalveolar lavage fluid, and immunofluorescence was performed to assess neutrophil extracellular traps. Lipopolysaccharides induced pulmonary congestion, interstitial edema, and alveolar wall thickening in the lungs, as well as upregulated lung histology score and wet/dry weight ratio. CTRP6 was reduced in lung tissues of septic mice. Injection with Ad-CTRP6 ameliorated extensive histopathological changes in LPS-induced mice and decreased lung histology score and wet/dry weight ratio. Overexpression of CTRP6 reduced the levels of TNF-α, IL-6, and IL-1β in septic mice. Injection with Ad-CTRP6 also decreased the number of neutrophils and downregulated Cit-H3 and myeloperoxidase polymers in septic mice. Protein expression of p-ERK in septic mice was reduced by overexpression of CTRP6. CTRP6 attenuated septic lung injury, exerted anti-inflammatory effect, and suppressed neutrophil extracellular traps formation against sepsis through inactivation of extracellular signal-regulated kinase signaling.

"Rogue" neutrophil-subset [DEspR+CD11b+/CD66b+] immunotype is an actionable therapeutic target for neutrophilic inflammation-mediated tissue injury - studies in human, macaque and rat LPS-inflammation models.

Background and objectiveThe correlation (Rs > 0.7) of neutrophils expressing the dual endothelin1/signal peptide receptor (DEspR+CD11b+/CD66b+) with severity of hypoxemia (SF-ratio) and multi-organ failure (SOFA-score) in patients with acute respiratory distress syndrome (ARDS) suggest the hypothesis that the DEspR+ neutrophil-subset is an actionable therapeutic target in ARDS. To test this hypothesis, we conducted in vivo studies to validate DEspR+ neutrophil-subset as therapeutic target and test efficacy of DEspR-inhibition in acute neutrophilic hyperinflammation models.MethodsWe performed tests in lipopolysaccharide (LPS)-induced acute neutrophilic inflammation in three species – human, rhesus macaque, rat – with increasing dose-dependent severity. We measured DEspR+CD66b+ neutrophils in bronchoalveolar lavage fluid (BALF) in healthy volunteers (HVs) 24-hours after segmental LPS-challenge by ChipCytometry, and DEspR+CD11b+ neutrophils in whole blood and BALF in an LPS-induced transient acute lung injury (ALI) model in macaques. We determined anti-DEspR antibody efficacy in vivo in LPS-ALI macaque model and in high-mortality LPS-induced encephalopathy in hypertensive rats.ResultsChipCytometry detected increased BALF total neutrophil and DEspR+CD66b+ neutrophil counts after segmental LPS-challenge compared to baseline (P =0.034), as well as increased peripheral neutrophil counts and neutrophil-lymphocyte ratio (NLR) compared to pre-LPS level (P <0.05). In the LPS-ALI macaque model, flow cytometry detected increased DEspR+ and DEspR[-] neutrophils in BALF, which was associated with moderate-severe hypoxemia. After determining pharmacokinetics of single-dose anti-DEspR[hu6g8] antibody, one-time pre-LPS anti-DEspR treatment reduced hypoxemia (P =0.03) and neutrophil influx into BALF (P =0.0001) in LPS-ALI vs vehicle mock-treated LPS-ALI macaques. Ex vivo live cell imaging of macaque neutrophils detected greater “intrinsic adhesion to hard-surface” in DEspR+ vs DEspR[-] neutrophils (P <0.001). Anti-DEspR[hu6g8] antibody abrogated intrinsic high adhesion in DEspR+ neutrophils, but not in DEspR[-] neutrophils (P <0.001). In the LPS-encephalopathy rat model, anti-DEspR[10a3] antibody treatment increased median survival (P =0.0007) and exhibited brain target engagement and bioeffects.ConclusionDetection of increased DEspR+ neutrophil-subset in human BALF after segmental LPS-challenge supports the correlation of circulating DEspR+ neutrophil counts with severity measure (SOFA-score) in ARDS. Efficacy and safety of targeted inhibition of DEspR+CD11b+ neutrophil-subset in LPS-induced transient-ALI and high-mortality encephalopathy models identify a potential therapeutic target for neutrophil-mediated secondary tissue injury.

Jian-Ti-Kang-Yi decoction alleviates poly(I:C)-induced pneumonia by inhibiting inflammatory response, reducing oxidative stress, and modulating host metabolism.

Jian-Ti-Kang-Yi decoction (JTKY) is widely used in the treatment of COVID-19. However, the protective mechanisms of JTKY against pneumonia remain unknown. In this study, polyinosinic-polycytidylic acid (poly(I:C)), a mimic of viral dsRNA, was used to induce pneumonia in mice; the therapeutic effects of JTKY on poly(I:C)-induced pneumonia model mice were evaluated. In addition, the anti-inflammatory and anti-oxidative potentials of JTKY were also investigated. Lastly, the metabolic regulatory effects of JTKY in poly(I:C)-induced pneumonia model mice were studied using untargeted metabolomics. Our results showed that JTKY treatment decreased the wet-to-dry ratio in the lung tissue, total protein concentration, and total cell count of the bronchoalveolar lavage fluid (BALF). Hematoxylin and Eosin (HE) and Masson staining indicated that the JTKY treatment alleviated the pathological changes and decreased the fibrotic contents in the lungs. JTKY treatment also decreased the expression of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α)] and increased the levels of immunomodulatory cytokines (IL-4 and IL-10) in the BALF and serum. Flow cytometry analysis showed that the JTKY treatment lowered the ratio of CD86+/CD206+ macrophages in the BALF, decreased inducible nitric oxide synthase (iNOS) level, and increased arginase 1 (Arg-1) level in lung. JTKY also lowered CD11b+Ly6G+ neutrophils in BALF and decreased myeloperoxidase (MPO) activity in lung. Moreover, it also elevated superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and decreased methane dicarboxylic aldehyde (MDA) level in lung. Untargeted metabolomic analysis showed that the JTKY treatment could affect 19 metabolites in lung, such as L-adrenaline, L-asparagine, ornithine, and alpha-ketoglutaric acid. These metabolites are associated with the synthesis and degradation of ketone bodies, butanoate, alanine, aspartate, and glutamate metabolism, and tricarboxylic acid (TCA) cycle processes. In conclusion, our study demonstrated that treatment with JTKY ameliorated poly(I:C)-induced pneumonia. The mechanism of action of JTKY may be associated with the inhibition of the inflammatory response, the reduction of oxidative stress, and the regulation of the synthesis and degradation of ketone bodies, TCA cycle, and metabolism of alanine, aspartate, glutamate, and butanoate processes in lung.

The phytochemical constituents and protective effect of Fritillaria hupehensis on acute lung injury

Acute lung injury (ALI), a severe respiratory disorder, frequently develops into acute respiratory distress syndrome (ARDS) without timely treatment and scores highly in terms of morbidity and mortality rates. Fritillaria hupehensis is a famous traditional Chinese medicine with antitussive, expectorant and anti-asthmatic effect. Here, the effects of F. hupehensis extracts on lipopolysaccharide (LPS)-induced ALI mice were evaluated for the first time. We showed ethyl acetate fraction (EAF) significantly reduced the leukocytes and neutrophils of bronchoalveolar lavage fluid (BALF) and the lung index as well as pro-inflammatory cytokines (TNF-α and IL-6) of lung homogenates but increasing the anti-inflammatory cytokines (IL-4 and IL-10). Additionally, the alleviation of EAF treatment on lung injury was verified through histopathological observations. Subsequent phytochemical investigation on bioactive fraction led to isolation of 17 compounds including two new, in which compounds 2, 5 and 6 exhibited better anti-inflammatory effect on LPS-induced 16 human airway epithelial (16HBE) cells model by inhibiting the production of CRP and PCT. Furthermore, compound 2 suppressed the LPS-induced upregulation of proteins containing p-p65, COX-2, Caspase-1 and IL-18. In summary, F. hupehensis alleviating LPS-induced ALI in mice may be associated with the anti-inflammatory activity of steroidal alkaloids by suppressing the NF-κB-regulated pro-inflammatory proteins.

Down-regulation of KLF9 ameliorates LPS-caused acute lung injury and inflammation in mice via reducing GSDMD expression

Acute lung injury (ALI) is considered as a severe respiratory disease with aggravated inflammatory responses. Krüppel-like factor 9 (KLF9), a member of KLF family, has been reported to be involved in inflammatory disorders. However, the effect of KLF9 in ALI has not been elucidated. Here the present study was to clarify the role of KLF9 and its mechanism in ALI. The ALI in vitro model was established with lipopolysaccharide (LPS)-treated RAW264.7 cells. Mice were injected with LPS to conduct an ALI in vivo model. The expression of KLF9 and gasdermin D (GSDMD) was examined using quantitative reverse transcription-PCR, haematoxylin-eosin/immunohistochemistry staining and western blot assays. Enzyme-linked immunosorbent assay was employed to detect the levels of inflammatory cytokines. JASPAR database was used to predict the recognition motif of KLF9, and the relationship between KLF9 and GSDMD was determined by luciferase reporter assay and chromatin immunoprecipitation analysis. The number of neutrophils in bronchoalveolar lavage fluid as well as the wet/dry weight ratio was caculated. The results showed that The expression of KLF9 in lung was significantly increased in LPS-stimulated mice. Moreover, KLF9 knockout relieved the lung injury in ALI mice. GSDMD is one of targets genes of the transcription factor KLF9. KLF9 knockout induced a decreased expression of GSDMD in LPS-treated mice. Furthermore, in RAW264.7 cells after LPS administration, KLF9 knockdown reduced the levels of inflammatory factors and suppressed the expression of GSDMD. In summarise, these findings exhibited that KLF9 knockout could mitigate the lung injury and inflammatory responses in ALI mice by directly regulating GSDMD.

Xiao-Chai-Hu Decoction Ameliorates Poly (I:C)-Induced Viral Pneumonia through Inhibiting Inflammatory Response and Modulating Serum Metabolism.

Viral pneumonia is widespread, progresses rapidly, and has a high mortality rate. Developing safe and effective therapies to treat viral pneumonia can minimize risks to public health and alleviate pressures on the associated health systems. Xiao-Chai-Hu (XCH) decoction can be used in the treatment of viral pneumonia. However, the mechanisms of XCH on viral pneumonia remain unclear. In this study, poly (I:C) was used to establish a mouse model of viral pneumonia, and the therapeutic effects of XCH on viral pneumonia were assessed. Furthermore, we evaluated the effects of XCH on inflammatory response. Lastly, untargeted metabolomics were used to study the metabolic regulatory mechanisms of XCH on viral pneumonia model mice. Our results showed that XCH treatment decreased the wet/dry ratio in lung tissue, total protein concentration, and total cell count in bronchoalveolar lavage fluid (BALF). H&E staining indicated that XCH treatment alleviated the pathological changes in lung. Moreover, XCH treatment decreased the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and lowered the ratio of CD86+/CD206+ macrophages and CD11b+LY6G+ neutrophils in BALF. XCH treatment also decreased the myeloperoxidase (MPO) and reduced the phosphorylations of PI3K, AKT, and NF-κB p65 in lung. Serum untargeted metabolomics analysis showed that XCH treatment could affect 18 metabolites in serum such as creatine, hydroxyproline, cortisone, hydrocortisone, corticosterone, hypotaurine, and taurine. These metabolites were associated with arginine and proline metabolism, steroid hormone biosynthesis, and taurine and hypotaurine metabolism processes. In conclusion, our study demonstrated that treatment with XCH can ameliorate viral pneumonia and reduce inflammatory response in viral pneumonia. The mechanism of action of XCH in the treatment of viral pneumonia may be associated with inhibiting the activation of PI3K/AKT/NF-κB signaling pathway in lung and regulating arginine and proline metabolism, steroid hormone biosynthesis, and taurine and hypotaurine metabolism in serum.

Budesonide/glycopyrronium/formoterol fumarate triple therapy prevents pulmonary hypertension in a COPD mouse model via NFκB inactivation

BackgroundChronic obstructive pulmonary disease (COPD) is a health problem that results in death, commonly due to the development of pulmonary hypertension (PH). Here, by utilizing a mouse model of intratracheal elastase-induced emphysema that presents three different phases of COPD, we sought to observe whether budesonide/glycopyrronium/formoterol fumarate (BGF) triple therapy could prevent COPD-PH in addition to ameliorating COPD progression.MethodsWe utilized intratracheal elastase-induced emphysema mouse model and performed experiments in three phases illustrating COPD progression: inflammatory (1 day post-elastase), emphysema (3 weeks post-elastase) and PH (4 weeks post-elastase), while treatments of BGF and controls (vehicle, one-drug, and two-drug combinations) were started in prior to elastase instillation (inflammatory phase), at day 7 (emphysema), or at day 14 (PH phase). Phenotype analyses were performed in each phase. In vitro, A549 cells or isolated mouse lung endothelial cells (MLEC) were treated with TNFα with/without BGF treatment to analyze NFκB signaling and cytokine expression changes.ResultsWe observed significant reductions in the proinflammatory phenotype observed in the lungs and bronchoalveolar lavage fluid (BALF) 1 day after elastase administration in mice treated with BGF compared with that in mice administered elastase alone (BALF neutrophil percentage, p = 0.0011 for PBS/Vehicle vs. PBS/Elastase, p = 0.0161 for PBS/Elastase vs. BGF). In contrast, only BGF treatment significantly ameliorated the elastase-induced emphysematous lung structure and desaturation after three weeks of elastase instillation (mean linear intercept, p = 0.0156 for PBS/Vehicle vs. PBS/Elastase, p = 0.0274 for PBS/Elastase vs. BGF). Furthermore, BGF treatment prevented COPD-PH development, as shown by improvements in the hemodynamic and histological phenotypes four weeks after elastase treatment (right ventricular systolic pressure, p = 0.0062 for PBS/Vehicle vs. PBS/Elastase, p = 0.027 for PBS/Elastase vs. BGF). Molecularly, BGF acts by inhibiting NFκB-p65 phosphorylation and subsequently decreasing the mRNA expression of proinflammatory cytokines in both alveolar epithelial and pulmonary endothelial cells.ConclusionOur results collectively showed that BGF treatment could prevent PH in addition to ameliorating COPD progression via the inhibition of inflammatory NFκB signaling.

Pulmonary exposure of mice to ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX) suppresses the innate immune response to carbon black nanoparticles and stimulates lung cell proliferation

Background Per- and polyfluoroalkyl substances (PFAS) have been associated with respiratory diseases in humans, yet the mechanisms through which PFAS cause susceptibility to inhaled agents is unknown. Herein, we investigated the effects of ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), an emerging PFAS, on the pulmonary immune response of mice to carbon black nanoparticles (CBNP). We hypothesized that pulmonary exposure to GenX would increase susceptibility to CBNP through suppression of innate immunity. Methods Male C57BL/6 mice were exposed to vehicle, 4 mg/kg CBNP, 10 mg/kg GenX, or CBNP and GenX by oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) was collected at 1 and 14 days postexposure for cytokines and total protein. Lung tissue was harvested for histopathology, immunohistochemistry (Ki67 and phosphorylated (p)-STAT3), western blotting (p-STAT3 and p-NF-κB), and qRT-PCR for cytokine mRNAs. Results CBNP increased CXCL-1 and neutrophils in BALF at both time points evaluated. However, GenX/CBNP co-exposure reduced CBNP-induced CXCL-1 and neutrophils in BALF. Moreover, CXCL-1, CXCL-2 and IL-1β mRNAs were increased by CBNP in lung tissue but reduced by GenX. Western blotting showed that CBNP induced p-NF-κB in lung tissue, while the GenX/CBNP co-exposed group displayed decreased p-NF-κB. Furthermore, mice exposed to GenX or GenX/CBNP displayed increased numbers of BALF macrophages undergoing mitosis and increased Ki67 immunostaining. This was correlated with increased p-STAT3 by western blotting and immunohistochemistry in lung tissue from mice co-exposed to GenX/CBNP. Conclusions Pulmonary exposure to GenX suppressed CBNP-induced innate immune response in the lungs of mice yet promoted the proliferation of macrophages and lung epithelial cells.

Delayed neutrophil apoptosis may enhance NET formation in ARDS

BackgroundAcute respiratory distress syndrome (ARDS) is a neutrophil-associated disease. Delayed neutrophil apoptosis and increased levels of neutrophil extracellular traps (NETs) have been described in ARDS. We aimed to investigate the relationship between these phenomena and their potential as inflammation drivers. We hypothesized that delayed neutrophil apoptosis might enhance NET formation in ARDS.MethodOur research was carried out in three aspects: clinical research, animal experiments, and in vitro experiments. First, we compared the difference between neutrophil apoptosis and NET levels in healthy controls and patients with ARDS and analyzed the correlation between neutrophil apoptosis and NET levels in ARDS. Then, we conducted animal experiments to verify the effect of neutrophil apoptosis on NET formation in Lipopolysaccharide-induced acute lung injury (LPS-ALI) mice. Furthermore, this study explored the relationship between neutrophil apoptosis and NETs at the cellular level. Apoptosis was assessed using morphological analysis, flow cytometry, and western blotting. NET formation was determined using immunofluorescence, PicoGreen assay, SYTOX Green staining, and western blotting.ResultsARDS neutrophils lived longer because of delayed apoptosis, and the cyclin-dependent kinase inhibitor, AT7519, reversed this phenomenon both in ARDS neutrophils and neutrophils in bronchoalveolar lavage fluid (BALF) of LPS-ALI mice. Neutrophils in a medium containing pro-survival factors (LPS or GM-CSF) form more NETs, which can also be reversed by AT7519. Tissue damage can be reduced by promoting neutrophil apoptosis.ConclusionsNeutrophils with extended lifespan in ARDS usually enhance NET formation, which aggravates inflammation. Enhancing neutrophil apoptosis in ARDS can reduce the formation of NETs, inhibit inflammation, and consequently alleviate ARDS.

Salidroside attenuates sepsis-associated acute lung injury through PPP1R15A mediated endoplasmic reticulum stress inhibition

BackgroundThis study explored the role and mechanism of salidroside in sepsis-associated acute lung injury (ALI). MethodSepsis-associated ALI in rats was induced by cecal ligation and perforation method, while lipopolysaccharide (LPS) was used to stimulate cell model. After the treatment of salidroside and dexamethasone (DEX), hematoxylin and eosin staining was applied to evaluate the rat lung injury. Next, the number of total cells and neutrophils in rat bronchoalveolar lavage fluid (BALF) was counted, and the lung wet/dry (W/D) ratio and water content were measured. The neutrophil elastase activity and inflammatory factor levels in BALF were detected by Elastase Assay kit and ELISA. The expressions of PPP1R15A and endoplasmic reticulum (ER) stress-related genes were detected by quantitative reverse transcription polymerase chain reaction and western blot. And the effects of silenced PPP1R15A and tauroursodeoxycholic acid (TUDCA) on cell viability, apoptosis and endoplasmic reticulum (ER) stress were determined by cell counting kit-8 assay, flow cytometry and western blot. ResultThe lung injury, inflammation and edema in sepsis model rats were alleviated by salidroside and DEX, meanwhile salidroside increased the viability and inhibited apoptosis in LPS-treated cells. The expression of PPP1R15A was decreased in sepsis models and increased by salidroside, and salidroside down-regulated the ER stress-related protein expressions in vitro and in vivo. Silenced PPP1R15A reversed the effect of salidroside on cell viability, apoptosis and ER stress, whereas TUDCA could counteract the above effect of silenced PPP1R15A. ConclusionSalidroside targeted PPP1R15A to ameliorate lung injury in sepsis through inhibiting ER stress.

Ablation of PDE4A Exacerbates Pseudomonas aeruginosa‐Induced Acute Lung Injury in Mice

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a family of four isoenzymes, PDE4A to D, that hydrolyze the second messenger cAMP. Three of these enzymes, PDE4A, PDE4B and PDE4D, are widely expressed throughout pulmonary and immune cell types, and their concurrent inhibition with non/PAN‐selective PDE4 inhibitors is well‐known to exert potent anti‐inflammatory benefits. To determine if PDE4 inhibition may represent a therapeutic approach for acute lung injury resulting from severe pneumonia, we explored the effect of genetic ablation of individual PDE4 subtypes, or their concurrent inhibition with PAN‐PDE4 inhibitors, in a model of acute intra‐tracheal Pseudomonas aeruginosa (PA) infection in mice. We have shown recently that selective ablation of PDE4B is sufficient to protect mice from lung injury in this model by reducing pulmonary and systemic levels of pro‐inflammatory cytokines, as well as pulmonary vascular leakage and mortality. With the present study we explored whether selective inactivation of PDE4A or treatment with PAN‐PDE4 inhibitors may provide additional therapeutic benefits.MethodsMice deficient in PDE4A (PDE4A‐KO) and their wildtype (WT) littermate controls were infected intratracheally with the PA lab strain PA01 and subjected 16 h later to bronchoalveolar lavage (BAL), cardiac blood draws, and tissue extractions. Alternatively, vascular/alveolar leakage was assessed by measuring Evans Blue Dye extravasation. To test the effect of PAN‐PDE4 inhibition, mice were treated with the PDE4 inhibitor Piclamilast (5 mg/kg, i.p.) at 2 h prior to PA01 infection.ResultsCompared to WT controls, PDE4A‐KO mice displayed a hyper‐inflammatory phenotype characterized by increased levels of pro‐inflammatory cytokines, immune cells (neutrophils) in BAL fluid, and vascular leakage. Despite these hyperinflammatory responses, the bacterial load in lungs was also increased in PDE4A‐KO mice compared to WT controls. Treatment with a PAN‐PDE4 inhibitor produced incongruent effects, with an increased bacterial load mirroring the effect of PDE4A ablation, whereas reduced levels of inflammatory cytokines such as TNFα mimicked the effect of PDE4B ablation.ConclusionsAblation of PDE4A produces detrimental effects in this model of acute PA‐lung infection. Current studies aim to determine whether an impaired bacterial killing, and the resulting increase in bacterial load, are responsible for the hyperinflammation, or whether these are independent effects of PDE4A ablation. PAN‐PDE4 inhibition mimics some of the beneficial effects of selective PDE4B ablation, but also some of the detrimental effects of PDE4A ablation. These findings suggest that selective inactivation of PDE4B may be a more effective therapeutic approach, compared to PAN‐PDE4 inhibition, in settings of acute PA‐lung infection.

Expression of S100A9 and KL-6 in common interstitial lung diseases.

By evaluating S100 calcium binding protein A9 (S100A9) and Klebs von den Lungen-6 (KL-6) expression in patients with 4 common interstitial lung diseases (ILDs), we aimed to investigate whether S100A9 or KL-6 can be of any value in the differential diagnosis of these ILDs and simultaneously signal the disease progression.We collected the data of patients diagnosed with the 4 ILDs and underwent fiber-optic bronchoscopy and BAL in the First Affiliated Hospital, China Medical University from January 2012 to December 2020. The data related to BGA, C-reactive protein, pulmonary function test, total number and fraction of cells, T lymphocyte subsets in bronchoalveolar lavage fluid (BALF), and the expression of S100A9 and KL-6 in BALF and serum were collected. We analyzed, whether S100A9 or KL-6 could serve as a biomarker for differential diagnosis between the 4 common ILDs; whether the levels of S100A9 and KL-6 correlated with each other; whether they were correlated with other clinical parameters and disease severity.This study included 98 patients, 37 patients with idiopathic pulmonary fibrosis (IPF), 12 with hypersensitivity pneumonitis, 13 with connective tissue disease-associated ILD, and 36 with sarcoidosis (SAR): stage I (18), stage II (9), stage III (5), and stage IV (4). The expression of KL-6 in BALF was significantly higher in IPF patients than other 3 groups (all P-value < .05). However, there was no significant difference in the levels of S100A9 in BALF and serum between the 4 groups (P-value > .05). The levels of S100A9 in BALF of IPF patients was positively and significantly correlated with KL-6 expression and the percentage of neutrophils in BALF (P-value < .05). Along with the stage increase of SAR patients, the level of S100A9 in BALF gradually increased, which was negatively and significantly correlated with the forced vital capacity/predicted, carbon monoxide diffusing capacity/predicted%, and PaO2 (all P-value < .05).The expression of KL-6 in BALF can be used as a biomarker to differentiate IPF from the other 3 common ILDs. While, this was not the case with expression of S100A9 in BALF and serum. However, the expression S100A9 in BALF is useful to indicate the progression of SAR. Thus, simultaneous measurement of KL-6 and S100A9 levels in BALF makes more sense in differential diagnosing of the 4 common ILDS.

Human placental hematopoietic stem cell derived natural killer cells (CYNK-001) mediate protection against influenza a viral infection

ABSTRACT Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells have been shown to play a pivotal role in reducing IAV-induced pulmonary infections in preclinical models; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Here, we investigated the effects of CYNK-001, human placental hematopoietic stem cell derived NK cells that exhibited strong cytolytic activity against a range of malignant cells and expressed high levels of activating receptors, against IAV infections. In a severe IAV-induced acute lung injury model, mice treated with CYNK-001 showed a milder body weight loss and clinical symptoms, which led to a delayed onset of mortality, thus demonstrating their antiviral protection in vivo. Analysis of bronchoalveolar lavage fluid (BALF) revealed that CYNK-001 reduced proinflammatory cytokines and chemokines highlighting CYNK-001’s anti-inflammatory actions in viral induced-lung injury. Furthermore, CYNK-001-treated mice had altered immune responses to IAV with reduced number of neutrophils in BALF yet increased number of CD8+ T cells in the BALF and lung compared to vehicle-treated mice. Our results demonstrate that CYNK-001 displays protective functions against IAV via its anti-inflammatory and immunomodulating activities, which leads to alleviation of disease burden and progression in a severe IAV-infected mice model. The potential of adoptive NK therapy for IAV infections warrants clinical investigation.