Activity Of Alveolar Macrophages Research Articles

Extracellular histones promote TWIK2-dependent potassium efflux and associated NLRP3 activation in alveolar macrophages during sepsis-induced lung injury.

Sepsis-induced acute lung injury (ALI) is a complex and life-threatening condition lacking specific and efficient clinical treatments. Extracellular histones, identified as a novel type of damage-associated molecular patterns, have been implicated in the inflammatory process of ALI. However, further elucidation is needed regarding the precise mechanism through which extracellular histones induce inflammation. The aim of this study was to investigate whether extracellular histones can activate NLRP3 inflammasome-mediated inflammation in alveolar macrophages (AMs) by affecting TWIK2-dependent potassium efflux. We conducted experiments using cecal ligation and puncture (CLP) C57BL/6 mice and extracellular histone-stimulated LPS-primed MH-Scells. The results demonstrated a significant increase in the levels of extracellular histones in the plasma and bronchoalveolar lavage fluid (BALF) of CLP mice. Furthermore, neutralizing extracellular histone mitigated lung injury and inflammation in CLP-induced ALI mice. In vitro studies confirmed that extracellular histones upregulated the expression of NLRP3 inflammasome activation-related proteins in MH-S cells, and this effect was dependent on increased potassium efflux mediated by the TWIK2 channel on the plasma membrane. Moreover, extracellular histones directly triggered a substantial influx of calcium, leading to increased Rab11 activity and facilitating the trafficking and location of TWIK2 to the plasma membrane. These findings underscore the critical role of extracellular histone-induced upregulation of TWIK2 expression on the plasma membrane of alveolar macrophages (AMs). This upregulation leads to potassium efflux and subsequent activation of the NLRP3 inflammasome, ultimately exacerbating lung inflammation and injury during sepsis.

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ Tcell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

Mechanistic insights into targeting caspase-3 activation and alveolar macrophage pyroptosis by Ephedra and bitter almond compounds for treating pediatric pneumonia via network pharmacology and bioinformatics.

This study investigates the molecular mechanism of Ma Huang-Ku Xing Ren, a traditional Chinese medicine formula, in treating pediatric pneumonia. The focus is on the regulation of caspase-3 activation and reduction of alveolar macrophage necrosis through network pharmacology and bioinformatics analyses of Ephedra and bitter almond components. Active compounds and targets from ephedrine and bitter almond were obtained using TCMSP, TCMID, and GeneCards databases, identifying pediatric pneumonia-related genes. A protein-protein interaction (PPI) network was constructed, and core targets were screened. GO and KEGG pathway enrichment analyses identified relevant genes and pathways. An acute pneumonia mouse model was created using the lipopolysaccharide (LPS) inhalation method, with caspase-3 overexpression induced by a lentivirus. The mice were treated with Ephedra and bitter almond through gastric lavage. Lung tissue damage, inflammatory markers (IL-18 and IL-1β), and cell death-related gene activation were assessed through H&E staining, ELISA, western blot, flow cytometry, and immunofluorescence. The study identified 128 active compounds and 121 gene targets from Ephedra and bitter almond. The PPI network revealed 13 core proteins, and pathway analysis indicated involvement in inflammation, apoptosis, and cell necrosis, particularly the caspase-3 pathway. Invivo results showed that Ephedra and bitter almond treatment significantly mitigated LPS-induced lung injury in mice, reducing lung injury scores and inflammatory marker levels. It also decreased caspase-3 activity and cell death in alveolar macrophages. In conclusion, the active ingredients of Ma Huang-Ku Xing Ren, particularly targeting caspase-3, may effectively treat pediatric pneumonia by reducing apoptosis in alveolar macrophages, as demonstrated by both network pharmacology, bioinformatics analyses, and experimental data.

DYNAMIC PORTRAIT OF ALVEOLAR MACROPHAGES IN EXPERIMENTAL DIABETES MELLITUS

SUMMARY. Diabetes mellitus (DM) is a globally prevalent metabolic disorder with profound implications for systemic organs, yet its intricate relationship with the respiratory system, particularly the alveolar macrophages (AM), remains underexplored. The aim – to comprehensively investigate the pathological alterations in AM during the progression of experimental diabetes mellitus. Material and Methods. An experimental investigation was conducted involving 88 male Wistar rats, categorized into intact (Group 1), control (Group 2), and experimental (Group 3) groups. Experimental diabetes was induced using streptozotocin (Sigma, USA), diluted in 0.1 M citrate buffer with a pH of 4.5, at a dose of 60 mg/kg of body weight. Tissue samples were collected at intervals of 14, 28, 42, and 70 days. Electron microscopy analysis was employed to examine lung tissue fragments. Results. On the 14th day, increased functional activity of AMs was observed, marked by distinct nuclear contours and shallow nucleolemic intussusceptions. Subsequent days revealed alterations in mitochondrial morphology, Golgi apparatus, and endoplasmic reticulum, culminating in dystrophic changes by the 70th day. The study period exhibited a dynamic temporal progression, showcasing the multifaceted responses of AMs to diabetic conditions. These responses include heightened functional activity, structural modifications in organelles, and a subsequent shift towards dystrophic changes, providing a comprehensive view of the evolving dynamics of AMs during the course of experimental diabetes mellitus. Conclusions. The study reveals a dynamic temporal progression in alveolar macrophages during the course of experimental diabetes mellitus. Early heightened functional activity transitions into dystrophic changes, providing insights into the evolving functional deficiency of macrophages as diabetes advances, contributing to the understanding of diabetes-related respiratory pathology.

Chronic TNF exposure induces glucocorticoid-like immunosuppression in the alveolar macrophages of aged mice that enhances their susceptibility to pneumonia.

Chronic low-grade inflammation, particularly elevated tumor necrosis factor (TNF) levels, occurs due to advanced age and is associated with greater susceptibility to infection. One reason for this is age-dependent macrophage dysfunction (ADMD). Herein, we use the adoptive transfer of alveolar macrophages (AM) from aged mice into the airway of young mice to show that inherent age-related defects in AM were sufficient to increase the susceptibility to Streptococcus pneumoniae, a Gram-positive bacterium and the leading cause of community-acquired pneumonia. MAPK phosphorylation arrays using AM lysates from young and aged wild-type (WT) and TNF knockout (KO) mice revealed multilevel TNF-mediated suppression of kinase activity in aged mice. RNAseq analyses of AM validated the suppression of MAPK signaling as a consequence of TNF during aging. Two regulatory phosphatases that suppress MAPK signaling, Dusp1 and Ptprs, were confirmed to be upregulated with age and as a result of TNF exposure both exvivo and invitro. Dusp1 is known to be responsible for glucocorticoid-mediated immune suppression, and dexamethasone treatment increased Dusp1 and Ptprs expression in cells and recapitulated the ADMD phenotype. In young mice, treatment with dexamethasone increased the levels of Dusp1 and Ptprs and their susceptibility to infection. TNF-neutralizing antibody reduced Dusp1 and Ptprs levels in AM from aged mice and reduced pneumonia severity following bacterial challenge. We conclude that chronic exposure to TNF increases the expression of the glucocorticoid-associated MAPK signaling suppressors, Dusp1 and Ptprs, which inhibits AM activation and increases susceptibility to bacterial pneumonia in older adults.

Inhalable hybrid nanovaccines with virus-biomimetic structure boost protective immune responses against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with different antigenic variants, has posed a significant threat to public health. It is urgent to develop inhalable vaccines, instead of injectable vaccines, to elicit mucosal immunity against respiratory viral infections. We reported an inhalable hybrid nanovaccine (NVRBD-MLipo) to boost protective immunity against SARS-CoV-2 infection. Nanovesicles derived from genetically engineered 293T cells expressing RBD (NVRBD) were fused with pulmonary surfactant (PS)-biomimetic liposomes containing MPLA (MLipo) to yield NVRBD-MLipo, which possessed virus-biomimetic structure, inherited RBD expression and versatile properties. In contrast to subcutaneous vaccination, NVRBD-MLipo, via inhalable vaccination, could efficiently enter the alveolar macrophages (AMs) to elicit AMs activation through MPLA-activated TLR4/NF-κB signaling pathway. Moreover, NVRBD-MLipo induced T and B cells activation, and high level of RBD-specific IgG and secretory IgA (sIgA), thus elevating protective mucosal and systemic immune responses, while reducing side effects. NVRBD-MLipo also demonstrated broad-spectrum neutralization activity against SARS-CoV-2 (WT, Delta, Omicron) pseudovirus, and protected immunized mice against WT pseudovirus infection. This inhalable NVRBD-MLipo, as an effective and safe nanovaccine, holds huge potential to provoke robust mucosal immunity, and might be a promising vaccine candidate to combat respiratory infectious diseases, including COVID-19 and influenza.

Smoking-Related Interstitial Fibrosis and Smoker's Macrophages.

Smoking-related interstitial lung diseases (SRILDs) are a group of heterogeneous diffuse pulmonary parenchymal diseases associated with tobacco exposure. Smoking-related interstitial fibrosis (SRIF) is relatively recent, a pathologically defined form of SRILDs. SRIF is characterized by the accumulation of macrophages in the alveolar spaces, which is associated with interstitial inflammation and fibrosis. The macrophages frequently contain light brown pigment and are called 'smoker's macrophages'. Patients with SRIF who have clinical evidence of interstitial lung disease are most commonly relatively young, heavy smokers with abnormalities on chest computed tomography showing ground-glass opacities, peripheral consolidation, and reticulation. Although SRIF is caused by cigarette smoking, the exact pathophysiological mechanisms by which smoking causes this type of interstitial fibrosis remain unknown. The degree of fibrosis and appearance of macrophage aggregates are important points of distinction when evaluating and diagnosing SRIF. Macrophage heterogeneity, particularly the activation and function of monocyte-derived alveolar macrophages (Mo-AMs) and interstitial macrophages (IMs), has important implications for the pathogenesis of SRIF and developing treatments. Further researches focused on smoker's macrophages are needed to understand of the pathogenesis of SRIF.

Asbestos Bodies in Human Lung: Localization of Iron and Carbon in the Coating

Asbestos is a term that includes six fibrous mineral phases related to different lung diseases, including asbestosis, lung cancer, and Malignant Pleural Mesothelioma (MPM). Since the last century, these minerals have been widely studied under their mineralogical/chemical and physical aspects with in vivo and in vitro studies to understand the mechanisms of their carcinogenicity. There are several techniques described in the literature, as optical and electron microscopies, for the identification of coated (asbestos bodies, ABs) and uncoated fibers, but only micro-Raman spectroscopy permits a sure characterization of these minerals—and of the related phases—directly in the histological sections of pulmonary parenchyma without any manipulation. In this case, the risk of the loss of associated inorganic phases from asbestos bodies (ABs) and fibers (e.g.: iron or carbonaceous micro-particles) is avoided. Asbestos bodies are produced by the activity of alveolar macrophages with degradation/inactivation of asbestos fibers. Inside the alveolar macrophages, organic and inorganic material settles on the foreign fibers forming an iron-rich proteic and carbonaceous coating. In this study, Variable Pressure Scanning Electron Microscopy with annexed Electron Dispersive Spectroscopy (VP-SEM/EDS) and micro-Raman spectroscopy were applied to the characterization of the phases in the ABs. Characterization of carbonaceous materials (CMs), observed in pristine asbestos phases in previous works, was therefore performed, addressing the micro-Raman laser beam on different points of the asbestos bodies, and Raman mappings on ABs were carried out for the first time. Coupling the data obtained by VP-SEM/EDS and micro-Raman spectroscopy, it was possible to collect information about the iron and carbonaceous phases adhered to the fibers, probably lost during the classical tissue digestion procedures. Information about both mineral and carbonaceous components might be useful to understand the whole structure of “asbestos bodies” and the inflammogenic and carcinogenic effects of the asbestos phases coupled to CMs, that might derive from cigarette smoke or from environmental pollution; this study might be useful to deepen also the possible detrimental role of ABs in the tissues.

BCG immunization induces CX3CR1hi effector memory T cells to provide cross-protection via IFN-γ-mediated trained immunity.

After a century of using the Bacillus Calmette-Guérin (BCG) vaccine, our understanding of its ability to provide protection against homologous (Mycobacterium tuberculosis) or heterologous (for example, influenza virus) infections remains limited. Here we show that systemic (intravenous) BCG vaccination provides significant protection against subsequent influenza A virus infection in mice. We further demonstrate that the BCG-mediated cross-protection against influenza A virus is largely due to the enrichment of conventional CD4+ effector CX3CR1hi memory αβ T cells in the circulation and lung parenchyma. Importantly, pulmonary CX3CR1hi T cells limit early viral infection in an antigen-independent manner via potent interferon-γ production, which subsequently enhances long-term antimicrobial activity of alveolar macrophages. These results offer insight into the unknown mechanism by which BCG has persistently displayed broad protection against non-tuberculosis infections via cross-talk between adaptive and innate memory responses.

Paralemmin-3 Augments Lipopolysaccharide-Induced Acute Lung Injury with M1 Macrophage Polarization via the Notch Signaling Pathway

BackgroundAcute lung injury (ALI) involves severe lung damage and respiratory failure, which are accompanied by alveolar macrophage (AM) activation. The aim of this article is to verify the influence of paralemmin-3 (PALM3) on alveolar macrophage (AM) polarization in ALI and the underlying mechanism of action. MethodsAn ALI rat model was established by successive lipopolysaccharide (LPS) inhalations. The influence of PALM3 on the survival rate, severity of lung injury, and macrophage polarization was analyzed. Furthermore, we explored the underlying mechanism of PALM3 in regulating macrophage polarization. ResultsPALM3 overexpression increased mortality of ALI rats, augmented lung pathological damage, and promoted AM polarization toward M1 cells. Conversely, PALM3 knockdown had the opposite effects. Mechanistically, PALM3 might promote M1 polarization by acting as an adaptor to facilitate transduction of Notch signaling. ConclusionPALM3 aggravates lung injury and induces macrophage polarization toward M1 cells by activating the Notch signaling pathway in LPS-induced ALI, which may shed light on ALI/ARDS treatments.

Secretion of related factors and AMPK/PGC-1 α signal pathway by alveolar macrophages in viral acute lung injury.

This study was to investigate the secretion of related factors by alveolar macrophages and AMPK/PGC-1 α signal pathway in viral acute lung injury (VALI) patients. 30 SD rats were randomly divided into blank control group (BCG) and VALI group (VALIG) with 15 rats in each group. The model of VALIG was induced by polyI: C and the BCG was given the same amount of normal saline. The IL-6, IL-8 and TNF-α levels in bronchoalveolar lavage fluid (BALF) were detected by ELISA. The protein, surface markers of alveolar macrophages, and related mRNA expression were detected by Western blot, ELISA, and qRT-PCR. IL-8, IL-6, TNF-α, MUC5AC and TLR4 levels in VALIG were raised than those in BCG (P<0.05), while the AQP5 level was reduced than that in the BCG (P<0.05). The Bcl-2 protein level in the VALIG was reduced than that in the BCG (P<0.05), while the Caspase3 protein level was raised than that in the BCG (P<0.05). The AMPK and PGC-1 α mRNA and protein expression level in the VALIG rat lung tissue was lower than that of the BCG (P<0.05) VALIG is related to inflammatory damage, activation of alveolar macrophages, and secretion of related factors by alveolar macrophages. This may be related to AMPK/PGC-1 α signal pathways.

Self-Propelled Proteomotors with Active Cell-Free mtDNA Clearance for Enhanced Therapy of Sepsis-Associated Acute Lung Injury.

Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis-associated ALI can help develop new therapeutic strategies. Herein, the crucial role of cell-free mitochondrial DNA (cf-mtDNA) in the regulation of alveolar macrophage activation during sepsis-associated ALI is identified. Most importantly, a biocompatible hybrid protein nanomotor (NM) composed of recombinant deoxyribonuclease I (DNase-I) and human serum albumin (HSA) via glutaraldehyde-mediated crosslinking is prepared to obtain an inhalable nanotherapeutic platform targeting pulmonary cf-mtDNA clearance. The synthesized DNase-I/HSA NMs are endowed with self-propulsive capability and demonstrate superior performances in stability, DNA hydrolysis, and biosafety. Pulmonary delivery of DNase-I/HSA NMs effectively eliminates cf-mtDNAs in the lungs, and also improves sepsis survival by attenuating pulmonary inflammation and lung injury. Therefore, pulmonary cf-mtDNA clearance strategy using DNase-I/HSA NMs is considered to be an attractive approach for sepsis-associated ALI.

Jingfang Granule alleviates bleomycin-induced acute lung injury via CD200-CD200R immunoregulatory pathway.

Jingfang granules (JF), one famous traditional Chinese formula in "She Sheng Zhong Miao Fang" written by Shi-Che Zhang during the Ming Dynasty era, has been widely used to prevent epidemic diseases in history and now was recommended for the treatment of coronavirus disease 2019 (COVID-19) in China. However, the roles of JF against acute lung injury and its mechanisms remain unclear. Acute lung injury (ALI) and its progressive acute respiratory distress syndrome (ARDS) are a continuum of lung inflammatory disease with high morbidity and mortality in clinic, especially in COVID-19 patients. The present study aims to investigate the effect of JF on ALI and clarify its underlying mechanisms for clinical application in COVID-19 control. Bleomycin-induced ALI mice were given oral gavage daily for seven days with or without Jingfang granules (2, 4g/kg). The body weight, lung wet/dry weight ratios, lung appearance and tissue histopathology were evaluated. Quantitative real-time PCR, biochemical bronchoalveolar lavage fluids analysis was used to determine the gene expression of proinflammation factor and infiltrated inflammatory cells in lung. Immunofluorescence image and western blot were used to detect the markers of alveolar macrophages (AMs), endothelial cell apoptosis and changes of CD200-CD200R pathway. Firstly, histopathological analysis showed that JF significantly attenuated pulmonary injury and inflammatory response in ALI mice. Then, cytokine detection, inflammatory cells assay, and JNKs and p38 pathway analysis indicated that the recruitment and activation of alveolar macrophages was the main reason to cause ALI and JF could reverse this variation. Next, immunofluorescence staining and TUNEL assay showed that JF upregulated the expression of CD200 and suppressed the apoptosis of alveolar endothelial cells. Finally, double immunofluorescence staining of CD200 and CD11c indicated that the seriously damaged tissue had the lower CD200 while more AMs infiltration, which was confirmed by RT-PCR analysis of CD200/CD200R. Jingfang granules can protect lung from acu te injury and mitigate the recruitment and overactive AMs-induced inflammation via CD200-CD200R immunoregulatory signal axis, which will provide an experimental basis for Jingfang granules clinical applications in COVID-19.

RNF128 regulates neutrophil infiltration and myeloperoxidase functions to prevent acute lung injury

Acute lung injury (ALI) is characterised by severe pulmonary inflammation, alveolar-capillary barrier disruption, and pulmonary oedema. Therefore, establishing effective therapeutic targets for ALI prevention is crucial. The present study reports a novel function of RNF128 in regulating LPS-induced ALI. Severe lung damage and increased immune cell infiltration were detected in RNF128-deficient mice. In vitro experiments revealed that RNF128 inhibits neutrophil activation by binding to myeloperoxidase (MPO) and reducing its levels and activity. Moreover, RNF128 regulates alveolar macrophage activation and neutrophil infiltration by interacting with TLR4, targeting it for degradation, and inhibiting NF-κB activation, hence decreasing pro-inflammatory cytokines. Our results demonstrate for the first time that RNF128 is a negative regulator of MPO and TLR4 in neutrophils and alveolar macrophages, respectively. However, AAV9-mediated RNF128 overexpression alleviated lung tissue damage and reduced inflammatory cell infiltration. Thus, RNF128 is a promising therapeutic candidate for pharmacological interventions in ALI.

Verbenalin alleviates acute lung injury induced by sepsis and IgG immune complex through GPR18 receptor

Acute lung injury is significantly associated with the aberrant activation and pyroptosis of alveolar macrophages. Targeting the GPR18 receptor presents a potential therapeutic approach to mitigate inflammation. Verbenalin, a prominent component of Verbena in Xuanfeibaidu (XFBD) granules, is recommended for treating COVID-19. In this study, we demonstrate the therapeutic effect of verbenalin on lung injury through direct binding to the GPR18 receptor. Verbenalin inhibits the activation of inflammatory signaling pathways induced by lipopolysaccharide (LPS) and IgG immune complex (IgG IC) via GPR18 receptor activation. The structural basis for verbenalin's effect on GPR18 activation is elucidated through molecular docking and molecular dynamics simulations. Furthermore, we establish that IgG IC induces macrophage pyroptosis by upregulating the expression of GSDME and GSDMD through CEBP-δ activation, while verbenalin inhibits this process. Additionally, we provide the first evidence that IgG IC promotes the formation of neutrophil extracellular traps (NETs), and verbenalin suppresses NETs formation. Collectively, our findings indicate that verbenalin functions as a “phytoresolvin” to promote inflammation regression and suggests that targeting the C/EBP-δ/GSDMD/GSDME axis to inhibit macrophage pyroptosis may represent a novel strategy for treating acute lung injury and sepsis.

Modulation of Alveolar Macrophage Activity by Eugenol Attenuates Cigarette-Smoke-Induced Acute Lung Injury in Mice.

This study investigates the role of eugenol (EUG) on CS-induced acute lung injury (ALI) and how this compound is able to modulate macrophage activity. C57BL/6 mice were exposed to 12 cigarettes/day/5days and treated 15 min/day/5days with EUG. Rat alveolar macrophages (RAMs) were exposed to CSE (5%) and treated with EUG. In vivo, EUG reduced morphological changes inflammatory cells, oxidative stress markers, while, in vitro, it induced balance in the oxidative stress and reduced the pro-inflammatory cytokine release while increasing the anti-inflammatory one. These results suggest that eugenol reduced CS-induced ALI and acted as a modulator of macrophage activity.

Alveolar macrophage-derived secretory autophagosomes exacerbate inflammation in lipopolysaccharide-induced acute respiratory distress syndrome

Background: Acute respiratory distress syndrome (ARDS) has a high incidence among patients in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) contribute to ARDS-associated lung injury; however, the mechanisms underlying the inflammatory process remain unclear. Purpose: This study investigated the effects and the mechanisms through which AM-derived secretory autophagosomes (SAPs) contribute to ARDS-associated lung injury. Methods: SAPs were isolated from cell culture supernatants of AMs treated or untreated with lipopolysaccharide (LPS) and intratracheally injected to determine whether SAP exacerbated lung injury in mice with ARDS. The SAP proteome was analysed using mass spectrometry. Lung fibroblasts were treated with SAPs and collected for transcriptome sequencing. A transwell assay was conducted to study the chemotactic activity of fibroblasts induced by SAPs. Results: We found that AMs contributed to ARDS-associated lung injury by releasing a novel type of pro-inflammatory vesicles named SAPs, which were characterised as double-membraned vesicles approximately 200 nm in diameter and light-chain-3 expression. Proteomic analysis of SAPs and gene ontology (GO) enrichment analysis revealed that LPS-SAPs contain several differentially expressed proteins involved in effecting an inflammatory response, including interleukin (IL)-1β. We confirmed this result by quantifying IL-1β in SAPs using ELISA. Moreover, administering SAPs from LPS-stimulated macrophages to mice with ARDS exacerbated lung injury, whereas IL-1 receptor antagonist (IL-1RA) pre-treatment significantly ameliorated SAP-induced lung injury. In addition, we conducted transcriptome sequencing of lung fibroblasts treated with SAPs to explore the effect of SAPs on fibroblasts in ARDS, which demonstrated that the expression of several chemokines (CXCL2, CXCL15, and so forth) significantly increased with SAP stimulation. Furthermore, the transwell assay indicated that SAPs could enhance the chemotactic activity of fibroblasts, suggesting that SAPs may exacerbate inflammation in ARDS by promoting neutrophil or monocyte recruitment. Conclusion: This is the first study to show that AM-derived SAPs contribute significantly to ARDS by secreting pro-inflammatory cytokines and promoting inflammatory cell recruitment by activating fibroblasts, which may be beneficial for therapeutic development of ARDS. National Natural Science Foundation of China (Grant numbers 81870066, 81670074, 81930058, 82270083 and 81971888), Six Talent Peaks Project in Jiangsu Province(Grant numbers TD-SWYY-003), and Social Development Specifc Projects of Jiangsu Province [Grant number BE2020786]. This is the full abstract presented at the American Physiology Summit 2023 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.

Mechanisms of pulmonary microvascular endothelial cells barrier dysfunction induced by LPS: The roles of ceramides and the Txnip/NLRP3 inflammasome.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are characterized by pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction and proinflammatory cytokine influx into lung tissue, resulting in pulmonary oedema. Ceramide overproduction is an important mediator of pulmonary hyperinflammation and pulmonary oedema in Acute lung injury (ALI). Ceramides induce NLRP3 inflammasome activation are essential for the hyperinflammatory response. However, the roles and specific mechanisms of ceramide-induced NLRP3 inflammasome activation, proinflammatory cytokine manufacturing and PMVECs barrier dysfunction in ALI are unclear. Herein, pretreatment with the acid sphingomyelinase (ASMase) inhibitor imipramine (but not a neutral sphingomyelinase (NSMase) inhibitor or de novo pathway inhibitor) significantly inhibited ceramide early production in rats with lipopolysaccharide (LPS)-induced ALI; Furthermore, the Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury were significantly decreased. Verapamil, a Txnip inhibitor, substantially inhibited Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury in rats with C8-ceramide-induced ALI. In vitro, short hairpin RNA-mediated Txnip silencing significantly inhibited C8-ceramide-induced Txnip/NLRP3 inflammasome activation in NR8383 alveolar macrophages (AMs) and early secretion of the proinflammatory cytokines IL-1β (4-12h) as well as IL-6 and TNF-α at subsequent times (later than 12h). However, C8-ceramide significantly increased the early secretion (within 8h) of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in a co-culture model of NR8383 AMs and PMVECs, and Txnip silencing of NR8383 AMs inhibited the secretion of pro-inflammatory cytokines and reduced cytoskeletal rearrangements, intercellular connection breakage and hyperpermeability in PMVECs. Overall, our results suggest that in LPS-induced ALI, ceramide-mediated Txnip/NLRP3 inflammasome activation in NR8383 AMs leads to early IL-1β release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI.

TLR2-hif1α-mediated glycolysis contributes to pyroptosis and oxidative stress in allergic airway inflammation.

As a pattern recognition receptor which activates innate immune system, toll-like receptor 2 (TLR2) has been reportedly mediates allergic airway inflammation (AAI), yet the underlying mechanism remains elusive. Here, in a murine AAI model, TLR2-/- mice showed decreased airway inflammation, pyroptosis and oxidative stress. RNA-sequencing revealed that allergen-induced hif1 signaling pathway and glycolysis were significantly downregulated when TLR2 was deficient, which were confirmed by lung protein immunoblots. Glycolysis inhibitor 2-Deoxy-d-glucose (2-DG) inhibited allergen-induced airway inflammation, pyroptosis, oxidative stress and glycolysis in wild type (WT) mice, while hif1α stabilizer ethyl 3,4-dihydroxybenzoate (EDHB) restored theses allergen-induced changes in TLR2-/- mice, indicating TLR2-hif1α-mediated glycolysis contributes to pyroptosis and oxidative stress in AAI. Moreover, upon allergen challenge, lung macrophages were highly activated in WT mice but were less activated in TLR2-/- mice, 2-DG replicated while EDHB reversed such effect of TLR2 deficiency on lung macrophages. Likewise, both in vivo and ex vivo WT alveolar macrophages (AMs) exhibited higher TLR2/hif1α expression, glycolysis and polarization activation in response to ovalbumin (OVA), which were all inhibited in TLR2-/- AMs, suggesting AMs activation and metabolic switch are dependent on TLR2. Finally, depletion of resident AMs in TLR2-/- mice abolished while transfer of TLR2-/- resident AMs to WT mice replicated the protective effect of TLR2 deficiency on AAI when administered before allergen challenge. Collectively, we suggested that loss of TLR2-hif1α-mediated glycolysis in resident AMs ameliorates allergic airway inflammation that inhibits pyroptosis and oxidative stress, therefore the TLR2-hif1α-glycolysis axis in resident AMs may be a novel therapeutic target for AAI.

Mechanical signaling regulates NLRP3 activation in macrophages

Abstract Macrophage controls infection and inflammation via releasing pro-inflammatory cytokine IL-1β. NLRP3 inflammasomes assembly triggers a signaling cascade to process pro-IL-1β in the active form via activating caspase-1. In the lungs, dysregulated NLRP3 activation escalates inflammatory diseases such as acute lung injury (ALI), ventilator-induced lung injury (VILI), and idiopathic pulmonary fibrosis (IPF). These inflammatory disorders are associated with changing tissue compliance/stiffness due to accumulated extracellular matrix (ECM). Tissue-resident macrophages interaction with ECM via integrins transduce “mechanotransduction” signaling. However, the molecular mechanism of how mechanotransduction cross-talks with NLRP3 pathways and inflammatory response is not clear yet. We are examining NLRP3 activation in lung resident alveolar macrophages (AMs) during mechanical stress. For that, we culture macrophages on collagen-coated surfaces with altered stiffness (elastic modulus of 0.2 to 64 Kilo Pascals (kPa)). We observed that the NLRP3 inflammasome pathway is responsive to surface stiffness and IL-1β processing suppressed by a stiffer surface (64 kPa) when compared to a softer surface (0.2 kPa). Similarly, culture surface stiffness also regulates monocyte-to-macrophage differentiation. AMs show altered transcriptome signatures with changing the stiffness and favored activated macrophage phenotype at 0.2 kPa. Further, activation of the integrin-induced signaling also reduced the NLRP3-induced IL-1β release. Future examination of adhesion-based integrin-induced mechanical signaling and NLRP3 pathway will reveal the therapeutic potential in associated inflammatory diseases such as ALI/fibrosis. National Institutes of Health grants R01- AI104732, R21EB030171, R01 AI118719, HL148033, R01AI139540, and R56 AI104732, National Science Foundation grants CBET-1900277 and CMMI1548571, American Lung Association grant ETRA 736343, Washington University in Saint Louis Children’s Discovery Institute grants CDI-CORE-2019-813 and CDI-CORE-2015-505, Foundation for Barnes-JewishHospital grants 3770 and 4642, American Association of Immunologists AAICareers in immunology fellowship.