Resistin alleviates lipopolysaccharide-induced inflammation in bovine alveolar macrophages by activating the AMPK/mTOR signaling pathway and autophagy.

Objective: Resistin (RETN) is an adipocyte-specific hormone that participates in metabolism and modulates cellular inflammation. Our study aimed to assess the effects of RETN treatment on autophagy and the underlying molecular and biological mechanisms in bovine alveolar macrophages (BAMs). Methods: The optimal concentration of RETN + lipopolysaccharide (LPS) on macrophages was screened and then used to co-culture with alveolar macrophages. Autophagosomes in BAMs were examined using a transmission electron microscope (TEM). Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of microtubule-associated protein light chain 3 (LC3) and p62. Western blot (WB) was used to detect the protein expressions of LC3 and p62. The distribution of LC3 and p62 proteins in the cells was observed by immunofluorescence (IF). The concentrations of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expression of adenosine-monophosphate-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), and p-mTOR was detected using WB. Results: The treatment of BAMs with RETN or LPS increased the number of autophagosomes and the ratio of LC3II/LC3I and decreased the expression level of p62 protein. RETN treatment significantly triggered autophagy compared to LPS treatment. Moreover, the ratios of p-AMPK/AMPK and p-mTOR/mTOR were upregulated and downregulated, respectively, after RETN treatment, suggesting that AMPK/mTOR signaling pathway activation is required for RETN-mediated autophagy in BAMs. Additionally, the ratio of LC3-II/LC3-I was lower, and the concentrations of IL-1ß, IL-6, and TNF-α significantly decreased in the LPS and RETN co-treatment groups compared to the single LPS treatment group. However, both autophagy- and LPS-induced inflammation were partially alleviated by RETN treatment. Conclusion: RETN can promote autophagy in BAMs by activating the AMPK/mTOR signaling pathway, it may help prevent LPS-induced inflammation.

Dietary Aflatoxin G1 exposure causes an imbalance between pulmonary tissue-resident alveolar macrophages and monocyte-derived macrophages in both mother and offspring mice.

Aflatoxin G1 (AFG1) is a mycotoxin commonly found in agricultural products, including dried fruits, meat, and milk products. Oral AFG1 administration induced tumor necrosis factor (TNF)-α-dependent chronic pulmonary inflammation, promoting AFG1-induced damage in alveolar epithelial cell, which is associated with lung adenocarcinoma. Pulmonary macrophages may be divided into tissue-resident alveolar macrophages (TRAMs) and monocyte-derived macrophages (MoMs), which involve in chronic lung inflammation. However, whether these macrophages contribute to AFG1-induced chronic pulmonary inflammation remains unknown. In this study, we found oral AFG1 administration disrupted the balance between TRAMs and MoMs, increasing MoMs infiltration and decreasing the number of TRAMs. AFG1 upregulated TNF-α expression in MoMs, but downregulated sialic acid binding Ig-like lectin F (Siglec-F) expression in TRAMs. Inhibition of TNF-α-dependent inflammation rescued the imbalance between TRAMs and MoMs in AFG1-treated lung tissues. Additionally, AFG1 stimulated MoMs differentiation to the proinflammatory M1 phenotype in vitro. Using a specific in vitro TRAM model, AFG1 downregulated Siglec-F and the M2 phenotypic markers arginase 1 and YM1, and upregulated the M1 phenotypic markers IL-6, iNOS and TNF-α, altering the TRAMs phenotype to the pro-inflammatory M1 phenotype in vitro. Additionally, mouse maternal dietary exposure to AFG1 caused an imbalance in pulmonary macrophages, decreasing TRAMs and increasing MoMs population in offspring, which was associated with proliferative lesions in the alveolar septa. Thus, dietary AFG1 exposure triggered an imbalance in pulmonary macrophages in both mother and offspring mice, and induced pro-inflammatory phenotypic alterations, which contributed to AFG1-induced chronic lung inflammation. These results provide clues to how AFG1-induced immunotoxicity and genotoxicity in humans might be prevented.

Nebulized Immunotherapy of Orthotopic Lung Cancer by Mild Magnetothermal-based Innate Immunity Activations.

Advances in adaptive immunity have greatly contributed to the development of cancer immunotherapy. However, its over-low efficacy and insufficient invasion of immune cells in the tumor tissue, and safety problems caused by cytokine storm, have seriously impeded further clinical application for solid tumor immunotherapy. Notably, the immune microenvironment of the lungs is naturally enriched with alveolar macrophages (AMs). Herein, we introduce a novel nebulized magnetothermal immunotherapy strategy to treat orthotopic lung cancer by using magnetothermal nanomaterial (Zn-CoFe2O4@Zn-MnFe2O4-PEG, named ZCMP), which can release iron ions via an acid/thermal-catalytic reaction to maximize the use of lung's immune environment through the cascade activations of AMs and natural killer (NK) cells. Nebulized administration greatly enhance drug bioavailability by localized drug accumulation at the lesion site. Upon mild magnetic hyperthermia, the released iron ions catalyze endogenous H2O2 decomposition to produce reactive oxygen species (ROS), which triggers the M1 polarization of AMs, and the resultant inflammatory cytokine IFN-ß, IL-1ß and IL-15 releases to activate c-Jun, STAT5 and GZMB related signaling pathways, promoting NK cells proliferation and activation. This innovative strategy optimally utilizes the lung's immune environment and shows excellent immunotherapeutic outcomes against orthotopic lung cancer.

Alveolar macrophage function is impaired following inhalation of berry e-cigarette vapor.

In the lower respiratory tract, the alveolar spaces are divided from the bloodstream and the external environment by only a few microns of interstitial tissue. Alveolar macrophages (AMs) defend this delicate mucosal surface from invading infections by regularly patrolling the site. AMs have three behavior modalities to achieve this goal: extending cell protrusions to probe and sample surrounding areas, squeezing the whole cell body between alveoli, and patrolling by moving the cell body around each alveolus. In this study, we found Rho GTPase, cell division control protein 42 (CDC42) expression significantly decreased after berry-flavored e-cigarette (e-cig) exposure. This shifted AM behavior from squeezing to probing. Changes in AM behavior led to a reduction in the clearance of inhaled bacteria, Pseudomonas aeruginosa. These findings shed light on pathways involved in AM migration and highlight the harmful impact of e-cig vaping on AM function.

Preconditioning with ß-hydroxybutyrate attenuates lung ischemia-reperfusion injury by suppressing alveolar macrophage pyroptosis through the SIRT1-FOXO3 signaling pathway.

The complex pathogenesis of lung ischemia-reperfusion injury (LIRI) was examined in a murine model, focusing on the role of pyroptosis and its exacerbation of lung injury. We specifically examined the levels and cellular localization of pyroptosis within the lung, which revealed alveolar macrophages as the primary site. The inhibition of pyroptosis by VX-765 reduced the severity of lung injury, underscoring its significant role in LIRI. Furthermore, the therapeutic potential of ß-hydroxybutyrate (ß-OHB) in ameliorating LIRI was examined. Modulation of ß-OHB levels was evaluated by ketone ester supplementation and 3-hydroxybutyrate dehydrogenase 1 (BDH-1) gene knockout, along with the manipulation of the SIRT1-FOXO3 signaling pathway using EX-527 and pCMV-SIRT1 plasmid transfection. This revealed that ß-OHB exerts lung-protective and anti-pyroptotic effects, which were mediated through the upregulation of SIRT1 and the enhancement of FOXO3 deacetylation, leading to decreased pyroptosis markers and lung injury. In addition, ß-OHB treatment of MH-S cells in vitro showed a concentration-dependent improvement in pyroptosis, linking its therapeutic benefits to specific cell mechanisms. Overall, this study highlights the significance of alveolar macrophage pyroptosis in the exacerbation of LIRI and indicates the potential of ß-OHB in mitigating injury by modulating the SIRT1-FOXO3 signaling pathway.

The Omega-6 Lipid pathway shift is associated with neutrophil influx and structural lung damage in early cystic fibrosis lung disease.

Objectives: In cystic fibrosis (CF), an imbalanced lipid metabolism is associated with lung inflammation. Little is known about the role that specific lipid mediators (LMs) exert in CF lung inflammation, and whether their levels change during early disease progression. Therefore, we measured airway LM profiles of young CF patients, correlating these with disease-associated parameters. Methods: Levels of omega (ω)-3/6 PUFAs and their LM derivatives were determined in bronchoalveolar lavage fluid (BALF) of children with CF ages 1-5 using a targeted high-performance liquid chromatography-tandem mass spectrometry approach. Hierarchical clustering analysis was performed on relative LM levels. Individual relative LM levels were correlated with neutrophilic inflammation (BALF %Neu) and structural lung damage (PRAGMA-CF %Disease). Significant correlations were included in a backward multivariate linear regression model to identify the LMs that are best related to disease progression. Results: A total of 65 BALF samples were analysed for ω-3/6 lipid content. LM profiles clustered into an arachidonic acid (AA)-enriched and a linoleic acid (LA)-enriched sample cluster. AA derivatives like 17-OH-DH-HETE, 5-HETE, 5,15-diHETE, 15-HETE, 15-KETE, LTB4 and 6-trans-LTB4 positively correlated with BALF %Neu and/or PRAGMA %Dis. Contrastingly, 9-HoTrE and the LA derivatives 9-HoDE, 9(10)-EpOME, 9(10)-DiHOME, 13-HoDE, 13-oxoODE and 12(13)-EpOME negatively correlated with BALF %Neu and/or PRAGMA %Dis. 6-trans-LTB4 was the strongest predictor for BALF %Neu. 5-HETE and 15-KETE contributed most to PRAGMA %Dis prediction. Conclusions: Our data provide more insight into the lung lipidome of infants with CF, and show that a shift from LA derivatives to AA derivatives in BALF associates with early CF lung disease progression.

Potential role of alveolar macrophages in HIV persistence and lung disease.

While antiretroviral therapy (ART) has revolutionized the management of human immunodeficiency virus (HIV) and has enabled people living with HIV (PLWH) to achieve near-normal life expectancies, an HIV cure remains elusive due to the presence of HIV reservoirs. Furthermore, compared with individuals in the general population, PLWH support a higher burden of multimorbidity, including pulmonary diseases of both an infectious and non-infection nature, which may be a consequence of the formation of HIV reservoirs. Their gut, lymph nodes, brain, testes and lungs constitute important anatomic sites for the reservoirs. While CD4+ T cells, and particularly memory CD4+ T cells, are the best characterized cellular HIV reservoirs, tissue resident macrophages (TRM) and alveolar macrophages (AM) also harbor HIV infection. AM are the most abundant cells in bronchoalveolar (BAL) fluid in healthy conditions, and act as sentinels in the alveolar space by patrolling and clearing debris, microbes and surfactant recycling. Long-lived tissue-resident AM of embryonic origin have the capacity of self-renewal without replenishment from peripheral monocytes. As in other tissues, close cell-cell contacts in lungs also provide a milieu conducive for cell-to-cell spread of HIV infection and establishment of reservoirs. As lungs are in constant exposure to antigens from the external environment, this situation contributes to pro-inflammatory phenotype rendering pulmonary immune cells exhausted and senescent-an environment facilitating HIV persistence. Factors such as tobacco and e-cigarette smoking, lung microbiome dysbiosis and respiratory coinfections further drive antigenic stimulation and HIV replication. HIV replication, in turn, contributes to ongoing inflammation and clonal expansion. Herein, the potential role of AM in HIV persistence is discussed. Furthermore, their contribution towards pulmonary inflammation and immune dysregulation, which may in turn render PLWH susceptible to chronic lung disease, despite ART, is explored. Finally, strategies to eliminate HIV-infected AM are discussed.

[Potential role of alveolar macrophages in HIV persistence and lung disease]. / Rôle potentiel des macrophages alvéolaires dans la persistance du VIH et les maladies pulmonaires.

While antiretroviral therapy (ART) has revolutionized the management of human immunodeficiency virus (HIV) and has enabled people living with HIV (PLWH) to achieve near-normal life expectancies, an HIV cure remains elusive due to the presence of HIV reservoirs. Furthermore, compared with individuals in the general population, PLWH support a higher burden of multimorbidity, including pulmonary diseases of both an infectious and non-infection nature, which may be a consequence of the formation of HIV reservoirs. Their gut, lymph nodes, brain, testes and lungs constitute important anatomic sites for the reservoirs. While CD4+ T-cells, and particularly memory CD4+ T-cells, are the best characterized cellular HIV reservoirs, tissue resident macrophages (TRM) and alveolar macrophages (AM) also harbor HIV infection. AM are the most abundant cells in bronchoalveolar (BAL) fluid in healthy conditions, and act as sentinels in the alveolar space by patrolling and clearing debris, microbes and surfactant recycling. Long-lived tissue-resident AM of embryonic origin have the capacity of self-renewal without replenishment from peripheral monocytes. As in other tissues, close cell-cell contacts in lungs also provide a milieu conducive for cell-to-cell spread of HIV infection and establishment of reservoirs. As lungs are in constant exposure to antigens from the external environment, this situation contributes to pro-inflammatory phenotype rendering pulmonary immune cells exhausted and senescent-an environment facilitating HIV persistence. Factors such as tobacco and e-cigarette smoking, lung microbiome dysbiosis and respiratory co-infections further drive antigenic stimulation and HIV replication. HIV replication, in turn, contributes to ongoing inflammation and clonal expansion. Herein, the potential role of AM in HIV persistence is discussed. Furthermore, their contribution towards pulmonary inflammation and immune dysregulation, which may in turn render PLWH susceptible to chronic lung disease, despite ART, is explored. Finally, strategies to eliminate HIV-infected AM are discussed.

Shengmai Powder regulates alveolar macrophage PPAR-γ and improves the chronic inflammatory state of chronic obstructive pulmonary disease.

This study examines the therapeutic effects of Shengmai Powder (SMP) on both in vitro and in vivo models of chronic obstructive pulmonary disease (COPD) and the underlying mechanisms. Cigarette smoke and cigarette extracts were used to create in vitro and in vivo models of COPD. ELISA was used to measure the levels of pro-inflammatory factors (IL-6, TNF-α, and IL-1ß) in mouse lung tissue and alveolar macrophages. Flow cytometry assessed the phagocytic capacity of alveolar macrophage. Western blotting was used to analyze the expression of RhoA, PPARγ, IκBα, p-IκBα, P65, and p-P65 in alveolar. The results show that SMP reversed the increased levels of pro-inflammatory factors (IL-6, TNF-α, and IL-1ß) in mouse lung tissue and alveolar macrophages induced by cigarette smoke and cigarette extract. SMP also restored the decreased fluorescence intensity and RhoA levels in alveolar macrophages caused by cigarette extract. Additionally, SMP increased PPARγ expression and decreased IκBα and P65 phosphorylation in alveolar macrophages exposed to cigarette extract. Also, the effects of SMP were reversed by PPARγ inhibitors. The study concluded that SMP regulates alveolar macrophage phagocytic function through the PPAR-γ/NF-κB pathway, thereby improving the chronic inflammatory state of COPD.

Spatial and phenotypic heterogeneity of resident and monocyte-derived macrophages during inflammatory exacerbations leading to pulmonary fibrosis.

Introduction: Genetic mutations in critical nodes of pulmonary epithelial function are linked to the pathogenesis of pulmonary fibrosis (PF) and other interstitial lung diseases. The slow progression of these pathologies is often intermitted and accelerated by acute exacerbations, complex non-resolving cycles of inflammation and parenchymal damage, resulting in lung function decline and death. Excess monocyte mobilization during the initial phase of an acute exacerbation, and their long-term persistence in the lung, is linked to poor disease outcome. Methods: The present work leverages a clinical idiopathic PF dataset and a murine model of acute inflammatory exacerbations triggered by mutation in the alveolar type-2 cell-restricted Surfactant Protein-C [SP-C] gene to spatially and phenotypically define monocyte/macrophage changes in the fibrosing lung. Results: SP-C mutation triggered heterogeneous CD68+ macrophage activation, with highly active peri-injured cells relative to those sampled from fully remodeled and healthy regions. Ingenuity pathway analysis of sorted CD11b-SigF+CD11c+ alveolar macrophages defined asynchronous activation of extracellular matrix re-organization, cellular mobilization, and Apolipoprotein E (Apoe) signaling in the fibrosing lung. Cell-cell communication analysis of single cell sequencing datasets predicted pro-fibrogenic signaling (fibronectin/Fn1, osteopontin/Spp1, and Tgfb1) emanating from Trem2/TREM2 + interstitial macrophages. These cells also produced a distinct lipid signature from alveolar macrophages and monocytes, characterized by Apoe expression. Mono- and di-allelic genetic deletion of ApoE in SP-C mutant mice had limited impact on inflammation and mortality up to 42 day after injury. Discussion: Together, these results provide a detailed spatio-temporal picture of resident, interstitial, and monocyte-derived macrophages during SP-C induced inflammatory exacerbations and end-stage clinical PF, and propose ApoE as a biomarker to identify activated macrophages involved in tissue remodeling.

Bone marrow mesenchymal stromal cells attenuate smoke inhalation injury by regulating the M1/M2-Th17/Treg immune homeostasis axis.

Smoke inhalation injury (SII) is the leading cause of death in fire burn patients. The inflammatory response induced by smoke inhalation is a significant factor in the development of acute lung injury or acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) can alleviate various inflammatory diseases by regulating the polarization of macrophages from the M1 to the M2 phenotype. Moreover, MSCs can facilitate the inflammatory response by regulating Th17/Treg homeostasis. However, little is known about the associations among MSCs, M1/M2 macrophages and Th17/Treg homeostasis. Therefore, the purpose of this study was to evaluate whether MSCs affect subsequent Th17/Treg differentiation and immune homeostasis by regulating M1/M2 polarization in SII. Our results showed that bone marrow mesenchymal stem cells (BMSCs) ameliorated lung inflammatory injury and fibrosis after SII by affecting the polarization of alveolar macrophages (AMs) from the M1 to the M2 phenotype. Moreover, BMSCs maintain Th17/Treg immune homeostasis by increasing the proportion of Treg cells and decreasing the proportion of Th17 cells. In vitro, we further demonstrated that BMSCs promoted the polarization of AMs from the M1 to the M2 phenotype and decreased IL-23 levels. Reduced IL-23 decreased Th17 differentiation and promoted Th17/Treg balance. Therefore, BMSCs ameliorate the inflammatory response and lung damage after SII through regulating M1/M2 polarization and subsequent Th17/Treg immune homeostasis, which are linked to alveolar macrophage-derived IL-23. These findings provide novel insight into how BMSCs regulate the M1/M2-Th17/Treg immune homeostasis axis and provide new therapeutic targets for more effective control of the inflammatory response after SII.

Solid Lipid Nanoparticles Loaded with Dexamethasone Palmitate for Pulmonary Inflammation Treatment by Nebulization Approach.

Pneumonia stands as the leading infectious cause of childhood mortality annually, underscoring its significant impact on pediatric health. Although dexamethasone (DXMS) is effective for treating pulmonary inflammation, its therapeutic potential is compromised by systemic side effects and suboptimal carrier systems. To address this issue, the current study introduces solid lipid nanoparticles encapsulating hydrophobic dexamethasone palmitate (DXMS-Pal-SLNs) as an anti-inflammatory nanoplatform to treat pneumonia. The specialized nanoparticle formulation is characterized by high drug loading efficiency, low drug leakage and excellent colloidal stability in particular during nebulization and is proficiently designed to target alveolar macrophages in deep lung regions via local delivery with the nebulization administration. In vitro analyses revealed substantial reductions in the secretions of tumor necrosis factor-α and interleukin-6 from alveolar macrophages, highlighting the potential efficacy of DXMS-Pal-SLNs in alleviating pneumonia-related inflammation. Similarly, in vivo experiments showed a significant reduction in the levels of these cytokines in the lungs of mice experiencing lipopolysaccharide-induced pulmonary inflammation after the administration of DXMS-Pal-SLNs via nebulization. Furthermore, the study demonstrated that DXMS-Pal-SLNs effectively control acute infections without causing pulmonary infiltration or excessive recruitment of immunocytes in lung tissues. These findings highlight the potential of nebulized DXMS-Pal-SLNs as a promising therapeutic strategy for mitigating pneumonia-related inflammations.

Extracellular vesicles from alveolar macrophages harboring phagocytosed methicillin-resistant Staphylococcus aureus induce necroptosis.

Methicillin-resistant Staphylococcus aureus (MRSA) infection, a major cause of hospital- and community-acquired pneumonia, still has a high mortality rate. Extracellular vesicles (EVs), as crucial mediators of intercellular communication, have a significant impact on infectious diseases. However, the role of EVs from alveolar macrophages (AMs) in MRSA pneumonia remains unclear. We report that AMs phagocytose MRSA and release more EVs in mice with MRSA pneumonia. EVs from AMs harboring phagocytosed MRSA exhibit significant proinflammatory effects and induce necroptosis by delivering tumor necrosis factor α (TNF-α) and miR-146a-5p. Mechanically, the upregulated miR-146a-5p in these EVs enhances the phosphorylation of RIPK1, RIPK3, and MLKL by targeting TNF receptor-associated factor 6 (TRAF6), thereby promoting TNF-α-induced necroptosis. The combination of a TNF-α antagonist and an miR-146a-5p antagomir effectively improves the outcomes of mice with MRSA pneumonia. Overall, we reveal the pronecrotic effect of EVs from MRSA-infected AMs and provide a promising target for the prevention and treatment of MRSA pneumonia.

A detailed insight into macrophages' role in shaping lung carcinogenesis.

Despite significant advancements in cancer treatment in recent decades, the high mortality rate associated with lung cancer remains a significant concern. The development and proper execution of new targeted therapies needs more deep knowledge regarding the lung cancer associated tumour microenvironment. One of the key component of that tumour microenvironment is the lung resident macrophages. Although in normal physiological condition the lung resident macrophages are believed to maintain lung homeostasis, but they may also initiate a vicious inflammatory response in abnormal conditions which is linked to lung cancer development. Depending on the activation pathway, the lung resident macrophages are either of M1 or M2 sub-type. The M1 and M2 sub-types differ significantly in various prospectuses, from phenotypic markers to metabolic pathways. In addition to this generalized classification, the recent advancement of the multiomics technology is able to identify some other sub-types of lung resident macrophages. Researchers have also observed that these different sub-types can manipulate the pathogenesis of lung carcinogenesis in a context dependent manner and can either promote or inhibit the development of lung carcinogenesis upon receiving proper activation. As proper knowledge about the role played by the lung resident macrophages' in shaping the lung carcinogenesis is limited, so the main purpose of this review is to bring all the available information under the same roof. We also elaborated the different mechanisms involved in maintenance of the plasticity of M1/M2 sub-type, as this plasticity can be a good target for lung cancer treatment.

Rifabutin loaded inhalable ß-glucan microparticle based drug delivery system for pulmonary TB.

Inhalable microparticle-based anti TB drug delivery systems are being investigated extensively for Tuberculosis [TB] treatment as they offer efficient and deep lung deposition with several advantages over conventional routes. It can reduce the drug dose, treatment duration and toxic effects and optimize the drug bioavailability. Yeast derived ß-glucan is a ß-[1-3/1-6] linked biocompatible polymer and used as carrier for various biomolecules. Due to presence of glucan chains, particulate glucans act as PAMP and thereby gets internalized via receptor mediated phagocytosis by the macrophages. In this study, ß-glucan microparticles were prepared by adding l-leucine as excipient, and exhibited 70% drug [Rifabutin] loading efficiency. Further, the sizing and SEM data of particles revealed a size of 2-4 µm with spherical dimensions. The FTIR and HPLC data confirmed the ß-glucan composition and drug encapsulations efficiency of the particles. The mass median aerodynamic diameter [MMAD] and geometric standard deviation [GSD] data indicated that these particles are inhalable in nature and have better thermal stability as per DSC thermogram. These particles were found to be non-toxic upto a concentration of 80 µg/ml and were found to be readily phagocytosed by human macrophage cells in-vitro as well as in-vivo by lung alveolar macrophage. This study provides a framework for future design of inhalable ß-glucan particle based host-directed drug delivery system against pulmonary TB.

Communication between alveolar macrophages and fibroblasts via the TNFSF12-TNFRSF12A pathway promotes pulmonary fibrosis in severe COVID-19 patients.

BACKGROUND: Severe COVID-19 infection has been associated with the development of pulmonary fibrosis, a condition that significantly affects patient prognosis. Understanding the underlying cellular communication mechanisms contributing to this fibrotic process is crucial. OBJECTIVE: In this study, we aimed to investigate the role of the TNFSF12-TNFRSF12A pathway in mediating communication between alveolar macrophages and fibroblasts, and its implications for the development of pulmonary fibrosis in severe COVID-19 patients. METHODS: We conducted single-cell RNA sequencing (scRNA-seq) analysis using lung tissue samples from severe COVID-19 patients and healthy controls. The data was processed, analyzed, and cell types were annotated. We focused on the communication between alveolar macrophages and fibroblasts and identified key signaling pathways. In vitro experiments were performed to validate our findings, including the impact of TNFRSF12A silencing on fibrosis reversal. RESULTS: Our analysis revealed that in severe COVID-19 patients, alveolar macrophages communicate with fibroblasts primarily through the TNFSF12-TNFRSF12A pathway. This communication pathway promotes fibroblast proliferation and expression of fibrotic factors. Importantly, silencing TNFRSF12A effectively reversed the pro-proliferative and pro-fibrotic effects of alveolar macrophages. CONCLUSION: The TNFSF12-TNFRSF12A pathway plays a central role in alveolar macrophage-fibroblast communication and contributes to pulmonary fibrosis in severe COVID-19 patients. Silencing TNFRSF12A represents a potential therapeutic strategy for mitigating fibrosis in severe COVID-19 lung disease.

Multiomic analysis of monocyte-derived alveolar macrophages in idiopathic pulmonary fibrosis.

BACKGROUND: Monocyte-derived alveolar macrophages (Mo_AMs) are increasingly recognised as potential pathogenic factors for idiopathic pulmonary fibrosis (IPF). While scRNAseq analysis has proven valuable in the transcriptome profiling of Mo_AMs, the integration analysis of multi-omics may provide additional dimensions of understanding of these cellular populations. METHODS: We performed multi-omics analysis on 116 scRNAseq, 119 bulkseq and five scATACseq lung tissue samples from IPF. We built a large-scale IPF scRNAseq atlas and conducted the Monocle 2/3 as well as the Cellchat to explore the developmental path and intercellular communication on Mo_AMs. We also reported the difference in metabolisms, tissue repair and phagocytosis between Mo_AMs and tissue-resident alveolar macrophages (TRMs). To determine whether Mo_AMs affected pulmonary function, we projected clinical phenotypes (FVC%pred) from the bulkseq dataset onto the scRNAseq atlas. Finally, we used scATATCseq to uncover the upstream regulatory mechanisms and determine key drivers in Mo_AMs. RESULTS: We identified three Mo_AMs clusters and the trajectory analysis further validated the origin of these clusters. Moreover, via the Cellchat analysis, the CXCL12/CXCR4 axis was found to be involved in the molecular basis of reciprocal interactions between Mo_AMs and fibroblasts through the activation of the ERK pathway in Mo_AMs. SPP1_RecMacs (RecMacs, recruited macrophages) were higher in the low-FVC group than in the high-FVC group. Specifically, compared with TRMs, the functions of lipid and energetic metabolism as well as tissue repair were higher in Mo_AMs than TRMs. But, TRMs may have higher level of phagocytosis than TRMs. SPIB (PU.1), JUNB, JUND, BACH2, FOSL2, and SMARCC1 showed stronger association with open chromatin of Mo_AMs than TRMs. Significant upregulated expression and deep chromatin accessibility of APOE were observed in both SPP1_RecMacs and TRMs. CONCLUSION: Through trajectory analysis, it was confirmed that SPP1_RecMacs derived from Monocytes. Besides, Mo_AMs may influence FVC% pred and aggravate pulmonary fibrosis through the communication with fibroblasts. Furthermore, distinctive transcriptional regulators between Mo_AMs and TRMs implied that they may depend on different upstream regulatory mechanisms. Overall, this work provides a global overview of how Mo_AMs govern IPF and also helps determine better approaches and intervention therapies.

Antimycobacterial and healing effects of Pranlukast against MTB infection and pathogenesis in a preclinical mouse model of tuberculosis.

It is essential to understand the interactions and relationships between Mycobacterium tuberculosis (Mtb) and macrophages during the infection in order to design host-directed, immunomodulation-dependent therapeutics to control Mtb. We had reported previously that ornithine acetyltransferase (MtArgJ), a crucial enzyme of the arginine biosynthesis pathway of Mtb, is allosterically inhibited by pranlukast (PRK), which significantly reduces bacterial growth. The present investigation is centered on the immunomodulation in the host by PRK particularly the activation of the host's immune response to counteract bacterial survival and pathogenicity. Here, we show that PRK decreased the bacterial burden in the lungs by upregulating the population of pro-inflammatory interstitial macrophages (IMs) and reducing the population of Mtb susceptible alveolar macrophages (AMs), dendritic cells (DCs), and monocytes (MO). Additionally, we deduce that PRK causes the host macrophages to change their metabolic pathway from fatty acid metabolism to glycolytic metabolism around the log phage of bacterial multiplication. Further, we report that PRK reduced tissue injury by downregulating the Ly6C-positive population of monocytes. Interestingly, PRK treatment improved tissue repair and inflammation resolution by increasing the populations of arginase 1 (Arg-1) and Ym1+Ym2 (chitinase 3-like 3) positive macrophages. In summary, our study found that PRK is useful not only for reducing the tubercular burden but also for promoting the healing of the diseased tissue.

MicroRNA Monitoring in Human Alveolar Macrophages from Patients with Smoking-Related Lung Diseases: A Preliminary Study.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is commonly considered to be a potent driver of non-small cell lung cancer (NSCLC) development and related mortality. A growing body of evidence supports a role of the immune system, mainly played by alveolar macrophages (AMs), in key axes regulating the development of COPD or NSCLC phenotypes in response to harmful agents. MicroRNAs (miRNAs) are small non-coding RNAs that influence most biological processes and interfere with several regulatory pathways. The purpose of this study was to assess miRNA expression patterns in patients with COPD, NSCLC, and ever- or never-smoker controls to explore their involvement in smoking-related diseases. Bronchoalveolar lavage (BAL) specimens were collected from a prospective cohort of 43 sex-matched subjects to determine the expressions of hsa-miR-223-5p, 16-5p, 20a-5p, -17-5p, 34a-5p and 106a-5p by RT-PCR. In addition, a bioinformatic analysis of miRNA target genes linked to cancer was performed. Distinct and common miRNA expression levels were identified in each pathological group, suggesting their possible role as an index of NSCLC or COPD microenvironment. Moreover, we identified miRNA targets linked to carcinogenesis using in silico analysis. In conclusion, this study identified miRNA signatures in AMs, allowing us to understand the molecular mechanisms underlying smoking-related conditions and potentially providing new insights for diagnosis or pharmacological treatment.

Transplantation of alveolar macrophages improves the efficacy of endothelial progenitor cell therapy in mouse model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm births, which develops due to exposure to supplemental oxygen and mechanical ventilation. Published studies demonstrated that the number of endothelial progenitor cells (EPC) is decreased in mouse and human BPD lungs and that adoptive transfer of EPC is an effective approach in reversing the hyperoxia-induced lung damage in mouse model of BPD. Recent advancements in macrophage biology identified the specific subtypes of circulating and resident macrophages mediating the developmental and regenerative functions in the lungs. Several studies reported the successful application of macrophage therapy in accelerating the regenerative capacity of damaged tissues and enhancing the therapeutic efficacy of other transplantable progenitor cells. In the present study, we explored the efficacy of combined cell therapy with EPC and resident alveolar macrophages (rAM) in hyperoxia-induced BPD mouse model. rAM and EPC were purified from neonatal mouse lungs and were used for adoptive transfer to the recipient neonatal mice exposed to hyperoxia. Adoptive transfer of rAM alone did not result in engraftment of donor rAM into the lung tissue but increased the mRNA level and protein concentration of proangiogenic CXCL12 chemokine in recipient mouse lungs. Depletion of rAM by chlodronate-liposomes decreased the retention of donor EPC after their transplantation into hyperoxia-injured lungs. Adoptive transfer of rAM in combination with EPC enhanced the therapeutic efficacy of EPC as evidenced by increased retention of EPC, increased capillary density, improved arterial oxygenation, and alveolarization in hyperoxia-injured lungs. Dual therapy with EPC and rAM has promise in human BPD.NEW & NOTEWORTHY Recent studies demonstrated that transplantation of lung-resident endothelial progenitor cells (EPC) is an effective therapy in mouse model of bronchopulmonary dysplasia (BPD). However, key factors regulating the efficacy of EPC are unknown. Herein, we demonstrate that transplantation of tissue-resident alveolar macrophages (rAM) increases CXCL12 expression in neonatal mouse lungs. rAM are required for retention of donor EPC in hyperoxia-injured lungs. Co-transplantation of rAM and EPC improves the efficacy of EPC therapy in mouse BPD model.