Pulmonary delivery of silver nanoparticles prevents influenza infection by recruiting and activating lymphoid cells.

Silver nanoparticles (AgNPs) are a potential antiviral agent due to their ability to disrupt the viral particle or alter the virus metabolism inside the host cell. In vitro, AgNPs exhibit antiviral activity against the most common human respiratory viruses. However, their capacity to modulate immune responses during respiratory viral infections has yet to be explored. This study demonstrates that administering AgNPs directly into the lungs prior to infection can reduce viral loads and therefore virus-induced cytokines in mice infected with influenza virus or murine pneumonia virus. The prophylactic effect was diminished in mice with depleted lymphoid cells. We showed that AgNPs-treatment resulted in the recruitment and activation of lymphocytes in the lungs, particularly natural killer (NK) cells. Mechanistically, AgNPs enhanced the ability of alveolar macrophages to promote both NK cell migration and IFN-γ production. By contrast, following infection, in mice treated with AgNPs, NK cells exhibited decreased activation, indicating that these nanoparticles can regulate the potentially deleterious activation of these cells. Overall, the data suggest that AgNPs may possess prophylactic antiviral properties by recruiting and controlling the activation of lymphoid cells through interaction with alveolar macrophages.

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.

Attenuation of PM2.5-Induced Lung Injury by 4-Phenylbutyric Acid: Maintenance of [Ca2+]i Stability between Endoplasmic Reticulum and Mitochondria.

Fine particulate matter (PM2.5) is a significant cause of respiratory diseases and associated cellular damage. The mechanisms behind this damage have not been fully explained. This study investigated two types of cellular damage (inflammation and pyroptosis) induced by PM2.5, focusing on their relationship with two organelles (the endoplasmic reticulum and mitochondria). Animal models have demonstrated that PM2.5 induces excessive endoplasmic reticulum stress (ER stress), which is a significant cause of lung damage in rats. This was confirmed by pretreatment with an ER stress inhibitor (4-Phenylbutyric acid, 4-PBA). We found that, in vitro, the intracellular Ca2+ ([Ca2+]i) dysregulation induced by PM2.5 in rat alveolar macrophages was associated with ER stress. Changes in mitochondria-associated membranes (MAMs) result in abnormal mitochondrial function. This further induced the massive expression of NLRP3 and GSDMD-N, which was detrimental to cell survival. In conclusion, our findings provide valuable insights into the relationship between [Ca2+]i dysregulation, mitochondrial damage, inflammation and pyroptosis under PM2.5-induced ER stress conditions. Their interactions ultimately have an impact on respiratory health.

RAMP1 Signaling Mitigates Acute Lung Injury by Distinctively Regulating Alveolar and Monocyte-Derived Macrophages.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury that induces cytokine hypersecretion. Receptor activity-modifying protein (RAMP) 1, a subunit of the calcitonin gene-related peptide (CGRP) receptor, regulates the production of cytokines. This study examined the role of RAMP1 signaling during lipopolysaccharide (LPS)-induced acute lung injury (ALI). LPS administration to wild-type (WT) mice depleted alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) and neutrophils. RAMP1-deficient (RAMP1-/-) mice exhibited higher lung injury scores, cytokine levels, and cytokine-producing neutrophil infiltration. RAMP1-deficient AMs produced more cytokines in response to LPS than WT AMs. Adoptive transfer of RAMP1-deficient AMs to RAMP1-/- mice increased cytokine levels and neutrophil accumulation compared to the transfer of WT AMs. RAMP1-/- mice had reduced MDM recruitment and lower pro-inflammatory and reparative macrophage profiles. Cultured bone marrow (BM)-derived RAMP1-deficient macrophages stimulated with LPS showed decreased expression of pro-inflammatory and pro-repairing genes. CGRP administration to WT mice reduced cytokine production and neutrophil accumulation. These findings indicate that RAMP1 signaling mitigates LPS-induced ALI by inactivating AMs and promoting inflammatory and repair activities of MDMs. Targeting RAMP1 signaling presents a potential therapeutic approach for the treatment of ARDS.

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.

Age-dependent changes in phagocytic activity: in vivo response of mouse pulmonary antigen presenting cells to direct lung delivery of charged PEGDA nanoparticles.

BACKGROUND: Current needle-based vaccination for respiratory viruses is ineffective at producing sufficient, long-lasting local immunity in the elderly. Direct pulmonary delivery to the resident local pulmonary immune cells can create long-term mucosal responses. However, criteria for drug vehicle design rules that can overcome age-specific changes in immune cell functions have yet to be established. RESULTS: Here, in vivo charge-based nanoparticle (NP) uptake was compared in mice of two age groups (2- and 16-months) within the four notable pulmonary antigen presenting cell (APC) populations: alveolar macrophages (AM), interstitial macrophages (IM), CD103+ dendritic cells (DCs), and CD11b+ DCs. Both macrophage populations exhibited preferential uptake of anionic nanoparticles but showed inverse rates of phagocytosis between the AM and IM populations across age. DC populations demonstrated preferential uptake of cationic nanoparticles, which remarkably did not significantly change in the aged group. Further characterization of cell phenotypes post-NP internalization demonstrated unique surface marker expression and activation levels for each APC population, showcasing heightened DC inflammatory response to NP delivery in the aged group. CONCLUSION: The age of mice demonstrated significant preferences in the charge-based NP uptake in APCs that differed greatly between macrophages and DCs. Carefully balance of the targeting and activation of specific types of pulmonary APCs will be critical to produce efficient, age-based vaccines for the growing elderly population.

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.

Role of sex as a biological variable in neonatal alveolar macrophages.

The lung macrophages play a crucial role in health and disease. Sexual dimorphism significantly impacts the phenotype and function of tissue-resident macrophages. The primary mechanisms responsible for sexually dimorphic outcomes in bronchopulmonary dysplasia (BPD) remain unidentified. We tested the hypothesis that biological sex plays a crucial role in the transcriptional state of alveolar macrophages, using neonatal murine hyperoxia-induced lung injury as a relevant model for human BPD. The effects of neonatal hyperoxia exposure (95 % FiO2, PND1-5: saccular stage) on the lung myeloid cells acutely after injury and during normoxic recovery were measured. Alveolar macrophages (AM) from room air- and hyperoxia exposed from male and female neonatal murine lungs were subjected to bulk-RNA Sequencing. AMs are significantly depleted in the hyperoxia-exposed lung acutely after injury, with subsequent recovery in both sexes. The transcriptome of the alveolar macrophages is impacted by neonatal hyperoxia exposure and by sex as a biological variable. Pathways related to DNA damage and interferon-signaling were positively enriched in female AMs. Metabolic pathways related to glucose and carbohydrate metabolism were positively enriched in the male AMs, while oxidative phosphorylation was negatively enriched. These pathways were shared with monocytes and airway macrophages from intubated male and female human premature neonates.

Role of Mitofusin 1 in mediating reactive oxygen species in alveolar macrophages during Streptococcuspneumoniae.

Alveolar macrophages (AM) are key effectors of the immune response and are essential for host responses to S. pneumoniae. Mitochondria are highly dynamic organelles whose function aids in regulating the cell cycle, innate immunity, autophagy, redox signaling, calcium homeostasis, and mitochondrial quality control in AM. In response to cellular stress, mitochondria can engage in stress-induced mitochondrial hyperfusion (SIMH). The current study aimed to investigate the role of Mfn1 on mitochondrial control of reactive oxygen species (ROS) in AMs and the role of Mfn1 deficiency on immune responses to S. pneumoniae. Compared to Mfn1FloxCre- controls, there were distinct histological differences in lung tissue collected from Mfn1Floxed; CreLysM mice, with less injury and inflammation observed in mice with Mfn1 deficient myeloid cells. There was a significant decrease in lipid peroxidation and ROS production in Mfn1 deficient AM that was associated with increased superoxide dismutase (SOD) and antioxidant activity. Our findings demonstrate that Mfn1 deficiency in myeloid cells decreased inflammation and lung tissue injury during S. pneumoniae infection.

Expression of human Interferon Regulatory Factor 3 (IRF-3) in alveolar macrophages relates to clinical and functional traits in COPD.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a frequent cause of morbidity and mortality. Dysregulated and enhanced immune-inflammatory responses have been described in COPD. Recent data showed impaired immune responses and, in particular, of interferon (IFNs) signaling pathway in these patients. AIM: To evaluate in peripheral lung of COPD patients, the expression of some of the less investigated key components of the innate immune responses leading to IFN productions including: IFN-receptors (IFNAR1/IFNAR2), IRF-3 and MDA-5. Correlations with clinical traits and with the inflammatory cell profile have been assessed. METHODS: Lung specimens were collected from 58 subjects undergoing thoracic surgery: 22 COPD patients, 21 smokers with normal lung function (SC) and 15 non-smoker controls (nSC). The expression of IFNAR1, IFNAR2, IRF-3 and MDA-5, of eosinophils and activated NK cells (NKp46+) were quantified in the peripheral lung by immunohistochemistry. RESULTS: A significant increase of IRF-3 + alveolar macrophages were observed in COPD and SC compared with nSC subjects. However, in COPD patients, the lower the levels of IRF-3 + alveolar macrophages the lower the FEV1 and the higher the exacerbation rate. The presence of chronic bronchitis (CB) was also associated with low levels of IRF-3 + alveolar macrophages. NKp46 + cells, but not eosinophils, were increased in COPD patients compared to nSC patients (p < 0.0001). CONCLUSIONS: Smoking is associated with higher levels of innate immune response as showed by higher levels of IRF-3 + alveolar macrophages and NKp46 + cells. In COPD, exacerbation rates, severe airflow obstruction and CB were associated with lower levels of IRF-3 expression, suggesting that innate immune responses characterize specific clinical traits of the disease.

Multimodal pulmonary clearance kinetics of carbon black nanoparticles deposited in the lungs of rats: the role of alveolar macrophages.

BACKGROUND: Alveolar macrophages (AMs) have been predicted to affect the pulmonary clearance of nanomaterials; however, their qualitative and quantitative roles are poorly understood. In this study, carbon black nanoparticles (CBNPs) were instilled into the lungs of Wistar rats at 30, 100, and 300 µg/rat. The concentrations of particles in organs, including the lung, lung-associated lymph nodes (LALN), liver, spleen, and kidney, were evaluated at days 0 (immediately after instillation), 1, 7, 28, 60, and 90 post-instillation. RESULTS: The results indicated a multimodal pulmonary clearance pattern for CBNPs: slow clearance until day 28, fast clearance from days 28 to 60, and slow clearance from days 60 to 90. To determine the mechanism of this unique clearance pattern, CBNPs were instilled into AM-depleted rats using clodronate liposomes (CLO). At 28 days after instillation, the CBNP levels in the lungs treated with CLO showed about 31% higher reduction than in normal rats. In addition, the concentration of CBNPs in LALN treated with CLO significantly increased on day 28, whereas in normal rats, no detectable levels were observed. CONCLUSIONS: This result highlights that the prolonged retention of poorly soluble NPs in the lung until day 28 is mediated by the phagocytosis of AMs, and the fast clearance between days 28-60 is due to the turnover time of AMs, estimated around 1-2 months after birth. Similarly, new generations of AMs mediate the slow phase between days 60 and 90. However, further studies are needed to understand the multimodal clearance mechanism and the modulation of pulmonary clearance of poorly soluble NPs.

SARS-CoV2 infection in whole lung primarily targets macrophages that display subset-specific responses.

Deciphering the initial steps of SARS-CoV-2 infection, that influence COVID-19 outcomes, is challenging because animal models do not always reproduce human biological processes and in vitro systems do not recapitulate the histoarchitecture and cellular composition of respiratory tissues. To address this, we developed an innovative ex vivo model of whole human lung infection with SARS-CoV-2, leveraging a lung transplantation technique. Through single-cell RNA-seq, we identified that alveolar and monocyte-derived macrophages (AMs and MoMacs) were initial targets of the virus. Exposure of isolated lung AMs, MoMacs, classical monocytes and non-classical monocytes (ncMos) to SARS-CoV-2 variants revealed that while all subsets responded, MoMacs produced higher levels of inflammatory cytokines than AMs, and ncMos contributed the least. A Wuhan lineage appeared to be more potent than a D614G virus, in a dose-dependent manner. Amidst the ambiguity in the literature regarding the initial SARS-CoV-2 cell target, our study reveals that AMs and MoMacs are dominant primary entry points for the virus, and suggests that their responses may conduct subsequent injury, depending on their abundance, the viral strain and dose. Interfering on virus interaction with lung macrophages should be considered in prophylactic strategies.

iRhom2 deficiency reduces sepsis-induced mortality associated with the attenuation of lung macrophages in mice.

Sepsis has a high mortality rate and leads to multi-organ failure, including lung injury. Inactive rhomboid protease family protein (iRhom2) has been identified as accountable for the release of TNF-α, a crucial mediator in the development of sepsis. This study aimed to evaluate the role of iRhom2 in sepsis and sepsis-induced acute lung injury (ALI). TNF-α and IL-6 secretion in vitro by peritoneal macrophages from wild-type (WT) and iRhom2 knoukout (KO) mice was assessed by enzyme-linked immunosorbent assay. Cecal ligation and puncture (CLP)-induced murine sepsis model was used for in vivo experiments. To evaluate the role of iRhom2 deficiency on survival during sepsis, both WT and iRhom2 KO mice were monitored for 8 consecutive days following the CLP. For histologic and biochemical examination, the mice were killed 18 h after CLP. iRhom2 deficiency improved the survival of mice after CLP. iRhom2 deficiency decreased CD68+ macrophage infiltration in lung tissues. Multiplex immunohistochemistry revealed that the proportion of Ki-67+ CD68+ macrophages was significantly lower in iRhom2 KO mice than that in WT mice after CLP. Moreover, CLP-induced release of TNF-α and IL-6 in the serum were significantly inhibited by iRhom2 deficiency. iRhom2 deficiency reduced NF-kB p65 and IκBα phosphorylation after CLP. iRhom2 deficiency reduces sepsis-related mortality associated with attenuated macrophage infiltration and proliferation in early lung injury. iRhom2 may play a pivotal role in the pathogenesis of sepsis and early stage of sepsis-induced ALI. Thus, iRhom2 may be a potential therapeutic target for the management of sepsis and sepsis-induced ALI.

Effects of isochlorogenic acid A on mitochondrial dynamics imbalance and RLR damage in PAM cells induced by combined mycotoxins.

Mycotoxins have strong immunotoxicity and can induce oxidative stress and mitochondrial dynamics imbalance. Mitochondrial antiviral signaling protein (MAVS) in the RIG-I like receptor (RLR) pathway of innate immunity is located on mitochondria, and whether it is affected by mycotoxins has not been reported yet. This experiment used porcine alveolar macrophages (PAM) to evaluate the antagonism of three isomers of chlorogenic acid (chlorogenic acid, isochlorogenic acid A, and neochlorogenic acid) against combined mycotoxins (Aflatoxin B1, Deoxynivalenol, and Ochratoxin A) induced mitochondrial damage and their effects on the RLR pathway, providing assistance for further elucidating the mechanism of mycotoxin immunotoxicity. Western blotting, enzyme linked immunosorbent assay (ELISA), and flow cytometry were used to detect relevant indicators. All three types of chlorogenic acid treatment can antagonize the cytotoxicity induced by combined mycotoxins, especially isochlorogenic acid A, which can protect cells from mycotoxins damage by maintaining mitochondrial dynamic homeostasis and improving innate immune function related to the RLR pathway.

Remimazolam Alleviates Ventilator-Induced Lung Injury by Activating TSPO to Inhibit Macrophage Pyroptosis.

BACKGROUND: Macrophages are activated in ventilator-induced lung injury (VILI), accompanied by macrophage pyroptosis. Remimazolam (Re) plays a role in inhibiting macrophage activation. In this study, we aimed to investigate the mechanism of Re in VILI. METHODS: A VILI model (20 mL/kg mechanical ventilation) was created using C57BL/6 mice. Alveolar macrophages were isolated from bronchoalveolar lavage fluid (BALF) and received mechanical stretching to simulate the mechanical ventilation in vitro. VILI model mice were treated with Re (16 mg/kg) to assess the alveolar structure, wet/dry (W/D) weight ratio, endothelial barrier antigen (EBA) permeability index, BALF protein content, inflammatory factors, macrophage pyroptosis, pyroptosis-related factors, and translocator protein (TSPO) level using a series of biological experiments. Whether Re alleviated macrophage pyroptosis by regulating TSPO was determined by rescue experiments. RESULTS: Re alleviated VILI, as evidenced by improvement of abnormal morphology of lung tissues during VILI and decreases in the lung W/D weight ratio, lung EBA permeability index, and BALF protein content. Re attenuated pulmonary inflammation and macrophage pyroptosis during VILI via down-regulation of inflammatory factors (myeloperoxidase, malondialchehyche, 8-hydroxy-2 deoxyguanosine, interleukin-6, tumor necrosis factor-α, macrophage inflammatory protein-2, interleukin-1ß, and interleukin-18), and pyroptosis factors (cleaved gasdermin D (GSDMD)/GSDMD value, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), and caspase-1). Re activated TSPO in macrophages. TSPO overexpression rescued the cell stretch-inhibited macrophage viability and cell stretch-induced macrophage pyroptosis. CONCLUSION: Re alleviates VILI by activating TSPO to inhibit macrophage pyroptosis.

Transcriptomic analysis reveals distinct effects of cigarette smoke on murine airspace and bone-marrow derived macrophages.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disease characterized by emphysema and chronic bronchitis and a leading cause of mortality worldwide. COPD is commonly associated with several comorbid diseases which contribute to exacerbated patient outcomes. Cigarette smoke (CS) is the most prominent risk factor for COPD development and progression and is known to be detrimental to numerous effector functions of lung resident immune cells, including phagocytosis and cytokine production. However, how CS mediates the various pathologies distant from the lung in COPD, and whether CS has a similar biological effect on systemic immune cells remains unknown. METHODS: C57BL/6 mice were exposed to 8 weeks of CS as an experimental model of COPD. Bone marrow cells were isolated from both CS-exposed and room air (RA) control mice and differentiated to bone marrow-derived macrophages (BMDMs). Airspace macrophages (AMs) were isolated from the same CS-exposed and RA mice and bulk RNA-Seq performed. The functional role of differentially expressed genes was assessed through gene ontology analyses. Ingenuity Pathway Analysis was used to determine the activation states of canonical pathways and upstream regulators enriched in differentially expressed genes in both cell types, and to compare the differences between the two cell types. RESULTS: CS induced transcriptomic changes in BMDMs, including an upregulation of genes in sirtuin signalling and oxidative phosphorylation pathways and a downregulation of genes involved in histone and lysine methylation. In contrast, CS induced decreased expression of genes involved in pathogen response, phagosome formation, and immune cell trafficking in AMs. Little overlap was observed in differentially expressed protein-coding genes in BMDMs compared to AMs and their associated pathways, highlighting the distinct effects of CS on immune cells in different compartments. CONCLUSIONS: CS exposure can induce transcriptomic remodelling in BMDMs which is distinct to that of AMs. Our study highlights the ability of CS exposure to affect immune cell populations distal to the lung and warrants further investigation into the functional effects of these changes and the ensuing role in driving multimorbid disease.

Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in interorgan communication in diverse pathological processes. ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of the alveolar macrophage (AM) markers CD11b, CD11c, and CD206 as well as ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction of inspired oxygen therapy (HO2⩾30%) had increased concentrations of AM-derived EV-ASC compared with infants on lower fraction of inspired oxygen (LO2<30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier, and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death, with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain cross-talk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

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.

Endogenous LXR signaling controls pulmonary surfactant homeostasis and prevents lung inflammation.

Lung type 2 pneumocytes (T2Ps) and alveolar macrophages (AMs) play crucial roles in the synthesis, recycling and catabolism of surfactant material, a lipid/protein fluid essential for respiratory function. The liver X receptors (LXR), LXRα and LXRß, are transcription factors important for lipid metabolism and inflammation. While LXR activation exerts anti-inflammatory actions in lung injury caused by lipopolysaccharide (LPS) and other inflammatory stimuli, the full extent of the endogenous LXR transcriptional activity in pulmonary homeostasis is incompletely understood. Here, using mice lacking LXRα and LXRß as experimental models, we describe how the loss of LXRs causes pulmonary lipidosis, pulmonary congestion, fibrosis and chronic inflammation due to defective de novo synthesis and recycling of surfactant material by T2Ps and defective phagocytosis and degradation of excess surfactant by AMs. LXR-deficient T2Ps display aberrant lamellar bodies and decreased expression of genes encoding for surfactant proteins and enzymes involved in cholesterol, fatty acids, and phospholipid metabolism. Moreover, LXR-deficient lungs accumulate foamy AMs with aberrant expression of cholesterol and phospholipid metabolism genes. Using a house dust mite aeroallergen-induced mouse model of asthma, we show that LXR-deficient mice exhibit a more pronounced airway reactivity to a methacholine challenge and greater pulmonary infiltration, indicating an altered physiology of LXR-deficient lungs. Moreover, pretreatment with LXR agonists ameliorated the airway reactivity in WT mice sensitized to house dust mite extracts, confirming that LXR plays an important role in lung physiology and suggesting that agonist pharmacology could be used to treat inflammatory lung diseases.

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with N in PRRSV-Infected PAMs.

One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus' assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein-protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus-host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines.