Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Introduction: Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. Methods: We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Results: Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Discussion: Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

Growth differentiation factor 15 (GDF15) as a stress response cytokine is involved in the development and progression of several diseases associated with metabolic disorders. However, the regulatory role and the underlying mechanisms of GDF15 in sepsis remain poorly defined. Our study analyzed the levels of GDF15 and its correlations with the clinical prognosis of patients with sepsis. In vivo and in vitro models of sepsis were applied to elucidate the role and mechanisms of GDF15 in sepsis-associated lung injury. We observed strong correlations of plasma GDF15 levels with the levels of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), and lactate as well as Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. In the mouse model of lipopolysaccharide-induced sepsis, recombinant GDF15 inhibited the proinflammatory responses and alleviated lung tissue injury. In addition, GDF15 decreased the levels of cytokines produced by alveolar macrophages (AMs). The anti-inflammatory effect of glycolysis inhibitor 2-DG on AMs during sepsis was mediated by GDF15 via inducing the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and the expression of activating transcription factor 4 (ATF4). Furthermore, we explored the mechanism underlying the beneficial effects of GDF15 and found that GDF15 inhibited glycolysis and mitogen-activated protein kinases (MAPK)/nuclear factor-κB (NF-κB) signaling via promoting AMPK phosphorylation. This study demonstrated that GDF15 inhibited glycolysis and NF-κB/MAPKs signaling via activating AMP-activated protein kinase (AMPK), thereby alleviating the inflammatory responses of AMs and sepsis-associated lung injury. Our findings provided new insights into novel therapeutic strategies for treating sepsis.

Metabolic changes enhance necroptosis of type 2 diabetes mellitus mice infected with Mycobacterium tuberculosis.

Previously, we found that Mycobacterium tuberculosis (Mtb) infection in type 2 diabetes mellitus (T2DM) mice enhances inflammatory cytokine production which drives pathological immune responses and mortality. In the current study, using a T2DM Mtb infection mice model, we determined the mechanisms that make T2DM mice alveolar macrophages (AMs) more inflammatory upon Mtb infection. Among various cell death pathways, necroptosis is a major pathway involved in inflammatory cytokine production by T2DM mice AMs. Anti-TNFR1 antibody treatment of Mtb-infected AMs from T2DM mice significantly reduced expression of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) (necroptosis markers) and IL-6 production. Metabolic profile comparison of Mtb-infected AMs from T2DM mice and Mtb-infected AMs of nondiabetic control mice indicated that 2-ketohexanoic acid and deoxyadenosine monophosphate were significantly abundant, and acetylcholine and pyridoxine (Vitamin B6) were significantly less abundant in T2DM mice AMs infected with Mtb. 2-Ketohexanoic acid enhanced expression of TNFR1, RIPK3, MLKL and inflammatory cytokine production in the lungs of Mtb-infected nondiabetic mice. In contrast, pyridoxine inhibited RIPK3, MLKL and enhanced expression of Caspase 3 (apoptosis marker) in the lungs of Mtb-infected T2DM mice. Our findings demonstrate that metabolic changes in Mtb-infected T2DM mice enhance TNFR1-mediated necroptosis of AMs, which leads to excess inflammation and lung pathology.

ANGPTL2 knockdown induces autophagy to relieve alveolar macrophage pyroptosis by reducing LILRB2-mediated inhibition of TREM2.

Acute lung injury (ALI) is featured with a robust inflammatory response. Angiopoietin-like protein 2 (ANGPTL2), a pro-inflammatory protein, is complicated with various disorders. However, the role of ANGPTL2 in ALI remains to be further explored. The mice and MH-S cells were administrated with lipopolysaccharide (LPS) to evoke the lung injury in vivo and in vitro. The role and mechanism of ANGPTL was investigated by haematoxylin-eosin, measurement of wet/dry ratio, cell count, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling, reverse transcription quantitative polymerase chain reaction, immunofluorescence, enzyme-linked immunosorbent assay, detection of autophagic flux and western blot assays. The level of ANGPTL2 was upregulated in lung injury. Knockout of ANGPTL2 alleviated LPS-induced pathological symptoms, reduced pulmonary wet/dry weight ratio, the numbers of total cells and neutrophils in BALF, apoptosis rate and the release of pro-inflammatory mediators, and modulated polarization of alveolar macrophages in mice. Knockdown of ANGPTL2 downregulated the level of pyroptosis indicators, and elevated the level of autophagy in LPS-induced MH-S cells. Besides, downregulation of ANGPTL2 reversed the LPS-induced the expression of leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) and triggering receptor expressed on myeloid cells 2 (TREM2), which was reversed by the overexpression of LILRB2. Importantly, knockdown of TREM2 reversed the levels of autophagy- and pyroptosis-involved proteins, and the contents of pro-inflammatory factors in LPS-induced MH-S cells transfected with si ANGPTL2, which was further inverted with the treatment of rapamycin. Therefore, ANGPTL2 silencing enhanced autophagy to alleviate alveolar macrophage pyroptosis via reducing LILRB2-mediated inhibition of TREM2.

Differential regulation of lung homeostasis and silicosis by the TAM receptors MerTk and Axl.

Introduction: TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. Methods: We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 µL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-ß and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. Conclusion: This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.

Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia.

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury.

Nebulized Inhalation of Peptide-Modified DNA Origami To Alleviate Acute Lung Injury.

Acute lung injury (ALI) is a severe inflammatory lung disease, with high mortality rates. Early intervention by reactive oxygen species (ROS) scavengers could reduce ROS accumulation, break the inflammation expansion chain in alveolar macrophages (AMs), and avoid irreversible damage to alveolar epithelial and endothelial cells. Here, we reported cell-penetrating R9 peptide-modified triangular DNA origami nanostructures (tDONs-R9) as a novel nebulizable drug that could reach the deep alveolar regions and exhibit an enhanced uptake preference of macrophages. tDONs-R9 suppressed the expression of pro-inflammatory cytokines and drove polarization toward the anti-inflammatory M2 phenotype in macrophages. In the LPS-induced ALI mouse model, treatment with nebulized tDONs-R9 alleviated the overwhelming ROS, pro-inflammatory cytokines, and neutrophil infiltration in the lungs. Our study demonstrates that tDONs-R9 has the potential for ALI treatment, and the programmable DNA origami nanostructures provide a new drug delivery platform for pulmonary disease treatment with high delivery efficiency and biosecurity.

Impact of Nebulization on the Physicochemical Properties of Polymer-Lipid Hybrid Nanoparticles for Pulmonary Drug Delivery.

Nanoparticles (NPs) have shown significant potential for pulmonary administration of therapeutics for the treatment of chronic lung diseases in a localized and sustained manner. Nebulization is a suitable method of NP delivery, particularly in patients whose ability to breathe is impaired due to lung diseases. However, there are limited studies evaluating the physicochemical properties of NPs after they are passed through a nebulizer. High shear stress generated during nebulization could potentially affect the surface properties of NPs, resulting in the loss of encapsulated drugs and alteration in the release kinetics. Herein, we thoroughly examined the physicochemical properties as well as the therapeutic effectiveness of Infasurf lung surfactant (IFS)-coated PLGA NPs previously developed by us after passing through a commercial Aeroneb® vibrating-mesh nebulizer. Nebulization did not alter the size, surface charge, IFS coating and bi-phasic release pattern exhibited by the NPs. However, there was a temporary reduction in the initial release of encapsulated therapeutics in the nebulized compared to non-nebulized NPs. This underscores the importance of evaluating the drug release kinetics of NPs using the inhalation method of choice to ensure suitability for the intended medical application. The cellular uptake studies demonstrated that both nebulized and non-nebulized NPs were less readily taken up by alveolar macrophages compared to lung cancer cells, confirming the IFS coating retention. Overall, nebulization did not significantly compromise the physicochemical properties as well as therapeutic efficacy of the prepared nanotherapeutics.

Dysregulation of TNF-induced protein 3 and CCAAT/enhancer-binding protein ß in alveolar macrophages: Implications for systemic sclerosis-associated interstitial lung disease.

OBJECTIVES: This study investigates the role of TNF-induced protein 3 (TNFAIP3) and CCAAT/enhancer-binding protein ß (C/EBPß) in alveolar macrophages (AMs) of patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) and their influence on pulmonary fibrosis. METHODS: Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPß was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPß, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPß, IL-10, and TGF-ß1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPß. RESULTS: TNFAIP3 expression was significantly reduced in SSc-ILD AMs, correlating with increased Col1 production in fibroblasts. Overexpression of TNFAIP3 inhibited this pro-fibrotic activity. Conversely, C/EBPß expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFß1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPß. CONCLUSIONS: This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPß expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.

Sialic Acid-Siglec-E Interactions Regulate the Response of Neonatal Macrophages to Group B Streptococcus.

The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung. In both neonatal and adult mice, GBS lung infection reduced Siglec1 expression, potentially delaying acquisition of immunity in neonates. Suppression of Siglec1 expression required interactions between sialic acid on the GBS capsule and the inhibitory host receptor Siglec-E. The Siglec1 gene contains multiple STAT binding motifs, which could regulate expression of sialoadhesin downstream of innate immune signals. Although GBS infection reduced STAT1 expression in the lungs of wild-type newborn mice, we observed increased numbers of STAT1+ cells in Siglece-/- lungs. To test if innate immune activation could increase sialoadhesin at birth, we first demonstrated that treatment of neonatal lung macrophages ex vivo with inflammatory activators increased sialoadhesin expression. However, overcoming the low sialoadhesin expression at birth using in vivo prenatal exposures or treatments with inflammatory stimuli were not successful. The suppression of sialoadhesin expression by GBS-Siglec-E engagement may therefore contribute to disease pathogenesis in newborns and represent a challenging but potentially appealing therapeutic opportunity to augment immunity at birth.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

Coordinated chemokine expression defines macrophage subsets across tissues.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

ZBP1 inhibits the replication of Senecavirus A by enhancing NF-κB signaling pathway mediated antiviral response in porcine alveolar macrophage 3D4/21 cells.

BACKGROUND: Senecavirus A (SVA) caused porcine idiopathic vesicular disease (PIVD) showing worldwide spread with economic losses in swine industry. Although some progress has been made on host factors regulating the replication of SVA, the role of Z-DNA binding protein 1 (ZBP1) remains unclear. METHODS: The expression of ZBP1 in SVA-infected 3D/421 cells was analyzed by quantitative real-time PCR (qRT-PCR) and western blot. Western blot and qRT-PCR were used to detect the effects of over and interference expression of ZBP1 on SVA VP2 gene and protein. Viral growth curves were prepared to measure the viral proliferation. The effect on type I interferons (IFNs), interferon-stimulated genes (ISGs), and pro-inflammatory cytokines in SVA infection was analyzed by qRT-PCR. Western blot was used to analysis the effect of ZBP1 on NF-κB signaling pathway and inhibitor are used to confirm. RESULTS: ZBP1 is shown to inhibit the replication of SVA by enhancing NF-κB signaling pathway mediated antiviral response. SVA infection significantly up-regulated the expression of ZBP1 in 3D4/21 cells. Infection of cells with overexpression of ZBP1 showed that the replication of SVA was inhibited with the enhanced expression of IFNs (IFN-α, IFN-ß), ISGs (ISG15, PKR, and IFIT1) and pro-inflammatory cytokines (IL-6, IL-8, and TNF-α), while, infected-cells with interference expression of ZBP1 showed opposite effects. Further results showed that antiviral effect of ZBP1 is achieved by activation the NF-κB signaling pathway and specific inhibitor of NF-κB also confirmed this. CONCLUSIONS: ZBP1 is an important host antiviral factor in SVA infection and indicates that ZBP1 may be a novel target against SVA.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Associations of cold-inducible RNA-binding protein with bacterial load, proinflammatory cytokines and mortality from pneumonia.

Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern that plays a critical role in triggering inflammatory responses. It remains unknown whether CIRP is strongly associated with bacterial load, inflammatory response, and mortality in sepsis model. Pneumonia was induced in specific pathogen-free 8-9-week old male rats by injecting bacteria via puncture of the tracheal cartilage. The expressions of CIRP and proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß] in lung tissues, alveolar macrophages (AMs), plasma, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The numbers of bacteria recovered from the lungs were correlated with the bacterial loads injected and mortality. The expressions of CIRP increased sharply as the bacterial loads increased in the lung tissues and AMs. The amounts of TNF-α, IL-6 and IL-1ß proteins synthesized were dependent on the bacterial load in the lung tissues. Releases of CIRP, TNF-α, IL-6, and IL-1ß increased with the bacterial load in the blood plasma. The proteins confirmed similar patterns in the BALF. CIRP was strongly associated with the releases of TNF-α, IL-6, and IL-1ß in the lung tissues, blood plasma, and BALF, and showed a close correlation with mortality. CIRP demonstrated a strong association with bacterial load, which is new evidence, and close correlations with proinflammatory cytokines and mortality of pneumonia in rats, suggesting that it might be an interesting pneumonic biomarker for monitoring host response and predicting mortality, and a promising target for immunotherapy.

The guardians of pulmonary harmony: alveolar macrophages orchestrating the symphony of lung inflammation and tissue homeostasis.

Recent breakthroughs in single-cell sequencing, advancements in cellular and tissue imaging techniques, innovations in cell lineage tracing, and insights into the epigenome collectively illuminate the enigmatic landscape of alveolar macrophages in the lung under homeostasis and disease conditions. Our current knowledge reveals the cellular and functional diversity of alveolar macrophages within the respiratory system, emphasising their remarkable adaptability. By synthesising insights from classical cell and developmental biology studies, we provide a comprehensive perspective on alveolar macrophage functional plasticity. This includes an examination of their ontology-related features, their role in maintaining tissue homeostasis under steady-state conditions and the distinct contribution of bone marrow-derived macrophages (BMDMs) in promoting tissue regeneration and restoring respiratory system homeostasis in response to injuries. Elucidating the signalling pathways within inflammatory conditions, the impact of various triggers on tissue-resident alveolar macrophages (TR-AMs), as well as the recruitment and polarisation of macrophages originating from the bone marrow, presents an opportunity to propose innovative therapeutic approaches aimed at modulating the equilibrium between phenotypes to induce programmes associated with a pro-regenerative or homeostasis phenotype of BMDMs or TR-AMs. This, in turn, can lead to the amelioration of disease outcomes and the attenuation of detrimental inflammation. This review comprehensively addresses the pivotal role of macrophages in the orchestration of inflammation and resolution phases after lung injury, as well as ageing-related shifts and the influence of clonal haematopoiesis of indeterminate potential mutations on alveolar macrophages, exploring altered signalling pathways and transcriptional profiles, with implications for respiratory homeostasis.

Porcine cis-acting lnc-CAST positively regulates CXCL8 expression through histone H3K27ac.

The chemokine CXCL8, also known as the neutrophil chemotactic factor, plays a crucial role in mediating inflammatory responses and managing cellular immune reactions during viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) primarily infects pulmonary alveolar macrophages (PAMs), leading to acute pulmonary infections. In this study, we explored a novel long non-coding RNA (lncRNA), termed lnc-CAST, situated within the Cxcl8 gene locus. This lncRNA was found to be highly expressed in porcine macrophages. We observed that both lnc-CAST and CXCL8 were significantly upregulated in PAMs following PRRSV infection, and after treatments with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Furthermore, we noticed a concurrent upregulation of lnc-CAST and CXCL8 expression in lungs of PRRSV-infected pigs. We then determined that lnc-CAST positively influenced CXCL8 expression in PAMs. Overexpression of lnc-CAST led to an increase in CXCL8 production, which in turn enhanced the migration of epithelial cells and the recruitment of neutrophils. Conversely, inhibiting lnc-CAST expression resulted in reduced CXCL8 production in PAMs, leading to decreased migration levels of epithelial cells and neutrophils. From a mechanistic perspective, we found that lnc-CAST, localized in the nucleus, facilitated the enrichment of histone H3K27ac in CXCL8 promoter region, thereby stimulating CXCL8 transcription in a cis-regulatory manner. In conclusion, our study underscores the pivotal critical role of lnc-CAST in regulating CXCL8 production, offering valuable insights into chemokine regulation and lung damage during PRRSV infection.

Modulation of alveolar macrophage and mitochondrial fitness by medicinal plant-derived nanovesicles to mitigate acute lung injury and viral pneumonia.

Acute lung injury (ALI) is generally caused by severe respiratory infection and characterized by overexuberant inflammatory responses and inefficient pathogens-containing, the two major processes wherein alveolar macrophages (AMs) play a central role. Dysfunctional mitochondria have been linked with distorted macrophages and hence lung disorders, but few treatments are currently available to correct these defects. Plant-derive nanovesicles have gained significant attention because of their therapeutic potential, but the targeting cells and the underlying mechanism remain elusive. We herein prepared the nanovesicles from Artemisia annua, a well-known medicinal plant with multiple attributes involving anti-inflammatory, anti-infection, and metabolism-regulating properties. By applying three mice models of acute lung injury caused by bacterial endotoxin, influenza A virus (IAV) and SARS-CoV-2 pseudovirus respectively, we showed that Artemisia-derived nanovesicles (ADNVs) substantially alleviated lung immunopathology and raised the survival rate of challenged mice. Macrophage depletion and adoptive transfer studies confirmed the requirement of AMs for ADNVs effects. We identified that gamma-aminobutyric acid (GABA) enclosed in the vesicles is a major molecular effector mediating the regulatory roles of ADNVs. Specifically, GABA acts on macrophages through GABA receptors, promoting mitochondrial gene programming and bioenergy generation, reducing oxidative stress and inflammatory signals, thereby enhancing the adaptability of AMs to inflammation resolution. Collectively, this study identifies a promising nanotherapeutics for alleviating lung pathology, and elucidates a mechanism whereby the canonical neurotransmitter modifies AMs and mitochondria to resume tissue homeostasis, which may have broader implications for treating critical pulmonary diseases such as COVID-19.

Lipid nanoparticle encapsulated large peritoneal macrophages migrate to the lungs via the systemic circulation in a model of clodronate-mediated lung-resident macrophage depletion.

Rationale: A mature tissue resident macrophage (TRM) population residing in the peritoneal cavity has been known for its unique ability to migrate to peritoneally located injured tissues and impart wound healing properties. Here, we sought to expand on this unique ability of large peritoneal macrophages (LPMs) by investigating whether these GATA6+ LPMs could also intravasate into systemic circulation and migrate to extra-peritoneally located lungs upon ablating lung-resident alveolar macrophages (AMs) by intranasally administered clodronate liposomes in mice. Methods: C12-200 cationic lipidoid-based nanoparticles were employed to selectively deliver a small interfering RNA (siRNA)-targeting CD-45 labeled with a cyanine 5.5 (Cy5.5) dye to LPMs in vivo via intraperitoneal injection. We utilized a non-invasive optical technique called Diffuse In Vivo Flow Cytometry (DiFC) to then systemically track these LPMs in real time and paired it with more conventional techniques like flow cytometry and immunocytochemistry to initially confirm uptake of C12-200 encapsulated siRNA-Cy5.5 (siRNA-Cy5.5 (C12-200)) into LPMs, and further track them from the peritoneal cavity to the lungs in a mouse model of AM depletion incited by intranasally administered clodronate liposomes. Also, we stained for LPM-specific marker zinc-finger transcription factor GATA6 in harvested cells from biofluids like broncho-alveolar lavage as well as whole blood to probe for Cy5.5-labeled LPMs in the lungs as well as in systemic circulation. Results: siRNA-Cy5.5 (C12-200) was robustly taken up by LPMs. Upon depletion of lung-resident AMs, these siRNA-Cy5.5 (C12-200) labeled LPMs rapidly migrated to the lungs via systemic circulation within 12-24 h. DiFC results showed that these LPMs intravasated from the peritoneal cavity and utilized a systemic route of migration. Moreover, immunocytochemical staining of zinc-finger transcription factor GATA6 further confirmed results from DiFC and flow cytometry, confirming the presence of siRNA-Cy5.5 (C12-200)-labeled LPMs in the peritoneum, whole blood and BALF only upon clodronate-administration. Conclusion: Our results indicate for the very first time that selective tropism, migration, and infiltration of LPMs into extra-peritoneally located lungs was dependent on clodronate-mediated AM depletion. These results further open the possibility of therapeutically utilizing LPMs as delivery vehicles to carry nanoparticle-encapsulated oligonucleotide modalities to potentially address inflammatory diseases, infectious diseases and even cancer.

Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis.

BACKGROUND: Influenza A viruses (IAV) are extremely common respiratory viruses for the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), in which IAV infection may further evoke abnormal macrophage polarization, amplify cytokine storms. Melatonin exerts potential effects of anti-inflammation and anti-IAV infection, while its effects on IAV infection-induced AECOPD are poorly understood. METHODS: COPD mice models were established through cigarette smoke exposure for consecutive 24 weeks, evaluated by the detection of lung function. AECOPD mice models were established through the intratracheal atomization of influenza A/H3N2 stocks in COPD mice, and were injected intraperitoneally with melatonin (Mel). Then, The polarization of alveolar macrophages (AMs) was assayed by flow cytometry of bronchoalveolar lavage (BAL) cells. In vitro, the effects of melatonin on macrophage polarization were analyzed in IAV-infected Cigarette smoking extract (CSE)-stimulated Raw264.7 macrophages. Moreover, the roles of the melatonin receptors (MTs) in regulating macrophage polarization and apoptosis were determined using MTs antagonist luzindole. RESULTS: The present results demonstrated that IAV/H3N2 infection deteriorated lung function (reduced FEV20,50/FVC), exacerbated lung damages in COPD mice with higher dual polarization of AMs. Melatonin therapy improved airflow limitation and lung damages of AECOPD mice by decreasing IAV nucleoprotein (IAV-NP) protein levels and the M1 polarization of pulmonary macrophages. Furthermore, in CSE-stimulated Raw264.7 cells, IAV infection further promoted the dual polarization of macrophages accompanied with decreased MT1 expression. Melatonin decreased STAT1 phosphorylation, the levels of M1 markers and IAV-NP via MTs reflected by the addition of luzindole. Recombinant IL-1ß attenuated the inhibitory effects of melatonin on IAV infection and STAT1-driven M1 polarization, while its converting enzyme inhibitor VX765 potentiated the inhibitory effects of melatonin on them. Moreover, melatonin inhibited IAV infection-induced apoptosis by suppressing IL-1ß/STAT1 signaling via MTs. CONCLUSIONS: These findings suggested that melatonin inhibited IAV infection, improved lung function and lung damages of AECOPD via suppressing IL-1ß/STAT1-driven macrophage M1 polarization and apoptosis in a MTs-dependent manner. Melatonin may be considered as a potential therapeutic agent for influenza virus infection-induced AECOPD.