Response Of Alveolar Macrophages Research Articles

EGR2 is an epigenomic regulator of phagocytosis and antifungal immunity in alveolar macrophages.

Alveolar macrophages (AMs) act as gatekeepers of the lung's immune responses, serving essential roles in recognizing and eliminating pathogens. The transcription factor (TF) early growth response 2 (EGR2) has been recently described as required for mature AMs in mice; however, its mechanisms of action have not been explored. Here, we identified EGR2 as an epigenomic regulator and likely direct proximal transcriptional activator in AMs using epigenomic approaches (RNA sequencing, ATAC sequencing, and CUT&RUN). The predicted direct proximal targets of EGR2 included a subset of AM identity genes and ones related to pathogen recognition, phagosome maturation, and adhesion, such as Clec7a, Atp6v0d2, Itgb2, Rhoc, and Tmsb10. We provided evidence that EGR2 deficiency led to impaired zymosan internalization and reduced the capacity to respond to Aspergillus fumigatus. Mechanistically, the lack of EGR2 altered the transcriptional response, secreted cytokines (i.e., CXCL11), and inflammation-resolving lipid mediators (i.e., RvE1) of AMs during in vivo zymosan-induced inflammation, which manifested in impaired resolution. Our findings demonstrated that EGR2 is a key proximal transcriptional activator and epigenomic bookmark in AMs responsible for select, distinct components of cell identity and a protective transcriptional and epigenomic program against fungi.

Testosterone and estradiol reduce inflammation of human macrophages induced by anti-SARS-CoV-2 IgG.

COVID-19, the disease caused by SARS-CoV-2, particularly causes severe inflammatory disease in elderly, obese, and male patients. Since both aging and obesity are associated with decreased testosterone and estradiol expression, we hypothesized that decreased hormone levels contribute to excessive inflammation in the context of COVID-19. Previously, we and others have shown that hyperinflammation in severe COVID-19 patients is induced by the production of pathogenic anti-spike IgG antibodies that activate alveolar macrophages. Therefore, we developed an in vitro assay in which we stimulated human macrophages with viral stimuli, anti-spike IgG immune complexes, and different sex hormones. Treatment with levels of testosterone reflecting young adults led to a significant reduction in TNF and IFN-γ production by human macrophages. In addition, estradiol significantly attenuated the production of a very broad panel of cytokines, including TNF, IL-1β, IL-6, IL-10, and IFN-γ. Both testosterone and estradiol reduced the expression of Fc gamma receptors IIa and III, the two main receptors responsible for anti-spike IgG-induced inflammation. Combined, these findings indicate that sex hormones reduce the inflammatory response of human alveolar macrophages to specific COVID-19-associated stimuli, thereby providing a potential immunological mechanism for the development of severe COVID-19 in both older male and female patients.

TGFβ primes alveolar-like macrophages to induce type I IFN following TLR2 activation.

Alveolar macrophages (AMs) are key mediators of lung function and are potential targets for therapies during respiratory infections. TGFβ is an important regulator of AM differentiation and maintenance, but how TGFβ directly modulates the innate immune responses of AMs remains unclear. This shortcoming prevents effective targeting of AMs to improve lung function in health and disease. Here we leveraged an optimized ex vivo AM model system, fetal-liver derived alveolar-like macrophages (FLAMs), to dissect the role of TGFβ in AMs. Using transcriptional analysis, we first globally defined how TGFβ regulates gene expression of resting FLAMs. We found that TGFβ maintains the baseline metabolic state of AMs by driving lipid metabolism through oxidative phosphorylation and restricting inflammation. To better understand inflammatory regulation in FLAMs, we next directly tested how TGFβ alters the response to TLR2 agonists. While both TGFβ (+) and TGFβ (-) FLAMs robustly responded to TLR2 agonists, we found an unexpected activation of type I interferon (IFN) responses in FLAMs and primary AMs in a TGFβ-dependent manner. Surprisingly, mitochondrial antiviral signaling protein and the interferon regulator factors 3 and 7 were required for IFN production by TLR2 agonists. Together, these data suggest that TGFβ modulates AM metabolic networks and innate immune signaling cascades to control inflammatory pathways in AMs.

Aspiration of acidified milk induces milk allergy by activating alveolar macrophages in mice

BackgroundEpidemiological studies have identified associations between gastroesophageal reflux (GER) and cow's milk allergy (CMA) in infants. However, the role of GER in the development of CMA remains poorly understood. Our primary objectives were to develop a mouse model that suggests GER as a potential pathogenic mechanism for CMA and to elucidate the immunological mechanisms that connect lung innate immunity with CMA. MethodsMice were exposed to cow's milk (CM) treated with hydrochloric acid through repeated aspiration into their airways. Subsequently, they were challenged by intraperitoneal injection of CM extract. The immunological mechanisms were investigated using comprehensive single-cell RNA sequencing (scRNA-seq) analysis of the lungs, combined with the use of genetically modified mice. ResultsMice exposed to CM mixed with hydrochloric acid via airway sensitization developed CMA, as evidenced by the production of antigen-specific IgE and IgG antibodies, and the induction of anaphylaxis upon systemic antigen administration. In contrast, aspiration of CM alone did not induce CMA. scRNA-seq analysis revealed potential roles of alveolar macrophages in response to hydrochloric acid. Mice lacking the TLR4 pathway were protected from developing CMA. ConclusionsWe have developed a novel mouse model for CMA that utilizes the natural antigen and follows the physiological airway sensitization pathway, thus potentially resembling clinical scenarios. This model, named the acidified milk aspiration-induced allergy model, has the potential to shed light on the role of early innate immunity by analyzing a more physiological model.

Human Alveolar and Monocyte-Derived Human Macrophage Responses to Mycobacterium tuberculosis.

Alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) mediate early lung immune responses to Mycobacterium tuberculosis. Differences in the response of these distinct cell types are poorly understood and may provide insight into mechanisms of tuberculosis pathogenesis. The objective of this study was to determine whether M. tuberculosis induces unique and essential antimicrobial pathways in human AMs compared with MDMs. Using paired human AMs and 5-d MCSF-derived MDMs from six healthy volunteers, we infected cells with M. tuberculosis H37Rv for 6 h, isolated RNA, and analyzed transcriptomic profiles with RNA sequencing. We found 681 genes that were M. tuberculosis dependent in AMs compared with MDMs and 4538 that were M. tuberculosis dependent in MDMs, but not AMs (false discovery rate [FDR] < 0.05). Using hypergeometric enrichment of DEGs in Broad Hallmark gene sets, we found that type I and II IFN Response were the only gene sets selectively induced in M. tuberculosis-infected AM (FDR < 0.05). In contrast, MYC targets, unfolded protein response and MTORC1 signaling, were selectively enriched in MDMs (FDR < 0.05). IFNA1, IFNA8, IFNE, and IFNL1 were specifically and highly upregulated in AMs compared with MDMs at baseline and/or after M. tuberculosis infection. IFNA8 modulated M. tuberculosis-induced proinflammatory cytokines and, compared with other IFNs, stimulated unique transcriptomes. Several DNA sensors and IFN regulatory factors had higher expression at baseline and/or after M. tuberculosis infection in AMs compared with MDMs. These findings demonstrate that M. tuberculosis infection induced unique transcriptional responses in human AMs compared with MDMs, including upregulation of the IFN response pathway and specific DNA sensors.

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.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

DYNAMIC PORTRAIT OF ALVEOLAR MACROPHAGES IN EXPERIMENTAL DIABETES MELLITUS

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

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

Unveiling Shared Immune Responses in Porcine Alveolar Macrophages during ASFV and PRRSV Infection Using Single-Cell RNA-seq.

African swine fever virus (ASFV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections lead to severe respiratory diseases in pigs, resulting in significant economic losses for the global swine industry. While numerous studies have focused on specific gene functions or pathway activities during infection, an investigation of shared immune responses in porcine alveolar macrophages (PAMs) after ASFV and PRRSV infections was lacking. In this study, we conducted a comparison using two single-cell transcriptomic datasets generated from PAMs under ASFV and PRRSV infection. Pattern recognition receptors (PRRs) RIG-I (DDX58), MDA5 (IFIH1), and LGP2 (DHX58) were identified as particularly recognizing ASFV and PRRSV, triggering cellular defense responses, including the upregulation of four cytokine families (CCL, CXCL, IL, and TNF) and the induction of pyroptosis. Through weighted gene co-expression network analysis and protein-protein interaction analysis, we identified thirteen gene and protein interactions shared by both scRNA-seq analyses, suggesting the ability to inhibit both ASFV and PRRSV viral replication. We discovered six proteins (PARP12, PARP14, HERC5, DDX60, RSAD2, and MNDA) in PAMs as inhibitors of ASFV and PRRSV replication. Collectively, our findings showed detailed characterizations of the immune responses in PAMs during ASFV and PRRSV infections, which may facilitate the treatments of these viral diseases.

Targeting the TRAF3-ULK1-NLRP3 regulatory axis to control alveolar macrophage pyroptosis in acute lung injury.

Acute lung injury (ALI) is a serious condition characterized by damage to the lungs. Recent research has revealed that activation of the NLRP3 inflammasome in alveolar macrophages, a type of immune cell in the lungs, plays a key role in the development of ALI. This process, known as pyroptosis, contributes significantly to ALI pathogenesis. Researchers have conducted comprehensive bioinformatics analyses and identified 15 key genes associated with alveolar macrophage pyroptosis in ALI. Among these, NLRP3 has emerged as a crucial regulator. This study further reveal that the ULK1 protein diminishes the expression of NLRP3, thereby reducing the immune response of alveolar macrophages and mitigating ALI. Conversely, TRAF3, another protein, is found to inhibit ULK1 through a process called ubiquitination, leading to increased activation of the NLRP3 inflammasome and exacerbation of ALI. This TRAF3-mediated suppression of ULK1 and subsequent activation of NLRP3 are confirmed through various in vitro and in vivo experiments. The presence of abundant M0 and M1 alveolar macrophages in the ALI tissue samples further support these findings. This research highlights the TRAF3-ULK1-NLRP3 regulatory axis as a pivotal pathway in ALI development and suggests that targeting this axis could be an effective therapeutic strategy for ALI treatment.

Decreased immune response in undernourished rats after air pollution exposure

Children are highly vulnerable subpopulation to malnutrition and air pollution. We investigate, in a rat nutritional growth retardation (NGR) model, the impact of Residual Oil Fly Ash (ROFA) on the lung immune response using in vitro and ex vivo methods. In vitro: Alveolar macrophages (AM) were isolated from Control (C) and NGR animals, cultured and treated with ROFA (1–100 µg/ml) for 24 h. Ex vivo: C and NGR rats were intranasally instilled with ROFA (1 mg/kg BW) or PBS. 24 h post-exposure AM were isolated and cultured. ROFA-treatment increased superoxide anion production and TNFα secretion in C-AM in vitro, though for NGR-AM this response was lower. A similar pattern was observed for TNFα and IL-6 secretion in ex vivo experiments. Regarding the antioxidant response, although NGR-AM showed increased Nrf2, after ROFA instillation an attenuated activation was observed. To conclude, chronic undernutrition altered AM response to ROFA affecting immune responsiveness to air pollutants.

Exposure to Mycobacterium remodels alveolar macrophages and the early innate response to Mycobacterium tuberculosis infection.

Alveolar macrophages (AMs) play a critical role during Mycobacterium tuberculosis (Mtb) infection as the first cells in the lung to encounter bacteria. We previously showed that AMs initially respond to Mtb in vivo by mounting a cell-protective, rather than pro-inflammatory response. However, the plasticity of the initial AM response was unknown. Here, we characterize how previous exposure to Mycobacterium, either through subcutaneous vaccination with Mycobacterium bovis (scBCG) or through a contained Mtb infection (coMtb) that mimics aspects of concomitant immunity, impacts the initial response by AMs. We find that both scBCG and coMtb accelerate early innate cell activation and recruitment and generate a stronger pro-inflammatory response to Mtb in vivo by AMs. Within the lung environment, AMs from scBCG vaccinated mice mount a robust interferon-associated response, while AMs from coMtb mice produce a broader inflammatory response that is not dominated by Interferon Stimulated Genes. Using scRNAseq, we identify changes to the frequency and phenotype of airway-resident macrophages following Mycobacterium exposure, with enrichment for both interferon-associated and pro-inflammatory populations of AMs. In contrast, minimal changes were found for airway-resident T cells and dendritic cells after exposures. Ex vivo stimulation of AMs with Pam3Cys, LPS and Mtb reveal that scBCG and coMtb exposures generate stronger interferon-associated responses to LPS and Mtb that are cell-intrinsic changes. However, AM profiles that were unique to each exposure modality following Mtb infection in vivo are dependent on the lung environment and do not emerge following ex vivo stimulation. Overall, our studies reveal significant and durable remodeling of AMs following exposure to Mycobacterium, with evidence for both AM-intrinsic changes and contributions from the altered lung microenvironments. Comparisons between the scBCG and coMtb models highlight the plasticity of AMs in the airway and opportunities to target their function through vaccination or host-directed therapies.

Estrogen contributes to sex differences in M2a macrophages during multi-walled carbon nanotube-induced respiratory inflammation.

Lung diseases characterized by type 2 inflammation are reported to occur with a female bias in prevalence/severity in both humans and mice. This includes previous work examining multi-walled carbon nanotube (MWCNT)-induced eosinophilic inflammation, in which a more exaggerated M2a phenotype was observed in female alveolar macrophages (AMs) compared to males. The mechanisms responsible for this sex difference in AM phenotype are still unclear, but estrogen receptor (ER) signaling is a likely contributor. Accordingly, male AMs downregulated ERα expression after MWCNT exposure while female AMs did not. Thus, ER antagonist Fulvestrant was administered prior to MWCNT instillation. In females, Fulvestrant significantly attenuated MWCNT-induced M2a gene expression and eosinophilia without affecting IL-33. In males, Fulvestrant did not affect eosinophil recruitment but reduced IL-33 and M2a genes compared to controls. Regulation of cholesterol efflux and oxysterol synthesis is a potential mechanism through which estrogen promotes the M2a phenotype. Levels of oxysterols 25-OHC and 7α,25-OHC were higher in the airways of MWCNT-exposed males compared to MWCNT-females, which corresponds with the lower IL-1β production and greater macrophage recruitment previously observed in males. Sex-based changes in cholesterol efflux transporters Abca1 and Abcg1 were also observed after MWCNT exposure with or without Fulvestrant. Invitro culture with estrogen decreased cellular cholesterol and increased the M2a response in female AMs, but did not affect cholesterol content in male AMs and reduced M2a polarization. These results reveal the modulation of (oxy)sterols as a potential mechanism through which estrogen signaling may regulate AM phenotype resulting in sex differences in downstream respiratory inflammation.

Age-Associated KLF9 Enhances the Inflammatory Response of Alveolar Macrophages Via Regulating TLR2 Expression.

Staphylococcus aureus pneumonia is a severe infection in infant and young children. Toll-like receptor 2 (TLR2)-mediated inflammation plays essential roles in S. aureus pneumonia. Krueppel-like factor 9 (KLF9) is a transcriptional factor participating in multiple cellular aspects including inflammation. In this study, the potential roles of KLF9 in S. aureus pneumonia were evaluated. The expression of KLF9 in peripheral blood mononuclear cells (PBMCs) from healthy donors with different ages and in alveolar macrophages from mice with different ages was measured. Pam3CK4-induced expression of inflammatory cytokines was compared in alveolar macrophages from young and old mice and in wild-type (WT) and KLF9-deficient macrophages. The survival rate, body weight loss, lung pathology were compared between WT and KLF9-deficient mice after S. aureus infection. The TLR2 expression was compared between WT and KLF9-deficient macrophages after Pam3CK4 treatment. Decreased expression of KLF9 was detected in PBMCs from elder donor and in macrophages from old mice. Impaired expression of pro-inflammatory cytokines was observed in macrophages from old mice and KLF9-deficient macrophages after Pam3CK4 treatment. KLF9-deficient mice had elevated survival rate, decreased lung injury after S. aureus infection. Decreased expression of TLR2 was detected in KLF9-deficient macrophages and overexpression of TLR2 rescued the impaired expression of inflammatory cytokines in KLF9-deficient macrophages. KLF9 regulated inflammatory responses in macrophages through TLR2.

Alveolar macrophages in lung cancer: opportunities challenges.

Alveolar macrophages (AMs) are critical components of the innate defense mechanism in the lung. Nestled tightly within the alveoli, AMs, derived from the yolk-sac or bone marrow, can phagocytose foreign particles, defend the host against pathogens, recycle surfactant, and promptly respond to inhaled noxious stimuli. The behavior of AMs is tightly dependent on the environmental cues whereby infection, chronic inflammation, and associated metabolic changes can repolarize their effector functions in the lungs. Several factors within the tumor microenvironment can re-educate AMs, resulting in tumor growth, and reducing immune checkpoint inhibitors (ICIs) efficacy in patients treated for non-small cell lung cancer (NSCLC). The plasticity of AMs and their critical function in altering tumor responses to ICIs make them a desirable target in lung cancer treatment. New strategies have been developed to target AMs in solid tumors reprograming their suppressive function and boosting the efficacy of ICIs. Here, we review the phenotypic and functional changes in AMs in response to sterile inflammation and in NSCLC that could be critical in tumor growth and metastasis. Opportunities in altering AMs' function include harnessing their potential function in trained immunity, a concept borrowed from memory response to infections, which could be explored therapeutically in managing lung cancer treatment.

Macrophage-Targeted Lipid Nanoparticle Delivery of microRNA-146a to Mitigate Hemorrhagic Shock-Induced Acute Respiratory Distress Syndrome.

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro coculture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro. Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose of miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a levels in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have the therapeutic potential to mitigate lung injury during mechanical ventilation.

Stimulation of lipopolysaccharide from Pseudomonas aeruginosa following H9N2 IAV infection exacerbates inflammatory responses of alveolar macrophages and decreases virus replication

H9N2 IAV infection contributed to P. aeruginosa coinfection, causing severe hemorrhagic pneumonia in mink. In this study, the in vitro alveolar macrophage models were developed to investigate the innate immune responses to P. aeruginosa LPS stimulation following H9N2 IAV infection, using MH-S cells. The cytokine levels, apoptosis levels and the viral nucleic acid levels were detected and analyzed. As a result, the levels of IFN-α, IL-1β, TNF-α, and IL-10 in MH-S cells with P. aeruginosa LPS stimulation following H9N2 IAV infection were significantly higher than those in MH-S cells with single H9N2 IAV infection and single LPS stimulation (P < 0.05), exacerbating inflammatory responses. LPS stimulation aggravated the apoptosis of MH-S cells with H9N2 IAV infection. Interestingly, LPS stimulation influences H9N2 IAV replication and indirectly reduced H9N2 IAV replications in in vitro AMs. It implied that LPS should play an important role in the pathogenesis of H9N2 IAV and P. aeruginosa coinfection.

Small Extracellular Vesicles Secreted by iPSC-Derived MSCs Ameliorate Pulmonary Inflammation and Lung Injury Induced by Sepsis through Delivery of miR-125b-5p.

Sepsis-induced acute lung injury is a common critical illness in intensive care units with no effective treatment is currently available. Small extracellular vesicles, secreted by mesenchymal stem cells (MSCs), derived from human-induced pluripotent stem cells (iMSC-sEV), possess striking advantages when incorporated MSCs and iPSCs, which are considered extremely promising cell-free therapeutic agents. However, no studies have yet been conducted to systemically examine the effects and underlying mechanisms of iMSC-sEV application on attenuated lung injury under sepsis conditions. iMSC-sEV were intraperitoneally administered in a rat septic lung injury model induced by cecal ligation and puncture (CLP). The efficacy of iMSC-sEV was assessed by histology, immunohistochemistry, and pro-inflammatory cytokines of bronchoalveolar lavage fluid. We also evaluated the in vitro effects of iMSC-sEV on the activation of the inflammatory response in alveolar macrophages (AMs). Small RNA sequencing was utilized to detect changes in the miRNA expression profile in lipopolysaccharide (LPS)-treated AMs after iMSC-sEV administration. The effects of miR-125b-5p on the function of AMs were studied. iMSC-sEV were able to attenuate pulmonary inflammation and lung injury following CLP-induced lung injury. iMSC-sEV were internalized by AMs and alleviated the release of inflammatory factors by inactivating the NF-κB signaling pathway. Moreover, miR-125b-5p showed a fold-change in LPS-treated AMs after iMSC-sEV administration and was enriched in iMSC-sEV. Mechanistically, iMSC-sEV transmitted miR-125b-5p into LPS-treated AMs to target TRAF6. Our findings demonstrated that iMSC-sEV treatment protects against septic lung injury and exerts anti-inflammatory effects on AMs at least partially through miR-125b-5p, suggesting that iMSC-sEV may provide a novel cell-free strategy for the treatment of septic lung injury.

Koyun Alveoler Makrofajlarında Mannheimia Haemolytica'ya Yanıt Olarak Bağışıklık ile İlişkili Genlerin İn Vitro mRNA Ekspresyon Dinamikleri

Alveolar macrophages (AMs) respond to these infections as the first line of defense and trigger the lung's immune response. Knowing the expression dynamics of key immune-related genes in alveolar macrophages against M. haemolytica challenge will help deepen the understanding of disease immunopathogenesis. For this purpose, first time an in vitro obtained from bronchoalveolar lavage (BAL) fluid and treated with three doses (T1: 1800, T2: 2700, T3: 5400 CFU) of M. haemolytica inoculum, while keeping one untreated as a control. Then, total RNA was extracted, and cDNA was synthesized. The real-time quantification data indicated that the expression of IL-1β, IL-6, IL-8, IL-10, and NF-κB genes were significantly changed as compared to control. Our study revealed that the exposure to M. haemolytica stimulates the immune response in the sheep alveolar macrophages in a dose and time-dependent manner.