Expression In Alveolar Macrophages Research Articles

Eclipta prostrata improves alveolar development of bronchopulmonary dysplasia via suppressing the NLRP3 inflammasome in a DLD-dependent manner.

Bronchopulmonary dysplasia (BPD), the most common late morbidity in preterm infants, is characterized by impaired alveolar development caused by persistent lung inflammation. Studies have shown that NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome-mediated inflammation is critically involved in the development of BPD. As a traditional Chinese medicinal herb, Eclipta prostrata (EAP) exhibits potent anti-inflammatory properties. Our study aims to investigate whether EAP could improve the lung development of BPD by suppressing the lung inflammatory response. The BPD rat model was established by intra-amniotic injection of lipopolysaccharide (LPS) and postnatal exposure to hyperoxia. Changes in the NLRP3 inflammasome and pyroptosis were assessed by treatment with EAP. The effect of EAP on the NLRP3 inflammasome was tested in vitro using the THP-1 cell line and primary alveolar macrophages. Proteomics analysis was used to elucidate the mechanism of action of EAP. Histopathological and immunofluorescence results of lung tissues revealed that LPS and hyperoxia induced lung injury and triggered NLRP3 inflammasome activation and pyroptosis in alveolar macrophages. EAP ameliorated BPD lung injury, inhibited NLRP3 inflammasome activation and reduced gasdermin D (GSDMD) expression in alveolar macrophages. EAP downregulated the expression of NLRP3 inflammasome pathway molecules (NLRP3, caspase-1, and IL-1β) and GSDMD in LPS-stimulated THP-1 macrophages and primary alveolar macrophages. In addition, proteomics analysis identified that dihydrolipoamide dehydrogenase (DLD) interacted with EAP. Inhibition of DLD activity abolished the protective effects of EAP. Our study suggested that EAP could attenuate arrest of alveolar development via inhibiting NLRP3 inflammasome in a DLD-dependent way, and could be a potential therapeutic method for BPD.

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

This study investigates the role of TNF-induced protein 3 (TNFAIP3) and CCAAT/enhancer-binding protein β (C/EBPβ) in alveolar macrophages (AMs) of patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) and their influence on pulmonary fibrosis. Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPβ was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPβ, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPβ, IL-10, and TGF-β1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPβ. 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/EBPβ expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFβ1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPβ. This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPβ expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.

Understanding the modulations of glycero-lysophospholipids in an elastase-induced murine emphysema model

BackgroundIncreasing evidence indicates that bioactive lipid mediators are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Recently, glycero-lysophospholipids, such as lysophosphatidic acid (LysoPA) and lysophosphatidylserine (LysoPS), have been recognized as significant inflammation-related lipid mediators. However, their association with COPD remains unclear. MethodsWe used an elastase-induced murine emphysema model to analyze the levels of lysophospholipids and diacyl-phospholipids in the lungs. Additionally, we assessed the expression of LysoPS-related genes and published data on smokers. ResultsIn the early phase of an elastase-induced murine emphysema model, the levels of LysoPS and its precursor (phosphatidylserine [PS]) were significantly reduced, without significant modulations in other glycero-lysophospholipids. Additionally, there was an upregulation in the expression of lysoPS receptors, specifically GPR34, observed in the lungs of a cigarette smoke-exposed mouse model and the alveolar macrophages of human smokers. Elastase stimulation induces GPR34 expression in a human macrophage cell line in vitro. ConclusionsElastase-induced lung emphysema affects the LysoPS/PS-GPR34 axis, and cigarette smoking or elastase upregulates GPR34 expression in alveolar macrophages. This novel association may serve as a potential pharmacological target for COPD treatment.

IL-27p28 specifically regulates MHC II expression in macrophages through CIITA

Antigen-presenting cells (APCs) constantly express major histocompatibility complex II (MHC II), including macrophages and dendritic cells (DCs) which deliver antigens to CD4+ T cells and play an important role in adaptive immunity. The expression of MHC II is controlled by the transcriptional coactivator CIITA. Interleukin-27 (IL-27), a newly discovered IL-12 family cytokine, is composed of p28 and EBI3 subunits. In this study, we used IL-27p28 conditional knock-out mice to investigate the regulatory effects of IL-27p28 on macrophage polarization and the expression of MHC II in macrophages. We found that MHC II expression was upregulated in the bone marrow-derived and peritoneal exudate macrophages (BMDMs; PEMs) from IL-27p28-deficient mice, with their inflammation regulating function unaffected. We also demonstrated that in the APCs, IL-27p28 selectively regulated MHC II expression in macrophages but not in dendritic cells. During Pseudomonas aeruginosa (P. aeruginosa) reinfection, higher survival rate, bacterial clearance, and ratio of CD4+/CD8+ T cells in the spleen during the specific immune phase were observed in IL-27p28 defect mice, as well as an increased MHC II expression in alveolar macrophages (AMs). But these did not occur in the first infection. For the first time we discovered that IL-27p28 specifically regulates the expression of MHC II in macrophages by regulating CIITA, while its absence enhances antigen presentation and adaptive immunity against P. aeruginosa.

A novel reporter mouse line for studying alveolar macrophages.

Alveolar macrophages (AMs) are self-maintained immune cells that play vital roles in lung homeostasis and immunity. Although reporter mice and culture systems have been established for studying macrophages, an accurate and specific reporter line for alveolar macrophage study is still not available. Here we reported a novel Rspo1-tdTomato gene reporter mouse line that could specifically label mouse AMs in a cell-intrinsic manner. Using this reporter system, we visualized the dynamics of alveolar macrophages intravitally under steady state and characterized the alveolar macrophage differentiation under in vitro condition. By performing ATAC-seq, we found that insertion of the tdTomato cassette in the Rspo1 locus increased the accessibility of a PPARE motif within the Rspo1 locus and revealed a potential regulation by key transcription factor PPAR-γ for alveolar macrophage differentiation in vitro and in vivo. Consistently, perturbation of PPAR-γ by its agonist rosiglitazone or inhibitor GW9662 resulted in corresponding alteration of tdTomato expression in alveolar macrophages together with the transcription of PPAR-γ downstream target genes. Furthermore, global transcriptomic analyses of AMs from the wild type mice and the Rspo1-tdTomato mice showed comparable gene expression profiles, especially those AM-specific genes, confirming that the insertion of the tdTomato cassette in the Rspo1 locus does not impact the cell identity and biological function of AMs under normal condition. Taken together, our study provides an alternative tool for in vivo and in vitro labeling of alveolar macrophages with high specificity which could also be utilized as an indicator of PPAR-γ activity for future development of PPAR-γ specific targeting drugs.

Pulmonary Expression of Interleukin-17 Contributes to Neutrophil Infiltration into the Lungs during Pneumonic Plague.

Inhalation of respiratory droplets infected with Yersinia pestis results in a rapidly progressing and lethal necrotic pneumonia called primary pneumonic plague. Disease manifests as biphasic, with an initial preinflammatory phase with rapid bacterial replication in the lungs absent readily detectable host immune responses. This is followed by the onset of a proinflammatory phase that sees the dramatic upregulation of proinflammatory cytokines and extensive neutrophil accumulation in the lungs. The plasminogen activator protease (Pla) is an essential virulence factor that is responsible for survival of Y. pestis in the lungs. Our lab recently showed that Pla functions as an adhesin that promotes binding to alveolar macrophages to facilitate translocation of effector proteins called Yops into the cytosol of target host cells via a type 3 secretion system (T3SS). Loss of Pla-mediated adherence disrupted the preinflammatory phase of disease and resulted in early neutrophil migration to the lungs. While it is established that Yersinia broadly suppresses host innate immune responses, it is not clear precisely which signals need to be inhibited to establish a preinflammatory stage of infection. Here, we show that early Pla-mediated suppression of Interleukin-17 (IL-17) expression in alveolar macrophages and pulmonary neutrophils limits neutrophil migration to the lungs and aids in establishing a preinflammatory phase of disease. In addition, IL-17 ultimately contributes to neutrophil migration to the airways that defines the later proinflammatory phase of infection. These results suggest that the pattern of IL-17 expression contributes to the progression of primary pneumonic plague.

An immunoregulator nanomedicine approach for the treatment of tuberculosis.

Introduction: A nanoparticle composed of a poly (lactic-co-glycolic acid) (PLGA) core and a chitosan (CS) shell with surface-adsorbed 1,3 β-glucan (β-glucan) was synthesized. The exposure response of CS-PLGA nanoparticles (0.1mg/mL) with surface-bound β-glucan at 0, 5, 10, 15, 20, or 25ng or free β-glucan at 5, 10, 15, 20, or 25ng/mL in macrophage in vitro and in vivo was investigated. Results: In vitro studies demonstrate that gene expression for IL-1β, IL-6, and TNFα increased at 10 and 15ng surface-bound β-glucan on CS-PLGA nanoparticles (0.1mg/mL) and at 20 and 25ng/mL of free β-glucan both at 24h and 48h. Secretion of TNFα protein and ROS production increased at 5, 10, 15, and 20ng surface-bound β-glucan on CS-PLGA nanoparticles and at 20 and 25ng/mL of free β-glucan at 24h. Laminarin, a Dectin-1 antagonist, prevented the increase in cytokine gene expression induced by CS-PLGA nanoparticles with surface-bound β-glucan at 10 and 15ng, indicating a Dectin-1 receptor mechanism. Efficacy studies showed a significant reduction in intracellular accumulation of mycobacterium tuberculosis (Mtb) in monocyte-derived macrophages (MDM) incubated with on CS-PLGA (0.1mg/ml) nanoparticles with 5, 10, and 15ng surface-bound β-glucan or with 10 and 15ng/mL of free β-glucan. β-glucan-CS-PLGA nanoparticles inhibited intracellular Mtb growth more than free β-glucan alone supporting the role of β-glucan-CS-PLGA nanoparticles as stronger adjuvants than free β-glucan. In vivo studies demonstrate that oropharyngeal aspiration (OPA) of CS-PLGA nanoparticles with nanogram concentrations of surface-bound β-glucan or free β-glucan increased TNFα gene expression in alveolar macrophages and TNFα protein secretion in bronchoalveolar lavage supernatants. Discussion: Data also demonstrate no damage to the alveolar epithelium or changes in the murine sepsis score following exposure to β-glucan-CS-PLGA nanoparticles only, indicating safety and feasibility of this nanoparticle adjuvant platform to mice by OPA.

Cigarette smoke increases susceptibility of alveolar macrophages to SARS-CoV-2 infection through inducing reactive oxygen species-upregulated angiotensin-converting enzyme 2 expression

Alveolar macrophages (AMs) are the drivers of pulmonary cytokine storm in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This study aimed to investigate clinical–regulatory factors for the entrance protein of SARS-CoV-2, angiotensin–converting enzyme 2 (ACE2) in AMs. Human AMs were collected from 56 patients using bronchoalveolar lavage. ACE2 expression in AMs was positively correlated with smoking pack-year (Spearman’s r = 0.347, P = 0.038). In multivariate analysis, current smoking was associated with increased ACE2 in AMs (β-coefficient: 0.791, 95% CI 0.019–1.562, P = 0.045). In vitro study, ex-vivo human AMs with higher ACE2 were more susceptible to SARS-CoV-2 pseudovirus (CoV-2 PsV). Treating human AMs using cigarette smoking extract (CSE) increases the ACE2 and susceptibility to CoV-2 PsV. CSE did not significantly increase the ACE2 in AMs of reactive oxygen species (ROS) deficient Cybb–/– mice; however, exogenous ROS increased the ACE2 in Cybb–/– AMs. N-acetylcysteine (NAC) decreases ACE2 by suppressing intracellular ROS in human AMs. In conclusion, cigarette smoking increases the susceptibility to SARS-CoV-2 by increasing ROS–induced ACE2 expression of AMs. Further investigation into the preventive effect of NAC on the pulmonary complications of COVID-19 is required.

Macrophages-derived Factor XIII links coagulation to inflammation in COPD.

The local, extravascular, activation of the coagulation system in response to injury is a key factor mediating the resulting inflammatory response. Coagulation Factor XIIIA (FXIIIA) found in alveolar macrophages (AM) and dendritic cells (DC), by influencing fibrin stability, might be an inflammatory modifier in COPD. To study the expression of FXIIIA in AM and Langerin+DC (DC-1) and their relation to the inflammatory response and disease progression in COPD. In 47 surgical lungs, 36 from smokers (22 COPD and 14 no-COPD) and 11 from non-smokers we quantified by immunohistochemistry FXIIIA expression in AM and DC-1 along with numbers of CD8+Tcells and CXCR3 expression in lung parenchyma and airways. Lung function was measured prior to surgery. The percentage of AM expressing FXIII (%FXIII+AM) was higher in COPD than no-COPD and non-smokers. DC-1 expressed FXIIIA and their numbers were higher in COPD than no-COPD and non-smokers. DC-1 positively correlated with %FXIII+AM (r=0.43; p<0.018). CD8+Tcells, which were higher in COPD than in no-COPD, were correlated with DC-1 (p<0.01) and %FXIII+AM. CXCR3+ cells were increased in COPD and correlated with %FXIII+AM (p<0.05). Both %FXIII+AM (r=-0.6; p=0.001) and DC-1 (r=-0.7; p=0.001) correlated inversely with FEV1. FXIIIA, an important link between the extravascular coagulation cascade and inflammatory response, is significantly expressed in alveolar macrophages and dendritic cells of smokers with COPD, suggesting that it could play an important role in the adaptive inflammatory reaction characteristic of the disease.

β-Catenin signaling in alveolar macrophages enhances lung metastasis through a TNF-dependent mechanism.

The main cause of malignancy-related mortality is metastasis. Although metastatic progression is driven by diverse tumor-intrinsic mechanisms, there is a growing appreciation for the contribution of tumor-extrinsic elements of the tumor microenvironment, especially macrophages, which correlate with poor clinical outcomes. Macrophages consist of bone marrow-derived and tissue-resident populations. In contrast to bone marrow-derived macrophages, the transcriptional pathways that govern the pro-metastatic activities of tissue-resident macrophages (TRMs) remain less clear. Alveolar macrophages (AMs) are a TRM population with critical roles in tissue homeostasis and metastasis. Wnt/β-catenin signaling is a hallmark of cancer and has been identified as a pathologic regulator of AMs in infection. We tested the hypothesis that β-catenin expression in AMs enhances metastasis in solid tumor models. Using a genetic β-catenin gain-of-function approach, we demonstrated that (a) enhanced β-catenin in AMs heightened lung metastasis; (b) β-catenin activity in AMs drove a dysregulated inflammatory program strongly associated with Tnf expression; and (c) localized TNF-α blockade abrogated this metastatic outcome. Last, β-catenin gene CTNNB1 and TNF expression levels were positively correlated in AMs of patients with lung cancer. Overall, our findings revealed a Wnt/β-catenin/TNF-α pro-metastatic axis in AMs with potential therapeutic implications against tumors refractory to the antineoplastic actions of TNF-α.

Extracorporeal Photopheresis Suppresses Transplant Fibrosis by Inducing Decorin Expression in Alveolar Macrophages.

Extracorporeal Photopheresis Suppresses Transplant Fibrosis by Inducing Decorin Expression in Alveolar Macrophages.

Stanniocalcin-1 secreted by human umbilical mesenchymal stem cells regulates interleukin-10 expression via the PI3K/AKT/mTOR pathway in alveolar macrophages.

Acute respiratory distress syndrome (ARDS) is a syndrome of acute respiratory failure caused by infection, trauma, shock, aspiration or drug reaction. The pathogenesis of ARDS is characterized as an unregulated inflammatory storm, which causes endothelial and epithelial layer damage, leading to alveolar fluid accumulation and pulmonary edema. Previous studies have shown the potential role of mesenchymal stem cells (MSC) in combating the inflammatory cascade by increasing the anti-inflammatory mediator interleukin-10 (IL-10). However, the involved mechanisms are unclear. Here we investigated whether a key immunomodulatory regulator, stanniocalcin-1 (STC-1), was secreted by MSC to activate phosphoinositide 3-kinase/protein kinase B (PI3K/AKT)/ mammalian target of rapamycin (mTOR) signaling pathway to increase IL-10 expression in alveolar macrophages. Lipopolysaccharide (LPS)-stimulated alveolar macrophages co-cultured with human umbilical mesenchymal stem cells (HUMSC) secreted high levels of IL-10. HUMSC co-cultured with alveolar macrophages expressed high STC-1 levels and increased PI3K, AKT and mTOR phosphorylation after LPS activation in alveolar macrophages. STC-1 knockdown in HUMSC decreased the phosphorylation of PI3K, AKT and mTOR and suppressed IL-10 expression in alveolar macrophages. Rapamycin (an mTOR inhibitor) reduced IL-10 secretion in alveolar macrophages. These results, together with our previous study and others, indicate that the PI3K/AKT/mTOR pathway is involved in the regulation of IL-10 production by STC-1 secreted by HUMSC in alveolar macrophages.

Replication-competent HIV-1 in human alveolar macrophages and monocytes despite nucleotide pools with elevated dUTP

BackgroundAlthough CD4+ memory T cells are considered the primary latent reservoir for HIV-1, replication competent HIV has been detected in tissue macrophages in both animal and human studies. During in vitro HIV infection, the depleted nucleotide pool and high dUTP levels in monocyte derived macrophages (MDM) leads to proviruses with high levels of dUMP, which has been implicated in viral restriction or reduced transcription depending on the uracil base excision repair (UBER) competence of the macrophage. Incorporated dUMP has also been detected in viral DNA from circulating monocytes (MC) and alveolar macrophages (AM) of HIV infected patients on antiretroviral therapy (ART), establishing the biological relevance of this phenotype but not the replicative capacity of dUMP-containing proviruses.ResultsAs compared to in vitro differentiated MDM, AM from normal donors had sixfold lower levels of dTTP and a sixfold increased dUTP/dTTP, indicating a highly restrictive dNTP pool for reverse transcription. Expression of uracil DNA glycosylase (UNG) was eightfold lower in AM compared to the already low levels in MDM. Accordingly, ~ 80% of HIV proviruses contained dUMP, which persisted for at least 14-days due to low UNG excision activity. Unlike MDM, AM expression levels of UNG and SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) increased over 14 days post-HIV infection, while dUTP nucleotidohydrolase (DUT) expression decreased. These AM-specific effects suggest a restriction response centered on excising uracil from viral DNA copies and increasing relative dUTP levels. Despite the restrictive nucleotide pools, we detected rare replication competent HIV in AM, peripheral MC, and CD4+ T cells from ART-treated donors.ConclusionsThese findings indicate that the potential integration block of incorporated dUMP is not realized during in vivo infection of AM and MC due to the near absence of UBER activity. In addition, the increased expression of UNG and SAMHD1 in AM post-infection is too slow to prevent integration. Accordingly, dUMP persists in integrated viruses, which based on in vitro studies, can lead to transcriptional silencing. This possible silencing outcome of persistent dUMP could promote viral latency until the repressive effects of viral dUMP are reversed.

Mesenchymal stromal cells-derived extracellular vesicles reprogramme macrophages in ARDS models through the miR-181a-5p-PTEN-pSTAT5-SOCS1 axis

RationaleA better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory...

MAVS Expression in Alveolar Macrophages Is Essential for Host Resistance against Aspergillus fumigatus.

Our recent data demonstrate a critical role of the RIG-I-like receptor family in regulating antifungal immunity against Aspergillus fumigatus in a murine model. However, the importance of this pathway in humans and the cell types that use this innate immune receptor family to detect A. fumigatus remain unresolved. In this study, using patients who underwent hematopoietic stem cell transplantation, we demonstrate that a polymorphism in human MAVS present in the donor genome was associated with the incidence of invasive pulmonary aspergillosis. Moreover, in a separate cohort of confirmed invasive pulmonary aspergillosis patients, polymorphisms in the IFIH1 gene alter the inflammatory response, including IFN-responsive chemokines. Returning to our murine model, we now demonstrate that CD11c+ Siglec F+ alveolar macrophages require Mavs expression to maintain host resistance against A. fumigatus. Our data support the role of MAVS signaling in mediating antifungal immunity in both mice and humans at least in part through the role of MAVS-dependent signaling in alveolar macrophages.

COPD lung studies of Nrf2 expression and the effects of Nrf2 activators

BackgroundNrf2 regulates cellular antioxidant defence in lung cells, including epithelial cells and alveolar macrophages (AM). The Nrf2/Keap-1 pathway can be modulated by activators with different modes of action; electrophilic compounds and protein–protein interaction (PPI) inhibitors.We assessed Nrf2 and Keap-1 protein and gene levels in COPD compared to controls and the effect of Nrf2 activators on COPD AM.MethodsLung resected tissue from non-smokers, smokers and COPD patients were analysed for epithelial and AM expression of Nrf2 and Keap-1 by imunoshistochemistry and by qPCR in isolated AM. AM were cultured with Nrf2 activators CDDO, C4X_6665, GSK7, MMF and Sulforaphane. Expression of Nrf2 target genes NQO1, HMOX1 SOD1 and TXNRD1 and NQO1 activity were assessed.ResultsNrf2 and Keap-1 expression was not altered in the epithelium or AM of COPD patients compared to controls. NQO1 activity was downregulated, while NQO1, HMOX1, SOD1 and TXNRD1 gene expression increased in COPD patients. All Nrf2 activators increased NQO1 activity, and NQO1, HMOX1, SOD1 and TXNRD1 expression in AMs from both COPD and smokers. The potency of C4X_6665 on NQO1 activity and regulation of Nrf2 target gene expression was higher than other compounds.ConclusionThere is evidence of dysregulation of the Nrf2 signalling pathway in AM from COPD patients. The higher potency of the novel PPI Nrf2 compound C4X_6665 for inducing antioxidant activity and gene expression compared to electrophilic and other PPI Nrf2 activators highlights the therapeutic potential of this compound to address Nrf2 pathway dysregulation in COPD AM.

CXCL10 conditions alveolar macrophages within the premetastatic niche to promote metastasis

Formation of the premetastatic niche is triggered by primary tumors and contributes to cancer metastasis. Evidence indicating the roles of macrophages in metastatic niche formation and organ-specific metastatic tropism has been steadily accumulating. However, the role of tissue-resident macrophages in the establishment of the premetastatic niche is not clearly defined. Here, we report that alveolar macrophages (AMs), which are lung tissue-resident macrophages, play a critical role in initiating the recruitment of monocytic myeloid-derived suppressor cells (mo-MDSCs) and the subsequent premetastatic niche formation by increasing CCL12 expression. We found that CXCL10 can induce CCL12 expression by activating CXCR3 and TLR4 in AMs. CXCR3/TLR4 deficiency or inhibition of its activity reduces CCL12 expression in AMs and subsequent mo-MDSC recruitment to the premetastatic niche, thereby attenuating lung metastasis. In addition, Ube2o is a negative modulator of CXCL10-induced CCL12 expression. Downregulation of Ube2o in AMs under tumor conditions enhances TAK1-NF-κB/ERK/JNK signaling and CXCL10-induced CCL12 expression by promoting TRAF6 polyubiquitination and inhibiting DDX3X degradation. Targeting mo-MDSC recruitment via the CXCL10-CXCR3/TLR4-CCL12 axis in AMs may have therapeutic potential for suppressing lung metastasis.

Abu Khaled Muhammed Akramul Haq

<h3>Rationale</h3> A better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute...

Flow cytometric analysis of bronchoalveolar lavage fluid immune dynamics in calves.

Understanding the immune dynamics in the respiratory mucosa of calves is necessary for a good management of bovine respiratory disease. Immune dynamics in the respiratory mucosa in humans and experimental animals has been assessed by flow cytometric analysis of bronchoalveolar lavage fluid (BALF); however, few reports have addressed this subject in calves. The aim of this study was to establish a universal method to analyze bronchoalveolar lavage fluid (BALF) by flow cytometry and to obtain basic knowledge of bovine respiratory mucosal immune dynamics. We investigated the immune cell populations in BALF and evaluated the surface antigen expression of alveolar macrophages in calves using flow cytometer. To further analyze the surface antigen variation observed in alveolar macrophages in detail, stimulation assays were performed in vitro. BALF cells were separated into three distinct populations based on their light scatter plot, which were considered to be macrophages, lymphocytes, and neutrophils. In most individuals, most of the BALF immune cells were alveolar macrophages, but an increased proportion of lymphocytes and neutrophils was observed in some individuals. Analysis of each surface antigen expression in alveolar macrophages showed that CD21 and MHC class II expression changed in response to changes in the leukocyte population. Moreover, when alveolar macrophages were stimulated with interferon-γ in vitro, the expression of CD21 was drastically reduced and MHC class II was increased, suggesting that functional changes in alveolar macrophages themselves are involved in the immune dynamics.

Surfactant Protein A Enhances the Degradation of LPS-Induced TLR4 in Primary Alveolar Macrophages Involving Rab7, β-arrestin2, and mTORC1.

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A-/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNF-α release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires β-arrestin2 and, in β-arrestin2-/- AMs and after intratracheal LPS challenge of β-arrestin2-/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A-/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires β-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, β-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.