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

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

Taz/Tead1 Promotes Alternative Macrophage Activation and Kidney Fibrosis via Transcriptional Upregulation of Smad3.

Macrophage alternative activation is involved in kidney fibrosis. Previous researches have documented that the transcriptional regulators Yes-associated protein (Yap)/transcriptional coactivator with PDZ-binding motif (Taz) are linked to organ fibrosis. However, limited knowledge exists regarding the function and mechanisms of their downstream molecules in regulating macrophage activation and kidney fibrosis. In this paper, we observed that the Hippo pathway was suppressed in macrophages derived from fibrotic kidneys in mice. Knockout of Taz or Tead1 in macrophages inhibited the alternative activation of macrophages and reduced kidney fibrosis. Additionally, by using bone marrow-derived macrophages (BMDMs), we investigated that knockout of Taz or Tead1 in macrophages impeded both cell proliferation and migration. Moreover, deletion of Tead1 reduces p-Smad3 and Smad3 abundance in macrophages. And chromatin immunoprecipitation (ChIP) assays showed that Tead1 could directly bind to the promoter region of Smad3. Collectively, these results indicate that Tead1 knockout in macrophages could reduce TGFß1-induced phosphorylation Smad3 via transcriptional downregulation of Smad3, thus suppressing macrophage alternative activation and IRI-induced kidney fibrosis.

SPI1-mediated macrophage polarization aggravates age-related macular degeneration.

Objective: This study revealed a core regulator and common upstream mechanisms for the multifaceted pathological processes of age-related macular degeneration (AMD) and provided proof-of-concept for this new therapeutic target. Methods: Comprehensive gene expression analysis was performed using RNA sequencing of eye cup from old mice as well as laser-induced choroidal neovascularization (CNV) mouse model. Through integrative analysis and protein-protein interaction (PPI) analysis, common pathways and key transcription factor was identified simultaneously engaged in age-related retinal degeneration and CNV, the two typical pathological process of AMD. Subsequently, the expression changes of Spi1, the key regulator, as well as the alternation of the downstream mechanisms were validated in both models through qRT-PCR, Elisa, flow cytometry and immunofluorescence. Further, we assessed the impact of Spi1 knockdown in vitro and in vivo using gene intervention vectors carried by adeno-associated virus or lentivirus to test its potential as a therapeutic target. Results: Compared to corresponding controls, we found 1,939 and 1,319 genes differentially expressed in eye cups of old and CNV mice respectively. The integrative analysis identified a total of 275 overlapping DEGs, of which 150 genes were co-upregulated. PPI analysis verified a central transcription factor, SPI1. The significant upregulation of Spi1 expression was then validated in both models, accompanied by macrophage polarization towards the M1 phenotype. Finally, SPI1 suppression significantly inhibited M1 polarization of BMDMs and attenuated neovascularization in CNV mice. Conclusion: This study demonstrates that SPI1 exerts a pivotal role in AMD by regulation of macrophage polarization and innate immune response, offering promise as an innovative target for treating AMD.

Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response.

Macrophages play a pivotal role in orchestrating the immune response against pathogens. While the intricate interplay between macrophage activation and metabolism remains a subject of intense investigation, the role of glutamate oxaloacetate transaminase 1 (Got1) in this context has not been extensively assessed. Here, we investigate the impact of Got1 on macrophage polarization and function, shedding light on its role in reactive oxygen species (ROS) production, pathogen defense, and immune paralysis. Using genetically modified mouse models, including both myeloid specific knockout and overexpression, we comprehensively demonstrate that Got1 depletion leads to reduced ROS production in macrophages. Intriguingly, this impairment in ROS generation does not affect the resistance of Got1 KO mice to pathogenic challenges. Furthermore, Got1 is dispensable for M2 macrophage differentiation and does not influence the onset of LPS-induced immune paralysis. Our findings underscore the intricate facets of macrophage responses, suggesting that Got1 is dispensable in discrete immunological processes.

Unveiling the Cutting-Edge Impact of Polarized Macrophage-Derived Extracellular Vesicles and MiRNA Signatures on TGF-ß Regulation within Lung Fibroblasts.

Depending on local cues, macrophages can polarize into classically activated (M1) or alternatively activated (M2) phenotypes. This study investigates the impact of polarized macrophage-derived Extracellular Vesicles (EVs) (M1 and M2) and their cargo of miRNA-19a-3p and miRNA-425-5p on TGF-ß production in lung fibroblasts. EVs were isolated from supernatants of M0, M1, and M2 macrophages and quantified using nanoscale flow cytometry prior to fibroblast stimulation. The concentration of TGF-ß in fibroblast supernatants was measured using ELISA assays. The expression levels of miRNA-19a-3p and miRNA-425-5p were assessed via TaqMan-qPCR. TGF-ß production after stimulation with M0-derived EVs and with M1-derived EVs increased significantly compared to untreated fibroblasts. miRNA-425-5p, but not miRNA-19a-3p, was significantly upregulated in M2-derived EVs compared to M0- and M1-derived EVs. This study demonstrates that EVs derived from both M0 and M1 polarized macrophages induce the production of TGF-ß in fibroblasts, with potential regulation by miRNA-425-5p.

TRIM59 deficiency promotes M1 macrophage activation and inhibits colorectal cancer through the STAT1 signaling pathway.

Tumor-associated macrophages play a crucial role in the tumor microenvironment. Tripartite motif 59 (TRIM59), a member of the tripartite motif (TRIM) family, is known to be associated with immunological diseases and macrophage activation. The functional and molecular mechanisms by which TRIM59 affects the occurrence and development of colorectal cancer (CRC) through macrophages are still not well understood. To address this, we generated macrophage-specific TRIM59 conditional knockout mice and utilized these mice to establish colitis-associated cancer and MC38 transplanted CRC models for further investigation. We found that the deficiency of TRIM59 in macrophages inhibited colorectal tumorigenesis in mice. This tumor-suppressive effect was achieved by promoting the activation of M1 macrophages via STAT1 signaling pathway. Further mechanistic studies revealed that TRIM59 could regulate macrophage polarization by ubiquitinating and degrading STAT1. These findings provide evidence that TRIM59 deficiency promotes M1 macrophage activation and inhibits CRC through the STAT1 signaling pathway, suggesting that the TRIM59/STAT1 signaling pathway may be a promising target for CRC.

miR-34a promotes the immunosuppressive function of multiple myeloma-associated macrophages by dampening the TLR-9 signaling.

BACKGROUND: Promising outcomes have been observed in multiple myeloma (MM) with the use of immunotherapies, specifically chimeric antigen receptor T (CAR-T) cell therapy. However, a portion of MM patients do not respond to CAR-T therapy, and the reasons for this lack of response remain unclear. The objective of this study was to investigate the impact of miR-34a on the immunosuppressive polarization of macrophages obtained from MM patients. METHODS: The levels of miR-34a and TLR9 (Toll-like receptor 9) were examined in macrophages obtained from both healthy individuals and patients with MM. ELISA was employed to investigate the cytokine profiles of the macrophage samples. Co-culture experiments were conducted to evaluate the immunomodulatory impact of MM-associated macrophages on CAR-T cells. RESULTS: There was an observed suppressed activation of macrophages and CD4+ T lymphocytes in the blood samples of MM patients. Overexpression of miR-34a in MM-associated macrophages dampened the TLR9 expression and impaired the inflammatory polarization. In both the co-culture system and an animal model, MM-associated macrophages suppressed the activity and tumoricidal effect of CAR-T cells in a miR-34a-dependent manner. CONCLUSION: The findings imply that targeting the macrophage miR-34a/TLR9 axis could potentially alleviate the immunosuppression associated with CAR-T therapy in MM patients.

Tumor-Derived Exosomal miR-143-3p Induces Macrophage M2 Polarization to Cause Radiation Resistance in Locally Advanced Esophageal Squamous Cell Carcinoma.

We aimed to determine whether monitoring tumor-derived exosomal microRNAs (miRNAs) could be used to assess radiotherapeutic sensitivity in patients with locally advanced esophageal squamous cell carcinoma (ESCC). RNA sequencing was employed to conduct a comparative analysis of miRNA expression levels during radiotherapy, focusing on identifying miRNAs associated with progression. Electron microscopy confirmed the existence of exosomes, and co-cultivation assays and immunofluorescence validated their capacity to infiltrate macrophages. To determine the mechanism by which exosomal miR-143-3p regulates the interplay between ESCC cells and M2 macrophages, ESCC cell-derived exosomes were co-cultured with macrophages. Serum miR-143-3p and miR-223-3p were elevated during radiotherapy, suggesting resistance to radiation and an unfavorable prognosis for ESCC. Increased levels of both miRNAs independently predicted shorter progression-free survival (p = 0.015). We developed a diagnostic model for ESCC using serum microRNAs, resulting in an area under the curve of 0.751. Radiotherapy enhanced the release of miR-143-3p from ESCC cell-derived exosomes. Immune cell infiltration analysis at the Cancer Genome Atlas (TCGA) database revealed that ESCC cell-derived miR-143-3p triggered M2 macrophage polarization. Mechanistically, miR-143-3p upregulation affected chemokine activity and cytokine signaling pathways. Furthermore, ESCC cell exosomal miR-143-3p could be transferred to macrophages, thereby promoting their polarization. Serum miR-143-3p and miR-223-3p could represent diagnostic and prognostic markers for patients with ESCC undergoing radiotherapy. Unfavorable prognosis could be linked to the increased levels of ESCC cell-derived exosomal miR-143-3p, which might promote tumor progression by interacting with macrophages.

Unifying considerations and evidence of macrophage activation mosaicism through human CSF1R and M1/M2 genes.

Addressing the mononuclear phagocyte system (MPS) and macrophage M1/M2 activation is important in diagnosing hematological disorders and inflammatory pathologies and designing therapeutic tools. CSF1R is a reliable marker to identify all circulating MPS cells and tissue macrophages in humans using a single surface protein. CSF1R permits the quantification and isolation of monocyte and dendritic cell (DC) subsets in conjunction with CD14, CD16, and CD1c and is stable across the lifespan and sexes in the absence of overt pathology. Beyond cell detection, measuring M1/M2 activation in humans poses challenges due to response heterogeneity, transient signaling, and multiple regulation steps for transcripts and proteins. MPS cells respond in a conserved manner to M1/M2 pathways such as interleukin-4 (IL-4), steroids, interferon-γ (IFNγ), and lipopolysaccharide (LPS), for which we propose an ad hoc modular gene expression tool. Signature analysis highlights macrophage activation mosaicism in experimental samples, an emerging concept that points to mixed macrophage activation states in pathology.

Transcriptome analysis of long non-coding RNAs in Mycobacterium avium complex-infected macrophages.

Mycobacterium avium complex (MAC) is a non-tuberculous mycobacterium widely distributed in the environment. Even though MAC infection is increasing in older women and immunocompromised patients, to our knowledge there has been no comprehensive analysis of the MAC-infected host-cell transcriptome-and particularly of long non-coding RNAs (lncRNAs). By using in vitro-cultured primary mouse bone-marrow-derived macrophages (BMDMs) and Cap analysis of gene expression, we analyzed the transcriptional and kinetic landscape of macrophage genes, with a focus on lncRNAs, during MAC infection. MAC infection of macrophages induced the expression of immune/inflammatory response genes and other genes similar to those involved in M1 macrophage activation, consistent with previous reports, although Nos2 (M1 activation) and Arg1 (M2 activation) had distinct expression profiles. We identified 31 upregulated and 30 downregulated lncRNA promoters corresponding respectively to 18 and 26 lncRNAs. Upregulated lncRNAs were clustered into two groups-early and late upregulated-predicted to be associated with immune activation and the immune response to infection, respectively. Furthermore, an Ingenuity Pathway Analysis revealed canonical pathways and upstream transcription regulators associated with differentially expressed lncRNAs. Several differentially expressed lncRNAs reported elsewhere underwent expressional changes upon M1 or M2 preactivation and subsequent MAC infection. Finally, we showed that expressional change of lncRNAs in MAC-infected BMDMs was mediated by toll-like receptor 2, although there may be other mechanisms that sense MAC infection. We identified differentially expressed lncRNAs in MAC-infected BMDMs, revealing diverse features that imply the distinct roles of these lncRNAs in MAC infection and macrophage polarization.

An integrative epigenome-based strategy for unbiased functional profiling of clinical kinase inhibitors.

More than 500 kinases are implicated in the control of most cellular process in mammals, and deregulation of their activity is linked to cancer and inflammatory disorders. 80 clinical kinase inhibitors (CKIs) have been approved for clinical use and hundreds are in various stages of development. However, CKIs inhibit other kinases in addition to the intended target(s), causing both enhanced clinical effects and undesired side effects that are only partially predictable based on in vitro selectivity profiling. Here, we report an integrative approach grounded on the use of chromatin modifications as unbiased, information-rich readouts of the functional effects of CKIs on macrophage activation. This approach exceeded the performance of transcriptome-based approaches and allowed us to identify similarities and differences among CKIs with identical intended targets, to recognize novel CKI specificities and to pinpoint CKIs that may be repurposed to control inflammation, thus supporting the utility of this strategy to improve selection and use of CKIs in clinical settings.

TM6SF1 suppresses the progression of lung adenocarcinoma and M2 macrophage polarization by inactivating the PI3K/AKT/mtor pathway.

Transmembrane 6 superfamily 1 (TM6SF1) is lowly expressed in lung adenocarcinoma (LUAD), but the function and mechanisms of TM6SF1 remain unclear. Thus, we attempt to explore the function of TM6SF1 and its underlying mechanisms in LUAD. qRT-PCR was used for detecting TM6SF1 mRNA expression. Immunohistochemistry staining was used for detecting the expression of MMP-2, TM6SF1, Ki67, MMP-9, and CD163 proteins. E-cadherin, p-PI3K, Vimentin, AKT, N-cadherin, PI3K, p-AKT, mTOR, p-mTOR, and marker proteins of M2 macrophages were evaluated using Western blot. CD206 protein expression was examined via immunofluorescence. The IL-10 concentration was measured via enzyme-linked immunosorbent assay (ELISA). Using CCK-8, colony formation and transwell assays, cell proliferation, migration, and invasion were assessed. A549 cells were injected into the mice's flank for establishing a mouse tumor model and into the tail vein for establishing the lung metastasis model. HE staining was performed to detect pathological changes in lung tissues. Decreased TM6SF1 expression was found in LUAD tissues and cells. TM6SF1 overexpression inhibited cell viability, proliferation, invasion, migration, EMT, and polarization of M2 macrophages in LUAD cells, along with tumor growth and metastasis in xenograft mice. Bioinformatics analysis demonstrated that TM6SF1 was correlated with the tumor microenvironment. TM6SF1 overexpression reduced expression levels of p-mTOR, p-PI3K, p-AKT, mTOR, and AKT. TM6SF1-caused inhibition of proliferation, migration, invasion and EMT, as M2 macrophage polarization was reversed by the PI3K activator in LUAD cells. TM6SF1 inactivated the PI3K/AKT/mTOR pathway to suppress LUAD malignancy and polarization of M2 macrophages, providing insight for developing new LUAD treatments.

Discovery and functional characterization of LncRNAs associated with inflammation and macrophage activation.

Long noncoding RNAs (lncRNA) are emerging players in regulation of gene expression and cell signaling and their dysregulation has been implicated in a multitude of human diseases. Recent studies from our laboratory revealed that lncRNAs play critical roles in cytokine regulation, inflammation, and metabolism. We demonstrated that lncRNA HOTAIR, which is a well-known regulator of gene silencing, plays critical roles in modulation of cytokines and proinflammatory genes, and glucose metabolism in macrophages during inflammation. In addition, we recently discovered a series of novel lncRNAs that are closely associated with inflammation and macrophage activation. We termed these as long-noncoding inflammation associated RNAs (LinfRNAs). We are currently engaged in the functional characterization of these hLinfRNAs (human LinfRNAs) with a focus on their roles in inflammation, and we are investigating their potential implications in chronic inflammatory human diseases. Here, we have summarized experimental methods that have been utilized for the discovery and functional characterization of lncRNAs in inflammation and macrophage activation.

Prognostic and immunological implications of glutathione metabolism genes in lung adenocarcinoma: A focus on the core gene SMS and its impact on M2 macrophage polarization.

Glutathione metabolism (GM) is a crucial part of various metabolic and pathophysiological processes. However, its role in lung adenocarcinoma (LUAD) has not been comprehensively studied. This study aimed to explore the potential relationship between GM genes, the prognosis, and the immune microenvironment of patients with LUAD. We constructed a risk signature model containing seven GM genes using Lasso combined Cox regression and validated it using six GEO datasets. Our analysis showed that it is an independent prognostic factor. Functional enrichment analysis revealed that the GM genes were significantly enriched in cell proliferation, cell cycle regulation, and metabolic pathways. Clinical and gene expression data of patients with LUAD were obtained from the TCGA database and patients were divided into high- and low-risk groups. The high-risk patient group had a poor prognosis, reduced immune cell infiltration, poor response to immunotherapy, high sensitivity to chemotherapy, and low sensitivity to targeted therapy. Subsequently, single-cell transcriptome analysis using the GSE143423 and GSE127465 datasets revealed that the core SMS gene was highly enriched in M2 Macrophages. Finally, nine GEO datasets and multiple fluorescence staining revealed a correlation between the SMS expression and M2 macrophage polarization. Our prognostic model in which the core SMS gene is closely related to M2 macrophage polarization is expected to become a novel target and strategy for tumor therapy.

Exploring the role of CITED transcriptional regulators in the control of macrophage polarization.

Macrophages are tissue resident innate phagocytic cells that take on contrasting phenotypes, or polarization states, in response to the changing combination of microbial and cytokine signals at sites of infection. During the opening stages of an infection, macrophages adopt the proinflammatory, highly antimicrobial M1 state, later shifting to an anti-inflammatory, pro-tissue repair M2 state as the infection resolves. The changes in gene expression underlying these transitions are primarily governed by nuclear factor kappaB (NF-κB), Janus kinase (JAK)/signal transducer and activation of transcription (STAT), and hypoxia-inducible factor 1 (HIF1) transcription factors, the activity of which must be carefully controlled to ensure an effective yet spatially and temporally restricted inflammatory response. While much of this control is provided by pathway-specific feedback loops, recent work has shown that the transcriptional co-regulators of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxy-terminal domain (CITED) family serve as common controllers for these pathways. In this review, we describe how CITED proteins regulate polarization-associated gene expression changes by controlling the ability of transcription factors to form chromatin complexes with the histone acetyltransferase, CBP/p300. We will also cover how differences in the interactions between CITED1 and 2 with CBP/p300 drive their contrasting effects on pro-inflammatory gene expression.

Targeting JUNB to modulate M2 macrophage polarization in preeclampsia.

Preeclampsia (PE) is a complex disorder affecting pregnant women, leading to significant maternal and fetal morbidity and mortality. Understanding the cellular dynamics and molecular mechanisms underlying PE is crucial for developing effective therapeutic strategies. This study utilized single-cell RNA sequencing (scRNA-seq) to delineate the cellular landscape of the placenta in PE, identifying 11 distinct cell subpopulations, with macrophages playing a pivotal role in mediating cell-cell communication. Specifically, the transcription factor JUNB was found to be a key gene in macrophages from PE samples, influencing the interaction between macrophages and both epithelial and endothelial cells. Functional experiments indicated that interference with JUNB expression promoted macrophage polarization towards an M2 phenotype, which facilitated trophoblast invasion, migration, and angiogenesis. Mechanistically, JUNB regulated the MIIP/PI3K/AKT pathway, as evidenced by gene expression analysis following JUNB knockdown. The study further demonstrated that targeting JUNB could activate the PI3K/AKT pathway by transcriptionally activating MIIP, thus promoting M2 polarization and potentially delaying the onset of PE. These findings present new insights into the pathogenesis of PE and suggest a novel therapeutic approach by modulating macrophage polarization.

Extracellular vesicle miRNAs drive aberrant macrophage responses in NSAID-exacerbated respiratory disease.

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.

Non-coding RNA LINC01010 regulates macrophage polarization and innate immune functions by modulating NFκB signaling pathway.

Innate immune response is regulated by tissue resident or infiltrating immune cells such as macrophages (Mφ) that play critical role in tissue development, homeostasis, and repair of damaged tissue. However, the epigenetic mechanisms that regulate Mφ plasticity and innate immune functions are not well understood. Long non-coding RNA (lncRNA) are among the most abundant class of transcriptome but their function in myeloid cell biology is less explored. In this study, we deciphered the regulatory role of previously uncharacterized lncRNAs in Mφ polarization and innate immune responses. Two lncRNAs showed notable changes in their levels during M1 and M2 Mφ differentiation. Our findings indicate that LINC01010 expression increased and AC007032 expression decreased significantly. LINC01010 exhibit myeloid cell-specificity, while AC007032.1 is ubiquitous and expressed in both myeloid and lymphoid (T cells, B cells and NK cells) cells. Expression of these lncRNAs is dysregulated in periodontal disease (PD), a microbial biofilm-induced immune disease, and responsive to lipopolysaccharide (LPS) from different oral and non-oral bacteria. Knockdown of LINC01010 but not AC007032.1 reduced the surface expression of Mφ differentiation markers CD206 and CD68, and M1Mφ polarization markers MHCII and CD32. Furthermore, LINC01010 RNAi attenuated bacterial phagocytosis, antigen processing and cytokine secretion suggesting its key function in innate immunity. Mechanistically, LINC01010 knockdown Mφ treated with Escherichia coli LPS exhibit significantly reduced expression of multiple nuclear factor kappa B pathway genes. Together, our data highlight functional role of a PD-associated lncRNA LINC01010 in shaping macrophage differentiation, polarization, and innate immune activation.

Getting everyone to agree on gene signatures for murine macrophage polarization in vitro.

Macrophages, key players in the innate immune system, showcase remarkable adaptability. Derived from monocytes, these phagocytic cells excel in engulfing and digesting pathogens and foreign substances as well as contributing to antigen presentation, initiating and regulating adaptive immunity. Macrophages are highly plastic, and the microenvironment can shaper their phenotype leading to numerous distinct polarized subsets, exemplified by the two ends of the spectrum: M1 (classical activation, inflammatory) and M2 (alternative activation, anti-inflammatory). RNA sequencing (RNA-Seq) has revolutionized molecular biology, offering a comprehensive view of transcriptomes. Unlike microarrays, RNA-Seq detects known and novel transcripts, alternative splicing, and rare transcripts, providing a deeper understanding of genome complexity. Despite the decreasing costs of RNA-Seq, data consolidation remains limited, hindering noise reduction and the identification of authentic signatures. Macrophages polarization is routinely ascertained by qPCR to evaluate those genes known to be characteristic of M1 or M2 skewing. Yet, the choice of these genes is literature- and experience-based, lacking therefore a systematic approach. This manuscript builds on the significant increase in deposited RNA-Seq datasets to determine an unbiased and robust murine M1 and M2 polarization profile. We now provide a consolidated list of global M1 differentially expressed genes (i.e. robustly modulated by IFN-γ, LPS, and LPS+ IFN-γ) as well as consolidated lists of genes modulated by each stimulus (IFN-γ, LPS, LPS+ IFN-γ, and IL-4).

Tumor Cell-Derived Exosomal miR-191-5p Activates M2-Subtype Macrophages Through SOCS3 to Facilitate Breast Cancer.

Differential activation of macrophages is associated with poor progression of breast cancer (BC). Many reports have elucidated the important involvement of exosomes produced by cancer cells in remodeling the macrophage activation phenotype to promote tumor expansion and invasion. However, the underlying mechanisms by which exosomes secreted by BC cells facilitate macrophage M2 polarization remain enigmatic and worth exploring. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate miR-191-5p expression in BC tumor tissues and cells. Cell counting kit 8 (CCK-8), transwell, and flow cytometry were applied to assess the functional role of miR-191-5p in BC. Isolated nano-vesicles were identified using transmission electron microscopy and western blotting. We also observed that miR-191-5p was significantly elevated in BC clinical samples and that inhibition of miR-191-5p hindered the growth and metastasis of BC cells. Importantly, BC cells successfully accelerated macrophage M2-like polarization by directly transferring exosomes to macrophages, resulting in increased miR-191-5p levels in macrophages. Mechanistically, exosomal miR-191-5p directly inhibited the suppressors of cytokine signaling 3 (SOCS3) expression in macrophages and aggravated macrophage M2 polarization. Similarly, si-SOCS3 transfected macrophages boosted BC cell migration and invasion in a positive feedback manner. Overall, our results manifested a pro-growth and pro-metastatic role between the two cells by elucidating the crucial role of exosomal miR-191-5p in stimulating M2 macrophage polarization and mediating communication between BC cells and macrophages. These findings opened up new horizons for the development of BC therapeutic strategies.