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.

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.

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.

Macrophage profiling in atherosclerosis: understanding the unstable plaque.

The development and rupture of atherosclerotic plaques is a major contributor to myocardial infarctions and ischemic strokes. The dynamic evolution of the plaque is largely attributed to monocyte/macrophage functions, which respond to various stimuli in the plaque microenvironment. To this end, macrophages play a central role in atherosclerotic lesions through the uptake of oxidized low-density lipoprotein that gets trapped in the artery wall, and the induction of an inflammatory response that can differentially affect the stability of the plaque in men and women. In this environment, macrophages can polarize towards pro-inflammatory M1 or anti-inflammatory M2 phenotypes, which represent the extremes of the polarization spectrum that include Mhem, M(Hb), Mox, and M4 populations. However, this traditional macrophage model paradigm has been redefined to include numerous immune and nonimmune cell clusters based on in-depth unbiased single-cell approaches. The goal of this review is to highlight (1) the phenotypic and functional properties of monocyte subsets in the circulation, and macrophage populations in atherosclerotic plaques, as well as their contribution towards stable or unstable phenotypes in men and women, and (2) single-cell RNA sequencing studies that have advanced our knowledge of immune, particularly macrophage signatures present in the atherosclerotic niche. We discuss the importance of performing high-dimensional approaches to facilitate the development of novel sex-specific immunotherapies that aim to reduce the risk of cardiovascular events.

Macrophage heterogeneity in atherosclerosis: A matter of context.

During atherogenesis, plaque macrophages take up and process deposited lipids, trigger inflammation, and form necrotic cores. The traditional inflammatory/anti-inflammatory paradigm has proven insufficient in explaining their complex disease-driving mechanisms. Instead, we now appreciate that macrophages exhibit remarkable heterogeneity and functional specialization in various pathological contexts, including atherosclerosis. Technical advances for studying individual cells, especially single-cell RNA sequencing, indeed allowed to identify novel macrophage subsets in both murine and human atherosclerosis, highlighting the existence of diverse macrophage activation states throughout pathogenesis. In addition, recent studies highlighted the role of the local microenvironment in shaping the macrophages' phenotype and function. However, this remains largely undescribed in the context of atherosclerosis. In this review we explore the origins of macrophages and their functional specialization, shedding light on the diverse sources of macrophage accumulation in the atherosclerotic plaque. Next, we discuss the phenotypic diversity observed in both murine and human atherosclerosis, elucidating their distinct functions and spatial distribution within plaques. Finally, we highlight the importance of the local microenvironment in both phenotypic and functional specialization of macrophages in atherosclerosis and elaborate on the need for spatial multiomics approaches to provide a better understanding of the different macrophage subsets' roles in the pathogenesis of atherosclerosis.

Macrophage activation drives ovarian failure and masculinization in zebrafish.

BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.

Copine 1 predicts poor clinical outcomes by promoting M2 macrophage activation in ovarian cancer.

OBJECTIVE: Copine 1 (CPNE1), a membrane-binding protein, influences the prognosis of various cancers. According to cBioPortal, CPNE1 amplification is a prevalent genetic mutation in ovarian cancer but with unknown oncogenic mechanism. METHODS: This study analysed the CPNE1 expression in ovarian cancer using online datasets, as validated by immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR) and western blotting. Concurrently, the prognostic value of CPNE1 was accessed. Cell Counting Kit-8, colony formation, transwells and xenograft experiments were performed to evaluate the functions of CPNE1 during ovarian cancer carcinogenesis. CPNE1 and its related genes were analysed by g:Profiler and Tumour Immune Estimation Resource. Furthermore, human monocytic THP-1 cells were co-cultured with ES2 cells to investigate the effect of CPNE1 on macrophage polarization. RESULTS: The results of bioinformatic analysis, IHC, qPCR and western blotting indicated a higher CPNE1 in ovarian cancer. CPNE1 overexpression demonstrated an association with a poor prognosis of ovarian cancer. Functionally, CPNE1 overexpression increased ES2 and SKOV3 cell proliferation, invasion and migration in vitro and promoted ovarian tumour xenograft growth in vivo, while CPNE1 knockdown led to opposite effects. Additionally, CPNE1 expression demonstrated an association with immune cell infiltration in ovarian cancer, especially macrophage. CPNE1 promoted protumour M2 macrophage polarization by upregulating cluster of differentiation 163 (CD163), CD206 and interleukin-10. CONCLUSIONS: Our study revealed that CPNE1 mediated M2 macrophage polarization and provided a therapeutic target for ovarian cancer.

GLUT3 promotes macrophage signaling and function via RAS-mediated endocytosis in atopic dermatitis and wound healing.

The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

The macrophage polarization by miRNAs and its potential role in the treatment of tumor and inflammation (Review).

The characteristics of monocyte/macrophage lineage are diversity and plasticity, mainly manifested by M1 and M2 subtypes in the body tissues, and playing different roles in the immunity. In the polarization process of macrophages, the classic molecular mechanism is related to sequential transcription factors. Whether in tumor or inflammatory local microenvironment, the pathological factors of the local microenvironment often affect the polarization of M1 and M2 macrophages, and participate in the occurrence and development of these pathological processes. In recent years, a growing number of research results demonstrated that non­coding RNA (ncRNA) also participates in the polarization process of macrophages, in addition to traditional cytokines and transcriptional regulation signal pathway molecules. Among numerous ncRNAs, microRNAs (miRNAs) have attracted more attention from scholars both domestically and internationally, and significant progress has been made in basic and clinical research. Therefore, for improved understanding of the molecular mechanism of miRNAs in macrophage polarization and analysis of the potential value of this regulatory pathway in tumor and inflammatory intervention therapy, a comprehensive review of the progress of relevant literature research was conducted and some viewpoints and perspectives were proposed.

MicroRNA: role in macrophage polarization and the pathogenesis of the liver fibrosis.

Macrophages, as central components of innate immunity, feature significant heterogeneity. Numerus studies have revealed the pivotal roles of macrophages in the pathogenesis of liver fibrosis induced by various factors. Hepatic macrophages function to trigger inflammation in response to injury. They induce liver fibrosis by activating hepatic stellate cells (HSCs), and then inflammation and fibrosis are alleviated by the degradation of the extracellular matrix and release of anti-inflammatory cytokines. MicroRNAs (miRNAs), a class of small non-coding endogenous RNA molecules that regulate gene expression through translation repression or mRNA degradation, have distinct roles in modulating macrophage activation, polarization, tissue infiltration, and inflammation regression. Considering the complex etiology and pathogenesis of liver diseases, the role and mechanism of miRNAs and macrophages in liver fibrosis need to be further clarified. We first summarized the origin, phenotypes and functions of hepatic macrophages, then clarified the role of miRNAs in the polarization of macrophages. Finally, we comprehensively discussed the role of miRNAs and macrophages in the pathogenesis of liver fibrotic disease. Understanding the mechanism of hepatic macrophage heterogeneity in various types of liver fibrosis and the role of miRNAs on macrophage polarization provides a useful reference for further research on miRNA-mediated macrophage polarization in liver fibrosis, and also contributes to the development of new therapies targeting miRNA and macrophage subsets for liver fibrosis.

A druggable copper-signalling pathway that drives inflammation.

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

[M2 macrophage-derived exosomal lncRNA NR_028113.1 promotes macrophage polarization possibly by activating the JAK2/STAT3 signaling pathway].

OBJECTIVE: To explore the effect of M2 macrophage-derived exosomal lncRNA NR_028113.1 on macrophage polarization and its possible mechanism. METHODS: Bone marrow-derived macrophages (BMDMs) from BALB/c mice were isolated and cultured in vitro. After IL-4 treatment to induce M2 macrophage polarization, exosomes (M2-exo) were extracted from the supernatant of M2 macrophages and identified. The expression of lncRNA in M2-exo was detected by qRT-PCR. BMDMs were co-cultured with M2-exo (100 µg/mL) or PBS for 48 h, and the changes in cellular expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α were detected using qRT-PCR and Western blotting. The percentage of CD206+ cells was analyzed using flow cytometry, and the phosphorylation levels of JAK2/STAT3 proteins were detected using Western blotting. A lncRNA smart silencer was designed to specifically inhibit the expression of lncRNA NR_028113.1 in the M2 macrophages, from which exosomes were extracted and co-cultured with BMDMs for 48 h. The mRNA expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α, CD206+ cell percentage and the phosphorylation levels of JAK2/STAT3 proteins were detected using qRT-PCR, flow cytometry and Western blotting. RESULTS: LncRNA NR_028113.1 was highly expressed in the exosomes of M2 macrophages (P < 0.05). Co-culture with M2-exo significantly increased mRNA expressions of M2 macrophage marker genes Arg1, YM-1 and FIZZ1 (P < 0.05), lowered the expressions of iNOS and TNF-α (P < 0.05), and increased CD206+ cell percentage and JAK2/STAT3 protein phosphorylation level in BMDMs (P < 0.05). After inhibiting the expression of lncRNA NR_028113.1 in M2 macrophages, the extracted M2-exo caused significant down-regulation of the mRNA expressions of Arg1, YM-1 and FIZZ1 and up-regulation of iNOS and TNF-α mRNA (P < 0.05), resulting also in signi-ficantly reduced CD206+ cell percentage and lowered phosphorylation levels of JAK2/STAT3 proteins in co-cultured BMDM (P < 0.05). CONCLUSIONS: M2 macrophage-derived exosomal lncRNA NR_028113.1 can significantly promote M2 polarization of macrophages possibly by activating the JAK2/STAT3 signaling pathway.

MicroRNAs in Macrophages: Regulators of Activation and Function.

Macrophages are sentinels of the innate immune system that maintain tissue homeostasis and contribute to inflammatory responses. Their broad scope of action depends on both functional heterogeneity and plasticity. Small noncoding RNAs called microRNAs (miRNAs) contribute to macrophage function as post-transcriptional inhibitors of target gene networks. Genetic and pharmacologic studies have uncovered genes regulated by miRNAs that control macrophage cellular programming and macrophage-driven pathology. miRNAs control proinflammatory M1-like activation, immunoregulatory M2-like macrophage activation, and emerging macrophage functions in metabolic disease and innate immune memory. Understanding the gene networks regulated by individual miRNAs enhances our understanding of the spectrum of macrophage function at steady state and during responses to injury or pathogen invasion, with the potential to develop miRNA-based therapies. This review aims to consolidate past and current studies investigating the complexity of the miRNA interactome to provide the reader with a mechanistic view of how miRNAs shape macrophage behavior.

ProCPU Is Expressed by (Primary) Human Monocytes and Macrophages and Expression Differs between States of Differentiation and Activation.

Carboxypeptidase U (CPU, TAFIa, CPB2) is a potent attenuator of fibrinolysis that is mainly synthesized by the liver as its inactive precursor proCPU. Aside from its antifibrinolytic properties, evidence exists that CPU can modulate inflammation, thereby regulating communication between coagulation and inflammation. Monocytes and macrophages play a central role in inflammation and interact with coagulation mechanisms resulting in thrombus formation. The involvement of CPU and monocytes/macrophages in inflammation and thrombus formation, and a recent hypothesis that proCPU is expressed in monocytes/macrophages, prompted us to investigate human monocytes and macrophages as a potential source of proCPU. CPB2 mRNA expression and the presence of proCPU/CPU protein were studied in THP-1, PMA-stimulated THP-1 cells and primary human monocytes, M-CSF-, IFN-γ/LPS-, and IL-4-stimulated-macrophages by RT-qPCR, Western blotting, enzyme activity measurements, and immunocytochemistry. CPB2 mRNA and proCPU protein were detected in THP-1 and PMA-stimulated THP-1 cells as well as in primary monocytes and macrophages. Moreover, CPU was detected in the cell medium of all investigated cell types and it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment. Comparison of CPB2 mRNA expression and proCPU concentrations in the cell medium between the different cell types provided evidence that CPB2 mRNA expression and proCPU secretion in monocytes and macrophages is related to the degree to which these cells are differentiated. Our results indicate that primary monocytes and macrophages express proCPU. This sheds new light on monocytes and macrophages as local proCPU sources.

UBC9 deficiency enhances immunostimulatory macrophage activation and subsequent antitumor T cell response in prostate cancer.

The role of tumor-associated macrophages (TAMs), along with the regulatory mechanisms underlying distinct macrophage activation states, remains poorly understood in prostate cancer (PCa). Herein, we report that PCa growth in mice with macrophage-specific Ubc9 deficiency is substantially suppressed compared with that in wild-type littermates, an effect partially ascribed to the augmented CD8+ T cell response. Biochemical and molecular analyses revealed that signal transducer and activator of transcription 4 (STAT4) is a crucial UBC9-mediated SUMOylation target, with lysine residue 350 (K350) as the major modification site. Site-directed mutation of STAT4 (K350R) enhanced its nuclear translocation and stability, thereby facilitating the proinflammatory activation of macrophages. Importantly, administration of the UBC9 inhibitor 2-D08 promoted the antitumor effect of TAMs and increased the expression of PD-1 on CD8+ T cells, supporting a synergistic antitumor efficacy once it combined with the immune checkpoint blockade therapy. Together, our results demonstrate that ablation of UBC9 could reverse the immunosuppressive phenotype of TAMs by promoting STAT4-mediated macrophage activation and macrophage-CD8+ T cell crosstalk, which provides valuable insights to halt the pathogenic process of tumorigenesis.