Macrophage Markers Research Articles

Detection of Tissue Macrophages in Different Organs Using Antibodies to the Microglial Marker Iba-1.

Resident macrophages of different organs have structural and functional features, which can complicate their identification and analysis. A promising candidate for the role of a universal immunohistochemical marker of resident macrophages is the calcium-binding protein Iba-1, a well-known marker of brain microglia. The purpose of this work was to study the possibility of using one variant of antibodies to the Iba-1 protein for the immunohistochemical detection of resident macrophages in the liver, myocardium, lung, and choroid plexus of the rat brain. The study was performed on male Wistar rats (n = 15). It was shown that the use of rabbit monoclonal antibodies against Iba-1 allows highly effective detection of Kupffer cells in the liver, resident macrophages in the myocardium, alveolar and interstitial macrophages in the lung, and Kolmer cells in the choroid plexus of the rat brain. In all cases, the reaction is characterized by a high specificity and the absence of background staining. In contrast to the classical marker of macrophages, the CD68 molecule, the Iba-1 protein is evenly distributed in the cytoplasm of cell bodies and processes. This makes it possible to more fully identify cells using immunostaining for Iba-1, carry out their three-dimensional reconstructions, and study their structural and functional organization. Immunohistochemical reaction against Iba-1 can be successfully used as a universal alternative to other common methods for identifying resident macrophages.

Accelerated BDNF expression in visceral white adipose tissues following high-fat diet feeding in mice.

Brain-derived neurotrophic factor (BDNF) is expressed in the white adipose tissues (WATs), and the expression increases during high-fat diet (HFD) feeding, implicating its role in obesity. Here, we focused on BDNF expression in epididymal WAT (eWAT), a visceral adipose tissue, in mice. During 2 weeks of HFD feeding, Bdnf mRNA expression in eWAT slightly increased, but a robust increase was observed after 8 weeks of HFD feeding. This upregulation of Bdnf mRNA was correlated with significant induction of hypoxia-inducible factor 1α (Hif1α) and platelet-derived growth factor subunit B (Pdgfb) mRNA in eWAT following 8 weeks of HFD feeding. Furthermore, the increased expression of the M1 macrophage markers was strongly correlated with the elevation of Bdnf mRNA in the eWAT. Notably, 8 weeks of HFD feeding significantly elevated Tnfα mRNA expression in eWAT, while no such induction was observed in inguinal WAT (iWAT). In contrast, the expression of Adipoq (adiponectin), implicated in improved insulin sensitivity and anti-inflammatory effects, was significantly upregulated in iWAT, but not in eWAT. Thus, our study may show the role of BDNF in eWAT in obesity models, potentially contributing to the pathological state of visceral adipose tissues.

Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma

BackgroundMacrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies.MethodsIn this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT.ResultsMacrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME.ConclusionsIn summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

CD44 is associated with muscle inflammation in polymyositis and skin damage in idiopathic inflammatory myopathy.

Idiopathic inflammatory myopathy (IIM) is a group of systemic autoimmune diseases characterised by muscle involvement. This study aims to reveal the characteristics of IIM subtypes and explore the molecular mechanisms underlying IIM. The STRING database was utilised to construct a protein-protein interaction network of differentially expressed genes obtained from the GSE128470, GSE3112, and GSE39454 datasets. The immune cell infiltration level was assessed by CIBERSORT in polymyositis (PM). Experimental autoimmune myositis (EAM) model mice were constructed for experimental verification. Serum levels of soluble CD44 (sCD44) were measured using enzyme-linked immunosorbent assay. The upregulated hub gene CD44 was highly expressed in inflammatory cells infiltrating the skeletal muscle of patients with PM and in EAM mice. CD44 was correlated with both M1 macrophages (r=0.57, p<0.0001) and M2 macrophages (r=0.57, p<0.0001) in PM. Additionally, CD44+F4/80+ macrophages in skeletal muscle were increased (p<0.0001) and CD44 showed a stronger association with markers of M1 macrophage in EAM mice. Moreover, serum sCD44 levels were elevated in patients with IIM (p=0.0024), PM (p=0.0332) and dermatomyositis (p=0.0001) notably in the anti-melanoma differentiation-associated gene 5 antibody positive subtype (p=0.0007). sCD44 levels also positively correlated with visual analogue score (r=0.4424, p=0.0013), myositis intention to treat activity index (r=0.3938, p=0.0047), skin damage score (r=0.3796, p=0.0101) and skin activity score (r=0.4625, p=0.0014) in patients with IIM. This study suggests that macrophages expressing CD44 may be involved in the pathogenesis of PM, and sCD44 could serve as a potential marker for skin damage and activity in IIM.

Reveal the pharmacodynamic substances and mechanism of an edible medicinal plant Platycodonis radix inhibits tumor

Accumulating evidence suggests that M2-polarized tumor-associated macrophages (TAMs) play a crucial role in cancer progression, making them a promising target for therapy. This study explored the molecular mechanisms by which Platycodon grandiflorum regulates M2 polarization of TAMs to exert anti-tumor effects. UPLC-MS identified the chemical composition and blood-absorbed components. Immunohistochemistry and Western blot detected TAM infiltration and proliferation pathways, while q-RT-PCR measured M2 macrophage marker expression. Our findings showed that PR reduces melanoma weight, inhibits the mTOR pathway, and suppresses M2-polarized macrophages. PR downregulates JAK2 and p-STAT3, promotes JAK2 degradation, and enhances ubiquitination. Molecular docking indicated strong affinities between Platycodin D components and JAK2/STAT3, highlighting PR’s potential in regulating TAMs and offering new clinical insights.

PLXDC1 serves as a potential prognostic marker and involves in malignant progression and macrophage polarization in colon cancer.

The malignant behavior and immune escape ability of cancer cells lead to therapeutic failure and poor prognosis for patients with various cancers, including colon cancer. Plexin domain containing 1 (PLXDC1) was initially identified to exert key roles in tumor by regulating angiogenesis and has recently proved to be involved in cell proliferation and migration of glioblastoma and gastric cancer cells. However, its roles in colon cancer remain unclear. In this study, the online bioinformatics databases confirmed high expression of PLXDC1 in colon cancer specimens, which was associated with cancer stages and nodal metastasis. Similarly, the increased expression of PLXDC1 was also validated in our collected samples and colon cancer cells. Moreover, patients with high expression of PLXDC1 had shorter survival, indicating that PLXDC1 might be a potential prognostic predictor for colon cancer patients. Notably, targeting PLXDC1 inhibited cancer cell viability and invasion, and enhanced cell apoptosis. Intriguingly, Tumor Immune Estimation Resource database confirmed that PLXDC1 expression was related to various tumor-infiltrating immune cells in colon adenocarcinoma including macrophages, and its expression was also correlated with M2-like macrophage markers. In vitro, colon cancer cells with PLXDC1 downregulation had a reduced ability to recruit and polarize macrophage towards M2 phenotype by decreasing the percentage of CD206+ cells and M2-like markers (CD206, CD163, arginase1, and interleukin 10 [IL-10]). Moreover, PLXDC1 knockdown attenuated M2 macrophage-mediated promotion in cancer cell viability and invasion. Mechanically, inhibition of PLXDC1 suppressed activation of the IL-6/Signal transducer and activator of transcription 3 (STAT3) signaling. Reactivating the above pathway by transfection with IL-6 plasmids reversed the suppressive effects of PLXDC1 knockdown on cancer cell malignant behaviors, macrophage recruitment and M2-like polarization. Thus, PLXDC1 downregulation may inhibit the malignancy of colon cancer cells and their ability to recruit and polarize macrophages towards M2 phenotype by blocking the IL-6/STAT3 pathway. Together, targeting PLXDC1 may attenuate the progression of colon cancer by direct roles in cancer cells and indirect roles in macrophage polarization, representing a promising therapeutic target for colon cancer patients.

Sulfated polysaccharide from Antrodia cinnamomea mycelium cultured with zinc sulfate stimulates M1 polarization of macrophages through AKT/mTOR pathways

Antrodia cinnamomea-derived sulfated polysaccharides (Ac-SPSs) have health benefits, but their yield is low. This study explores a strategy to increase Ac-SPS yield and elucidates the biofunctions of Ac-SPS. For this, A. cinnamomea mycelia were treated with zinc sulfate (ZnSO4) administered at 1, 10, and 100 μM. Firstly, functional assay indicated that ZnSO4 increases the Ac-SPS yield by 20 %–30 % compared with the control treatment. ZnSO4 engenders a population of middle-molecular-weight (~200 kDa) Ac-SPSs. Ac-SPS (ASZ-10) from A. cinnamomea treated with 10 μM ZnSO4 exhibits the best anti-proliferation ability against lung cancer A549 cells. Co-treatment of ASZ-10 does not inhibit lipopolysaccharide-induced inflammation but does induce M1-related markers of macrophage RAW264.7 cells. Secondly, immunomodulatory properties showed that ASZ-10 increases the expression of CD80+ and CD86+ in M-CSF-stimulated bone-marrow-derived macrophages. ASZ-10 induces M1 polarization through up-regulation of the AKT/mTOR pathway as confirmed by AKT and mTOR inhibitors eliminating ASZ-10-induced M1-like markers of macrophages. Through systemic chemical and functional analysis, this study shows that trace amounts (10 μM) of ZnSO4 increase Ac-SPS yield and it reveals that ASZ-10 exhibits anti-cancer activity and acts as a stimulator for M1 macrophages by stimulation of AKT and mTOR.

SIRT1 mediates the inflammatory response of macrophages and regulates the TIMP3/ADAM17 pathway in atherosclerosis

ObjectiveMacrophage polarization and the resulting phenotype have versatile roles in atherosclerosis. The study aims to decipher the role of SIRT1 in regulating macrophage phenotypes and atherosclerosis development. MethodsTwo mouse lines of SIRT1△Mac/ApoE−/− and SIRT1fl/fl/ApoE−/− were fed with high-fat diet to generate atherosclerotic lesion. Mouse peritoneal macrophages were isolated and transfected with SIRT1-overexpressing vector or vector-null. ResultsThe SIRT1△Mac/ApoE−/− mice exhibited greater atherosclerotic lesions, stronger immunofluorescence staining for M1-like macrophage marker, iNOS, and weaker immunofluorescence staining for M2-like macrophage marker, Arginase-1, than the SIRT1fl/fl/ApoE−/− littermates. The gene expressions of M1 markers (IL-1β, IL-6, and iNOS) were increased and those of M2 markers (IL-10 and Arg-1) decreased in both aortic roots and peritoneal macrophages from SIRT1△Mac/ApoE−/− mice, whereas SIRT1 overexpression rectified the changes in M1/M2 expression. A declined expression of TIMP3 with an increased expression of ADAM17 was noted in SIRT1△Mac/ApoE−/− macrophages, whereas SIRT1 overexpression rescued TIMP3 expression and inhibited ADAM17 expression. ConclusionOur data suggest that SIRT1 deficiency may promote macrophage M1 polarization and regulate the TIMP3/ADAM17 pathway thus favoring atherosclerosis development, indicating an anti-atherosclerotic role of macrophage SIRT1.

Construction and validation of a TAMRGs prognostic signature for gliomas by integrated analysis of scRNA and bulk RNA sequencing data

BackgroundThis study aimed to construct and validate a prognostic model based on tumor associated macrophage-related genes (TAMRGs) by integrating single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) data. MethodsThe scRNA-seq data of three inhouse glioma tissues were used to identify the tumor-associated macrophages (TAMs) marker genes, the DEGs from the The Cancer Genome Atlas (TCGA) − Genotype-Tissue Expression (GTEx) dataset were used to further select TAMs marker genes. Subsequently, a TAMRG-score was constructed by Least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analysis in the TCGA dataset and validated in the Chinese Glioma Genome Atlas (CGGA) dataset. ResultsWe identified 186 TAMs marker genes, and a total of 6 optimal prognostic genes including CKS2, LITAF, CTSB, TWISTNB, PPIF and G0S2 were selected to construct a TAMRG-score. The high TAMRG-score was significantly associated with worse prognosis (log-rank test, P<0.001). Moreover, the TAMRG-score outperformed the other three models with AUC of 0.808. Immune cell infiltration, TME scores, immune checkpoints, TMB and drug susceptibility were significantly different between TAMRG-score groups. In addition, a nomogram were constructed by combing the TAMRG-score and clinical information (Age, Grade, IDH mutation and 1p19q codeletion) to predict the survival of glioma patients with AUC of 0.909 for 1-year survival. ConclusionThe high TAMRG-score group was associated with a poor prognosis. A nomogram by incorporating TMARG-score could precisely predict glioma survival, and provide evidence for personalized treatment of glioma.

Cinobufagin inhibits M2‑like tumor‑associated macrophage polarization to attenuate the invasion and migration of lung cancer cells.

Macrophages have crucial roles in immune responses and tumor progression, exhibiting diverse phenotypes based on environmental cues. In the present study, the impact of cinobufagin (CB) on macrophage polarization and the consequences on tumor‑associated behaviors were investigated. Morphological transformations of THP‑1 cells into M0, M1 and M2 macrophages were observed, including distinct changes in the size, shape and adherence properties of these cells. CB treatment inhibited the viability of A549 and LLC cells in a concentration‑dependent manner, with an IC50 of 28.8 and 30.12 ng/ml, respectively. CB at concentrations of <30 ng/ml had no impact on the viability of M0 macrophages and lung epithelial (BEAS‑2B) cells. CB influenced the expression of macrophage surface markers, reducing CD206 positivity in M2 macrophages without affecting CD86 expression in M1 macrophages. CB also altered certain expression profiles at the mRNA level, notably downregulating macrophage receptor with collagenous structure (MARCO) expression in M2 macrophages and upregulating tumor necrosis factor‑α and interleukin‑1β in both M0 and M1 macrophages. Furthermore, ELISA analyses revealed that CB increased the levels of pro‑inflammatory cytokines in M1 macrophages and reduced the levels of anti‑inflammatory factors in M2 macrophages. CB treatment also attenuated the migration and invasion capacities of A549 and LLC cells stimulated by M2 macrophage‑conditioned medium. Additionally, CB modulated peroxisome proliferator‑activated receptor γ (PPARγ) and MARCO expression in M2 macrophages and epithelial‑mesenchymal transition in A549 cells, which was partially reversed by rosiglitazone, a PPARγ agonist. Finally, CB and cisplatin treatments hindered tumor growth in vivo, with distinct impacts on animal body weight and macrophage marker expression in tumor tissues. In conclusion, the results of the present study demonstrated that CB exerted complex regulatory effects on macrophage polarization and tumor progression, suggesting its potential as a modulator of the tumor microenvironment and a therapeutic for cancer treatment.

Similarities and differences in morphology, CD31 and CD68 expression of male vs. female sinoatrial node and its surrounding atrial muscle in ageing and obesity

Research purposeThe sinoatrial node (SN) is the main pacemaker site, and it is located in the junctional area of the superior vena cava within the right atrium (RA). The precise micro-anatomy of the SN in males and females in ageing and obesity remains unclear. Basic proceduresHuman SN/RA specimens were dissected from 25 post-mortem hearts (preserved in 4 % formaldehyde solution), under Polish local ethical rules. The SN/RA tissue blocks were embedded in paraffin. Masson's Trichrome staining and immunohistochemistry for CD31 (a marker of endothelial cells) and CD68 (a marker of macrophages) were performed. Images at different magnifications were taken and analysed. 12-lead ECGs from 24 patients under Polish local ethical rules were obtained. Heart rate and P wave morphologies from lead II, lead III and lead aVF were analysed. Statistical analysis was performed with the unpaired t-test. Principal resultsHeart weight to body weight ratio was significantly higher in aged obese males vs. their female counterparts. In the RA samples, there was an increase in connective tissue and decreased myocyte content from aged obese females compared to aged obese males. Aged non-obese males had significantly increased cellular hypertrophy than the aged non-obese females. Both the aged obese and aged non-obese females showed more CD3 but less CD68 expressing cells than males. In the SN samples, CD31 and CD68 expressing cells were higher in both aged non-obese and aged obese males than their female counterparts. Major conclusionsAgeing and/or obesity are more likely to impact these cardiac tissues through increased inflammation, increased immune response and hypertrophy.

LncRNA-MM2P regulates retinal neovascularization through M2 macrophage polarization

The study aims to investigate the effects and potential mechanisms of lncRNA-MM2P on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). The OIR model was established in C57BL/6J mice. RAW264.7 cell line and bone marrow-derived macrophages (BMDMs) from mice were used for in vitro studies. RT-qPCR was used to analyze the expressions of lncRNA and mRNAs. The protein expression levels were determined by western blotting. The size of avascular areas and neovascular tufts were assessed based on isolectin B4 immunofluorescence staining images. The human retinal endothelial cells (HRECs) were used to evaluate the proliferation, migration, and tube formation of endothelial cells. The expression of lncRNA-MM2P was significantly upregulated from P17 to P25 in OIR retinas. Knockdown of lncRNA-MM2P levels in vivo led to a significant reduction in the neovascular tufts and avascular areas in the retinas of OIR mice. Knockdown of lncRNA-MM2P levels in vitro suppressed the expression of M2 markers in macrophages. Moreover, we found a significant inhibition of avascular areas and neovascular tufts in OIR mice injected intravitreally with M2 macrophages treated by shRNA-MM2P. The cellular functions of proliferation, migration, and tube formation were significantly attenuated in HRECs cultured with a supernatant of shRNA-MM2P-treated M2 macrophages. Our results indicate that lncRNA-MM2P regulates retinal neovascularization by inducing M2 polarization of macrophages in OIR mice. Therefore, lncRNA-MM2P may be a potential molecular target for immunoregulation of retinal neovascularization.

Comprehensive scRNA-seq analysis to identify new markers of M2 macrophages for predicting the prognosis of prostate cancer

Background Prostate cancer (PCa) has become the highest incidence of malignant tumor among men in the world. Tumor microenvironment (TME) is necessary for tumor growth. M2 macrophages play an important role in many solid tumors. This research aimed at the role of M2 macrophages’ prognosis value in PCa. Methods Single-cell RNA-seq (scRNA-seq) data and mRNA expression data were obtained from the Gene Expression Omnibus database (GEO) and The Cancer Genome Atlas (TCGA). Quality control, normalization, reduction, clustering, and cell annotation of scRNA-seq data were preformed using the Seruat package. The sub-populations of the tumor-associated macrophages (TAMs) were analysis and the marker genes of M2 macrophage were selected. Differentially expressed genes (DEGs) in PCa were identified using limma and the immune infiltration was detected using CIBERSORTx. Then, a weighted correlation network analysis (WGCNA) was constructed to identify the M2 macrophage-related modules and genes. Integration of the marker genes of M2 macrophage from scRNA-seq data analysis and hub genes from WGCNA to select the prognostic gene signature based on Univariate and LASSO regression analysis. The risk score was calculated, and the DEGs, biological function, immune characteristics related to risk score were explored. And a predictive nomogram was constructed. CCK8, Transwell, and wound healing were used to verify cell phenotype changes after co-cultured. Results A total of 2431 marker genes of M2 macrophage and 650 hub M2 macrophage-related genes were selected based on scRNA-seq data and WGCNA. Then, 113 M2 macrophage-related genes were obtained by overlapping the scRNA-seq data and WGCNA results. Nine M2 macrophage-related genes (SMOC2, PLPP1, HES1, STMN1, GPR160, ABCG1, MAZ, MYC, and EPCAM) were screened as prognostic gene signatures. M2 risk score was calculated, the DEGs, Immune score, stromal score, ESTIMATE score, tumor purity, and immune cell infiltration, immune checkpoint expression, and responses of immunotherapy and chemotherapy were identified. And a predictive nomogram was constructed. CCK8, Transwell invasion, and wound healing further verified that M2 macrophages promoted the proliferation, invasion, and migration of PCa (p < 0.05). Conclusions We uncovered that M2 macrophages and relevant genes played key roles in promoting the occurrence, development, and metastases of PCa and played as convincing predictors in PCa.

Identification of potential diagnostic biomarkers and therapeutic targets in patients with hypoxia pulmonary hypertension

BackgroundPulmonary hypertension is a serious disease. Emerging studies have shown that M2 macrophages play an essential role in pulmonary hypertension; however, their mechanism of action is uncertain. MethodsFour GEO datasets were downloaded. The differentially expressed genes (DEGs) were obtained using the limma package. Simultaneously, the Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm and weighted gene co-expression network analysis (WGCNA) were used to get the information about M2 macrophage–related modules. Potential key genes were obtained by intersecting DEGs with M2 macrophage–related module genes (M2MRGs), and finally the area under the curve (AUC) was calculated. Rats were exposed to hypoxia condition (10 % O2) for 4 weeks to induce PH. Subsequently, potential key genes with AUC>0.7 were analyzed by quantitative real-time polymerase chain reaction and Western blot using normoxia and hypoxia rat lungs. We knocked down EPHA3 in Raw264.7 cells and detected the protein expression of M2 macrophage markers including arginase 1 (ARG1) and interleukin 10 (IL-10), phospho-protein kinase B (P-Akt), and protein kinase B (Akt) to explore the downstream pathways of EPHA3. ResultsSeven potential hub genes were detected by intersecting M2MRGs and DEGs. Six genes with AUC values above 0.7 were used for further exploration. The expression of EPHA3 mRNA and protein was significantly more upregulated in rats with hypoxia than in rats with normoxia. The expression levels of IL10, ARG1, and P-Akt/Akt decreased after knocking down EPHA3. ConclusionsThis study suggested that the activation of the P-Akt/Akt signaling pathway promoted by EPHA3 played an essential role in the progression of pulmonary hypertension.

Role of ACSL4 in modulating farnesoid X receptor expression and M2 macrophage polarization in HBV-induced hepatocellular carcinoma.

The intricate relationship between bile acid (BA) metabolism, M2 macrophage polarization, and hepatitis B virus-hepatocellular carcinoma (HBV-HCC) necessitates a thorough investigation of ACSL4's (acyl-CoA synthetase long-chain family member 4) role. This study combines advanced bioinformatics and experimental methods to elucidate ACSL4's significance in HBV-HCC development. Using bioinformatics, we identified differentially expressed genes in HBV-HCC. STRING and gene set enrichment analysis analyses were employed to pinpoint critical genes and pathways. Immunoinfiltration analysis, along with in vitro and in vivo experiments, assessed M2 macrophage polarization and related factors. ACSL4 emerged as a pivotal gene influencing HBV-HCC. In HBV-HCC liver tissues, ACSL4 exhibited upregulation, along with increased levels of M2 macrophage markers and BA. Silencing ACSL4 led to heightened farnesoid X receptor (FXR) expression, reduced BA levels, and hindered M2 macrophage polarization, thereby improving HBV-HCC conditions. This study underscores ACSL4's significant role in HBV-HCC progression. ACSL4 modulates BA-mediated M2 macrophage polarization and FXR expression, shedding light on potential therapeutic targets and novel insights into HBV-HCC pathogenesis.

Abstract P335: An innate immune component in hypertension-induced cardiac dysfunction Christoph Mora, Katherine Deck, Yunmeng Liu, Lance Benson, Tonya Rafferty, &amp; Shengyu Mu

Hypertension affects nearly half of American adults, inevitably leading to cardiac dysfunction and heart failure. Cardiac fibrosis is a major factor contributing to cardiac dysfunction during hypertension progression. However, the mechanisms involved in cardio-fibrogenesis remain to be elucidated. In the past decade, studies revealed that macrophages could differentiate into professional fibrotic cells, myofibroblasts, through a process termed macrophage to myofibroblast transition (MMT), helping maintain the integrity of major organs, such as the kidney. However, it is unknown whether MMT contributes to fibrotic remodeling in hypertensive hearts. Here, we aim to identify this phenomenon in the hearts of hypertensive animals and determine the role it plays in the pathological progression from hypertension to cardiac diastolic dysfunction. In this study we adopt the classic DOCA + Salt model of hypertension and utilized cardiac ultrasound and direct left ventricular (LV) pressure catheterization to gauge diastolic function. Heart sections from hypertensive and sham mice were stained with Masson’s trichrome to assess the extent of cardiac fibrosis. Furthermore, flow cytometry was utilized to identify MMT cells, which express both CD68 (monocytes) and α-SMA (myofibroblast) markers. These methods gave insight into the role that MMT plays in the degradation of heart function during hypertension. Direct left ventricle pressure catheter revealed that DOCA + salt mice exhibited impaired diastolic function. This result is supported by cardiac ultrasound showing that the compliance of the heart had significantly decreased, as evidenced by the lowering of the efferent/atrial (E/A) ratio and deceleration time (DcT). In addition to this evidence of mechanical cardiac degradation, leukocytes isolated from the heart and stained with macrophage and myofibroblast markers revealed a significant increase in CD68+ &amp; αSMA+ cells—a result reflected by increased fibrotic remodeling via Masson’s trichrome stain, in the hypertensive animals. This study demonstrated an onset of diastolic dysfunction in the heart of DOCA + salt hypertensive mice. Moreover, we found a positive association between MMT cells and fibrosis in the hearts of hypertensive animals. Future studies will aim to parse out both the gravity of MMT in hypertensive heart dysfunction and the mechanism by which macrophages undergo this transition to find new targets against this debilitating disease.

Allogenic Vertebral Body Adherent Mesenchymal Stromal Cells Promote Muscle Recovery in Diabetic Mouse Model of Limb Ischemia

Chronic limb threatening ischemia (CLTI) carries a significant risk for amputation especially in diabetic patients with poor options for revascularization. Phase I trials have demonstrated efficacy of allogeneic mesenchymal stromal cells (MSC) in treating diabetic CLTI. Vertebral bone adherent mesenchymal stromal cells (vBA-MSC) are derived from vertebral bodies of deceased organ donors which offer the distinct advantage of providing a 1,000x greater yield compared to that of living donor bone aspiration. This study describes the effects of intramuscular injection of allogenic vBA-MSC in promoting limb perfusion and muscle recovery in a diabetic CLTI mouse model. A CLTI mouse model was created through unilateral ligation of the femoral artery in male polygenic diabetic TALLYHO mice. Treated mice were injected with vBA-MSC into the gracilis muscle of the ischemic limb 7 days post ligation. Gastrocnemius or tibialis muscle was assessed post-mortem for fibrosis by collagen staining, capillary density via immunohistochemistry, and mRNA by quantitative real time PCR. Laser Doppler perfusion imaging and plantar flexion muscle testing were performed to quantify changes in limb perfusion and muscle function. Compared to vehicle control, treated mice demonstrated indicators of muscle recovery including decreased fibrosis, increased perfusion, muscle torque, and angiogenesis. PCR analysis of muscle obtained 7- and 30-days post vBA-MSC injection showed an upregulation in expression of MyoD1 (p = 0.03) and MyH3 (p = 0.008) mRNA representing muscle regeneration, VEGF-A (p = 0.002 ; p = 0.004) signifying angiogenesis as well as IL-10 (p < 0.001), T regulatory cell marker Foxp3 (p = 0.04), and M2-biased macrophage marker Mrc1 (CD206) (p = 0.02). These findings indicate human allogeneic vBA-MSC ameliorate ischemic muscle damage and rescue muscle function. These results in a murine model will enable further studies to develop potential therapies for diabetic CLTI patients.

Knockdown of HM13 Inhibits Metastasis, Proliferation, and M2 Macrophage Polarization of Non-small Cell Lung Cancer Cells by Suppressing the JAK2/STAT3 Signaling Pathway.

An upregulated histocompatibility minor 13 (HM13) has been studied in various tumors, yet the exact mechanism of HM13 in non-small cell lung cancer (NSCLC) is unclear. In view of same, the present study investigates crucial role and action mechanism of HM13 in human NSCLC. HM13 expression was higher in NSCLC tissue and cells through the Western blotting technique along with qRT-PCR. As per data from The Cancer Genome Atlas (TCGA), NSCLC patients having high HM13 expression show lower overall survival. 5-ethynyl-2-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and transwell tests were assessed for NSCLC cell growth, and invasion, and we found that silencing of HM13 inhibited the NSCLC cell proliferation, invasion. Additionally, to investigate the effects of HM13 on THP-1 macrophage polarization, a co-culture model of NSCLC and THP-1 macrophages were used. The CD206 + macrophages were examined using flow cytometry. As the markers of M2 macrophage, the mRNA levels of IL-10 and TGF-β of THP-1 cells were also detected by qRT-PCR. Knockdown of HM13 could inhibit the M2 polarization. Further experiments demonstrated that downregulated HM13 could inhibit the JAK2/STAT3 signaling pathway. RO8191 (activator of JAK/STAT3 pathway) influenced the invasion, proliferation, and expression of JAK2/STAT3 signaling pathway and Epithelial-mesenchymal transition (EMT) markers induced by HM13 silencing. HM13 knockdown also inhibited the tumor growth in vivo by xenograft nude mouse model. By inhibiting JAK2/STAT3 signaling pathway, HM13 knockdown inhibited the NSCLC cell proliferation, metastasis tumor growth, and tumor-associated macrophage M2 polarization. In NSCLC, HM13 could be a therapeutic target to treat the NSCLC.

Bioinformatic analysis reveals the relationship between macrophage infiltration and Cybb downregulation in hyperoxia-induced bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is the most common sequela of prematurity and is characterized by alveolar simplification and lung angiogenesis failure. The aim of this study was to explore the immune signatures of BPD. Differentially expressed gene analysis and immune infiltration analysis were conducted to identify key immune cell types and related genes by using the mRNA-seq dataset GSE25286. The expression patterns of key genes were validated in the scRNA-seq dataset GSE209664 and in experiments. The cell–cell crosstalk of key immune cells was explored with CellChat. We found that differentially expressed genes between BPD mice and controls were mostly enriched in leukocyte migration and M1 macrophages were highly enriched in BPD lungs. Hub genes (Cybb, Papss2, F7 and Fpr2) were validated at the single-cell level, among which the downregulation of Cybb was most closely related to macrophage infiltration. The reduced mRNA and protein levels of Cybb were further validated in animal experiments. Colocalization analysis of Cybb and macrophage markers demonstrated a significant decrease of Cybb in M1 macrophages. Cell–cell crosstalk found that alveolar epithelial cells interacted actively with macrophages through MIF-(CD74 + CD44) signalling. In conclusion, M1 macrophages played important roles in promoting BPD-like lung injury, which was correlated with a specific reduction of Cybb in macrophages and the potential activation of MIF signalling.

Retinal perivascular macrophages regulate immune cell infiltration during neuroinflammation in mouse models of ocular disease.

The blood-retina barrier (BRB), which is disrupted in diabetic retinopathy (DR) and uveitis, is an important anatomical characteristic of the retina, regulating nutrient, waste, water, protein, and immune cell flux. The BRB is composed of endothelial cell tight junctions, pericytes, astrocyte end feet, a collagen basement membrane, and perivascular macrophages. Despite the importance of the BRB, retinal perivascular macrophage function remains unknown. We found that retinal perivascular macrophages resided on postcapillary venules in the superficial vascular plexus and expressed MHC class II. Using single-cell RNA-Seq, we found that perivascular macrophages expressed a prochemotactic transcriptome and identified platelet factor 4 (Pf4, also known as CXCL4) as a perivascular macrophage marker. We used Pf4Cre mice to specifically deplete perivascular macrophages. To model retinal inflammation, we performed intraocular CCL2 injections. Ly6C+ monocytes crossed the BRB proximal to perivascular macrophages. Depletion of perivascular macrophages severely hampered Ly6C+ monocyte infiltration. These data suggest that retinal perivascular macrophages orchestrate immune cell migration across the BRB, with implications for inflammatory ocular diseases including DR and uveitis.