M1 Macrophage Content Research Articles

M1 macrophages as promising agents for cell therapy of endometriosis

Endometriosis is a chronic estrogen-dependent disease characterized by the presence of endometrial glands and stroma outside their normal anatomical location. While laparoscopic removal of foci remains the gold standard therapy, it has limited efficacy and certain risks. However, cell therapy using pro-inflammatory M1 macrophages presents a promising and minimally invasive alternative for treating endometriosis. This approach showcases the potential for innovative and effective treatments for this condition. This study aims to explore the anti-endometriosis properties of M1 macrophages. A reproducible syngeneic mouse model of endometriosis was utilized, revealing that formed foci are primarily composed of macrophages with an anti-inflammatory M2 phenotype rather than M1 macrophages. To investigate further, chemically reprogrammed M1 macrophages were labeled with the membrane fluorescent tag PKH26 and administered to animals with endometriosis. Therapy resulted in a decrease in the number and size of foci, accompanied by a shift in the phenotypic composition of peritoneal macrophages. Specifically, the content of M2 macrophages decreased while that of M1 macrophages increased, resembling the composition of healthy animals. Our study conclusively demonstrates the anti-endometriosis properties of M1 macrophages, providing a strong foundation for future research in the cell therapy of endometriosis.

Zhiqiao Gancao decoction regulated JAK2/STAT3/ macrophage M1 polarization to ameliorate intervertebral disc degeneration

BackgroundZhiqiao Gancao decoction (ZQGCD) was created by Professor Gong Zhengfeng, a renowned Chinese medicine expert. Clinical studies have shown its efficacy in alleviating pain and enhancing lumbar function in intervertebral disc degeneration (IDD) patients. However, the precise mechanism of ZQGCD in treating IDD remains unclear. MethodsThe active components of ZQGCD were identified using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A rat model of intervertebral disc degeneration was established, and rats in each group received ZQGCD for three weeks. Assessment parameters included hyperalgesia status, observation of intervertebral disc tissue degeneration and macrophage infiltration, and analysis of JAK2/STAT3 pathway protein expression in the intervertebral disc. Primary macrophage M1 polarization was induced using LPS, with cells treated using the JAK2 inhibitor (AZD1480) and ZQGCD to evaluate macrophage polarization, cellular supernatant inflammatory factors, and JAK2/STAT3 pathway expression. Macrophage supernatant served as a conditioned medium to observe its effects on the proliferation of nucleus pulposus cells (NPCs) and the expression of collagen II and MMP3 proteins. ResultsA total of 81 active components were identified in ZQGCD. Following ZQGCD treatment, infiltrating macrophages in intervertebral disc tissues of model rats decreased, the content of M1 macrophages decreased, while the content of M2 macrophages increased, the expression of proinflammatory factors and pain-inducing factors in serum decreased, and the expression of substance P in intervertebral disc tissue decreased. Consequently, the intervertebral disc degeneration and hyperalgesia of rats were improved. In vitro studies revealed that LPS induced M1 macrophage polarization. By inhibiting the JAK2/STAT3 pathway, both JAK2 inhibitors and ZQGCD effectively suppressed M1 polarization, resulting in decreased levels of IL-1β, IL-6, TNF-α, and various other inflammatory factors. Consequently, this inhibition led to a delay in the degeneration of NPCs. ConclusionThere is macrophage infiltration in the intervertebral disc tissue of IDD rats, and JAK2/STAT3 pathway is activated, macrophages are polarized to M1 type, resulting in inflammatory microenvironment, leading to intervertebral disc degeneration and hyperalgesia. ZQGCD exhibited a delaying effect on IDD and improved hyperalgesia by inhibiting the JAK2/STAT3/macrophage M1 polarization pathway.

SMPDL3B contributes to gastric adenocarcinoma cells progression by promoting the infiltration of M2 macrophages.

The common disease gastric adenocarcinoma (GAC) has a high morbidity and mortality, so there is an urgent need for research to explore new diagnostic markers and therapeutic targets. This investigation was carried out to investigate the expression of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3B) in GAC and its effects on tumor progression. Samples were collected from patients who underwent radical gastrectomy from January 2021 to December 2022. Along with the normal gastric epithelial cell lines GES-1 and SGC-7901, the AGS, MGC-803, and MSN-45 human gastric cancer cell lines were used to confirm SMPDL3B expression. RT-qPCR, Western blot, immunohistochemical, cell proliferation, assay of wound healing, transwell migration assay, invasion assay, flow cytometry, and immune evaluation experiments were carried out. SMPDL3B was found to be substantially expressed in GAC, and this condition has a bad prognosis. By establishing SMPDL3B knockdown and overexpression of GAC cell lines, this study confirmed that SMPDL3B promoted tumor cell proliferation, migration, and invasion. Additional bioinformatics research revealed a connection between SMPDL3B and immune cell infiltration in the GAC immunological microenvironment, which enhanced tumor cell proliferation by promoting the infiltration content of M2 macrophages. This study determined the function of SMPDL3B for the clinical diagnosis, prediction, and novel management of GAC.

Peroxiredoxin 1 aggravates acute kidney injury by promoting inflammation through Mincle/Syk/NF-κB signaling

Damage-associated molecular patterns (DAMPs) are a cause of acute kidney injury (AKI). Our knowledge of these DAMPs remains incomplete. Here, we report serum peroxiredoxin 1 (Prdx1) as a novel DAMP for AKI. Lipopolysaccharide (LPS) and kidney ischemia/reperfusion injury instigated AKI with concurrent increases in serum Prdx1 and reductions of Prdx1 expression in kidney tubular epithelial cells. Genetic knockout of Prdx1 or use of a Prdx1-neutralizing antibody protected mice from AKI and this protection was impaired by introduction of recombinant Prdx1 (rPrdx1). Mechanistically, lipopolysaccharide increased serum and kidney proinflammatory cytokines, macrophage infiltration, and the content of M1 macrophages. All these events were suppressed in Prdx1-/- mice and renewed upon introduction of rPrdx1. In primary peritoneal macrophages, rPrdx1 induced M1 polarization, activated macrophage-inducible C-type lectin (Mincle) signaling, and enhanced proinflammatory cytokine production. Prdx1 interacted with Mincle to initiate acute kidney inflammation. Of note, rPrdx1 upregulated Mincle and the spleen tyrosine kinase Syk system in the primary peritoneal macrophages, while knockdown of Mincle abolished the increase in activated Syk. Additionally, rPrdx1 treatment enhanced the downstream events of Syk, including transcription factor NF-κB signaling pathways. Furthermore, serum Prdx1 was found to be increased in patients with AKI; the increase of which was associated with kidney function decline and inflammatory biomarkers in patient serum. Thus, kidney-derived serum Prdx1 contributes to AKI at least in part by activating Mincle signaling and downstream pathways.

Periodontitis Promotes the Progression of Oral Squamous Cell Carcinoma by Inducing Macrophage M2 Polarization

To investigate the role of periodontitis in the development of oral squamous cell carcinoma (OSCC) and to determine whether periodontitis microorganisms induce M2 macrophage (M2) polarization and promote tumor progression. The tumor tissues of OSCC patients with periodontitis and those without periodontitis were collected and immunohistochemistry tests were done to validate the trend of changes in M2 macrophages. A mouse model of OSCC accompanied by periodontitis was established by treating mice with drinking water containing four antibiotics for three consecutive days, applying in the mouths of the mice a coat of bacteria collected from the saliva of patients with periodontitis once every other day for five times, and injecting in their buccal mucosa OSCC cells (SCC7). We observed the effect of periodontitis on the development of OSCC, analyzed the M2 macrophage content in the tumor tissues, and analyzed salivary microbiota structure, and examined the pathological changes in the spleen and colon tissues of the mice. Finally, we collected saliva from patients with periodontitis, co-cultured it with mice peripheral blood mononuclear cells (PBMC) and SCC7 cells, and examined M2 macrophage percentage by flow cytometry. Immunohistochemical findings from the clinical samples showed that M2-polarized macrophages in OSCC patients with periodontitis were more enriched (27.01%±2.12%) compared with those of OSCC patients without periodontitis (17.00%±3.66%). The OSCC mice with periodontitis (PO group) had tumors of larger size and lower survival rate than OSCC mice (O group) did. Furthermore, the expression rate of Ki67-positive cells (35.49%±5.00%) was significantly higher than that of O group (23.89%±4.13%) ( P<0.05). According to the results of flow cytometry, M2 macrophage expression (24.97%±4.41%) in PO group was higher than that of O group (5.75%±0.52%) ( P<0.05). In addition, qPCR results showed that gene expression of M2 macrophage-related factors, Arg1, IL-10, and CD206, showed an overall upward trend. Immunohistochemistry results showed that the positive expression of M2 macrophages was significantly increased in the PO group (21.82%±4.16%) compared to that of the O group (9.64%±0.60%) ( P<0.05). Mice in the PO group showed changes in their oral flora structure, exhibiting increased bands and diversity. The white pulp in their spleen tissue decreased and the boundary of the red pulp became indistinct with severe bleeding. The morphology of the colon glands was abnormal and the U-shaped crypt was damaged rather seriously. According to the results of cell experiment, when co-culturing PBMC with SCC7 cells, the presence of periodontitis microorganisms increased the polarization of M2 macrophages (71.00%±0.66%). Periodontitis promotes the development of OSCC by inducing M2 polarization in tumor-associated macrophages. Hence, periodontitis treatment holds important values for OSCC patients.

Effective breast cancer therapy based on palmitic acid-loaded PLGA nanoparticles

Although new strategies for breast cancer treatment have yielded promising results, most drugs can lead to serious side effects when applied systemically. Doxorubicin (DOX), currently the most effective chemotherapeutic drug to treat breast cancer, is poorly selective towards tumor cells and treatment often leads to the development of drug resistance. Recent studies have indicated that several fatty acids (FAs) have beneficial effects on inhibiting tumorigenesis. The saturated FA palmitic acid (PA) showed anti-tumor activities in several types of cancer, as well as effective repolarization of M2 macrophages towards the anti-tumorigenic M1 phenotype. However, water insolubility and cellular impermeability limit the use of PA in vivo. To overcome these limitations, here, we encapsulated PA into a poly(d,l-lactic co-glycolic acid) (PLGA) nanoparticle (NP) platform, alone and in combination with DOX, to explore PA's potential as mono or combinational breast cancer therapy. Our results showed that PLGA-PA-DOX NPs and PLGA-PA NPs significantly reduced the viability and migratory capacity of breast cancer cells in vitro. In vivo studies in mice bearing mammary tumors demonstrated that PLGA-PA-NPs were as effective in reducing primary tumor growth and metastasis as NPs loaded with DOX, PA and DOX, or free DOX. At the molecular level, PLGA-PA NPs reduced the expression of genes associated with multi-drug resistance and inhibition of apoptosis, and induced apoptosis via a caspase-3-independent pathway in breast cancer cells. In addition, immunohistochemical analysis of residual tumors showed a reduction in M2 macrophage content and infiltration of leukocytes after treatment of PLGA-PA NPs and PLGA-PA-DOX NPs, suggesting immunomodulatory properties of PA in the tumor microenvironment. In conclusion, the use of PA alone or in combination with DOX may represent a promising novel strategy for the treatment of breast cancer.

Heme-heme oxygenase-2 reduces the atherosclerosis by preventing inflammation

ObjectiveHeme oxygenase (HO) has been shown to have important antioxidant and anti-inflammatory properties, resulting in a vascular antitherogenic effect. This study was undertaken to evaluate the role of HO-2 in atherosclerosis. Method and resultsThe expression levels of HO-2 were evaluated in M1 and M2 bone marrow macrophage induced by LPS and IL4. The expression of HO-2 was significantly higher in M2 macrophage than in M1 macrophage. Western diet (WD) caused a significant increase in HO-2 expression in ApoE−/− mice. The adeno-associated viral (AAV) vectors expressing HO-2 was constructed, and the mice were received saline (ApoE−/−), AAV (ApoE−/−), AAV–HO–2 (ApoE−/−) on WD at 12 weeks and their plasma lipids, inflammatory cytokines, atherosclerosis were evaluated for 16 weeks. The results showed AAV–HO–2 was robust, with a significant decrease in the en face aortas, lipids levels, inflammatory cytokines and M1 macrophage content in AAV–HO–2 ApoE−/− compared to control AAV-ApoE−/−. ConclusionHO-2 expression in macrophages plays an important role of the antiatherogenic effect, decreasing the inflammatory component of atherosclerotic lesions. These results suggest that HO-2 may be a novel therapeutic target for cardiovascular diseases.

Role of ARRB1 in prognosis and immunotherapy: A Pan-Cancer analysis.

Background: β-arrestin1 (ARRB1), was originally identified as a multifunctional adaptor protein. Although ARRB1 has recently been shown to also play an important role in tumor growth, metastasis, inflammation, and immunity, its relationship with distinct tumor types and the tumor immune microenvironment remains unclear. Methods: We analyzed the ARRB1 expression profile and clinical characteristics in 33 cancer types using datasets from The Cancer Genome Atlas (TCGA) database. Clinical parameters such as patient survival, tumor stage, age, and gender were used to assess the prognostic value of ARRB1. The Human Protein Atlas (HPA) database was used to explore ARRB1 protein expression data. ESTIMATE and CIBERSORT algorithms were performed to assess immune infiltration. Furthermore, putative correlations between ARRB1 and tumor-infiltrating immune cells, the signatures of T-cell subtypes, immunomodulators, the tumor mutation burden (TMB), Programmed cell death ligand 1 (PD-L1), and microsatellite instability (MSI) were also explored. Gene functional enrichment was determined using GSEA. GSE40435 and GSE13213 cohorts were used to validate the correlation of ARRB1 with KIRC and LUAD clinicopathological parameters. Finally, the relationship between ARRB1 and immunotherapeutic responses was assessed using three independent immunotherapy cohorts, namely, GSE67501, GSE168204, and IMvigor210. Results: We found that ARRB1 expression levels were lower in 17 tumor tissues than in the corresponding normal tissues. We further found that ARRB1 expression was significantly correlated with tumor stage in BRCA, ESCA, KIRC, TGCT, and THCA, while in some tumors, particularly KIRC and LUAD, ARRB1 expression was associated with better prognosis. ARRB1 expression was also positively correlated with the stromal score or the immune score in some tumors. Regarding immune cell infiltration, ARRB1 expression in DLBC was positively correlated with M1 macrophage content and negatively correlated with B-cell infiltration. Additionally, there was a broad correlation between ARRB1 expression and three classes of immunomodulators. Furthermore, high ARRB1 expression levels were significantly correlated with some tumor immune-related pathways. Finally, ARRB1 expression was significantly associated with MSI, PD-L1, and TMB in some tumors and with the efficacy of immune checkpoint inhibitors (ICIs) in melanoma. Conclusion: ARRB1 has prognostic value in malignant tumors, especially in KIRC and LUAD. At the same time, ARRB1 was closely correlated with the tumor immune microenvironment and indicators of immunotherapy efficacy, indicating its great potential as a reliable marker for predicting the efficacy of immunotherapy.

Tumor-Infiltrating Myeloid Cells Confer De Novo Resistance to PD-L1 Blockade through EMT-Stromal and Tgfβ-Dependent Mechanisms.

Most patients with bladder cancer do not respond to ICB targeting of the PD-L1 signaling axis. Our modeling applied a de novo resistance signature to show that tumor-infiltrating myeloid cells promote poor treatment response in a TGFβ-dependent mechanism.

Is cell regeneration and infiltration a double edged sword for porcine aortic valve deterioration? A large cohort of histopathological analysis

Background and objectiveBioprostheses are the most common prostheses used for valve replacement in the Western medicine. The major flaw of bioprostheses is the occurrence of structural valve deterioration (SVD). This study aimed to assess the pathological features of porcine aortic valve (PAV)-SVD based on histomorphological and immunopathological characteristics of a large cohort of patients.MethodsHistopathological data of 109 cases with resected PAV were collected. The type and amount of infiltrated cells were evaluated in the different types of bioprosthetic SVD by immunohistochemical staining.ResultsThe most common cause of SVD was calcification, leaflet tear, and dehiscence (23.9%, 19.3%, and 18.3%, respectively). Immunohistochemical staining demonstrated that macrophages were infiltrated in the calcified, lacerated and dehiscence PAV, in which both M1 and M2 macrophages were existed in the calcified PAV. Importantly, the higher content of M1 macrophages and less content of M2 macrophages were found in the lacerated and dehiscence PAV, and MMP-1 expression was mainly found in the lacerated PAV. The endothelialization rate of leaflet dehiscence was higher than that of calcified and lacerated leaflets. A large number of CD31+/CD11b+ cells was aggregated in the spongy layer in the lacerated and dehiscence PAV.ConclusionCell regeneration and infiltration is a double edged sword for the PAV deterioration. Macrophage infiltration is involved in the different types of SVD, while only MMP-1 expression is involved in lacerated leaflets. The macrophage subtype of circulating angiogenic cells in dehiscence and tear PAV could be identified, which could reserve macrophages in the PAV-SVD.

The Correlation between the Inflammatory Effects of Activated Macrophages in Atherosclerosis and Aortic Dissection

To study the correlation between lipid metabolism index, inflammatory factor index, and M1 macrophage content and aortic dissection. Patients with only basic atherosclerotic diseases were selected as the control group, and patients with only basic atherosclerotic diseases and aortic dissection were set as the experiment group. Blood of patients was collected. Chemiluminescent immunoassay was applied to determine the concentration of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), apolipoprotein A1 (ApoA1), and apolipoprotein B1 (ApoB1). Enzyme-linked immunosorbent assay was applied to measure the concentration of tumor necrosis factor-α (TNF-α), Interleukin 1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). The content of M1 macrophages in blood was measured with the flow cytometry (FCM) method. The correlation between the inflammatory effects of activated macrophages in atherosclerosis and aortic dissection was analyzed by a simple linear regression analysis. Concentration of TC, TG, LDL-C, and ApoB and the concentration of TNF-α, IL-1β, and IL-6 in the experiment group were markedly higher compared to those in the control group, whereas the concentration of HDL-C and ApoA1 and IL-10 concentration in the experiment group was markedly lower. The content of M1 macrophage in the control group was significantly lower compared to the experiment group. The proportion of M1 macrophages, concentration of TC, TG, LDL, HDL, ApoA1, and ApoB1, and concentration of TNF-α, IL-1β, IL-6, and IL-10 were all significantly correlated to the occurrence of aortic dissection. The proportion of M1 macrophages, concentration of TC, TG, LDL, HDL, ApoA1, and ApoB1, and concentration of TNF-α, IL-1β, IL-6, and IL-10 are significantly correlated with the occurrence of aortic dissection.

Construction and Analysis of an Immune-Related Gene Prognostic Index for Bladder Cancer

Objective: To construct an Immune-Related Gene Prognostic Index (IRGPI) for bladder cancer using a bioanalytical approach to analyze its molecular and immunological characteristics, as well as to assess the benefit of Immune Checkpoint Inhibitor (ICI) therapy in the IRGPI-defined bladder cancer subgroup. Methods: Twenty-nine immune-related pivotal genes were identified by Weighted Gene Co-expression Network Analysis (WGCNA) based on The Cancer Genome Atlas (TCGA) bladder cancer immune dataset (n = 433). Six genes were identified using a multifactorial Cox regression approach to construct the IRGPI and validated against the Gene Expression Omnibus (GEO) dataset (n = 256). Then, molecular and immunological features in the subgroups defined by IRGPI were synthesized by GSEA, Kaplan-Meier survival curves, and other methods, and the benefit of ICI treatment was assessed. Results: IRGPI was constructed based on six genes including AHNAK, ILK, OGN, PDGFD, PPARGC1B, and JAM3. Patients with low IRGPI had better Overall Survival (OS) than those with high IRGPI, which was confirmed in the validation cohort of GEO. Pooled analysis showed that the low IRGPI subgroup was associated with higher infiltration of CD8 T cells, activated memory CD4 T cells, and could benefit from ICI treatment. Meanwhile, high IRGPI subgroups were associated with higher resting memory CD4T cells, M0 macrophages, and M2 macrophage content, immunosuppression, and benefited less from ICI treatment. Conclusion: IRGPI is a novel biomarker with better efficacy in differentiating the prognosis of bladder cancer, molecular and immune features, and evaluation of ICI therapy for individualized treatment of bladder cancer.

Alternative Splicing Events in Immune Infiltration of Lung Adenocarcinoma

BackgroundThe exact mechanisms of lung adenocarcinoma (LUAD) etiology and pathogenesis remain unclear.Material/MethodsIn this study, we evaluated alternative splicing (AS) events in LUAD. We separately analyzed LUAD data from The Cancer Genome Atlas (TCGA) database and AS-related feature lists from SpliceSeq to develop risk models for AS events and further validated risk models for AS events. The association between immune-related features and the risk model of AS events was evaluated.ResultsWe found that exon skip (ES) and mutually exclusive exons (ME) exhibited the most and least AS events, respectively. The risk score and stage of LUAD patients were strongly associated with prognosis and were independent influences on the prognosis of LUAD. The pathological stage (stage, T, N) and risk model were incorporated to construct a column line graph with better predictive ability. Risk models were associated with tumor microenvironment, and higher risk score values were associated with higher M2 macrophage content and lower M0 macrophage and helper T lymphocyte content. The correlation between core genes and immunity was further assessed by analyzing differentially-expressed genes between risk models. These results suggest an association between the level of risk for AS events and the density of immune cell infiltration.ConclusionsOur findings suggest a clear association between AS risk model and patient prognosis, and was performed to confirm the biological relationship between AS and immunity.

HOXA9 inhibitors promote microcirculation of coronary arteries in rats via downregulating E-selectin/VCAM-1.

Atherosclerosis (AS) is a chronic pathophysiological process that causes high mortality and morbidity. It has previously been reported that homeobox A9 (HOXA9) may participate in regulation of the cardiovascular system and the pathology of AS by upregulating E-selectin and vascular cell adhesion molecule-1 (VCAM-1). Thus, inhibiting HOXA9 could promote microcirculation of coronary arteries and could act as a potential therapy for AS treatment. Sprague-Dawley rats were randomly divided into four groups, as follows: i) AS; ii) AS + HOXA9 inhibitor; iii) AS + small interfering RNA-HOXA9 and iv) normal control. ELISA was used to measure the levels of TNF-α, IL-1β, IL-12, C-C motif chemokine ligand 25 (CCL25), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL). Flow cytometry was employed to detect the content of M1 macrophages. Hematoxylin & eosin (H&E) staining was performed to observe the morphology of the coronary arteries. Oil red O staining was conducted for the evaluation of lipid accumulation. Immunohistochemistry was used to visualize the protein expression levels of HOXA9 in the coronary arteries. Western blotting was utilized to determine the protein expression levels of HOXA9, platelet factor-4 (PF4), E-selectin and VCAM-1. HOXA9 inhibitors were found to downregulate the levels of TNF-α, IL-1β, IL-12, CCL25, LDL and VLDL, and upregulate HDL levels in the blood of AS rats. The content of M1 macrophages was also decreased following injection of HOXA9 inhibitors in the AS group. H&E and oil red O staining analysis indicated that HOXA9 inhibitors attenuated vascular symptoms and lipid formation in AS rats. Furthermore, western blotting suggested that inhibition of HOXA9 reduced the expression levels of PF4, E-selectin and VCAM-1, while overexpression of PF4 resulted in the opposite effects. The present study revealed that inhibiting HOXA9 alleviated the symptoms of AS via downregulation of the PF4 and E-selectin/VCAM-1 pathway to promote microcirculation in the coronary arteries of AS rats. These findings indicated that HOXA9 inhibitors may have the potential to succeed in the treatment of AS.

Islet transplantation modulates macrophage to induce immune tolerance and angiogenesis of islet tissue in type I diabetes mice model.

Objective To investigate the dual mechanism of islet transplantation in T1D by regulating the immune tolerance of macrophages and inducing the neovascularization.Methods NC group, T1D model group and T1D model + islet group were constructed. Then, the abdominal aorta blood of abdominal aorta and islet tissue were collected. ELISA was performed to detect the level of IL-1Rα, IL-1α, IL-1β, CXCL2, MCP1, TNF-α and IL-10. Flow cytometry was used to measure the content of M1 and M2 macrophages. HE staining indicated the pathological characteristics of islet. IHC and WB were applied to determine the protein levels of IGF1R, FGFR2 or VEGFA as well as IGF1R, GRB2, EGFR, PTPN1, JAK2, STAT3, Caspase-1, Bcl2 respectively.Results Islet transplantation in T1D stimulated the expression of IL-1Rα, IL-1α, IL-1β, CXCL2, MCP1, TNF-α and IL-10 in abdominal aorta blood, changed the content of MHCII+CD206-M1 and MHCII+CD206+M2 macrophages, reduced the pathological features and the infiltration of immunocytes, promoted the expression of IGF1R, FGFR2 and VEGFA, eliminated cell apoptosis and induced the neovascularization in islet grafts.Conclusions Islet transplantation is an effective strategy for the treatment of T1D. It can increase the content of M2 macrophages whose immune tolerance can elevate the survival of islet grafts, reduce the inflammatory responses mediated by macrophages, promote the neovascularization and eliminate the cell apoptosis of islet grafts.

Tumor Immune Microenvironment Clusters in Localized Prostate Adenocarcinoma: Prognostic Impact of Macrophage Enriched/Plasma Cell Non-Enriched Subtypes.

Background: Prostate cancer (PCa) is characterized by significant heterogeneity in its molecular, genomic, and immunologic characteristics. Methods: Whole transcriptome RNAseq data from The Cancer Genome Atlas of prostate adenocarcinomas (n = 492) was utilized. The immune microenvironment was characterized using the CIBERSORTX tool to identify immune cell type composition. Unsupervised hierarchical clustering was performed based on immune cell type content. Analyses of progression-free survival (PFS), distant metastases, and overall survival (OS) were performed using Kaplan–Meier estimates and Cox regression multivariable analyses. Results: Four immune clusters were identified, largely defined by plasma cell, CD4+ Memory Resting T Cells (CD4 MR), and M0 and M2 macrophage content (CD4 MRHighPlasma CellHighM0LowM2Mid, CD4 MRLowPlasma CellHighM0LowM2Low, CD4 MRHighPlasma CellLowM0HighM2Low, and CD4 MRHighPlasma CellLowM0LowM2High). The two macrophage-enriched/plasma cell non-enriched clusters (3 and 4) demonstrated worse PFS (HR 2.24, 95% CI 1.46–3.45, p = 0.0002) than the clusters 1 and 2. No metastatic events occurred in the plasma cell enriched, non-macrophage-enriched clusters. Comparing clusters 3 vs. 4, in patients treated by surgery alone, cluster 3 had zero progression events (p < 0.0001). However, cluster 3 patients had worse outcomes after post-operative radiotherapy (p = 0.018). Conclusion: Distinct tumor immune clusters with a macrophage-enriched, plasma cell non-enriched phenotype and reduced plasma cell enrichment independently characterize an aggressive phenotype in localized prostate cancer that may differentially respond to treatment.

TGF-β inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity.

Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored.Methods: Here, we re-purposed the TGF-β inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors.Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4.Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

185PD - Immunological differences between immune-rich estrogen receptor-positive and -negative breast cancers

BackgroundA subset of estrogen receptor-positive (ER+) breast cancers have significant tumor infiltrating lymphocytes (TILs), similar to triple-negative breast cancer (TNBC). We examined differences in the immune microenvironment of immune-rich ER+ and immune-rich TNBC to find out if similar or different immunotherapy strategies are appropriate for these distinct disease types. MethodsER+/HER2-negative and TNBC cases were obtained from The Cancer Genome Atlas (TCGA) (n=818 RNA Seq) and METABRIC (n=1465 microarray). An immune gene expression score, as surrogate for TILs, was calculated for each case (Danaher et al). Signature scores were correlated with histologic TILs (R=0.44, p<0.001) for available cases. All cases in the top 25% of signature scores were considered immune-rich. We compared 22 immune cell populations between immune-rich TNBC (n=86) and ER+(n=119) using CIBERSORT (Student’s t-test, FDR adjusted). We examined differential expression of 770 immune-related genes and 137 immuno-oncology (IO) drug targets. Macrophage abundance was measured by quantitative immunofluorescence (QIF) using pan-macrophage marker CD68 in 169 TNBC and 87 ER+ FFPE tissues. ResultsIn TCGA and METABRIC, CIBERSORT showed more M0 (p=0.015, p=0.0043) and M1 macrophages (p=9.4e-08, p=6.24e-11) in immune-rich TNBC compared to ER+. Mast cells (p=0.0093, p=4.09e-15) and M2 macrophages (p=4.4e-05, p=0.053) were more abundant in immune-rich ER+. QIF confirmed higher macrophage content in TNBC (p=0.0001). In both datasets, 36 IO targets were higher expressed in TNBC and 15 in ER+ cases (Table). Notably, coordinated high expression of TGFb pathway members TGFb3, TGFb-R2, and LRRC32 was seen in ER+ cancers and correlated positively with M2 and negatively with M1 macrophage content across all cases.Table185PDTablePotential ER+ Immuno- Oncology TargetsTNBC Mean ExpressionER+ Mean ExpressionFold-Change Mean Expressionp-valueFDR-adjustedIL6ST2,71711,8154.355.13E-212.32E-19TGF-b32,0854,7452.281.32E-248.97E-23CXCR1 (IL-8 receptor A)4399882.251.04E-097.46E-09CSF3R1,0502,0251.938.46E-127.67E-11RORC1,0081,8551.843.65E-082.26E-07ADORA2A2,1633,4421.596.18E-083.65E-07LRRC32 (TGFb activator)7,54911,1521.481.09E-075.31E-07TLR38801,3001.481.65E-055.92E-05CXCL121,0551,5351.458.44E-052.53E-04CLEC14A4927051.432.68E-059.11E-05TGFb-R27,5599,8451.302.08E-045.90E-04TNFSF147998721.092.94E-036.89E-03MICA3493741.077.54E-031.51E-29NLRP33,0153,1451.043.12E-037.20E-03JAK112,68313,0581.030.0190.035 ConclusionsIO drugs targeting TGFb, M2 macrophages and mast cells are attractive therapeutic candidates in immune-rich ER+ breast cancer based on the expression characteristics of these targets. Legal entity responsible for the studyThe authors. FundingHoward Hughes Medical Institute Medical Fellows Grant. DisclosureV. Pelenkanou: Full / Part-time employment: Sanofi, US. D. Rimm: Advisory / Consultancy: Amgen; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy: Agendia; Advisory / Consultancy: Biocept; Advisory / Consultancy: BMS; Advisory / Consultancy: Cell Signaling Technology; Advisory / Consultancy, Research grant / Funding (institution): Cepheid; Advisory / Consultancy: Daiichi Sankyo; Advisory / Consultancy: GSK; Advisory / Consultancy: InVicro/Konica/Minolta; Advisory / Consultancy: Merck; Advisory / Consultancy, Research grant / Funding (institution): NanoString; Advisory / Consultancy, Research grant / Funding (institution): Perkin Elmer; Advisory / Consultancy: PAIGE.AI; Advisory / Consultancy, Research grant / Funding (institution): Ventana; Advisory / Consultancy, Research grant / Funding (institution): Ultivue; Shareholder / Stockholder / Stock options: PixelGear; Research grant / Funding (institution): Navigate/Novartis; Research grant / Funding (institution): NextCure; Research grant / Funding (institution): Lilly. L. Pusztai: Honoraria (self), Advisory / Consultancy: Merck; Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Seattle Genetics; Honoraria (self), Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: Almac. All other authors have declared no conflicts of interest.

IL-6 knockout ameliorates myocardial remodeling after myocardial infarction by regulating activation of M2 macrophages and fibroblast cells.

To investigate the effects of interleukin-6 (IL-6) gene knockout on myocardial remodeling after myocardial infarction (MI) in mice and the potential mechanism, to provide certain references for the prevention and treatment of MI in clinic. A total of 40 male C57 mice were divided into two groups, namely Sham group (n=20) and MI group (n=20), using a random number table. Another 20 mice with IL-6 gene knockout were enrolled into the MI + IL-6 KO group. The MI model was established by means of ligating the left anterior descending coronary artery of the mice. 28 d later, the survival status of the three groups of mice was recorded. In addition, the cardiac functions of each group of mice, including two-dimensional echocardiography, ejection fraction (EF%) and fractional shortening (FS%), were measured. The cross-sectional area and pathological change of the myocardial cells in cardiac tissues of each group of mice were detected via hematoxylin and eosin (H&E) staining. Immunohistochemistry was applied to determine the expression of tumor necrosis factor-alpha (TNF-α) in each group of mouse cardiac tissues. Moreover, immunofluorescent staining was utilized to measure the content of M2 macrophages in each group of mouse cardiac tissues. The 28-d survival rate of the mice with IL-6 gene knockout was remarkably higher than that of the wild-type mice (p<0.05). Furthermore, the cardiac functions of the mice in the MI + IL-6 KO group were superior to those in the MI group, with markedly improved FS% and EF% (p<0.05). According to the H&E staining results, the cross-sectional areas of the heart and myocardial cells were decreased notably in MI + IL-6 KO group compared with those in the MI group (p<0.05). The immunohistochemical staining results showed that IL-6 knockout could lower the MI-induced high expression of TNF-α (p<0.05), and Masson's trichrome staining indicated that IL-6 knockout could also repress the degree of cardiac fibrosis. Moreover, it was discovered through immunofluorescent staining that the mice in the MI + IL-6 KO group had markedly elevated content of M2 macrophages in cardiac tissues than those in the MI group (p<0.05). Inhibiting IL-6 gene expression can prominently ameliorate the MI-induced myocardial remodeling, whose mechanism is possibly associated with the activation of M2 macrophages and reduced collagen production in fibroblast cells.

Exosomal communication by metastatic osteosarcoma cells contributes to the modulation of alveolar macrophages to an M2 tumor-promoting phenotype

Abstract Osteosarcoma (OS) metastasizes to the lung and there is a link between tumor-promoting M2 macrophage content and poorer patient survival. Conversely, high M1 content correlates with improved survival. M2 macrophages are immunosuppressive, contribute to tumor progression and metastasis, and can be induced by various stimuli in the tumor microenvironment, including exosomes (Exo). Exo are small 40–150 nm vesicles that are involved in inter-cellular communication, and contribute to tumor progression, increased tumor survival and immune evasion. Because of the link between M2 content and inferior patient survival, we evaluated the effect of Exo from non-metastatic K7 OS cells and the metastatic subline K7M3 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophage cells with K7M3 Exo induced mRNA expression of IL10, TGFB2, IL1rα, and CCL22, (markers of M2 macrophages), and decreased phagocytosis, efferocytosis and macrophage-mediated tumor cell killing. In contrast, Exo from non-metastatic K7 or 3T3 normal fibroblast cells did not inhibit phagocytosis, efferocytosis or macrophage-mediated tumor cell killing. Our data suggest that the Exo from metastatic OS cells can modulate cellular signaling of tumor associated macrophages, thereby promoting the M2 phenotype and inhibiting their phagocytic and cytotoxic abilities. Identifying the factors within Exo from metastatic OS cells and the mechanisms responsible for Exo communication between metastatic OS cells and macrophages could reveal new therapeutic targets and opportunities to improve the treatment of patients with metastatic disease in the lung.