Proportion Of M2 Macrophages Research Articles

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages

To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

The clinical impact of macrophage polarity after Kasai portoenterostomy in biliary atresia.

Biliary atresia (BA) is a cholestatic hepatopathy caused by fibrosing destruction of intrahepatic and extrahepatic bile ducts, and its etiology has not been clearly revealed. In BA, liver fibrosis progression is often observed even after Kasai portoenterostomy (KPE), and more than half of cases require liver transplantation in their lifetime in Japan. Macrophages play an important role in liver fibrosis progression and are classically divided into proinflammatory (M1) and fibrotic macrophages (M2), whose phenotypic transformation is called "macrophage polarity." The polarity has been reported to reflect the tissue microenvironment. In this study, we examined the relationship between macrophage polarity and the post-KPE clinical course. Thirty BA patients who underwent KPE in our institution from 2000 to 2020 were recruited. Multiple immunostainings for CD68, CD163, CK19, and α-SMA were carried out on liver biopsy specimens obtained at KPE. ROC curves were calculated based on each clinical event, and the correlation with the clinical data was analyzed. The M2 ratio, defined as the proportion of M2 macrophages (CD163-positive cells), was correlated inversely with the occurrence of postoperative cholangitis (AUC: 0.7602). The patients were classified into M2 high (n = 19) and non-high (n = 11) groups based on an M2 ratio value obtained from the Youden index ( = 0.918). As a result, pathological evaluations (Metavir score, αSMA area fraction, and CK19 area fraction) were not significantly different between these groups. In mild liver fibrosis cases (Metavir score = 0-2), the M2 non-high group had a significantly lower native liver survival rate than the high group (p = 0.02). Moreover, 4 out of 8 cases in the M2 non-high group underwent early liver transplantation within 2 years after KPE. Non-M2 macrophages, including M1 macrophages, may be correlated with postoperative cholangitis, and the M2 non-high group in mild liver fibrosis cases had a significantly lower native liver survival rate than the high group, requiring early liver transplantation in this study. Preventing advanced liver fibrosis is a key factor in improving native liver survival for BA patients, and liver macrophages may play important roles in liver homeostasis and the promotion of inflammation and fibrosis.

Carnitine functions as an enhancer of NRF2 to inhibit osteoclastogenesis via regulating macrophage polarization in osteoporosis

Osteoporosis, which manifests as reduced bone mass and deteriorated bone quality, is common in the elderly population. It is characterized by persistent elevation of macrophage-associated inflammation and active osteoclast bone resorption. Currently, the roles of intracellular metabolism in regulating these processes remain unclear. In this study, we initially performed bioinformatics analysis and observed a significant increase in the proportion of M1 macrophages in bone marrow with aging. Further metabolomics analysis demonstrated a notable reduction in the expression of carnitine metabolites in aged macrophages, while carnitine was not detected in osteoclasts. During the differentiation process, osteoclasts took up carnitine synthesized by macrophages to regulate their own activity. Mechanistically, carnitine enhanced the function of Nrf2 by inhibiting the Keap1-Nrf2 interaction, reducing the proteasome-dependent ubiquitination and degradation of Nrf2. In silico molecular ligand docking analysis of the interaction between carnitine and Keap1 showed that carnitine binds to Keap1 to stabilize Nrf2 and enhance its function. In this study, we found that the decrease in carnitine levels in aging macrophages causes overactivation of osteoclasts, ultimately leading to osteoporosis. A decrease in serum carnitine levels in patients with osteoporosis was found to have good diagnostic and predictive value. Moreover, supplementation with carnitine was shown to be effective in the treatment of osteoporosis.

Upregulation of TCPTP in Macrophages Is Involved in IL-35 Mediated Attenuation of Experimental Colitis.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease with complex etiology. Interleukin-35 (IL-35), as a cytokine with immunomodulatory function, has been shown to have therapeutic effects on UC, but its mechanism is not yet clear. Therefore, we constructed Pichia pastoris stably expressing IL-35 which enables the cytokines to reach the diseased mucosa, and explored whether upregulation of T-cell protein tyrosine phosphatase (TCPTP) in macrophages is involved in the mechanisms of IL-35-mediated attenuation of UC. After the successful construction of engineered bacteria expressing IL-35, a colitis model was successfully induced by giving BALB/c mice a solution containing 3% dextran sulfate sodium (DSS). Mice were treated with Pichia/IL-35, empty plasmid-transformed Pichia (Pichia/0), or PBS by gavage, respectively. The expression of TCPTP in macrophages (RAW264.7, BMDMs) and intestinal tissues after IL-35 treatment was detected. After administration of Pichia/IL-35, the mice showed significant improvement in weight loss, bloody stools, and shortened colon. Colon pathology also showed that the inflammatory condition of mice in the Pichia/IL-35 treatment group was alleviated. Notably, Pichia/IL-35 treatment not only increases local M2 macrophages but also decreases the expression of inflammatory cytokine IL-6 in the colon. With Pichia/IL-35 treatment, the proportion of M1 macrophages, Th17, and Th1 cells in mouse MLNs were markedly decreased, while Tregs were significantly increased. In vitro experiments, IL-35 significantly promoted the expression of TCPTP in macrophages stimulated with LPS. Similarly, the mice in the Pichia/IL-35 group also expressed more TCPTP than that of the untreated group and the Pichia/0 group.

ASB1 inhibits prostate cancer progression by destabilizing CHCHD3 via K48-linked ubiquitination.

Prostate cancer is a major contributor to male mortality worldwide. In this study, we revealed that Ankyrin Repeat and SOCS Box Containing 1 (ASB1) expression was significantly decreased in prostate cancer tissues, correlating strongly with poor patient prognosis. Notably, the group with low ASB1 expression exhibited an increased proportion of M2 macrophages and showed resistance to immune checkpoint inhibitors and cisplatin, but remained sensitive to androgen-receptor-targeting drug bicalutamide. Silencing ASB1 enhanced prostate cancer cell proliferation, clonogenicity, and migration, whereas its overexpression exerted the opposite effects. Through quantitative mass spectrometry interactome analysis, we identified 37 novel proteins interacting with ASB1, including CHCHD3. Subsequent experiments including co-immunoprecipitation, cycloheximide treatment, and ubiquitination assays, revealed that ASB1 interacts with CHCHD3, promoting its degradation via K48-linked ubiquitination. Cell rescue experiments further demonstrated that ASB1 inhibits prostate cancer cell through the CHCHD3/reactive oxygen species (ROS) pathway. Taken together, our study indicated that ASB1 functions as a tumor suppressor by inhibiting CHCHD3/ROS signaling, thereby playing a vital part in prevention of prostate cancer proliferation, clonogenicity, and migration.

Role of tenascin C in lesion formation in early peritoneal endometriosis

To identify cytokines or extracellular matrix components that contribute to adhesion to, and invasion of, the peritoneum, proximal to lesions in the early phase of endometriosis. Laboratory-based study. University Hospital and Laboratory of Animal Science. Five women with ovarian endometrioma, 138 wild-type (WT) C57BL/6N mice, and 48 Tenascin C (Tnc) knockout (TncKO) mice. To establish a murine endometriosis model, 20 pieces of minced uterine tissue fragments from each horn were administered intraperitoneally to syngeneic mice. Three days later, endometriotic lesions and peritoneal tissues were collected. Separately, we transfected human peritoneal mesothelial cells (HMrSV5) or human endometrial stromal cells (hESCs) with Tnc small interfering ribonucleic acid. We employed a polymerase chain reaction array to profile gene expression in the murine peritoneum, in both peritoneum distal to lesions and peritoneum surrounding lesions (PSL). The expression of upregulated genes in the PSL was verified in the peritoneal samples by real-time reverse transcription-polymerase chain reaction. TncKO mice were used to investigate the role of Tnc in the development of endometriosis. We evaluated the proliferative activity or inflammatory state of lesions by Ki67 or CD3 immunostaining. Intraperitoneal distribution of macrophages was assessed by fluorescence-activated cell sorting. Using Tnc small interfering ribonucleic acid, we examined the invasive capacity of hESCs in a coculture system with HMrSV5. Tnc gene expression was significantly higher in PSL than in peritoneum distal to lesions. The weight and number of TncKO lesions in TncKO hosts were lower than those of WT lesions in WT hosts. In contrast, the weight and number of nonattached TncKO lesions in TncKO hosts were higher than those of nonattached WT lesions in WT hosts. We observed decreased Ki67-positive cells or H-scores for CD3, a lower proportion of M1 macrophages, and a higher proportion of M2 macrophages in TncKO lesions in TncKO recipients. Silencing of Tnc expression in hESCs and HMrSV5 diminished the invasivity of hESCs. Tnc may be a crucial factor in the development of early peritoneal endometriosis.

Substance P Increases STAT6-Mediated Transcription Activation of Lymphocyte Cytosolic Protein 2 to Sustain M2 Macrophage Predominance in Pediatric Asthma

Substance P (SP) is a neuropeptide released by neurons and participates in various biological processes, including inflammation. M2 macrophages are major immune cells associated with type 2 inflammation in asthma. This study investigated the effect of SP on macrophage phenotype in pediatric asthma and the underpinning factors. Asthmatic children exhibited an increased level of SP, along with a higher proportion of M2 macrophages in their bronchoalveolar lavage fluid. Flow cytometry revealed that SP treatment enhanced the M2 polarization of 12-O-tetradecanoylphorbol 13-acetate-treated THP-1 cells (macrophages) invitro. By contrast, the administration of a neutralizing antibody of SP reduced the M2 macrophage population, mitigated inflammatory cell infiltration in mouse lung tissues, and decreased the population of immune cells in the mouse bronchoalveolar lavage fluid. SP up-regulated the expression of STAT6, which, in turn, activated the transcription of lymphocyte cytosolic protein 2 (LCP2). The population of macrophages and allergic inflammatory responses in mice were reduced by STAT6 inhibition but restored by LCP2 overexpression. Collectively, the present study demonstrated that SP sustains M2 macrophage predominance and allergic inflammation in pediatric asthma by enhancing STAT6-dependent transcription activation of LCP2.

Qijia rougan formula ameliorates ECM deposition in hepatic fibrosis by regulating the JAK1/STAT6-microRNA-23a feedback loop in macrophage M2 polarization

Hepatic fibrosis is the critical pathological stage in the progression of chronic liver disease to cirrhosis and hepatocellular carcinoma (HCC). However, no approved anti-hepatic fibrosis drugs are available currently. Qijia Rougan Formula (QRF) is a traditional Chinese medicine (TCM) with significant clinical efficacy on hepatic fibrosis. It was derived from Sanjiasan, a famous decoction documented in the Book of Treatise on the Pestilence in the Ming Dynasty of China. However, the underlying regulatory mechanisms remain elusive. This study further confirmed the therapeutic effects of QRF on hepatic fibrosis and dissected its underlying molecular mechanisms from the perspective of macrophage M2 polarization, one of the critical events in hepatic fibrosis. Experimentally, QRF significantly improved extracellular matrix (ECM) deposition and fibrosis in the liver of model rats. QRF diminished the proportion of M2 macrophages, decreased the levels of TGF-β, PDGFB and IL-10, and regulated the expression of p-JAK1, p-STAT6, JAK1 and microRNA-23a both in vitro and in vivo. Collectively, it was confirmed that QRF effectively improves liver function and hepatocyte damage, and reduces ECM deposition. QRF ameliorates hepatic fibrosis by regulating JAK1/STAT6-microRNA-23a negative feedback loop to inhibit macrophage M2 polarization and thus reduce ECM deposition. Our study illustrates the potential of QRF for hepatic fibrosis therapy, suggesting that QRF is a promising anti-hepatic fibrosis drug candidate.

Machine learning-based metabolism-related genes signature, single-cell RNA sequencing, and experimental validation in hypersensitivity pneumonitis.

Metabolism is involved in the pathogenesis of hypersensitivity pneumonitis. To identify diagnostic feature biomarkers based on metabolism-related genes (MRGs) and determine the correlation between MRGs and M2 macrophages in patients with hypersensitivity pneumonitis (HP). We retrieved the gene expression matrix from the Gene Expression Omnibus database. The differentially expressed MRGs (DE-MRGs) between healthy control (HC) and patients with HP were identified using the "DESeq2" R package. The "clusterProfiler" R package was used to perform "Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses" on DE-MRGs. We used machine learning algorithms for screening diagnostic feature biomarkers for HP. The "receiver operating characteristic curve" was used to evaluate diagnostic feature biomarkers' discriminating ability. Next, we used the "Cell-type Identification by Estimating Relative Subsets of RNA Transcripts" algorithm to determine the infiltration status of 22 types of immune cells in the HC and HP groups. Single-cell sequencing and qRT-PCR were used to validate the diagnostic feature biomarkers. Furthermore, the status of macrophage polarization in the peripheral blood of patients with HP was determined using flow cytometry. Finally, the correlation between the proportion of M2 macrophages in peripheral blood and the diagnostic biomarker expression profile in HP patients was determined using Spearman analysis. We identified a total of 311 DE-MRGs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that DE-MRGs were primarily enriched in processes like steroid hormone biosynthesis, drug metabolism, retinol metabolism, etc. Finally, we identified NPR3, GPX3, and SULF1 as diagnostic feature biomarkers for HP using machine learning algorithms. The bioinformatic results were validated using the experimental results. The CIERSORT algorithm and flow cytometry showed a significant difference in the proportion of M2 macrophages in the HC and HP groups. The expression of SULF1 was positively correlated with the proportion of M2-type macrophages. In addition, a positive correlation was observed between SULF1 expression and M2 macrophage proportion. Finally, we identified NPR3, GPX3, and SULF1 as diagnostic feature biomarkers for HP. Further, a correlation between SULF1 and M2 macrophages was observed, providing a novel perspective for treating patients with HP and future studies.

Radiotherapy Combined with PD-L1 Antibody Exerts a Synergistic Anti-tumor Effect in a Mouse Model of Esophageal Squamous Cell Carcinoma

The combination of radiotherapy and immune checkpoint inhibitors (ICIs) has been shown to exert synergistic anti-tumor effects in various tumors. In esophageal squamous cell carcinoma (ESCC), several clinical trials of radiotherapy combined with ICIs are undergoing or have been finished. However, the efficacy and action mechanisms of radiotherapy combined with ICIs is still not clear in ESCC. Our study aimed to investigate whether radiotherapy improved the anti-cancer effect of PD-L1 antibody and the effect of this combination therapy on tumor-immune microenvironment. The mouse esophageal cancer cell line mEC25 was implanted into the armpits of female C57 mice. When the tumor volume grew to 150-250 mm3, the mice were randomly divided into different groups including control group (administration of IgG antibody), radiotherapy group (12 Gy at 4 Gy per fraction), PD-L1 antibody group (200 mg/kg i.p. for three times) and combination group (fractionated radiation combined with PD-L1 antibody was delivered on day 1, day 4 and day 7, the day when fractionated radiation began was recorded as day 1).The mice were sacrificed on the 1st, 3rd and 7th day after the treatment was ended. The tumor, spleen and draining lymph nodes of the mice were collected and analyzed using flow cytometry (FCM), and the tumor volume and survival time of mice were calculated. Compared with radiotherapy or PD-L1 antibody alone, the combination therapy significantly prolonged the survival time of mice and decreased the growth rate of xenograft tumors FCM analysis showed that the combination therapy significantly increased the infiltration and cytotoxicity of effector T cells and reduced the proportion of M2 macrophages in tumor, spleen and lymph nodes. In addition, in tumor and spleen, the proportion of regulatory T cells (Tregs) decreased in the group of combination therapy. In tumor, spleen, and lymph nodes, the proportion of central memory T cell (TCM) was increased in the group of combination therapy. The anti-tumor effect of radiotherapy combined with PD-L1 antibody is superior to single treatment in the mouse model of esophageal cancer. Radiotherapy can shape tumor-immune microenvironment, allowing the infiltration of effector T cells and exclusion of immune-suppressive cells such as M2 macrophages and Tregs.

PLEKHA4 is a novel prognostic biomarker that reshapes the tumor microenvironment in lower-grade glioma.

Lower-grade glioma (LGG) is a primary intracranial tumor that carry a high risk of malignant transformation and limited therapeutic options. Emerging evidence indicates that the tumor microenvironment (TME) is a superior predictor for tumor progression and therapy response. PLEKHA4 has been demonstrated to be a biomarker for LGG that correlate with immune infiltration. However, the fundamental mechanism by which PLEKHA4 contributes to LGG is still poorly understood. Multiple bioinformatic tools, including Tumor Immune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA2), Shiny Methylation Analysis Resource Tool (SMART), etc., were incorporated to analyze the PLEKHA4. ESTIMATE, ssGSEA, CIBERSORT, TIDE and CellMiner algorithms were employed to determine the association of PLEKHA4 with TME, immunotherapy response and drug sensitivities. Immunohistochemistry (IHC)-based tissue microarrays and M2 macrophage infiltration assay were conducted to verify their associations. PLEKHA4 expression was found to be dramatically upregulated and strongly associated with unfavorable overall survival (OS) and disease-specific survival (DSS) in LGG patients, as well as their poor clinicopathological characteristics. Cox regression analysis identified that PLEKHA4 was an independent prognostic factor. Methylation analysis revealed that DNA methylation correlates with PLEKHA4 expression and indicates a better outcome in LGG. Moreover, PLEKHA4 was remarkably correlated with immune responses and TME remodeling, as evidenced by its positive correlation with particular immune marker subsets and the putative infiltration of immune cells. Surprisingly, the proportion of M2 macrophages in TME was strikingly higher than others, inferring that PLEKHA4 may regulate the infiltration and polarization of M2 macrophages. Evidence provided by IHC-based tissue microarrays and M2 macrophage infiltration assay further validated our findings. Moreover, PLEKHA4 expression was found to be significantly correlated with chemokines, interleukins, and their receptors, further supporting the critical role of PLEKHA4 in reshaping the TME. Additionally, we found that PLEKHA4 expression was closely associated with drug sensitivities and immunotherapy responses, indicating that PLEKHA4 expression also had potential clinical significance in guiding immunotherapy and chemotherapy in LGG. PLEKHA4 plays a pivotal role in reshaping the TME of LGG patients, and may serve as a potential predictor for LGG prognosis and therapy.

Overexpression of CD2/CD27 could inhibit the activation of nitrogen metabolism pathways and suppress M2 polarization of macrophages, thereby preventing brain metastasis of breast cancer

ObjectiveOur study aimed to reveal the possible molecular mechanisms of CD2 and CD27 in influencing the tumor microenvironment of breast cancer (BC) brain metastasis based on the TCGA (The Cancer Genome Atlas) and SRA (Sequence Read Archive) databases. MethodsWe calculated the proportions of tumor-infiltrating immune cells and the immune and stromal cell scores in 1222 BC samples from the TCGA-BRCA dataset, followed by identification of candidate DEGs. We further screened for BC brain metastasis-related DEGs in the BC brain metastasis dataset SUB12911144 from the SRA database. Finally, we established a mouse breast cancer brain metastasis model for in vivo validation. ResultsWe further screened two immune-regulatory DEGs (CD2 and CD27). GSEA analysis showed that the downregulation of CD2 and CD27 expression was closely related to the activation of nitrogen metabolism pathways. CIBERSORT algorithm analysis showed a correlation between the expression of 16 types of tumor-infiltrating immune cells and CD2 and 19 types of tumor-infiltrating immune cells and CD27. In addition, CD2 and CD27 expression were negatively associated with the proportion of M2 macrophages. In vivo experimental results demonstrated that overexpression of CD2/CD27 could suppress the M2 polarization of macrophages and inhibit breast cancer brain metastasis. ConclusionIn the tumor microenvironment, overexpression of CD2/CD27 inhibited the activation of nitrogen metabolism pathways and suppressed M2 polarization of macrophages, thereby preventing brain metastasis of breast cancer.

Spatial proteomics enables identification of prognostic biomarkers in papillary renal cell carcinoma

Abstract Background Papillary renal cell carcinoma (PRCC) is the second most common adult kidney cancer histology, constituting 15-20% of cases. While some patients may have indolent PRCC tumors that grow slowly, other tumors rapidly metastasize. Some PRCC tumors respond to immune checkpoint inhibitors. Thus, understanding the immune tumor microenvironment and how it correlates with patient outcomes in this relatively rare disease is a critical need. Spatial interrogation of patient samples has the potential to offer novel insights into the tumor-immune axis and provide avenues for enhanced diagnosis and treatment. Addressing questions of spatial arrangement within tumors has remained a technical and biological challenge, but emerging spatial biology technologies provide molecular data at single cell resolution. We hypothesized that the spatial interaction of immune, stromal and tumor cells would be prognostic for PRCC patients. Methods A tissue microarray was assembled from an archive of ~100 patients presenting with PRCC. This dataset was assayed with PhenoCycler/CODEX (Akoya Biosciences) using a 31-antibody panel with immune- and cancer-related proteins. We have developed methodology and novel algorithms to perform signal normalization, cell segmentation, and cell typing. We computed neighborhoods for each of 2.5 million cells and performed network analysis to identify spatial clusters. This method allowed us to identify clusters consistently present across multiple TMA spots from the same patient and across multiple patients. Results Using spatial neighborhood analysis, we have identified diverse spatial clusters of potential clinical relevance, including five distinct M2 macrophage spatial clusters. We have described one of the clusters as being physically associated with helper T cells, which is visualized on a PRCC spot and shows co-occurence of M2-macrophages (CD163) and helper T cells (CD4). This pattern was replicated in additional TMA spots from the same patient. In contrast, other clusters of M2-macrophages (M2-M2 spatial cluster and M2-M1 macrophages) are visualized to show vastly different cellular neighborhoods. Clinical associations show that the patients with presence of the M2-T helper cluster have a poor cancer-associated survival (p=0.005). In comparison, the total proportion of M2 macrophages is not associated with survival (p=0.4), highlighting the importance of characterizing spatial interactions beyond cell type quantitation. Conclusions In summary, we highlight the utility of spatial biology to explain the heterogeneity in patients’ tumors and to uncover novel correlates of clinical phenotypes, establishing a platform for future discovery in this field and the identification of additional spatial correlates of patient outcome and clinical response. CDMRP DOD Funding: yes

Identification of genes related to glucose metabolism and analysis of the immune characteristics in Alzheimer's disease

ObjectiveGlucose metabolism plays a crucial role in the progression of Alzheimer's disease (AD). The purpose of this study is to identify genes related to glucose metabolism in AD by bioinformatics, construct an early AD prediction model from the perspective of glucose metabolism, and analyze the characteristics of immune cell infiltration. MethodsAD-related modules and genes were screened by weighted gene co-expression network analysis (WGCNA). The GO and KEEG enrichment analysis were used to explore the potential biological functions of glucose metabolism related genes (GMRGs) in AD. The Least Absolute Shrinkage Selection Operator (LASSO) method was used to construct an early AD prediction model based on GMRGs. Then, the receiver operating characteristic curve (ROC) and nomogram were introduced to evaluate the effectiveness of this model. Finally, CIBERSORT and single-cell analysis were applied for illustrating the immune characteristics in AD patients. ResultsA total of 462 differential expressed genes (DEGs) were obtained between Non-Alzheimer's disease (ND,) and AD groups. The genes in the blue module had the highest correlation with AD by WGCNA analysis. We found 18 intersected genes among DEGs, blue model genes and GMRGs according to the Venn diagram. The GO and KEEG enrichment analysis showed that these 18 genes were mainly involved in the production of metabolites and energy, glycolysis, amino acid biosynthesis and so on. The early AD prediction model including ENO2, TPI1, AEBP1, HERC1, PCSK1, PREPL, SLC25A4, UQCRC2, CHST6, DDIT4, ACSS1 and SUCLA2 was constructed by LASSO analysis. The area under the curve (AUC) of this model in brain tissues was 0.942. Then, we draw the nomogram of this model and the C-index was 0.942. The model was further validated in blood samples and the AUC was 0.644. Immune cell infiltration analysis showed that the proportion of plasma cells, T cells follicular helper and activated NK cells in AD group were significantly lower than ND group, while the proportion of M1 macrophages, neutrophils, T cells CD4 naive and γ-δ T cells was significantly increased when compared with the ND group. Additionally, the specific GMRGs such as ENO2, DDIT4, and SUCLA2 are significantly correlated with certain immune cells such as plasma cells, follicular helper T cells, and M1 macrophages. Single-cell analysis results suggested that the increased macrophages in AD was associated with the up-regulation of AEBP1, DDIT4 and ACSS1. ConclusionsThe diagnosis model based on the twelve GMRGs has strong predictive ability and can be used as early diagnosis biomarkers for AD. In addition, these GMRGs closely associate with AD development by influencing the glucose metabolism of immune cells.

Nocardia rubra cell wall skeleton regulates tumour-associated macrophage polarization by reprogramming M2 macrophages into M1 macrophages via STAT1/STAT6 pathways.

Targeted therapy with tumour-associated macrophages (TAMs) has emerged as a new paradigm for immunotherapy of cervical cancer. Nocardia rubra cell wall skeleton (Nr-CWS) for external use is an immunotherapeutic agent. In this study, we aimed to explore the effects of Nr-CWS on TAMs and the potential mechanisms. Cervical tissue samples were collected before and after Nr-CWS treatment from patients with high-risk HPV infection and cervical intraepithelial neoplasia (CIN). The effect of Nr-CWS on macrophages in vivo was examined by immunohistochemistry and double-labeling immunofluorescence histochemistry. In vitro experiments were performed using a TAM model established by THP-1 cells under Nr-CWS treatment. We found that Nr-CWS treatment significantly reduced the numbers of total macrophages and M2 macrophages, increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages in cervical tissues. After Nr-CWS treatment in vitro, the expression levels of the M1 macrophage markers were increased, while the expression levels of the M2 macrophage markers were decreased. Nr-CWS treatment also activated STAT1 pathways but inhibited STAT6 pathways. These results indicated that Nr-CWS may improve local immune response and reverse immunosuppression by regulating the M2 to M1 polarization of TAMs via STAT1/STAT6 pathways.

Periostin-An inducer of pro-fibrotic phenotype in monocytes and monocyte-derived macrophages in systemic sclerosis.

Enhanced circulating blood periostin levels positively correlate with disease severity in patients with systemic sclerosis (SSc). Monocytes/macrophages are predominantly associated with the pathogenesis of SSc, but the effect of periostin on immune cells, particularly monocytes and macrophages, still remains to be elucidated. We examined the effect of periostin on monocytes and monocyte-derived macrophages (MDM) in the pathogenesis of SSc. The modified Rodnan total skin thickness score in patients with dcSSc was positively correlated with the proportion of CD80-CD206+ M2 cells. The proportion of M2 macrophages was significantly reduced in rPn-stimulated MDMs of HCs compared to that of SSc patients. The mRNA expression of pro-fibrotic cytokines, chemokines, and ECM proteins was significantly upregulated in rPn-stimulated monocytes and MDMs as compared to that of control monocytes and MDMs. A similar trend was observed for protein expression in the respective MDMs. In addition, the ratio of migrated cells was significantly higher in rPn-stimulated as compared to control monocytes. These results suggest that periostin promotes inflammation and fibrosis in the pathogenesis of SSc by possible modulation of monocytes/macrophages.

Expression of spleen macrophages in a mouse model of alveolar bone resorption periodontitis.

It has been shown that macrophages can be endotoxin-tolerant under the stimulation of continuous endotoxin of Porphyromonas gingivalis. Macrophage transforms into M2-type which inhibits inflammation, and its pro-inflammatory cytokine secretion is reduced to avoid the tissue damaged by inflammation. This experiment established the corresponding animal model to explore the relative number, phenotypic proportion, and function of spleen macrophages in mice with chronic periodontitis. Twenty 16-week-old mice were randomly divided into a true ligation group (LFP group) and a pseudo-ligation group (LFC group). The periodontitis in the LFP group was induced by experimental ligation, and the LFC group was treated as a control. After 10 days of ligation, the maxilla was taken, IHC and HE staining were performed to observe the pathological changes of periodontal tissues, and IHC staining was performed to observe the RANKL/OPG ratio. Spleen mononuclear cells were isolated and counted. The ratio of M1 and M2 phenotypes was determined by fluorescence-activated cell sorting (FACS) in the spleen. The relative expression levels of macrophage-associated inflammatory cytokine TNF-a, IL-1β and anti-inflammatory cytokine IL-10 mRNA were detected by real-time PCR. Compared with the control group (LFC:M2/M110.04%), the M2 ratio among spleen mature macrophages in the periodontitis group (LFP: M2/M135.86%) was significantly increased (P<0.01) in the spleen. The proportion of M1 macrophages was not significantly different, and the ratio of M1/M2 was significantly decreased (P<0.05) in the spleen. But the expression level of M1-type macrophage inflammatory factor TNF-a mRNA was inclined. Chronic periodontitis can up-regulate the proportion of M2 macrophages, decrease the ratio of macrophage phenotype M1/M2, and incline the expression of pro-inflammatory factor TNF-a mRNA.

Personal immune profiles: Diversity and prognostic value for oral tongue squamous cell carcinoma evaluated by comprehensive immune parameter analyses with multiplex immunofluorescence

ObjectivesUnderstanding the tumor immune microenvironment is becoming increasingly necessary for risk prediction and treatment selection. In particular, oral cancer has various immunosuppressive characteristics in the tumor microenvironment. Therefore, we comprehensively assessed the immune profiles of oral tongue squamous cell carcinoma (OTSCC). Materials and methodsMultiplex immunofluorescence and tissue imaging analyses were performed to evaluate immune profiles at the invasive tumor front of 60 OTSCC surgical specimens. We analyzed 58 immune parameters including the density and proportion (%) of total leukocytes (Leu) and T cells, six subsets of T and myeloid cells, and the expression of programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1). ResultsThe density, proportion, and location of CD45+ Leu, three T cell subsets (CD8+, Foxp3–CD4+ conventional, and Foxp3+CD4+ regulatory T cells), CD163-CD68+ M1 and CD163+CD68+ M2 macrophages, and neutrophils were highly variable at the individual level. The density and proportion of M2 macrophages were significantly lower in the T1 stage group. Risk prediction analyses for recurrence and/or metastasis (R/M) showed that R/M (+) T1 cases had significantly higher M2 density and percentages. ConclusionsThe immune profiles of OTSCC patients are diverse and cannot be predicted from clinicopathological information alone. The M2 macrophage abundance is a potential candidate biomarker for R/M in the early stage of OTSCC. Personal immune profiling may provide beneficial information for risk prediction and treatment selection.

Tumor-associated M2 macrophages in the immune microenvironment influence the progression of renal clear cell carcinoma by regulating M2 macrophage-associated genes.

Renal clear cell carcinoma (RCC) has negative prognosis and high mortality due to its early diagnosis difficulty and early metastasis. Although previous studies have confirmed the negative progression of RCC is closely related to M2 macrophages in tumor-associated macrophages (TAMs), the specific mechanism is still unknown. We used immunofluorescence labeling and flow cytometry to detect the proportion of M2 macrophages in RCC tissues. And bioinformatics technique was used to obtain 9 M2 macrophage-related model genes, including SLC40A1, VSIG4, FUCA1, LIPA, BCAT1, CRYBB1, F13A, TMEM144, COLEC12. Using these genes, model formulas are constructed to devide samples into high and low risk groups, and then the overall survival (OS), progression-free survival (PFS) and Gene set enrichment analysis (GSEA) of the high and low risk groups were analyzed. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of model genes between normal kidney tissue and RCC tissue, as well as between HK-2 cell and 786-O cell. Besides, we induced the M2 differentiation of THP-1 cell, and then co-cultured with the RCC cell 786-O in transwell to observe what effect M2 macrophages will cause on the invasion, migration and the expression of model genes of RCC. Our study demonstrated M2 macrophages in RCC was about 2 folds that of normal renal tissue (P<0.0001) and M2 macrophages affected the prognosis of patients with RCC by affecting the co-expressed genes, which were mainly enriched in immune-related pathways. The results of in vitro experiments showed that in RCC tissues and 786-O cells, the model gene FUCA1 was down-regulated, and SLC40A1, VSIG4, CRYBB1 and LIPA were up-regulated. Besides, the results of co-culture showed that after 786-O co-culture with M2 macrophages, the ability of migration and invasion was promoted and the expressions of FUCA1, SLC40A1, VSIG4, CRYBB1, LIPA and TMEM144 were all up-regulated. The proportion of tumor-associated M2 macrophages in RCC tissues is upregulated, and M2 macrophages promote the progression of RCC by regulating the expression of SLC40A1, VSIG4, FUCA1, LIPA, BCAT1, CRYBB1, F13A, TMEM144, COLEC12 genes, thereby affecting the prognosis of patients with RCC.

Human mesenchymal stem-derived extracellular vesicles improve body growth and motor function following severe spinal cord injury in rat.

Spinal cord injury (SCI) in young adults leads to severe sensorimotor disabilities as well as slowing of growth. Systemic pro-inflammatory cytokines are associated with growth failure and muscle wasting. Here we investigated whether intravenous (IV) delivery of small extracellular vesicles (sEVs) derived from human mesenchymal stem/stromal cells (MSC) has therapeutic effects on body growth and motor recovery and can modulate inflammatory cytokines following severe SCI in young adult rats. Contusional SCI rats were randomized into three different treatment groups (human and rat MSC-sEVs and a PBS group) on day 7 post-SCI. Functional motor recovery and body growth were assessed weekly until day 70 post-SCI. Trafficking of sEVs after IV infusions in vivo, the uptake of sEVs in vitro, macrophage phenotype at the lesion and cytokine levels at the lesion, liver and systemic circulation were also evaluated. An IV delivery of both human and rat MSC-sEVs improved functional motor recovery after SCI and restored normal body growth in young adult SCI rats, indicating a broad therapeutic benefit of MSC-sEVs and a lack of species specificity for these effects. Human MSC-sEVs were selectively taken up by M2 macrophages in vivo and in vitro, consistent with our previous observations of rat MSC-sEV uptake. Furthermore, the infusion of human or rat MSC-sEVs resulted in an increase in the proportion of M2 macrophages and a decrease in the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-6 at the injury site, as well as a reduction in systemic serum levels of TNF-α and IL-6 and an increase in growth hormone receptors and IGF-1 levels in the liver. Both human and rat MSC-sEVs promote the recovery of body growth and motor function after SCI in young adult rats possibly via the cytokine modulation of growth-related hormonal pathways. Thus, MSC-sEVs affect both metabolic and neurological deficits in SCI.