[A risk scoring model based on M2 macrophage-related genes for predicting prognosis of HBV-related hepatocellular carcinoma].

OBJECTIVE: To investigate the prognostic value of M2 macrophage-related genes (MRG) in hepatitis B virus (HBV)- related hepatocellular carcinoma (HCC). METHODS: The transcriptome data of 73 patients with HBV-related HCC were obtained from TCGA database, and the MRG modules were identified by WGCNA. The MRG-based risk scoring model was constructed by LASSO regression analysis and validated using an external dataset. The correlation of the risk score with immune cell infiltration and drug sensitivity of HCC were analyzed with CIBERSORT and R. pRRophetic. The signaling pathways of the differential genes between the high- and low-risk groups were investigated using GSVA and GSEA enrichment analyses, and MRG expressions at the single cell level were validated using R.Seurat. The cell interaction intensity was analyzed by R.Cellchat to identify important cell types related to HCC progression. MRG expression levels were detected by RT-qPCR in THP-1 cells with HCC-conditioned medium-induced M2 polarization and in HBV-positive HCC cells. RESULTS: A high M2 macrophage infiltration level was significantly correlated with a poor prognosis of HCC, and 5 hub MRG (VTN, GCLC, PARVB, TRIM27, and GMPR) were identified. The overall survival of HCC patients was significantly lower in the high-risk than in the low-risk group. The high- and the low-risk groups showed significant enrichment of M2 macrophages and na?ve B cells, respectively, and were sensitive to BI. 2536 and to AG. 014699, AKT. inhibitor. Ⅷ, AZD. 0530, AZD7762, and BMS. 708163, respectively. The proliferation-related and metabolism-related pathways were enriched in the high-risk group, where monocytes showed the most active cell interactions during HCC progression. VTN was significantly upregulated in HCC cell lines, while GCLC, PARVB, TRIM27, and GMPR were upregulated in M2 THP-1 cells. CONCLUSION: The MRG-based risk scoring model can accurately predict the prognosis of HBV-related HCC and reveal the differences in tumor microenvironment to guide precision treatment of the patients.

GJA4 expressed on cancer associated fibroblasts (CAFs)-A 'promoter' of the mesenchymal phenotype.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide. Connexin is a transmembrane protein involved in gap junctions (GJs) formation. Our previous study found that connexin 37 (Cx37), encoded by gap junction protein alpha 4 (GJA4), expressed on fibroblasts acts as a promoter of CRC and is closely related to epithelial-mesenchymal transition (EMT) and tumor immune microenvironment. However, to date, the mechanism concerning the malignancy of GJA4 in tumor stroma has not been studied. METHODS: Hematoxylin-eosin (HE) and immunohistochemical (IHC) staining were used to validate the expression and localization of GJA4. Using single-cell analysis, enrichment analysis, spatial transcriptomics, immunofluorescence staining (IF), Sirius red staining, wound healing and transwell assays, western blotting (WB), Cell Counting Kit-8 (CCK8) assay and in vivo experiments, we investigated the possible mechanisms of GJA4 in promoting CRC. RESULTS: We discovered that in CRC, GJA4 on fibroblasts is involved in promoting fibroblast activation and promoting EMT through a fibroblast-dependent pathway. Furthermore, GJA4 may act synergistically with M2 macrophages to limit T cell infiltration by stimulating the formation of an immune-excluded desmoplasic barrier. Finally, we found a significantly correlation between GJA4 and pathological staging (P < 0.0001) or D2 dimer (R = 0.03, P < 0.05). CONCLUSION: We have identified GJA4 expressed on fibroblasts is actually a promoter of the tumor mesenchymal phenotype. Our findings suggest that the interaction between GJA4+ fibroblasts and M2 macrophages may be an effective target for enhancing tumor immunotherapy.

Genistein Promotes M2 Macrophage Polarization via Aryl Hydrocarbon Receptor and Alleviates Intestinal Inflammation in Broilers with Necrotic Enteritis.

The aryl hydrocarbon receptor (AhR) is a transcription factor that regulates the immune system through complicated transcriptional programs. Genistein, an AhR ligand, exhibits anti-inflammatory properties. However, its role in modulating immune responses via the AhR signaling pathway remains unclear. In this study, 360 male Arbor Acre broilers (1-day-old) were fed a basal diet supplemented with 40 or 80 mg/kg genistein and infected with or without Clostridium perfringens (Cp). Our results demonstrated that genistein ameliorated Cp-induced intestinal damage, as reflected by the reduced intestinal lesion scores and improved intestinal morphology and feed-to-gain ratio. Moreover, genistein increased intestinal sIgA, TGF-ß, and IL-10, along with elevated serum IgG, IgA, and lysozyme levels. Genistein improved intestinal AhR and cytochrome P450 family 1 subfamily A member 1 (CYP1A1) protein levels and AhR+ cell numbers in Cp-challenged broilers. The increased number of AhR+CD163+ cells in the jejunum suggested a potential association between genistein-induced AhR activation and anti-inflammatory effects mediated through M2 macrophage polarization. In IL-4-treated RAW264.7 cells, genistein increased the levels of AhR, CYP1A1, CD163, and arginase (Arg)-1 proteins, as well as IL-10 mRNA levels. This increase was attenuated by the AhR antagonist CH223191. In summary, genistein activated the AhR signaling pathway in M2 macrophages, which enhanced the secretion of anti-inflammatory cytokines and attenuated intestinal damage in Cp-infected broilers Cp.

Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications.

BACKGROUND: Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ. AIM OF THE STUDY: To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. METHODS: Differentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. RESULTS: Mreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased. CONCLUSION: Exposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

M2 macrophage-derived exosomes promote angiogenesis and improve cardiac function after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. The intercellular communication in post-infarction angiogenesis remains unclear. METHODS: In this study, we explored the role and mechanism of action of M2 macrophage-derived exosomes (M2-exos) in angiogenesis after MI. M2-exos were harvested and injected intramyocardially at the onset of MI. Two distinct endothelial cells (ECs) were cultured with M2-exos to explore the direct effects on angiogenesis. RESULTS: We showed that M2-exos improved cardiac function, reduced infarct size, and enhanced angiogenesis after MI. Moreover, M2-exos promoted angiogenesis in vitro; the molecules loaded in the vesicles were responsible for its proangiogenic effects. We further validated that higher abundance of miR-132-3p in M2-exos, which recapitulate their functions, was required for the cardioprotective effects exerted by M2-exos. Mechanistically, miR-132-3p carried by M2-exos down-regulate the expression of THBS1 through direct binding to its 3´UTR and the proangiogenic effects of miR-132-3p were largely reversed by THBS1 overexpression. CONCLUSION: Our findings demonstrate that M2-exos promote angiogenesis after MI by transporting miR-132-3p to ECs, and by binding to THBS1 mRNA directly and negatively regulating its expression. These findings highlight the role of M2-exos in cardiac repair and provide novel mechanistic understanding of intercellular communication in post-infarction angiogenesis.

Identification of a ferroptosis-related prognostic signature and validation of ITGA6-AS1 in enhancing cell proliferation, migration and invasion in glioma.

Glioma is the most common malignant tumor of the adult central nervous system. In this study, we aimed to identify a novel model for predicting glioma prognosis and a potential therapeutic target. Here, lncRNAs related to prognosis and ferroptosis were analyzed and screened through R software and online websites. A nomogram model was established and evaluated with calibration curve, receiver operating characteristic curve and decision curve analysis. Further, an enrichment analysis and immune infiltration analysis were performed. In addition, the expression level and biological function of ITGA6-AS1 were verified in vitro. We obtained a ferroptosis-related 7-lncRNA signature, and constructed a nomogram prognostic model with good predictability for 1-, 3- and 5-year overall survival of glioma patients. The enrichment analysis indicated potential involvement of certain pathways and suggested a correlation between the high-risk group and infiltration of M2 macrophages and MDSCs. Furthermore, the expression level of ITGA6-AS1 in the U118, U87, and LN229 cells was upregulated compared to the H1800 cell. Interestingly, knockdown of ITGA6-AS1 may inhibit U118 cells' proliferation, migration and invasion in vitro. while overexpression of ITGA6-AS1 in LN229 cells plays a promoting role. This study implies that the 7-lncRNA signature may contribute to the stratification of glioma prognosis, and the immune suppressive microenvironment may be associated with macrophage-ferroptosis crosstalk. Furthermore, ITGA6-AS1 may be a potential therapeutic target for patients with glioma.

CD248-expressing cancer-associated fibroblasts induce epithelial-mesenchymal transition of non-small cell lung cancer via inducing M2-polarized macrophages.

Non-small cell lung cancer (NSCLC)-originating cancer-associated fibroblasts (CAFs) expressing CD248 regulate interaction with immune cells to accelerate cancer progression. Epithelial-mesenchymal transition (EMT) is a key feature of metastatic cells. In our pervious study, we found that CD248+CAFs activated M2-polarized macrophages, enhancing the progression of NSCLC. However, it is yet unclear how CD248+CAFs inducing M2-polarized macrophages induce EMT program in NSCLC cells. Herein, we examined CD248 expression from CAFs derived from NSCLC patient tumour tissues. Furthermore, we determined the influence of CD248 knock down CAFs on macrophages polarization. Next, we explored the influences of CD248-harboring CAFs-mediated M2 macrophage polarization to promote NSCLC cells EMT in vitro. We constructed fibroblasts specific CD248 gene knock out mice to examine the significance of CD248-harboring CAFs-induced M2-polarized macrophages to promote NSCLC cells EMT in vivo. Based on our analysis, CD248 is ubiquitously expressed within NSCLC-originating CAFs. CD248+CAFs mediated macrophages polarized to M2 type macrophages. CD248+CAFs induced M2 macrophage polarization to enhance NSCLC cells EMT both in vivo and in vitro. Our findings indicate that CD248-harboring CAFs promote NSCLC cells EMT by regulating M2-polarized macrophages.

SIRT5 exacerbates eosinophilic chronic rhinosinusitis by promoting polarization of M2 macrophage.

BACKGROUND: Previous studies implied that local M2 polarization of macrophage promoted mucosal edema and exacerbated TH2 type inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP). However, the specific pathogenic role of M2 macrophages and the intrinsic regulators in the development of CRS remains elusive. OBJECTIVE: We sought to investigate the regulatory role of SIRT5 in the polarization of M2 macrophages and its potential contribution to the development of CRSwNP. METHODS: Real-time reverse transcription-quantitative PCR and Western blot analyses were performed to examine the expression levels of SIRT5 and markers of M2 macrophages in sinonasal mucosa samples obtained from both CRS and control groups. Wild-type and Sirt5-knockout mice were used to establish a nasal polyp model with TH2 inflammation and to investigate the effects of SIRT5 in macrophage on disease development. Furthermore, in vitro experiments were conducted to elucidate the regulatory role of SIRT5 in polarization of M2 macrophages. RESULTS: Clinical investigations showed that SIRT5 was highly expressed and positively correlated with M2 macrophage markers in eosinophilic polyps. The expression of SIRT5 in M2 macrophages was found to contribute to the development of the disease, which was impaired in Sirt5-deficient mice. Mechanistically, SIRT5 was shown to enhance the alternative polarization of macrophages by promoting glutaminolysis. CONCLUSIONS: SIRT5 plays a crucial role in promoting the development of CRSwNP by supporting alternative polarization of macrophages, thus providing a potential target for CRSwNP interventions.

Shikonin reduces M2 macrophage population in ovarian cancer by repressing exosome production and the exosomal galectin 3-mediated ß-catenin activation.

BACKGROUND: Shikonin (SK), a naphthoquinone with anti-tumor effects, has been found to decrease production of tumor-associated exosomes (exo). This study aims to verify the treatment effect of SK on ovarian cancer (OC) cells, especially on the production of exo and their subsequent effect on macrophage polarization. METHODS: OC cells SKOV3 and A2780 were treated with SK. The exo were isolated from OC cells with or without SK treatment, termed OC exo and SK OC exo, respectively. These exo were used to treat PMA-induced THP-1 cells (M0 macrophages). M2 polarization of macrophages was determined by measuring the M2 specific cell surface markers CD163 and CD206 as well as the secretion of M2 cytokine IL-10. The functions of galectin 3 (LGALS3/GAL3) and ß-catenin in macrophage polarization were determined by gain- or loss-of-function assays. CB-17 SCID mice were subcutaneously injected with SKOV3 cells to generate xenograft tumors, followed by OC exo or SK OC exo treatment for in vivo experiments. RESULTS: SK suppressed viability, migration and invasion, and apoptosis resistance of OC cells in vitro. Compared to OC exo, SK OC exo reduced the M2 polarization of macrophages. Regarding the mechanism, SK reduced exo production in cancer cells, and it decreased the protein level of GAL3 in exo and recipient macrophages, leading to decreased ß-catenin activation. M2 polarization of macrophages was restored by LGALS3 overexpression but decreased again by the ß-catenin inhibitor FH535. Compared to OC exo, the SK OC exo treatment reduced the xenograft tumor growth in mice, and it decreased the M2 macrophage infiltration within tumor tissues. CONCLUSION: This study suggests that SK reduces M2 macrophage population in OC by repressing exo production and blocking exosomal GAL3-mediated ß-catenin activation.

MIF as a potential diagnostic and prognostic biomarker for triple-negative breast cancer that correlates with the polarization of M2 macrophages.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a crucial role in antitumor immunity. However, the role of MIF in influencing the tumor microenvironment (TME) and prognosis of triple-negative breast cancer (TNBC) remains to be elucidated. Using R, we analyzed single-cell RNA sequencing (scRNA-seq) data of 41 567 cells from 10 TNBC tumor samples and spatial transcriptomic data from two patients. Relationships between MIF expression and immune cell infiltration, clinicopathological stage, and survival prognosis were determined using samples from The Cancer Genome Atlas (TCGA) and validated in a clinical cohort using immunohistochemistry. Analysis of scRNA-seq data revealed that MIF secreted by epithelial cells in TNBC patients could regulate the polarization of macrophages into the M2 phenotype, which plays a key role in modulating the TME. Spatial transcriptomic data also showed that epithelial cells (tumor cells) and MIF were proximally located. Analysis of TCGA samples confirmed that tumor tissues of patients with high MIF expression were enriched with M2 macrophages and showed a higher T stage. High MIF expression was significantly associated with poor patient prognosis. Immunohistochemical staining showed high MIF expression was associated with younger patients and worse clinicopathological staging. MIF secreted by epithelial cells may represent a potential biomarker for the diagnosis and prognosis of TNBC and may promote TNBC invasion by remodeling the tumor immune microenvironment.

Integrated analysis of tumor-associated macrophages and M2 macrophages in CRC: unraveling molecular heterogeneity and developing a novel risk signature.

BACKGROUND: Emerging investigations have increasingly highlighted the critical role of tumor-associated macrophages (TAMs) and M2 macrophages in cancer development, progression, and metastasis, marking them as potential targets in various cancer types. The main objective of this research is to discover new biomarkers associated with TAM-M2 macrophages in colorectal cancer (CRC) and to dissect the molecular heterogeneity of CRC by combining single-cell RNA sequencing and bulk RNA-seq data. METHODS: By utilizing weighted gene co-expression network analysis (WGCNA), we acquired TAM-M2-associated genes by intersecting TAM marker genes obtained from scRNA-seq data with module genes of M2 macrophages derived from bulk RNA-seq data. We employed least absolute shrinkage and selection operator (LASSO) Cox analysis to select predictive biomarkers from these TAM-M2-related genes. Quantitative polymerase chain reaction (qPCR) was employed to validate the mRNA expression levels of the genes identified in the screening. This led to the development of the TAM-M2-related signature (TAMM2RS). We also conducted functional and immune landscape analyses of different risk groups. RESULTS: The combination of scRNA-seq and bulk RNA-seq analyses yielded 377 TAM-M2-related genes. DAPK1, NAGK, and TRAF1 emerged as key prognostic genes in CRC, which were identified through LASSO Cox analysis. Utilizing these genes, we constructed and validated the TAMM2RS, demonstrating its effectiveness in predicting survival in CRC patients. CONCLUSION: Our research offers a thorough investigation into the molecular mechanisms associated with TAM-M2 macrophages in CRC and unveils potential therapeutic targets, offering new insights for treatment strategies in colorectal cancer.

The Immune Microenvironment Landscape of Pituitary NeuroEndocrine Tumors, a Transcriptomic Approach.

Pituitary neuroendocrine tumors (PitNET) are known to be variably infiltrated by different immune cells. Nonetheless, their role in pituitary oncogenesis has only begun to be unveiled. The immune microenvironment could determine the biological and clinical behavior of a neoplasm and may have prognostic implications. To evaluate the expression of immune-related genes and to correlate such expression with the presence of infiltrating immune cells in forty-two PitNETs of different lineages, we performed whole transcriptome analysis and RT-qPCR. Deconvolution analysis was carried out to infer the immune cell types present in each tumor and the presence of immune cells was confirmed by immunofluorescence. We found characteristic expression profiles of immune-related genes including those encoding interleukins and chemokines for each tumor lineage. Genes such as IL4-I1, IL-36A, TIRAP, IL-17REL, and CCL5 were upregulated in all PitNETS, whereas IL34, IL20RA, and IL-2RB characterize the NR5A1-, TBX19-, and POU1F1-derived tumors, respectively. Transcriptome deconvolution analysis showed that M2 macrophages, CD4+ T cells, CD8+ T cells, NK cells, and neutrophils can potentially infiltrate PitNET. Furthermore, CD4+ and CD8+ T cells and NK cells infiltration was validated by immunofluorescence. Expression of CCL18, IL-5RA, and HLA-B as well as macrophage tumor infiltration could identify patients who can potentially benefit from treatment with immune checkpoint inhibitors.

Quercetin Inhibits Neuronal Pyroptosis and Ferroptosis by Modulating Microglial M1/M2 Polarization in Atherosclerosis.

Atherosclerosis (AS) with iron and lipid overload and systemic inflammation is a risk factor for Alzheimer's disease. M1 macrophage/microglia participate in neuronal pyroptosis and recently have been reported to be the ferroptosis-resistant phenotype. Quercetin plays a prominent role in preventing and treating neuroinflammation, but the protective mechanism against neurodegeneration caused by iron deposition is poorly understood. ApoE-/- mice were fed a high-fat diet with or without quercetin treatment. The Morris water maze and novel object recognition tests were conducted to assess spatial learning and memory, and nonspatial recognition memory, respectively. Prussian blue and immunofluorescence staining were performed to assess the iron levels in the whole brain and in microglia, microglia polarization, and the degree of microglia/neuron ferroptosis. In vitro, we further explored the molecular biological alterations associated with microglial polarization, neuronal pyroptosis, and ferroptosis via Western blot, flow cytometry, CCK8, LDH, propidium iodide, and coculture system. We found that quercetin improved brain lesions and spatial learning and memory in AS mice. Iron deposition in the whole brain or microglia was reversed by the quercetin treatment. In the AS group, the colocalization of iNOS with Iba1 was increased, which was reversed by quercetin. However, the colocalization of iNOS with PTGS2/TfR was not increased in the AS group, suggesting a character resisting ferroptosis. Quercetin induced the expression of Arg-1 and decreased the colocalizations of Arg-1 with PTGS2/TfR. In vitro, ox-LDL combined with ferric ammonium citrate treatment (OF) significantly shifted the microglial M1/M2 phenotype balance and increased the levels of free iron, ROS, and lipid peroxides, which was reversed by quercetin. M1 phenotype induced by OF caused neuronal pyroptosis and was promoted to ferroptosis by L-NIL treatment, which contributed to neuronal ferroptosis as well. However, quercetin induced the M1 to M2 phenotype and inhibited M2 macrophages/microglia and neuron pyroptosis or ferroptosis. In summary, quercetin alleviated neuroinflammation by inducing the M1 to M2 phenotype to inhibit neuronal pyroptosis and protected neurons from ferroptosis, which may provide a new idea for neuroinflammation prevention and treatment.

M2 macrophages participate in ILC2 activation induced by Helicobacter pylori infection.

Helicobacter pylori (H. pylori) causes a diversity of gastric diseases. The host immune response evoked by H. pylori infection is complicated and can influence the development and progression of diseases. We have reported that the Group 2 innate lymphocytes (ILC2) were promoted and took part in building type-2 immunity in H. pylori infection-related gastric diseases. Therefore, in the present study, we aim to clarify how H. pylori infection induces the activation of ILC2. It was found that macrophages were necessary for activating ILC2 in H. pylori infection. Mechanistically, H. pylori infection up-regulated the expression of indoleamine 2,3-dioxygenase (IDO) in macrophages to induce M2 polarization, and the latter secreted the alarmin cytokine Thymic Stromal Lymphopoietin (TSLP) to arouse ILC2.

BACH1 transcriptionally upregulates FOSL2 to induce M2 macrophages phenotype by activating TGFß/SMAD signaling to promote the transformation of lung fibroblasts into myofibroblasts.

Pulmonary fibrosis is a chronic and progressive lung disorder. The pro-fibrosis factors induced by M2 macrophage phenotype promote the differentiation of fibroblasts into myofibroblasts, which is essential for pulmonary fibrosis. We aimed to explore the role and mechanism of BTB domain and CNC homology 1 (BACH1) in pulmonary fibrosis. BACH1 was knocked down in THP-1 polarized M2 macrophages with or without FOS-like antigen 2 (FOSL2) overexpression, the expression of M2 macrophage markers was detected. Cell viability, migration, invasion and extracellular matrix (ECM) accumulation were estimated by CCK-8, wound healing, transwell, western bot and immunofluorescence staining. Luciferase reporter and chromatin immunoprecipitation assays were used to verify the binding of BACH1 to FOSL2 promotor region. In vivo, a bleomycin (BLM)-induced pulmonary fibrosis mice model was established to evaluate the effect of BACH1 silencing on the histopathological changes, M2 macrophage phenotype and extracellular matrix (ECM) deposition. Expression of proteins was assessed with western blot. Results indicated that BACH1 expression was upregulated in M2 macrophages polarized from THP-1 cells. BACH1 deficiency inhibited the polarization of THP-1 to the M2 macrophage phenotype to promote the transformation of lung fibroblasts into myofibroblasts. Additionally, BACH1 could transcriptionally activate FOSL2 expression in THP-1-derived macrophages to upregulate TGFß/SMAD signaling in HFL-1 cells. The animal experiments indicated that BACH1 knockdown alleviated BLM-induced pulmonary fibrosis, M2 macrophage polarization and inactivated FOSL2/TGFß/SMAD signaling in mice lung tissues. Together, this finding suggests BACH1/FOSL2 may be useful therapeutic targets for the treatment of pulmonary fibrosis.

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

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

Mitochondrial electron transport chain in macrophage reprogramming: Potential role in antibacterial immune response.

Macrophages restrain microbial infection and reinstate tissue homeostasis. The mitochondria govern macrophage metabolism and serve as pivot in innate immunity, thus acting as immunometabolic regulon. Metabolic pathways produce electron flows that end up in mitochondrial electron transport chain (mtETC), made of super-complexes regulating multitude of molecular and biochemical processes. Cell-intrinsic and extrinsic factors influence mtETC structure and function, impacting several aspects of macrophage immunity. These factors provide the macrophages with alternate fuel sources and metabolites, critical to gain functional competence and overcoming pathogenic stress. Mitochondrial reactive oxygen species (mtROS) and oxidative phosphorylation (OXPHOS) generated through the mtETC are important innate immune attributes, which help macrophages in mounting antibacterial responses. Recent studies have demonstrated the role of mtETC in governing mitochondrial dynamics and macrophage polarization (M1/M2). M1 macrophages are important for containing bacterial pathogens and M2 macrophages promote tissue repair and wound healing. Thus, mitochondrial bioenergetics and metabolism are intimately coupled with innate immunity. In this review, we have addressed mtETC function as innate rheostats that regulate macrophage reprogramming and innate immune responses. Advancement in this field encourages further exploration and provides potential novel macrophage-based therapeutic targets to control unsolicited inflammation.

Unveiling the Role of Endoplasmic Reticulum Stress Pathways in Canine Demodicosis.

Canine demodicosis is a prevalent skin disease caused by overpopulation of a commensal species of Demodex mite, yet its precise cause remains unknown. Research suggests that T-cell exhaustion, increased immunosuppressive cytokines, induction of regulatory T cells and increased expression of immune checkpoint inhibitors may contribute to its pathogenesis. This study aimed to gain a deeper understanding of the molecular changes occurring in canine demodicosis using mass spectrometry and pathway enrichment analysis. The results indicate that endoplasmic reticulum stress promotes canine demodicosis through regulation of three linked signalling pathways: eIF2, mTOR, and eIF4 and p70S6K. These pathways are involved in the modulation of Toll-like receptors, most notably TLR2, and have been shown to play a role in the pathogenesis of skin diseases in both dogs and humans. Moreover, these pathways are also implicated in the promotion of immunosuppressive M2 phenotype macrophages. Immunohistochemical analysis, utilising common markers of dendritic cells and macrophages, verified the presence of M2 macrophages in canine demodicosis. The proteomic analysis also identified immunological disease, organismal injury and abnormalities and inflammatory response as the most significant underlying diseases and disorders associated with canine demodicosis. This study demonstrates that Demodex mites, through ER stress, unfolded protein response and M2 macrophages contribute to an immunosuppressive microenvironment, thereby assisting in their proliferation.

Gut microbially produced tryptophan metabolite melatonin ameliorates osteoporosis via modulating SCFA and TMAO metabolism.

Osteoporosis (OP) is a severe global health issue that has significant implications for productivity and human lifespan. Gut microbiota dysbiosis has been demonstrated to be closely associated with OP progression. Melatonin (MLT) is an important endogenous hormone that modulates bone metabolism, maintains bone homeostasis, and improves OP progression. Multiple studies indicated that MLT participates in the regulation of intestinal microbiota and gut barrier function. However, the promising effects of gut microbiota-derived MLT in OP remain unclear. Here, we found that OP resulted in intestinal tryptophan disorder and decreased the production of gut microbiota-derived MLT, while administration with MLT could mitigate OP-related clinical symptoms and reverse gut microbiota dysbiosis, including the diversity of intestinal microbiota, the relative abundance of many probiotics such as Allobaculum and Parasutterella, and metabolic function of intestinal flora such as amino acid metabolism, nucleotide metabolism, and energy metabolism. Notably, MLT significantly increased the production of short-chain fatty acids and decreased trimethylamine N-oxide-related metabolites. Importantly, MLT could modulate the dynamic balance of M1/M2 macrophages, reduce the serum levels of pro-inflammatory cytokines, and restore gut-barrier function. Taken together, our results highlighted the important roles of gut microbially derived MLT in OP progression via the "gut-bone" axis associated with SCFA metabolism, which may provide novel insight into the development of MLT as a promising drug for treating OP.

Rejuvenating fecal microbiota transplant enhances peripheral nerve repair in aged mice by modulating endoneurial inflammation.

Peripheral nerve injury (PNI) resulting from trauma or neuropathies can cause significant disability, and its prognosis deteriorates with age. Emerging evidence suggests that gut dysbiosis and reduced fecal short-chain fatty acids (SCFAs) contribute to an age-related systemic hyperinflammation (inflammaging), which hinders nerve recovery after injury. This study thus aimed to evaluate the pro-regenerative effects of a rejuvenating fecal microbiota transplant (FMT) in a preclinical PNI model using aged mice. Aged C57BL/6 mice underwent bilateral crush injuries to their sciatic nerves. Subsequently, they either received FMT from young donors at three and four days after the injury or retained their aged gut microbiota. We analyzed gut microbiome composition and SCFA concentrations in fecal samples. The integrity of the ileac mucosal barrier was assessed by immunofluorescence staining of Claudin-1. Flow cytometry was utilized to examine immune cells and cytokine production in the ileum, spleen, and sciatic nerve. Various assessments, including behavioural tests, electrophysiological studies, and morphometrical analyses, were conducted to evaluate peripheral nerve function and repair following injury. Rejuvenating FMT reversed age-related gut dysbiosis by increasing Actinobacteria, especially Bifidobacteriales genera. This intervention also led to an elevation of gut SCFA levels and mitigated age-related ileac mucosal leakiness in aged recipients. Additionally, it augmented the number of T-helper 2 (Th2) and regulatory T (Treg) cells in the ileum and spleen, with the majority being positive for anti-inflammatory interleukin-10 (IL-10). In sciatic nerves, rejuvenating FMT resulted in increased M2 macrophage counts and a higher IL-10 production by IL-10+TNF-α- M2 macrophage subsets. Ultimately, restoring a youthful gut microbiome in aged mice led to improved nerve repair and enhanced functional recovery after PNI. Considering that FMT is already a clinically available technique, exploring novel translational strategies targeting the gut microbiome to enhance nerve repair in the elderly seems promising and warrants further evaluation.