M1 Polarization Markers Research Articles

Tryptophan metabolism regulates inflammatory macrophage polarization as a predictive factor for breast cancer immunotherapy

Metabolic reprogramming plays a pivotal role in regulating macrophage polarization and function. However, the impact of macrophage tryptophan metabolism on polarization within the breast cancer microenvironment remains elusive. In this study, we used single-cell transcriptome analysis and found that macrophages had the highest tryptophan metabolic activity in breast cancer, melanoma, and head and neck squamous cell carcinoma (HNSC). Further analysis revealed that the tryptophan metabolic activity of macrophages was positively correlated with the M1 macrophage scores in breast cancer. Pancancer analysis found positive correlations between tryptophan metabolism and the M1 macrophage score in almost all tumor types. Spatial transcriptome analysis revealed higher tryptophan metabolism in regions with higher M1 macrophage score in breast cancer tissues. Immune infiltration analysis revealed that the high tryptophan metabolism group exhibited a higher immune score, an increased proportion of CD8+ T cells, augmented cytolytic activity mediated by CD8+ T cells, and elevated expression of immune checkpoint molecules. Spatial immunophenotype cohort analysis exhibited that breast cancer patients expected to respond to immunotherapy had stronger tryptophan metabolism, with a 73.8 % area under the ROC curve. Single-cell transcriptome analysis of the immunotherapy cohort found that patients responding to immunotherapy had higher macrophage tryptophan metabolism prior to treatment initiation. Finally, in vitro experiments demonstrated elevated expression of tryptophan metabolic enzymes in M1 macrophages. Moreover, tryptophan facilitated the expression of M1 polarization markers, whereas inhibitors of tryptophan metabolic enzymes, such as NLG919, LM10, and Ro 61–8048, inhibited the expression of M1 polarization markers. In conclusion, this study identified a dual role for macrophage tryptophan metabolism in breast cancer; on the one hand, it promotes macrophage M1 polarization, while on the other hand, it serves as a promising predictor for the effectiveness of immunotherapy in breast cancer.

The transcription factor ELF4 alleviates inflammatory bowel disease by activating IL1RN transcription, suppressing inflammatory TH17 cell activity, and inducing macrophage M2 polarization.

Inflammatory bowel disease (IBD) is a chronic immune-mediated disorder affecting millions worldwide. Due to the complexity of its pathogenesis, the treatment options for IBD are limited. This study focuses on ELF4, a member of the ETS transcription factor family, as a target to elucidate its role in IBD and investigate its mechanism of action in alleviating IBD symptoms by activating IL1RN transcription to suppress the activity of inflammatory TH17 cells. Using the GEO database, this study examined LPS-induced intestinal inflammatory genes and their regulation mechanisms. We examined the colon length of LPS-treated mice and derived the Disease Activity Index (DAI). H&E staining, ELISA, and flow cytometry were used to detect mice colon tissue damage, inflammatory factor levels in mouse serum, mouse macrophage types and inflammatory TH17 cell activity. RT-qPCR and Western blot detected ELF4, IL1RN, M1, and M2 polarization markers. In Vitro, using dual-luciferase and ChIP assays, we tested mouse bone marrow-derived macrophages (BMDMs) and mouse intestinal epithelial cells for IL1RN promoter activity and ELF4 enrichment. Bioinformatics showed that LPS-induced colitis animals have reduced ELF4 expression in their colon tissue. In vivo tests confirmed reduced ELF4 expression in mice with LPS-induced colitis. ELF4 overexpression reduced mouse intestinal inflammation. ELF4 activated IL1RN transcription in bioinformatics and in vitro tests. ELF4 promoted IL1RN transcription and macrophage M2 polarization to limit intestinal epithelial cell death and inflammation and reduce mouse intestinal inflammation in vitro. ELF4 also reduced the Th17/Treg ratio by increasing IL1RN transcription. ELF4 activates IL1RN transcription, suppresses inflammatory TH17 cells, and induces macrophage M2 polarization to treat IBD.

Advantages and disadvantages of treatment of experimental ARDS by M2-polarized RAW 264.7 macrophages

Innate immunity reactions are core to any immunological process, including systemic inflammation and such extremes as acute respiratory distress syndrome (ARDS) and cytokine storm. Macrophages, the key cells of innate immunity, show high phenotypic plasticity: depending on microenvironmental cues, they can polarize into M1 (classically activated, pro-inflammatory) or M2 (alternatively activated, anti-inflammatory). The anti-inflammatory M2 macrophage polarization-based cell therapies constitute a novel prospective modality. Systemic administration of ‘educated’ macrophages is intended at their homing in lungs in order to mitigate the pro-inflammatory cytokine production and reduce the risks of ‘cytokine storm’ and related severe complications. Acute respiratory distress syndrome (ARDS) is the main mortality factor in pneumonia including SARS-CoV-associated cases. This study aimed to evaluate the influence of infusions of RAW 264.7 murine macrophage cell line polarized towards M2 phenotype on the development of LPS-induced ARDS in mouse model. The results indicate that the M2-polarized RAW 264.7 macrophage infusions in the studied model of ARDS promote relocation of lymphocytes from their depots in immune organs to the lungs. In addition, the treatment facilitates expression of M2-polarization markers Arg1, Vegfa and Tgfb and decreases of M1-polarization marker Cd38 in lung tissues, which can indicate the anti-inflammatory response activation. However, treatment of ARDS with M2-polarized macrophages didn't change the neutrophil numbers in the lungs. Moreover, the level of the Arg1 protein in lungs decreased throughtout the treatment with M2 macrophages, which is probably because of the pro-inflammatory microenvironment influence on the polarization of macrophages towards M1. Thus, the chemical polarization of macrophages is unstable and depends on the microenvironment. This adverse effect can be reduced through the use of primary autologous macrophages or some alternative methods of M2 polarization, notably siRNA-mediated.

Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate neuropathic pain.

(Switching from the microglial M1 phenotype to the M2 phenotype is a promising therapeutic strategy for neuropathic pain (NP). This study aimed to investigate the potential use of stigmasterol for treating NP. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, chronic constriction injury (CCI) group, CCI + ibuprofen group, and CCI + stigmasterol group. We performed behavioral tests, enzyme-linked immunosorbent assay, hematoxylin-esoin staining (H&E) staining and immunohistochemistry, immunofluorescence, and Western blotting. In cell experiments, we performed flow cytometry, immunofluorescence, Western blotting, and qRT-PCR. Stigmasterol reduced thermal and mechanical hyperalgesia and serum IL-1β and IL-8 levels and increased serum IL-4 and TGF-β levels in CCI rats. Stigmasterol reduced IL-1β, COX-2, and TLR4 expression in the right sciatic nerve and IL-1β expression in the spinal cord. Stigmasterol reduced the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, COX-2, IL-1β, and CD32 in the spinal cord of CCI rats while increasing the expression of IL-10 and CD206. Stigmasterol decreased M1 polarization markers and increased M2 polarization markers in lipopolysaccharide (LPS)-induced microglia and decreased the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, iNOS, COX-2, and IL-1β in LPS-treated microglia while increasing the expression of Arg-1 and IL-10. Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate NP.

CD137L Inhibition Ameliorates Hippocampal Neuroinflammation and Behavioral Deficits in a Mouse Model of Sepsis-Associated Encephalopathy

Anxiety manifestations and cognitive dysfunction are common sequelae in patients with sepsis-associated encephalopathy (SAE). Microglia-mediated inflammatory signaling is involved in anxiety, depression, and cognitive dysfunction during acute infection with bacterial lipopolysaccharide (LPS). However, the molecular mechanisms underlying microglia activation and behavioral and cognitive deficits in sepsis have not been in fully elucidated. Based on previous research, we speculated that the CD137 receptor/ligand system modulates microglia function during sepsis to mediate classical neurological SAE symptoms. A murine model of SAE was established by injecting male C57BL/6 mice with LPS, and cultured mouse BV2 microglia were used for in vitro assays. RT-qPCR, immunofluorescence staining, flow cytometry, and ELISA were used to assess microglial activation and the expression of CD137L and inflammation-related cytokines in the mouse hippocampus and in cultured BV2 cells. In addition, behavioral tests were conducted in assess cognitive performance and behavioral distress. Immunofluorescence and RT-qPCR analyses showed that hippocampal expression of CD137L was upregulated in activated microglia following LPS treatment. Pre-treatment with the CD137L neutralizing antibody TKS-1 significantly reduced CD137L levels, attenuated the expression of M1 polarization markers in microglia, and inhibited the production of TNF-α, IL-1β, and IL-6 in both LPS-treated mice and BV2 cells. Conversely, stimulation of CD137L signaling by recombinant CD137-Fc fusion protein activated the synthesis and release of pro-inflammatory cytokines in cultures BV2 microglia. Importantly, open field, elevated plus maze, and Y-maze spontaneous alternation test results indicated that TKS-1 administration alleviated anxiety-like behavior and spatial memory decline in mice with LPS-induced SAE. These findings suggest that CD137L upregulation in activated microglia critically contributes to neuroinflammation, anxiety-like behavior, and cognitive dysfunction in the mouse model of LPS-induced sepsis. Therefore, therapeutic modulation of the CD137L/CD137 signaling pathway may represent an effective way to minimize brain damage and prevent cognitive and emotional deficits associated with SAE.

Nintedanib ameliorates osteoarthritis in mice by inhibiting synovial inflammation and fibrosis caused by M1 polarization of synovial macrophages via the MAPK/PI3K-AKT pathway.

Synovial inflammation and fibrosis are important pathological changes associated with osteoarthritis (OA). Herein, we investigated if nintedanib, a drug specific for pulmonary fibrosis, plays a positive role in osteoarthritic synovial inflammation and fibrosis. We assessed the effect of nintedanib on osteoarthritic synovial inflammation and fibrosis in a mouse model of OA created by destabilization of the medial meniscus and a macrophage M1 polarization model created by stimulating RAW264.7 cells with lipopolysaccharide. Histological staining showed that daily gavage administration of nintedanib significantly alleviated articular cartilage degeneration, reduced the OARSI score, upregulated matrix metalloproteinase-13 and downregulated collagen II expression, and significantly reduced the synovial score and synovial fibrosis in a mouse OA model. In addition, immunofluorescence staining showed that nintedanib significantly decreased the number of M1 macrophages in the synovium of a mouse model of OA. In vitro results showed that nintedanib downregulated the phosphorylation levels of ERK, JNK, p38, PI3K, and AKT while inhibiting the expression of macrophage M1 polarization marker proteins (CD86, CD80, and iNOS). In conclusion, this study suggests that nintedanib is a potential candidate for OA treatment. The mechanisms of action of nintedanib include the inhibition of M1 polarization in OA synovial macrophages via the MAPK/PI3K-AKT pathway, inhibition of synovial inflammation and fibrosis, and reduction of articular cartilage degeneration.

Integrating Network Pharmacology and Component Analysis to Study the Potential Mechanisms of Qi-Fu-Yin Decoction in Treating Alzheimer's Disease.

To elucidate the potential mechanisms of QFY for the treatment of Alzheimer's Disease (AD), and explore the effective substances of QFY. UPLC-LTQ-Orbitrap-MS was used to identify the chemical constituents of the serum samples and the cerebrospinal fluid samples of rats after QFY administration. Network pharmacology was used to predict potential targets and pathways of QFY against AD. The AD mice model was established by subcutaneous injection of D-gal for 8 consecutive weeks. New object recognition (NOR) and Morris water maze test (MWM) were used to evaluate the learning and memory abilities of mice. Moreover, the levels of TNF-α, IL-1β, and IL-18 in the brain hippocampus of mice were determined by ELISA. The expression of Bax, Bcl-2, Caspase-1, PSD95, SYP, ICAM-1 and MCP-1 proteins in the hippocampus was detected by Western blotting. Furthermore, qRT-PCR was used to detect the gene expressions of PSD95, SYP, M1 and M2 polarization markers of microglia, including iNOS, CD16, ARG-1, and IL-10 in the hippocampus. A total of 51 prototype compounds were detected in rat serum and 15 prototype components were identified in rat cerebrospinal fluid. Behavioral experiments revealed that QFY significantly increased the recognition index, decreased the escape latency, increased the platform crossing times and increased the residence time in the target quadrant. QFY also could alleviate the ultrastructural pathological changes in the hippocampus of AD mice. Meanwhile, QFY treatment suppressed the expression of inflammatory factors, such as TNF-α, IL-1β, and IL-18. QFY improved the synaptic plasticity of the hippocampus in D-gal model mice by significantly increasing the expression of proteins and mRNAs of PSD95 and SYP. QFY could effectively improve the learning and memory impairment of D-gal-induced AD mice by inhibiting the excessive activation of microglia, enhancing the expression of M2 microglia, inhibiting the increase of inflammatory factors, cell adhesion factors and chemokines, anti-apoptosis, and improving synaptic plasticity.

Paeonol accelerates skin wound healing by regulating macrophage polarization and inflammation in diabetic rats.

Diabetic ulcer is usually seen in people with uncontrolled blood sugar. Reportedly, many factors such as impaired glucose metabolism, and macrovascular and microvascular diseases caused angiogenesis disorders and delayed the healing of diabetic ulcers, thus affecting the body's metabolism, nutrition, and immune function. This study aimed to explore the effect of paeonol on skin wound healing in diabetic rats and the related mechanism. A rat model of diabetic ulcer was established. High glucose-treated mouse skin fibroblasts were co-cultured with M1 or M2-polarized macrophages treated with or without paeonol. H&E and Masson staining were used to reveal inflammatory cell infiltration and collagen deposition, respectively. Immunohistochemistry visualized the expression of Ki67, CD31, and vascular endothelial growth factor (VEGF). Western blot was used to detect interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-4, IL-10, CD31, VEGFA, and collagen I/III. The expression of iNOS and arginase 1 was revealed by immunofluorescence staining. Paeonol treatment augmented collagen deposition and the expression of Ki67, CD31, VEGF, and macrophage M2 polarization markers (IL-4 and IL-10) and reduced wound area, inflammatory cell infiltration, and macrophage M1 polarization markers (IL-1β and TNF-α) in the ulcerated area. In vitro, paeonol treatment promoted M2-polarization and repressed M1-polarization in macrophages, thereby improving the repair of cell damage induced by high glucose. Paeonol accelerates the healing of diabetic ulcers by promoting M2 macrophage polarization and inhibiting M1 macrophage polarization.

LncRNA MEG3 Inhibits Tumor Progression by Modulating Macrophage Phenotypic Polarization via miR-145-5p/DAB2 Axis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the predominant histological type of primary liver cancer, which ranks sixth among the most common human tumors. Tumor-associated macrophages (TAMs) are an important component of tumor microenvironment (TME) and the M2 macrophage polarization substantially contributes to tumor growth and metastasis. Long non-coding RNA (lncRNA) MEG3 was reported to restrain HCC development. However, whether MEG3 regulates macrophage phenotypic polarization in HCC remains unclear. Bone marrow derived macrophages (BMDMs) were treated with LPS/IFNγ and IL4/IL13 to induce the M1 and M2 macrophage polarization, respectively. M2-polarized BMDMs were simultaneously transfected with adenovirus vector overexpressing MEG3 (Adv-MEG3). Subsequently, M2-polarized BMDMs were cultured for 24 h with serum-free medium, the supernatants of which were harvested as conditioned medium (CM). HCC cell line Huh7 was cultured with CM for 24 h. F4/80+CD68+ and F4/80+CD206+ cell percentages in M1-and M2-polarized BMDMs were calculated using flow cytometry. Huh7 cell migration, invasion and angiogenesis were determined via Transwell assay and tube formation experiment. Nude mice were implanted with Huh7 cells and Adv-MEG3-transfected M2-polarizd BMDMs, and tumor growth and M2 macrophage polarization markers were assessed. The binding between miR-145-5p and MEG3 or disabled-2 (DAB2) was verified by luciferase reporter assay. MEG3 presented lower expression in HCC tissues than in normal controls, and low expression of MEG3 was correlated to poorer prognosis of HCC patients. MEG3 expression was enhanced during LPS/IFNγ-induced M1 polarization, but was reduced during IL4/IL13-induced M2 polarization. MEG3 overexpression inhibited the expression of M2 polarization markers in both M2-polarized BMDMs and mice. Mechanically, MEG3 bound with miR-145-5p to regulate DAB2 expression. Overexpressing MEG3 suppressed M2 polarization-induced HCC cell metastasis and angiogenesis by upregulating DAB2 and inhibited in vivo tumor growth. LncRNA MEG3 curbs HCC development by repressing M2 macrophage polarization via miR-145-5p/DAB2 axis.

C/EBPα involvement in microglial polarization via HDAC1/STAT3 pathway aggravated sevoflurane-induced cognitive impairment in aged rats.

Postoperative cognitive dysfunction (POCD) is a clinically frequent postoperative complication in the elderly, which is mainly manifested by the occurrence of cognitive dysfunction after anesthetized surgery in patients. To explore the involvement of C/EBPα in microglial polarization in sevoflurane anesthesia induced cognitive impairment in aged rats. Sprague-Dawley (SD) rats were anesthetized by inhalation of 3% sevoflurane for 6 h to establish the POCD model. The histopathological structure of hippocampus was observed by hematoxylin and eosin (HE) staining. Associative learning and memory function and spatial learning and memory function were assessed by conditioned fear test and water maze test. The concentrations of inflammatory factors in the hippocampus were measured by ELISA. The levels of microglial activation marker (Iba1) and microglial M1 (CD86) and M2 (CD206) polarization markers were determined by immunofluorescence staining and RT-qPCR, respectively. The transcriptional regulation of HDAC1 by C/EBPα was confirmed by dual luciferase reporter assay and ChIP assay. Sevoflurane-induced pathomorphological damage in the hippocampal tissue of aged rats, accompanied by elevated expression of C/EBPα. Silencing of C/EBPα alleviated hippocampal histopathological injury, inhibited M1 microglial activation and the expression of M1 marker CD86, enhanced the expression of M2 marker CD206. C/EBPα transcriptionally activated HDAC1. Knockdown of C/EBPα downregulated the expression of HDAC1 and STAT3 phosphorylated proteins, which inhibited the pro-inflammatory factors (IL-6 and TNF-α) and accelerated anti-inflammatory factors (IL-10 and TGF-β) secretion. In addition, silencing of C/EBPα caused rats to have a delayed freezing time in contextual conditioned fear, a shorter escape latency, and an increased number of platform crossings. Inhibition of C/EBPα promotes the M2 polarization of microglia and reduces the production of pro-inflammatory cytokines to alleviate the cognitive dysfunction of sevoflurane-induced elderly rats by HDAC1/STAT3 pathway.

Cigarette smoke prevents M1 polarization of alveolar macrophages by suppressing NLRP3

Chronic obstructive pulmonary disease (COPD) is an inflammatory condition mainly caused by cigarette smoke (CS). Alveolar macrophages (AMs) contribute to its development, although the polarization of AMs is controversial. This study explored the polarization of AMs and mechanisms underlying their involvement in COPD. AM gene expression data from non-smokers, smokers, and COPD patients were downloaded from the GSE13896 and GSE130928 datasets. Macrophage polarization was evaluated by CIBERSORT and gene set enrichment analysis (GSEA). Polarization-related differentially expressed genes (DEGs) were identified in GSE46903. KEGG enrichment analysis and single sample GSEA were performed. M1 polarization levels were decreased in smokers and COPD patients, whereas M2 polarization did not change. In the GSE13896 and GSE130928 datasets, 27 and 19 M1-related DEGs, respectively, showed expression changes opposite to those in M1 macrophages in smokers and COPD patients compared with the control group. These M1-related DEGs were enriched in NOD-like receptor signaling pathway. Next, C57BL/6 mice were divided into control, lipopolysaccharide (LPS), CS, and LPS + CS groups, and cytokine levels in bronchoalveolar lavage fluid (BALF) and AM polarization were determined. The expression of macrophage polarization markers and NLRP3 was determined in AMs treated with CS extract (CSE), LPS, and an NLRP3 inhibitor. Cytokines levels and the percentage of M1 AMs in BALF were lower in the LPS + CS group than in the LPS group. Exposure to CSE downregulated the expression of M1 polarization markers and NLRP3 induced by LPS in AMs. The present results indicate that M1 polarization of AMs is repressed in smokers and COPD patients, and CS may inhibit LPS-induced M1 polarization of AMs by suppressing NLRP3.

Exosome-derived miR-142-5p from liver stem cells improves the progression of liver fibrosis by regulating macrophage polarization through CTSB.

This study aims to explore the effect of liver stem cells (LSCs)-derived exosomes and the miR-142a-5p carried by them on the process of fibrosis by regulating macrophages polarization. In this study, CCL4 was used to establish liver fibrosis model. The morphology and purity of exosomes (EVs) were verified by transmission electron microscopy, western blotting (WB) and nanoparticle tracing analysis (NTA). Real-time quantitative PCR (qRT-PCR), WB and enzyme-linked immunoadsorption (ELISA) were used to detect liver fibrosis markers, macrophage polarization markers and liver injury markers. Histopathological assays were used to verify the liver injury morphology in different groups. The cell co-culture model and liver fibrosis model were constructed to verify the expression of miR-142a-5p and ctsb. Immunofluorescence of LSCs markers CK-18, epithelial cell adhesion molecule (EpCam), and AFP showed that these markers were up-regulated in LSCs. In addition, we evaluated the ability of LSCs to excrete EVs by labeling LSCs-EVs with PKH67. We found that CCL4 and EVs were simultaneously treated at 50 and 100 μg doses, and both doses of EVs could reduce the degree of liver fibrosis in mice. We tested markers of M1 or M2 macrophage polarization and found that EVs reduced M1 marker expression and promoted M2 marker expression. Further, ELISA was used to detect the secreted factors related to M1 and M2 in tissue lysates, which also verified the above views. Further analysis showed that the expression of miR-142a-5p increased significantly with the increase of EVs treatment concentration and time. Further, in vitro and in vivo LSCs-EVs regulate macrophage polarization through miR-142a-5p/ctsb pathway and affect the process of liver fibrosis. Our data suggest that EVs-derived miR-142-5p from LSCs improves the progression of liver fibrosis by regulating macrophage polarization through ctsb.

MAGL regulates synovial macrophage polarization vis inhibition of mitophagy in osteoarthritic pain.

Pain is the hallmark symptom of osteoarthritis (OA), and current analgesic treatments may be insufficient or have potentially adverse effects. The inhibition of Monoacylglycerol lipase (MAGL) produces anti‑inflammatory and anti‑nociceptive effects. However, the potential mechanism of MAGL in OA pain remains unclear. In the present study, the synovial tissues were removed from OA patients and mice. Immunohistochemical staining and western blotting were used to detect the expression of MAGL. M1 and M2 polarization markers were detected by flow cytometry and western blotting, and the mitophagy levels were detected by the immunofluorescence staining of mitochondrial autophagosomes with lysosomes and western blotting. The OA mice were intraperitoneally injected with MJN110 to inhibit MAGL once a day for a week. Mechanical and thermal pain thresholds were detected by electronic Von Frey and hot plate methods on days0, 3, 7, 10, 14, 17, 21, and 28. The accumulation of MAGL in the synovial tissues of OA patients and mice promoted the polarization of macrophages towards an M1 phenotype. Pharmacological inhibition and siRNA knockdown of MAGL promoted polarization of M1 macrophages towards an M2 phenotype. MAGL inhibition increased the mechanical and thermal pain thresholds of OA mice and enhanced the mitophagy levels of M1 macrophages. In conclusion, in the present study, it was shown that MAGL regulated synovial macrophage polarization by inhibiting mitophagy in OA.

The inflammatory injury in the striatal microglia-dopaminergic-neuron crosstalk involved in Tourette syndrome development.

Tourette syndrome (TS) is associated with immunological dysfunction. The DA system is closely related to TS development, or behavioral stereotypes. Previous evidence suggested that hyper-M1-polarized microglia may exist in the brains of TS individuals. However, the role of microglia in TS and their interaction with dopaminergic neurons is unclear. In this study, we applied iminodipropionitrile (IDPN) to establish a TS model and focused on the inflammatory injury in the striatal microglia-dopaminergic-neuron crosstalk. Male Sprague-Dawley rats were intraperitoneally injected with IDPN for seven consecutive days. Stereotypic behavior was observed to verify the TS model. Striatal microglia activation was evaluated based on different markers and expressions of inflammatory factors. The striatal dopaminergic neurons were purified and co-cultured with different microglia groups, and dopamine-associated markers were assessed. First, there was pathological damage to striatal dopaminergic neurons in TS rats, as indicated by decreased expression of TH, DAT, and PITX3. Next, the TS group showed a trend of increased Iba-1 positive cells and elevated levels of inflammatory factors TNF-α and IL-6, as well as an enhanced M1-polarization marker (iNOS) and an attenuated M2-polarization marker (Arg-1). Finally, in the co-culture experiment, IL-4-treated microglia could upregulate the expression of TH, DAT, and PITX3 in striatal dopaminergic neurons vs LPS-treated microglia. Similarly, the TS group (microglia from TS rats) caused a decreased expression of TH, DAT, and PITX3 compared with the Sham group (microglia from control rats) in the dopaminergic neurons. In the striatum of TS rats, microglia activation is M1 hyperpolarized, which transmits inflammatory injury to striatal dopaminergic neurons and disrupts normal dopamine signaling.

RNA binding protein RPS3 mediates microglial polarization by activating NLRP3 inflammasome via SIRT1 in ischemic stroke

BackgroundIschemic stroke is the obstruction of cerebral blood flow with a high morbidity. Microglial polarization is a contributing factor for ischemic stroke-induced injury. Here, we focused on function and mechanism of RNA binding protein RPS3 in microglial polarization after ischemic stroke. MethodsTransient middle cerebral artery occlusion (tMCAO) was conducted in SD rats. Infarct area was detected by TTC staining and neurological score was assessed. Fluorescence staining tested neuronal apoptosis and microglial differentiation. Oxygen and glucose deprivation/reoxygenation (OGD/R) was applied for treating microglia. Levels of RPS3, SIRT1, M1 and M2 polarization markers (CD86, iNOS, CD206, Arg-1) were determined by RT-qPCR. Western blot detected RPS3, SIRT1, NLRP3, ASC and Cleaved-caspase-1 expression. RIP assay validated that RPS3 interacted with SIRT1. CCK-8 measured cell viability. Flow cytometry and ELISA assessed M1 and M2 polarization markers. LDH release was detected using colorimetric CytoTox 96 Cytotoxicity kit. ResultsRPS3 depletion improved neurological dysfunction and reduced infarction area in rats after tMCAO. Knockdown of RPS3 resulted in increased SIRT1 expression and decreased NLRP3 inflammasome activation, and induced microglia M2 polarization after ischemia-reperfusion (I/R). Besides, RPS3 directly targeted SIRT1 and reduced its expression in microglia. RPS3 silencing suppressed OGD/R-triggered neuronal and microglial cell death through SIRT1. Moreover, RPS3 activated NLRP3 inflammasome and regulated microglial polarization via SIRT1. ConclusionRPS3 regulates microglial polarization and neuronal injury through SIRT1/NLRP3 pathway, suggesting a novel target for ischemic stroke treatment.

Chitinase 3 like 1 deficiency ameliorates lipopolysaccharide-induced acute liver injury by inhibition of M2 macrophage polarization

Chitinase 3-like-1 protein (CHI3L1) is involved in various infectious diseases, especially sepsis. Aberrant CHI3L1 expression potentially plays a critical role in chronic inflammation because a considerable number of macrophages are associated with immune/inflammatory diseases. In this study, we examined the effect of CHI3L1 on hepatic sepsis injury using a lipopolysaccharide (LPS)-induced model. LPS-treated CHI3L1 knockout (KO) mice exhibited a higher survival rate than LPS-treated CHI3L1 wild-type (WT) mice. In addition, hepatic injury-related enzyme levels (aspartate transaminase, alanine transaminase, and lactate dehydrogenase) decreased in CHI3L1 KO mice sera, suggesting attenuated LPS-induced septic liver damage in CHI3L1 KO mice. A greater reduction in the mRNA and protein expressions of M2 polarization markers, such as MRC1, ARG1, IL-10, and IL-4, was observed in LPS-induced CHI3L1 KO mice livers than in LPS-induced WT mice livers. Nonetheless, no change in the mRNA and protein expressions of M1 polarization markers, such as INOS, CD86, TNF-α, and IL6, was noted in LPS-induced CHI3L1 KO mice livers compared with those in LPS-induced WT and KO mice. Similar to the in vivo scenario, liver CHI3L1 depletion in LPS-treated HEP3B cells significantly decreased M2 polarization marker protein expression. However, M1 polarization marker protein expression did not differ significantly. These results suggest that CHI3L1 depletion decreases M2 macrophage polarization, and this effect is potentially associated with the alleviation of liver sepsis in CHI3L1 KO mice.

MP05-02 TISSUE-RESIDENT MACROPHAGES ARE ASSOCIATED WITH RANDALL'S PLAQUES

You have accessJournal of UrologyCME1 Apr 2023MP05-02 TISSUE-RESIDENT MACROPHAGES ARE ASSOCIATED WITH RANDALL'S PLAQUES Heiko Yang, Hanbing Song, Tom Chi, Marshall Stoller, Sunita Ho, and Franklin W. Huang Heiko YangHeiko Yang More articles by this author , Hanbing SongHanbing Song More articles by this author , Tom ChiTom Chi More articles by this author , Marshall StollerMarshall Stoller More articles by this author , Sunita HoSunita Ho More articles by this author , and Franklin W. HuangFranklin W. Huang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The cellular and molecular mechanisms involved in Randall’s plaque formation are poorly understood. Macrophages are closely associated with Randall’s plaques and are hypothesized to be an effector cell type, but their role in biomineralization remains unclear. The objective of this study was to better understand the origin and nature of Randall’s plaque-associated macrophages using single nucleus RNA sequencing (snRNAseq). METHODS: Randall’s plaque and renal papillary tissues were collected from adult patients undergoing stone surgery. Randall’s plaque tissue was obtained from endoscopic biopsies while whole renal papilla tissue were obtained from fresh nephrectomy specimens. snRNAseq was performed using the 10X Genomics platform. Additional tissue was used for immunohistologic staining. Analysis was conducted using R Studio and Seurat. Intercellular molecular interactions were predicted using CellphoneDB. RESULTS: A total of 34,468 nuclei from 24 patients (9 targeted Randall’s plaque biopsies) were included in the final analytic dataset. Myeloid cells were annotated using commonly expressed markers. We identified a macrophage population enriched in Randall’s plaque tissue consistent with previously described tissue-resident macrophages. Chemokine signaling was predicted to originate from epithelial cells in the loop of Henle rather than vascular endothelial cells. In addition to expressing both M1 and M2 polarization markers, which was confirmed with immunofluorescent staining (Figure 1), these macrophages expressed multiple isoforms of collagen and had ligand-receptor interactions with fibroblasts in fibrosis-promoting pathways. CONCLUSIONS: Tissue-resident macrophages, rather than monocyte-derived macrophages, are associated with Randall’s plaques and may contribute to collagen deposition and fibrosis. Further investigation is required to determine whether these cells play a causative role in Randall’s plaque formation. Source of Funding: California Urology Foundation (HY), UCSF Department of Medicine (FWH) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e43 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Heiko Yang More articles by this author Hanbing Song More articles by this author Tom Chi More articles by this author Marshall Stoller More articles by this author Sunita Ho More articles by this author Franklin W. Huang More articles by this author Expand All Advertisement PDF downloadLoading ...

Atf4 regulates angiogenic differences between alveolar bone and long bone macrophages by regulating M1 polarization, based on single-cell RNA sequencing, RNA-seq and ATAC-seq analysis

In the repair of maxillofacial bone defects, autogenous craniofacial bone can often provide superior clinical results over long bone grafts. Most current studies have focused on the osteogenic differences between alveolar bone marrow (ABM) and long bone marrow (LBM), however, studies about the angiogenic differences between the two are currently lacking. We downloaded single-cell RNA sequencing (scRNA-seq) of mouse ABM and LBM respectively from the public database, and the data were processed by using Seurat package. CellphoneDB2 results showed that macrophages had the strongest interaction with mesenchymal stem cells (MSCs) and endothelial cells (ECs). ELISA results confirmed that ABM macrophages secreted a higher level of vascular endothelial growth factor A (Vegfa) compared to LBM macrophages, which further promoted angiogenesis of ECs and MSCs. Using SCENIC package, six key transcription factors (TFs) were identified to regulate the difference between ABM and LBM macrophages, and activating transcription factor 4 (Atf4) was confirmed to be more expressed in ABM macrophages by polymerase chain reaction (PCR) and western blot (WB), with predicted target genes including Vegfa. Besides, the result of scRNA-seq implied ABM macrophages more in M1 status than LBM macrophages, which was confirmed by the following experiments. From the results of another assay for transposase accessible chromatin sequencing (ATAC-seq) and RNA-seq about M1 macrophages, Atf4 was also confirmed to regulate the M1 polarization. So, we suspected that Atf4 regulated the different expression of Vegfa between ABM and LBM macrophages by activating M1 polarization. After knocking down Atf4, the expression of M1 polarization markers and Vegfa were downregulated and vasculogenic differences were eliminated, which were subsequently reversed by the addition of LPS/IFN-γ. Our study might provide a new idea to improve the success rate of autologous bone grafting and treatment of oral diseases.

Discovery of microglia gonadotropin‑releasing hormone receptor and its potential role in polycystic ovarian syndrome.

Hypothalamic inflammation is a pathophysiological basis of polycystic ovarian syndrome (PCOS), while overactivated and/or excess M1 polarized microglia are considered to be the main reason for the occurrence of hypothalamic inflammation. Therefore, invitro and invivo experiments were performed to assess the relationships between microglia‑mediated inflammatory reactions and endocrine functions in the PCOS hypothalamus. The expression of gonadotropin‑releasing hormone (GnRH) receptor (GnRHR) was demonstrated in hypothalamic microglia, and it was found that low concentration, GnRH agonist, leuprolide acetate accelerated the expression of M2 polarization marker CD206, while high concentration leuprolide acetate increased the expression of M1 polarization marker CD86 invitro. Furthermore, aerobic exercise not only reduced the levels of serum testosterone, luteinizing hormone and GnRH and the amount of overactivated microglia, but also increased the number of M2 microglia in the hypothalamus of letrozole‑induced PCOS rats. In combination, these results not only demonstrated the expression of GnRHR in hypothalamic microglia, but also demonstrated that GnRH can induce microglial polarization, while aerobic exercise may improve the microglia‑mediated inflammatory reaction by reducing the expression of GnRHR in the hypothalamic microglia of PCOS rats.

Inhibition of Chitinase-3-like-1 expression by K284 ameliorates lipopolysaccharide-induced acute liver injury through down regulation of CXCL3

Chitinase 3 like 1 protein (CHI3L1) is involved in various types of infectious disease, especially sepsis. Aberrant expression of CHI3L1 could play a critical role in inflammation and immune responses. Previously, we found that compound K284 has the highest binding activity to CHI3L1. In this study, we investigated the effect of K284 on liver injury during sepsis using a lipopolysaccharide (LPS)-induced C57BL/6 mouse model. We found that the survival rate was a 65 % in LPS + K284 injected mice whereas it was 0% in LPS-treated mice. The serum levels of hepatic injury-related enzymes were decreased in K284-injected mice, indicating that septic liver damage was alleviated by K284. In addition, expression levels of M2 polarization markers were reduced in liver tissues of K284-injected mice. Depletion of CHI3L1 in LPS-treated THLE-2 cells decreased the expression of M2 polarization marker proteins. We investigated the major pathway involved in the observed effects and found that CXCL3 expression is correlated with that of CHI3L1. We found that the expression of CXCL3 was lowered in liver tissues of LPS + K284-treated mice compared with LPS-treated mice. M2 polarization-related cytokine levels were increased by the combination of LPS and recombinant CXCL3, but decreased by K284. M2 polarization-related cytokines, CHI3L1 and CXCL3 expression levels were decreased by CXCL3 siRNA, and further decreased by combination treatment of CXCL3 siRNA and K284. Therefore, CXCL3 might be a significant chemokine involved in the reduction of LPS-induced liver injury during sepsis by K284. Our findings indicate that K284 could be a suitable treatment for sepsis like hepatic injury.