TRIM59 deficiency promotes M1 macrophage activation and inhibits colorectal cancer through the STAT1 signaling pathway.

Tumor-associated macrophages play a crucial role in the tumor microenvironment. Tripartite motif 59 (TRIM59), a member of the tripartite motif (TRIM) family, is known to be associated with immunological diseases and macrophage activation. The functional and molecular mechanisms by which TRIM59 affects the occurrence and development of colorectal cancer (CRC) through macrophages are still not well understood. To address this, we generated macrophage-specific TRIM59 conditional knockout mice and utilized these mice to establish colitis-associated cancer and MC38 transplanted CRC models for further investigation. We found that the deficiency of TRIM59 in macrophages inhibited colorectal tumorigenesis in mice. This tumor-suppressive effect was achieved by promoting the activation of M1 macrophages via STAT1 signaling pathway. Further mechanistic studies revealed that TRIM59 could regulate macrophage polarization by ubiquitinating and degrading STAT1. These findings provide evidence that TRIM59 deficiency promotes M1 macrophage activation and inhibits CRC through the STAT1 signaling pathway, suggesting that the TRIM59/STAT1 signaling pathway may be a promising target for CRC.

A Novel Mechanism Linking Melatonin, Ferroptosis and Microglia Polarization via the Circodz3/HuR Axis in Subarachnoid Hemorrhage.

A subarachnoid hemorrhage (SAH) is life-threatening bleeding into the subarachnoid space that causes brain damage. Growing evidence has suggested that melatonin provides neuroprotection following SAH. Exploring the mechanisms underlying melatonin-mediated neuroprotection contributes to its clinical application in SAH. The plasma and cerebrospinal fluid (CSF) were collected from SAH patients, and SAH mice were established via pre-chiasmatic injection. Circodz3 expression, levels of IL-1ß and TNF-α, brain water content, neurological and beam-waling scores were determined. Ferroptosis was evaluated by analyzing levels of iron, lipid ROS, MDA, and GSH. The colocalization of circodz3 and Iba-1 was analyzed by immunofluorescence staining. Interaction of circodz3 and HuR was determined with RNA pull-down and RNA immunoprecipitation assays. Herein, we found that circodz3 was highly abundant in SAH patients and mice. Colocalization of circodz3 and Iba-1 in the left hemisphere of SAH mice suggested the implication of circodz3 in regulating microglia activation following SAH. Melatonin alleviated brain edema, neurological impairment, and microglia activation and inhibited circodz3 expression in SAH mice. Moreover, melatonin inhibited M1 polarization, oxidative stress and ferroptosis and restrained circodz3 expression in primary microglia following SAH. These effects were abrogated by circodz3 overexpression. Circodz3 knockdown inhibited ferroptosis and M1 polarization of BV2 microglia after SAH. Circodz3 interacted with HuR to facilitate ß-Trcp1-mediated ubiquitination and degradation, thus restraining the expression of SLC7A11 and GPX4. Collectively, melatonin exerted neuroprotection following SAH via inhibiting ferroptosis and M1 polarization through the circodz3/HuR axis. Our study suggests potential application of melatonin in the treatment of SAH.

Rhein inhibits M1 polarization of BV2 microglia through MAPK/IκB signalling pathway and reduces neurotoxicity caused by neuroinflammation.

BACKGROUND: Rhein is an anthraquinone compound with anti-inflammatory pharmacological activity. It has been found to play a neuroprotective role in neurological diseases, but the neuroprotective mechanism of rhein remains unclear. METHODS: SH-SY5Y cells serving as neuron-like cells and BV2 microglia were used. The toxicity of rhein on BV2 microglia and the viability of SH-SY5Y cells were measured by CCK-8 assay. The mRNA expression and secretion of pro-inflammatory cytokines were detected by qPCR and ELISA. Iba1, CD86 and pathway signalling protein in BV2 microglia were assessed by Western blot and immunofluorescence. Apoptosis of SH-SY5Y cells exposed to neuroinflammation was analysed through flow cytometry. RESULTS: Rhein inhibited MAPK/IκB signalling pathways. Further studies revealed that rhein inhibited the production of pro-inflammatory cytokines TNF-α, IL-6, IL-1ß and iNOS in BV2 cells and also inhibited the expression of M1 polarization markers Iba1 and CD86 in BV2 cells. Furthermore, rhein reduced the apoptotic rate and restored cell viability of SH-SY5Y cells exposed to neuroinflammation. CONCLUSIONS: Our study demonstrated that rhein inhibited microglia M1 polarization via MAPK/IκB signalling pathway and protected nerve cells through suppressing neuroinflammation.

Corrigendum: Sulforaphane diminishes moonlighting of pyruvate kinase M2 and interleukin 1ß expression in M1 (LPS) macrophages.

[This corrects the article DOI: 10.3389/fimmu.2022.935692.].

Pathogenic Autoimmunity in Atherosclerosis Evolves from HSP60-Reactive CD4 + T Cells.

Clinical evidence suggests anti-Hsp60 antibodies could contribute to atherosclerosis (AS) development, with unclear mechanisms. This study aims to explore the role of anti-HSP60-mediated autoimmunity in AS progression. HSP60-MHC tetramers were used to characterize HSP60-specific CD4 + T cells and assess TCR responses in mice. These cells were transplanted into AS mice to examine immune cell differentiation and infiltration in plaques and blood. Mice were injected with recombinant HSP60 or anti-HSP60 sera to evaluate effects on plaque progression and macrophage activity. Experiments with muMT-/-Apoe-/- mice examined humoral immunity's role in this autoimmunity. HSP60-reactive CD4 + T cells in AS mice differentiated into follicular helper cells, not Th1/Th17. Anti-HSP60 treatments increased macrophage infiltration and M1 polarization, indicating an anti-HSP60-driven inflammatory progression, dependent on humoral immunity. Anti-HSP60 influences macrophage infiltration, polarization, and plaque formation via humoral immunity, shedding light on its potential role in AS progression.

Activation of the cGAS-STING pathway by viral dsDNA leading to M1 polarization of macrophages mediates antiviral activity against hepatitis B virus.

BACKGROUND AND AIMS: Activation of the cGAS-STING pathway induces the production of type I interferons, initiating the antiviral immune response, which contributes to the clearance of pathogens. Previous studies have shown that STING agonists promote hepatitis B virus (HBV) clearance; however, few studies have investigated the effect of activating the cGAS-STING pathway in macrophages on HBV. METHODS: The polarization status of HBV particle-stimulated RAW264.7 macrophages was analyzed. After stimulation with HBV particles, the analysis focused on determining whether the DNA sensors in RAW264.7 macrophages recognized the viral double-stranded DNA (dsDNA) and evaluating the activation of the cGAS-STING pathway. Coculture of mouse macrophages and hepatocytes harboring HBV was used to study the antiviral activity of HBV-stimulated RAW264.7 macrophages. RESULTS: After stimulation with HBV particles, HBV relaxed circular DNA (rcDNA) was detected in RAW264.7 macrophages, and the protein expression of phospho-STING, phospho-TBK1, and phospho-IRF3 in the STING pathway was increased, as shown by Western blot analysis, which revealed that M1 polarization of macrophages was caused by increased expression of CD86. RT-PCR analyses revealed elevated expression of M1 macrophage polarization-associated cytokines such as TNFα, IL-1ß, iNOS, and IFNα/ß. In the coculture experiment, both HBsAg and HBeAg expression levels were significantly decreased in AML12-HBV1.3 cells cocultured with the supernatants of HBV-stimulated RAW264.7 macrophages. CONCLUSION: The results suggest that macrophages can endocytose HBV particles. Additionally, viral dsDNA can be recognized by DNA pattern recognition receptors, which in turn activate the cGAS-STING pathway, promoting the M1 polarization of macrophages, while no significant M2 polarization is observed. Macrophages stimulated with HBV particles exhibit enhanced antiviral activity against HBV.

MCP-1 controls IL-17-promoted monocyte migration and M1 polarization in osteoarthritis.

Osteoarthritis (OA) is a low-grade inflammatory joint illness in which monocytes migrate and infiltrate synovial tissue, differentiating into the pro-inflammatory M1 macrophage phenotype. IL-17 is a proinflammatory mediator principally generated by Th17 cells, which is elevated in OA patients; nevertheless, investigators have yet to elucidate the function of IL-17 in M1 polarization during OA development. Our analysis of clinical tissues and results from the open online dataset discovered that the level of M1 macrophage markers is elevated in human OA tissue samples than in normal tissue. High-throughput screening demonstrated that MCP-1 is a potential candidate factor after IL-17 treatment in OA synovial fibroblasts (OASFs). Immunohistochemistry data revealed that the level of MCP-1 is higher in humans and mice with OA than in normal tissues. IL-17 stimulation facilitates MCP-1-dependent macrophage polarization to the M1 phenotype. It also appears that IL-17 enhances MCP-1 synthesis in human OASFs, enhancing monocyte migration via the JAK and STAT3 signaling cascades. Our findings indicate the IL-17/MCP-1 axis as a novel strategy for the remedy of OA.

Isobavachalcone alleviates ischemic stroke by suppressing HDAC1 expression and improving M2 polarization.

Ischemic stroke is a serious cerebrovascular condition. Isobavachalcone (ISO) has been documented to exhibit an anti-inflammatory effect across a variety of diseases; however, its protective impact on ischemic stroke remains unexplored. In this study, we evaluated the influence of ISO in both transient middle cerebral artery occlusion/reperfusion (tMCAO/R) rat models and oxygen-glucose deprivation/reperfusion (OGD/R) cell models. We observed that pretreatment with 50 mg/kg ISO diminished the volume of brain infarction, reduced brain edema, and ameliorated neurological deficits in rats. A reduction in Nissl bodies was noted in the tMCAO/R group, which was reversed following treatment with 50 mg/kg ISO. TUNEL/NeuN double staining revealed a decrease in TUNEL-positive cells in tMCAO/R rats treated with ISO. Furthermore, ISO treatment suppressed the expression of cleaved caspase-3 and BAX, while elevating the expression of BCL-2 in tMCAO/R rats. The levels of CD86 and iNOS were elevated in tMCAO/R rats; conversely, ISO treatment enhanced the expression of CD206 and Arg-1. Additionally, the expression of TNF-α, IL-6, and IL-1ß was elevated in tMCAO/R rats, whereas ISO treatment counteracted this effect. ISO treatment also increased the expression of TGF-ß and IL-10 in the ischemic penumbra of tMCAO/R rats. It was found that ISO treatment hindered microglial M1 polarization and favored M2 polarization. Histone Deacetylase 1 (HDAC1) is the downstream target protein of ISO, with ISO treatment resulting in decreased HDAC1 expression in both tMCAO/R rats and OGD/R-induced cells. Overexpression of HDAC1 was shown to promote microglial M1 polarization and inhibit M2 polarization in OGD/R+ISO cells. Overall, ISO treatment mitigated brain damage following ischemic stroke by promoting M2 polarization and attenuated ischemic injury by repressing HDAC1 expression.

mCRP-Induced Focal Adhesion Kinase-Dependent Monocyte Aggregation and M1 Polarization, Which Was Partially Blocked by the C10M Inhibitor.

Monomeric C-reactive protein (mCRP) has recently been implicated in the abnormal vascular activation associated with development of atherosclerosis, but it may act more specifically through mechanisms perpetuating damaged vessel inflammation and subsequent aggregation and internalization of resident macrophages. Whilst the direct effects of mCRP on endothelial cells have been characterized, the interaction with blood monocytes has, to our knowledge, not been fully defined. Here we showed that mCRP caused a strong aggregation of both U937 cell line and primary peripheral blood monocytes (PBMs) obtained from healthy donors. Moreover, this increase in clustering was dependent on focal adhesion kinase (FAK) activation (blocked by a specific inhibitor), as was the concomitant adhesive attachment to the plate, which was suggestive of macrophage differentiation. Confocal microscopy confirmed the increased expression and nuclear localization of p-FAK, and cell surface marker expression associated with M1 macrophage polarization (CD11b, CD14, and CD80, as well as iNOS) in the presence of mCRP. Inclusion of a specific CRP dissociation/mCRP inhibitor (C10M) effectively inhibited PBMs clustering, as well as abrogating p-FAK expression, and partially reduced the expression of markers associated with M1 macrophage differentiation. mCRP also increased the secretion of pro-inflammatory cytokines Interleukin-8 (IL-8) and Interleukin-1ß (IL-1ß), without notably affecting MAP kinase signaling pathways; inclusion of C10M did not perturb or modify these effects. In conclusion, mCRP modulates PBMs through a mechanism that involves FAK and results in cell clustering and adhesion concomitant with changes consistent with M1 phenotypical polarization. C10M has potential therapeutic utility in blocking the primary interaction of mCRP with the cells-for example, by protecting against monocyte accumulation and residence at damaged vessels that may be predisposed to plaque development and atherosclerosis.

CD274 (PD-L1) negatively regulates M1 macrophage polarization in ALI/ARDS.

Background: Acute lung injury (ALI)/severe acute respiratory distress syndrome (ARDS) is a serious clinical syndrome characterized by a high mortality rate. The pathophysiological mechanisms underlying ALI/ARDS remain incompletely understood. Considering the crucial role of immune infiltration and macrophage polarization in the pathogenesis of ALI/ARDS, this study aims to identify key genes associated with both ALI/ARDS and M1 macrophage polarization, employing a combination of bioinformatics and experimental approaches. The findings could potentially reveal novel biomarkers for the diagnosis and management of ALI/ARDS. Methods: Gene expression profiles relevant to ALI were retrieved from the GEO database to identify co-upregulated differentially expressed genes (DEGs). GO and KEGG analyses facilitated functional annotation and pathway elucidation. PPI networks were constructed to identify hub genes, and differences in immune cell infiltration were subsequently examined. The expression of hub genes in M1 versus M2 macrophages was evaluated using macrophage polarization datasets. The diagnostic utility of CD274 (PD-L1) for ARDS was assessed by receiver operating characteristic (ROC) analysis in a validation dataset. Experimental confirmation was conducted using two LPS-induced M1 macrophage models and an ALI mouse model. The role of CD274 (PD-L1) in M1 macrophage polarization and associated proinflammatory cytokine production was further investigated by siRNA-mediated silencing. Results: A total of 99 co-upregulated DEGs were identified in two ALI-linked datasets. Enrichment analysis revealed that these DEGs were mainly involved in immune-inflammatory pathways. The following top 10 hub genes were identified from the PPI network: IL-6, IL-1ß, CXCL10, CD274, CCL2, TLR2, CXCL1, CCL3, IFIT1, and IFIT3. Immune infiltration analysis revealed a significantly increased abundance of M1 and M2 macrophages in lung tissue from the ALI group compared to the control group. Subsequent analysis confirmed that CD274 (PD-L1), a key immunological checkpoint molecule, was highly expressed within M1 macrophages. ROC analysis validated CD274 (PD-L1) as a promising biomarker for the diagnosis of ARDS. Both in vitro and in vivo experiments supported the bioinformatics analysis and confirmed that the JAK-STAT3 pathway promotes CD274 (PD-L1) expression on M1 macrophages. Importantly, knockdown of CD274 (PD-L1) expression potentiated M1 macrophage polarization and enhanced proinflammatory cytokines production. Conclusion: This study demonstrates a significant correlation between CD274 (PD-L1) and M1 macrophages in ALI/ARDS. CD274 (PD-L1) functions as a negative regulator of M1 polarization and the secretion of proinflammatory cytokines in macrophages. These findings suggest potential new targets for the diagnosis and treatment of ALI/ARDS.

Cynaroside improved depressive-like behavior in CUMS mice by suppressing microglial inflammation and ferroptosis.

Depression is a common mental health disorder, and in recent years, the incidence of various forms of depression has been on the rise. Most medications for depression are highly dependency-inducing and can lead to relapse upon discontinuation. Therefore, novel treatment modalities and therapeutic targets are urgently required. Traditional Chinese medicine (TCM) offers advantages in the treatment of depression owing to its multi-target, multi-dimensional approach that addresses the root cause of depression by regulating organ functions and balancing Yin and Yang, with minimal side effects. Cynaroside (CNS), an extract from the traditional Chinese herb honeysuckle, is a flavonoid compound with antioxidant properties. In this study, network pharmacology identified 44 potential targets of CNS associated with depression and several highly correlated inflammatory signaling pathways. CNS alleviated LPS-induced M1 polarization and the release of inflammatory factors in BV-2 cells. Transcriptomic analysis and validation revealed that CNS reduced inflammatory polarization, lipid peroxidation, and ferroptosis via the IRF1/SLC7A11/GPX4 signaling pathway. In vivo experiments showed that CNS treatment had effects similar to those of fluoxetine (FLX). It effectively ameliorated anxiety-, despair-, and anhedonia-like states in chronic unpredictable mild stress (CUMS)-induced mice and reduced microglial activation in the hippocampus. Thus, we conclude that CNS exerts its therapeutic effect on depression by inhibiting microglial cells from polarizing into the M1 phenotype and reducing inflammation and ferroptosis levels. This study provides further evidence that CNS is a potential antidepressant, offering new avenues for the treatment of depression.

Brucine alleviates fibroblast-like synoviocytes dysfunction and inflammation by regulating YY1 during rheumatoid arthritis.

Brucine is a weak alkaline indole alkaloid with wide pharmacological activities and has been identified to protect against rheumatoid arthritis (RA) process. Circular RNAs (circRNAs) are also reported to be involved in the pathogenesis of RA. Here, we aimed to probe the role and mechanism of Brucine and circ_0139658 in RA progression. The fibroblast-like synoviocytes of RA (RA-FLSs) were isolated for functional analysis. Cell proliferation, apoptosis, invasion, migration, as well as inflammatory response were evaluated by CCK-8 assay, EdU assay, flow cytometry, transwell assay, and ELISA analysis, respectively. qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. The binding between miR-653-5p and circ_0139658 or Yin Yang 1 (YY1), was verified using dual-luciferase reporter and RNA pull-down assays. Brucine suppressed the proliferation, migration, and invasion of RA-FLSs, and alleviated inflammation by reducing the release of pro-inflammatory factors and macrophage M1 polarization. RA-FLSs showed increased circ_0139658 and YY1 levels and decreased miR-653-5p levels. Circ_0139658 is directly bound to miR-653-5p to regulate YY1 expression. Brucine treatment suppressed circ_0139658 and YY1 expression but increased YY1 expression in RA-FLSs. Functionally, circ_0139658 overexpression reversed the suppressing effects of Brucine on RA-FLS dysfunction and inflammation. Moreover, circ_0139658 silencing alleviated the dysfunction and inflammation in RA-FLSs, which were reverted by YY1 overexpression. Brucine suppressed the proliferation, migration, invasion, and inflammation in RA-FLSs by decreasing YY1 via circ_0139658/miR-653-5p axis.

ARC@DPBNPs suppress LPS-induced acute lung injury via inhibiting macrophage pyroptosis and M1 polarization by ERK pathway in mice.

AIM OF THE STUDY: Exploring the protective effect of ARC@DPBNP on lipopolysaccharides (LPS)-induced ALI and its underlying mechanism. MATERIALS AND METHODS: ALI model was established by intransally administrating LPS (4 mg/kg) into C57BL/6 mice. The suppression effects of ALI was first compared between ARC (intragastric administrated, with doses ranging from 10 to 80 mg/kg) and ARC@BPBNPs (intratracheally administrated, with doses ranging from 1 to 4 mg/kg). Changes in lung histology post intratracheal intervention of 3 mg/kg ARC@DPBNPs were detected. The expression of pyrotosis pathway-related proteins in lungs as well as in RAW264.7 cells was detected by western blotting. The ASC expression in lung macrophages was examined using immune-fluorescent staining. The polarization of RAW264.7 cells and lung macrophages were detected by flow cytometry. The network pharmacology was constructed by Cytoscape, and the molecular docking was perfomed by AutoDock Vina. RESULTS: Docking predicted the high affinity of ARC to MAPK1 (ERK2). HE staining showed that ARC@DPBNPs attenuated LPS-induced ALI at a remarkably lower dose than ARC. The improved histopathological changes, lung W/D weight ratio, and decreased of inflammatory factor levels in lung collectively demonstrated the alleviation effects of ARC@DPBNPs. Compared with the LPS group, ARC@DPBNPs down-regulated the ERK pathway, resulted in a suppression of the macrophage pyroptosis and M1 polarization. This suppression effects could be removed by the ERK activator Ro 67-7476. CONCLUSION: ARC@DPBNPs attenuated ALI by suppressing LPS-induced macrophage pyroptosis and polarization, probably through down-regulation of the ERK pathway.

Neonatal microglia transplantation at early stage but not late stage after traumatic brain injury shows protective effects in mice.

The transplantation of neonatal microglia suppresses neuroinflammation caused by traumatic brain injury (TBI). This research aimed to explore the optimal time point of neonatal microglia transplantation for the best effect on the improvement of long-term cognitive function and inflammatory response in mouse models. qPCR and immunoblotting showed that the level of Iba1 gradually increased to the highest on day 7 and then gradually declined in TBI mice. Furthermore, it was observed that the level of CD86 and TNF-α increased to the highest after 7 days and subsequently was maintained until day 21, whereas the level of CD206 and IL-10 increased to the highest after 24 h and subsequently decreased until day 21 by qPCR and enzyme-linked immunosorbent assay. Afterward, it was shown that the neonatal microglia transplantation within 1 h significantly attenuated anxiety-like behavior and improved cognitive impairments in TBI mice. Mechanism exploration showed that the neonatal microglia could significantly decrease the level of cleaved caspase-3, M1/M2 polarization, and inflammatory cytokine (TNF-α) while increasing the level of anti-inflammatory factor IL-10 in TBI mice after transplantation within 1 h. Here, our findings demonstrated that neonatal microglia transplantation within 1 h significantly attenuated anxiety-like behavior and cognitive impairments caused by TBI.NEW & NOTEWORTHY The study demonstrated that neonatal microglia transplantation within 1 h significantly inhibited the pathogenesis of traumatic brain injury (TBI) in mouse models through inhibition of M1 polarization and promotion of M2 polarization.

Adaptor protein HIP-55 promotes macrophage M1 polarization through promoting AP-1 complex activation.

Overwhelming macrophage M1 polarization induced by malfunction of the renin-angiotensin-aldosterone system (RAAS) initiates inflammatory responses, which play a crucial role in various cardiovascular diseases. However, the underlying regulatory mechanism remains elusive. Here, we identified adaptor protein HIP-55 as a critical regulator of macrophage M1 polarization. The expression of HIP-55 was upregulated in M1 macrophage induced by Ang II. Overexpression of HIP-55 significantly promoted Ang II-induced macrophage M1 polarization, whereas genetic deletion of HIP-55 inhibited the Ang II-induced macrophage M1 polarization. Mechanistically, HIP-55 facilitated activator protein-1 (AP-1) complex activation induced by Ang II via promoting ERK1/2 and JNK phosphorylation. Moreover, blocking AP-1 complex activation can attenuate the function of HIP-55 in macrophage polarization. Collectively, our results reveal the role of HIP-55 in macrophage polarization and provide potential therapeutic insights for cardiovascular diseases associated with RAAS dysfunction.

Esketamine Prevents Postoperative Emotional and Cognitive Dysfunction by Suppressing Microglial M1 Polarization and Regulating the BDNF-TrkB Pathway in Ageing Rats with Preoperative Sleep Disturbance.

Postoperative depression (POD) and postoperative cognitive dysfunction (POCD) have placed heavy burden on patients' physical and mental health in recent years. Sleep disturbance before surgery is a common phenomenon that has been increasingly believed to affect patients' recovery, especially in aged patients, while little attention has been paid to sleep disruption before surgery and the potential mechanism remains ambiguous. Ketamine has been reported to attenuate POCD after cardiac surgery and elicit rapid-acting and sustained antidepressant actions. The present study aimed to clarify the effect of esketamine's (the S-enantiomer of ketamine) protective effects and possible mechanisms of action in POCD and POD. Our results showed that sleep disturbance before surgery exacerbated microglial M1 polarization and microglial BDNF-TrkB signalling dysfunction induced by surgery, resulting in postoperative emotional changes and cognitive impairments. Notably, treatment with esketamine reversed the behavioural abnormalities through inhibiting the M1 polarization of microglia and the inflammatory response thus improving BDNF-TrkB signalling in vivo and vitro. In addition, esketamine administration also reversed the impaired hippocampal synaptic plasticity which has been perturbed by sleep disturbance and surgery. These findings warrant further investigations into the interplay of esketamine and may provide novel ideas for the implication of preoperative preparations and the prevention of postoperative brain-related complications.

TRIM65 knockout inhibits the development of HCC by polarization tumor-associated macrophages towards M1 phenotype via JAK1/STAT1 signaling pathway.

BACKGROUND & AIMS: Tumor-associated macrophages (TAMs) are main components of immune cells in tumor microenvironment (TME), and play a crucial role in tumor progression. Tripartite motif-containing protein 65 (TRIM65) has been associated with tumor progression. However, whether TRIM65 regulate the interaction of tumor cell and TAMs in HCC and the underlying mechanisms remain unknown. In this study, we investigated the role of TRIM65 in TME of HCC and explored its underlying mechanisms. METHODS: The relation of TRIM65 expression level with tumor grades, TNM stages, and worse prognosis of HCC patients was evaluated by bioinformatics analysis, as well as immune infiltration level of macrophages. TRIM65 shRNA was transfected into HepG2 cells, and TRIM65 overexpression plasmid was transfected into Huh7 cells, and the effect of TRIM65 on cell growth was examined by EdU assay. The mouse subcutaneous Hep1-6 tumor-bearing model with WT and TRIM65-/- mice was established to study the role of TRIM65 in HCC. Immunohistochemistry staining, Immunofluorescence staining, qRT-PCR and western blot were performed to evaluate the effect of TRIM65 on TAM infiltration, TAM polarization and JAK1/STAT1 signaling pathway. RESULTS: Bioinformatics analysis revealed that TRIM65 was upregulated in 16 types of cancer especially in HCC, and high level of TRIM65 was strongly correlated with higher tumor grades, TNM stages, and worse prognosis of patients with HCC as well as immune infiltration level of macrophages (M0, M1, and M2). Moreover, we observed that TRIM65 shRNA-mediated TRIM65 knockdown significantly inhibited the HepG2 cells growth while TRIM65 overexpression highly increased the Huh7 cells growth in vitro. TRIM65 knockout significantly inhibited the tumor growth as well as macrophages polarization towards M2 but promoted macrophages polarization towards M1 in vivo. Mechanistically, the results demonstrate that TRIM65 knockout promoted macrophage M1 polarization in conditioned medium-stimulated peritoneal macrophages and in tumor tissues by activating JAK1/STAT1 signaling pathway. CONCLUSIONS: Taken together, our study suggests that tumor cells utilize TRIM65-JAK1/STAT1 axis to inhibit macrophage M1 polarization and promote tumor growth, reveals the role of TRIM65 in TAM-targeting tumor immunotherapy.

Brain microvascular endothelial cell-derived exosomes transmitting circ_0000495 promote microglial M1-polarization and endothelial cell injury under hypoxia condition.

Human brain microvascular endothelial cells (HBMVECs) and microglia play critical roles in regulating cerebral homeostasis during ischemic stroke. However, the role of HBMVECs-derived exosomes in microglia polarization after stroke remains unknown. We isolated exosomes (Exos) from oxygen glucose deprivation (OGD)-exposed HBMVECs, before added them into microglia. Microglia polarization markers were tested using RT-qPCR or flow cytometry. Inflammatory cytokines were measured with ELISA. Endothelial cell damage was assessed by cell viability, apoptosis, apoptosis-related proteins, oxidative stress, and angiogenic activity using CCK-8, flow cytometry, western blot, ELISA, and endothelial tube formation assay, respectively. We also established middle cerebral artery occlusion (MCAO) mice model to examine the function of circ_0000495 on stroke in vivo. Our study found that HBMVECs-Exos reduced M2 markers (IL-10, CD163, and CD206), increased M1 markers (TNF-α, IL-1ß, and IL-12), CD86-positive cells, and inflammatory cytokines (TNF-α and IL-1ß), indicating the promotion of microglial M1-polarization. Microglial M1-polarization induced by HBMVECs-Exos reduced viability and promoted apoptosis and oxidative stress, revealing the aggravation of endothelial cell damage. However, circ_0000495 silencing inhibited HBMVECs-Exos-induced alterations. Mechanistically, circ_0000495 adsorbed miR-579-3p to upregulate toll-like receptor 4 (TLR4) in microglia; miR-579-3p suppressed HBMVECs-Exos-induced alterations via declining TLR4; furthermore, Yin Yang 1 (YY1) transcriptionally activated circ_0000495 in HBMVECs. Importantly, circ_0000495 aggravated ischemic brain injury in vivo via activating TLR4/nuclear factor-κB (NF-κB) pathway. Collectively, OGD-treated HBMVECs-Exos transmitted circ_0000495 to regulate miR-579-3p/TLR4/NF-κB axis in microglia, thereby facilitating microglial M1-polarization and endothelial cell damage.

HgCl2 exposure mediates pyroptosis of HD11 cells and promotes M1 polarization and the release of inflammatory factors through ROS/Nrf2/NLRP3.

Mercury (Hg) is a serious metal environmental pollutant. HgCl2 exposure causes pyroptosis. When macrophages are severely stimulated, they often undergo M1 polarization and release inflammatory factors. However, the mechanisms by which mercuric chloride exposure induces macrophage apoptosis, M1 polarization, and inflammatory factors remain unclear. HD11 cells were exposed to different concentrations of Hg chloride (180, 210 and 240 nM HgCl2). The results showed that mercury chloride exposure up-regulated ROS, C-Nrf2 and its downstream factors (NQO1 and HO-1), and down-regulated N-Nrf2. In addition, the expressions of focal death-related indicators (Caspase-1, NLRP3, GSDMD, etc.), M1 polarization marker CD86 and inflammatory factors (TNF-α, IL-1ß) increased, and the above changes were related to mercury. Oxidative stress inhibitor (NAC) can block ROS/ NrF2-mediated oxidative stress, inhibit mercury-induced pyroptosis and M1 polarization, and effectively reduce the release of inflammatory factors. The addition of Vx-765 to inhibit pyroptosis can effectively alleviate M1 polarization of HD11 cells and reduce the expression of inflammatory factors. HgCl2 mediates pyroptosis of HD11 cells by regulating ROS/Nrf2/NLRP3, promoting M1 polarization and the release of inflammatory factors.

LncRNA-AC020978 Promotes Metabolic Reprogramming in M1 Microglial Cells in Postoperative Cognitive Disorder via PKM2.

The present work aimed to explore the role of long non-coding RNA (lncRNA)-AC020978 in postoperative cognitive disorder (POCD) and the underlying mechanism. The POCD mouse model was constructed through isoflurane anesthesia + abbreviated laparotomy. The AC020978 expression in brain tissue was silenced after lentivirus injection, then Morris water maze test was conducted to detect the cognitive disorder level, flow cytometry was performed to analyze M1 macrophage level, ELISA was carried out to measure inflammatory factor levels, H&E, Nissl and immunohistochemical staining was performed to detect the pathological changes in brain tissue, and Western blotting assay was adopted to detect protein expression. In addition, microglial cells were cultured in vitro, after lentivirus infection, the effect of AC020978 on the M1 polarization of microglial cells and glycolysis was observed. AC020978 overexpression promoted POCD progression and aggravated cognitive disorder in mice; in addition, the proportion of peripheral and central M1 cells increased, the inflammatory factor levels were upregulated, and microglial cells were activated. By contrast, AC020978 silencing led to cognitive disorder in mice and suppressed microglial cell activation and M1 polarization. In vitro experimental results indicated that AC020978 promoted the expression and phosphorylation of PKM2, which promoted inflammatory response through enhancing microglial cell glycolysis and M1 polarization. AC020978 interacts with PKM2 to promote the glycolysis and M1 polarization of microglial cells, thus regulating cognitive disorder and central inflammation in POCD.