Induced M2 Polarization Research Articles

Extracellular peroxiredoxin 6 released from alveolar epithelial cells as a DAMP drives macrophage activation and inflammatory exacerbation in acute lung injury.

Acute respiratory distress syndrome (ARDS) is featured with acute lung inflammatory injury. Our prospective study found that higher levels of peroxiredoxin 6(PRDX6) were detected in bronchoalveolar lavage (BAL) fluid from ARDS patients. Elevated PRDX6 was also correlated with monocytic activation and poor prognosis in ARDS patients. To investigate the origin of extracellular PRDX6, we conducted in vitro and in vivo experiments, demonstrating that PRDX6 can be actively released from alveolar epithelial cells under stress conditions. Our study demonstrated that it could be released from injured lung epithelial cells into the bronchoalveolar interstitial space in mice with acute lung injury and in vitro experiments. Moreover, exogenous PRDX6 was shown to activate the TLR4/NF-κB signalling pathway and induce M1 polarization of macrophages. Notably, the inflammatory effects of PRDX6 were mitigated by specific inhibition of the TLR4 (Toll-like receptor 4)-MD2 (Myeloid differentiation factor 2) complex. Using molecular docking simulations and in vitro binding assays, we confirmed a direct interaction between PRDX6 and MD2, further supporting its role as a damage-associated molecular patterns (DAMP) in ARDS. Our findings suggest that extracellular PRDX6 in bronchoalveolar lavage fluid could be a new DAMP factor in ALI, providing new insights into the pathogenesis of secondary hit in ALI/ARDS and highlighting PRDX6 as a potential therapeutic target for mitigating lung inflammation.

Dipyridamole Attenuates Experimental Periodontitis by Regulating M1 Macrophage Polarization via PKA/PKG Pathways.

Periodontitis is a chronic inflammatory disease initiated by dysbiosis of the local microbial community. As a non-specific phosphodiesterase inhibitor, dipyridamole features anti-oxidant and anti-inflammatory properties. This study aimed to investigate the effects of dipyridamole in an experimental rat model of periodontitis. Thirty rats were divided randomly into three groups (n = 10): non-ligature group (NL), ligature-induced periodontitis group (L), and ligature-induced periodontitis with dipyridamole administered group (L + D). All rats were euthanized on Day 14. Alveolar bone resorption was analyzed by microcomputed tomography. The mRNA levels of Il1b, Il6, tumor necrosis factor alpha (Tnfa), and inducible nitric oxide synthase (iNos) in gingival tissue were assessed by real-time quantitative polymerase chain reaction (qRT-PCR). Inflammation level, osteoclasts, and macrophages infiltration were analyzed histologically. RAW264.7 macrophages were stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) to induce M1 polarization. Different concentration of dipyridamole (0/2/10 μM) was added simultaneously. To explore the role of PKA/PKG pathways, RAW 264.7 macrophages were pretreated with 10 μM H-89 (PKA inhibitor) or 1 μM KT-5823 (PKG inhibitor), respectively. Expression of pro-inflammatory cytokines and M1 markers were detected by qRT-PCR, ELISA, and flow cytometry. Dipyridamole administration reduced alveolar bone loss, protein levels of inflammatory cytokines, and osteoclastogenesis in rats with experimental periodontitis. It also showed a tendency to decrease mRNA levels of Il1b, Il6, and Tnfa but without significant differencesin gingival tissues. Moreover, the infiltration of macrophage and M1 macrophage polarization in gingival tissue of periodontitis rats were inhibited by dipyridamole administration. In addition, dipyridamole could downregulate the gene expression of Il1b and Tnfa, as well as the protein level of TNF-α, CD86, and iNOS in RAW264.7 treated with P.g. LPS. When PKA/PKG pathways were blocked, the suppression of TNF-α, CD86, and iNOS was reversed significantly. Dipyridamole alleviated experimental periodontitis in rat models by regulating M1 polarization via activation of PKA/PKG pathways and emerges as a hopeful remedy for periodontitis.

RNA-seq shows Angiopoietin-like 4 promotes hepatocellular carcinoma progression by inducing M2 polarization of tumor-associated macrophages.

Hepatocellular carcinoma (HCC) represents a particularly aggressive form of cancer, characterized by its rapid progression and a complex interplay with the surrounding immune cellular environment. The primary objective of this study was to comprehensively investigate the role of ANGPTL4 in the context of HCC, utilizing RNA sequencing (RNA-seq) techniques to explore its impact on the M2 polarization of tumor-associated macrophages (TAM) and to uncover potential mechanisms driving HCC progression. To achieve this, we performed a transcriptome analysis of HCC cell lines, alongside cells obtained after co-culturing these lines with macrophages. By comparing gene expression profiles between the experimental groups exposed to ANGPTL4 and control groups, we aimed to identify specific molecular pathways associated with ANGPTL4's function. In addition to gene expression analysis, we employed flow cytometry to assess the polarization status of TAM. Furthermore, we utilized immunohistochemistry to evaluate the distribution of macrophages within HCC tissues and to quantify the expression levels of M2 macrophage markers. The results derived from RNA-seq analysis were particularly revealing; treatment with ANGPTL4 led to a significant upregulation of genes linked to M2 polarization, notably including CD206 and Arg1. In subsequent experimental observations, it became evident that ANGPTL4 not only facilitated the M2 polarization of macrophages but also enhanced the proliferation and migratory capacity of HCC cells through the upregulation of these same cytokines.

CHD1L accelated the progression of cutaneous squamous cell carcinoma via promoting PI3K/PD-L1 signaling pathway induced M2 polarization of TAMs

To investigate CHD1L’s impacts and molecular processes in hypoxic cutaneous squamous cell carcinoma. Monoclonal proliferation assays and CCK-8 were used to detect the proliferation capacity of A431 cells and Colon16 cells; wound healing experiments and Transwell assays were used to examine the migration and invasion capacity of A431 cells and Colon16 cells; angiogenesis experiments were conducted to assess the influence of A431 cells on angiogenesis; a nude mouse tumor xenograft experiment and HE staining were utilized to evaluate the impact of CHD1L on the progression of cutaneous squamous cell carcinoma; western blot analysis was performed to detect the expression of p-PI3K, p-AKT, and PD-L1 in A431 cells, as well as CD9, TSG101, PD-L1 in exosomes, and CD206, Arginase-1, iNOS, IL-1β, p-AKT, p-mTOR, VEGF, COX-2, MMP2, MMP9, p-ERK1/2 in tumor-associated macrophages. Under hypoxic conditions, CHD1L promoted the proliferation, migration, invasion, and angiogenesis of cutaneous squamous cell carcinoma. Furthermore, CHD1L facilitated the progression of cutaneous squamous cell carcinoma. CHD1L also increased the relative protein expression of p-PI3K, p-AKT, and PD-L1 in A431 cells, as well as CD9, TSG101, PD-L1 in exosomes, CD206, Arginase-1, p-AKT, p-mTOR, VEGF, COX-2, MMP2, MMP9, and p-ERK1/2 in tumor-associated macrophages, while inhibiting the relative protein expression of iNOS and IL-1β. Under hypoxic conditions, CHD1L can promote the proliferation and migration of cutaneous squamous cell carcinoma.

Masticatory Stress Maintains Mucosal Homeostasis via M2 Polarization.

In addition to breaking down food, mastication plays regulatory roles in tissue homeostasis. During mastication, the oral mucosa is subjected to masticatory stress, and the maintenance between mucosal damage and repair is a key process. Despite rapid healing in the oral mucosa during chewing, the molecular mechanisms underlying repair remain unclear. In this study, we investigated the impact of masticatory stress on masticatory mucosal wound healing. Our data showed that reduced masticatory stress on the oral mucosa in mice fed a soft food diet resulted in decelerated hard palate mucosal wound healing and decreased numbers of Ki67-positive cells as compared with the hard food diet group. An RNA sequencing analysis revealed lower expression levels of the mechanosensitive gene Piezo1 in the hard palate mucosa, as well as lower levels of transforming growth factor β1 (Tgf-β1) and Tgf-β receptor 2 (Tgf-βr2), in the soft food diet group than the hard food diet group. Immunofluorescence staining, flow cytometry, and polymerase chain reaction analyses demonstrated that masticatory stress induced M2 polarization of macrophages surrounding the wound in the hard food diet group, leading to increased Tgf-β1 secretion. The specific deletion of Piezo1 in macrophages (Piezo1DLysm) attenuated masticatory stress-induced accelerated healing in mice. These findings reveal the crucial role of masticatory stress-induced Piezo1 expression in tissue repair, potentially influencing M2 polarization of mucosal macrophages and Tgf-β1 secretion. These findings underscore the pivotal role of physical stimulation in the immune response and tissue repair and may provide important insights into therapeutic interventions for tissue repair.

Spermine synthase engages in macrophages M2 polarization to sabotage antitumor immunity in hepatocellular carcinoma.

Disturbances in tumor cell metabolism reshape the tumor microenvironment (TME) and impair antitumor immunity, but the implicit mechanisms remain elusive. Here, we found that spermine synthase (SMS) was significantly upregulated in tumor cells, which correlated positively with the immunosuppressive microenvironment and predicted poor survival in hepatocellular carcinoma (HCC) patients. Via "subcutaneous" and "orthotopic" HCC syngeneic mouse models and a series of in vitro coculture experiments, we identified elevated SMS levels in HCC cells played a role in immune escape mainly through its metabolic product spermine, which induced M2 polarization of tumor-associated macrophages (TAMs) and subsequently corresponded with a decreased antitumor functionality of CD8+ T cells. Mechanistically, we discovered that spermine reprogrammed TAMs mainly by activating the PI3K-Akt-mTOR-S6K signaling pathway. Spermine inhibition in combination with immune checkpoint blockade effectively diminished tumor burden in vivo. Our results expand the understanding of the critical role of metabolites in regulating cancer progression and antitumor immunity and open new avenues for developing novel therapeutic strategies against HCC.

Creatine promotes endometriosis progression by inducing M2 polarization of peritoneal macrophages.

Endometriosis is a chronic inflammatory disease characterized by the growth of endometrium-like tissues outside the uterine cavity, with an unclear pathogenesis. Analysis of single-cell sequencing data revealed the pivotal role of peritoneal macrophages in the development of endometriosis. We noted significant creatine enrichment and synthesis in peritoneal macrophages of patients with endometriosis compared to women without endometriosis. To further investigate the mechanisms of creatine in endometriosis, we performed RNA sequencing and in vitro experiments. We found that creatine reprograms M2 polarization by enhancing matrix metalloproteinases and anti-inflammatory cytokines, which are involved in angiogenesis, fibrogenesis, cell adhesion, and tissue repair. The co-culture of creatine-treated macrophages promoted migration and fibrogenesis of endometrial stromal cells, as well as angiogenesis of HUVECs in vitro. In summary, this article reveals that creatine might polarize M2 macrophages, promoting the initiation, fibrosis, and angiogenesis of ectopic endometrial lesions, ultimately resulting in the development of endometriosis. These findings underscore the crucial immunomodulatory role of creatine in the pathogenesis of endometriosis, offering a promising target for therapeutic intervention.

P2Y6R Inhibition Induces Microglial M2 Polarization by Promoting PINK1/Parkin-Dependent Mitophagy After Spinal Cord Injury.

Secondary injury presents a significant hurdle to neural regeneration following spinal cord injury (SCI), primarily driven by inflammation in which microglial cells play a crucial role. Despite the growing interest in mitophagy, studies on its occurrence post-spinal cord injury, particularly within microglial cells, are scarce. While P2Y6R has been implicated in inflammation regulation in various neurological conditions, its specific role in SCI remains uncertain. Our study revealed an upregulation of P2Y6R expression following SCI notably in microglial cells. Treatment with the P2Y6R-specific inhibitor, MRS2578, in mice facilitated M2 polarization of microglial cells and alleviated secondary damage, ultimately enhancing neural regeneration and functional recovery. In an in vitro BV2 inflammation model, our findings indicate that P2Y6R inhibition induced M2 polarization of BV2 cells and reduced neuroinflammation through PINK/Parkin-dependent mitophagy activation. In summary, our results underscore the potential of P2Y6R inhibition in promoting mitophagy-induced M2 polarization of microglial cells, thereby ameliorating secondary injury following spinal cord injury.

Mincle Maintains M1 Polarization of Macrophages and Contributes to Renal Aging Through the Syk/NF-κB Pathway.

Kidney is a classic organ undergoing senescence, and chronic inflammation has an important effect in cellular senescence. Mincle has been shown to be vital for maintaining the M1 phenotype of macrophages, but its role in regulating renal aging has yet to be explored. Young (2 months of age) and old (24 months of age) mice were used to analyze the changes of kidney damage during natural aging. Mice were subcutaneously injected with D-galactose (D-gal) to establish a renal aging model, and miR-6948-3p mimic and Mincle siRNA were administered via the tail vein every 3 days. Aged kidney and experimental aging kidney were characterized by decreased renal function and structural damage, and upregulated expression of senescence-related proteins and SPAP components. The ratio of M1 macrophages was increased in the aged kidney, and Mincle accumulated in the aged kidney macrophages. Administration of miR-6948-3p mimic or Mincle siRNA alleviated D-gal-induced renal senescence. LPS was used to induce M1 polarization of bone marrow-derived macrophages, and a coculture system of M1 macrophages and mouse renal tubular epithelial cells (TCMK-1) was established. Mincle was upregulated in LPS-induced M1 macrophages in vitro, and silencing Mincle in M1 macrophages attenuated M1 macrophage-induced TCMK-1 cell senescence. Mechanistically, Mincle was regulated by miR-6948-3p and maintained the M1 phenotype of macrophages through the Syk/NF-κB pathway. In conclusion, Mincle, posttranscriptionally suppressed by miR-6948-3p, modulated renal senescence by maintaining the phenotype of M1 macrophages through the Syk/NF-κB pathway.

Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

BackgroundTriple-negative breast cancer (TNBC) is characterized by high invasiveness and metastasis potential. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is strongly associated with breast cancer progression, although the underlying mechanisms are largely unknown.MethodsThe gene expression profiles of TNBC samples were downloaded from the TCGA database, and ubiquitination enzymes related to immune regulation were screened. UCHL1 expression in the TNBC tissues and in adipose-derived mesenchymal stem cells (ADSCs) stimulated in vitro with pro-inflammatory cytokines were analyzed. Exosomes were isolated from these stimulated ADSCs and transfected with scrambled (si-NC) or UCHL1-specific (si-UCHL1) siRNA constructs. TNBC cells were treated with the ADSCs-derived exosomes (ADSCs-Exos) and then co-cultured with macrophages or T cells. Finally, the tumorigenic potential of the ADSCs-Exos was evaluated by injecting the exosomes into mice bearing TNBC xenografts.ResultsUCHL1 was highly expressed in TNBC tissues and the stimulated ADSCs. The exosomes derived from stimulated ADSCs increased the viability and migration capacity of TNBC cells in vitro, and significantly increased Ki-67 expression through UCHL1. Furthermore, ADSCs-Exos induced M2 polarization of THP-1 monocytes by upregulating CD206 and Arg-1, and downregulating TNF-α and iNOS, and also decreased the proportion of CD3+CD8+ T cells. Mechanistically, UCHL1 regulated the STAT3 and PD-L1 signaling pathways through HDAC6. Exosomes derived from the control and cytokine-stimulated ADSCs also promoted tumor growth in vivo, and increased the expression of UCHL1, CD206, HDAC6, STAT3, and PD-L1. However, UCHL1 knockdown reversed the pro-tumorigenic effects of the ADSCs-derived exosomes in vivo and in vitro.ConclusionPro-inflammatory factors (IFN-γ + TNF-α) stimulating ADSCs-Exos enhance immune evasion in triple-negative breast cancer by regulating the HDAC6/STAT3/PD-L1 pathway via UCHL1 transporter. Thus, UCHL1 inhibition may enhance the response of TNBC to immunotherapy.Graphical

Exosome-derived circ-001422 promotes tumor-associated macrophage M2 polarization to accelerate the progression of glioma

Cytokines, tumor cells, and tumor-associated macrophages play crucial roles in the composition of glioma tissue. Studies have demonstrated that certain cytokines can induce M2 polarization of tumor-associated macrophages and contribute to the progression of glioma. Nonetheless, the intricate molecular interactions among cytokines, glioma cells, and tumor-associated macrophages remain largely unexplored. To investigate this cross-talk, a combination of RNA-sequencing, chromatin immunoprecipitation, immunoprecipitation, exosome isolation, and biological experiments were employed. Treatment with IL-6 significantly increased circ-001422 expression in glioma cells. A poorer prognosis was associated with elevated levels of circ-001422 in glioma tissues. Circ-001422 was transcribed directly by STAT3 through binding to its promoter. Circ-001422 exerted cancer-promoting functions when co-cultured with M2 macrophages. Furthermore, glioma cells were found to transfer circ-001422 to macrophages via an exosomal pathway, promoting M2 polarization. Mechanically, circ-001422 interacted with p300, resulting in STAT3 acetylation, thus promoting nuclear localization and transcriptional activity of STAT3/NF-κB and M2 macrophage polarization. In conclusion, glioma cells released exosomes enriched with circ-001422, which in turn induce M2 macrophage polarization by activating the STAT3/NF-κB pathway, thereby enhancing the aggressive characteristics of glioma cells. Targeting circ-001422 may represent a potential therapeutic approach for glioma.

An immunoregulatory and metabolism-improving injectable hydrogel for cardiac repair after myocardial infarction.

The hypoxia microenvironment post-myocardial infarction (MI) critically disturbs cellular metabolism and inflammation response, leading to scarce bioenergy supplying, prolonged inflammatory phase and high risk of cardiac fibrosis during cardiac restoration. Herein, an injectable hydrogel is prepared by Schiff base reaction between fructose-1,6-bisphosphate (FBP)-grafted carboxymethyl chitosan (CF) and oxidized dextran (OD), followed by loading fucoidan-coated baicalin (BA)-encapsulated zein nanoparticles (BFZ NPs), in which immunoregulatory and metabolism improving functions are integrally included. The grafted FBP serves to enhance glycolysis and provide more bioenergy for cardiomyocytes survival under hypoxia microenvironment, and elevating cellular antioxidant capacity via pentose phosphate pathway. OD with intrinsic anti-inflammatory effect can induce M2 polarization of macrophages to accelerate inflammatory elimination. While facing the possibility of endothelial-to-mesenchymal transition (EndoMT) caused by excessive expressed TGF-β1 secreted from M2 macrophages, BFZ NPs can target endothelia cells and intracellularly release BA to regulate the level of fatty acid oxidation, resulting in retained endothelial features and decreased risk of cardiac fibrosis. After being injecting the hydrogel into rats' infarcted cardiac, the 28-day-post surgical outcomes demonstrate its benign effects on restoring cardiac functions and attenuating adverse left ventricular remodeling. This study shows a promising measure for MI treatment with immunoregulating and metabolism regulation comprehensively.

Jing Si Herbal Tea Modulates Macrophage Polarization and Inflammatory Signaling in LPS-Induced Inflammation.

Background: Sepsis is a lethal disease due to uncontrolled inflammatory responses. Macrophages play an important role in sepsis-associated inflammation. Jing Si Herbal Tea (JSHT) is a plant-based regimen with anti-inflammatory properties designed to treat respiratory diseases; however, its underlying therapeutic mechanism remains unclear. This study aimed to investigate the effects of JSHT on macrophage polarization and inflammatory signaling in lipopolysaccharide (LPS)-induced inflammation to provide therapeutic approaches for inflammatory diseases. Methods: RAW264.7 cells were stimulated with LPS (1 µg/mL for 16 h) to induce inflammatory responses and treated by JSHT (0.0125% concentration for 4 h) in the experimental groups (Control, JSHT, LPS, Pre-JSHT, and Post-JSHT groups). We investigated the protein and cytokine expression levels using western blotting and enzyme-linked immunosorbent assay (ELISA). Macrophage morphology was observed using immunofluorescence staining. The polarization surface markers were detected by flow cytometry. Results: In the LPS group, the expressions of the inflammatory signaling (pERK, pJNK, and nuclear NFκB) and the pro-inflammatory cytokine levels (TNF-α, IL-1β, and IL-6) were significantly increased, with M1 polarization (CD68+/CD80+) compared to the Control group. In the Pre-JSHT and Post-JSHT groups, the expressions of the inflammatory signaling and the pro-inflammatory cytokine levels were significantly decreased, with a higher M2 polarization ratio (CD163+/CD206+) compared to the LPS group. RAW264.7 cells exhibited filopodia protruding from the cell surface in the LPS group, which were inhibited in the Pre-JSHT and Post-JSHT groups. Conclusions: LPS induced M1 polarization with elevated inflammatory signaling and cytokine levels, while JSHT not only decreased M1 polarization but also promoted M2 polarization with decreased inflammatory responses. We propose JSHT as a potential anti-inflammatory agent against LPS-induced inflammation.

Tarm1 may affect colitis by regulating macrophage M1 polarization in a mouse colitis model.

In this study, we aimed to explore the role of Tarm1 in juvenile mice with dextran sulfate sodium (DSS)-induced colitis and elucidate the mechanisms that affect intestinal barrier function. A DSS-induced pediatric inflammatory bowel disease mouse model was established using 4-week-old juvenile mice. Disease activity index and histopathological damage scores were determined using hematoxylin and eosin (H&E) staining. Tarm1, F4/80, CD68, and CD86 levels were detected using qPCR, western blotting, and immunofluorescence. Trans epithelial electric resistance (TEER) was detected using the transwell assay. Results revealed that juvenile colitis mice fed 4% DSS drinking water had increased Tarm1 expression in the colon tissue, increased macrophage M1 polarization, higher expression of pro-inflammatory cytokines, and an impaired intestinal mucosal barrier, compared with the control group. Tarm1-knockdown RAW264.7 cells inhibited lipopolysaccharide (LPS)-induced M1 polarization and attenuated barrier damage in co-cultured intestinal epithelial cells. Tarm1 expression was increased in colonic tissues of juvenile mice with colitis, and LPS-induced M1 polarization and intestinal barrier damage were attenuated in Tarm1-knockdown RAW264.7 cells. This suggests that attenuation of Tarm1 expression is a potential target for pediatric inflammatory bowel disease therapy.

Graft-derived extracellular vesicles transport miRNAs to modulate macrophage polarization after heart transplantation

Heart transplantation, a crucial intervention for saving lives of those with end-stage cardiac failure, often faces complications from acute allograft rejection. This study focuses on the intricate dynamics of immune cell interactions and specific communication pathways between organs, which are not yet well understood. Our study investigates this interplay using a murine heterotopic transplant model, using single-cell RNA sequencing to examine CD45+ immune cells from both the heart grafts and spleens. We conduct a comprehensive analysis focused on functional enrichment, cell trajectory, and interorgan communication in heart transplants, highlighting dynamic interactions between monocyte/macrophage subtypes that is mediated by extracellular vesicles (EVs). We use unsupervised clustering and elucidate the complex cellular interactions that influence allograft outcomes. Notably, we discovered that microRNA-363 and microRNA-709, carried by EVs from CD63+ graft macrophages, can induce M1 polarization within the recipient’s spleen via the Fcho2/Notch1 signaling pathway. These insights illuminate the nuanced immune responses during acute cardiac rejection and suggest that targeting EVs from graft-resident macrophages may offer a new strategy to mitigate transplant rejection.

MiR-126 accelerates renal injury induced by UUO via inhibition PI3K/ IRS-1/ FAK signaling induced M2 polarization and endocytosis in macrophages

To investigate the role and molecular mechanism of miR-126 in unilateral ureteral occlusion (UUO). We used bioinformatics to analyse miRNAs specifically expressed in UUO. The mouse model of UUO was established using RAW264.7 cells cultured in vitro and in vivo. The mice were divided into control group, miR-126-NC (negative control) group and miR-126-KD (knockdown) group. Then the relative expression of miR-126 was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the renal fibrosis was detected by Masson staining, and the protein expression of CD68, collagen I and collagen III in the kidney was detected by immunofluorescence assay. Immunohistochemistry detects α-SMA expression. Moreover, Western blotting was performed to measure the expressions of p-PI3K, CD163, CD206, CD86, iNOS, IL-1β, p-FAK, p-Rac-1, p-IRS-1 and MMP9. The relative fluorescence intensity of F-actin and p-FAK was detected by immunofluorescence assay, and the phagocytosis ability of macrophages was determined by phagocytosis assay with fluorescent microspheres. Bioinformatics analysis reveals miR-126-specific overexpression in UUO. Successful transfection of miR-126-NC and miR-126-KD was confirmed by RT-PCR. The selective reduction of miR-126 was validated by Masson, immunohistochemistry and immunofluorescence staining to decrease the area of UUO-induced renal fibrosis and to lower the expression of CD68, α-SMA, collagen I, and collagen III. The reduction of iNOS expression may also be achieved with selective knockdown of miR-126, as verified by cell tests. enhances the phagocytic ability of macrophages and the expression of p-PI3K, CD206, p-FAK, F-actin, p-Rac-1, p-IRS-1 and MMP9. MiR-126 can inhibit the PI3K signaling pathway, promote M1 macrophage polarization, and suppress the activation of FAK and Rac-1, thus accelerating the progression of UUO.

MiR-1226-5p is involved in radioresistance of colorectal cancer by activating M2 macrophages through suppressing IRF1

BackgroundAlthough the representative treatment for colorectal cancer (CRC) is radiotherapy, cancer cells survive due to inherent radioresistance or resistance acquired after radiation treatment, accelerating tumor malignancy and causing local recurrence and metastasis. However, the detailed mechanisms of malignancy induced after radiotherapy are not well understood. To develop more effective and improved radiotherapy and diagnostic methods, it is necessary to clearly identify the mechanisms of radioresistance and discover related biomarkers.MethodsTo analyze the expression pattern of miRNAs in radioresistant CRC, sequence analysis was performed in radioresistant HCT116 cells using Gene Expression Omnibus, and then miR-1226-5p, which had the highest expression in resistant cells compared to parental cells, was selected. To confirm the effect of miR-1226-5 on tumorigenicity, Western blot, qRT-PCR, transwell migration, and invasion assays were performed to confirm the expression of EMT factors, cell mobility and invasiveness. Additionally, the tumorigenic ability of miR-1226-5p was confirmed in organoids derived from colorectal cancer patients. In CRC cells, IRF1, a target gene of miR-1226-5p, and circSLC43A1, which acts as a sponge for miR-1226-5p, were discovered and the mechanism was analyzed by confirming the tumorigenic phenotype. To analyze the effect of tumor-derived miR-1226-5p on macrophages, the expression of M2 marker in co-cultured cells and CRC patient tissues were confirmed by qRT-PCR and immunohistochemical (IHC) staining analyses.ResultsThis study found that overexpressed miR-1226-5p in radioresistant CRC dramatically promoted epithelial-mesenchymal transition (EMT), migration, invasion, and tumor growth by suppressing the expression of its target gene, IRF1. Additionally, we discovered circSLC43A1, a factor that acts as a sponge for miR-1226-5p and suppresses its expression, and verified that EMT, migration, invasion, and tumor growth are suppressed by circSLC43A1 in radioresistant CRC cells. Resistant CRC cells-derived miR-1226-5p was transferred to macrophages and contributed to tumorigenicity by inducing M2 polarization and secretion of TGF-β.ConclusionsThis study showed that the circSLC43A1/miR-1226-5p/IRF1 axis is involved in radioresistance and cancer aggressiveness in CRC. It was suggested that the discovered signaling factors could be used as potential biomarkers for diagnosis and treatment of radioresistant CRC.

Role of cuproptosis in mediating the severity of experimental malaria-associated acute lung injury/acute respiratory distress syndrome

BackgroundMalaria-associated acute lung injury/acute respiratory distress syndrome (MA-ALI/ARDS) is a fatal complication of Plasmodium falciparum infection that is partially triggered by macrophage recruitment and polarization. As reported, copper exposure increases the risk of malaria infection, and copper accumulation-induced cuproptosis triggers M1 macrophage polarization. It is thus hypothesized that cuproptosis could act as a critical mediator in the pathogenesis of MA-ALI/ARDS, but its underlying mechanism remains unclear. The present study aimed to explore the role of cuproptosis in the severity of murine MA-ALI/ARDS.MethodsWe utilized an experimental model of MA-ALI/ARDS using female C57BL/6 mice with P. berghei ANKA infection, and treated these animals with the potent copper ion carrier disulfiram (DSF) or copper ion chelator tetrathiomolybdate (TTM). The RAW 264.7 macrophages, which were stimulated with infected red blood cells (iRBCs) in vitro, were also targeted with DSF-CuCl2 or TTM-CuCl2 to further investigate the underlying mechanism.ResultsOur findings showed a dramatic elevation in the amount of copper and the expression of SLC31A1 (a copper influx transporter) and FDX1 (a key positive regulator of cuproptosis) but displayed a notable reduction in the expression of ATP7A (a copper efflux transporter) in the lung tissue of experimental MA-ALI/ARDS mice. Compared to the P. berghei ANKA-infected control group, mice that were administered DSF exhibited a remarkable increase in parasitemia/lung parasite burden, total protein concentrations in bronchoalveolar lavage fluid (BALF), lung wet/dry weight ratio, vascular leakage, and pathological changes in lung tissue. Strikingly, the experimental MA-ALI/ARDS mice with DSF treatment also demonstrated dramatically elevated copper levels, expression of SLC31A1 and FDX1, numbers of CD86+, CD68+, SLC31A1+-CD68+, and FDX1+-CD68+ macrophages, and messenger RNA (mRNA) levels of pro-inflammatory cytokines (tumor necrosis factor [TNF-α] and inducible nitric oxide synthase [iNOS]) in lung tissue, but showed a remarkable decrease in body weight, survival time, expression of ATP7A, number of CD206+ macrophages, and mRNA levels of anti-inflammatory cytokines (transforming growth factor beta [TGF-β] and interleukin 10 [IL-10]). In contrast, TTM treatment reversed these changes in the infected mice. Similarly, the in vitro experiment showed a notable elevation in the mRNA levels of SLC31A1, FDX1, CD86, TNF-α, and iNOS in iRBC-stimulated RAW 264.7 cells targeted with DSF-CuCl2, but triggered a remarkable decline in the mRNA levels of ATP7A, CD206, TGF-β, and IL-10. In contrast, TTM-CuCl2 treatment also reversed these trends in the iRBC-stimulated RAW 264.7 cells.ConclusionsOur data demonstrate that the activation of cuproptosis with DSF aggravated the severity of MA-ALI/ARDS by partially inducing M1 polarization of pulmonary macrophages, while inhibition of cuproptosis with TTM contrarily ameliorated the severity of MA-ALI/ARDS by promoting macrophage M2 polarization. Our findings suggest that blockage of cuproptosis could be a potential therapeutic strategy for treatment of MA-ALI/ARDS.Graphical

Periostin+ myeloid cells improved long bone regeneration in a mechanosensitive manner

Myeloid cells are pivotal in the inflammatory and remodeling phases of fracture repair. Here, we investigate the effect of periostin expressed by myeloid cells on bone regeneration in a monocortical tibial defect (MTD) model. In this study, we show that periostin is expressed by periosteal myeloid cells, primarily the M2 macrophages during bone regeneration. Knockout of periostin in myeloid cells reduces cortical bone thickness, disrupts trabecular bone connectivity, impairs repair impairment, and hinders M2 macrophage polarization. Mechanical stimulation is a regulator of periostin in macrophages. By activating transforming growth factor-β (TGF-β), it increases periostin expression in macrophages and induces M2 polarization. This mechanosensitive effect also reverses the delayed bone repair induced by periostin deficiency in myeloid cells by strengthening the angiogenesis-osteogenesis coupling. In addition, transplantation of mechanically conditioned macrophages into the periosteum over a bone defect results in substantially enhanced repair, confirming the critical role of macrophage-secreted periostin in bone repair. In summary, our findings suggest that mechanical stimulation regulates periostin expression and promotes M2 macrophage polarization, highlighting the potential of mechanically conditioned macrophages as a therapeutic strategy for enhancing bone repair.

A multifunctional self-reinforced injectable hydrogel for enhancing repair of infected bone defects by simultaneously targeting macrophages, bacteria, and bone marrow stromal cells

Injectable hydrogels (IHs) have demonstrated huge potential in promoting repair of infected bone defects (IBDs), but how to endow them with desired anti-bacterial, immunoregulatory, and osteo-inductive properties as well as avoid mechanical failure during their manipulation are challenging. In this regard, we developed a multifunctional AOHA-RA/Lap nanocomposite IH for IBDs repair, which was constructed mainly through two kinds of reversible cross-links: (i) the laponite (Lap) crystals mediated electrostatic interactions; (ii) the phenylboronic acid easter bonds between the 4-aminobenzeneboronic acid grafted oxidized hyaluronic acid (AOHA) and rosmarinic acid (RA). Due to the specific structural composition, the AOHA-RA/Lap IH demonstrated superior injectability, self-recoverability, spatial adaptation, and self-reinforced mechanical properties after being injected to the bone defect site. In addition, the RA molecules could be locally released from the hydrogel following a Weibull model for over 10 days. Systematic in vitro/vivo assays proved the strong anti-bacterial activity of the hydrogel against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Moreover, its capability of inducing M2 polarization of macrophages (Mφ) and osteogenic differentiation of bone marrow stromal cells (BMSCs) was verified either, and the mechanism of the former was identified to be related to the JAK1-STAT1 and PI3K-AKT signaling pathways and that of the latter was identified to be related to the calcium signaling pathway, extracellular matrix (ECM) receptor interaction and TGF-β signaling pathway. After being implanted to a S. aureus infected rat skull defect model, the AOHA-RA/Lap IH significantly accelerated repair of IBDs without causing significant systemic toxicity. Statement of significanceRosmarinic acid and laponite were utilized to develop an injectable hydrogel, promising for accelerating repair of infected bone defects in clinic. The gelation of the hydrogel was completely driven by two kinds of reversible cross-links, which endow the hydrogel superior spatial adaption, self-recoverability, and structural stability. The as-prepared hydrogel demonstrated superior anti-bacterial/anti-biofilm activity and could induce M2 polarization of macrophages and osteogenic differentiation of BMSCs. The mechanism behind macrophages polarization was identified to be related to the JAK1-STAT1 and PI3K-AKT signaling pathways. The mechanism behind osteogenic differentiation of BMSCs was identified to be related to the ECM receptor interaction and calcium signaling/TGF-β signaling pathways.