Macrophage Polarization Research Articles

Refining prognostic assessment of diffuse large B-cell lymphoma: insights from multi-omics and single-cell analysis unveil SRM as a key target for regulating immunotherapy

PurposesPrevious studies have demonstrated that proliferation, stroma or immunity strongly influence the prognosis and therapeutic resistance of diffuse large B-cell lymphoma (DLBCL). Herein, we aimed to integrate proliferation, stromal, and immune (PSI) features to systematically evaluate the risk stratification and explore novel therapeutic targets in DLBCL.MethodsUsing data from multiple researches, we comprehensively evaluated the characteristics and prognostic impact of PSI features in DLBCL, and developed a novel risk stratification model (PSI score) with a consistent cutoff value to stratify the risk of 3,229 DLBCL patients from different cohorts. Mechanisms underlying adverse prognosis in the high-risk DLBCLs were investigated through transcriptomic (n = 3,229), genomic (n = 576), and scRNA-seq (n = 20) analyses.ResultsWe identified a high-risk DLBCL subgroup (HPSI, 36.1% of DLBCL). HPSI was characterized by upregulation of spermidine synthase (SRM) and cold tumor microenvironment (TME). Compared to low-risk group, HPSI exhibited poorer prognosis, with lower 3-year OS (51.7% vs. 78.1%, P < 0.0001) and PFS (48.9% vs. 72.6%, P < 0.0001) rates. HPSI shared malignant proliferative phenotype resembling Burkitt lymphoma. Genomic analysis revealed extensive copy-number loss in the chemokine and interleukin coding regions within HPSI. Bulk and scRNA-seq analyses indicated that upregulation of SRM might mediate cold TME in DLBCL, potentially through suppressing immune activation pathways, promoting dendritic cells (DCs) transformation into tolerogenic DCs, and facilitating M2 polarization of macrophages. Finally, for eventual clinical translation, we integrated the model with other clinical features to develop a comprehensive database for DLBCL.ConclusionOur study effectively simplifies risk stratification of DLBCL, revealing that immune microenvironment and SRM jointly shape a subgroup of DLBCL with extremely poor prognosis. Targeting SRM may become a potential strategy for modulating immunotherapy in DLBCL, providing new insight for immunotherapy.

CD271high cancer stem cells regulate macrophage polarization in head and neck squamous cell carcinoma.

Cancer stem cells (CSCs) are considered key drivers of progression in head and neck squamous cell carcinoma (HNSCC). Our single-cell RNA sequencing (scRNA-seq) analysis revealed predominant expression of CD271 in CSCs, however, its role as a CSC marker in HNSCC requires further elucidation. We investigated the stemness characteristics of CD271high HNSCC cells and their interactions with the tumor immune microenvironment. scRNA-seq data from hypopharyngeal squamous cell carcinoma (HPSCC) tissues were analyzed to identify expression profile of CSCs. Overall survival was compared between CD271high and CD271low patients based on immunostaining of HPSCC samples. The stemness of CD271high HNSCC cells was evaluated via an in vivo limiting dilution assay. In a C57BL/6 mice model, the percentage of immune cells and macrophage subtypes were analyzed by flow cytometry. The role of CD271 in macrophage polarization was further examined by in vitro coculture of CD271high cells with CD14+ monocytes. Gene expressions were analyzed by qPCR. CD271 is predominantly expressed in CSCs identified by scRNA-seq analysis. CD271 enhances HNSCC cell proliferation and is negatively correlated with patient prognosis in HPSCC. CD271 knockdown suppressed HNSCC tumor growth and regulated macrophage polarization within the TME. CD271high cells exhibited stemness features and enhanced tumor growth in vivo. CD271high HNSCC cells exhibit CSC characteristics and regulate macrophage polarization. Targeting CD271 may improve the immunosuppressive TME to inhibit tumor growth. Combining CD271-targeting agents with other therapies presents a promising strategy that may enhance therapeutic efficacy and prognosis in HNSCC.

Hepatic Abnormal Secretion of Apolipoprotein C3 Promotes Inflammation in Aortic Dissection.

Apolipoprotein C3 (apo C3) is primarily secreted by the liver and is involved in promoting sterile inflammation and organ damage under pathological conditions. Previous studies have shown that apo C3 is abundant in the plasma exosomes of patients with aortic dissection (AD), but its specific role in AD remains unclear. In vivo, adeno-associated virus was used to knock down hepatic apo C3 expression in an AD mouse model to assess the impact of liver-derived apo C3 on the development of AD. Invitro, recombinant apo C3 protein was added to the culture medium of J774A.1 macrophages to evaluate its effect on macrophage polarization and to identify the underlying mechanisms. Additionally, the effect of apo C3 on the function of aortic endothelial and smooth muscle cells was explored. Apo C3 in the aortas of AD mice was found to originate from abnormal hepatic secretion, which enters the bloodstream and subsequently deposits in the aorta. Adeno-associated virus-mediated hepatic apo C3 knockdown significantly reduced AD incidence (P=0.0036), macrophage infiltration (P=0.0004), and collagen deposition in the aorta (P=0.0016). Similarly, inhibiting the apo C3 receptor Toll-like receptor 2 significantly lowered AD incidence (P=0.0352). Invitro, recombinant apo C3 protein promoted M1 polarization and matrix metalloproteinase secretion in macrophages by activating the Toll-like receptor 2/NLR family pyrin domain containing 3 pathway. Additionally, apo C3 increased adhesion molecule expression in endothelial cells and induced inflammation, chemotaxis, and apoptosis in vascular smooth muscle cells. Our findings highlight the role of abnormally secreted hepatic apo C3 in promoting aortic inflammation.

Remodeling the Proinflammatory Microenvironment in Osteoarthritis through Interleukin-1 Beta Tailored Exosome Cargo for Inflammatory Regulation and Cartilage Regeneration.

Osteoarthritis (OA) presents a significant therapeutic challenge, with few options for preserving joint cartilage and repairing associated tissue damage. Inflammation is a pivotal factor in OA-induced cartilage deterioration and synovial inflammation. Recently, exosomes derived from human umbilical cord mesenchymal stem cells (HucMSCs) have gained recognition as a promising noncellular therapeutic modality, but their use is hindered by the challenge of harvesting a sufficient number of exosomes with effective therapeutic efficacy. Given that HucMSCs are highly sensitive to microenvironmental signals, we hypothesized that priming HucMSCs within a proinflammatory environment would increase the number of exosomes secreted with enhanced anti-inflammatory properties. Subsequent miRNA profiling and pathway analysis confirmed that interleukin-1 beta (IL-1β)-induced exosomes (C-Exos) exert positive effects through miRNA regulation and signaling pathway modulation. In vitro experiments revealed that C-Exos enhance chondrocyte functionality and cartilage matrix production, as well as macrophage polarization, thereby enhancing cartilage repair. C-Exos were encapsulated in hyaluronic acid hydrogel microspheres (HMs) to ensure sustained release, leading to substantial improvements in the inflammatory microenvironment and cartilage regeneration in a rat OA model. This study outlines a strategy to tailor exosome cargo for anti-inflammatory and cartilage regenerative purposes, with the functionalized HMs demonstrating potential for OA treatment.

Synergistic anti-oxidative/anti-inflammatory treatment for acute lung injury with selenium based chlorogenic acid nanoparticles through modulating Mapk8ip1/MAPK and Itga2b/PI3k-AKT axis

Oxidative stress and inflammatory dysregulation play crucial roles in pathogenesis of acute lung injury (ALI), and their cyclic synergy drives excessive inflammatory responses and further exacerbates ALI. Therefore, new effective strategies to treat ALI are urgently needed. Herein, a novel synergistic selenium based chlorogenic acid nanoparticle was developed to disrupt the cyclic synergistic effect between oxidative stress and inflammatory response in ALI. The chlorogenic acid, a polyphenol commonly found in herb, had been effectively conjugated with human serum albumin and coated on selenium nanoparticles (Se NPs) to create CHSe NPs. The CHSe NPs exhibited superoxide dismutase(SOD) like and glutathione peroxidase(GPX) like activities, effectively scavenging various types of reactive oxygen species (ROS), and inhibited the inflammatory response of macrophages. Additionally, with excellent biosafety, CHSe NPs exhibited superior therapeutic effects in ALI mice models in vivo, surpassing the performance of the clinic drug dexamethasone. They remarkably reduced ROS levels, and elevated the SOD and GPX enzyme activities in lung tissue to exert antioxidant effects. In addition, the CHSe NPs modulated the immune microenvironment of ALI by reversing M1 macrophage polarization, downregulating the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-a), nitric oxide synthase (iNOS), and myeloperoxidase (MPO), and upregulating anti-inflammatory cytokine (IL-10) levels, thereby alleviating excessive inflammation and decreasing neutrophil infiltration. Further mechanistic research revealed that CHSe NPs directly acted on and modulated the expression of Mapk8ip1 and Itga2b, which were upstream proteins of MAPK signaling pathway and PI3K-Akt signaling pathway, therefore impeding the cyclic synergy between oxidative stress and inflammatory dysregulation. In summary, CHSe NPs synergistically exert antioxidant and anti-inflammatory effects by regulating the MAPK signaling pathway and PI3K-Akt signaling pathway, showing enormous potential in the treatment of ALI.Graphical

Reactive Oxygen Species-Responsive Gel-Based Microneedle Patches with Antimicrobial and Immunomodulating Properties for Oral Mucosa Disease Treatment.

Oral ulcer wounds are difficult to heal due to bacterial infections, persistent inflammatory responses, and excessive reactive oxygen species (ROS). Therefore, the elimination of bacteria, removal of ROS, and reduction of inflammation are prerequisites for the treatment of mouth ulcer wounds. In this study, oligomeric proanthocyanidins (OPC) and 3-(aminomethyl)phenylboronic acid-modified hyaluronic acid (HP) were used to form polymer gels through dynamic covalent borate bonds. Minocycline hydrochloride (MH) was then loaded into the polymer gel, and a multifunctional MH/OPC-HP microneedles (MNs) with ROS-responsive properties was prepared using a vacuum method. The MH/OPC-HP MNs can rapidly release MH in a diffusive manner and sustainably release OPC in response to ROS. The gel-based MH/OPC-HP MNs extended the retention of OPC in oral ulcers, leading to prolonged ROS scavenging effects. Cytocompatibility and hemocompatibility tests showed that MH/OPC-HP MNs had good biocompatibility. Antibacterial experiments demonstrated that MNs loaded with MH exhibited excellent antibacterial effects. In vitro experiments indicated that MH/OPC-HP MNs could effectively clear ROS, reduce oxidative stress damage, inhibit M1-type macrophage polarization, and induce M2-type polarization. Furthermore, in vivo experiments revealed that MH/OPC-HP MNs could inhibit pro-inflammatory cytokines, promote neovascularization, accelerate epithelial healing of ulcers, and significantly promote healing in a rat model of oral ulcer wound infection. In summary, MH/OPC-HP MNs hold promise as a therapeutic strategy for enhancing the healing of oral ulcer wounds.

Akkermansia muciniphila relieves inflammatory response in DSS-induced ulcerative colitis in mice through regulating macrophage polarization via SCFAs-SLC52A2/FFAR2 pathway.

Ulcerative colitis (UC) remains an intractable and relapsing disease featured by intestinal inflammation. The anti-UC activity of Akkermansia muciniphila (AKK), an intestinal microorganism, has been widely investigated. The current work is to explore the impacts of AKK on UC and its possible reaction mechanism. In vivo UC model was induced by dextran sulfate sodium (DSS) and phorbol-12-myristate-13-acetate (PMA)-induced THP-1-M0 and raw264.7 macrophages were treated by lipopolysaccharide (LPS). H&E staining evaluated tissue damage. Inflammatory and oxidative stress levels were assessed by relevant kits. The high-throughput analysis of fatty acids was performed by the LC/MS method. RT-qPCR and Western blot detected related gene expression. Flow cytometry measured cell apoptosis and macrophage polarization. Energy metabolism was detected by ELISA, related assay kits, JC-1 staining, and Western blot. AKK reduced the pathological damage of mice colon tissues, alleviated oxidative stress and inflammatory response, upregulated the expression of Occludin-1 and SCFAs receptors, and stimulated M1 to M2 macrophage polarization in vivo. After FFAR2 was silenced, the promoting role of AKK in the viability and M1 to M2 macrophage polarization and the inhibitory role in oxidative stress, inflammation, apoptosis, energy metabolism disorder, necroptosis, and pyroptosis were both reverted. Conclusively, AKK might mediate SCFAs-SLC52A2/FFAR2 pathways to exert protective activities against intestinal inflammatory response in UC, suggesting that AKK might represent a novel and promising candidate for UC therapy.

P0023 Methionine Restriction Promotes Intestinal Mucosal Repair by Regulating Selenbp1 and Modulating Macrophage Polarization

Abstract Background Intestinal epithelial homeostasis is critical for maintaining gut integrity and function. Methionine is an essential amino acid involved in various cellular processes, and its dietary regulation may impact intestinal injury and repair mechanisms. Selenbp1, an oxidase of the methionine metabolite methanethiol, plays an important role in this process. This study investigates the role of methionine intake and Selenbp1 in colonic injury and epithelial repair. Methods Animal models with radiation-induced intestinal injury were used to assess the effects of methionine restriction and supplementation. Intestinal epithelial-specific knockout of Selenbp1 and control mice were used for in vivo experiments. Colonic organoids, macrophage co-culture assays, and biochemical assays, including qPCR, immunoblotting, and flow cytometry, were employed to study epithelial proliferation, SUMOylation of Satb2, and macrophage polarization. Clinical samples from patients with Crohn's disease were also analyzed for macrophage markers, as well as Satb2 and SUMO2/3 expression levels. Results Methionine restriction alleviated radiation-induced colonic injury in mice, as evidenced by improved histological scores and reduced inflammatory markers. In contrast, high methionine levels decreased Selenbp1 expression in the colon. Intestinal epithelial-specific Selenbp1 knockout exacerbated radiation-induced colonic mucosal injury, with significantly lower organoid survival rates and budding efficiency post-irradiation. However, this detrimental effect of Selenbp1 knockout was reversed upon methionine restriction, demonstrating the potential of dietary modulation. Furthermore, methionine supplementation led to a reduction in the SUMOylation of Satb2, a key regulator of intestinal epithelial homeostasis. The lower the level of Satb2 SUMOylation, the higher the clinical pathological features of Crohn's disease, such as disease activity. In co-culture assays, methionine supplementation enhanced M1 polarization of macrophages, further indicating a role of methionine in modulating immune responses. In clinical Crohn's disease (CD) specimens, we also found that a higher proportion of M1 macrophages correlated with lower expression levels of Selenbp1. Conclusion Methionine restriction promotes intestinal mucosal repair by regulating Selenbp1 and macrophage polarization, suggesting its potential as a therapeutic strategy for colonic repair.

Single-cell profiling of SLC family transporters: uncovering the role of SLC7A1 in osteosarcoma

BackgroundOsteosarcoma is the most common malignant bone tumor in children and adolescents, characterized by high disability and mortality rates. Over the past three decades, therapeutic outcomes have plateaued, underscoring the critical need for innovative therapeutic targets. Solute carrier (SLC) family transporters have been implicated in the malignant progression of a variety of tumors, however, their specific role in osteosarcoma remains poorly understood.MethodsThe single-cell sequencing data from GSE152048 and GSE162454, along with RNA-seq from the TARGET and GSE21257 cohorts, were utilized for the analysis in this study. LASSO regression analysis was conducted to identify prognostic genes and construct an SLC-related prognostic signature. Survival analysis and ROC analysis evaluated the validity of the prognostic signature. The ESTIMATE and CIBERSORT Packages were utilized to assess the immune infiltration status. Pseudotime and CellChat analyses were performed to investigate the relationship between SLC7A1, malignant phenotypes, and the immune microenvironment. CCK8 assays, EdU staining, colony formation assays, Transwell assays, and co-culture systems were used to assess the effects of SLC7A1 on cell proliferation, metastasis, and macrophage polarization. Finally, virtual docking identified potential drugs targeting SLC7A1.ResultsSLCs displayed distinct expression patterns across various cell types within the osteosarcoma microenvironment, with myeloid cells exhibiting a preference for amino acid uptake. A prognostic model comprising nine genes was constructed via LASSO regression, with SLC7A1 showing the highest hazard ratio. Multiple analytical algorithms indicated that SLCs were associated with immune cell infiltration and immune checkpoint gene expression. Single-cell analysis indicated that SLC7A1 was predominantly expressed in osteosarcoma cells and correlated with various malignant tumor characteristics. SLC7A1 also regulate interactions between tumor cells and macrophages, as well as modulate macrophage function through multiple pathways. In vitro assays and survival analysis demonstrated that inhibition of SLC7A1 suppressed the malignant phenotype of osteosarcoma cells, with SLC7A1 expression correlating with poor prognosis. Co-culture models confirmed the involvement of SLC7A1 in macrophage polarization. Finally, virtual screening and CETSA identified Cepharanthine as potential inhibitors of SLC7A1.ConclusionSLC-related prognostic signatures can be utilized for the prognostic evaluation of osteosarcoma. Pharmacological inhibition of SLC7A1 may be a feasible therapeutic approach for osteosarcoma.

DOP032 Extracellular vesicles from Bifidobacterium longum Subspecies infantis attenuate intestinal inflammation via macrophage polarization

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by chronic intestinal inflammation driven by a complex interplay of genetic, environmental, and immune interactions1. Despite significant advancements in IBD therapies, many patients still face challenges in achieving complete remission or maintaining long-term treatment efficacy. We hypothesize that extracellular vesicles from Bifidobacterium longum subspecies infantis (B. infantis-EVs) can regulate immune responses, promote balanced macrophage polarization, and modulate the gut microbiota, ultimately attenuating intestinal inflammation. Methods B. infantis-EVs were isolated via tangential flow filtration (TFF)2. In vitro studies examined cellular morphology, expression of inflammatory cytokines, and immunofluorescence staining of activated RAW 264.7 macrophage cells34. In a 3% dextran sulfate sodium (DSS) mouse model, disease severity was assessed using the Disease Activity Index (DAI), colon length, survival rate, and metagenome analysis. To confirm the targeting capability of B. infantis-EVs for inflamed lesions, cellular uptake was analyzed using confocal microscopy, and biodistribution was assessed with an In Vivo Imaging System (IVIS). Results To investigate the effects of B. infantis-EVs on macrophage polarization, we examined the cellular morphology and macrophage markers of RAW 264.7 cells. B. infantis-EVs suppress macrophage cells from driving an M1-like phenotype, and downregulated M1 markers (iNOS and CD86) while upregulating M2 markers (Arg1 and CD200R) (Fig. 1a-c). They also decreased the expression of LPS-induced pro-inflammatory cytokines (IL-1β, IL-2, IL-6, and TNF-α) while increasing anti-inflammatory cytokines (IL-10 and TGF-β) (Fig. 1d). In vivo, oral administration of B. infantis-EVs mitigated weight loss (p=0.0120), preserved colon length (p=0.0018), reduced DAI (p=0.0079), lowered histological score (p=0.0003), and modulated gut microbiota composition in DSS-treated mice compared to the DSS + PBS groups. Additionally, fluorescence intensities were stronger in the inflamed group treated with Cy5-labeled B. infantis-EVs compared to non-inflamed group at all-time points tested both cellular uptake and biodistribution (Fig. 1e). These findings suggest that B. infantis-EVs have the capability to target inflamed lesions and exhibit significant anti-inflammatory effects. Conclusion In this study, B. infantis-EVs attenuated intestinal inflammation via the modulation of macrophage polarization with its targeting capability to inflamed lesions. B. infantis-EVs could emerge as an innovative and promising therapeutic option for future IBD treatment.

P0176 Single-cell RNA sequencing reveals the immunological mechanisms underlying Anti-saccharomyces cerevisiae antibody positivity in both non-inflamed and inflamed mucosal tissues of IBD patients

Abstract Background Previous studies have shown anti-saccharomyces cerevisiae antibody (ASCA) positivity in inflammatory bowel disease (IBD) patients is associated with increased risk of developing IBD and poor prognosis. However, no comprehensive study has elucidated the ASCA positive-related immunological phenotype in non-inflamed and inflamed colonic mucosa and its clinical implications. Methods We collected endoscopic biopsies from 43 ulcerative colitis [28 non-inflamed sites and 43 inflamed sites] and 20 Crohn’s disease at Seoul National University Hospital. Single-cell RNA sequencing (scRNA-seq) was performed on these samples according to the ASCA positivity. First, we compared ASCA-positive and -negative samples obtained from non-lesion sites of UC patients. Second, we investigated immunologic phenotypes in inflamed mucosa based on the disease activity (remission vs active). Results We conducted scRNA-seq analysis of over 130,000 single cells from 28 biopsies obtained from non-inflamed tissues of UC patients. Substantial reduction in the frequency of type 3 innate lymphoid cells (ILC3) and expression of IL18 and IL22 in ILC3s, along with a significant decrease in tight junction-associated genes (TJP1, CLDN3, CLDN4) within epithelial cells were observed in the positive group[P &amp;lt;0.05]. In addition, data from positive group exhibited decreased levels of CSF2 in ILC3s, which is likely contributing to reduced M2 macrophage polarization. We performed scRNA-seq analysis of over 123,673 single cells from 63 biopsies obtained from inflamed tissues. In active inflamed status, the expression of IL-17A - a cytokine that helps maintain tight-junction barrier in the intestinal epithelium during inflammation was decreased in tissue-resident memory T cells (Tc17 TRM) [P 0.026]. In addition, ASCA positive resulted in decreased CD4 central memory T cells (CD4 TCM) [P 0.046], suggesting impaired gut barrier function and subsequent rapid response to reencountered antigens. Cytotoxic gene expression, including granzyme and perforin 1, was increased in CD8 T effector memory cells (CD8 TEM) [P&amp;lt;0.05] during remission, suggesting that ongoing subclinical inflammation may develop and contribute to tissue damage. Conclusion These findings suggest that Saccharomyces cerevisiae infection induces immunological changes characterized by gut barrier dysfunction, rapid responses to commensal antigens, and increased cytotoxic gene expression. Based on these findings, we believe that Saccharomyces cerevisiae may serve as a potential target for preventing disease progression in patients with IBD.

The Metabolite Indole-3-Acetic Acid of Bacteroides Ovatus Improves Atherosclerosis by Restoring the Polarisation Balance of M1/M2 Macrophages and Inhibiting Inflammation.

Emerging research has highlighted the significant role of the gut microbiota in atherosclerosis (AS), with microbiota-targeted interventions offering promising therapeutic potential. A central component of this process is gut-derived metabolites, which play a crucial role in mediating the distal functioning of the microbiota. In this study, a comprehensive microbiome-metabolite analysis using fecal and serum samples from patients with atherosclerotic cardiovascular disease and volunteers with risk factors for coronary heart disease and culture histology is performed, and identified the core strain Bacteroides ovatus (B. ovatus). Fecal microbiota transplantation experiments further demonstrated that the gut microbiota significantly influences AS progression, with B. ovatus alone exerting effects comparable to volunteer feces from volunteers. Notably, B. ovatus alleviated AS primarily by restoring the intestinal barrier and enhancing bile acid metabolism, particularly through the production of indole-3-acetic acid (IAA), a tryptophan-derived metabolite. IAA inhibited the TLR4/MyD88/NF-κB pathway in M1 macrophages, promoted M2 macrophage polarisation, and restored the M1/M2 polarisation balance, ultimately reducing aortic inflammation. These findings clarify the mechanistic interplay between the gut microbiota and AS, providing the first evidence that B. ovatus, a second-generation probiotic, can improve bile acid metabolism and reduce inflammation, offering a theoretical foundation for future AS therapeutic applications involving this strain.

P0144 Unveiling the role of the MFSD2A protein in contributing to the resolution of colitis-associated cancer inflammation

Abstract Background The intrinsic connection between inflammation and tumor promotion is well-established in colorectal cancer (CRC), where chronic inflammation, like ulcerative colitis (UC), plays a significant role in intestinal carcinogenesis (1). UC patients are at higher risk of colitis-associated cancer, emphasizing the inflammation-cancer connection. Previous studies from our laboratory revealed that UC patients with active inflammation have impaired production of inflammation-resolving DHA-derived metabolites due to a defect in the endothelial Major Facilitator Superfamily Domain containing 2A (MFSD2A) (2). Given the MFSD2A’s role in reducing colonic inflammation, we hypothesize it also counteracts cancer-associated inflammation. Methods Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with lentiviruses carrying either the GFP-tagged MFSD2A encoding sequence (MFSD2A-OE), or the relative GFP control, or the MFSD2A-targeting shRNAs, or the scramble as control, then analyzed by transcriptomics and lipidomics. A colon-adenocarcinoma cell line (Caco-2) was cocultured with either MFSD2A-OE or GFP CRC HIMEC to evaluate their proliferation rate. An orthotopic model of CRC was performed by intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and CRC HIMEC overexpressing either MFSD2A or GFP as control. Collected tumors were analyzed by flow cytometry and lipidomics (3). Results CRC HIMEC displayed a pro-resolving signature at the lipidomic level, consistently with higher MFSD2A expression as compared to the healthy. However, gene expression analysis shows a pro-inflammatory profile in CRC HIMEC, suggesting that MFSD2A levels alone are insufficient to induce a fully pro-resolving phenotype. We performed loss of function experiments to assess whether the pro-resolving profile in CRC HIMEC depends on MFSD2A. MFSD2A silencing reduces pro-resolving functions, confirming its role in balancing pro-inflammatory and pro-resolving signals in CRC regarding lipid production. Caco-2 co-cultured with MFSD2A-OE CRC HIMEC displayed a decrease in their proliferation rate after 48 and 72 hours of co-culture, indicating that endothelial MFSD2A overexpression reduces tumor cell proliferation. By comparison with animals injected with Caco-2 and GFP HIMEC, mice injected with a Caco-2/MFSD2A-OE CRC HIMEC mixture developed tumors with reduced weight and size, and increased pro-resolving lipid release. Additionally, flow cytometry showed increased M2 macrophage polarization, indicating a shift toward a pro-resolving immune response. Conclusion These data suggested that MFSD2A contrasts cancer-associated inflammation, ensuring the correct balance between pro-inflammatory and pro-resolving milieu.

Targeting MAPK14 by Lobeline Upregulates Slurp1-Mediated Inhibition of Alternative Activation of TAM and Retards Colorectal Cancer Growth.

Colorectal cancer (CRC) usually creates an immunosuppressive microenvironment, thereby hindering immunotherapy response. Effective treatment options remain elusive. Using scRNA-seq analysis in a tumor-bearing murine model, it is found that lobeline, an alkaloid from the herbal medicine lobelia, promotes polarization of tumor-associated macrophages (TAMs) toward M1-like TAMs while inhibiting their polarization toward M2-like TAMs. Additionally, lobeline upregulates mRNA expression of secreted Ly-6/UPAR-related protein 1 (Slurp1) in cancer cells. The inhibitory effects of lobeline on tumor load and TAM polarization are almost completely eliminated when Slurp1-deficient MC38 cells are subcutaneously injected into mice, suggesting that lobeline exerts an antitumor effect in a Slurp1-dependent manner. Furthermore, using target-responsive accessibility profiling, MAPK14 is identified as the direct target protein of lobeline. Mechanistically, upon binding to MAPK14 in colon cancer cells, lobeline prevents nuclear translocation of MAPK14, resulting in decreased levels of phosphorylated p53. Consequently, negative transcriptional regulation of SLURP1 by p53 is suppressed, leading to enhanced transcription and secretion of SLURP1. Finally, combination therapy using lobeline and anti-PD1 exhibits stronger antitumor effects. Taken together, these findings suggest that remodeling the immunosuppressive microenvironment using small-molecule lobeline may represent a promising therapeutic strategy for CRC.

ZnFe Layered Double Hydroxide Nanosheets Loaded with Cu Single-Atom Nanozymes with Multi-Enzyme-Like Catalytic Activities as an Effective Treatment for Bacterial Keratitis.

Bacterial keratitis (BK) is a type of corneal inflammation resulting from bacterial infection in the eye. Although nanozymes have been explored as promising materials in corneal wound healing, currently available nanozymes lack sufficient catalytic activity and the ability to penetrate bacterial biofilms, limiting their efficacy against the treatment of BK. To remedy this, ZnFe layered double hydroxide (ZnFe-LDH) nanosheets are loaded with Cu single-atom nanozymes (Cu-SAzymes) and aminated dextran (Dex-NH2), resulting in the formation of the nanozyme DT-ZnFe-LDH@Cu, which possesses peroxidase (POD)-, oxidase (OXD)-, and catalase (CAT)-like catalytic activities. This enables the nanozyme to generate reactive oxygen species (ROS), such as hydroxyl radicals (•OH), superoxide anion radical (O2 •-), and singlet oxygen (1O2) from hydrogen peroxide (H2O2), thereby killing the bacteria causing the infections. The surface Dex-NH2 enabled the DT-ZnFe-LDH@Cu to penetrate the biofilm and adsorb onto extracellular polymeric substances (EPS) produced by bacteria in the biofilm. Additionally, the DT-ZnFe-LDH@Cu successfully repaired P. aeruginosa-infected corneal injury in a BK rabbit model more effectively than commercially available tobramycin eye drops. This was enabled, in part, by the ability of DT-ZnFe-LDH@Cu to reduce inflammation by promoting the polarization of pro-inflammatory macrophages (M1) to anti-inflammatory macrophages (M2) and decrease the expression of α-smooth muscle actin (α-SMA) to promote wound healing without scar formation. This study provides an innovative concept for the treatment of BK and holds great scientific value and clinical application potential.

A Baicalin-Based Functional Polymer in Dynamic Reversible Networks Alleviates Osteoarthritis by Cellular Interactions.

Osteoarthritis (OA) is increasingly recognized as a whole-organ disease predominantly affecting the elderly, characterized by typical alterations in subchondral bone and cartilage, along with recurrent synovial inflammation. Despite the availability of various therapeutics and medications, a complete resolution of OA remains elusive. In this study, novel functional hydrogels are developed by integrating natural bioactive molecules for OA treatment. Specifically, baicalin (Bai) is combined with 2-hydroxyethyl acrylate (HEA) to form a polymerizable monomer (HEA-Bai) through esterification, which is subjected to reversible addition-fragmentation chain transfer (RAFT) polymerization to produce Bai-based polymer (Pm). These macromolecules are incorporated into Schiff-base hydrogels, which demonstrate excellent mechanical properties and self-healing performance. Notably, the Bai-based formulations are taken up by fibroblast-like synoviocytes (FLSs), where they regulate glycolysis. Mechanistically, inhibition of yes-associated protein 1 (YAP1) by the formulations suppressed the FLSs glycolysis and reduced the secretion of inflammatory factors, including interleukin 1β (IL-1β), IL-6, and IL-8. Furthermore, the functional hydrogel (AG-Pm)-OC, severing as a lubricant and nutrient, prolonged joint retention of Bai, thereby reducing cartilage degradation and synovial inflammation. Meanwhile, (AG-Pm)-OC alleviated joint pain by targeting the YAP1 signaling and inhibiting macrophage recruitment and polarization. Taken together, this flavonoid-based injectable hydrogel exhibits enhanced biocompatibility and efficacy against OA.

Tumor and intratumoral pathogen cascade-targeting photothermal nanotherapeutics for boosted immunotherapy of colorectal cancer.

Clinical benefits of immunotherapy in colorectal cancer (CRC) are limited due to the low immunogenicity and immunosuppressive tumor microenvironment. Fusobacterium nucleatum (Fn) is discovered to colonize CRC tumors and dampen immunotherapy by fostering an immunosuppressive TME. Herein, a controllable "Shielding-deshielding" N-acetylgalactosamine (GalNAc)-derived photothermal nanotherapeutic is developed to mediate cascade targeting toward tumor and intratumoral Fn for enhanced photothermal-immunotherapy. This nanotherapeutic can in situ generate near infrared-II laser-activatable photothermal agent by reacting with endogenous hydrogen sulfide in CRC. The Schiff bond-tethered hyaluronic acid coating not only facilitates precise localization within CRC but shieldes GalNAc-mediated liver targeting, which can be deshielded upon a slightly acidic TME to anchor Fn by binding to its lectin Fap2. This cascade-targeting nanotherapeutic enables efficacious tumor accumulation and reinforces photothermal therapy (PTT) efficacy. Notably, PTT efficiently induces immunogenic cell death in CRC cells, leading to augmented immunogenicity and CD8+ T cell activation. Meanwhile, synchronous eradication of Fn facilitates M1 macrophage polarization, and promotes intratumoral infiltration of CD8+ T cell by reducing succinic acid level, thereby further boosting antitumor immunity against both primary and distant tumors. Overall, this study involving cascade targeting-reinforced PTT and intratumoral microorganism modulation offers new insight into effective CRC immunotherapy.

Targeting ARPC1B Overcomes Immune Checkpoint Inhibitor Resistance in Glioblastoma by Reversing Pro-tumorigenic Macrophage Polarization.

Immunotherapy has elicited significant improvements in outcomes for patients with several tumor types. However, the immunosuppressive microenvironment in glioblastoma restricts the therapeutic efficacy of immune checkpoint blockade (ICB). In this study, we investigated which components of the immune microenvironment contribute to ICB failure in glioblastoma to elucidate the underlying causes of immunotherapeutic resistance. Macrophages were identified as a main contributor to ICB resistance. Expression of ARPC1B, a regulatory subunit of the Arp2/3 complex, was elevated in glioblastoma and correlated with macrophage enrichment and prognosis. ARPC1B in tumor cells increased STAT1 expression and subsequent IL10 production, which induced a pro-tumorigenic macrophage state. Mechanistically, ARPC1B inhibited the ubiquitination and degradation of STAT1 by preventing the E3 ubiquitin ligase NEDD4L from binding to STAT1 and by supporting the interaction between STAT1 and the deubiquitinase USP7. Inhibiting ARPC1B reshaped the immunosuppressive microenvironment and increased the efficacy of ICB in glioblastoma models. This study highlights the important role of ARPC1B in macrophage-mediated immunosuppression and proposes a combination treatment regimen for glioblastoma immunotherapy.

P1350 The Role of oral Porphyromonas gingivalis and Its Outer Membrane Vesicles in Inflammatory Bowel Disease

Abstract Background The etiology of Inflammatory bowel disease (IBD) remains elusive, but emerging evidence suggests a potential link between oral microbiota and IBD. Porphyromonas gingivalis(Pg.), a major pathogen in periodontal disease, and its outer membrane vesicles(OMVs) may play significant roles in the development and progression of IBD. Methods Animal models of IBD were established to observe the impact of Pg. and its OMVs on intestinal inflammation. In vitro cell culture was conducted to explore the interaction between Pg.-OMVs and intestinal epithelial cells and immune cells. Results Through the analysis of salivary microbiota in IBD patients and healthy individuals, we observed a significant increase in the abundance of Pg. in the saliva of IBD patients. OMVs may serve as a novel medium for oral bacteria to communicate with the gut. Our findings indicate that Pg.-OMVs can promote the development of IBD through various mechanisms. Firstly, they can disrupt intestinal barrier function by promoting inflammation in intestinal epithelial cells and degrading tight junction proteins. Additionally, Pg.-OMVs carry pathogenic factors and pro-inflammatory molecules, which activate intestinal macrophages to polarize towards the M1 phenotype, inducing inflammatory responses. Beyond systemic effects, we discovered that the key virulence factor LPS of Pg. can stimulate oral epithelial cells to secrete exosomes. These exosomes exhibit pro-inflammatory properties, including the disruption of the intestinal mucosal barrier and the promotion of macrophage polarization towards the M1 phenotype, with subsequent secretion of inflammatory factors. Conclusion Porphyromonas gingivalis and its outer membrane vesicles play important roles in the pathogenesis of IBD. Understanding these mechanisms provides new insights into the relationship between oral health and IBD and may lead to the development of novel diagnostic and therapeutic strategies for IBD.

Suberosin attenuates rheumatoid arthritis by repolarizing macrophages and inhibiting synovitis via the JAK/STAT signaling pathway

BackgroundRheumatoid arthritis (RA) is a systemic disease that primarily manifests as chronic synovitis of the symmetric small joints. Despite the availability of various targeted drugs for RA, these treatments are limited by adverse reactions, warranting new treatment approaches. Suberosin (SBR), isolated from Plumbago zeylanica—a medicinal plant traditionally used to treat RA in Asia—possesses notable biological activities. This study aimed to investigate the effects and potential underlying pathways of SBR on RA.MethodsTumor necrosis factor-alpha (TNF-α) induced inflammation in RA-derived fibroblast-like synoviocytes (RA-FLS), and the expression of proinflammatory mediators was assessed using q-RT PCR and ELISA after treatment with various SBR concentrations. Bone marrow-derived macrophages (BMDMs) were induced to differentiate into M1 and M2 macrophages, followed by treatment with various SBR concentrations and macrophage polarization assessment. Low-dose (0.5 mg/kg/d) and high-dose (2 mg/kg/d) SBR regimens were administered to a collagen-induced arthritis (CIA) mouse model for 21 days, and the anti-arthritic effects of SBR were evaluated. Network pharmacology and molecular docking analyses were used to predict the anti-arthritic targets of SBR. The effect of SBR on the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was evaluated.ResultsSBR suppressed macrophage polarization toward the M1 phenotype while enhancing their polarization toward the M2 phenotype. SBR reduced the levels of proinflammatory mediators in TNF-α-induced RA-FLS. Mechanistically, SBR inhibited the phosphorylation of the JAK1/STAT3 signaling pathway in RA-FLS and M1 macrophages and promoted the phosphorylation of the JAK1/STAT6 pathway in M2 macrophages, enhancing M2 polarization. In vivo, prophylactic treatment of low-dose SBR reduced M1 macrophage infiltration into synovial tissue, increased the proportion of M2 macrophages, and decreased the expression of inflammatory mediators in the serum and synovial tissue, alleviating synovial inflammation. SBR significantly alleviated arthritis in CIA mice through macrophage repolarization and inhibition of inflammation.ConclusionSBR significantly reduced clinical symptoms, joint pathological damage, and expression inflammatory cytokine expression in CIA mice. SBR exhibited anti-arthritic effects via the JAK1/STAT3 and JAK1/STAT6 signaling pathways, inhibiting synovial tissue inflammation and M1 macrophage polarization while promoting M2 macrophage polarization. Therefore, SBR may be an effective candidate for RA treatment.