Background Pulmonary macrophages are central orchestrators of inflammatory resolution and tissue repair in the lung. Whether vaping-associated macrophage dysregulation is spatially organized, sex-dimorphic, or detectable by bulk transcriptomic approaches remains poorly understood. Methods We performed a targeted re-analysis of a published spatial transcriptomics dataset of vaping-exposed murine lung, isolating macrophage-associated spots and applying unsupervised Leiden clustering to identify transcriptionally distinct macrophage microenvironments. Macrophage polarization was assessed using SMaRTspot, a novel adaptation of the validated SMaRT framework enabling polarization scoring at the spatial microenvironment level. Cell-type-specific differential gene expression analysis was performed against a purpose-built curated macrophage gene universe. Results In the vaped animals, the conserved homeostatic macrophage program of non-vaped lung was replaced with multiple transcriptionally distinct microenvironments. These microenvironments exhibited strong sexual dimorphism and were undetectable by bulk transcriptomic analysis. Vaped male microenvironments showed progressive homeostatic identity loss, inflammatory polarization signatures, senescence-associated biology, and fibrotic remodeling potential. Suppression of IFN-γ-responsive genes, including Ciita was a consistent feature of all vaped male-containing microenvironments regardless of polarization state. Vaped female macrophage microenvironments showed preserved homeostatic identity with alternative activation marked by a de novo lipid synthesis signature. A shared cross-sex microenvironment exhibited TLR4-associated inflammasome priming, senescence-associated secretory phenotype activation, and estrogen receptor upregulation in both sexes. Spatially distinct distributions between sexes suggested convergent responses to distinct local stimuli rather than a shared paracrine mechanism. Conclusions Vaping was associated with spatially organized, sex-dimorphic macrophage microenvironmental reprogramming invisible to bulk approaches. SMaRTspot extends validated macrophage polarization scoring to spatial transcriptomics and reveals opposing sex-specific polarization trajectories: male-associated suppression of IFN-γ responsiveness and antigen presentation capacity, and female-associated lipid-reprogrammed alternative activation with preserved homeostatic identity. These alterations may play a key role in vaping-associated pulmonary injury. As a re-analysis of a single-animal-per-condition dataset, these findings define testable hypotheses for confirmation in larger cohorts.
Aim The aim of our research was to investigate the potential effect of pristimerin (Pris) on atherosclerosis (AS), and clarify its underlying molecular mechanisms. Methods The effect of Pris on atherosclerotic lesions were assessed by using HE staining and Oil red O staining. Moreover, serum lipid profiles also detected using the commercial reagent kits. Inflammatory factors expression was determined employing qRT-PCR. The proteins associated with the phenotypic transformation of VSMCs and JAK2/STAT3 pathway-related proteins were assessed using western blot, immunohistochemistry and immunofluorescence. The proliferation and migration of VSMCs were determined utilizing EdU and Transwell assay. Results We found that Pris could suppress atherosclerotic lesions in mice. Moreover, Pris ameliorated lipid metabolism disorders and inflammation, and inhibited the phenotypic transformation of VSMCs in mice. In ox-LDL-treated VSMCs, Pris could also inhibit the phenotypic transformation and inflammatory responses. Subsequently, Pris was demonstrated to inhibit JAK2/STAT3 pathway in both HFD-caused AS mice and ox-LDL-treated VSMCs. The inhibition of Pris on the phenotypic transformation and inflammatory responses in VSMCs could be reversed by Colivelin TFA. Conclusion Pris could alleviate the progression of AS by inhibiting the phenotypic transformation of VSMCs and inflammatory responses via suppressing the activation of JAK2/STAT3 pathway.
Purpose Aberrant activation of insulin-like growth factor 1 receptor (IGF1R) in hepatocellular carcinoma (HCC) is closely associated with resistance to targeted therapies. However, the mechanism by which IGF1R transcriptionally upregulates programmed death-ligand 1 (PD-L1) to drive sorafenib resistance remained unclear. This study aimed to elucidate the molecular pathway through which IGF1R upregulates PD-L1 via the PI3K/AKT/NF-κB signaling cascade, thereby promoting sorafenib resistance. Methods The regulatory network of the IGF1R-PI3K/AKT/NF-κB-PD-L1 axis was analyzed using immunohistochemistry (20 HCC clinical samples), Western blotting (THLE-2 and Huh7/Hep3B/SNU-387 cells), functional assays (CCK8, colony formation, Transwell, JC-1, Annexin V-FITC/PI), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. Two independent in vivo cohorts were established: Cohort 1(SCID mice bearing Huh7 xenografts, IGF1 + sorafenib (SFB)) to assess IGF1R-driven resistance; Cohort 2 (NOD-SCID mice/SNU-387, PD-L1 knockdown/overexpression ± sorafenib) to validate PD-L1 as the downstream effector. Results IGF1R was significantly overexpressed in HCC. IGF1R activation increased the sorafenib IC50 by 2.3-fold and activated PI3K/AKT/NF-κB signaling. NF-κB directly bound to the PD-L1 promoter to drive its transcription, as confirmed by ChIP and dual-luciferase assays. PI3K inhibition with LY294002 blocked IGF1R-induced PD-L1 upregulation and restored sorafenib sensitivity. PD-L1 overexpression promoted HCC proliferation, migration, and cell-cycle progression while suppressing apoptosis; PD-L1 knockdown yielded opposite effects. In vivo, Cohort 1 confirmed that IGF1R activation promoted tumor growth, attenuated sorafenib efficacy, and elevated p-AKT, p-NF-κB, and PD-L1 in tumor tissues. Cohort 2 demonstrated that PD-L1 knockdown augmented sorafenib-induced tumor suppression, whereas PD-L1 overexpression blunted sorafenib efficacy, with reciprocal changes in Ki-67 and pathway phosphorylation. Conclusion IGF1R upregulates PD-L1 via the PI3K/AKT/NF-κB axis through direct NF-κB-mediated transcriptional activation, and PD-L1 serves as a critical downstream effector driving sorafenib resistance in HCC.
BACKGROUND:Septic cardiomyopathy (SCM) is a severe manifestation of sepsis characterized by myocardial dysfunction and systemic inflammation. The NLRP3 inflammasome-driven pyroptosis plays a pivotal role in SCM pathogenesis. This study aimed to elucidate the upstream molecular regulators of pyroptosis in SCM and investigate the functional role of serine/threonine kinase 39 (STK39) in modulating NLRP3 inflammasome activation. METHODS:Peripheral blood samples were collected from 44 patients with sepsis (19 with SCM) and 30 healthy controls. A murine cecal ligation and puncture (CLP) model and LPS-stimulated human cardiomyocytes (HCMs) were employed to simulate SCM. Cardiac function was assessed via echocardiography, and pyroptosis markers were quantified using ELISA, qRT-PCR, and western blot. Protein interactions and transcriptional regulation were evaluated by co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), immunofluorescence, and dual-luciferase reporter assays. RESULTS:STK39 expression was upregulated in SCM patients and septic mice. STK39 knockdown significantly attenuated cardiac pyroptosis in LPS-treated HCMs, and improved myocardial function in CLP-induced mice. Mechanistically, STK39 interacted with MAPK14 to activate p38 MAPK signaling, promoting ELK1-mediated transcriptional upregulation of NLRP3 and subsequent pyroptotic cell death. Rescue assay demonstrated that activation of p38 MAPK signaling greatly diminished the protective roles of STK39 silence on cardiomyocyte pyroptosis. CONCLUSION:This study identifies STK39 as a novel upstream regulator of NLRP3 inflammasome-mediated pyroptosis in SCM through the MAPK14/ELK1 signaling axis. These findings suggest that STK39 may serve as a potential molecular target for SCM.
Chronic prostatitis (CP) is common in young males. The etiology is unclear and treatment options are limited. Pyroptosis, a newly identified type of cell death, has been linked to CP pathogenesis, warranting further investigation. Here, the therapeutic effects of artemisinin (ART) on chronic non-specific inflammation were assessed. Comprehensive analyses, including evaluation of chronic pain progression, histopathology, and cytokine levels, revealed that ART significantly and dose-dependently suppressed NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis and alleviated pathological damage in experimental autoimmune prostatitis. Mechanistic studies indicated that ART markedly reduced oxidative stress in autoimmune mouse models by stimulating the Nrf2/HO-1 axis. The effects of ART were blocked by a specific Nrf2/HO-1 inhibitor, preventing inflammasome-mediated induction of pyroptosis. Cellular experiments showed that ART elevated Nrf2 levels and suppressed NLRP3 inflammasome-mediated pyroptosis in RAW264.7 macrophages following lipopolysaccharide (LPS) stimulation. In summary, ART mitigates CP by modulating the Nrf2/HO-1 pathway, thereby reducing oxidative stress and suppressing NLRP3-inflammasome-induced pyroptosis.
Antibody polyreactivity, defined as the ability of a single antibody molecule to recognize multiple unrelated antigens, is a common feature of the human antibody repertoire and contributes to immune defense and homeostasis. In addition to naturally occurring polyreactivity, antibodies can acquire inducible polyreactivity following exposure to environmental factors such as heme, ferrous ions, reactive oxygen species, or protein-destabilizing conditions. Although this "cryptic" polyreactivity is thought to arise in inflammatory and hemolytic settings, its relationship with natural polyreactivity remains poorly understood. In this commentary, we examine whether cryptic polyreactivity represents an amplification of pre-existing antigen-binding promiscuity or instead emerges de novo in otherwise monospecific antibodies. Analyses of clinical-stage therapeutic antibody repertoires indicate that heme- or ferrous ion-induced polyreactivity does not correlate with baseline natural polyreactivity, suggesting that these phenomena are largely mechanistically distinct. However, heme can enhance the promiscuous antigen recognition of certain naturally polyreactive broadly neutralizing anti-HIV-1 antibodies, underscoring the complexity of this phenomenon. A better understanding of the relationship between natural and cryptic antibody polyreactivity may help identify hidden liabilities in therapeutic antibodies and provide new insight into the adaptive flexibility of humoral immunity.
BACKGROUND:Pyoderma gangrenosum (PG), a refractory inflammatory disorder, remains a condition with incompletely understood pathogenesis. Currently, no FDA-approved treatments are available. Janus kinase (JAK) inhibitors have been reported sporadically in case studies as effective treatments for PG. OBJECTIVE:To characterize the cellular and molecular landscape associated with JAK/STAT (signal transducer and activator of transcription) pathway overactivation in PG lesions, and investigate the potential effects of tofacitinib on PG. METHODS:Single-cell RNA sequencing (scRNA-seq) and multiplex immunohistochemistry (mIHC) were employed to characterize the cellular and molecular landscape of JAK/STAT pathway overactivation in PG. In vitro experiments (immunostaining, qPCR, western blot, flow cytometry) were performed to further validate the potential effects of tofacitinib on PG. RESULTS:We identified significant overactivation of the JAK/STAT pathway in PG lesions-particularly in advanced stages-which, in terms of immune inflammation, is primarily driven by myeloid cells and T cells. This activation was associated with enhanced neutrophil extracellular trap (NET) formation in myeloid cells and aberrant differentiation/plasticity of Th17 and Th17.1 (IL-17/IFN-γ double-producing) cells. In vitro cell experiments further demonstrated that the JAK inhibitor tofacitinib suppresses STAT phosphorylation in myeloid and T cells, myeloid NETosis, and IL-17A production. CONCLUSION:Our study investigated immunological profiling of PG lesions via scRNA-seq and mIHC, along with in vitro validation. These findings delineate a multi-axis cellular and molecular landscape of PG linked to aberrant JAK/STAT signaling, and provide preliminary in vitro evidence supporting potential inhibitory effects of tofacitinib on key pathological processes of PG.
BACKGROUND:Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with limited treatment options. Guchang capsule (GC) has been used in traditional Chinese medicine, but its effects on IBD and the underlying molecular mechanisms remain unclear. METHODS:A dextran sulfate sodium (DSS)-induced colitis model in mice and an H₂O₂-induced Caco-2 cell injury model were established. Network pharmacology, Transwell co-culture, lncRNA THRIL overexpression/knockdown, and pathway intervention approaches were employed to systematically investigate the mechanism of GC in IBD. RESULTS:GC attenuated colonic inflammation and tissue damage in vivo, suppressed pro-inflammatory cytokines while restoring anti-inflammatory cytokines, and exhibited a dose-dependent protective effect on Caco-2 cells in vitro. Network pharmacology suggested that the p38 MAPK and NF-κB pathways might serve as key candidate pathways of GC. Mechanistically, p38 MAPK appeared to act upstream of NF-κB, and GC showed inhibitory effects on the activation of both pathways. In the intestinal epithelial-macrophage co-culture system, GC tended to restore barrier function and promote M1-to-M2 macrophage polarization. GC downregulated THRIL expression both in vivo and in vitro. THRIL overexpression activated p38 MAPK and NF-κB and partially attenuated the protective effects of GC, while THRIL knockdown partially mimicked the anti-inflammatory action of GC and exhibited a certain synergistic tendency. CONCLUSION:GC may alleviate DSS-induced colitis by downregulating THRIL and inhibiting the p38 MAPK and NF-κB pathways, providing a molecular basis for its therapeutic application in inflammatory bowel disease.
BACKGROUND:Intestinal ischemia-reperfusion (I/R) injury is a critical clinical condition whose pathogenesis is closely associated with immune-inflammatory responses. In this process, macrophage polarization plays a pivotal role in the initiation and progression of inflammation. While lactate, a microbial metabolite, has been shown to exert significant immunomodulatory effects and can influence macrophage polarization. Therefore, this study aimed to investigate the function of lactate and macrophage polarization in I/R injury. OBJECTIVE:This study aimed to investigate whether gut microbiota-derived lactate regulates macrophage M1 polarization via the NF-κB signaling pathway, thereby mitigating intestinal I/R injury. METHODS:A rat model of intestinal I/R injury was established. A time gradient of reperfusion was set to determine the optimal time point for observation. Interventions including macrophage depletion, antibiotic treatment, lactate administration, and NF-κB inhibition were employed. Intestinal injury severity, macrophage polarization status, and NF-κB pathway activity were analyzed using histopathology, immunofluorescence, Western blot, and qPCR. For in vitro experiments, macrophages were treated with LPS and/or lactate, with polarization phenotypes assessed by flow cytometry and immunofluorescence. RESULTS:Comparative analysis of Sham, I/R, and macrophage-depleted (I/R+MPD) groups revealed an increased proportion of M1 macrophages following I/R, while macrophage depletion attenuated intestinal injury, demonstrating the involvement of M1 polarization in I/R pathology. Time-course analysis demonstrated that the proportion of M1 macrophages peaked at 2-6 h of reperfusion, whereas intestinal lactate content reached its minimum at 2 h after reperfusion. Comparison among Sham, I/R, and antibiotic-treated (I/R+ABX) groups showed that gut microbiota depletion exacerbated intestinal injury and increased M1 macrophage proportion, indicating a protective role of gut microbiota against I/R injury. Furthermore, in vitro lactate treatment reduced M1 polarization in RAW264.7 cells. In vivo lactate administration alleviated intestinal tissue damage and decreased serum levels of pro-inflammatory cytokines secreted by M1 macrophages. Finally, Western blot analysis confirmed that lactate treatment suppressed phosphorylation of NF-κB pathway proteins p65 and IκBα. CONCLUSION:The gut microbiota metabolite lactate may alleviate intestinal I/R injury by inhibiting the NF-κB pathway and reducing macrophage M1 polarization.
Hypertrophic cardiomyopathy (HCM), the most prevalent inherited cardiovascular disease, is strongly linked to progressive heart failure and sudden cardiac death (SCD). However, its underlying pathogenic mechanisms remain incompletely understood, and effective therapeutic strategies are still lacking. Here, we established two murine HCM models harboring high SCD risk-associated mutations. Single-cell RNA sequencing revealed immune activation and enhanced fibrotic remodeling in the myocardium of these models. Therefore, we hypothesized that colchicine, a widely used anti-inflammatory drug known to reduce cardiovascular events in multiple cardiac disorders, may also represent a promising therapeutic candidate for HCM. As we expected, colchicine treatment attenuated pathological remodeling in our study, as evidenced by reduced cardiomyocyte hypertrophy, decreased fibrosis, and downregulation of cardiac stress markers (Anp, Bnp) and fibrotic mediators (Ctgf, Col1a1, Col3a1). In addition, colchicine attenuated pro-inflammatory macrophage populations and suppressed IL-6 expression, thereby contributing to the preservation of cardiac function. These findings provide the first preclinical evidence that colchicine alleviates myocardial inflammation and fibrosis in HCM, underscoring its potential as a novel therapeutic strategy to reduce fibrosis, lower SCD risk, and improve patient outcomes.
Atopic dermatitis (AD) is a self-reinforcing epithelial-immune disorder in which barrier failure, alarmin release, type 2 cytokines, oxidative stress and dysbiosis converge on interconnected signaling circuits. This review critically evaluates natural products by mode of action rather than by pathway name alone. The available evidence supports coordinated suppression of NF-kB/MAPK-driven inflammatory cascades, indirect attenuation of JAK/STAT amplification, activation of Nrf2/HO-1 and AHR-dependent barrier programs, and upstream modulation of microbiota-metabolite-immune signaling. We distinguish system-level upstream processes (barrier injury, dysbiosis and epithelial alarmins), inflammatory convergence hubs (NF-kB/MAPKs), cytokine amplifiers (JAK/STAT), and counter-regulatory nodes (Nrf2/AHR). Importantly, most natural-product studies rely on endpoint assays in immortalized keratinocytes or hapten-induced murine models; direct target engagement, kinetic selectivity and achievable skin exposure are rarely established. Translation therefore requires chemical standardization, human-relevant models, pharmacokinetic-pharmacodynamic analysis, sensitization testing, and evaluation under the altered pH and microbial conditions of AD skin. Rational adjunctive use with biologics or JAK inhibitors also demands formal assessment of CYP-mediated interactions and combined safety rather than an assumption that natural origin confers tolerability.
Peri-implantitis is caused by bacterial infection. Melatonin (Mel) is effective on treating periodontitis, but its efficacy on peri-implantitis remains unclear. The present study assessed the therapeutic effects of Mel on peri-implantitis and its mode of action. Peri-implant in vivo model was generated by implanting titanium implants and feeding rats with sugar water. Human periodontal ligament stem cells (hPDLSCs) were treated with lipopolysaccharide (LPS) to induce inflammation in vitro. Inflammation was evaluated by measuring cytokines contents. Osteogenic differentiation was assessed by Alkaline phosphatase and Alizarin red S staining, and measurements of RUNX2, OCN and OPN through quantitative real-time PCR. Effect of Mel in vivo was evaluated by hematoxylin-eosin staining. Results showed that Mel significantly inhibited inflammation but facilitated osteogenic differentiation in LPS-induced hPDLSCs. In LPS-treated hPDLSCs, Mel enhanced protein succinylation while elevating HAT1 expression. Notably, the beneficial effects of Mel on suppressing inflammation and promoting osteogenic differentiation were reversed by HAT1 knockdown. Mechanically, HAT1 knockdown inhibited succinylation and stability of p65 protein, but enhanced phosphorylation of p65 and p65 nuclear translocation at K311 site. Consistently, in the rat peri-implantitis model, Mel treatment suppressed inflammatory infiltration and promoted osteogenic differentiation, effects which were dependent on HAT1 expression and associated with increased p65 succinylation and decreased phosphorylation. In conclusion, we demonstrated that Mel exerts therapeutic effects on peri-implantitis in vivo, and our in vitro data suggests this may be mediated by promoting p65 succinylation by upregulating HAT1, thereby inhibiting p65 nuclear translocation.
BACKGROUND:Gastric cancer (GC) is one of the most common malignancies, characterized by high mortality and recurrence rates. Quercetin, a natural flavonoid found in various herbs and foods, exhibits potential therapeutic effects against GC. This study aims to explore the potential molecular mechanisms of quercetin in the treatment of GC. METHODS:Bioinformatic analyses were performed using the TCGA-STAD dataset to evaluate SERPINE1 expression, prognosis, angiogenesis-related pathway enrichment, and correlations with angiogenic factors. Molecular docking was employed to predict the binding affinity between quercetin and Serpine1, followed by validation using cellular thermal shift assay (CETSA). Cell proliferation and cytotoxicity were assessed by CCK-8 assays in GC cells and normal gastric epithelial GES-1 cells, and IC50 values were calculated. Serpine1 overexpression and siRNA-mediated knockdown were performed to investigate its functional role. Colony formation assays were used to evaluate clonogenic ability, apoptosis was analyzed by flow cytometry, and Transwell and wound healing assays were used to assess invasion and migration. Tube formation assays were conducted to examine angiogenic potential in HUVECs. Western blotting and qPCR were used to detect Serpine1, angiogenesis-related genes (VEGFA, FGF-2, FGF-7), and HUVEC angiogenesis markers (VEGFR-2, CD31, VE-cadherin). VEGFA secretion in cell supernatants was quantified by ELISA. RESULTS:Bioinformatic analysis showed that Serpine1 was highly expressed in GC tissues, associated with poor prognosis, and positively correlated with angiogenesis-related signatures and factors, including VEGFA, FGF-2, and FGF-7. Quercetin suppressed proliferation, invasion, and migration while promoting apoptosis in GC cells. At the selected treatment concentration, quercetin showed stronger inhibitory effects on GC cells than on GES-1 cells. Serpine1 expression was upregulated in GC cells and downregulated after quercetin treatment. Serpine1 knockdown inhibited GC cell proliferation, colony formation, migration, invasion, and angiogenic potential, while promoting apoptosis. Conversely, Serpine1 overexpression enhanced GC proliferation, invasion, and migration and inhibited apoptosis, whereas quercetin counteracted these effects. Quercetin also reduced angiogenesis-related factors (VEGFA, FGF-2, and FGF-7) in GC cells and decreased HUVEC angiogenesis-associated proteins (VEGFR-2, CD31, and VE-cadherin). These findings suggest that the anti-GC and anti-angiogenic effects of quercetin are mediated, at least in part, through targeting Serpine1. CONCLUSION:Our experimental results demonstrate that quercetin inhibits GC progression by targeting Serpine1 to suppress angiogenesis in vitro. Serpine1 represents a potential therapeutic target for GC treatment with quercetin, providing a new research direction for future treatment strategies.
Cannabis sativa L. produces a wide range of bioactive metabolites, including phytocannabinoids such as tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabigerol (CBG), which exhibit antiviral activity and modulate innate immune responses through Toll-like receptors (TLRs), particularly TLR4 and TLR7. The cross-regulation between cannabinoids and TLRs can influence the production of cytokines and antimicrobial peptides. Given their key role in orchestrating innate immunity, particularly inflammatory responses and antiviral activity, understanding these processes in bovine immune cells is essential. This study evaluated the immunomodulatory and antiviral effects of extracts from C. sativa chemotypes - THC-dominant (I), intermediate THC:CBD (II), CBD-dominant (III) and CBG-dominant (IV) - in bovine cells. In peripheral blood mononuclear cells, chemotype I induced an enhanced inflammatory response, increasing TLR4 and BMAP28 transcription and pro-inflammatory cytokine expression at both transcriptional and protein levels. Similarly, chemotype IV promoted a pro-inflammatory profile characterised by increased TLR4, BMAP28 and IFNβ expression, as well as elevated TNFα protein levels. In contrast, chemotypes II and III elicited anti-inflammatory effects. Chemotype III decreased TLR4, TLR7, BMAP28, TNFα and IFNβ transcription, although IFNγ protein levels increased. Chemotype II produced a comparable, although less pronounced, anti-inflammatory pattern, reducing TLR4, TLR7, TNFα and IFNβ while increasing BMAP28. Additionally, chemotypes II, III, and IV exhibited antiviral activity in BoAHV-1-infected MDBK cells, significantly reducing viral titres at 48 h post-infection. Overall, these findings demonstrate that C. sativa chemotypes differentially modulate bovine innate immune gene expression and may also exert antiviral effects, highlighting their potential as dual immunomodulatory and antiviral agents in bovine infectious contexts.
Acalabrutinib (ACP-196), a second-generation BTK inhibitor, is an FDA-approved drug for the treatment of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL). Beyond targeting malignant B cells, BTK inhibitors can modulate the abundance and functional properties of certain myeloid cell subsets in patients with CLL or MCL. In this study, we investigated the effects of acalabrutinib on the frequency, number, and phenotype of myeloid cell subsets using a mouse model of chronic inflammation induced by repeated injections of heat-killed Bacillus Calmette-Guérin (BCG). We found that acalabrutinib did not inhibit the expansion of myeloid-derived suppressor cells (MDSCs) or affect macrophage abundance in wild-type (WT) or myeloid cell-specific Traf3-deficient (M-Traf3-/-) mice, a mouse model that exhibits MDSC hyperexpansion during chronic inflammation. Interestingly, acalabrutinib significantly inhibited the expression of the immunoinhibitory ligand PD-L1 on monocytic MDSCs (M-MDSCs), macrophages, and dendritic cells (DCs) in vivo. Using cultured MDSCs and macrophages, we demonstrated that acalabrutinib suppressed Toll-like receptor 4 (TLR4)-induced transcript expression of genes encoding PD-L1 and other immunosuppressive molecules, including IL-10, Cox2, and iNos. Mechanistically, acalabrutinib attenuated TLR4-induced activation of NF-κB and Akt signaling. In summary, acalabrutinib inhibits the expression of PD-L1 and other immunosuppressive mediators in MDSCs and macrophages during inflammatory responses by suppressing TLR-driven signaling pathways. Our findings provide novel mechanistic insights and support a potentially broader therapeutic use of acalabrutinib in infectious and inflammatory diseases.
BACKGROUND:Colorectal cancer (CRC) is the third most common gastrointestinal cancer in terms of morbidity and mortality worldwide. Reprogramming of glucose metabolism will directly affect the energy supply pattern of tumor cells and determine their malignant biological behavior. Mitofusin-2 (MFN2) has been shown to be associated with glycolysis. Therefore, targeting MFN2 may be a potential therapeutic approach for CRC. METHODS:Firstly, GSE143939, GSE81558 dataset and GSE184093 dataset were used to screen differentially expressed mRNAs in CRC. The expression of MFN2 in colorectal cancer was detected by TCGA website, Immunohistochemistry and western blot analysis. The impacts of MFN2 on CRC malignant biological behavior were evaluated by a series of in vitro assays. The potential molecular mechanism of MFN2 was elucidated by co-immunoprecipitation, immunofluorescence staining, western blotting and rescue experiment. The effect of MFN2 on tumor growth capacity was assessed in xenograft model. RESULTS:TCGA and GEO data analysis showed that MFN2 was differentially expressed in CRC, which was significantly lowly expressed in CRC cell lines and tissues. Aberrant expression of MFN2 could reduce glycolysis of CRC cells in vitro and subsequently activate apoptosis and hinder proliferation and migration in vitro. In vivo, MFN2 overexpression retarded tumor growth rate. Mechanistically, MFN2 interacted with PFKP and facilitated the binding of TRIM21 to PFKP to mediate its degradation, thereby hindering the malignant progression of CRC. CONCLUSIONS:Overexpression of MFN2 suppressed CRC cell glycolysis and tumor growth by enhancing TRIM21-mediated degradation of PFKP. Targeting the MFN2/TRIM21/PFKP axis may serve as a new direction for CRC treatment.
BACKGROUND:Inflammatory oral ulcers (RAS, Behçet's oral lesions) severely impair quality of life. Local Th1 cytokine upregulation has been reported, yet systemic Th1 polarization, myeloid mediators and clinical correlations remain undefined. While apremilast and anti-TNF-α biologics aid systemic Behçet manifestations, no agents target isolated oral ulcers specifically. METHODS:Thirty-five ulcer patients (22 RAS, 8 Behçet's with sole oral lesions, 5 traumatic) and 30 matched healthy controls were enrolled. We performed plasma cytokine arrays, PBMC flow cytometry, HOK epithelial functional assays, and correlated Th1 markers with the validated Oral Ulcer Severity Score (OUSS). Longitudinal Th1 changes after topical steroids were assessed in an open-label pilot cohort of 18 patients. RESULTS:Inflammatory ulcer patients showed a dominant systemic Th1 signature (elevated IFN-γ, TNF-α, IL-12; expanded T-bet+ CD4 + T cells), absent in traumatic ulcers and controls. Mild plasma IL-17 elevation lacked matching Th17 cell expansion or epithelial toxicity; Th2 markers were unaltered. Activated CD14 + monocytes drove IL-12 release. Combined IFN-γ/TNF-α synergistically damaged keratinocytes, while IL-4/IL-17 had no effect. T-bet + CD4 + T cells correlated with OUSS (r = 0.68, p < 0.0001), and IFN-γ correlated with ulcer size (r = 0.54, p < 0.01). Post-treatment OUSS dropped by 65%, accompanied by separate significant reductions in T-bet + cells (p < 0.05) and IFN-γ (p < 0.01). Limitations include small subgroups, no ulcer biopsy validation, and uncontrolled longitudinal data prone to spontaneous remission bias. CONCLUSIONS:Myeloid-initiated systemic Th1 hyperactivity is a lineage-dominant feature of inflammatory oral ulcers, driving epithelial injury and correlating with severity. Though causal inference is constrained by study limitations, the Th1 pathway represents a rational target for precision therapy.
Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder characterized by persistent airflow limitation, and its pathogenesis is closely associated with cigarette smoke (CS) exposure. Neutrophil extracellular trap (NET) formation contributes critically to airway inflammation and tissue damage in COPD. However, the upstream regulators of NETosis remain poorly defined. In this study, a CS-induced mouse model of COPD was established, and transcriptomic sequencing of lung tissues was performed to identify differentially expressed genes. CXCL3 was significantly upregulated in COPD mouse lungs, a finding confirmed by qPCR, Western blot, and immunofluorescence, which localized CXCL3 predominantly to airway epithelial cells. Analysis of a public human single-cell RNA-seq dataset further validated CXCL3 enrichment in both immune and epithelial cells in COPD lungs. In vitro, knockdown of CXCL3 in BEAS-2B epithelial cells stimulated with cigarette smoke extract (CSE) reduced the ability of these cells to promote NETosis in neutrophil-like HL-60 cells. In vivo functional knockdown of CXCL3 attenuated NETs formation, pulmonary inflammation, and emphysema in CS-exposed mice. Mechanistically, Gene Set Enrichment Analysis (GSEA) revealed enrichment of the MAPK pathway, and Western blot analysis showed that CXCL3 knockdown suppressed CSE-induced phosphorylation of p38, ERK, and JNK. Pharmacological inhibition of each MAPK component (SB203580, U0126, SP600125) phenocopied the effect of CXCL3 knockdown, confirming that CXCL3 acts upstream of MAPK signaling to promote NETs formation. Collectively, this study identifies CXCL3 as a critical regulator of CS-induced NETosis and COPD progression through activation of the MAPK pathway, and highlights epithelial-derived CXCL3 as a promising therapeutic target for COPD intervention.
