Dual knockdown of Scn1b and Gsdmd induces an M2-like functional state in macrophages and modulates inflammation and fibrosis in a rat model of acute respiratory distress syndrome
The potential use of adoptive transfer of siRNA-modified macrophages to influence the progression of ARDS is investigated and dual knockdown of Scn1b and Gsdmd leads to the formation of an M2-like macrophage phenotype in vitro.
BACKGROUND
Sepsis-induced acute kidney injury (S-AKI) is marked by tubular damage, inflammation, and dysregulated autophagy. N6-methyladenosine (m6A) RNA modification has emerged as an important regulator of mRNA stability and cellular stress responses; however, its involvement and related regulatory mechanisms in S-AKI remain incompletely understood.
METHODS
Human HK-2 proximal tubular epithelial cells were stimulated with LPS to establish an in vitro S-AKI model, while CLP-induced septic C57BL/6J mice were used as an in vivo model. Global m6A levels, METTL3, and FOSL1 expression were assessed by ELISA, qRT-PCR, and Western blot. Functional roles of METTL3 and FOSL1 were evaluated using siRNA-mediated knockdown, plasmid-driven overexpression, and pharmacological inhibitors. MeRIP-qPCR and RIP-qPCR were performed to evaluate m6A-related enrichment and the association between METTL3 and FOSL1 mRNA. Inflammatory cytokines, autophagy-related markers, NF-κB and mTOR signaling alterations, renal function, and histopathological changes were assessed.
RESULTS
LPS stimulation increased global m6A levels and upregulated METTL3 and FOSL1 expression in HK-2 cells, accompanied by enhanced inflammatory responses and autophagy-related alterations. FOSL1 knockdown attenuated LPS-induced inflammation and autophagy-related changes, whereas METTL3 overexpression increased FOSL1 expression and exacerbated these effects. Further analyses indicated that METTL3 was associated with FOSL1 mRNA stability in an m6A-related manner. Alterations in FOSL1 expression were associated with changes in NF-κB and mTOR-related signaling responses under S-AKI conditions. In CLP-induced S-AKI mice, METTL3 knockdown reduced FOSL1 expression, alleviated inflammatory responses and autophagy-related alterations, and improved renal function and histopathological injury.
CONCLUSION
Our findings suggest that METTL3/FOSL1-associated regulatory responses may participate in S-AKI through m6A-related regulation, contributing to inflammatory and autophagy-related alterations. These findings provide further insight into the involvement of m6A-related regulation in the pathogenesis of S-AKI.
Feifei Shao, Junhao Pan, Qing-qing Yan et al.· Archives of Biochemistry and...· 0 citations
BACKGROUND
To investigate the protective effect of M2 macrophage-derived exosomes (M2-Exo) on high glucose-induced podocyte injury and the underlying molecular mechanism.
METHODS
THP-1 cells were induced to differentiate into M2 macrophages using PMA combined with IL-4/IL-13, and exosomes were extracted and identified. A high glucose-induced podocyte injury model was established in vitro, and a streptozotocin (STZ)-induced diabetic nephropathy (DN) mouse model was established in vivo to evaluate the protective effect and mechanism of M2-Exo.
RESULTS
M2-Exo were successfully isolated and identified. M2-Exo intervention restored podocyte viability, inhibited apoptosis, and suppressed NLRP3-mediated pyroptosis. SET-binding factor 2-antisense RNA 1 (SBF2-AS1) was highly expressed in M2-Exo and functioned as a competing endogenous RNA (ceRNA) by competitively binding to miR-650, thereby upregulating mouse double minute 2 (MDM2) expression. MDM2, acting as an E3 ubiquitin ligase, promoted NIMA-related kinase 7 (NEK7) ubiquitination and degradation, subsequently inhibiting NOD-like receptor family pyrin domain containing 3 (NLRP3) pyroptosis pathway activation. In vivo experiments confirmed that SBF2-AS1-overexpressing M2-Exo improved renal function and alleviated renal pathological injury in DN mice.
CONCLUSION
M2-Exo deliver SBF2-AS1 to regulate the miR-650/MDM2/NEK7 axis, inhibit NLRP3-mediated podocyte pyroptosis, and ameliorate the progression of DN.
Yan Zhang, Qing Zhan, Min Zhao et al.· Cellular Signalling· 0 citations
BACKGROUND
In non-small cell lung cancer (NSCLC), M2-type tumor-associated macrophages (M2-TAMs) promote malignancy development, and long non-coding RNA FGD5 antisense RNA 1 (FGD5-AS1) is upregulated. However, it remains unclear whether M2-TAMs exert their effects by regulating FGD5-AS1.
METHODS
THP-1 cells differentiation into M2-type macrophages was induced, before 48 h of NSCLC cell co-culture to simulate M2-TAMs. The functions of M2-TAMs in NSCLC cell biological behavior following FGD5-AS1 silencing were assessed through loss-of-function experiments, such as CCK-8, scratch, Transwell, and nude mouse tumorigenesis assays. Epithelial-mesenchymal transition (EMT)-related protein levels were measured by Western blotting, whereas FGD5-AS1 levels within cells were determined through qRT-PCR assay. The subcellular localization of FGD5-AS1 in NSCLC cells was determined using fluorescence in situ hybridization and subcellular fractionation assays. Finally, bioinformatics analysis was conducted to predict miRNAs that potentially bind to FGD5-AS1, followed by experimental validation using a dual-luciferase assay.
RESULTS
Under co-culture conditions with M2-TAMs, the viability, migratory capacity, invasive potential, and EMT of NSCLC cells were significantly enhanced, accompanied by an upregulation in the expression level of FGD5-AS1. However, silencing of FGD5-AS1 in NSCLC cells led to inhibition of proliferation, migration, invasion, and EMT in cells co-cultured with M2-TAMs, along with a reduction in tumorigenic potential. In NSCLC cells, FGD5-AS1 interacted with miR-22-3p by acting as a sponge, thereby modulating the expression level of miR-22-3p.
CONCLUSION
M2-TAMs promote the invasive phenotype of NSCLC cells via upregulating the expression of FGD5-AS1 in NSCLC cells.
Han-Gui Wang, Ning Wang, Lijie Yang et al.· Tissue & Cell· 0 citations
Acute Respiratory Distress Syndrome (ARDS) is a severe inflammatory lung disorder associated with high mortality. Currently, there are no FDA-approved pharmaceuticals approved for treatment of this disorder. This study evaluated the therapeutic potential of Δ8-Tetrahydrocannabinol (Δ8-THC), a cannabinoid that is less psychoactive than Δ9-THC, in a murine model of LPS-induced ARDS.
Female C57BL/6 mice received LPS (10 mg/kg, intratracheally) to induce ARDS, followed by Δ8-THC or vehicle treatment (i.p.). After 48h, lung tissue was collected for scRNA-seq analysis.
scRNA-seq analysis of vehicle & Δ8-THC treated lungs identified sixteen transcriptionally dis-tinct clusters across stromal, endothelial, myeloid, and lymphoid lineages. Δ8-THC significantly reduced infiltration of neutrophils, while increasing the populations of polymorphonuclear MDSCs, immune endothelial cells and macrophages compared to vehicle. Neutrophil clusters from vehicle group showed enrichment of ribosomal and translational activity reflecting inflammatory stress which were significantly downregulated in Δ8-THC treated group, and instead shifted gene expression toward interferon-responsive, immunoregulatory programs. Δ8-THC-enriched clusters exhibited increased interactions via COLLAGEN, APP, CypA and CDH5 pathways, consistent with enhanced endothelial stabilization.
Δ8-THC alleviated lung inflammation in LPS-induced ARDS by reprogramming immune and endothelial responses toward interferon-driven, regulatory states. These findings suggest a potential therapeutic role for Δ8-THC in restoring lung homeostasis during ARDS.
This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896
Immune Response Regulation: Cellular Mechanisms (IRC)
Shruthi Thada, Ahmed K. Aladhami, Chloe Nichols et al.· Journal of Immunology· 0 citations
Sepsis-associated acute respiratory distress syndrome (S-ARDS) is often followed by immunoparalysis, leaving patients vulnerable to secondary infection and adverse outcomes. Lactate is widely used as a severity marker in sepsis, but its direct contribution to immune paralysis remains unclear. We examined lactate-driven immune dysfunction in mouse models, bone marrow-derived macrophages and human CD14+ monocyte-derived macrophages, and tested C646-loaded mesenchymal stromal cell-derived extracellular vesicles (EVs-C646) as an experimental intervention. Lactate elevation blunted cytokine responses after rechallenge, reduced macrophage phagocytosis, shifted macrophages toward an M2-like phenotype, expanded regulatory T cells and decreased interferon-γ (IFN-γ) responses in CD8+ T cells. Mechanistically, lactate activated a p300-histone H3 lysine 18 lactylation (H3K18la) program and upregulated proteasome 26S subunit, non-ATPase 14 (PSMD14). Integrated RNA sequencing, H3K18la chromatin immunoprecipitation sequencing, public sepsis transcriptomic analysis and perturbation experiments identified PSMD14 as a lactylation-linked effector that strengthened AKT/mTOR signalling and promoted macrophage dysfunction. EVs-C646 decreased H3K18la and PSMD14 expression, restored macrophage inflammatory responsiveness, improved bacterial clearance, reduced lung injury and increased survival in experimental S-ARDS-related immunoparalysis models. These findings define a lactate-p300-H3K18la-PSMD14-AKT/mTOR pathway in immunoparalysis and support further preclinical evaluation of lactylation-targeted extracellular vesicle therapy.
Yuan-Yuan Zhang, Jia-Xu Guo, Jia-Ming Zhang et al.· International Immunopharmaco...· 0 citations
Combination therapies targeting multiple of these glucose signaling pathway proteins, together with glucose and N-acetylcysteine, yielded superior therapeutic benefit in complex I disease cell and C. elegans models.
Kelsey Keith, Min Peng, Cristina Remes et al.· bioRxiv· 0 citations
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