Pyroptosis is a lytic and inflammatory form of programmed cell death that is typically initiated by inflammasome activation and executed by gasdermin proteins. It is characterized by cellular swelling, plasma membrane perforation, and the release of intracellular contents. Pyroptosis functions as a "double-edged sword" and plays an essential role in defending the host against pathogen invasion when properly regulated; however, excessive or dysregulated pyroptotic activity can contribute to severe inflammatory pathologies. Aberrant pyroptosis is associated with a range of diseases, including sepsis, inflammatory disorders, cancer, atherosclerosis and neurodegenerative disorders. N6-methyladenosine (m6A) modification is among the most abundant and widespread epigenetic modifications in eukaryotic RNAs and influences multiple stages of gene regulation, from messenger RNA processing to protein synthesis. Emerging evidence indicates that m6A modification plays a regulatory role in pyroptosis, suggesting promising avenues for therapeutic intervention in pyroptosis-related diseases. This review provides a systematic delineation of the signalling pathways regulating pyroptosis and a comprehensive analysis of how m6A modification dynamically regulates this form of inflammatory cell death through its writer, eraser, and reader proteins and explores potential molecular triggers of disease progression. We further aimed to elucidate the pathophysiological significance of m6A-mediated regulation of pyroptosis in disease and identify novel therapeutic targets.

Introduction: Neutrophils are the most abundant innate immune cells in the peripheral blood and eliminate bacteria through phagocytosis and antimicrobial mechanisms. Early in infection, they often encounter high bacterial loads before full recruitment. Individual neutrophils can ingest many bacteria, but it remains unclear how high bacterial loads per neutrophil affect intracellular killing. Methods: Neutrophils were isolated from healthy donor blood by fluorescence-activated cell sorting. Intracellular bacterial load was quantified using imaging flow cytometry to measure spot counts and green fluorescent protein (GFP) intensity after exposure to GFP-expressing Staphylococcus aureus. A single-cell killing assay assessed intracellular killing across bacterial load categories by sorting individual GFP+ neutrophils into 384-well plates and counting wells with outgrowth after 100 h. Phagolysosomal acidification was measured using dual-labeled (pH-sensitive pHrodo and pH-insensitive PromoFluor 520 LSS NHS ester [PF520]) S. aureus bioparticles. Results: Bacterial uptake by neutrophils was highly heterogeneous in vivo and in vitro. GFP spot counts strongly correlated with GFP intensity (R2 = 0.66), allowing stratification into GFP fluorescence intensity categories. In the single-cell killing assay, higher bacterial loads per neutrophil were associated with reduced intracellular killing (χ2(4) = 11.72, p = 0.0003). Higher bacterial loads per neutrophil corresponded with diminished phagolysosomal acidification capacity (χ2(4) = 24.00, p < 0.0001). Conclusion: Neutrophils ingesting higher bacterial loads exhibit reduced intracellular killing, likely due to decreased phagolysosomal acidification. These findings highlight how bacterial load per neutrophil shapes antimicrobial capacity and early infection control.

Introduction: Pseudomonas aeruginosa is a significant pathogen associated with acute and chronic infections, particularly in immunocompromised individuals. Its capacity for biofilm formation, combined with antibiotic resistance, plays a critical role in the persistence of these infections. Peptidyl-arginine deiminases (PADs), including PAD2 and PAD4 isoforms, are involved in neutrophil phagocytic killing of P. aeruginosa. This study aimed to investigate the impact of PAD enzymes on biofilm development and virulence factor production in P. aeruginosa, with focus on the multidrug resistant strain, PGO2330. Methods: Biofilm formation was assessed using crystal violet assays and confocal scanning laser microscopy. Quorum sensing (QS) gene expression and QS-related virulence factor production were quantified using qPCR and virulence factor assays. Results: Exposure to 20 nm of PAD2 or PAD4 reduced PGO2330 surface attachment (p < 0.0001) and biofilm formation to 67.9 ± 5.6% (p < 0.0001) and 68.2 ± 4.2% (p = 0.0004), respectively. Moreover, rPAD2 and rPAD4 citrullinated multiple protein substrates of P. aeruginosa, yet citrullination activity was not required by rPADs to reduce P. aeruginosa biofilm formation. PGO2330 exposed to PAD2 and PAD4 showed reduced lasR, lasI, rhlR, rhlI, and mvfR gene expression and reduced levels of extracellular DNA, rhamnolipids, pyocyanin, and protease activity. Conclusion: These results demonstrate that PADs inhibit P. aeruginosa biofilm formation and decrease the production of QS-related virulence factors, highlighting their potential as novel antimicrobials and supporting further research into the development of PAD-based therapeutics.

Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by dysregulated immune responses in the gut. Macrophages, as key innate immune cells in the intestinal mucosa, play a central role in both maintaining homeostasis and driving pathology in UC. Summary: Under physiological conditions, intestinal macrophages exhibit a unique “inflammatory anergy” phenotype, supporting epithelial integrity and immune tolerance. In UC, however, persistent inflammatory signals promote monocyte recruitment and their polarization into pro-inflammatory M1-like macrophages. These cells secrete cytokines such as TNF-α, IL-1β, IL-6, and IL-12/23, produce reactive oxygen species and reactive nitrogen species, and release matrix metalloproteinases, collectively driving epithelial barrier disruption, tissue damage, and sustained inflammation. This review comprehensively discusses the origin, heterogeneity, and functional plasticity of intestinal macrophages, their dynamic interactions with other cells, and key regulatory signaling pathways – such as NF-κB, JAK-STAT, and the NLRP3 inflammasome – in UC. Key Messages: We evaluate current and emerging macrophage-targeted therapies, including cytokine blockade, chemokine receptor antagonism, phenotypic reprogramming, nanomedicine, and cell-based interventions. Furthermore, we highlight the limitations of the M1/M2 dichotomy and emphasize the need for single-cell and spatial transcriptomic approaches to better define macrophage subsets in human disease. Advancing the understanding of macrophage biology in UC will facilitate the development of precise immunomodulatory strategies and biomarker-based diagnostics, ultimately aiming to bridge the gap between mechanistic discovery and improved patient care.

Introduction: This study investigated the role of FCGR3A in pediatric inflammatory bowel disease (IBD) through integrated transcriptomic analysis and experimental validation, aiming to provide new mechanistic insights and therapeutic strategies. Methods: Transcriptomic datasets of pediatric IBD were obtained from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified. Weighted gene co-expression network analysis (WGCNA) was applied to screen disease-related modules, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of overlapping DEGs. Hub genes were determined using protein-protein interaction networks, least absolute shrinkage and selection operator regression, and random forest algorithms. Their expression was verified by meta-analysis across multiple datasets, and diagnostic value was assessed using receiver operating characteristic (ROC) curves. The relationship between hub genes and immune infiltration was further explored. Functional assays included lipopolysaccharide (LPS)-stimulated intestinal epithelial and THP-1 macrophage models, as well as a dextran sulfate sodium (DSS)-induced murine colitis model. Results: WGCNA revealed 370 module genes associated with pediatric IBD, enriched in inflammatory pathways. Seven candidate genes were identified, among which FCGR3A showed a strong association with pediatric IBD. Immune infiltration analysis demonstrated that FCGR3A expression correlated with M1 macrophage enrichment. In vitro, FCGR3A was upregulated in LPS-induced epithelial cells, and its knockdown inhibited M1 macrophage polarization. In vivo, FCGR3A was highly expressed in DSS-induced murine colitis, promoting M1 polarization and disease progression. ROC analysis indicated strong predictive value (AUC = 0.968). Conclusion: FCGR3A may serve as a pivotal regulator in pediatric IBD by driving M1 macrophage polarization, representing a promising biomarker and potential therapeutic target.

Introduction: Cystic fibrosis (CF) is an inherited disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). It is characterized by progressive decline in lung function, often driven by chronic respiratory infections, particularly with Staphylococcus aureus and Pseudomonas aeruginosa. MicroRNAs (miRNAs), small noncoding regulatory RNAs that negatively regulate protein expression by binding to mRNA, are altered in people with CF and potentially contribute to the pulmonary manifestations of CF. The management of CF lung infections is further complicated by the formation of bacterial biofilms and the emergence of antimicrobial resistance which renders conventional treatments ineffective. Methods: In silico analysis identified hsa-miR.101.3p as a promising miRNA with potential targets including genes associated with beta-lactam resistance and biofilm formation in P. aeruginosa, as well as genes involved in the overall growth of S. aureus. To facilitate delivery, miRNA mimic DNA oligonucleotides were conjugated to DNA tetrahedrons (DNAtds). The structural integrity of the DNAtd-miRNA complexes was confirmed via transmission electron microscopy, characterized by nanoparticle tracking analysis, and successful bacterial uptake was verified using fluorescence microscopy. Results: DNAtd-miR.101.3p significantly reduced the viability of both S. aureus and P. aeruginosa. Furthermore, DNAtd-miR.101-3p enhanced the activity of the beta-lactam antibiotic cefotaxime against both non-mucoid and mucoid planktonic and biofilm-forming P. aeruginosa. The mechanisms involve DNAtd-miR.101.3p targeting of ampC, fleN, and pslK. Conclusion: DNAtd-miR.101.3p displays unique inhibition properties against P. aeruginosa and S. aureus in the exponential phase of bacterial growth in vitro and increases the rate of the bactericidal activity of cefotaxime against P. aeruginosa.

Introduction: Group B Streptococcus (GBS) asymptomatic colonizes the female genital tract (FGT) but can contribute to adverse pregnancy outcomes including preterm birth, chorioamnionitis, and neonatal infection. We previously observed that GBS elicits FGT cytokine responses, including IL-17, during murine vaginal colonization; yet the anti-GBS cellular immune response during colonization remained unknown. We hypothesized that GBS may induce cellular immunity, resulting in FGT clearance. Methods: Herein, we utilize depleting antibodies and knockout mice and performed flow cytometry to investigate cellular immune responses during GBS colonization. Results: We found that neutrophils (effectors of the IL-17 response) are important for GBS mucosal control as neutrophil depletion promoted increased GBS burdens in FGT tissues. Flow cytometric analysis of immune populations in the vagina, cervix, and uterus revealed, however, that GBS colonization did not induce a marked increase in FGT CD45+ immune cells. We also found that Vγ6+ γδ T cells comprise a primary source of FGT IL-17. Finally, using knockout mice, we observed that IL-17-producing γδ T cells are important for the control of GBS in the FGT during murine colonization. Conclusions: Taken together, this work characterizes FGT cellular immunity and suggests that GBS colonization does not elicit a significant immune response, which may be a bacterial directed adaptive outcome. However, certain FGT immune cells, such as neutrophils and γδ T cells, contribute to host defense and control of GBS colonization.

In the article “Plasma Levels of Hepcidin and Reticulocyte Haemoglobin during Septic Shock” [J Innate Immun. 2020;12(6):448–460. https://doi.org/10.1159/000508561] by Olinder et al., the authors would like to clarify that it was serum hepcidin and not plasma hepcidin that was analysed. The term “serum” was inadvertently changed to “plasma” by the authors during the final manuscript adjustments. The authors would like to apologise for this error and clarify that this error does not affect the results of the study.

Introduction: Escherichia coli (E. coli) is a significant commensal gram-negative bacterium that can give rise to various diseases. The roles of Toll-like receptor 2 (TLR2) and the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in sepsis induced by E. coli infection remain unclear. Methods: In vivo, we investigated differences in mortality, production of inflammatory mediators, organ damage, neutrophil count, and bacterial load during E. coli infection in C57BL/6J mice, as well as in mice deficient in TLR2 or NLRP3. In vitro, we investigated the impact of E. coli on the activation of TLR2 and NLRP3 in macrophages and the influence of TLR2 and NLRP3 on the activation of inflammatory signaling pathways and the secretion of inflammatory mediators in macrophages induced by E. coli infection. Results: TLR2-deficient (TLR2−/−) and NLRP3-deficient (NLRP3−/−) mice exhibit significantly increased mortality and organ damage after E. coli infection. These mice also show elevated levels of TNF-α and IL-10 in serum and peritoneal lavage fluid. Additionally, TLR2−/− and NLRP3−/− mice display heightened neutrophil recruitment and increased bacterial load in the blood. Furthermore, macrophages from these mice demonstrate a significant reduction in the activation of the MAPK signaling pathway. Conclusion: TLR2 and NLRP3 play crucial roles in modulating inflammatory mediator expression, immune cell recruitment, and bactericidal activity, thereby preventing excessive tissue damage and reducing mortality in E. coli-induced sepsis.