Consecutive palmitoylation and phosphorylation orchestrates NLRP3 membrane trafficking and inflammasome activation.

NLRP3 inflammasome activation, essential for cytokine secretion and pyroptosis in response to diverse stimuli, is closely associated with various diseases. Upon stimulation, NLRP3 undergoes subcellular membrane trafficking and conformational rearrangements, preparing itself for inflammasome assembly at the microtubule-organizing center (MTOC). Here, we elucidate an orchestrated mechanism underlying these ordered processes using human and murine cells. Specifically, NLRP3 undergoes palmitoylation at two sites by palmitoyl transferase zDHHC1, facilitating its trafficking between subcellular membranes, including the mitochondria, trans-Golgi network (TGN), and endosome. This dynamic trafficking culminates in the localization of NLRP3 to the MTOC, where LATS1/2, pre-recruited to MTOC during priming, phosphorylates NLRP3 to further facilitate its interaction with NIMA-related kinase 7 (NEK7), ultimately leading to full NLRP3 activation. Consistently, Zdhhc1-deficiency mitigated LPS-induced inflammation and conferred protection against mortality in mice. Altogether, our findings provide valuable insights into the regulation of NLRP3 membrane trafficking and inflammasome activation, governed by palmitoylation and phosphorylation events.

The anti-inflammatory role of zDHHC23 through the promotion of macrophage M2 polarization and macrophage necroptosis in large yellow croaker (Larimichthys crocea).

Zinc finger Asp-His-His-Cys motif-containing (zDHHC) proteins, known for their palmitoyltransferase (PAT) activity, play crucial roles in diverse cellular processes, including immune regulation. However, their non-palmitoyltransferase immunomodulatory functions and involvement in teleost immune responses remain underexplored. In this study, we systematically characterized the zDHHC family in the large yellow croaker (Larimichthys crocea), identifying 22 members. Phylogenetic analysis unveiled that each of the 22 LczDHHCs formed distinct clusters with their orthologues from other teleost species. Furthermore, all LczDHHCs exhibited a highly conserved DHHC domain, as confirmed by tertiary structure prediction. Notably, LczDHHC23 exhibited the most pronounced upregulation following Pseudomonas plecoglossicida (P. plecoglossicida) infection of macrophage/monocyte cells (MO/MΦ). Silencing LczDHHC23 led to heightened pro-inflammatory cytokine expression and diminished anti-inflammatory cytokine levels in MO/MΦ during infection, indicating its anti-inflammatory role. Functionally, LczDHHC23 facilitated M2-type macrophage polarization, as evidenced by a significant skewing of MO/MΦ towards the pro-inflammatory M1 phenotype upon LczDHHC23 knockdown, along with the inhibition of MO/MΦ necroptosis induced by P. plecoglossicida infection. These findings highlight the non-PAT immunomodulatory function of LczDHHC23 in teleost immune regulation, broadening our understanding of zDHHC proteins in host-pathogen interactions, suggesting LczDHHC23 as a potential therapeutic target for immune modulation in aquatic species.

Prior exposure to ciprofloxacin disrupts intestinal homeostasis and predisposes ayu ( Plecoglossus altivelis) to subsequent Pseudomonas plecoglossicida-induced infection.

With the rapid development of intensive farming, the aquaculture industry uses a great many antibiotics for the prevention and treatment of bacterial diseases. Despite their therapeutic functions, the overuse and accumulation of antibiotics also pose a threat to aquaculture organisms. In the present study, ayu ( Plecoglossus altivelis) was used as a fish model to study the impacts of ciprofloxacin (CIP) overuse on intestinal homeostasis and immune response during subsequent Pseudomonas plecoglossicida infection. Based on 16S rRNA gene amplification and Illumina sequencing, we found that CIP pre-exposure caused significant variation in intestinal microbiota, including increased species richness, altered microbiota composition and interaction networks, and increased metabolic dysfunction. Furthermore, immunohistochemical analysis indicated that CIP pre-exposure resulted in severe mucosal layer damage, goblet cell reduction, and epithelial cell necrosis of the intestinal barrier in infected ayu. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that disruption of intestinal homeostasis impaired systemic anti-infection immune responses in the intestine, gill, spleen, and head kidney, while inhibiting IL-1ß, TNF-α, and IL-10 expression and promoting TGF-ß expression. Our findings indicated that CIP administration can directly affect intestinal microbiota composition and intestinal integrity in ayu fish. This perturbation of intestinal homeostasis is likely responsible for the lower survival rate of hosts following subsequent infection as the capacity to mount an effective immune response is compromised. This study also provides preliminary clues for understanding the effects of antibiotic overuse on higher vertebrates through trophic transfer.

Identification and Verification of Feature Biomarkers Associated With Immune Cells in Dilated Cardiomyopathy by Bioinformatics Analysis.

Objective: To explore immune-related feature genes in patients with dilated cardiomyopathy (DCM). Methods: Expression profiles from three datasets (GSE1145, GSE21610 and GSE21819) of human cardiac tissues of DCM and healthy controls were downloaded from the GEO database. After data preprocessing, differentially expressed genes (DEGs) were identified by the 'limma' package in R software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were then performed to identify biological functions of the DEGs. The compositional patterns of stromal and immune cells were estimated using xCell. Hub genes and functional modules were identified based on protein-protein interaction (PPI) network analysis by STRING webtool and Cytoscape application. Correlation analysis was performed between immune cell subtypes and hub genes. Hub genes with |correlation coefficient| > 0.5 and p value <0.05 were selected as feature biomarkers. A logistic regression model was constructed based on the selected biomarkers and validated in datasets GSE5406 and GSE57338. Results: A total of 1,005 DEGs were identified. Functional enrichment analyses indicated that extracellular matrix remodeling and immune and inflammation disorder played important roles in the pathogenesis of DCM. Immune cells, including CD8+ T-cells, macrophages M1 and Th1 cells, were proved to be significantly changed in DCM patients by immune cell infiltration analysis. In the PPI network analysis, STAT3, IL6, CCL2, PIK3R1, ESR1, CCL5, IL17A, TLR2, BUB1B and MYC were identified as hub genes, among which CCL2, CCL5 and TLR2 were further screened as feature biomarkers by using hub genes and immune cells correlation analysis. A diagnosis model was successfully constructed by using the three biomarkers with area under the curve (AUC) scores 0.981, 0.867 and 0.946 in merged dataset, GSE5406 and GSE57338, respectively. Conclusion: The present study identified three immune-related genes as diagnostic biomarkers for DCM, providing a novel perspective of immune and inflammatory response for the exploration of DCM molecular mechanisms.

Plasma protein profiling analysis in patients with atrial fibrillation before and after three different ablation techniques.

Background: There are controversies on the pathophysiological alteration in patients with atrial fibrillation (AF) undergoing pulmonary vein isolation using different energy sources. Objectives: We evaluated the changes in plasma proteins in acute phase post-ablation in patients receiving cryoballoon ablation, radiofrequency balloon ablation, or radiofrequency ablation. Methods: Blood samples from eight healthy controls and 24 patients with AF were taken on the day of admission, day 1, and day 2 post-ablation and analyzed by the Olink proximity extension assay. Proteins were identified and performed with enrichment analysis. Protein-protein interaction network and module analysis were conducted using Cytoscape software. Results: Of 181 proteins, 42 proteins in the cryoballoon group, 46 proteins in the radiofrequency balloon group, and 43 proteins in the radiofrequency group significantly changed after ablation. Most of the proteins altered significantly on the first day after ablation. Altered proteins were mainly involved in cytokine-cytokine receptor interaction. Both balloon-based ablations showed a similar shift toward enhancing cell communication and regulation of signaling while inhibiting neutrophil chemotaxis. However, radiofrequency ablation presented a different trend. Seed proteins, including osteopontin, interleukin-6, interleukin-10, C-C motif ligand 8, and matrix metalloproteinase-1, were identified. More significant proteins associated with hemorrhage and coagulation were selected in balloon-based ablations by machine learning. Conclusion: Plasma protein response after three different ablations in patients with AF mainly occurred on the first day. Radiofrequency balloon ablation shared similar alteration in protein profile as cryoballoon ablation compared with radiofrequency ablation, suggesting that lesion size rather than energy source is the determinant in pathophysiological responses to the ablation.

RGD-binding integrins mediated phagocytosis involved in the entry of Edwardsiella tarda into mudskipper MO/MФ.

The versatile fish pathogen Edwardsiella tarda is an intracellular pathogen with the ability to invade and replicate in host phagocytes. However, the mechanism mediating the uptake of E. tarda in fish monocytes/macrophages (MO/MΦ) is not yet understood. Generating mudskipper kidney-derived MO/MФ transcriptomic resources from mudskipper challenged by E. tarda is crucial for understanding the molecular mechanisms underlying the mudskipper invasion process. In the present study, a total of 1185 up-regulated and 885 down-regulated differentially expressed genes (DEGs) were identified using RNA-seq. Enrichment and pathway analysis of DEGs revealed the centrality of the phagosome and regulation of actin cytoskeleton pathways in pathogen entry. The progress of phagosome formation was observed by transmission electron microscopy. Eight conserved integrin (ITG) subunit genes, belonging to the phagocytic receptors, were found in the transcriptomic sequence data. Additionally, quantitative real-time PCR showed that the mRNA expressions of most ITG subunit genes were related to the different infection times of E. tarda and the different bacterial pathogens. Further assays demonstrated that phagocytosis of FITC-labeled E. tarda by mudskipper MO/MФ was significantly reduced by the tetrapeptide Asp-Gly-Arg-Ser (RGDS). In summary, phagocytosis is one of the entry pathways into mudskipper MO/MΦ, and RGD-binding ITGs are involved in the phagosome formation process.

Bmi-1 high-expressing cells enrich cardiac stem/progenitor cells and respond to heart injury.

Bmi-1 gene is well recognized as an oncogene, but has been recently demonstrated to play a role in the self-renewal of tissue-specific stem cells. By using Bmi-1GFP/+ mice, we investigated the role of Bmi-1 in cardiac stem/progenitor cells and myocardial repair. RT-PCR and flow cytometry analysis indicated that the expression of Bmi-1 was significantly higher in cardiac side population than the main population from CD45- Ter119- CD31- heart cells. More Sca-1+ cardiac stem/progenitor cells were found in Bmi-1 GFPhi subpopulation, and these Bmi-1 GFPhi heart cells showed the potential of differentiation into SMM+ smooth muscle-like cells and TnT+ cardiomyocyte-like cells in vitro. The silencing of Bmi-1 significantly inhibited the proliferation and differentiation of heart cells. Otherwise, myocardial infarction induced a significantly increase (2.7-folds) of Bmi-1 GFPhi population, mainly within the infarction and border zones. These preliminary data suggest that Bmi-1hi heart cells are enriched in cardiac stem/progenitor cells and may play a role in myocardial repair.