Journal of Innate Immunity最新文献

Introduction: Diabetic cardiomyopathy (DCM) is a common complication of diabetes characterized by chronic low-grade inflammation. Exercise has been recognized as an effective intervention for DCM; however, its underlying mechanisms remain incompletely understood.

Methods: Pyroptosis regulation by exercise was examined using DCM and HFD mouse models. In vitro, H9C2 cardiomyocytes were exposed to high glucose or palmitic acid to mimic diabetic condition. The AMPK agonist AICAR was applied to H9C2 cells to reproduce the molecular effects of exercise. WB and caspase-1 activity assays analyzed protein expression and inflammasome activation. Cell death was assessed by cell viability, LDH release, and microscopy.

Results: Exercise attenuated pyroptosis in DCM, prevented the upregulation of P2X4 and PANX1, inhibited NLRP3 activation, and reduced GSDMD and IL-1β cleavage in DCM mouse hearts, without altering P2X7 levels. In HFD-fed obese mice, exercise suppressed P2X4 expression, which correlated with NLRP3 activation, whereas P2X7 remained elevated. In vitro, high glucose alone did not induce significant H9C2 cell death or upregulate P2X4/P2X7 expression, whereas palmitic acid promoted concentration-dependent increases in P2X4 and NLRP3 expression, caspase-1 activity, and necrosis. Exercise and AICAR share a common mechanism in inhibiting NLRP3 inflammasome activation through P2X4, while AICAR exhibits a partial effect on P2X7 compared with exercise.

Conclusion: These findings highlight P2X4 as a critical mediator of NLRP3-driven pyroptosis in the diabetic heart. Exercise ameliorates myocardial inflammation in DCM and appears to act primarily through suppressing P2X4, providing new insight into its cardioprotective mechanisms and suggesting P2X4 as a potential therapeutic target.

Introduction: Macrophages abundantly express liver X receptors (LXRs), which are ligand-dependent transcription factors and sensors of several cholesterol metabolites. In response to agonists, LXRs promote the expression of key lipid homeostasis regulators. Cross talk between LXRs and inflammatory signals exists in a cell type- and gene-specific manner. A common feature in the macrophage response to inflammatory mediators is the induction of CCAAT/enhancer-binding protein beta (C/EBPβ), a master transcriptional regulator and lineage-determining transcription factor in monocytes/macrophages.

Methods: Quantitative real-time PCR in control and C/EBPβ-deficient macrophages was used to explore the role of C/EBPβ in the cross talk between inflammatory mediators and the macrophage response to pharmacological LXR activation. The functional interaction between C/EBPβ and LXRs on selected genomic regions was further characterized by chromatin-immunoprecipitation (ChIP) and gene reporter studies.

Results: Whereas inflammatory signaling repressed several LXR-regulated genes involved in lipid metabolism, these effects were conserved after deletion of C/EBPβ. In contrast, inflammatory mediators and LXRs synergistically induced the expression of the multifunctional protein CD38 in a C/EBPβ-dependent manner. C/EBPβ and LXRs bound to several regions with enhancer activity upstream and within the mouse Cd38 gene and their functional cooperation in macrophages required intact binding sites for LXR and C/EBPβ.

Conclusion: This study reveals positive cross talk between C/EBPβ and LXRs during the macrophage inflammatory response, which selectively impacts CD38 expression.

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: The alternative pathway of complement activation is consistently active, keeping the complement system primed for immediate response. This constant "tick-over" mechanism is regulated by the factor H (FH) protein family, which encompasses seven highly related proteins: FH, FHL-1, and five FH-related (FHR-1 to -5) proteins. The current model is that the FHRs compete with FH and FHL-1 to fine-tune their activities. Genetic studies of this complex locus have revealed distinct haplotypes associating with a wide array of human diseases, underscoring its significant role in complement regulation. Nevertheless, a comprehensive analysis of systemic concentrations of all FH protein family members, accounting for known genetic variability within the population, is still lacking.

Methods: Systemic levels of each member of the FH protein family were quantified with the use of recently developed target specific ELISAs. Next, a genetic analysis focused on the chromosome 1q31.3 region was performed using next generation sequencing and multiplex ligase probe-dependent amplification.

Results: We report systemic protein levels of each member of the FH protein family found in vivo and demonstrate common haplotypes within the CFH locus give rise to classifiable protein expression patterns, establishing distinct ratios between FH, FHL-1, and the FHRs.

Conclusions: The established reference intervals and identified genetic effects provide a benchmark for further research and emphasize the importance of including all family members when studying their role in both health and disease.

Introduction: In severe COVID-19, excessive cytokine release may be driven by SARS-CoV-2. We investigated the modulatory effect of probiotics taking into consideration direct interaction with the immune gut cells.

Methods: Fifty-five patients with confirmed COVID-19 infection were classified by the presence of acute respiratory distress syndrome (ARDS) or not. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with lipopolysaccharide (LPS), a preparation of four probiotics (LactoLevure® containing Saccharomyces boulardii, Bifidobacterium lactis BB-12, Lactobacillus acidophilus LA-5, and L. plantarum) and/or recombinant human interferon-gamma (rhIFNγ) and tocilizumab. Cytokine concentrations were measured in cell supernatants. Gene expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) was performed by quantitative real-time polymerase chain reaction (RT-PCR). Results were associated with the level of viremia.

Results: Probiotics decreased tumor necrosis factor-alpha (TNFα) production by the PBMCs of both ARDS and non-ARDS patients. LPS stimulated the production of interleukin (IL)-1β, IL-6 in non-ARDS patients. IL-6 production was maintained in the presence of probiotics. rhIFNγ enhanced LPS-stimulated cytokine production by PBMCs; this was not the case when PBMCs were stimulated by probiotics. Probiotics upregulated TLR2 and LPS downregulated TLR4 in the PBMCs of patients with ARDS. PBMCs from patients with viremia had more cytokine production by probiotic stimulation.

Conclusion: Probiotics interact with the immune system of COVID-19 patients by modulating the production of TNFα, IL-1β, and IL-6 in an IFNγ-independent mechanism.

Introduction: Toll-like receptor (TLR) engagement on macrophages can improve responsiveness to infection. TNF is upregulated following TLR2 or TLR4 stimulation. We sought to determine whether and how the two bioactive forms of TNF, soluble (sTNF) and transmembrane (tmTNF), may be contributing to macrophage priming, which improved responsiveness to subsequent Staphylococcus aureus infection.

Methods: RNA sequencing and cytokine quantification assays identified differentially upregulated cytokines in response to TLR2 stimulation. Immortalized and primary bone marrow-derived macrophages (BMDMs) coupled with receptor blocking and cytokine supplementation were used to investigate whether/how prior TLR-primed macrophages improved S. aureus clearance.

Results: TLR2 or TLR4 stimulated TNF-/- BMDMs failed to efficiently clear a subsequent S. aureus infection compared to TLR-stimulated wild-type (WT) BMDMs. Depletion of sTNF from TLR-stimulated WT BMDMs retained their improved S. aureus clearance. Exogenous sTNF supplementation to TNF-/- BMDMs did not rescue improved S. aureus clearance. Cell density assays showed cell-to-cell contact was important for TLR-induced improvement of S. aureus clearance. Conversely, blocking TNFR2 reduced BMDM clearance of S. aureus, despite TLR2 stimulation.

Conclusions: Our results demonstrated that TNF produced in response to TLR stimulated BMDMs was required for improved clearance of a subsequent S. aureus infection. We found that sTNF did not contribute to this priming, which suggested that tmTNF may be critical for BMDM priming which leads to improved S. aureus clearance.

Introduction: COVID-19 is highly heterogeneous, ranging from cases with mild disease with an almost asymptomatic carrier to severe cases, in which the disease evolves rapidly. A better understanding of monocyte response during SARS-CoV-2 infection would highlight potential biomarkers and establish other possible approaches for severe cases.

Methods: The study group consisted of 32 COVID-19 patients and 18 health controls from June 2023 to March 2024. The COVID-19 patients were further classified as mild and severe illnesses based on World Health Organization (WHO) criteria. For flow cytometric analysis, 50 µL of peripheral blood and 1 µL of specific monoclonal antibodies were added to each cytometric tube for surface marker detection.

Results: Here, the promising finding was that the blood non-classical/classical monocyte (NC/CL) subset was skewed toward NChighCLlow and NClowCLhigh clusters among the severe COVID-19 patients. The NChighCLlow cluster in severe COVID-19 displayed a distinct clinical phenotype, implying a higher 7-day disease progression rate (p = 0.019) and a worse 28-day survival (p = 0.026). Moreover, the secretion of IL-1β and IFN-γ was primarily attributed to CL subset in monocytes, while IL-6 was secreted mainly by NC subset.

Conclusion: As supported, regarding cytokine profile in context of SARS-CoV-2 infection, it was identified that circulating NC cells are proinflammatory cells most related to regulatory cells, while CL subset displayed an effective capacity to virus. These findings have implications toward optimizing evaluation in severe COVID-19, and developing strategies that target altered balance of NC/CL cell subsets.

Introduction: Streptococcus pyogenes (group A streptococcus, GAS) is an exclusively human pathogen. It causes a wide spectrum of diseases, ranging from mild infections such as pharyngitis to severe life-threatening conditions such as streptococcal toxic shock syndrome (STSS). Thrombocytopenia is a common feature of STSS and is associated with severe outcome. GAS produce a plethora of virulence factors, including streptolysin S (SLS), which has lytic as well as immunomodulatory properties. However, its role in platelet activation remains unclear.

Methods: Washed human platelets were infected with GAS wild-type and SLS-deficient mutant (ΔsagA) strains. Platelet activation was assessed by measuring degranulation (CD62P expression). The role of calcium influx and the involvement of purinergic type 2 receptors (P2R) in platelet activation by GAS were assessed using chemical antagonists and calcium chelators.

Results: GAS activate human platelets via SLS-mediated calcium influx, marked by increased surface expression of CD62P. IVIG treatment improved platelet viability in wild-type infections but failed to prevent SLS-mediated activation. Blocking of P2 receptors via suramin or NF449 as well as the use of calcium chelators reduced SLS-mediated platelet activation.

Conclusion: This study identified SLS as an M-protein and consequently a serotype-independent activator of human platelets. While IVIG partially improved platelet viability in GAS infections, its inability to prevent excessive platelet activation underscores the need for additional treatment options in severe GAS infections.

Introduction: Streptococcus pyogenes (group A streptococcus, GAS) is an exclusively human pathogen. It causes a wide spectrum of diseases, ranging from mild infections such as pharyngitis to severe life-threatening conditions such as streptococcal toxic shock syndrome (STSS). Thrombocytopenia is a common feature of STSS and is associated with severe outcome. GAS produce a plethora of virulence factors, including streptolysin S (SLS), which has lytic as well as immunomodulatory properties. However, its role in platelet activation remains unclear.

Methods: Washed human platelets were infected with GAS wild-type and SLS-deficient mutant (ΔsagA) strains. Platelet activation was assessed by measuring degranulation (CD62P expression). The role of calcium influx and the involvement of purinergic type 2 receptors (P2R) in platelet activation by GAS were assessed using chemical antagonists and calcium chelators.

Results: GAS activate human platelets via SLS-mediated calcium influx, marked by increased surface expression of CD62P. IVIG treatment improved platelet viability in wild-type infections but failed to prevent SLS-mediated activation. Blocking of P2 receptors via suramin or NF449 as well as the use of calcium chelators