Background: Neutrophil-infiltrating inflammation is closely linked to severe asthma in teenagers, and its mechanism needs to be investigated. CEACAM6 is a critical molecule that regulates neutrophil function and is significantly expressed in the airways of asthmatic patients, but its precise role in the disease and downstream signaling mechanisms remain unknown. The goal of this study was to investigate the role and mechanism of CEACAM6 in neutrophil airway inflammation. Methods: The GSE188424 data set was analyzed using bioinformatics. An OVA-induced juvenile asthma mouse model was created utilizing CEACAM6 gene knockout mice. CEACAM6 was knocked down in BEAS-2B cells using siRNA, and LPS was utilized to produce inflammation. Molecular and phenotypic studies were carried out using Western blot, Co-IP, ELISA, and flow cytometry. Results: CEACAM6 expression in asthmatic lung tissue was significantly elevated. In mice, knocking down CEACAM6 drastically reduces airway neutrophil inflammation and inflammatory factor release. Cell tests demonstrated that CEACAM6 knockdown reduced LPS-induced epithelial cell damage and inflammation. CEACAM6 binds to elastase, neutrophil expressed (ELANE), activating the PI3K/AKT/NF-κB signaling cascade. ELANE supplementation reversed the anti-inflammatory effects of CEACAM6 knockdown. Conclusion: CEACAM6 increases neutrophil airway inflammation by binding to ELANE and activating the PI3K/AKT/NF-κB pathway. Targeting the CEACAM6-ELANE axis could be a novel method for treating neutrophilic asthma.
INTRODUCTION:Macrophages are essential components of innate immunity, serving as a frontline defense against pathogens and maintaining tissue homeostasis. Human induced pluripotent stem cell (iPSC)-derived macrophages (iMacs) provide a powerful platform for studying human innate immunity and macrophage biology. Here, we describe a robust, reproducible, efficient serum-free and feeder-free protocol for generating functional iMacs and characterizing their innate immune properties. METHODS:A 30-day monolayer culture system was utilized to continually generate hematopoietic progenitor cells (HPCs) from iPSCs starting on day 9, followed by macrophage differentiation over 21 days. Macrophage identity was assessed by flow cytometry, while functional assays evaluated phagocytosis and cytokine production, including interferons (IFNs). Transcriptomic profiling was performed by RNAseq across differentiation stages and following IFN stimulation. RESULTS:The optimized protocol consistently yielded iMacs with >99% purity, expressing canonical macrophage markers including CD14, CD16, CD163, HLA-DR, and CD11b. iMacs demonstrated robust phagocytic capacity and cytokine production in response to microbial stimuli. RNA sequencing revealed distinct gene signatures during differentiation, highlighting key transitions from pluripotency to progenitors, then to mature macrophages. iMac transcriptomes aligned with tissue-resident macrophage profiles, supporting their relevance for modelling tissue-specific immunity. iMacs displayed differential interferon responses, with a strong response to type I IFNs. CONCLUSION:This study establishes a highly efficient and robust protocol for generating functional human iPSC-derived macrophages, providing a versatile model for investigating innate immunity, host-pathogen interactions, and interferon signaling.
Introduction: Lambda interferons signal through the interferon lambda receptor 1 (IFNLR1) and IL10RB heterodimer to induce interferon stimulated genes (ISGs). We previously showed that proteins derived from distinct IFNLR1 splice isoforms uniquely influence gene expression and hepatitis B virus replication in stem cell-derived hepatocytes (iHeps). Here, we evaluated signal transduction mechanisms of full-length IFNLR1 (variant 1) and a truncated variant missing part of the cytoplasmic JAK1-interacting domain (variant 2). Methods: We evaluated HEK293T cells, wild-type (WT), and IFNLR1 knockout (KO) iHeps with doxycycline-inducible expression of FLAG-tagged IFNLR1 variants using the Duolink proximity ligation assay, ImageStream flow cytometry, Western blotting, susceptibility to JAK1 and TYK2 inhibitors, and gene expression profiling. Results: Each variant colocalized with IL10RB after IFNL3 treatment, but variant 1 was more rapidly and extensively internalized. In WT iHeps, overexpression of variant 1 or variant 2 enabled higher IFNL-induced JAK1, TYK2, STAT1, and STAT2 phosphorylation, yet variant 2 supported less robust ISG induction. In KO iHeps, variant 2 supported less JAK1 and TYK2 phosphorylation and ISG induction than variant 1, yet supported comparable STAT1 and STAT2 phosphorylation. In iHeps expressing variant 1, WT iHeps were more resistant than KO iHeps to TYK2 inhibition of antiviral ISG induction, yet were more susceptible to TYK2 inhibition of proinflammatory ISG induction, suggesting endogenous noncanonical variants influence TYK2-signaling dependence. Conclusion: IFNLR1 variants promote differential utilization of signaling mediators to influence IFNL-induced gene expression patterns, indicating a putative role in pathway regulation. Lambda interferons are soluble proteins produced by cells to help combat viral infections. Lambda interferons function by binding receptor proteins on the cell surface to activate a signaling pathway that results in increased expression of hundreds of genes that encode for proteins with antiviral properties. In this report, we studied how distinct forms of the lambda interferon receptor differ in their capacity to transmit signal within cells. We evaluated the full-length receptor and a receptor variant that is missing part of a domain that binds and interacts with other signaling proteins in the cytoplasm of cells. We identified differences in how these receptor variants are internalized, in how they activate cytoplasmic signaling mediators, and in their susceptibility to inhibitors of signaling. The results provide insight into how these distinct receptor variants fine-tune how cells respond to lambda interferon exposure to influence antiviral and inflammatory cellular responses.
INTRODUCTION:Bruton Tyrosine kinase inhibitors (BTKi) impair anti-aspergillus immune responses and increase the risk of invasive fungal infections (IFI). More selective second generation BTKi have fewer off-target effects thus a better tolerance than ibrutinib. Though clinical data suggest the incidence of IFI is lower with second generation BTKi, whether they have less impact on anti-fungal immunity remain unclear. METHODS:We studied the activation phenotype and behavior of human neutrophils in vitro with fungal extracts or living Aspergillus fumigatus in the presence of either ibrutinib or zanubrutinib, a more selective second generation BTKi, using various approaches, including live microscopy. RESULTS:Both molecules inhibited the activation-induced phenotypic changes in neutrophils following exposure to hyphae. Similarly, ibrutinib and zanubrutinib impaired neutrophil-mediated inhibition of conidia germination. By contrast, live neutrophil imaging showed that neutrophils exposed to ibrutinib but not zanubrutinib kept a round morphology, suggesting a defective activation, were less mobile and showed impaired conidia phagocytosis. CONCLUSION:We confirm here that ibrutinib and zanubrutinib inhibit neutrophil antifungal activity, strongly suggesting that BTK plays a central role. However, subtle differences between ibrutinib and zanubrutinib were evidenced using live microscopy, which might indicate additional off-target effects, and potentially partially explain the lower incidence of IFI with zanubrutinib; the clinical relevance remains to be determined.
