Modulatory Effect of Alarmins in Immune and Structural Cells: Implication for Asthma Pathogenesis and Clinical Relevance

Abstract

Background: Asthma is a heterogeneous disease characterized by airway hyperresponsiveness (AHR) and inflammation. Alarmins, including TSLP, IL-33, and IL-25 (IL-17E), are released by epithelial cells in response to environmental triggers and play a crucial role in shaping immune responses. These cytokines contribute to airway inflammation and have emerged as potential therapeutic targets. While their role in Th2 inflammation is well established, their effects on airway smooth muscle (ASM) responsiveness, immune cell activation (PBMCs, CD4+T cells, and CD8+T cells), and the transcriptional regulation of CD4+T cells remain incompletely understood. Methods: (i) Tracheal rings from C57BL/6 mice, divided into upper and lower parts, were cultured in DMEM with recombinant mouse TSLP, IL-33, and/or IL-17E (IL-25) at 100 ng/ml for 24 hours. Contraction to carbachol (CCh) and relaxation to formoterol were then assessed using the DMT organ bath system. (ii) Human PBMCs, CD4+T cells, and CD8+T cells from healthy donors were pre-treated with recombinant human TSLP, IL-33, and /or IL-17E (IL-25) 100 ng/ml for 24 hours prior to TCR engagement aCD3 (anti-CD3) 1 µg/ml/ aCD28 (anti-CD28) 2 µg/ml for 4 days, and the anti-inflammatory effect of dexamethasone were assessed at different time point during stimulation. (iii) Transcriptome responses in isolated healthy CD4+T cells, with or without combined alarmin cytokines stimulation (100ng/ml), were assessed by RNA-seq. Results: (i) Contractile responses to carbachol (CCh) were enhanced in tracheal ring treated with TSLP, while IL-25 decreased CCh-induced ASM contractility in both upper and lower tracheal rings. IL-33 had no effect on CCh-evoked responses. Notably, incubation with combined alarmins significantly reduced contractile responses in the lower parts. (ii) in immune cells, treatment of isolated CD4+T cells with alarmins led to a significant alter the TCR-induced cytokine production. All alarmins, either alone or in combination, enhanced IL-5 production. TSLP and combined alarmins significantly increased IL-10 and IFNg production, while TSLP and IL-33 significantly enhanced TNFa production. (iii) RNA-seq analysis revealed distinct gene expression changes in isolated healthy CD4+T cells treated with combined alarmins (100 ng/ml) under TCR activation conditions. Upregulated genes included SMN2, AFF3, GSTM1, and IRS1, whereas HLA-C, HLA-DQB1, HLA-DQA1, and AOAH were downregulated. Gene ontology analysis further showed that alarmins supressed pathways related to metabolism and immune activation, while enhancing those associated with protein transport and secretion compared with TCR activation alone. Conclusion: These findings demonstrate that alarmins differentially regulate ASM contractility, modulate TCR-induced cytokine production in immune cells, and reshape the transcriptional profile of CD4+T cells. Collectively, this highlights their pivotal role in orchestrating airway inflammation and immune responses and provides insight into their potential as therapeutic targeting in asthma.