Deciphering human lung mast cell heterogeneity

<p dir="ltr">Mast cells are tissue-resident immune effector cells that play critical roles in allergic responses and pulmonary pathologies. Traditional protease-based classifications vastly underestimate mast cell heterogeneity, and systematic characterization of their functional diversity, particularly across anatomical compartments and in response to epithelial-derived alarmins, has been lacking. This thesis comprehensively characterizes human lung mast cell heterogeneity, functional properties, and alarmin responsiveness through single-cell transcriptomics, optimized functional assays, and multi-omics profiling.</p><p dir="ltr">Study I employed single-cell RNA sequencing analysis of sorted human lung mast cells to investigate mast cell heterogeneity beyond traditional classifications. The analysis revealed that mast cell heterogeneity manifests primarily as continuous variation in gene expression rather than discrete subpopulations. The traditional MC_T/MC_TC classification was challenged by striking discordance between mRNA and protein levels, particularly the ubiquitous CPA3 mRNA expression despite variable protein detection. These findings demonstrated that transcriptomic approaches alone are insufficient for defining classical mast cell subsets and highlighted the importance of post- transcriptional regulatory mechanisms in mast cell phenotype determination.</p><p dir="ltr">Study II performed large-scale single-cell RNA sequencing of 19,232 FACS- sorted mast cells from four anatomical compartments (bronchi, parenchyma, arteries, and veins) across five donors. This comprehensive analysis revealed five transcriptional clusters/states (MC1-MC5) with distinct gene-expression programs, establishing anatomical context as a key organizing principle of mast- cell heterogeneity. MC1 specialized in stress responses, with high expression of SDF2L1, MT2A, and BCL2A1. MC2 displayed broad transcriptional activation, characterized by elevated immediate early response genes such as FOS, JUN, and the growth factor AREG. MC3 showed selective activation with increased expression of signal termination factors DUSP1 and RGS1, together with enrichment for TGF-β-responsive signatures. MC4 represented a pro- inflammatory effector state expressing TNF, IL13, and multiple chemokines, showing preferential localization to bronchi and arteries and enrichment for IL- 33-responsive programs. MC5 exhibited a regulatory phenotype dominated by long non-coding RNAs MEG3 and MEG8. Compartment-resolved analysis demonstrated that bronchial mast cells functioned as inflammatory sentinels optimized for pathogen defense, whereas parenchymal populations displayed homeostatic programs associated with tissue maintenance. Cross-dataset validation confirmed the reproducibility of the MC1-MC4 transcriptional states across independent studies.</p><p dir="ltr">Study III systematically optimized functional assay conditions to enable robust and reproducible assessment of mast cell activation. The study identified serum-free culture conditions as critical for enhanced IgE-mediated degranulation responses. Pre-incubation in fresh serum-free media for 30 minutes significantly enhanced degranulation, while serum presence abolished this effect, suggesting serum-derived inhibitory factors. CD63 and CD164 were identified as the most stable and reliable degranulation markers. Human lung mast cells did not respond to MRGPRX2 agonist compound 48/80, confirming their distinct phenotype from skin mast cells and highlighting tissue-specific functional adaptations.</p><p dir="ltr">Study IV investigated the regulation of IL-2 receptor expression by IL-33 in human lung mast cells. Global transcriptomic analysis revealed that IL-33 potently upregulated CD25 (IL-2Rα) and CD132 (γc) without affecting CD122 (IL- 2Rβ), creating a non-functional IL-2 receptor complex. Despite robust CD25 expression and enhanced IL-2 binding capacity, mast cells failed to respond to IL-2, showing no STAT5 phosphorylation or transcriptional changes. Analysis of publicly available single-cell RNA-seq datasets revealed that IL2RA-positive mast cells increased dramatically from 2.0% in healthy control to 23.8% in emphysema patients, with gene expression signatures substantially overlapping IL-33-induced transcriptional profiles. These findings suggest that CD25- positive mast cells may function as IL-2 sinks, potentially modulating local T cell responses and the cytokine microenvironment. Immunofluorescence analysis confirmed heterogeneous CD25 protein expression on mast cells in lung tissues from healthy controls, COPD and IPF patients.</p><p dir="ltr">Study V performed comprehensive multi-omics profiling to characterize the transcriptional and functional responses of human lung mast cells to the epithelial-derived alarmins IL-33 and TSLP. IL-33 established itself as a potent mast cell activator, inducing over 600 differentially expressed genes at 2 hours, with "signaling by interleukins" emerging as the most enriched pathway. In stark contrast, TSLP failed to elicit transcriptional changes despite inducing STAT5 phosphorylation, revealing a dissociation between signaling and transcriptional outcomes. PTGS2 (COX-2) emerged as the most highly upregulated gene following IL-33 stimulation, yet IL-33-enhanced eicosanoid production remained paradoxically COX-1-dependent, demonstrating the complexity of inflammatory mediator regulation. Comprehensive secretome profiling identified IL-5, IL-13, CSF2/GM-CSF, CCL2, CCL4, and CXCL8/IL-8 as dominant secreted mediators exceeding 1000 pg/mL, with IL-5 and IL-13 particularly enriched in IL-33 responses. Several mediators including GZMA, GZMB, TNFSF12, TGFA, HGF, LIF, and AREG were identified as granule-stored and rapidly released upon IgE- receptor induced degranulation.</p><p dir="ltr">Collectively, these studies establish that human lung mast cells exhibit substantial heterogeneity organized by anatomical compartmentalization and functional specialization. The IL-33/ST2 axis emerges as the predominant epithelial-mast cell communication pathway, with IL-33 functioning as a master regulator of mast cell phenotypic plasticity and inflammatory responses. The identification of distinct mast cell clusters with specialized functions, combined with comprehensive characterization of IL-33-driven CD25 expression and mediator production, provides mechanistic insights for precision therapeutic approaches targeting specific mast cell subsets and signaling pathways in airway inflammatory diseases.</p><h3>List of scientific papers</h3><p dir="ltr">I. Rönnberg, E., Ravindran, A., Mazzurana, L., <b>Gong, Y.</b>, Säfholm, J., Lorent, J., Dethlefsen, O., Orre, A. C., Al-Ameri, M., Adner, M., Dahlén, S. E., Dahlin, J. S., Mjösberg, J., & Nilsson, G. Deciphering human lung mast cell heterogeneity by single cell RNA sequencing. Front Immunol. 2023;14(March):1-9. <a href="https://doi.org/10.3389/fimmu.2023.1151754" rel="noreferrer" target="_blank">https://doi.org/10.3389/fimmu.2023.1151754</a></p><p dir="ltr">II. <b>Gong, Y.</b>, Boey, D., Atanasoai, I., Al-Ameri, M., Sachs, E., Vali, K., Adner, M., Säfholm, J., Dahlin, J., Nilsson, G., & Rönnberg, E. Single-Cell Transcriptomic Profiling Reveals Heterogeneity of Human Lung Mast Cells Across Anatomical Compartments. [Manuscript]</p><p dir="ltr">III. <b>Gong, Y.</b>, Johnsson, A. K., Säfholm, J., Al-Ameri, M., Sachs, E., Vali, K., Nilsson, G., & Rönnberg, E. An optimized method for IgE-mediated degranulation of human lung mast cells. Front Immunol. 2024; May31; 15: 1393802. <a href="https://doi.org/10.3389/fimmu.2024.1393802" rel="noreferrer" target="_blank">https://doi.org/10.3389/fimmu.2024.1393802</a></p><p dir="ltr">IV. <b>Gong, Y.</b>, Atanasoai, I., Siddhuraj, P., Johnsson, A .- K., Peng, Y., Al- Ameri, M., Sachs, E., Vali, K., Adner, M., Säfholm, J., Erjefält, J. S., Nilsson, G., & Rönnberg, E. Expression of CD25 on Human lung Mast Cells and its Regulation by IL-33. [Manuscript]</p><p dir="ltr">V. Atanasoai, I., <b>Gong, Y.</b>, Johnsson, A .- K., Kolmert, J., Al-Ameri, M., Sachs, E., Vali, K., Adner, M., Wheelock, C. E., Säfholm, J., Rönnberg, E., & Nilsson, G. Selective Mast Cell Responsiveness to IL-33, but Not TSLP, Suggests a Dominant Axis of Innate Immune Activation in Airway Inflammation. [Manuscript]</p>