Understanding how the type 2 cytokines interleukin 4 and 13 shape the function of dendritic cells

Abstract

Conventional dendritic cell type 2 (cDC2) populations play an important role in type 2 immunity by responding to signals from the tissue environment and shaping CD4⁺ T helper cell responses. In the skin, IL-13 and IL-4 have been linked to the development of CD11b low DC2 populations, but these cells are difficult to study directly in vivo because they are rare and are influenced by many local tissue signals. This project used FLT3L bone marrow-derived dendritic cells (BMDCs) as an in vitro model to test how IL-4 and IL-13 directly condition SIRPα⁺ cDC-like cells, and to assess which features of in vivo DC2 biology this model captures. Flow cytometry showed that IL-4 and IL-13 did not substantially alter the overall proportions of major dendritic cell populations within BMDC cultures. Instead, both cytokines shifted the SIRPα⁺ cDC2 compartment toward a CD11b low phenotype. This supported previous evidence that IL-4 and IL-13 can induce features associated with CD11b low DC2s. To define the transcriptional changes associated with this response, SIRPα⁺ BMDCs were isolated by fluorescence-activated cell sorting and analysed by bulk RNA sequencing. Both cytokines shifted SIRPα⁺ BMDCs away from the unstimulated state and increased expression of genes associated with antigen presentation, chemokine signalling, NF-κB related pathways, T cell interaction, and cholesterol biosynthesis. Although IL-4 and IL-13 induced overlapping transcriptional responses, direct comparison showed that each cytokine produced a distinct transcriptional signature, indicating that these cytokines are not interchangeable regulators of SIRPα⁺ BMDC state. Comparison with published in vivo CD11b low and CD11b high DC2 signatures from skin showed that cytokine-treated BMDCs shared only modest transcriptional overlap with either skin DC2 population. IL-4 and IL-13 treated SIRPα⁺ BMDCs shared 19 genes with the published CD11b low signature and 20 genes with the published CD11b high signature. This shows that the FLT3L BMDC model did not reproduce either in vivo transcriptional identity. However, the model still captured direct effects of IL-4 and IL-13 on cDC-like BMDCs, including the shift toward lower CD11b expression and shared cytokine-responsive transcriptional changes. Because IL-4 and IL-13 altered expression of cholesterol biosynthesis genes, we next examined whether these transcriptional changes were reflected in Filipin III staining or BMDC morphology. Filipin III flow cytometry showed increased fluorescence in cytokine-treated BMDC subsets, particularly within cDC2 populations. Confocal microscopy showed that IL-4 and IL-13 treated BMDCs were larger and less circular, while mean Filipin III intensity was not significantly increased. These findings suggest that the increased Filipin III signal measured by flow cytometry may reflect changes in cell size or structure rather than a simple increase in free cholesterol concentration. A multiplexed Cell Painting workflow was therefore adapted to visualise multiple cellular structures within the same BMDCs and provide a basis for future analysis of cytokine-induced morphology and cholesterol localisation. In addition to the shared increase in cell area and reduction in circularity observed after both cytokines, IL-13 treated cultures contained multinucleated cells that were not detected in the unstimulated or IL-4 treated images analysed. This identified multinucleation as an additional IL-13 associated cellular phenotype that requires further investigation. Whether IL-4 and IL-13 alter free cholesterol distribution, accessibility, or localisation within BMDCs remains to be determined. Overall, this thesis shows that IL-4 and IL-13 condition SIRPα⁺ DC2 like BMDCs in related but distinct ways. Both cytokines shifted cells toward a CD11b low phenotype, increased expression of genes linked to antigen presentation, chemokine signalling, immune activation, and cholesterol biosynthesis, and altered BMDC morphology. However, comparison with published in vivo DC2 signatures showed that the model only partially reflected skin DC2 transcriptional identity. Therefore, FLT3L-derived BMDCs provide a useful model for investigating direct IL-4 and IL-13 effects on cDC-like cells, which exhibit overlapping but distinct responses to these cytokines. However, these responses do not fully recapitulate the transcriptional characteristics of skin CD11b low DC2s in vivo.