D1.427 - Exploring the role of lipids on initiation of allergenic response in innate lymphoid cells

Allergic sensitization and reactions can be triggered by various factors, such as protein or lipid compounds that are co-present or attached to allergens. Here, we aimed to investigate the allergy-inducing potential of lipids, as known for birch pollen derived lipids, and their effect on innate lymphoid cells (ILCs). The hypothesis was that T2 polarization potential may be assessed via the application of nut-associated lipids on ILC3 cells and assess their plasticity towards T2 as a surrogate of pro-allergenic properties.

We took advantage of a mouse ILC3 cell line (MNK3) and performed in vitro stimulation assays with different lipids derived from walnut, peanut and birch pollen. Subsequent changes in gene expression were assessed by microarray and RT-PCR as well as cytometry bead assays. To validate the functional relevance of the discovered target genes we used CRISPR/Cas9-mediated knock outs.

Lipid stimulation significantly increased expression of T2 and T3 inflammatory cytokines, including IL-2, IL-5, IL-9, IL-13, IL-17a, and IL-22, as shown by RT-PCR and a cytometric bead assay. Additionally, microarray data revealed increased expression of the NR4A1 transcription factor, a member of the nuclear receptor superfamily, in stimulated MNK3 cells. Signaling of these transcription factors was initiated by lipid-derived molecules and plays a critical role in the regulation of inflammatory responses of both myeloid and lymphoid cells. 

To demonstrate the functional importance of NR4A1 in the context of lipids stimulation in MNK3, we performed CRISPR/Cas9-mediated knock out (KO) of NR4A1 in MNK3 cells. Upon lipid stimulation, these NR4A1-KO ILCs produced significantly less Th2 cytokines, such as IL-4, IL-5, and IL-13, but elevated levels of IL-17, Gata3, and Rorc2. Our results demonstrate that lipid-derived mediators trigger robust inflammatory responses in ILCs and the NR4A1 transcription factor may play a central role in this mechanism. This highlights lipids as potential facilitators of allergic sensitization and provides new insights into the molecular regulation of ILC-driven inflammation.