Gli3 Regulates PD-L1 Expression in Macrophages and Alters Maturation and Cytokine Profiles in Plasmacytoid Dendritic Cells 2260198

Abstract

Abstract Introduction Our lab previously demonstrated that TLR—TRIF—IRF3 signaling induces the transcription factor GLI3, revealing a non-canonical regulatory axis beyond Hedgehog (HH) signaling. However, how GLI3 influences myeloid cell function downstream of innate activation remains unclear. Methods Bone-marrow-derived macrophages (BMDM) and dendritic cells (DC) were generated from wild-type (Gli3fl/fl LysMcre-/-) and myeloid-specific Gli3 knockout (Gli3fl/fl LysMcre+/-, referred to as M-Gli3-/-) mice. Macrophages were in vitro polarized to M2 and analyzed for Cd274 (PD-L1) expression by qPCR and flow cytometry. DC subsets (moDCs, cDC1, cDC2, pDC) were in vitro derived and assessed for lineage differentiation, CD86 expression, and cytokine production. Results M2 macrophages from WT mice showed robust PD-L1 expression, whereas MGli3⁻/⁻ macrophages exhibited markedly reduced PD-L1 transcript and surface expression by flow, independent of HH/SMO signaling, as supplementation with sonic hedgehog (Shh) ligand did not restore PD-L1 levels. GLI3 deletion did not impair DC subset differentiation, indicating normal myeloid development. However, matured pDCs from MGli3⁻/⁻ cultures displayed reduced TNF-α and CD86 expression compared with WT DCs, suggesting a selective role for GLI3 in pDC activation rather than lineage specification. Conclusion Building upon our prior demonstration that TLR-TRIF—IRF3 signaling induces GLI3, these findings reveal that GLI3 functions downstream to regulate macrophage PD-L1 and pDC activation markers, positioning GLI3 as a context-dependent modulator of myeloid immunoregulatory functions independent of canonical Hedgehog signaling. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)