Flightless I Negatively Regulates Macrophage Surface TLR4, Delays Early Inflammation, and Impedes Wound Healing

TLR4 plays a pivotal role in orchestrating inflammation and tissue repair. Its expression has finally been balanced to initiate the early, robust immune response necessary for efficient repair without excessively amplifying and prolonging inflammation, which impairs healing. Studies show Flightless I (Flii) is an immunomodulator that negatively regulates macrophage TLR4 signalling. Using macrophages from Flii^+/-, WT, and Flii^Tg/Tg mice, we have shown that elevated Flii reduces early TLR4 surface expression, delaying and reducing subsequent TNF secretions. In contrast, reduced Flii increases surface TLR4, leading to an earlier robust TNF peak. In Flii^+/- mice, TLR4 levels peak earlier during wound repair, and overall healing is accelerated. Fewer neutrophils, monocytes and macrophages are recruited to Flii^+/- wounds, leading to fewer TNF-positive macrophages, alongside an early peak and a robust shift to M2 anti-inflammatory, reparative Ym1⁺ and IL-10⁺ macrophages. Importantly, in diabetic mice, high Flii levels are found in plasma and unwounded skin, with further increases observed in their wounds, which have impaired healing. Lowering Flii in diabetic mice results in an earlier shift to M2 macrophages and improved healing. Overall, this suggests Flii regulation of TLR4 reduces early inflammation and decreases the M2 macrophage phenotype, leading to impaired healing.