Wound healing is a complex process consisting of a series of overlapping events which include inflammation, matrix deposition and remodelling. Any perturbations in this process can result in delayed or incomplete healing, leading to chronic wounds. These wounds often have a prolonged or incomplete inflammatory phase of healing. In this state the majority of macrophages present remain in their M1 phenotype, where activation of Toll-like receptor 4 (TLR4) and signalling through MyD88, results in an increase in TNF expression, further exacerbating the pro-inflammatory reaction. Flightless I (Flii) is a protein know to inhibit TLR4 signalling via two mechanisms: sequestering the TLR4 activator LPS and by binding MyD88 preventing TLR4 signalling. This mode of action suggests that Flii could potentially inhibit inflammation and promote M1 to M2 switching.
To investigate this hypothesis we used in vitro and in vivo approaches with three Flii lines of genetic mice containing low (Flii+/-), normal (WT) or high (FliiTg/Tg) levels of Flii. Incisional dorsal wounds were created on these mice and the effect of Flii on TLR signalling, cytokine production and inflammation were determined. Macrophages were also collected from the mice lines and stimulated with LPS. The media was collected and TNF production quantified by ELISA.
Our data showed that increasing levels of Flii hindered the onset of inflammation and the overall rate of healing. Increased Flii levels delayed TNF production, but contrary to its known role in TLR4 signalling, increased TNF levels overall. It impeded monocyte to macrophage transition and delayed M1 to M2 switching. In mice with reduced Flii levels there was an earlier differentiation of monocytes to macrophages, an increased ratio of M1:M2 macrophages and a shortened inflammatory response, with rapid healing. These studies illustrate that Flii has important functions during inflammation and reducing its activity leads to improved healing outcomes.