Spry2 Downregulation Shifts Macrophages Toward an M2 Phenotype by Stimulation with Interferon γ and Porphyromonas Gingivalis Lipopolysaccharide

Objectives: Periodontitis is a chronic inflammation caused by specific bacteria residing in a biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway, We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, whereas it diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increased periodontal ligament cell proliferation and migration accompanied by the prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro.
Methods: J774.1 murine macrophage-like cells were stimulated with IFNγ and Pg LPS after transfection of Spry2 siRNA. Measurement of cytokine production, arginase activity assay, efferocyotosis assay, Rac1 assay, real-time PCR, and Western blot analysis were performed.
Results: Transfection of macrophages with Spry2 small-interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)-10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3-kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFκB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation.
Conclusions: Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage-based anti-inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration.