The Influence of Thymic Stromal Lymphopoietin on Macrophage Polarization via the Nuclear Factor Kappa B Signaling Pathway in Fungal Keratitis

Abstract

Background: Fungal keratitis (FK) is characterized by corneal inflammation induced by fungal infection. Thymic stromal lymphopoietin (TSLP) is crucial in FK pathogenesis. This study aimed to explore the impact of TSLP on FK and elucidate the mechanism of TSLP signaling on macrophage-mediated inflammation. Methods: Transformed human corneal epithelial cells (THCEs) were stimulated with heat-killed fragments of Aspergillus fumigatus hyphae. TSLP secretion was assessed using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and western blotting (WB). THCEs stimulated with hyphae or recombinant thymic stromal lymphopoietin (rTSLP) were co-cultured with tohoku hospital pediatrics-1 (THP-1) cell-derived macrophages. The inflammatory activity of rTSLP was evaluated in THP-1 macrophages through real-time PCR, ELISA, WB, and flow cytometry. Additionally, high-throughput transcriptome sequencing was employed to elucidate the underlying mechanisms. Results: Secretion of TSLP by A. fumigatus-stimulated THCEs was correlated with increased inflammation in THP-1 macrophage. Treatment with rTSLP resulted in elevated expression of proinflammatory markers associated with macrophage type 1 (M1) phenotype, including tumor necrosis factor-alpha (TNF-α) (p < 0.01), interleukin-1 beta (IL-1β) (p < 0.01), inducible nitric oxide synthase (iNOS) (p < 0.001), and CD86 (p < 0.001) at 24 hours. Exposure of THP-1 macrophages to rTSLP induced activation of the nuclear factor kappa B (NF-κB) signaling pathway. The improved level of phosphorylated NF-κB and inflammatory factors (iNOS, IL-1β, TNF-α) were attenuated by a specific NF-κB inhibitor. Conclusions: In A. fumigatus keratitis, TSLP secreted by THCEs promoted TNF-α, IL-1β, and iNOS secretion in THP-1-derived macrophages by upregulating the NF-κB signaling pathway. This led to dysregulation of the pro-inflammatory (M1)/alternative (M2) macrophage ratio and polarization of THP-1 macrophages towards the pro-inflammatory phenotype.