Role of toll‐like receptor 2 and 4 of keratocytes on corneal innate immunity

Abstract

Purpose To investigate the role of keratocytes in corneal innate immune system and cross‐talk of keratocytes with resident antigen presenting cells (APCs), especially through toll‐like receptor (TLR)2 and TLR4. Methods Firstly, primary cultivated C57BL/6 (B6) mouse keratocytes were stimulated by Pam3CSK4 (TLR2 agonist) or lipopolysaccharide (LPS; TLR4 agonist) and then were evaluated for their cytokine secretion. To demonstrate the cross‐talk between B6 keratocytes and APCs, cytokine changes under Pam3CSK4 or LPS challenge were assessed in co‐culture condition of B6 keratocytes with mouse dendritic cell line (DC 2.4) or mouse macrophage cell line (Raw 264.7). The reversal effect was checked out using keratocytes from TLR2 knockout (KO) or TLR4KO mice. Results Primary cultivated keratocytes from B6 mice per se actively secreted pro‐inflammatory cytokines, especially interleukin (IL)‐6, with a dose‐dependent manner in response to Pam3CSK4 or LPS challenge. With co‐culture of keratocytes with APCs, secretion of IL‐6 and tumor necrosis factor (TNF)‐α was markedly increased and it was counterbalanced by concurrent increase in IL‐10 and tumor growth factor (TGF)‐β1. After Pam3CSK4 or LPS stimulation, this cytokine balance was completely broken down by overwhelming amplification of IL‐6 and TNF‐α secretion, especially in co‐culture of keratocytes with macrophages, rather than with dendritic cells. Using keratocytes from TLR2KO or TLR4KO mice, we could find the reversal of Pam3CSK4 or LPS‐responsive dose‐dependent increment pattern in IL‐6 and TNF‐α. Conclusion These results implied that keratocytes and their TLRs could be key components for the ocular homeostasis and pathogen‐associated ocular innate immunity.