Expression And Regulation Mechanism Of IL-1β In Fungal Keratitis

Objective To investigate the expression and molecular mechanisms of inflammatory cytokine IL-1β in fungal keratitis.Methods Mouse model of fungal keratitis established by release Aspergillus fumigatus spores into the corneal stroma with injection is used for in vivo experiments. Bone marrow cells isolated from mice are used for in vitro experiments.(1) To investigate the expression of IL-1β in the fungal keratitis model of C57BL/6mice with flow cytometry and confocal microscope and to explore the major cellular source of IL-1β in fungal keratitis.(2) To investigate the expression of IL-1β in the neutrophils of C57BL/6, TLR4-/-and TRIF-/-mice treated with Aspergillus fumigatus by ELISA and Western-blot and to explore the regulation for IL-1β by the classic Signal1pathway in fungal keratitis.(3) To investigate the expression of IL-1β in the fungal keratitis model of C57BL/6, NLRP3-/-, ASC-/-and Caspase-1-/-mice and in the neutrophils of C57BL/6, Dectin-1-/-, NLRP3-/-, ASC-/-and Caspase-1-/-mice treated with Aspergillus fumigatus by ELISA and Western-blot and to explore the regulation for IL-1β by the classic Signal2pathway in fungal keratitis.(4) To investigate the expression of IL-1β, Caspase-11and Caspase-1in the fungal keratitis model of C57BL/6and Caspase-1-/-mice and in the neutrophils of C57BL/6, Dectin-1-/-, TLR4-/-, TRIF-/-and Caspase-1-/-mice treated with Aspergillus fumigatus by Western-blot and to explore the regulation for IL-1β by Caspase-11in fungal keratitis.Results (1) There is a pronounced neutrophil infiltration to the corneal stroma of mouse fungal keratitis model in24h.92.2%cells in cornea of fungal keratitis which can express IL-1β are neutrophils and85.4%neutrophils can express IL-1β. There are many macrophages recruitment to the corneal stroma in48h.29.7%cells in cornea which can express IL-1β are macrophages and96.6%macrophages can express IL-1β.(2) The expression of pro-IL-1β in bone marrow neutrophils of C57BL/6mice treated with Aspergillus fumigatus spores increased when the priming signal was activated in advance. The expression of pro-IL-1β also increased without the priming, but was slightly less than priming group. The expression of pro-IL-1β in neutrophils of TLR4-/-or TRIF-/-mice treated with Aspergillus fumigatus spores increased in4h and18h, but was not different from C57BL/6mice.(3) The expression of pro-IL-1β in neutrophils of Dectin-1-/-mice or C57BL/6mice added syk inhibitor in advance treated with Aspergillus fumigatus spores was less than C57BL/6mice in4h. The expression of pro-IL-1β and mature IL-1β in the corneas of fungal keratitis model of NLRP3-/-or ASC-/-mice was not different from C57BL/6mice in24h. The expression of pro-IL-1β in the corneas of fungal keratitis model of Caspase-1-/-mice was not different from C57BL/6mice, but mature IL-1β was significantly less than C57BL/6mice in24h. The expression of pro-IL-1β and mature IL-1β in neutrophils of NLRP3-/-or ASC-/-mice treated with Aspergillus fumigatus spores was not different from C57BL/6mice in4h. The expression of pro-IL-1β in neutrophils of Caspase-1-/-mice or C57BL/6mice added Caspase-1inhibitor in advance treated with Aspergillus fumigatus spores was not different from C57BL/6mice, but mature IL-1β was significantly less than C57BL/6mice in4h.(4) The expression of pro-IL-1β in the corneas of fungal keratitis model of C57BL/6mice added Caspase-11inhibitor in advance was not different from C57BL/6mice, but mature IL-1β was significantly less than C57BL/6mice in24h. The expression of pro-IL-1β in neutrophils of C57BL/6mice added Caspase-11inhibitor in advance treated with Aspergillus fumigatus spores was not different from C57BL/6mice, but mature IL-1β and mature Caspase-1were significantly less than C57BL/6mice in4h. The expression of Caspase-11was time-dependent in neutrophils of C57BL/6mice treated with Aspergillus fumigatus spores. The expression of Caspase-11in neutrophils of TLR4-/-or TRIF-/-mice treated with Aspergillus fumigatus spores was not different from C57BL/6mice in4h. The expression of pro-Caspase-11in neutrophils of Dectin-1-/-mice treated with Aspergillus fumigatus spores was slightly less than C57BL/6mice in4h. But the expression of pro-Caspase-11in neutrophils of C57BL/6mice added Caspase-11inhibitor in advance treated with Aspergillus fumigatus spores was significantly less than C57BL/6mice in4h. Conclusions (1) IL-1β has a role in the innate immunity of fungal keratitis, and neutrophil is its major cellular source.(2) IL-lp can be expressed to against fungal infection without the priming signal, and the expression of pro-IL-1β is independent of TLR4/TRIF.(3) The expression of IL-1β is dependent on Dectin-1/syk in the innate immunity of fungal keratitis. Caspase-1leads to maturation of the proinflammatory cytokines IL-1β in response to fungal infection, but NLRP3inflammasome and ASC have no roles in this activation process.