A Preliminary Study Of The Molecular Mechanism Of Dectin-1 In Experimental Fungal Keratitis

Objective To identify the response and the underlying response pathways of Dectin-1 on macrophages to Fusarium solani and Aspergillus fumigatus. And then we sought to determine whether Dectin-1 plays a similar role in beta-glucan-induced activation in fungal keratitis.Methods Peritoneal macrophages isolated after intraperitoneal injection of sodium thioglycollate were co-cultured with Laminarin, a purified beta-glucan and spores of Fusarium solani and Aspergillus fumigatus for 12 hours. The expression level of Dectin-1 and several cytokines was evaluated by immunofluorescence and real-time quantitative polymerase chain reaction (RTQ-PCR). Then mouse fungal keratitis model were establish by injection of inoculation fungal spores substromally. corneal lesions and inflammatory responses were observed by slit-lamp and histopathology at one, two, three, five and seven days post-infection. RTQ-PCR was used to detect the levels of Dectin-1,CARD9, CXCL1/KC and interleukin (IL)-1β,IL-2,1L-6, macrophage inflammatory protein-2 (MIP-2),IL-10,1L-12,1L-17, tumor necrosis factor a (TNF-a) and monocyte chemotactic protein-1 (MCP-1). For the intervention experiment with neutralizing antibody, macrophages were elicited and cultured for 24 hours and then co-cultured with Dectin-1 antibody (25μg/ml) for 1 hour at 37℃before stimulated with Laminarin and spores. The level of IL-1βwas measured by ELISA.Results Expression of Dectin-1 was upregulated significantly on macrophages stimulated by spores of Fusarium solani and Aspergillus fumigatus, but not by Laminarin(P=0.261). Dectin-1 was not detected in normal corneas of C57BL/6 mouse. But it was detected in infected corneas from the first day post-infection and the mRNA level was highest on the second and third days. Dectin-1 was expressed on the surface of macrophages (F4/80+) and neutrophilic granulocytes (Gr-1+) presence in the infected cornea. CARD9, a key transducer of Dectin-1 signalling, was also upregulated at the same time with Dectin-1 on macrophages and the infected corneas. IL-17 was not detected on macrophages with or without stimulation by Laminarin. Expression of the other examined cytokines were upregulated on macrophages stimulated by Laminarin except TNF-a (P=0.096). The cytokines which were upregulated most significantly were IL-1β、IL-6 and IL-12. All of the examined proinflammatory cytokines and inflammatory factors were produced in detectable in murine fungal keratitis. And,the predominances were IL-1β、MIP-2、CXCL1/KCand IL-12. The produced cytokines were similar in fungal keratitis caused by the two filamentous fungi. Upon administration of Dectin-1 polyclonal antibodies on macrophages, the decrease in mRNA level of IL-1βwas apparent (p=0.016).Conclusion Fusarium solani and Aspergillus fumigatus up-regulate the expression of Dectin-1 in mouse peritoneal macrophages. Dectin-1 could be detected in corneas infected by Fusarium solani and Aspergillus fumigatus but not in normal and mock-infected corneas. CARD9 is activated in FK and in vivo production of IL-1β, MIP-2, CXCL1/KC and IL-12 was significantly increased as compared to control groups. Dectin-1 polyclonal antibodies can reduce IL-1βproduced by macrophages. Thus, Dectin-1 may play an important role in FK and further study is still required about the molecular mechanisms.