Immune Responses And Mechanism Of CpG Oligodeoxynucleotides In The Treatment For Experimental Fungal Keratitis

ObjectiveTo investigate the immune responses and the possible mechanism of CpG oligodeoxynucleotides (CpG ODN) in the treatment of fungal keratitis in mice.MethodsExperimental murine fungal keratitis was induced by intrastromal injection of Candida albicans and Fusarium solani spores separately. According to the different methods of treatment, mice were divided into five groups as following, Group A:No treatment before or after fungal injection, Group B and Group C:CpG and non-CpG oligodeoxynucleotides separately was injected subconjunctivally after fungal injection instantly, Group D:CpG oligodeoxynucleotides was injected subconjunctivally 2 days before fungal injection, Group E:non-CpG oligodeoxynucleotides was injected subconjunctivally 2 days before fungal injection. Clinical scores and fungal load were measured regularly. Serum, cornea and proliferative spleen cells ex vivo, were colleted at different times in Candida albicans infected mice for measurement of interleukin (IL)-12,IFN-γ,IL-10,MCP-1 and Candida-specific-antibody IgG,IgM,and IgA. Immunohistochemistry was used to detect leukocyte, phagocyte, and lymphocyte infiltration in diseased corneas.ResultsIn the corneas infected by both Candida albicans and Fusarium solani, CpG ODN pretreatment could significantly decreased the clinical scores and fungal load in the diseased corneas as compared with that in other groups (p<0.05). No significant difference was found in the clinical scores and fungal load among groups A,B,C and E (p>0.05). In the mice with cornea infected by Candida albicans and CpG pretreatment subconjunctivally, the levels of IL-12,IFN-γ,IL-10 and MCP-1 in both the serum and the cornea increased after infection in group D to some extents, in those the levels of IL-12 and IFN-y elevated most significantly (p<0.05). IL-12 level sharply increased at the first day after infection and persisted at a higher level to at least 14 days post-infection, which was significantly higher than that in any other groups (p<0.05). The level of IL-10 sharply increased at the first day and then reached to the normal level at 14 days post-infection, which was lower than that in group B (p>0.05), but much higher than that in group A,group C and group E on day 1 post infection (p<0.05). The levels of IFN-y increased at the fist day post-infection and elevated slightly later. While in group B, the levels of IL-12 and IFN-y in both the serum and the cornea sharply elevated as well when compared with that in mice without CpG ODN treatment (p<0.05). However, the IL-12 level in mice of group B was still significantly lower than that in group D (p<0.05). In addition, both IL-10 and MCP-1 in serum and cornea in mice of group B showed higher levels as compared with that in group A, group C, group D and group E on day 1 after infection (p<0.05). In total, the IL-12 and IFN-y in cornea and serum had increased significantly after infection in mice with-CpG ODN treatment when compared with that got no CpG ODN intervention (p<0.05). In the cornea and serum of CpG treatment groups (groups B and D), Thl cytokines of IL-12 and IFN-y elevated significantly than that in groups got no CpG ODN intervention (groups A, C and E) (p<0.05). The cytokines assay in the spleen cells ex vivo showed that the immune responses induced by pretreatment of CpG ODN were significantly biased toward the T-helper 1 type as well. No elevating of Candida-specific antibody was detected in the corneas, serum and spleen cells in any of the groups. The immunohistochemistry assay showed that more infiltration of immune cells, including leukocytes, macrophage, and lymphocytes, were found in the corneas of mice with subconjunctival injection of CpG ODN.Conclusions The present study suggested that CpG ODN pretreatment activated the host anti-fungal immune responses and showed more effectiveness to prevent the fungal keratitis. The immune responses against fungal keratitis in mice with CpG ODN pretreatment were mediated by significant T-helper 1 cytokines, which benefit for the clearance of fungal pathogens and healing up of corneal ulcers and thus contribute to a favourable perspective in the treatment of fungal keratitis.