Effects Of Lacrimal Androgen-binding Proteins On Aspergillus Fumigatus Keratitis In Mice

Aim: To clarify the regulatory mechanisms of lacrimal androgen-binding proteins(ABPs)in mice with keratitis caused by Aspergillus fumigatus(A.fumigatus).Methods: 1.The lacrimal glands are altered in mice with A.fumigatus keratitis: The lacrimal glands of female mice were removed 24 h after A.fumigatus keratitis infection.The length,weight and lacrimal gland/body weight ratio of normal mice and mice were measured.Hematoxylin-eosin Staining was used to determine the histological changes of lacrimal glands.2.The expression of ABP ε and ABP δ increased in mice with A.fumigatus keratitis: Images taken via slit lamp microscopy and clinical scores showed the disease responses to A.fumigatus infection in male and female mice after 24 h.Mice were killed and their lacrimal glands were removed.The expressions of ABP α,ABP η,ABP ζ,ABP ε and ABP δ in lacrimal gland of mice were detected by q RT-PCR and corneal tissue protein mass spectrometry.3.Effects of glucocorticoid on lacrimal gland and lacrimal ABPs in mice with A.fumigatus keratitis: Mice were divided into four groups: the normal group(Group A),A.fumigatus keratitis infection group(Group B),subconjunctival injection of glucocorticoid before A.fumigatus keratitis infection group(Group C),and lacrimal gland injection of glucocorticoid before A.fumigatus keratitis infection group(Group D).Dexamethasone(1mg/ml)5 μL was injected into the conjunctiva or lacrimal gland respectively in group C and group D.The lacrimal gland/body weight ratio was measured.The expression of ABP ε and ABP δ in lacrimal gland was detected by q RT-PCR.4.Changes to inflammatory mediators in mice with A.fumigatus keratitis: The expression of inflammatory mediators IL-1β,IL-8,IL-10 and TNF α in lacrimal gland were detected by q RT-PCR and Western blot.Immunofluorescence was used to detect the distribution of neutrophils and macrophages and the expression of IL-1β in lacrimal gland.5.Effect of IL-1β on the lacrimal glands and ABPs in mice with A.fumigatus keratitis:Mice were divided into three groups:the first group was the normal control group,the second group had PBS injected into the lacrimal gland,and the third group had IL-1βinjected into the lacrimal gland.In the second and third groups,5 μL PBS or IL-1β(50ng/ml,100ng/ml or 400ng/ml)were injected into lacrimal gland respectively.After 24 hours,mice were killed and lacrimal glands were taken.Lacrimal gland length,weight and lacrimal gland/body weight ratio were recorded.The expression of ABP ε and ABP δm RNA was detected by q RT-PCR.Results: 1.The lacrimal glands are altered in mice with A.fumigatus keratitis: The volume of the lacrimal glands in the infected group were larger than those in the normal group.In addition,the ratios of length,weight,and lacrimal gland/ body weight of the infected mice were higher than those in normal group.It could be seen from HE staining that the structure of lacrimal gland in the infection group became disorganized compared with the normal group,but we didn’t observe the infiltration of inflammatory cells.2.The expression of ABP ε and ABP δ increased in mice with A.fumigatus keratitis: There was no significant difference between the clinical scores between genders.Compared with the normal group,the expression of ABP α,ABP η,and ABP ζ in the lacrimal gland were not significantly altered,while the expression of ABP ε and ABP δ increased significantly in the mice with A.fumigatus keratitis.Protein mass spectrometry also confirmed that the expression of ABP ε and ABP δ increased in the A.fumigatus keratitis group.Moreover,there was no significant difference in the expression of the five ABPs between male and female mice.3.Effects of glucocorticoid on lacrimal gland and lacrimal ABPs in mice with A.fumigatus keratitis: Compared with group B,corneal clinical scores of group C were decreased,but there was no obvious difference between group B and group D.As for the lacrimal gland/body weight ratio,the ratio in group C was lower than group B and there was no difference between group D and group B.Furthermore,the expression of ABP ε and ABP δ m RNAs in the lacrimal gland decreased after subconjunctival pretreatment with glucocorticoid,while there were no meaningful changes following lacrimal gland injection.4.Changes to inflammatory mediators in mice with A.fumigatus keratitis: q RT-PCR results showed that the expression of IL-1β increased in the lacrimal gland of mice infected with A.fumigatus keratitis,while the expression of the other three mediators were unaltered.Western blots also indicated that the expression of IL-1βincreased in the A.fumigatus keratitis group.Positive staining for IL-1β in the lacrimal glands of infected mice was higher compared to the normal mice 24 h after A.fumigatus keratitis infection.However,there was no increase in the infiltration of neutrophils or macrophages.5.Effect of IL-1β on the lacrimal glands and ABPs in mice with A.fumigatus keratitis: The length ratio,weight ratio,and lacrimal gland/body weight ratio in mice injected with IL-1β were higher than the other two groups.q RT-PCR analysis showed that the expression of ABP δ and ABP ε decreased after IL-1β was injected into the lacrimal gland.With the increase in IL-1β,the expression of ABP δ and ABP εdecreased gradually.Conclusion: 1.Lacrimal glands contributed to protection in fungal keratitis,which was not due to the involvement of inflammatory cells in mice.2.ABP δ and ABP ε of mice were involved in reducing the severity of corneal damage in mice with A.fumigatus keratitis.3.The expression of IL-1β and ABP δ and ABP ε were intrinsically linked.