Abstract
PurposeCorneal infection in humans caused by Pythium insidiosum can lead to blindness and the host ocular immune response to it is less studied. Herein, we investigate the expression of mediators of innate and adaptive immune responses in a rabbit model.MethodsP. insidiosum zoospores were injected intracorneally in right eye of the nine New Zealand White rabbits while left eye was injected with 1XPBS. RT-qPCR and multiplex ELISA (mELISA) were used to study the expression of antimicrobial peptides (AMPs) and immune mediators in infected cornea on 3rd, 7th and 9th day of post-infection(PI). STRING-11.0 analysis was used to predict the interactions of immune mediators. mRNA expressions of pathogen recognition receptors (PRRs) were determined in human corneal epithelial cells (HCECs) stimulated with P. insidiosum zoospores. Data was analyzed using one-way ANOVA with Post-Hoc Tukey HSD test and p-value <0.05 was considered significant.ResultsmRNA expression assay for IL-1β, IL-6, IL-8 and Cathelicidin antimicrobial peptide (CAP)-18 showed significant upregulation (p-value < 0.05) on 7th day post-infection (PI) compared to 3rd and 9th day while Leukocyte protein (LeukoP) was elevated significantly on 3rd day followed by 7th and 9th day PI . Only IL-17A among other adaptive immune cytokines showed significant upregulation on 7th day compared to 9th day PI. Expressed in pg/mL, mELISA showed significant higher levels (p-value < 0.05) of IL-1β, IL-8 in infected tissue in each of the time points compared to control. STRING analysis revealed co-expression of IL-1β, IL-8 and IL-6. Among PRRs, Dectin 1 and TLR4 showed significant upregulation in HCECs at 12 hrs compared to 6 hrs.ConclusionIn the rabbit P. insidiosum keratitis model, innate immune mediators: IL-1β, IL-8, IL-6, AMPs: LeukoP and CAP-18 are strongly associated in early to mid-stage of corneal infection. Dectin 1 and TLR4 were observed to be associated with recognition of P. insidiosum.
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