Abstract
Purpose :
Aspergillus flavus is the most common etiology of fungal endophthalmitis in India leading to severe vision impairment, while Candia albicans is the causative agent in the West. The host immune response is a critical line of defence against these fungal pathogens and in this study we explored the role of microglial cells in mitigating inflammation, following infection with A. flavus and C. albicans strains isolated from patients with endophthalmitis.
Methods :
Immortalized human microglia (CHME-3) were infected with A. flavus and C. albicans and quantitative real time-PCR (qRT-PCR) was performed to study the expression of Toll Like Receptors (TLR), cytokines and Matrix metalloproteinases (MMP) at various time intervals. All qRT-PCR assays were performed in triplicate and the relative expression of each gene was determined after normalization to b-actin transcript levels.
Results :
C. albicans infected microglia exhibited induction of innate immune response via upregulation of TLR-1, -2 and -9 at 3 hrs while A. flavus showed significant expression (p<0.001) of TLR-1,-2,-5, -6,-7 and -9 at 12 hrs. Similarly while C. albicans infection led to expression of Interleukins (IL)-10 (p< 0.001) and IL-17 (p< 0.05) at 24 hrs, A. flavus infection concomitantly showed expression of IL-1a, TNFa and IL-8 along with IL-10 and IL-17 (p< 0.001) at 12 hrs. Significant expression of immune modulator IL-6 was observed in microglia infected with C. albicans at 3 hrs (p< 0.01) and A. flavus at 12 hrs. IL-1b was however induced by both C. albicans and A. flavus by 6 hrs. Differential responses was also observed in the activation of MMP-2 by cells infected by C. albicans at 6 hrs, and activation of MMP-9 (p< 0.05) by cells stimulated with A. flavus. Interestingly TIMP-1 and M-CSF (p< 0.01) was upregulated in cells infected with A. flavus and downregulated in cells infected with C. albicans. The protein expression of MMPs, IL-6, -8 and -10 were validated by ELISA while expression of TLR4, IL-17 and IL-1β was confirmed by Immunofluorescence assay.
Conclusions :
These results corroborate the relevance of microglial cells within the antifungal immune response, in the outcome of endophthalmitis. Together, these results help in understanding the differential responses involved in moulds and filamentous fungi for construction of better immunomodulatory strategies.
This is a 2020 ARVO Annual Meeting abstract.