Persistence of Innate Immune Pathways in Late Stage Human Bacterial and Fungal Keratitis: Results from a Comparative Transcriptome Analysis.

Jaya D Chidambaram,Julien Bauer,Palepu Srikanthi,Martin J Holland,Namperumalsamy V Prajna,Prajna Lalitha,Shichina Kannambath,Shanmugam Elakkiya,Matthew J Burton,Manisha Shah

doi:10.3389/fcimb.2017.00193

Abstract

Microbial keratitis (MK) is a major cause of blindness worldwide. Despite adequate antimicrobial treatment, tissue damage can ensue. We compared the human corneal transcriptional profile in late stage MK to normal corneal tissue to identify pathways involved in pathogenesis. Total RNA from MK tissue and normal cadaver corneas was used to determine transcriptome profiles with Illumina HumanHT-12 v4 beadchips. We performed differential expression and network analysis of genes in bacterial keratitis (BK) and fungal keratitis (FK) compared with control (C) samples. Results were validated by RTqPCR for 45 genes in an independent series of 183 MK patients. For the microarray transcriptome analysis, 27 samples were used: 12 controls, 7 BK culture positive for Streptococcus pneumoniae (n = 6), Pseudomonas aeruginosa (n = 1), and 8 FK, culture positive for Fusarium sp. (n = 5), Aspergillus sp. (n = 2), or Lasiodiplodia sp. (n = 1). There were 185 unique differentially expressed genes in BK, 50 in FK, and 339 common to both [i.e., genes with fold-change (FC) < −4 or ≥4 and false discovery rate (FDR) adjusted P < 0.05]. MMP9 had the highest FC in BK (91 FC, adj p = 3.64 E-12) and FK (FC 64, adj. p = 6.10 E-11), along with other MMPs (MMP1, MMP7, MMP10, MMP12), pro-inflammatory cytokines (IL1B, TNF), and PRRs (TLR2, TLR4). HIF1A and its induced genes were upregulated uniquely in BK. Immune/defense response and extracellular matrix terms were the most enriched Gene Ontology terms in both BK and FK. In the network analysis, chemokines were prominent for FK, and actin filament reorganization for BK. Microarray and RTqPCR results were highly correlated for the same samples tested with both assays, and with the larger RTqPCR series. In conclusion, we found a great deal of overlap in the gene expression profile of late stage BK and FK, however genes unique to fungal infection highlighted a corneal epithelial wound healing response and for bacterial infection the prominence of HIF1A-induced genes. These sets of genes may provide new targets for future research into therapeutic agents.

Highlights

Microbial keratitis (MK) is a major cause of blindness worldwide, and affects an estimated 840,000 people per year in India alone (Whitcher et al, 2001)
In order to better understand the immunopathogenesis of this disease, we have investigated the human corneal transcriptome in bacterial keratitis (BK) and fungal keratitis (FK) compared with the normal non-infected cadaveric cornea as the control (C), using Illumina HumanHT12 v4 microarrays
For RNA extracted from corneal tissue used in both the microarray and real time quantitative reverse transcriptase PCR (RTqPCR) experiments, we found a high correlation between expression values for the genes tested (Spearman’s rho 0.90 for BK and 0.90 for FK samples, p < 0.0001 for both; Figure 2)

Summary

Introduction

Microbial keratitis (MK) is a major cause of blindness worldwide, and affects an estimated 840,000 people per year in India alone (Whitcher et al, 2001). Some pathogens are able to produce enzymes that can damage the cornea, much of the corneal destruction in MK is likely due to an excessive host inflammatory response (Steuhl et al, 1987; Gopinathan et al, 2001). Additional sources of MMPs during MK include host corneal epithelial cells and activated keratocytes, causing an excess of these enzymes that can contribute to further tissue destruction, and even cornea perforation (Matsubara et al, 1991). By using a transcriptomic approach, Huang et al investigated the entire murine corneal response to early P. aeruginosa infection and found increased gene expression for pro-inflammatory cytokines (e.g., IL1B, TNF), chemokines (e.g., CXCL2), in the corneas that developed perforation (Huang and Hazlett, 2003). Genes that protected against apoptosis, e.g., BCL2, were upregulated in perforated corneas, implying prolonged survival of immune effector cells and an extended inflammatory response (Huang and Hazlett, 2003)

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Results

Conclusion