Epidemic keratoconjunctivitis (EKC), an important human eye infection, is caused by human adenovirus species D (HAdV-D) types 8, 37, 53, 54, 56, and 64. The clinical manifestations of this infection include severe membranous conjunctivitis and epithelial keratitis, followed by multifocal subepithelial (stromal) corneal infiltrates that cause photophobia and reduced vision and may persist or recur for months to years after the acute infection. We have shown previously that HAdV-D infection of human keratocytes induces intracellular signaling and robust expression of neutrophil chemokines, and that physical components of the adenovirus (viral capsid and DNA) initiate distinct cytokine responses in the corneal stroma. However, characterization of responses to infection of other ocular surface cell types such as corneal and conjunctival epithelial cells, is lacking,.
The specific aims of this proposal are to test the hypotheses that 1) HAdV-D37 infection of ocular surface cells induces cytokine signatures distinguishable by cell type, 2) HAdV-D37 infection of ocular surface cells activates unique molecular pattern recognition receptors distinguishable by cell type, and 3) cell type-specific pattern recognition receptor activation by HAdV-D37 and intracellular trafficking of HAdV-D37 are interdependent. We will apply intracellular cytokine staining with flow cytometry along with cytokine protein array analysis of the various cell types on the human ocular surface to identify cell type-specific patterns of cytokine expression upon HAdV-D37 infection. Because CXCL8 has been shown to increase subsequent adenoviral entry by increasing the expression and apical localization of host cell receptors, we will compare infectivity of corneal cells in the presence of cytokines expressed during infection of conjunctival and immune cells from the ocular surface. We will apply Western blot, immuno-confocal microscopy, ELISA, cytokine arrays, and siRNA knockdown, to determine the molecular pattern receptors activated in human ocular surface and bone marrow derived cells by HAdV-D37 infection. Finally, with methods that allow differentiation of intact virus versus capsid-free viral DNA and endosomal versus cytoplasmic virus, with confocal microscopy, we will characterize viral trafficking and its kinetics across ocular surface cell types, identify loci of interaction between viral DNA and specific pattern recognition receptors, and evaluate the effect of pattern recognition receptor knockdown on viral trafficking. The study of cytokine expression, pattern recognition receptor activation and signaling, and intracellular trafficking across a broad spectrum of ocular surface cell types, will take us closer to our goal of effective information-based therapies against HAdV eye infections. EKC is a common affliction, and the proposed study addresses a major public health concern.

Public Health Relevance

Adenovirus Is the most common cause of human eye infection. This project will dissect cytokine expression, pattern recognition receptor activation and signaling, and intracellular trafficking of human adenovirus type 37, associated with epidemic ocular surface infection and the most virulent adenoviral eye pathogen.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY013124-19
Application #
9893874
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mckie, George Ann
Project Start
2001-06-01
Project End
2021-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
19
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Massachusetts Eye and Ear Infirmary
Department
Type
DUNS #
073825945
City
Boston
State
MA
Country
United States
Zip Code
02114
Ismail, Ashrafali M; Cui, Tiange; Dommaraju, Kalpana et al. (2018) Author Correction: Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens. Emerg Microbes Infect 7:208
Lee, Cecilia S; Lee, Aaron Y; Akileswaran, Lakshmi et al. (2018) Determinants of Outcomes of Adenoviral Keratoconjunctivitis. Ophthalmology 125:1344-1353
Uchino, Yuichi; Woodward, Ashley M; Mauris, Jérôme et al. (2018) Galectin-3 is an amplifier of the interleukin-1?-mediated inflammatory response in corneal keratinocytes. Immunology 154:490-499
Deiner, Michael S; McLeod, Stephen D; Chodosh, James et al. (2018) Clinical Age-Specific Seasonal Conjunctivitis Patterns and Their Online Detection in Twitter, Blog, Forum, and Comment Social Media Posts. Invest Ophthalmol Vis Sci 59:910-920
Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C et al. (2018) Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection. mSphere 3:
Ismail, Ashrafali M; Lee, Ji Sun; Lee, Jeong Yoon et al. (2018) Corrigendum: Adenoviromics: Mining the Human Adenovirus Species D Genome. Front Microbiol 9:3005
Ismail, Ashrafali M; Lee, Ji Sun; Lee, Jeong Yoon et al. (2018) Adenoviromics: Mining the Human Adenovirus Species D Genome. Front Microbiol 9:2178
Pan, Haibin; Yan, Yuqian; Zhang, Jing et al. (2018) Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly. Viruses 10:
Ismail, Ashrafali M; Cui, Tiange; Dommaraju, Kalpana et al. (2018) Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens. Emerg Microbes Infect 7:10
Sié, Ali; Diarra, Abdramane; Millogo, Ourohiré et al. (2018) Seasonal and Temporal Trends in Childhood Conjunctivitis in Burkina Faso. Am J Trop Med Hyg 99:229-232

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