How the microbiome powerfully modulates our ocular health Harvey A. - PowerPoint PPT Presentation

How the microbiome powerfully modulates our ocular health Harvey A. Fishman, MD, PhD Comprehensive Ophthalmology 706 Webster St. Palo Alto, CA

Financial Disclosures • Co-Founder of EyeCareLive • Co-Founder of TearBio • Digital Health Patents • Speaking Consultant with Lumenis • Consultant with 23andMe • Past Research Consulting or Speaker for AMO, Allergan, Zeiss, OptoVue, Bio-Tissue

Overview I. Definitions: “High-Yield Biology Primer” II. Human Microbiome Project (HMP) III. Genetic techniques for microbiome analysis IV. Non-ocular microbiome and Ocular health V. Ocular Microbiome and Ocular health VI. Future directions DNA Chip Technology

“High-Yield Biology Primer” What is a microbiome? • Nobel Prize Laureate, Dr. Joshua Lederberg, coined “ microbiome” in 2001 to signify the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease. “ Ome sweet 'omics: -- A genealogical treasury of words. The Scientist. 2001; 15:8.

“High-Yield Biology Primer” • Phylogenetics - the study of the evolutionary history and relationships among individuals or groups of organisms (e.g. species, or populations) • Taxonomy - the identification, naming and classification of organisms- Taxon (plural taxa)- is a group of one or more populations of an organism or organisms to form a unit) • “ Microbiota ” - the microbial taxa associated with humans • “ Microbiome ” - the catalog of these microbes and their genes • “ Metagenomics ” - originally referred to shotgun characterization of total DNA, although now it is being applied to studies of marker genes such as the 16S rRNA gene • Ribosome - complex molecular machine, found within all living cells, that serves as the site of biological protein synthesis (translation) • “ OTUs ” (Operational Taxonomic Units) - clusters of (uncultivated or unknown) organisms, grouped by DNA sequence similarity of a specific taxonomic marker gene- pragmatic proxies for microbial "species" at different taxonomic levels, in the absence of traditional systems of biological classification as are available for macroscopic organisms. Typically, OTUs are based on similar 16S rRNA sequences

NIH Human Microbiome Project (HMP) The overall mission of the HMP is to generate resources to facilitate characterization of the human microbiota to further our understanding of how the microbiome impacts human health and disease a. Pregnancy & Preterm Birth: How microbiome and host profiles change throughout pregnancy and influence the establishment of the nascent microbiome in neonates b. Onset of Inflammatory Bowel Disease (IBD): How the human gut microbiome changes over time in adults and children with IBD c. Onset of Type 2 Diabetes Type 2 diabetes mellitus (T2D): Performing a detailed analysis of the biological processes that occur in the microbiome and human host by longitudinal profiling of patients at risk for T2D.

Genetic Techniques for Microbiome Analysis 16S ribosomal RNA (or 16S rRNA) sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories a. The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria and archaea b. Archaea constitute a domain of single-celled microorganisms. These microbes are prokaryotes, meaning they have no cell nucleus c. 16S rRNA gene sequences contain hypervariable regions that can provide Pearson Education, Inc. species-specific signature sequences useful for identification of bacteria.

Genetic Techniques for Microbiome Analysis 16S ribosomal RNA (or 16S rRNA) sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories d. 16S rRNA is the component of the 30S small subunit of a prokaryotic ribosome that binds to the Shine-Dalgarno sequence. e .The genes coding for it are referred to as 16S rRNA gene and are used in reconstructing phylogenies, due to the slow rates of evolution of this region of the gene. f. 16S rDNA sequencing is particularly important for identification of bacterial with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections.

Genetic Techniques for Microbiome Analysis Ilumina Chip Technology: Oligonucleotides, cDNA or small fragments of PCR products corresponding to specific genes are spotted on the chip.

Non-ocular microbiome and ocular health • Human microbiota consists of the 10-100 trillion symbiotic microbial cells harbored by each person, primarily bacteria in the gut; the human microbiome consists of the genes these cells harbor • Meta- HIT consortium reported a gene catalog of 3.3 million non- redundant genes in the human gut microbiome alone as compared to the ∼ 22,000 genes present in the entire human genome • Decreased Bacteriodetes and increased Firmicutes have been found in genetically obese mice (ob/ob) when compared to their lean counterparts, and the obesity phenotype can even be transferred to a germ- free but genetically wild-type mouse by way of the microbiota • Most of the 10–100 trillion microorganisms in the human gastrointestinal tract live in the colon. • More than 90% of all phylogenetic types (phylotypes) of colonic bacteria belong to just 2 of the 70 known divisions (phyla) in the domain Bacteria: the Firmicutes and the Bacteroidetes. 19th January 2018 by Laura Berry

Non-ocular microbiome and ocular health Microbiome dysbiosis and ocular disease AMD- EFEI • Macular degeneration • Uveitis • Non-specific autoimmune ocular disease • Dry eye disease Uveitis - Medicine.net • Glaucoma

The severity of SS ocular and systemic disease was inversely correlated with microbial diversity. These findings suggest that SS is marked by a dysbiotic intestinal microbiome driven by low relative abundance of commensal bacteria and high relative abundance of potentially pathogenic genera that is associated with worse ocular mucosal disease in a mouse model of SS and in SS patients.

Highlights • T cell activation in the gut correlates with spontaneous uveitis in R161H mice • Elimination of gut microbiota attenuates disease and reduces T cell activation in the gut • Presence of endogenous antigen is not required for intestinal T cell activation • Extracts of intestinal contents signal via retina-specific TCR and trigger uveitis

Chronic inflammation and tissue damage may be the result of an exaggerated or dysregulated host response to the infection and the microbiome might be a significant source of infectious antigen and antigen-specific T cells. Thus, chronic or recurrent uveitic disease may be caused by local reactivations of persistent microbial agents or inadequately cleared antigen, including retroviral antigen, which intermittently disrupt the Treg/ T-effector cell ratio. In addition, a dysregulated microbiome may predispose to, or even be the source of, uveitogenic pathogens or adjuvants.

The role of IM in ocular Inflammatory disease Certain bacterial strains, segmented filamentous bacteria found in rodents and analogous bacterial strains found in human can promote differentiation of T helper 17 cells (Th17) in the gut.

Five main therapeutic strategies for targeting the intestinal microbiome for treatment of ocular inflammatory disease 1. Targeting specific causative bacteria 2. Target the intestinal microbiome could be through the administration of oral live bacterial strains that are known to promote immune homeostasis by enhancing, for instance, regulatory T cell differentiation. 3. Chemical drugs- antibiotics that are not broad-spectrum but instead are chemicals designed to target the metabolic pathways of only a specific community of bacteria. 4. Dietary modifications, for example exposing individuals to a diet high in nondigestible fibres to enhance the produc- tion of endogenous short chain fatty acids by the intestinal microbiota 5. Supplant an entire community of intestinal bacteria with a normal community using a faecal microbial transplant

Controversy surrounding the existence of the ocular microbiome

Gut- Skin Conection

“These studies, and many similar, have given rise to 2 views. In the first, microbial DNA and organisms may be isolated sporadically from the ocular surface, but without the implication that they are stable colonizers. Rather, their fate is to be killed or removed from the eye” “By contrast, others suggest that, similar to other mucosal sites, there is a normal consortium of microbes that colonize the ocular surface”

CONCLUSIONS: Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.)

Using culture dependent and independent methods, the ocular surface does not appear to support a substantial core microbiome. However, consistently present taxa could be observed within individuals suggesting the possibility of individual-specific core microbiomes.