ARVO 2024   12

Advancements in Ocular Immunobiology and Microbiology—Insights from Next-Gen Researchers

Young researchers in vision science presented their discoveries regarding ocular immunobiology and microbiology, while sharing factors that contribute to the success of their research endeavors during the Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO 2024).

Flow cytometry-based profiling of corneal immune cells

First on the agenda was Pratima Suvas, PhD (Wayne State University School of Medicine, USA), who presented on the use of flow cytometry to assess the diversity of Langerhans cells in the corneal epithelium under both normal conditions and after corneal HSV-1 infection. 

Dr. Suvas began by outlining the gating strategy used for analyzing cells in the epithelium and stroma, highlighting variations observed in infected tissues. Langerhans cells in the corneal epithelium are found to exhibit two distinct populations: CD11c low cells and CD11c high cells, with differing expression levels of key markers indicating their maturity. Following HSV-1 infection, changes in the frequency and total number of Langerhans cells are observed.

The origin of Langerhans cells is investigated, revealing subsets with monocytic and non-monocytic origins in uninfected corneal epithelium. Similar analysis is conducted for myeloid cell subsets, showing shifts in cell populations post-infection.

Furthermore, the study examined tissue-resident memory T cells in the epithelium and stroma following HSV-1 infection, with a higher abundance observed in the epithelium. Functional analysis via cytokine secretion assays revealed the activity of CD4 and CD8 tissue-resident memory T cells.

Dr. Suvas said that the flow cytometry approach enables the quantification and characterization of non-immune cell types, such as vascular endothelial cells and hematopoietic stem progenitor cells in corneal tissues. “Our approach of separating intact corneal epithelium and stroma has broad applications in different ocular inflammation and infection models,” she concluded. 

NETs: An emerging new target for herpetic keratitis 

Next, Chandrashekhar D. Patil, PhD (University of Illinois, USA), shared his research on keratitis development in patients, primarily using animal models due to their similarity to human corneas in pathology. The study focused on neutrophil responses to HSV infection, noting their role in forming extracellular traps to control the virus. Observations revealed rapid DNA release from infected neutrophils, leading to neutrophil extracellular trap (NET) formation. Further experiments suggested a protective role of NETs in controlling virus levels. Transcriptome analysis of human corneal samples confirmed increased NET-associated markers in HSV keratitis patients. The study also explored therapeutic avenues involving glycosaminoglycan-degrading enzymes and heparin. Overall, the research provided insights into neutrophil behavior and potential therapeutic targets for keratitis treatment.

Functional in vivo confocal microscopy

Meanwhile, Mengliang Wu, PhD (University of Melbourne, Australia), talked about a novel technique he and his team had developed, called the Functional In Vivo Confocal Microscopy (Fun-IVCM), which allows for dynamic intravital imaging to assess corneal immune cell behaviors in living humans. 

He noted that the cornea, being transparent, vascular and highly innervated, has garnered significant interest in corneal immunology, particularly in understanding immune cell roles in homeostasis and diseases. While previous knowledge of corneal immune cells mainly stemmed from animal studies, Fun-IVCM provides a way to visualize immune cell dynamics in human corneas.

Using Fun-IVCM, the researchers could observe immune cell density, morphology and interactions with nerves, offering insights into tissue inflammatory status and bridging preclinical and clinical findings. This technique has been applied in various studies to investigate various ocular and systemic conditions, such as dry eye disease, corneal infections, post-refractive surgeries, diabetes, multiple sclerosis and central nervous system diseases.

Fun-IVCM imaging has revealed morphologically distinct immune cells in the human cornea, with some resembling dendritic cells and others lacking dendrites, possibly indicating different activation statuses. By capturing the motility of these cells, the researchers classified them into mature and immature Langerhans cells. Additionally, dynamic imaging has shown the behavior of small, highly motile cells resembling T cells and stromal microphages, contributing to our understanding of corneal immune cell subsets and their activities.

To acquire these dynamic videos, the researchers repeat imaging of the same corneal region at different time points, typically 4 to 6 minutes apart, and process the images using automated techniques. Various dynamic parameters are evaluated, such as T cell speed, dendritic cell behavior (including dendrite extension and probing) and microphage dancing index, providing insights into immune cell activities and interactions with the neuroimmune system.

“The Fun-IVCM technique is a noninvasive, intravital, and clinical available technique. It enables us to evaluate the dynamic features of different corneal immune cell substances and assess the neuroimmune in action. And based on this, the cornea can be a window to immunity and neuroinflammation, and these immune cell dynamic parameters may serve as potential biomarkers for the diagnosis, evaluation and treatment of different diseases,” he concluded. 

Discussion: Factors that contribute to research success

After the presentations, the researchers shed light on several key aspects that contribute to their professional development beyond just financial support. 

A supportive research environment emerged as a critical factor, encompassing factors like the research culture within academia and the collaborative spirit fostered within departments and institutions. The importance of collaboration opportunities was emphasized, allowing researchers to engage with colleagues from different disciplines and broaden their perspectives.

Mentorship emerged as a cornerstone of career advancement, with mentors playing pivotal roles in providing guidance, fostering skill development, and creating opportunities for growth. Effective mentorship involved a delicate balance between providing autonomy for learning and offering support when needed, as highlighted by personal anecdotes shared by the investigators.

Furthermore, the discussion delved into the role of institutions in supporting early investigators. Beyond financial resources, institutions can facilitate interdisciplinary interactions, provide grant writing support, and ensure access to necessary research facilities and equipment. Additionally, creating dedicated time for research amidst clinical responsibilities is identified as a challenge that institutions could address to support early-stage researchers more effectively.

The process of finding a mentor was also explored, with speakers sharing their experiences and strategies for identifying suitable mentors. Systematic approaches involving thorough research into a mentor’s background, publication record, and mentoring style were highlighted as essential steps in this process.

Looking beyond individual mentorship relationships, the conversation touched upon the potential for peer mentorship and community-building within professional societies like ARVO. Creating platforms for early-career researchers to connect, share experiences and support each other could foster a sense of belonging and facilitate collaborative opportunities.

Editor’s Note: The Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO 2024) is being held from 5-9 May in Seattle, Washington, USA. Reporting for this story took place during the event. 

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