Immunoregulatory strategies in corneal transplantation and dry eye disease

Despite substantial advances in our understanding of the cellular and molecular factors that regulate immune homeostasis at the ocular surface and anterior segment, important questions remain. In this thesis I present investigations of two phases of the adaptive immune response – the antigen recognition phase, and the final memory phase. In my experiments I employ murine models of corneal transplantation and dry eye disease. Specifically, I investigate how the activation of antigen-presenting cells is modulated by purinergic signaling and by mesenchymal stem cells in a model of corneal transplantation. Furthermore, I investigate how IL-15 signaling regulates the memory T helper 17 cell pool in age-related dry eye disease. In addition to providing useful models to investigate immunoregulation in the eye, studies of corneal transplantation and dry eye disease are of high translational potential. Corneal transplantation is the most common form of tissue grafting performed worldwide. Despite high success rates in low-risk recipients, failure rates exceed 50% in patients with a history of graft rejection, or with vascularized and inflamed host beds. Dry eye disease is an extremely common chronic condition of the ocular surface, with prevalence estimated to be more than 10% for ages greater than 50 years. Dry eye disease significantly impairs quality of life and is associated with a substantial socioeconomic burden. Accordingly, there is an unmet clinical need for novel therapeutic strategies to improve corneal allograft survival and treat dry eye disease. The data presented in this thesis indicate that both the purinergic receptor antagonist oxidized adenosine triphosphate and mesenchymal stem cell-derived hepatocyte growth factor promote corneal allograft survival by regulating antigen-presenting cell maturation. In addition, the data presented indicate that targeting interleukin-15 signaling is an effective strategy to deplete the memory T helper 17 pool in aged mice and reduce the severity of age-related dry eye disease

Inflammaging at ocular surface: clinical and biomolecular analyses in healthy volunteers

PURPOSE. To assess the ocular surface in volunteers who consider themselves as healthy, in order to evaluate how para-inflammatory mechanisms fail with age, and thus investigate the phenomenon of "InflammAging.''METHODS. In this observational prospective cohort study, volunteers were categorized into three groups according to age: young (19-40 years), middle-aged (41-60 years), and older adults (61-93 years). Clinical assessments included tear breakup time (T-BUT) and Schirmer test type I. Dry eye symptoms were evaluated by the Ocular Surface Disease Index (OSDI) questionnaire. Conjunctival mRNA and protein expression of intercellular adhesion molecule-1 (ICAM-1), MUC5AC, and IL-8 were measured by real-time PCR and immunofluorescence.RESULTS. A total of 82 volunteers (38 males and 44 females) were enrolled. T-BUT decreased significantly with increasing age (young: 11.13 +/- 0.18 seconds; middle-aged: 10.83 +/- 0.56 seconds; older: 9.00 +/- 1.00 seconds, P < 0.05). Schirmer test values decreased significantly with age (young: 20.6 +/- 1.0 mm; middle-aged: 19.2 +/- 1.2 mm; older: 16.0 +/- 1.1 mm, P < 0.05). OSDI scores increased with age in both groups, but they were substantially higher in women. Conjunctival expression of inflammatory markers ICAM-1, IL-8, and MUC5AC increased with age.CONCLUSIONS. Clinical signs, symptoms, and biomarkers of chronic inflammation increased with age in a cohort of volunteers who considered themselves healthy, indicating an age-related progressive impairment of ocular surface system function

Age-Related Changes to Human Tear Composition.

Purpose We characterize age-associated alterations in the expression of inflammatory mediators and tissue remodeling factors in human tears. Methods A total of 75 consecutive volunteers (32 male/44 female; 19-93 years) underwent clinical assessment of ocular surface status, ocular surface disease index (OSDI) grading and tear sampling. The volunteers were categorized into three groups: young (18-40 years), middle-aged (41-60 years), and old (>60 years). Total protein profiles and chip-based protein array evaluations were conducted to investigate the expression of 60 potential candidates, including pro-/anti-inflammatory mediators and tissue remodeling factors. Appropriate validations were performed using conventional assays. Multiple comparisons for regression between potential candidates and age were performed, as well as statistical analyses among the three age groups. Nonpooled samples were used for quantifications. Results Pearson analysis of chip-arrays identified 9 of 60 potential candidates. Specifically, IL-8, IL-6, and regulated on activation, normal T cell expressed and secreted (RANTES; P < 0.0083) protein as well as matrix metalloproteinase (MMP)-1, IL-3, and TNF-α (P < 0.05) correlated positively with aging. MIP-3β showed an opposite tendency. Western blot and ELISA analysis corroborated the array data. OSDI grading did not correlate with aging. Conclusions Dynamic changes to tear protein profiles occur with aging. Our study identifies the expression of IL-8, IL-6, RANTES, MMP-1, and MIP-3β as increasing with age. These select inflammatory and matrix remodeling factors may be relevant to the development of novel diagnostic tools and therapeutics in the context of age-related ocular surface disease

Keratoconus progression associated with hormone replacement therapy

Purpose: To report a postmenopausal patient with keratoconus who experienced significant progression after using hormone replacement therapy. Observations: A 51-year-old woman with previously stable keratoconus presented with acute disease progression following hormone replacement therapy in the context of prophylactic hysterectomy and bilateral ovariosalpingectomy. Over a 14-month period after starting hormone therapy, the steepest K increased from 63.7D to 71.5D in the right eye and from 65.8D to 78.1D in the left eye. Conclusions: Hormone replacement therapy may amplify progression of keratoconus

Visual implications of digital device usage in school children: a cross-sectional study

Abstract Purpose To evaluate the use of digital devices, reading habits and the prevalence of eyestrain among urban Indian school children, aged 11–17 years. Methods The study included 576 adolescents attending urban schools who were surveyed regarding their electronic device usage. Additional information on the factors that may have an effect on ocular symptoms was collected. Results Twenty percent of students aged 11 in the study population use digital devices on a daily basis, in comparison with 50% of students aged 17. In addition to using these devices as homework aids, one third of study participants reported using digital devices for reading instead of conventional textbooks. The majority of students preferred sitting on a chair while reading (77%; 445 students), with only 21% (123 students) preferring to lie on the bed and 8 students alternating between chair and bed. There was a significant association between the students who preferred to lie down and those who experienced eyestrain, as reported by a little over one fourth of the student population (27%). Out of 576 students, 18% (103) experienced eyestrain at the end of the day after working on digital devices. Conclusions The increased use of digital devices by adolescents brings a new challenge of digital eyestrain at an early age. Our study reports the patterns of electronic device usage by school children, evaluates factors associated with eyestrain and highlights the need for further investigation of these issues

When Clarity Is Crucial: Regulating Ocular Surface Immunity

The ocular surface is a unique mucosal immune compartment in which anatomical, physiological, and immunological features act in concert to foster a particularly tolerant microenvironment. These mechanisms are vital to the functional competence of the eye, a fact underscored by the devastating toll of excessive inflammation at the cornea - blindness. Recent data have elucidated the contributions of specific anatomical components, immune cells, and soluble immunoregulatory factors in promoting homeostasis at the ocular surface. We highlight research trends at this distinctive mucosal barrier and identify crucial gaps in our current knowledge

Review: The function of regulatory T cells at the ocular surface

Regulatory T cells (Tregs) are critical modulators of immune homeostasis. Tregs maintain peripheral tolerance to self-antigens, thereby preventing autoimmune disease. Furthermore, Tregs suppress excessive immune responses deleterious to the host. Recent research has deepened our understanding of how Tregs function at the ocular surface. This manuscript describes the classification, the immunosuppressive mechanisms, and the phenotypic plasticity of Tregs. We review the contribution of Tregs to ocular surface autoimmune disease, as well as the function of Tregs in allergy and infection at the ocular surface. Finally, we review the role of Tregs in promoting allotolerance in corneal transplantation. (C) 2017 Elsevier Inc. All rights reserved

Pathological conversion of regulatory T cells is associated with loss of allotolerance

CD4+CD25+Foxp3+ Regulatory T cells (Tregs) play a critical role in immune tolerance. The plasticity and functional adaptability of Tregs in an inflammatory microenvironment has been demonstrated in autoimmunity. Here, using a double transgenic mouse model that permits Foxp3 lineage tracing, we investigated the phenotypic plasticity of Foxp3+ Tregs in a well-characterized murine model of corneal transplantation. In order to subvert the normal immune privilege of the cornea and foster an inflammatory milieu, host mice were exposed to desiccating stress prior to transplantation. Treg frequencies and function were decreased following desiccating stress, and this corresponded to decreased graft survival. A fraction of Tregs converted to IL-17+ or IFNγ+ ‘exFoxp3’ T cells that were phenotypically indistinguishable from effector Th17 or Th1 cells, respectively. We investigated how Foxp3 expression is modulated in different Treg subsets, demonstrating that neuropilin-1− peripherally-derived Tregs are particularly susceptible to conversion to IL-17+/IFNγ+ exFoxp3 cells in response to cues from their microenvironment. Finally, we show that IL-6 and IL-23 are implicated in the conversion of Tregs to exFoxp3 cells. This report demonstrates that the pathological conversion of Tregs contributes to the loss of corneal immune privilege

Mesenchymal Stromal Cells Modulate Corneal Alloimmunity via Secretion of Hepatocyte Growth Factor

Abstract Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and possess considerable immunomodulatory potential. MSCs have been shown to promote allograft survival, yet the mechanisms behind this phenomenon have not been fully defined. Here, we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation, we report that MSCs promote graft survival in an HGF‐dependent manner. Moreover, our data indicate that topically administered recombinant HGF (a) suppresses antigen‐presenting cell maturation in draining lymphoid tissue, (b) limits T‐helper type‐1 cell generation, (c) decreases inflammatory cell infiltration into grafted tissue, and (d) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF‐based therapeutics. Stem Cells Translational Medicine 2019;8:1030–104

Mast Cells Initiate the Recruitment of Neutrophils Following Ocular Surface Injury

Purpose The purpose of this study was to investigate the contribution of mast cells to early neutrophil recruitment during ocular inflammation. Methods: In a murine model of corneal injury, the epithelium and anterior stroma were removed using a handheld motor brush. Cromolyn sodium (2% in PBS) eye drops were administered topically for mast cell inhibition. In vitro, bone marrow–derived mast cells were cultured alone or with corneal tissue. The frequencies of CD45+ inflammatory cells, CD11b+Ly6G+ neutrophils, and ckit+FcεR1+ mast cells in the cornea were assessed by flow cytometry. mRNA expression of CXCL2 was evaluated by real-time PCR and protein expression by ELISA. β-Hexosaminidase assays were performed to gauge mast cell activation. Results: Neutrophil infiltration of the cornea was observed within 1 hour of injury, with neutrophil frequencies increasing over subsequent hours. Concurrent expansion of mast cell frequencies at the cornea were observed, with mast cell activation (assessed by β-hexosaminidase levels) peaking at 6 hours after injury. Evaluation of CXCL2 mRNA and protein expression levels demonstrated augmented expression by injured corneal tissue relative to naïve corneal tissue. Mast cells were observed to constitutively express CXCL2, with significantly higher expression of CXCL2 protein compared with naïve corneal tissue. Culture with harvested injured corneas further amplified CXCL2 expression by mast cells. In vivo, mast cell inhibition was observed to decrease CXCL2 expression, limit early neutrophil infiltration, and reduce inflammatory cytokine expression by the cornea. Conclusions: Our data suggest that mast cell activation after corneal injury amplifies their secretion of CXCL2 and promotes the initiation of early neutrophil recruitment