William Stevenson

Expression of toll-like receptor 4 contributes to corneal inflammation in experimental dry eye disease.

Purpose. To investigate the corneal expression of toll-like receptor (TLR) 4 and determine its contribution to the immunopathogenesis of dry eye disease (DED). Methods. Seven to 8-week-old female C57BL/6 mice were housed in a controlled environment chamber and administered scopolamine to induce experimental DED. Mice received intravenous TLR4 inhibitor (Eritoran) to block systemic TLR4-mediated activity. The expression of TLR4 by the corneal epithelium and stroma was evaluated using real-time polymerase chain reaction and flow cytometry. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease severity. The corneal expression of proinflammatory cytokines (IL-1β, IL-6, TNF, and CCL2), corneal infiltration of CD11b(+) antigen-presenting cells, and lymph node frequency of mature MHC-II(hi) CD11b(+) cells were assessed. Results. The epithelial cells of normal corneas expressed TLR4 intracellularly; however, DED significantly increased the cell surface expression of TLR4. Similarly, flow cytometric analysis of stromal cells revealed a significant increase in the expression of TLR4 proteins by DED-induced corneas as compared with normal corneas. DED increased the mRNA expression of TLR4 in corneal stromal cells, but not epithelial cells. TLR4 inhibition decreased the severity of CFS and significantly reduced the mRNA expression of IL-1β, IL-6, and TNF. Furthermore, TLR4 inhibition significantly reduced the corneal infiltration of CD11b(+) cells and the lymph node frequency of MHC-II(hi) CD11b(+) cells. Conclusions. These results suggest that DED increases the corneal expression of TLR4 and that TLR4 participates in the inflammatory response to ocular surface desiccating stress.

Short-term topical bevacizumab in the treatment of stable corneal neovascularization.

PURPOSE To evaluate the safety and efficacy of topical bevacizumab in the treatment of corneal neovascularization. DESIGN Prospective, nonrandomized, interventional case series. METHODS setting: Institutional, multicenter clinical trial. study population: Twenty eyes from 20 patients with stable corneal neovascularization. intervention procedures: Patients were treated with topical 1.0% bevacizumab for 3 weeks and were monitored for a total of 24 weeks. main outcome measures: Primary outcome measures included: neovascular area, defined as the area of the corneal vessels themselves; vessel caliber, defined as the mean corneal vessel diameter; and invasion area, defined as the fraction of the total cornea into which the vessels extended. The occurrence of ocular and systemic adverse events was monitored closely. RESULTS As compared with the baseline visit, patients exhibited a statistically significant improvement in neovascular area by week 6 (P = .007) and in vessel caliber by week 12 (P = .006). At the final visit, neovascular area, vessel caliber, and invasion area were reduced by 47.5%, 36.2%, and 20%, respectively. The decreases in neovascular area and vessel caliber were statistically significant (P < .001 and P = .003, respectively); however, the reduction in invasion area did not reach statistical significance (P = .06). There were no significant changes in the secondary outcomes, and there were no adverse events. CONCLUSIONS Short-term topical bevacizumab treatment reduced the extent of stable corneal neovascularization as measured by neovascular area and vessel caliber with no associated adverse events. Interestingly, the degree of treatment efficacy was inversely proportional to the baseline invasion area.

Effect of desiccating environmental stress versus systemic muscarinic AChR blockade on dry eye immunopathogenesis.

PURPOSE A majority of experimental data on dry eye disease (DED) immunopathogenesis have been derived from a murine model of DED that combines desiccating environmental stress with systemic muscarinic acetylcholine receptor (mAChR) inhibition. However, to our knowledge the effects of pharmacologic mAChR blockade on the pathogenesis of experimental DED have not been evaluated systemically. The purpose of our study was to investigate the differential effects of desiccating environmental stress and mAChR inhibition on the pathogenesis of DED. METHODS DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled-environment chamber or to systemic scopolamine, or by performing extraorbital lacrimal gland excision. Clinical disease was assessed using corneal fluorescein staining (CFS) and the cotton thread test (CTT). Corneal CD11b(+) and conjunctival CD3(+) T-cell infiltration were evaluated by flow cytometry. T-cells from draining cervical lymph nodes (CLN) and distant inguinal lymph nodes (ILN) were analyzed for Th1, Th2, Th17, and Treg responses by flow cytometry and ELISA. RESULTS Desiccating environmental stress and systemic mAChR blockade induced similar clinical signs of DED. However, desiccating environmental stress imparted higher conjunctival CD3(+) T-cell infiltration, and greater Th17-cell activity and Treg dysfunction than mAChR blockade, while mAChR blockade decreased tear secretion to a greater extent than desiccating environmental stress. Systemic mAChR blockade attenuated Th17 activity and enhanced Th2 and Treg responses without affecting Th1 activity. CONCLUSIONS In vivo inhibition of mAChRs variably affects CD4(+) T-cell subsets, and desiccating environmental stress and systemic mAChR blockade induce DED through different primary pathogenic mechanisms.

PDE4 Inhibition Suppresses IL-17–Associated Immunity in Dry Eye Disease

PURPOSE To determine the effect of phosphodiesterase type-4 (PDE4) inhibition on IL-17-associated immunity in experimental dry eye disease (DED). METHODS Murine DED was induced, after which a PDE4 inhibitor (cilomilast), dexamethasone, cyclosporine, or a relevant vehicle was administered topically. Real-time PCR, immunohistochemical staining, and flow cytometry were employed to evaluate the immuno-inflammatory parameters of DED with a focus on IL-17-associated immunity. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease progression. RESULTS DED induction increased proinflammatory cytokine expression, pathogenic immune cell infiltration, and CFS scores. Cilomilast significantly decreased the expression of TNF-α in the cornea (P ≤ 0.05) and IL-1α, IL-1β, and TNF-α in the conjunctiva (P ≤ 0.05) as compared with vehicle control. Cilomilast treatment markedly decreased the presence of CD11b+ antigen-presenting cells in the central and peripheral cornea (P ≤ 0.05), and led to decreased conjunctival expression of cytokines IL-6, IL-23, and IL-17 (P ≤ 0.05). Moreover, cilomilast decreased the expression of IL-17 and IL-23 in the draining lymph nodes (P ≤ 0.05). Topical cilomilast was significantly more effective than vehicle at reducing CFS scores (P ≤ 0.05). The therapeutic efficacy of cilomilast was comparable or superior to that of dexamethasone and cyclosporine in all tested measures. CONCLUSIONS Topical cilomilast suppresses the generation of IL-17-associated immunity in experimental DED.

Graft Site Microenvironment Determines Dendritic Cell Trafficking Through the CCR7-CCL19/21 Axis

Purpose The graft site microenvironment has a profound effect on alloimmunity and graft survival. We aimed to study the kinetics and phenotype of trafficking antigen-presenting cells (APC) to the draining lymph nodes (DLNs) in a mouse model of corneal transplantation, and to evaluate the homing mechanisms through which graft site inflammation controls APC trafficking. Methods Allogeneic donor corneas were transplanted onto inflamed or quiescent graft beds. Host- (YAe+) and donor (CD45.1+ or eGFP+)-derived APCs were analyzed by flow cytometry. Protein and mRNA expression of the CC chemokine receptor (CCR)7 ligands CCL19 and CCL21 were assessed using ELISA and Real-Time qPCR, respectively. Transwell migration assay was performed to assess the effect of DLNs isolated from hosts with inflamed graft beds on mature bone marrow–derived dendritic cells (BMDCs). Results We found that inflamed graft sites greatly promote the trafficking of both recipient- and graft-derived APCs, in particular mature CCR7+ CD11c+ dendritic cells (DC). CCL19 and CCL21 were expressed at significantly higher levels in the DLNs of recipients with inflamed graft beds. The supernatant of DLNs from recipients with inflamed graft beds induced a marked increase in mature DC migration compared with supernatant from recipients with quiescent graft beds in a transwell assay. This effect was abolished by neutralizing CCL19 or CCL21. These data suggest that graft site inflammation increases the expression of CCR7 ligands in the DLNs, which promote mature DC homing and allorejection. Conclusions We conclude that the graft site microenvironment plays a critical role in alloimmunity by determining DC trafficking through the CCR7-CCL19/21 axis.

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.