Simona L. Schlereth

Blocking CCR7 at the Ocular Surface Impairs the Pathogenic Contribution of Dendritic Cells in Allergic Conjunctivitis

CCR7 plays a key role in mobilizing tissue dendritic cells (DCs) to the lymphoid compartment for consequent elicitation of adaptive immunity. Interfering with CCR7 function therapeutically would therefore be anticipated to inhibit the progression of atopic conditions, for example, allergic conjunctivitis (AC). However, the CCR7-CCL19/CCL21 system in the ocular surface is poorly understood as is the precise role of DCs in AC immunopathogenesis. T cells from ovalbumin (OVA)-primed mice were adoptively transferred into wild-type (WT) hosts. Exogenous WT (eGFP(+)) versus CCR7(-/-) DCs were engrafted subconjunctivally (SCJ), and hosts were challenged with OVA (Texas-Red+) eye drops. AC immunopathogenesis was evaluated via clinical examinations, infiltration of mast cells and eosinophils, Th2 reactivity, and serum IgE levels. AC was also assessed in actively immunized mice challenged with OVA eye drops containing 1% anti-CCR7 antibody or isotype control. In eye-draining lymph nodes (LNs), OVA(+) SCJ engrafted WT DCs conferred upregulated CCR7 and caused augmentation of clinical signs. This result was corroborated by increased conjunctival infiltration, Th2 cytokines in LNs, and serum OVA-specific IgE. Strikingly, this was completely reversed with SCJ engrafted CCR7(-/-) DCs in all parameters tested. Furthermore, topical antibody blockade of CCR7 in actively immunized mice significantly inhibited AC. Ocular surface DCs via CCR7 expression contribute to the immunopathogenesis of AC, thereby allowing significant inhibition of this experimental condition via topical CCR7 antibody blockade.

Ocular Allergy Modulation to Hi-Dose Antigen Sensitization Is a Treg-Dependent Process

A reproducible method to inhibit allergic immune responses is accomplished with hi-dose Ag sensitization, via intraperitoneal (IP) injection. However, the role of CD4+ CD25+ FoxP3+ T regulatory cells (Treg) in this process is unknown, as is whether such modulation extends to ocular allergy. We therefore determined herein whether hi-dose sensitization modulates ocular allergy, and whether CD4+ CD25+ FoxP3+ Treg are involved. C57BL/6 mice were IP sensitized via low-dose (100 µg) versus hi-dose (1000 µg) ovalbumin (OVA), in aluminum hydroxide (1 mg) and pertussis-toxin (300 ng). Other mice received anti-CD25 Ab (PC61) to ablate Treg during sensitization. In another experiment, Treg from hi-dose sensitized mice were adoptively transferred into low-dose sensitized mice. Once daily OVA challenges were administered. Clinical signs, IgE, T cell cytokines, and eosinophils were assessed. Data revealed that hi-dose, but not low-dose, sensitization led to allergy modulation, indicated by decreased clinical signs, serum IgE levels, Th2 recall responses, and eosinophil recruitment. T cells from hi-dose sensitized mice showed a robust increase in TGF-b production, and Treg from these mice were able to efficiently suppress effector T cell proliferation in vitro. In addition, in vivo Treg ablation in hi-dose sensitized mice revoked allergy modulation. Lastly, Treg from hi-dose sensitized mice were able to adoptively transfer allergy modulation to their low-dose sensitized counterparts. Collectively, these findings indicate that modulation to hi-dose sensitization, which is extended to ocular allergy, occurs in a Treg-dependent manner. In addition, our data suggest that hi-dose sensitization may henceforth facilitate the further examination of CD4+ CD25+ FoxP3+ Treg in allergic disease.

Secondary allergic T cell responses are regulated by dendritic cell-derived thrombospondin-1 in the setting of allergic eye disease

Allergic eye disease, as in most forms of atopy, ranges in severity among individuals from immediate hypersensitivity to a severe and debilitating chronic disease. Dendritic cells play a key role in stimulating pathogenic T cells in allergen re‐exposure, or secondary responses. However, molecular cues by dendritic cells underpinning allergic T cell response levels and the impact that this control has on consequent severity of allergic disease are poorly understood. Here, we show that a deficiency in thrombospondin‐1, a matricellular protein known to affect immune function, has subsequent effects on downstream T cell responses during allergy, as revealed in an established mouse model of allergic eye disease. More specifically, we demonstrate that a thrombospondin‐1 deficiency specific to dendritic cells leads to heightened secondary T cell responses and consequent clinical disease. Interestingly, whereas thrombospondin‐1‐deficient dendritic cells augmented activity of allergen‐primed T cells, this increase was not recapitulated with naïve T cells in vitro. The role of dendritic cell‐derived thrombospondin‐1 in regulating secondary allergic T cell responses was confirmed in vivo, as local transfer of thrombospondin‐1‐sufficient dendritic cells to the ocular mucosa of thrombospondin‐1 null hosts prevented the development of augmented secondary T cell responses and heightened allergic eye disease clinical responses. Finally, we demonstrate that topical instillation of thrombospondin‐1‐derived peptide reduces T cell activity and clinical progression of allergic eye disease. Taken together, this study reveals an important modulatory role of dendritic cell‐derived thrombospondin‐1 on secondary allergic T cell responses and suggests the possible dysregulation of dendritic cell‐derived thrombospondin‐1 expression as a factor in allergic eye disease severity.

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and VII). Due to its autofluorescence, opaqueness, and thickness, it has not been found suitable for confocal microscopy. An alternative approach to the one presented here, which uses formalin-fixed sclera embedded in paraffin for immunohistochemistry, has technical challenges, especially when preheating the tissue for antigen retrieval. Since the sclera is relatively poor in both cells and vessels, the use of larger tissue samples was explored to help prevent overlooking cells and to understand their localization in relation to vessels and other anatomical sites. To allow for the analysis of larger tissue samples under the confocal microscope, a laminating technique was performed to create thin layers from the sclera. Following the analysis of results of CD31 blood vessels and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) positive cells, for which approval for scientific examination was obtained, the advantages and limitations of this method are discussed.