Nancy J. Reyes

Ocular mucosal CD11b+ and CD103+ mouse dendritic cells under normal conditions and in allergic immune responses.

Steady state dendritic cells (DC) found in non-lymphoid tissue sites under normal physiologic conditions play a pivotal role in triggering T cell responses upon immune provocation. CD11b+ and CD103+ DC have received considerable attention in this regard. However, still unknown is whether such CD11b+ and CD103+ DC even exist in the ocular mucosa, and if so, what functions they have in shaping immune responses. We herein identified in the ocular mucosa of normal wild-type (WT) and Flt3-/- mice the presence of a CD11b+ DC (i.e., CD11c+ MHCII+ CD11b+ CD103- F4/80+ Sirp-a+). CD103+ DC (i.e. CD11c+ MHCII+ CD11b low CD103+ CD8a+ DEC205+ Langerin+) were also present in WT, but not in Flt3-/- mice. These CD103+ DC expressed high levels of Id2 and Flt3 mRNA; whereas CD11b+ DC expressed high Irf4, Csfr, and Cx3cr1 mRNA. Additionally, the functions of these DC differed in response to allergic immune provocation. This was assessed utilizing a previously validated model, which includes transferring specific populations of exogenous DC into the ocular mucosa of ovalbumin (OVA)/alum-primed mice. Interestingly, in such mice, topical OVA instillation following engraftment of exogenous CD11b+ DC led to dominant allergic T cell responses and clinical signs of ocular allergy relative to those engrafted with CD103+ DC. Thus, although CD11b+ and CD103+ DC are both present in the normal ocular mucosa, the CD11b+ DC subset plays a dominant role in a mouse model of ocular allergy.

Two different regulatory T cell populations that promote corneal allograft survival

PURPOSE To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts. METHODS Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated. RESULTS Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID. CONCLUSIONS Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.

Involvement of corneal lymphangiogenesis in a mouse model of allergic eye disease.

PURPOSE The contribution of lymphangiogenesis (LA) to allergy has received considerable attention and therapeutic inhibition of this process via targeting VEGF has been considered. Likewise, certain inflammatory settings affecting the ocular mucosa can trigger pathogenic LA in the naturally avascular cornea. Chronic inflammation in allergic eye disease (AED) impacts the conjunctiva and cornea, leading to sight threatening conditions. However, whether corneal LA is involved is completely unknown. We addressed this using a validated mouse model of AED. METHODS Allergic eye disease was induced by ovalbumin (OVA) immunization and chronic OVA exposure. Confocal microscopy of LYVE-1-stained cornea allowed evaluation of corneal LA, and qRT-PCR was used to evaluate expression of VEGF-C, -D, and -R3 in these mice. Administration of VEGF receptor (R) inhibitor was incorporated to inhibit corneal LA in AED. Immune responses were evaluated by in vitro OVA recall responses of T cells, and IgE levels in the serum. RESULTS Confocal microscopy of LYVE-1-stained cornea revealed the distinct presence of corneal LA in AED, and corroborated by increased corneal expression of VEGF-C, -D, and -R3. Importantly, prevention of corneal LA in AED via VEGFR inhibition was associated with decreased T helper two responses and IgE production. Furthermore, VEGFR inhibition led a significant reduction in clinical signs of AED. CONCLUSIONS Collectively, these data reveal that there is a distinct involvement of corneal LA in AED. Furthermore, VEGFR inhibition prevents corneal LA and consequent immune responses in AED.

New Twists to an Old Story: Novel Concepts in the Pathogenesis of Allergic Eye Disease

Abstract The prevalence of allergy is rising globally at a very significant rate, which is currently at 20–40% of individuals in westernized nations. In the eye, allergic conditions can take on the acute form such as in seasonal and perennial allergic conjunctivitis, or a more severe and debilitating chronic form such as in vernal and atopic keratoconjunctivitis. Indeed, some key aspects of allergic eye disease pathophysiology are understood, such as the role of mast cells in the acute allergic reaction, and the contribution of eosinophils in late-onset and chronic allergy. However, recent developments in animal models and clinical studies have uncovered new and important roles for previously underappreciated players, including chemokine receptors on ocular surface dendritic cells such as CCR7, the contribution of conjunctival epithelium to immunity, histamine and leukotriene receptors on conjunctival goblet cells and a role for mast cells in late-onset manifestations. Furthermore, recent work in animal models has delineated the contribution of IL-4 in the increased incidence of corneal graft rejection in hosts with allergic conjunctivitis. Recent studies such as these mean that conventional paradigms and concepts should be revisited. The aim of this review is to highlight some of the most recent advances and insights on newly appreciated players in the pathogenesis of allergic eye disease.

NKT cells are necessary for maximal expression of allergic conjunctivitis1

Allergic conjunctivitis (AC) is elicited by immediate hypersensitivity responses to environmental agents. It is initiated by a T(h)2-dominated immune response that is characterized by production of IgE antibodies and eosinophilic infiltration. By using an experimental mouse model of AC induced by short ragweed (SRW) pollen, we show that sensitized Jalpha18(-/-) mice, which lack type I NKT cells, and CD1d(-/-) mice, which lack type I and type II NKT cells, exhibited a decrease in tearing, lid edema, conjunctival edema and vasodilatation and eosinophil infiltration into the conjunctiva when compared with wild-type (WT) mice in both T(h)1- and T(h)2-prone hosts (C57BL/6 and BALB/c mice, respectively). This demonstrates that NKT cells are needed for both the early and late phases of AC. Adoptive transfer of SRW-primed CD4(+) T cells from Jalpha18(-/-) mice into naive WT BALB/c mice revealed that NKT cells were needed for the maximal induction of allergen-specific T(h)2 cells. Results from adoptive transfer of SRW-primed CD4(+) T cells from WT BALB/c mice to naive Jalpha18(-/-) mice indicated that NKT cells were also needed for the expression of AC produced by allergen-primed CD4(+) T cells. The decreased expression of AC in NKT cell-deficient mice was correlated with significant reduction in the production of T(h)2 cytokines in SRW pollen-sensitized mice compared with WT mice and in the capacity of SRW pollen-sensitized CD4(+) T cells to mediate ocular inflammation when the hosts were confronted with SRW pollen at the ocular surface.

New insights into mononuclear phagocyte biology from the visual system

Major advances in mononuclear phagocyte biology have been made but key questions pertinent to their roles in health and disease remain, including in the visual system. One problem concerns how dendritic cells can trigger immune responses from certain tightly regulated immune- privileged sites of the eye. Another, albeit separate, problem involves whether there are functional specializations for microglia versus monocytes in retinal neurodegeneration. In this Review, we examine novel insights in eye immune privilege and, separately, we discuss recent inroads concerning retinal degeneration. Both themes have been extensively studied in the visual system and show parallels with recent findings concerning mononuclear phagocytes in the central nervous system and in the periphery.

T helper subsets in allergic eye disease.

Purpose of reviewOcular allergy is an IgE-mediated disease that results in inflammation of the conjunctiva and, in more severe cases, the cornea. This is driven by an immediate hypersensitivity response via mast cells, followed by a late phase response mediated by eosinophils both of which are indeed dependent on T helper (Th) lymphocyte activity. Here, we provide an update on Th subsets [Th1, Th2, Th17, and T regulatory (Treg)] and their relevance in ocular allergy. Recent findingsRecent evidence in ocular allergy points to an involvement of other Th subsets, in addition to Th2. However, how these subsets are activated and their role in mediating the different clinical forms is poorly understood. Novel mouse models may facilitate addressing such unknowns, and future challenges will involve how to translate such findings into more effective and ‘patho-specific’ treatments. SummaryOcular allergy, especially in severe forms, involves subsets other than Th2. Th1 cells have been detected in mild and severe forms, and recent evidence points to a possible role for IL-17 in severe disease. Tregs, on the other hand, dampen pathogenic Th cell function and allergy immunotherapy is associated with Treg augmentation in disease management. Further understanding of Th biology is warranted and may lead to better therapies.

γδ T Cells Are Required for Maximal Expression of Allergic Conjunctivitis

PURPOSE To determine the function of γδ T cells in early- and late-phase responses in allergic conjunctivitis. METHODS Wild-type (WT) C57BL/6 and γδ T cell-deficient (TCR-δ(-/-)) mice were immunized intraperitoneally and challenged topically for 7 consecutive days with short ragweed pollen. Natural killer T (NKT) and γδ T cell-double-deficient mice were generated by treating TCR-δ(-/-) mice with anti-CD1d antibody. Allergic conjunctivitis was evaluated clinically, and the late-phase response was assessed by histopathology. Cytokine profiles were evaluated by ELISA. The afferent and efferent arms of allergic conjunctivitis were assessed by adoptive transfer of CD4(+) T cells from WT or TCR-δ(-/-) mice into naive TCR-δ(-/-) or WT mice. RESULTS TCR-δ(-/-) mice had decreased clinical manifestations of allergic conjunctivitis compared with WT mice. TCR-δ(-/-) mice had decreased eosinophilic infiltration compared with WT mice. TCR-δ(-/-) mice produced less Th2-associated cytokines interleukin (IL)-4, -5, and -13 compared with WT mice. Clinical manifestations of allergic conjunctivitis were lowest in NKT cell-depleted TCR-δ(-/-) mice. However, late-phase allergic conjunctivitis in NKT cell-depleted, TCR-δ(-/-) mice was the same as TCR-δ(-/-) mice. Adoptive transfer of CD4(+) T cells revealed that γδ T cells are needed for the afferent and efferent arms of allergic conjunctivitis. CONCLUSIONS γδ T cells are needed for full expression of both the clinical manifestations and the late phase of allergic conjunctivitis. Thus, γδ T cells have an important impact in the expression of allergic conjunctivitis and are a potential therapeutic target in the management of allergic diseases of the ocular surface.

Classical dendritic cells mediate fibrosis directly via the retinoic acid pathway in severe eye allergy

Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b+ DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b+ DC-derived ALDH was associated with 9-cis-retinoic acid ligation to retinoid x receptor (RXR), which induced conjunctival fibroblast activation. In vivo, stimulating RXR led to rapid onset of ocular mucosal fibrosis, whereas inhibiting ALDH activity in DCs or selectively depleting DCs markedly reduced fibrosis. Collectively, these data reveal a profibrotic ALDH-dependent pathway by DCs and uncover a role for DC retinoid metabolism.

Allergic conjunctivitis renders CD4(+) T cells resistant to t regulatory cells and exacerbates corneal allograft rejection.

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4+ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4+ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα−/− CD4+ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα−/− mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.