Lucia Kuffova

Dendritic cell physiology and function in the eye

Summary:  The eye and the brain are immunologically privileged sites, a property previously attributed to the lack of a lymphatic circulation. However, recent tracking studies confirm that these organs have good communication through classical site‐specific lymph nodes, as well as direct connection through the blood circulation with the spleen. In addition, like all tissues, they contain resident myeloid cell populations that play important roles in tissue homeostasis and the response to foreign antigens. Most of the macrophage and dendritic cell (DC) populations in the eye are restricted to the supporting connective tissues, including the cornea, while the neural tissue (the retina) contains almost no DCs, occasional macrophages (perivascularly distributed), and a specialized myeloid cell type, the microglial cell. Resident microglial cells are normally programmed for immunological tolerance. The privileged status of the eye, however, is relative, as it is susceptible to immune‐mediated inflammatory disease, both infectious and autoimmune. Intraocular inflammation (uveitis and uveoretinitis) and corneal graft rejection constitute two of the more common inflammatory conditions affecting the eye leading to considerable morbidity (blindness). As corneal graft rejection occurs almost exclusively by indirect allorecognition, host DCs play a major role in this process and are likely to be modified in their behavior by the ocular microenvironment. Ocular surface disease, including allergy and atopy, also comprise a significant group of immune‐mediated eye disorders in which DCs participate, while infectious disease such as herpes simplex keratitis is thought to be initiated via corneal DCs. Intriguingly, some more common conditions previously thought to be degenerative (e.g. age‐related macular degeneration) may have an autoimmune component in which ocular DCs and macrophages are critically involved. Recently, the possibility of harnessing the tolerizing potential of DCs has been applied to experimental models of autoimmune uveoretinitis with good effect. This approach has considerable potential for use in translational clinical therapy to prevent sight‐threatening disease caused by ocular inflammation.

Uveitis in Mouse and Man

Uveitis is underappreciated as a sight-threatening cause of blindness. There are two broad causative classes of uveitis: infectious and non-infectious. Non-infectious uveitis is considered a prototypical autoimmune disorder based mainly on data from experimental models in the mouse. Several different experimental models exist that reflect the different types of uveitis in man (anterior, intermediate, and posterior uveitis). These models have demonstrated that uveitis is predominantly a Th1/Th17 mediated disease, although innate immune cells play a significant role both in induction of disease and in tissue damage. Most experimental models of uveitis rely on activation of the innate immune system by use of adjuvants that activate a range of pathogen recognition receptors (PRRs). This begs the question of the underlying role of initial and/or persistent infection, including latent infection, in immune-mediated uveitis in which active infection cannot be demonstrated. This further raises the possibility of pathogenic mechanisms such as antigenic cross-reactivity and molecular mimicry. Alternatively, residual/latent antigen from infectious agents may act as “endogenous” adjuvants for induction of immune reactions to damaged/altered self antigen, suggesting a commonality in pathogenesis for both infectious and non-infectious uveitis in man.

Cross Presentation of Antigen on MHC Class II via the Draining Lymph Node after Corneal Transplantation in Mice

We investigated Ag trafficking from the cornea and T effector cell activation in secondary lymphoid tissue after corneal transplantation. In preliminary experiments, the central cornea was shown to contain a population of CD45+, CD11b+, CD11c- cells, with a few MHC class II+ cells, and F4/80+ cells. However, MHC class II+ passenger leukocytes in donor cornea after allografting did not traffic to the draining lymph node. Instead, Ag (plasmid) delivered to the eye via the donor cornea during allograft was detected in host CD11c+ and F4/80+ APC in the draining lymph nodes and spleen. The earliest detection of APC-associated Ag was at 6 h in the draining lymph node and 24 h in the spleen. After 48 h Ag was not detected in the draining lymph node but was still present in the spleen. Ag applied to the donor corneal epithelium before allografting induced Ag-specific T cell activation and expansion in the draining lymph node with a peak response at 4–6 days, indicating that cross-presentation of Ag had occurred. We conclude therefore, that Ag is transported from the donor cornea within host APC and that this event occurs within hours after grafting. Ag is cross-presented to host CD4+ T cells on MHC class II and leads to the activation of Ag-specific effector T cells and clonal expansion in the draining lymph node.

Evaluation of corneal graft rejection in a mouse model

Corneal graft rejection presents clinically and in experimental models as opacification and is considered to be the result of endothelial cell dysfunction or loss. However, recovery from opacification can occur suggesting either (a) that new endothelial cells can regenerate if the original cells were lost, or (b) that sufficient numbers of original cells can regain function if the opacification was due to temporary dysfunction. In this perspective, previous experimental studies of allograft rejection plus some new data are reviewed to support the latter mechanism.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind‐ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell–cell or bridging MNTs and blind‐ended MNTs was greatest between major histocompatibility complex (MHC) class II+ cells during corneal injury or TLR ligand‐mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid‐derived cells in inflamed corneal explants from Cx3cr1GFP and CD11ceYFP transgenic mice revealed that MNTs form de novo at a rate of 15.5 μm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell–cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus‐1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

Cytokine polymorphism in noninfectious uveitis.

PURPOSE Noninfectious uveitis is a sight-threatening immune-mediated intraocular inflammatory disorder. The inheritance of uveitis in multiplex families and its association with known monogenic and polygenic immunologic disorders suggests that common genetic variants underlie susceptibility to uveitis as well as to other immunologic disorders. TNFA and IL10 are strong candidate genes, given the influence of these cytokines on inflammation, immune tolerance, and apoptosis. METHODS The role of 12 polymorphisms spanning the TNFA and IL10 genomic regions was investigated in 192 uveitis patients and 92 population control subjects from four regional centers in the United Kingdom and Republic of Ireland. RESULTS The results demonstrate that uveitis is associated with three haplotype-tagging SNPs (htSNPs) in the IL10 gene: htSNP2 (rs6703630), htSNP5 (rs2222202), and htSNP6 (rs3024490). IL10htSNP2AG/htSNP5TC was the most significantly associated haplotype (P = 0.00085), whereas the LTA+252AA/TNFhtSNP2GG haplotype was protective (P = 0.00031). Furthermore, subgroup analysis showed that the frequency of the TNFd4 allele was higher in patients with nonremitting ocular disease and/or those requiring higher levels of maintenance immunosuppression. Although these associations lost significance after Bonferroni correction, they infer a relationship that may be validated by a larger study. CONCLUSIONS Since these variants are implicated in the susceptibility and severity of several immunologic disorders, the results support the hypothesis that common genetic determinants influence shared mechanisms of autoimmunity.

Regeneration of corneal cells and nerves in an implanted collagen corneal substitute.

Purpose: Our objective was to evaluate promotion of tissue regeneration by extracellular matrix (ECM) mimics, by using corneal implantation as a model system. Methods: Carbodiimide cross-linked porcine type I collagen was molded into appropriate corneal dimensions to serve as substitutes for natural corneal ECM. These were implanted into corneas of mini-pigs after removal of the host tissue, and tracked over 12 months, by clinical examination, slit-lamp biomicroscopy, in vivo confocal microscopy, topography, and esthesiometry. Histopathology and tensile strength testing were performed at the end of 12 months. Other samples were biotin labeled and implanted into mice to evaluate matrix remodeling. Results: The implants promoted regeneration of corneal cells, nerves, and the tear film while retaining optical clarity. Mechanical testing data were consistent with stable, seamless host-graft integration in regenerated corneas, which were as robust as the untreated fellow corneas. Biotin conjugation is an effective method for tracking the implant within the host tissue. Conclusions: We show that a simple ECM mimetic can promote regeneration of corneal cells and nerves. Gradual turnover of matrix material as part of the natural remodeling process allowed for stable integration with host tissue and restoration of mechanical properties of the organ. The simplicity in fabrication and shown functionality shows potential for ECM substitutes in future clinical applications.

Regenerative Approaches as Alternatives to Donor Allografting for Restoration of Corneal Function

A range of alternatives to human donor tissue for corneal transplantation are being developed to address the shortfall of good quality tissues as well as the clinical conditions for which allografting is contraindicated. Classical keratoprostheses, commonly referred to as artificial corneas, are being used clinically to replace minimal corneal function. However, they are used only as last resorts, as they are associated with significant complications, such as extrusion/rejection, glaucoma, and retinal detachment. The past few years have seen significant developments in technologies designed to replace part or the full thickness of damaged or diseased corneas with materials that encourage regeneration to different extents. This review describes selected examples of these corneal substitutes, which range from cell-based regenerative strategies to keratoprostheses with regenerative capabilities via tissue-engineered scaffolds pre-seeded with stem cells. It is unlikely that one corneal substitute will be best for all indications, but taken together, the various approaches may soon be able to supplement the supply of human donor corneas for transplantation or allow restoration of diseased or damaged corneas that cannot be treated by currently available techniques.

Punctate inner choroidopathy and multifocal choroiditis with panuveitis share haplotypic associations with IL10 and TNF Loci

PURPOSE The white-dot syndromes are a heterogenous group of chorioretinal disorders that have many common clinical features. Whether these disorders represent distinct clinical entities or different manifestations of the same disease warrants further interrogation. Two white-dot syndromes were investigated, with closely overlapping phenotypes--multifocal choroiditis with panuveitis (MFCPU) and punctate inner choroidopathy (PIC)--for differences in clinical course and genotype frequency at IL10 and TNF loci, known to be associated with noninfectious uveitis. METHODS Twelve polymorphisms were genotyped, spanning the TNFA and IL10 genomic regions, in 61 patients with MFCPU or PIC and 92 population controls from the United Kingdom and Republic of Ireland. RESULTS There were clear differences in clinical course between patients with MFCPU and PIC which had prognostic significance. However, both patient groups demonstrated similar associations with the IL10 haplotype, IL10htSNP2(-2849)AX/htSNP5(+434)TC and negative associations with the TNF haplotype, LTA+252A/TNFhtSNP1(-308)G/TNFhtSNP2(-238)G/TNFhtSNP3(+488)A/TNFd3. CONCLUSIONS Despite clear differences in clinical course and outcome, MFCPU and PIC may still represent two manifestations of the same disease, given their similar genetic associations with IL10 and TNF loci, which are known to be associated with noninfectious uveitis and autoimmunity, in general. Definitive proof will necessitate genomewide sequence analysis. However, the data also support the notion that epigenetic factors have a strong effect on clinical phenotype.

Quantitative evaluation of the corneal endothelium in the mouse after grafting

Background/aim: Corneal graft survival depends critically on the quality of the endothelium. In this study the authors aimed to evaluate corneal endothelium in mice at different times after transplantation and to correlate endothelial integrity with corneal graft survival. Methods: Syngeneic and allogeneic corneal grafts at various times (days 0–60) after engraftment were examined in flat mount preparation by confocal microscopy, by evaluating the hexagonal pattern of the endothelial monolayer using actin staining of the cell cortex. Corneas from untreated mice and from mice, who were grafted after removal of draining lymph nodes served as controls. Results: In control corneas, more than 90% of the posterior surface was covered by endothelium. Syngeneic grafts were always covered by 54–99% of endothelium. In contrast, the posterior surface of corneal allografts showed great variation in the degree of endothelial cell coverage (0–98%). In addition, clinical opacity grading measure was not a reliable predictor of endothelial coverage. Conclusion: In corneal allografts there is progressive loss of endothelium over time, unlike with syngeneic grafts. However, in the early stages of allograft rejection, the grade of graft opacity does not accurately reflect the degree of endothelial cell coverage. Although corneal opacity grade is considered the main determinant of graft rejection, the data suggest that both the grade of corneal opacity plus a sufficient post-graft time duration (>8 weeks in the mouse) are required for the diagnosis of irreversible graft rejection.