Conjunctival Inflammation in Thrombospondin-1 Deficient Mouse Model of Sjögren’s Syndrome

Lacrimal gland inflammation during autoimmune Sjögren’s syndrome (SS) leads to ocular surface inflammation – Keratoconjunctivitis sicca (KCS). This condition afflicts both the cornea and conjunctiva that form the ocular surface. Thrombospondin-1 (TSP-1) deficiency in mice results in lacrimal gland and corneal inflammation that resembles the human disease. In this study we report conjunctival pathology in this mouse model of SS. We found that TSP-1 null mice develop inflammation in the conjunctiva and associated loss of goblet cell function similar to that seen in patients with SS. Increased expression of Th1 (IFN-γ, TNF-α) and Th17 (IL-6, IL-17A) inflammatory cytokines and related transcription factors (Tbet and RORγt) were detected in TSP-1 null conjunctiva as well as their draining lymph nodes (LNs). The conjunctival inflammation was also accompanied by an increase in local lymphatic vessels. Interestingly, migration of antigen-bearing dendritic cells (DCs) from the ocular surface to the LNs was dependent on the TSP-1 available in the tissue. These results not only reveal potential immunopathogenic mechanisms underlying KCS in SS but also highlight the therapeutic potential of TSP-1

Topical application of TGF-β-activating peptide, KRFK, prevents inflammatory manifestations in the TSP-1-deficient mouse model of chronic ocular inflammation

Producción CientíficaChronic inflammation of the ocular surface poses a risk of vision impairment. The understanding of the molecular mechanisms that are involved in the inflammatory response is critical to identify novel molecular targets. Recently, thrombospondin-1 (TSP-1) has emerged as a key player in ocular surface homeostasis that efficiently activates the TGF-β2 isoform that is predominantly expressed in the ocular mucosa. Here, the potential of the peptide derived from TSP-1 (KRFK), that can activate TGF-β, is proposed as a potentially applicable therapeutic for chronic ocular surface inflammatory disorders. Our in vitro results confirm that the chosen peptide activates TGF-β, reducing the expression of co-stimulatory molecules on dendritic cells, driving them towards a tolerogenic phenotype. For the in vivo studies, the TSP-1−/− mouse is used as a pre-clinical model of chronic ocular inflammation. We observe that the topical application of KRFK alters the peripheral balance of effectors by reducing the proportion of pathogenic Th1 and Th17 cells while increasing Treg cell proportion in cervical lymph nodes. In line with these findings, the development of chronic ocular surface inflammation is significantly prevented in KRFK-treated TSP-1−/− mice, as assessed by clinical parameters and inflammatory cytokine expression in conjunctival and lacrimal gland tissues. Together, our results identify the KRFK peptide as a novel therapeutic option to prevent the development of chronic inflammatory manifestations of the ocular surface.Ministerio de Economía, Industria y Competitividad (FEDER-CICYT grant MAT2013-47501-C02-1-R)Massachusetts Lions Eye Research Fund (MLERF, S.M.) and NEI grant EY015472 (S.M.)Junta de Castilla y León - Fondo Social Europeo (project VA098-12

The Role of ACAID and CD4+CD25+FOXP3+ Regulatory T Cells on CTL Function Against MHC Alloantigens

Purpose: Anterior chamber associated immune deviation (ACAID) is an antigen-specific form of peripheral immune tolerance that is induced to exogenous antigens placed in the ocular anterior chamber, which leads to a suppression in delayed-type hypersensitivity (DTH). Considerable work has been done on ACAID induction to major histocompatibility (MHC) alloantigens. However, its role on cytotoxic T lymphocyte (CTL) activity is currently unknown. Methods: C57BL/6 (H-2b) mice received an intracameral (IC) inoculation with BALB/c (H-2d) splenocytes. Splenic CD4+ and CD8+ T cell populations were characterized by flow cytometry and proliferation assays during induction and expression phases of ACAID. Percentages of CD4+CD25+FoxP3+ T regulatory cells (Treg) were also followed. Lastly, CTL function was measured at various time points during ACAID expression, and Treg were added to identify potential alterations in CTL function. Results: CD4+ and CD8+ T cell percentages and proliferation increased in the spleen during ACAID induction but then sharply decreased in response to an allospecific immunization. Expression of ACAID also exhibited a significant drop in CTL function. However, while Treg expansion was observed, these cells did not directly mediate the CTL inhibition. Conclusions: ACAID mediates an inhibition of CTL function against MHC alloantigens. Furthermore, we found that ACAID induction leads to the expansion and proliferation of CD4+ and CD8+ T cells while ACAID expression is associated with a diminishment in T cell percentages due to proliferation impairment. Lastly, Treg also expand during ACAID induction. However, our data suggest that Treg do not directly inhibit CTL activity

GpnmbR150X allele must be present in bone marrow derived cells to mediate DBA/2J glaucoma

<p>Abstract</p> <p>Background</p> <p>The <it>Gpnmb </it>gene encodes a transmembrane protein whose function(s) remain largely unknown. Here, we assess if a mutant allele of <it>Gpnmb </it>confers susceptibility to glaucoma by altering immune functions. DBA/2J mice have a mutant <it>Gpnmb </it>gene and they develop a form of glaucoma preceded by a pigment dispersing iris disease and abnormalities of the immunosuppressive ocular microenvironment.</p> <p>Results</p> <p>We find that the <it>Gpnmb </it>genotype of bone-marrow derived cell lineages significantly influences the iris disease and the elevation of intraocular pressure. GPNMB localizes to multiple cell types, including pigment producing cells, bone marrow derived F4/80 positive antigen-presenting cells (APCs) of the iris and dendritic cells. We show that APCs of DBA/2J mice fail to induce antigen induced immune deviation (a form of tolerance) when treated with TGFβ2. This demonstrates that some of the immune abnormalities previously identified in DBA/2J mice result from intrinsic defects in APCs. However, the tested APC defects are not dependent on a mutant <it>Gpnmb </it>gene. Finally, we show that the <it>Gpnmb </it>mediated iris disease does not require elevated IL18 or mature B or T lymphocytes.</p> <p>Conclusion</p> <p>These results establish a role for <it>Gpnmb </it>in bone marrow derived lineages. They suggest that affects of <it>Gpnmb </it>on innate immunity influence susceptibility to glaucoma in DBA/2J mice.</p

Thrombospondin 1 inhibits inflammatory lymphangiogenesis by CD36 ligation on monocytes

Lymphangiogenesis plays an important role in tumor metastasis and transplant outcome. Here, we show that thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein and naturally occurring inhibitor of angiogenesis inhibits lymphangiogenesis in mice. Compared with wild-type mice, 6-mo-old TSP-1–deficient mice develop increased spontaneous corneal lymphangiogenesis. Similarly, in a model of inflammation-induced corneal neovascularization, young TSP-1–deficient mice develop exacerbated lymphangiogenesis, which can be reversed by topical application of recombinant human TSP-1. Such increased corneal lymphangiogenesis is also detected in mice lacking CD36, a receptor for TSP-1. In these mice, repopulation of corneal macrophages with predominantly WT mice via bone marrow reconstitution ameliorates their prolymphangiogenic phenotype. In vitro, exposure of WT macrophages to TSP-1 suppresses expression of lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and VEGF-D, but not of a primarily hemangiogenic factor VEGF-A. Inhibition of VEGF-C is not detected in the absence or blockade of CD36. These findings suggest that TSP-1, by ligating CD36 on monocytic cells, acts as an endogenous inhibitor of lymphangiogenesis

Thrombospondin 1 inhibits inflammatory lymphangiogenesis by CD36 ligation on monocytes

By engaging CD36 on murine macrophages, thrombospondin-1 prevents excessive macrophage VEGF-C production and corneal neovascularization

Cultured conjunctival goblet cells express TGF-ß2, TSP-1 and CD36.

<p>(a) The expression of TGF-ß1 and TGF-ß2 isoforms in primary cultures of conjunctival goblet cells was assessed using flow cytometry. Representative histograms from 3 independent experiments: unstained cells (empty histogram), isotype controls (grey histogram), and cultured goblet cells (solid histogram). The mean fluorescence Intensity (MFI) of TGF-ß2 staining was significantly increased as compared to TGF-ß1. (b) Expression of TGF-ß1 and -ß2 isoforms was evaluated in cultured goblet cells using real time PCR. Results are presented as relative expression to that of the housekeeping gene GAPDH. (*P < 0.05 compared to TGF-ß1). (c) Immunostaining for TSP-1 and its receptor CD36 was detected in cultured goblet cells using flow cytometry.</p

Matricellular Protein Thrombospondins: Influence on Ocular Angiogenesis, Wound Healing and Immuneregulation

Thrombospondins are a family of large multi-domain glycoproteins described as matricelluar proteins based on their ability to interact with a broad range of receptors, matrix molecules, growth factors or proteases, and to modulate array of cellular functions including intracellular signaling, proliferation and migration. Two members of the thrombospondin family, thrombospondin 1 (TSP-1) and thrombospondin 2 (TSP-2) are studied extensively to determine their structure and function. While expressed at low levels in normal adult tissues, their increased expression is seen predominantly in response to cellular perturbations. Despite structural similarities, a notable functional difference between TSP-1 and TSP-2 includes the ability of former to activate of latent TGF-beta and its competitive inhibition by the latter. Both these thrombospondins are reported to play important roles in TGF-b rich ocular environment with most reports related to TSP-1. They are expressed by many ocular cell types and detectable in the aqueous and vitreous humor. TSP-1 and TSP-2 influence many cellular interactions in the eye such as angiogenesis, cell migration, wound healing, TGF-beta activation and regulation of inflammatory immune responses. Together, these processes are known to contribute to the immune privilege status of the eye. Emerging roles of TSP-1 and TSP-2 in ocular functions and pathology are reviewed here

Immunomodulatory function of conjunctival goblet cells in ocular surface mucosa.

<p>In response to an LPS-mediated stimulus, conjunctival goblet cells increase expression of TGF-ß2, and activate it in a TSP-1 dependent manner via a TSP-1 receptor, CD36. Such goblet cell derived active TGF-ß2 modulates the phenotype of surrounding dendritic cells toward an immature or tolerogenic state, decreasing the expression of activation markers, such as MHC class II, CD80, CD86 and CD40.</p