Sharmila Masli

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.

Suppression of Inflammatory Corneal Lymphangiogenesis by Application of Topical Corticosteroids

OBJECTIVESnTo analyze whether topical application of corticosteroids inhibits inflammatory corneal lymphangiogenesis and to study the potential underlying antilymphangiogenic mechanisms.nnnMETHODSnInflammatory corneal neovascularization was induced by suture placement, and the corneas were then treated with topical fluorometholone, prednisolone acetate, or dexamethasone sodium phosphate. After 1 week, the corneas were stained with lymphatic vessel endothelial hyaluronan receptor 1 for detection of pathological corneal lymphangiogenesis. The effect of these corticosteroids on macrophage recruitment was assessed via fluorescence-activated cell sorting analysis. The effect of these corticosteroids on proinflammatory cytokine expression by peritoneal exudate cells was tested via real-time polymerase chain reaction. Furthermore, the effect of steroid treatment on the proliferation of lymphatic endothelial cells was assessed via enzyme-linked immunosorbent assay.nnnRESULTSnTreatment with corticosteroids resulted in a significant reduction of inflammatory corneal lymphangiogenesis. The antilymphangiogenic effect of fluorometholone was significantly weaker than that of prednisolone and dexamethasone. Corneal macrophage recruitment was also significantly inhibited by the application of topical steroids. Treatment of peritoneal exudate cells with corticosteroids led to a significant downregulation of the RNA expression levels of tumor necrosis factor and interleukin 1β. Additionally, proliferation of lymphatic endothelial cells was also inhibited.nnnCONCLUSIONSnCorticosteroids are strong inhibitors of inflammatory corneal lymphangiogenesis, with significant differences between various corticosteroids in terms of their antilymphangiogenic potency. The main mechanism of the antilymphangiogenic effect seems to be through the suppression of macrophage infiltration, proinflammatory cytokine expression, and direct inhibition of proliferation of lymphatic endothelial cells.nnnCLINICAL RELEVANCEnSteroids block corneal lymphangiogenesis, the main risk factor for immune rejections after corneal transplantation. The different antilymphangiogenic potency of these drugs should be taken into account when using steroids in clinical practice (eg, after keratoplasty).

Immunomodulatory cross-talk between conjunctival goblet cells and dendritic cells.

Goblet cells are secretory epithelial cells of mucosal tissues that confer protection from environmental agents or pathogens via expression and secretion of soluble mucins. Loss of these cells is associated with several chronic inflammatory disorders of the mucosa. Although demonstrated to transfer antigens from the luminal surface to stromal cells in the intestinal mucosa, it is not known if goblet cells contribute to the regulation of an immune response. In this study we report that similar to intestinal and respiratory mucosal epithelia, mouse ocular surface epithelia predominantly express the TGF-ß2 isoform. Specifically, we demonstrate the ability of goblet cells to express TGF-ß2 and increase it in response to Toll-Like Receptor 4 mediated stimulus in cultures. Goblet cells not only express TGF-ß2, but are also able to activate it in a thrombospondin-1 (TSP-1) dependent manner via their cell surface receptor CD36. Furthermore, goblet cell derived soluble factors that possibly include TGF-ß2, alter dendritic cell (DC) phenotype to a tolerogenic type by downregulating DC expression of MHC class II and co-stimulatory molecules CD80, CD86 and CD40. Thus our study demonstrates goblet cells as a cellular source of active TGF-ß2 in ocular mucosa and implicates their immunomodulatory function in maintaining mucosal immune homeostasis.

Conjunctival epithelial and goblet cell function in chronic inflammation and ocular allergic inflammation.

Purpose of reviewAlthough conjunctival goblet cells are a major cell type in ocular mucosa, their responses during ocular allergy are largely unexplored. This review summarizes the recent findings that provide key insights into the mechanisms by which their function and survival are altered during chronic inflammatory responses, including ocular allergy. Recent findingsConjunctiva represents a major component of the ocular mucosa that harbors specialized lymphoid tissue. Exposure of mucin-secreting goblet cells to allergic and inflammatory mediators released by the local innate and adaptive immune cells modulates proliferation, secretory function, and cell survival. Allergic mediators like histamine, leukotrienes, and prostaglandins directly stimulate goblet cell mucin secretion and consistently increase goblet cell proliferation. Goblet cell mucin secretion is also detectable in a murine model of allergic conjunctivitis. Additionally, primary goblet cell cultures allow evaluation of various inflammatory cytokines with respect to changes in goblet cell mucin secretion, proliferation, and apoptosis. These findings in combination with the preclinical mouse models help understand the goblet cell responses and their modulation during chronic inflammatory diseases, including ocular allergy. SummaryRecent findings related to conjunctival goblet cells provide the basis for novel therapeutic approaches, involving modulation of goblet cell mucin production, to improve treatment of ocular allergies.

Polymorphism in THBS1 Gene Is Associated with Post-Refractive Surgery Chronic Ocular Surface Inflammation

PURPOSEnTo determine the association of single nucleotide polymorphisms (SNPs) of the thrombospondin 1 (THBS1) gene with development of chronic ocular surface inflammation (keratoconjunctivitis) after refractive surgery.nnnDESIGNnRetrospective cohort study.nnnPARTICIPANTSnActive duty U.S. Army soldiers (n = 143) who opted for refractive surgery.nnnMETHODSnConjunctival impression cytology samples collected from participants before the surgery were used to harvest DNA for genotyping 5 THBS1 SNPs (rs1478604, rs2228262, rs2292305, rs2228262, and rs3743125) using the Sequenom iPLEX Gold platform (Sequenom, San Diego, CA). Samples collected after surgery were used to harvest RNA for gene expression analysis by real-time polymerase chain reaction (PCR). Participants were followed for 1 year after surgery to monitor the status of keratoconjunctivitis.nnnMAIN OUTCOME MEASURESnGenetic basis of the development of chronic keratoconjunctivitis after refractive surgery.nnnRESULTSnCarriers of minor alleles of 3 SNPs each were found to be more susceptible to developing chronic keratoconjunctivitis (rs1478604: odds ratio [OR], 2.5; 95% confidence interval [CI], 1.41-4.47; P = 2.5 × 10(-3); rs2228262 and rs2292305: OR, 1.9; 95% CI, 1.05-3.51; P = 4.8 × 10(-2)). Carriers of the rs1478604 minor allele expressed significantly reduced levels of thrombospondin 1 (TSP1) (P = 0.042) and increased levels of an inflammatory cytokine associated with keratoconjunctivitis, interleukin-1β (P = 0.025), in their ocular surface epithelial cells compared with homozygous major allele controls.nnnCONCLUSIONSnGenetic variation in the THBS1 gene that results in decreased expression of the encoded glycoprotein TSP1 in ocular surface epithelial cells significantly increases the susceptibility to develop chronic ocular surface inflammation after refractive surgery. Further investigation of THBS1 SNPs in a larger sample size is warranted.

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

Abstract 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-β and its competitive inhibition by the latter. Both these thrombospondins are reported to play important roles in TGF-β 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-β 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.

Thrombospondin-1-dependent immune regulation by transforming growth factor-β2-exposed antigen-presenting cells

An important role of transforming growth factor‐β (TGF‐β) in the development of regulatory T cells is well established. Although integrin‐mediated activation of latent TGF‐β1 is considered essential for the induction of regulatory T (Treg) cells by antigen‐presenting cells (APCs), such an activation mechanism is not applicable to the TGF‐β2 isoform, which lacks an integrin‐binding RGD sequence in its latency‐associated peptide. Mucosal and ocular tissues harbour TGF‐β2‐expressing APCs involved in Treg induction. The mechanisms that regulate TGF‐β activation in such APCs remain unclear. In this study, we demonstrate that murine APCs exposed to TGF‐β2 in the environment predominantly increase expression of TGF‐β2. Such predominantly TGF‐β2‐expressing APCs use thrombospondin‐1 (TSP‐1) as an integrin‐independent mechanism to activate their newly synthesized latent TGF‐β2 to induce Foxp3+ Treg cells both in vitro and in vivo. Expression of Treg induction by TGF‐β2‐expressing APCs is supported by a TSP‐1 receptor, CD36, which facilitates activation of latent TGF‐β during antigen presentation. Our results suggest that APC‐derived TSP‐1 is essential for the development of an adaptive regulatory immune response induced by TGF‐β2‐expressing APCs similar to those located at mucosal and ocular sites. These findings introduce the integrin‐independent mechanism of TGF‐β activation as an integral part of peripheral immune tolerance associated with TGF‐β2‐expressing tissues.

Determining immune components necessary for progression of pigment dispersing disease to glaucoma in DBA/2J mice

BackgroundThe molecular mechanisms causing pigment dispersion syndrome (PDS) and the pathway(s) by which it progresses to pigmentary glaucoma are not known. Mutations in two melanosomal protein genes (Tyrp1b and GpnmbR150X) are responsible for pigment dispersing iris disease, which progresses to intraocular pressure (IOP) elevation and subsequent glaucoma in DBA/2J mice. Melanosomal defects along with ocular immune abnormalities play a role in the propagation of pigment dispersion and progression to IOP elevation. Here, we tested the role of specific immune components in the progression of the iris disease and high IOP.ResultsWe tested the role of NK cells in disease etiology by genetically modifying the B6.D2-GpnmbR150XTyrp1b strain, which develops the same iris disease as DBA/2J mice. Our findings demonstrate that neither diminishing NK mediated cytotoxic activity (Prf1 mutation) nor NK cell depletion (Il2rg mutation) has any influence on the severity or timing of GpnmbR150XTyrp1b mediated iris disease. Since DBA/2J mice are deficient in CD94, an important immune modulator that often acts as an immune suppressor, we generated DBA/2J mice sufficient in CD94. Sufficiency of CD94 failed to alter either the iris disease or the subsequent IOP elevation. Additionally CD94 status had no detected effect on glaucomatous optic nerve damage.ConclusionOur previous data implicate immune components in the manifestation of pigment dispersion and/or IOP elevation in DBA/2J mice. The current study eliminates important immune components, specifically NK cells and CD94 deficiency, as critical in the progression of iris disease and glaucoma. This narrows the field of possible immune components responsible for disease progression.

TSP-1 Deficiency Alters Ocular Microbiota: Implications for Sjögren's Syndrome Pathogenesis

PURPOSEnThe potential role of commensals as triggering factors that promote inflammation in dry eye disease has not been explored. The objective of this study was to evaluate whether ocular microbiota changes with the onset of dry eye disease in thrombospondin-1-deficient (TSP-1(-/-)) mice, a strain that develops Sjögrens syndrome-like disease.nnnMETHODSnConjunctival swabs were collected from TSP-1(-/-) and C57BL/6 mice and analyzed for bacterial presence. Opsonophagocytosis of the bacterial conjunctival isolates derived from the aged TSP-1(-/-) mice by neutrophils derived from either TSP-1(-/-) or C57BL/6 bone marrow was evaluated. The bactericidal activities of TSP-1-derived peptide were examined.nnnRESULTSnWe found that in TSP-1(-/-) mice, the conjunctival colonization with Staphylococcus aureus and coagulase negative staphylococci sp (CNS) species was significantly increased with aging and preceded that of the wild-type C57BL/6 control mice. This correlated with increased neutrophil infiltration into the conjunctiva of the TSP-1(-/-) mice, suggesting that TSP-1 plays a significant role in regulating immunity to commensals. Accordingly, the TSP-1(-/-) PMNs opsonophagocytozed the ocular commensals less efficiently than the TSP-1-sufficient neutrophils. Furthermore, a TSP-1-derived peptide, 4N1K, exhibited significant antimicrobial activity when compared to a control peptide against commensal sp.nnnCONCLUSIONnThese studies illustrate that alterations in the commensal frequency occur in the early stages of development of Sjögrens-like pathology and suggest that interventions that limit commensal outgrowth such as the use of TSP-1-derived peptides could be used for treatment during the early stages of the disease to reduce the commensal burden and ensuing inflammation.

Thrombospondin derived peptide attenuates Sjögren's syndrome‐associated ocular surface inflammation in mice

Sjögrens syndrome is the second most common rheumatic disease in which autoimmune response targets exocrine glands (salivary and lacrimal glands) result in clinical symptoms of dry mouth and dry eye. Inflammation of the lacrimal gland induces tear abnormalities that contribute to the inflammation of the ocular surface, which includes ocular mucosa. Thrombospondin‐1 (TSP‐1) plays a critical regulatory role in the ocular mucosa and as such TSP‐1–/– mice develop spontaneously chronic ocular surface inflammation associated with Sjögrens syndrome. The autoimmune pathology is also accompanied by a peripheral imbalance in regulatory (Treg) and inflammatory Th17 effectors. In this study, we demonstrate an in‐vitro effect of a CD47‐binding TSP‐derived peptide in the induction of transforming growth factor (TGF)‐β1‐secreting forkhead box protein 2 (Foxp3+) Tregs from activated CD4+CD25– T cells and the inhibition of pathogenic T helper type 17 (Th17)‐promoting interleukin (IL)‐23 derived from antigen‐presenting cells. The in‐vivo administration of this peptide promotes Foxp3+ Treg induction and inhibition of Th17 development. Consistent with these results, topical administration of CD47‐binding TSP peptide, both before and after the onset of the disease, attenuates clinical symptoms of SS‐associated dry eye in TSP‐1–/– mice. Augmented expression of Foxp3 detected in the draining lymph nodes of TSP peptide ‐treated mice compared to those treated with control peptide suggests the ability of TSP peptide to restore peripheral immune imbalance. Thus, our results suggest that TSP‐derived peptide attenuates Sjögrens syndrome‐associated dry eye and autoimmune inflammation by preventing Th17 development while promoting the induction of Tregs. Collectively, our data identify TSP‐derived peptide as a novel therapeutic option to treat autoimmune diseases.