Bruce Turpie

Sjögren’s Syndrome-Like Ocular Surface Disease in Thrombospondin-1 Deficient Mice

Thrombospondin-1 (TSP-1) is a major activator of latent transforming growth factor-beta in vitro as well as in vivo. Mice deficient in TSP-1, despite appearing normal at birth, develop a chronic form of ocular surface disease that is marked by increased apoptosis and deterioration in the lacrimal gland, associated dysfunction, and development of inflammatory infiltrates that result in abnormal tears. The increase in CD4(+) T cells in the inflammatory infiltrates of the lacrimal gland, and the presence of anti-Sjögrens syndrome antigen A and anti-Sjögrens syndrome antigen B antibodies in the serum resemble autoimmune Sjögrens syndrome. These mice develop an ocular surface disorder dry eye that includes disruption of the corneal epithelial layer, corneal edema, and a significant decline in conjuctival goblet cells. Externally, several mice develop dry crusty eyes that eventually close. The inflammatory CD4(+) T cells detected in the lacrimal gland, as well as those in the periphery of older TSP-1 null mice, secrete interleukin-17A, a cytokine associated with chronic inflammatory diseases. Antigen-presenting cells, derived from TSP-1 null, but not from wild-type mice, activate T cells to promote the Th17 response. Together, these results indicate that TSP-1 deficiency results in a spontaneous form of chronic dry eye and aberrant histopathology associated with Sjögrens syndrome.

Expression of Thrombospondin in TGFβ-Treated APCs and Its Relevance to Their Immune Deviation-Promoting Properties

APCs deployed within iris/ciliary body are responsible for promoting anterior chamber-associated immune deviation following injection of Ag into the eye. TGFβ-2, a constituent of the ocular microenvironment, converts conventional APCs that are pulsed with Ag into cells that induce immune deviation when injected into naive mice. TGFβ-2-treated APCs under-express IL-12 and CD40, and over-express active TGFβ. We have examined transcriptional changes within macrophage hybridoma no. 59, which promotes Th1 cell differentiation, and TGFβ-2-treated no. 59 as well as macrophage hybridoma no. 63, both of which induce immune deviation similar to anterior chamber-associated immune deviation. Immune deviation-inducing hybridomas up-regulated expression of thrombospondin, TGFβ, IFN-α and β, murine macrophage elastase, and macrophage-inflammatory protein-2 genes, while down-regulating expression of the genes for NF-κB and CD40. Based on the known properties of these gene products, a model is proposed in which these gene products, alone and through interacting signaling pathways, confer upon conventional APCs the capacity to create and surround themselves with an immunomodulatory microenvironment. The model proposes that the pleiotropic effects of thrombospondin are primarily responsible for creating this microenvironment that is stabile, rich in active TGFβ and IFN-α and β, deficient in IL-12, and chemoattractant via macrophage-inflammatory protein-2 for NK T cells. It is further proposed that presentation of Ag to T cells in this microenvironment leads to their differentiation into regulatory cells that suppress Th1 cell-dependent immunogenic inflammation.

Anti-inflammatory effects of tumour necrosis factor (TNF)-α are mediated via TNF-R2 (p75) in tolerogenic transforming growth factor-β-treated antigen-presenting cells

Exposure of macrophages to transforming growth factor (TGF)‐β is known to alter their functional phenotype such that antigen presentation by these cells leads to tolerance rather than an inflammatory immune response. Typically, eye‐derived antigen‐presenting cells (APCs) exposed to TGF‐β in the local environment are known to induce a form of peripheral tolerance and protect the eye from inflammatory immune effector‐mediated damage. In response to TGF‐β, APCs increase their expression of tumour necrosis factor (TNF)‐α and TNF receptor 2 (TNF‐R2). Although TNF‐α has been implicated in tolerance and the associated regulation of the inflammatory immune response, its source and the receptors involved remain unclear. In this report we determined the contribution of TNF‐α and TNF‐R2 expressed by TGF‐β‐treated APCs to their anti‐inflammatory tolerogenic effect. Our results indicate that APC‐derived TNF‐α is essential for the ability of APCs to regulate the immune response and their IL‐12 secretion. Moreover, in the absence of TNF‐R2, APCs exposed to TGF‐β failed to induce tolerance or regulatory cells known to participate in this tolerance. Also, blocking of TNF‐R1 signalling enhanced the ability of the APCs to secrete increased TGF‐β in response to TGF‐β exposure. Together our results support an anti‐inflammatory role of TNF‐α in regulation of an immune response by TGF‐β‐treated APCs and suggest that TNF‐R2 contributes significantly to this role.

Increased IκBα expression is essential for the tolerogenic property of TGF-β-exposed APCs

IκBα is an inhibitor of the transcrip tional factor NF‐κ B, and it is an essential component of the signaling pathways that lead to expression of inflammatory molecules. These include cytokines and costimulatory molecules associated with antigen presentation in an inflammatory immune response. In this study, we report that antigen‐presenting cells exposed to TGF‐β induce peripheral tolerance by increasing IκBα expression. Exposure of antigen presenting cells (APCs) to TGF‐β is known to impair their ability to secrete IL‐12, and such impairment correlated with reduced NF‐κB activity as indicated by significantly reduced nuclear levels of p50, an essential subunit of NF‐κB for IL‐12 transcription. Blockade of increased nuclear IκBα in APCs by expression of small interfering RNA molecules (siRNAs) targeting IκBα transcripts prevented IL‐12 impairment and the decline in nuclear p50 levels. Furthermore, such IκBα blockade also interfered with the tolerogenic property of TGF‐β‐ exposed APCs. However, increased expression of IκBα in APCs, independent of TGF‐β exposure, reduced nuclear p50 levels and permitted tolerance induction by APCs. Thus, our findings attribute a direct and significant role to IκBα in the tolerogenic potential of APCs. Increased IκBα expression in APCs may therefore offer a therapeutic approach to achieve antigen‐specific immunomodulation.—Ghafoori, P.,Yoshimura, T., Turpie, B., Masli, S. Increased IκBα expression is essential for the tolerogenic property of TGF‐β‐exposed APCs. FASEB J. 23, 2226–2234 (2009)

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.

Alteration in cellular turnover and progenitor cell population in lacrimal glands from thrombospondin 1−/− mice, a model of dry eye

The purpose of this study was to investigate the changes that occur in the lacrimal glands (LGs) in female thrombospondin 1 knockout (TSP1-/-) mice, a mouse model of the autoimmune disease Sjogrens syndrome. The LGs of 4, 12, and 24 week-old female TSP1-/- and C57BL/6J (wild type, WT) mice were used. qPCR was performed to measure cytokine expression. To study the architecture, LG sections were stained with hematoxylin and eosin. Cell proliferation was measured using bromo-deoxyuridine and immunohistochemistry. Amount of CD47 and stem cell markers was analyzed by western blot analysis and location by immunofluorescence microscopy. Expression of stem cell transcription factors was performed using Mouse Stem Cell Transcription Factors RT2 Profiler PCR Array. Cytokine levels significantly increased in LGs of 24 week-old TSP1-/- mice while morphological changes were detected at 12 weeks. Proliferation was decreased in 12 week-old TSP1-/- mice. Three transcription factors were overexpressed and eleven underexpressed in TSP1-/- compared to WT LGs. The amount of CD47, Musashi1, and Sox2 was decreased while the amount of ABCG2 was increased in 12 week-old TSP1-/- mice. We conclude that TSP1 is necessary for maintaining normal LG homeostasis. Absence of TSP1 alters cytokine levels and stem cell transcription factors, LG cellular architecture, decreases cell proliferation, and alters amount of stem cell markers.