EFFECTS OF DESICCATING STRESS ON THROMBOSPONDIN-1 KNOCK-OUT MICE

Gandhi NB, Volpe E, Zhang X, Su Z, Farley WJ, Li DQ, Pflugfelder SC, De Paiva CS Ocular Surface Center, Department of Ophthalmology, Baylor College of Medicine, Houston, TX

PURPOSE

METHODS CONCLUSION

REFERENCES

INTRODUCTION RESULTS II RESULTS I

The purpose of this study was to evaluate the ocular surface response to desiccating stress in thrombospondin-1 knock-out (TSP-1KO) mice.

Dry eye, a common ophthalmologic problem, often leads to an autoimmune reaction on the ocular surface, particularly in Sjögren’s Syndrome. One pathway of ocular surface inflammation consists of production and release of interleukin-17A (IL-17A) by T-helper 17 (Th-17) cells, which contribute to the disruption of epithelial barrier function and consequently corneal surface irregularities with reduced optical quality. Correspondingly, desiccating stress has resulted in a significant upregulation of Th-17 promoters on the ocular surface as well as an increase in IL-17A in the corneal epithelium and conjunctiva.

Thrombospondin-1 is a potent activator of Transforming Growth Factor-β1 (TGF-β1), especially on and around the corneal surface and conjunctiva. TGF-β1 has both immunosuppressive effects (by blocking proliferation of T cells via inhibiting interleukin-2 generation) and immunostimulatory effects (by encouraging the development of Th-17 cells, in conjunction with interleukin-6).

Prior work in the field has implicated the absence of TSP-1 to the development of lacrimal keratoconjunctivitis (an immunosuppressive effect of TSP-1) as well as to the attenuation of encephalomyelitis (an immunostimulatory effect of TSP-1).

Desiccating stress (DS) was induced by subcutaneous injection of scopolamine and exposure to a drafty, low humidity (30%) environment in TSP-1KO and wild-type (C57BL/6; B6) mice, aged 12 weeks, for 5 and 10 days. Non-stressed (NS) control mice were maintained in a separate room containing 50–75% relative humidity without exposure to forced air.

Conjunctival goblet cell density was counted in periodic acid Schiff (PAS) stained sections using NIS Elements software.

Immunohistochemistry was used to identify and count positively stained cells in corneal and conjunctival tissue sections.

Corneal smoothness was examined using images taken of an illuminated ring reflection of off the corneal surface. Degree of smoothness was assessed in NIS Elements software. Corneal barrier function was assessed by staining corneas using Oregon green dextran.

RNA from corneal epithelium and conjunctiva were analyzed using qPCR. CD11c+/CD11b+ reconstitution was performed via ex vivo bone marrow expansion

and grown with GM-CSF and IL-4 stimulation. CD4+ cells for adoptive transfer were isolated from donor spleens and cervical lymph nodes and selected using magnetic beads. Cells were injected intraperitoneally into recipient mice.

Flow cytometric analyses were performed on conjunctival and cervical lymph node sections using fluourescently-labeled antibodies. PI was used as a live/dead cell discriminator.

TGF-β bioactivity level was measured using a PAI/L transfected MLEC line via a Luciferase Assay System.

1. Behrens,A., Doyle,J.J., Stern,L., Chuck,R.S., McDonnell,P.J., Azar,D.T., Dua,H.S., Hom,M., Karpecki,P.M., Laibson,P.R. et al. (2006) Dysfunctional tear syndrome: a Delphi approach to treatment recommendations. Cornea, 25, 900-907. 2. Niederkorn,J.Y., Stern,M.E., Pflugfelder,S.C., de Paiva,C.S., Corrales,R.M., Gao,J. and Siemasko,K. (2006) Desiccating Stress Induces T Cell-Mediated Sjogren's Syndrome-Like Lacrimal Keratoconjunctivitis. J. Immunol., 176, 3950-3957. 3. Dong,C. (2008) TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat. Rev. Immunol., 8, 337-348. 4. Weaver,C.T., Hatton,R.D., Mangan,P.R. Harrington,L.E. (2007) IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu. Rev. Immunol., 25, 821-852. 5. Chen,Z. and O'Shea,J.J. (2008) Th17 cells: a new fate for differentiating helper T cells. Immunol. Res., 41, 87-102. 6. Luo,L., Li,D.Q., Corrales,R.M. Pflugfelder,S.C. (2005) Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface. Eye & Contact Lens, 31(5): 186-93. 7. Stockinger,B., Veldhoen,M. Martin,B. (2007) Th17 T cells: linking innate and adaptive immunity. Semin. Immunol., 19, 353-361. 8. Chauhan,S.K., El,A.J., Ecoiffier,T., Goyal,S., Zhang,Q., Saban,D.R. and Dana,R. (2009) Autoimmunity in dry eye is due to resistance of Th17 to Treg suppression. J. Immunol., 182, 1247-1252. 9. Murphy-Ullrich,J.E. Poczatek,M. (2000) Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology. Cytokine Growth Factor Rev., 11, 59-69. 10. Turpie,B., Yoshimura,T., Gulati,A., Rios,J.D., Dartt,D.A. Masli,S. (2009) Sjogren's syndrome-like ocular surface disease in thrombospondin-1 deficient mice. Am. J. Pathol., 175, 1136-1147. 11. Lutz,M.B., Kukutsch,N., Ogilvie,A.L., Rossner,S., Koch,F., Romani,N. Schuler,G. (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods, 223, 77-92. 12. Pflugfelder,S.C., Farley,W., Luo,L., Chen,L.Z., de Paiva,C.S., Olmos,L.C., Li,D.Q. Fini,M.E. (2005) Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye. Am. J. Pathol., 166, 61-71. 13. Veldhoen,M., Hocking,R.J., Flavell,R.A. Stockinger,B. (2006) Signals mediated by transforming growth factor-beta initiate autoimmune encephalomyelitis, but chronic inflammation is needed to sustain disease. Nat. Immunol., 7, 1151-1156. 14. de,L.N., Gallardo,E., Sonnet,C., Chazaud,B., Dominguez-Perles,R., Suarez-Calvet,X., Gherardi,R.K. Illa,I. (2010) Role of thrombospondin 1 in macrophage inflammation in dysferlin myopathy. J. Neuropathol. Exp. Neurol., 69, 643-653.

Supported by: NIH Grant EY018888 (CSDP), EY11915 (SCP),

An unrestricted Grant from Research to Prevent Blindness, The Oshman Foundation and

The William Stamps Farish Fund.

TSP-1KO mice exhibited an inverse immune phenotype secondary to desiccation than WT Improvement in goblet cell density and decrease in infiltrating CD4+ T cells in the CJ No disruption of corneal barrier function Failure to upregulate IL-17A, IL-6, IL-13 and IFN-γ mRNA in the cornea and conjunctiva

TSP-1 is essential to the generation of pathogenic CD4+ T-cells Dendritic cell TSP-1 is crucial to the ocular surface response secondary to desiccation Taken together, these results indicate that the interplay between TSP-1 and TGF-β1 is

critical to the immune phenotype in dry eye

Adoptive transfer of WT or TSP-1KO dendritic cells (DC) into WT and TSP-1KO mice. Corneal permeability to Oregen green dextran dye (LEFT). Levels of CD4+ cells infiltrating the conjunctival epithelium (MIDDLE). Goblet cell density in the conjunctival epithelium (RIGHT).

* p < 0.05 ** p < 0.01 *** p < 0.001

Localization of thrombospondin-1 activity. Immunohistochemistry staining of TSP-1 in corneal and conjunctival sections of nonstressed (NS) and stressed (5D, 10D) B6 mice, aged 12 weeks, highlighting that TSP-1 is localized in the corneal and conjunctival stroma and that its levels are unaffected by desiccation (LEFT). Levels of bioactive TGF-β found in the tears and lacrimal gland tissue of NS mice (RIGHT).

* p < 0.05 ** p < 0.01 *** p < 0.001

Desiccating stress effects. Levels of CD4+ cells infiltrating the conjunctival epithelium of WT and TSP-1KO mice subjected to desiccation (A). Goblet cell density of the conjunctival epithelium of WT and TSP-1KO mice subjected to desiccation (B). Evaluation of smoothness and regularity of the cornea among WT and TSP-1KO mice subjected to desiccation. Values normalized to non-stressed control mice (C). Corneal permeability to Oregen green dextran dye in WT and TSP-1KO subjected to desiccating stress (D). Images (LEFT) with corresponding graphical representation (RIGHT).

* p < 0.05 ** p < 0.01 *** p < 0.001

1. TSP-1 is expressed in corneal and conjunctival stroma and TSP-1KO mice have reduced TGF-β levels vs. wildtype

2. TSP-1 is critical to ocular surface inflammation secondary to desiccating stress

5. Dendritic cell associated TSP-1 is critical to the ocular surface response secondary to desiccating stress

Quantitative RT-PCR analyses. Corneal mRNA transcripts of MMP3 (A), IL-17a (B), MMP9 (D) and IFN-γ (E) and conjunctival mRNA transcripts of IL-17a (C) and IFN-γ (F) of WT and TSP-1KO mice subjected to desiccating stress. Fold of expression presented as relative to NS control. Ratio of IL-13:IFN-γ mRNA transcripts in the conjunctiva of WT and TSP-1KO mice subjected to desiccating stress (G).

* p < 0.05 ** p < 0.01 *** p < 0.001

3. TSP-1KO mice do not upregulate inflammatory cytokines secondary to desiccating stress

4. TSP-1 is essential for the generation of pathogenic CD4+ T-cells

Adoptive transfer of WT and TSP-1KO CD4+ cells into RAG-1KO mice. Levels of CD4+ cells infiltrating the conjunctival epithelium (A). Goblet cell density of the conjunctival epithelium (B). Quantitative RT-PCR analysis of corneal mRNA transcripts of MMP3 (C), IL-17a (D), MMP9 (F), and IFN-γ (G) and conjunctival mRNA transcripts of IL-17a (E) and IFN-γ (H). Fold of expression presented as relative to NS control. Ratio of IL-13:IFN-γ mRNA transcripts in the conjunctiva (I).

* p < 0.05 ** p < 0.01 *** p < 0.001

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