M.Sc Thesis

M.Sc StudentHaves Dana
SubjectCathepsins and their Endogenous Inhibitors - Cystatins:
Expression and Modulation in Immune Cells and in
Multiple Sclerosis
DepartmentDepartment of Medicine
Supervisor PROF. Ariel Miller
Full Thesis textFull thesis text - English Version


Background: Cathepsins are proteolytic enzymes that are involved in a variety of physiological processes including antigen presentation, ECM degradation and cell migration, and thus may be involved in pathological processes such as immunity to infection and autoimmune diseases. Recent pharmacogenetics studies detected associations between the gene encoding Cathepsin S and response to immunotherapies for relapsing Multiple Sclerosis (MS).

Aims: A. To compare levels of cathepsin S (CTSS), cathepsin B (CTSB) and their inhibitors cystatin C (CSTC) and B (CSTB) in peripheral blood leukocytes (PBLs) and serum of healthy individuals versus MS patients at various disease states; B. To examine the effect of MS drugs interferon β (IFN-β) and glucocorticoids (GCs) on cathepsin and cystatin expression; C. To test for association between MS patients’ response to IFN-β treatment and cathepsin S levels; D. To study the effects of GCs on cathepsin and cystatin levels in-vitro.

Methods: Expression levels of the cathepsins and cystatins were analyzed in RNA of PBL from MS patients and matched healthy individuals, using Real-Time PCR, and in their serum proteins, using ELISA. U937 macrophages were treated with GCs, mRNA expression and supernatant protein levels were analyzed using Real-Time PCR and western blots.

Results: CTSS RNA expression was significantly elevated in MS patients compared to healthy controls, with higher levels observed in relapse state versus remission. Serum levels of cathepsin S, and cathepsin S to cystatin C ratio, were also significantly elevated in relapse. GCs appeared to reduce the relapse levels of cathepsin S, both in PBL RNA and in serum protein, concomitant with elevation of its inhibitor cystatin C levels. Similar results were obtained in vitro in macrophages, where high dose of GC reduced the mRNA and supernatant protein levels of cathepsin S and pro cathepsin B.  IFN-β significantly elevated the mRNA levels of CSTC and CSTB in patients' PBLs, and reduced serum cathepsin S. A correlation between patients' response phenotype to IFN-β and serum cathepsin S protein levels was observed.

Conclusions: Cathepsin and cystatin levels appear to be correlated with MS disease activity. IFN‑β and GC treatments exert their beneficial effect in part by shifting the balance between cathepsins and their inhibitors. Cathepsin S levels are correlated with IFN-β response phenotype. In vitro findings suggest that the effect of GCs on cathepsins and cystatins levels observed ex vivo, is mediated, at least partially, through macrophage function. We suggest cathepsin activity may contribute to MS pathology and response to medication.