NOD males exhibit the autoimmune dacryoadenitis and reduced tear flow that constitute the principal ocular signs in SS patients22, and develop increased tear CTSS4, similar to SS patients. tears compared to other groups. While tear CTSS activity remained the strongest discriminator of SS in autoimmune populations, combining LF and CTSS improved discrimination of SS beyond CTSS in DE patients. Reductions in Cys C and other endogenous proteases may enhance CTSS activity in SS tears. Tear CTSS activity is reconfirmed as a putative biomarker of SS in an independent patient cohort while combined LF and CTSS measurements may distinguish SS from DE patients. Introduction Sj?grens syndrome (SS) is a systemic autoimmune disease characterised by lymphocytic infiltration and loss of secretory function of lacrimal glands (LG) and salivary glands. In addition to keratoconjunctivitis sicca and xerostomia, constitutional symptoms are common, and may involve pulmonary, neurological, vascular, and renal systems1. However, onset of sicca and systemic symptoms is often not parallel, making early diagnosis a challenge in patients presenting primarily with either dry eye or systemic symptoms2,3. The lack of specificity of currently-used biomarkers combined with the invasive nature of minor salivary gland biopsies have prompted efforts to identify new non-invasive biomarkers for diagnosis of SS. Tear and salivary biomarkers may be especially relevant, since alterations in the composition of these fluids may mirror the pathological processes and functional status of these glands affected by SS. Tear cathepsin S (CTSS) activity may be one such biomarker. CTSS activity in tears of male NOD mice, a murine model of SS, is greater than that in tears of healthy BALB/c mice4, while CTSS activity in tears of SS patients is greater than that in tears of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), non-autoimmune dry eye (DE) disorders, and healthy controls5. CTSS Mefloquine HCl is an intriguing protein with functions linked to inflammation including regulation of major histocompatibility complex class II-mediated antigen presentation6,7. Furthermore, it also has important functions in extracellular Mefloquine HCl matrix degradation8 and activation of the protease activated receptor 29 which mediates inflammatory pain10,11, triggering cytokine production and itchiness12. Moreover, since CTSS exhibits potent activity at both acid and neutral pH levels13, elevated tear CTSS activity may alter tear composition, thereby affecting the ocular surface. Indeed, proteoglygan 4/lubricin, a glycoprotein important for lubrication and smooth movement of the eyelids, is degraded by tear CTSS14. The activities of cysteine cathepsins, including CTSS, are physiologically regulated Mefloquine HCl by a family of protease inhibitors, cystatins15. Cystatin C (Cys C) is the most abundant and potent inhibitor of CTSS, and changes in its levels in the extracellular environment are implicated in formation of atherosclerotic plaques and tumor metastases16C18. Accordingly, changes in tear Cys C levels could affect tear CTSS activity. In this study, we hypothesized that tears of SS patients may contain reduced levels of Cys C, and possibly other endogenous protease inhibitors as well, thereby allowing tear CTSS to directly or indirectly enhance the degradation of other tear proteins. We demonstrate that reduced Cys C tear levels are correlated with increased CTSS activity in LG and tears of SS-model mice and in tears of SS patients. Furthermore, we demonstrate that CTSS, at activity levels found in tears of SS patients but not in tears of healthy controls, promotes degradation of two other abundant tear proteins, lactoferrin (LF) and secretory IgA (sIgA), both of which play fundamental roles in ocular defense against pathogens19C21. Finally, we demonstrate remarkably reduced levels of Cys C, LF, and sIgA in combination with elevated CTSS activity in SS patient tears relative to levels in Rabbit polyclonal to CyclinA1 tears from patients with other rheumatic diseases, non-autoimmune DE, and healthy controls. Intriguingly, while these decreases do not offer the ability to further discriminate.Early observations in SS patient tears reported a decrease in total IgA rather than sIgA43. sIgA levels were significantly decreased, in SS tears compared to other groups. While tear CTSS activity remained the strongest discriminator of SS in autoimmune populations, combining LF and CTSS improved discrimination of SS beyond CTSS in DE patients. Reductions in Cys C and other endogenous proteases may enhance CTSS activity in SS tears. Tear CTSS activity is reconfirmed as a putative biomarker of SS in an independent patient cohort while combined LF and CTSS measurements may distinguish SS from DE patients. Introduction Sj?grens syndrome (SS) is a systemic autoimmune disease characterised by lymphocytic infiltration and loss of secretory function of lacrimal glands (LG) Mefloquine HCl and salivary glands. In addition to keratoconjunctivitis sicca and xerostomia, constitutional symptoms are common, and may involve pulmonary, neurological, vascular, and renal systems1. However, onset of sicca and systemic symptoms is definitely often not parallel, making early diagnosis challenging in individuals presenting primarily with either dry attention or systemic symptoms2,3. The lack of specificity of currently-used biomarkers combined with the invasive nature of small salivary gland biopsies have prompted efforts to identify new non-invasive biomarkers for analysis of SS. Tear and salivary biomarkers may be especially relevant, since alterations in the composition of these fluids may mirror the pathological processes and functional status of these glands affected by SS. Tear cathepsin S (CTSS) activity may be one such biomarker. CTSS activity in tears of male NOD mice, a murine model of SS, is definitely greater than that in tears of healthy BALB/c mice4, while CTSS activity in tears of SS individuals is definitely greater than that in tears of individuals with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), non-autoimmune dry attention (DE) disorders, and healthy settings5. CTSS is an intriguing protein with functions linked to swelling including rules of major histocompatibility complex class II-mediated antigen demonstration6,7. Furthermore, it also has important functions in extracellular matrix degradation8 and activation of the protease triggered receptor 29 which mediates inflammatory pain10,11, triggering cytokine production and itchiness12. Moreover, since CTSS exhibits potent activity at both acid and neutral pH levels13, elevated tear CTSS activity may alter tear composition, thereby influencing the ocular surface. Indeed, proteoglygan 4/lubricin, a glycoprotein important for lubrication and clean movement of the eyelids, is definitely degraded by tear CTSS14. The activities of cysteine cathepsins, including CTSS, are physiologically regulated by a family of protease inhibitors, cystatins15. Cystatin C (Cys C) is the Mefloquine HCl most abundant and potent inhibitor of CTSS, and changes in its levels in the extracellular environment are implicated in formation of atherosclerotic plaques and tumor metastases16C18. Accordingly, changes in tear Cys C levels could affect tear CTSS activity. With this study, we hypothesized that tears of SS individuals may contain reduced levels of Cys C, and possibly additional endogenous protease inhibitors as well, thereby allowing tear CTSS to directly or indirectly enhance the degradation of additional tear proteins. We demonstrate that reduced Cys C tear levels are correlated with increased CTSS activity in LG and tears of SS-model mice and in tears of SS individuals. Furthermore, we demonstrate that CTSS, at activity levels found in tears of SS individuals but not in tears of healthy settings, promotes degradation of two additional abundant tear proteins, lactoferrin (LF) and secretory IgA (sIgA), both of which play fundamental tasks in ocular defense against pathogens19C21. Finally, we demonstrate amazingly reduced levels of Cys C, LF, and sIgA in combination with elevated CTSS activity in SS patient tears relative to levels in tears from individuals with additional rheumatic diseases, non-autoimmune DE, and healthy settings. Intriguingly, while these decreases do not offer the ability to further discriminate SS within the autoimmune disease human population beyond that of CTSS activity, the combination of LF and CTSS measurements may allow better discrimination between those individuals with SS and those with non-autoimmune DE, therefore potentially offering a fresh means to fix an ongoing medical challenge. Results Cystatin C is definitely reduced in tears and LG of male NOD mice We 1st analysed levels of Cys C in tears and LG of 12-week male healthy (BALB/c) and SS-model (NOD) mice. NOD males show the autoimmune dacryoadenitis and reduced tear circulation that constitute the principal ocular indications in SS individuals22, and develop improved tear CTSS4, much like SS individuals. Western blotting exposed that Cys C was present in tears and LG.