4 biomarkers may improve Sjögren's syndrome treatment

Take-home: Research on the composition of tears and their secretory processes is generating insights that may lead to earlier detection and treatment of Sjögren’s syndrome.

Reviewed by Maria C. Edman, PhD, and Sarah F. Hamm-Alvarez, PhD

Los Angeles—Ongoing research on the secretory processes of lacrimal glands continues to yield findings that may facilitate earlier diagnosis of Sjögren’s syndrome and more effective treatment.

One of the recent developments is the discovery that four tear biomarkers distinguish patients with Sjögren’s syndrome from patients with other autoimmune diseases, said Sarah F. Hamm-Alvarez, PhD, professor and vice chairman, basic research, Department of Ophthalmology, Roski Eye Institute, University of Southern California, and professor of pharmacology and pharmaceutical sciences (secondary), USC School of Pharmacy.

For more than 20 years, Dr. Hamm-Alvarez and colleagues have focused their research on the process of secretion by the lacrimal glands, which are affected by Sjögren’s syndrome. The team has studied molecular processes for sorting and release of proteins, how these processes go awry, and therapeutic development by targeting anti-inflammatory agents to the glands and the tears.

The focus has shifted increasingly to Sjögren’s syndrome, specifically the defects in the secretory process that may emerge early in the disease and characterize the diseased lacrimal gland, according to Dr. Hamm-Alvarez.

About 10 years ago, the researchers discovered in a mouse model of Sjögren’s syndrome that the protein cathepsin S (CTSS) was increased significantly in the tears and lacrimal glands. Moving the work to the clinic, they found that CTSS levels were also elevated in the patients with Sjögren’s syndrome. These findings were published in 2014.

The new findings

In 2015, Dr. Hamm-Alvarez, Maria C. Edman, PhD, research associate, USC Roski Eye Institute, and team released new findings on tear biomarkers that may help diagnose patients with Sjögren’s syndrome.

“We were very interested to begin with CTSS, a protease that is usually present in the lysosomes and helps to degrade proteins,” Dr. Edman said. “Given that we were studying the trafficking processes, we were very curious why the CTSS ended up in the tears of Sjögren’s syndrome mice and also in the tears in the Sjögren’s syndrome patients.”

After finding alterations in the trafficking processes for this protein, the researchers expanded their study to other proteins present in the tear film that also might serve as biomarkers: secretory IgA (sIgA), lactoferin (LF), and cystatin C (CysC).

Secretory IgA is the main antibody in the tear fluid and is very active in the defense of mucosal membranes, while LF is decreased in tears of patients with both non-Sjögren’s syndrome dry eye and Sjögren’s syndrome-mediated dry eye. CysC was chosen as the third marker because it is an active inhibitor of CTSS and could be used to detect any shift in the balance between it and CTSS, Dr. Edman explained.

Female patients with primary or secondary Sjögren’s syndrome (n=28), rheumatoid arthritis (n=31), or other autoimmune disease (n=31) were recruited for the study. An anesthetized Schirmer’s test was performed on both eyes of each patient, followed by analysis for CTSS activity and concentrations of the three other proteins.

Greater CTSS activity

Greater CTSS activity

The investigators found that tear CTSS activity was 6 times as great in patients with Sjögren’s syndrome as in those with rheumatoid arthritis and 8 times as great as in patients with other autoimmune diseases. However, levels of the three other tear proteins were reduced in in Sjögren’s patients compared to the other two groups.

sIgA was 6-fold lower in Sjögren’s syndrome patient tears compared to tears from those patients with rheumatoid arthritis or other autoimmune diseases, LF was 5-fold lower in Sjögren’s syndrome patient tears than in rheumatoid arthritis patient tears and 8-fold lower than in tears of those patients with other autoimmune diseases. CysC was 4-fold lower in Sjögren’s syndrome patient tears than in rheumatoid arthritis patient tears and 6-fold lower than in tears of those patients with other autoimmune diseases.

Because CysC regulates CTSS in an equimolar fashion, higher levels of CysC result in lower activity of CTSS and vice versa.

“If you see the concentration levels of CTSS go up in the tear film, while the CysC levels go down, that probably means that the capability of CysC to regulate the CTSS activity as it does in the tear film of normal patients is disrupted,” Dr. Edman pointed out.

The researchers continue to study these mechanisms in a mouse-disease model, further exploring the hypothesis that both an increase in the gene expression of CTSS in the lacrimal glands and its increased recovery in secretory granules in tears, as well as the decrease in CysC, are involved in increased disease activity.

They also are exploring whether these proteins may not be just passive biomarkers of Sjögren’s syndrome but may reflect the level of inflammation of the lacrimal gland.

“It’s both a decrease in the ratio and an increase in the protein abundance,” Dr. Hamm-Alvarez outlined. “We go back and forth between the studies where we are identifying the biomarkers and then trying to figure out what does that illustrate about defective function in the glands. We’re very interested in using the biomarkers diagnostically, but then trying to deduce changes in molecular function that represent opportunities to intervene or to restore function.”

Confirming the relationship

Confirming the relationship

Dr. Edman is recruiting additional patients into the study to confirm the relationship between the biomarkers and to determine whether all four would be needed to develop a successful diagnostic tool as well as to understand the statistical power of using one or combinations of the proteins.

The team has also conducted research on identifying threshold values, particularly for CTSS that might be useful in a future diagnostic test, and help doctors determine when to refer dry eye patients to a rheumatologist for a full work-up for symptoms of Sjögren’s syndrome.

Yet another avenue of research is a potential correlation between levels of CTSS in tears and the degree of inflammation in the lacrimal gland. Evidence suggesting a relationship has been found in a mouse model, noted Dr. Hamm-Alvarez, and if confirmed, might be used to evaluate the efficacy of treatment aimed at reducing inflammation.

This ongoing research has produced additional discoveries pertaining to molecular changes in glands in mice that can result in increased release of CTSS and appear to be related to disease processes. Focusing on the proteins Rab3D and Rab27, the research team found that genetically modified mice lacking Rab3D had more CTSS in their tears, while mice lacking Rab27 had lower levels of CTSS.

The two proteins counteracted each other, and it appears that the loss of Rab3D may increase the influence of Rab27 and contribute to the release of CTSS in patients with Sjögren’s syndrome. This study was published in the American Journal of Physiology—Cell Physiology.¹

References

1.  Meng Z, Edman MC, Hsueh PY, Chen CY, Klinngam W, Tolmachova T, Okamoto CT, Hamm-Alvarez SF. Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's syndrome. Am J Physiol Cell Physiol. April 13, 2016.

Maria C. Edman, PhD

e: edman@usc.edu

Sarah F. Hamm-Alvarez, PhD

e: sarah.hamm-alvarez@med.usc.edu

This article was adapted from a poster presented at the 2015 meeting of the Association for Research in Vision and Ophthalmology.