Findings provide foundation for the development of novel therapies
Sjögren’s syndrome is an autoimmune disease in which the immune system mistakenly attacks the glands that produce tears and saliva. One of the most common autoimmune disorders, Sjögren’s is thought to affect 1 to 4 million people in the United States, most of them women older than 40.
The most common symptoms of Sjögren’s are dry, itchy eyes, and dry mouth, which can make it difficult to speak and swallow. People with the syndrome can develop visual problems like sensitivity to light and blurry vision, and are at increased risk of tooth decay and infections in the mouth. Sjögren’s is often accompanied by other autoimmune diseases such as rheumatoid arthritis or lupus, but it can occur on its own as well.
The causes of Sjögren’s have been somewhat elusive, but it is thought to involve both environmental and genetic factors. For example, an infection may trigger the syndrome in people who are genetically predisposed. While several viruses have been associated with Sjögren’s, their roles in the syndrome have been unclear. Now, two new studies by researchers at the NIH National Institute of Dental and Craniofacial Research (NIDCR), provide insight into the roles played by two viruses. Their findings were published in the journals, EBioMedicine and Pathogens and Immunity.
Epstein-Barr virus: targeting key salivary gland genes
Epstein-Barr virus (EBV) is a widespread virus that infects most people in childhood or early adulthood. Most individuals recover quickly from an EBV infection, but in some cases it causes infectious mononucleosis. Following infection, the virus persists in the body in a dormant form. EBV has been linked to Sjögren’s, as well as other autoimmune diseases.
Previous studies revealed the presence of a type of small RNA molecule called a microRNA from EBV in salivary glands of Sjögren’s patients. The EBV microRNA, ebv-miR-BART13-3p, had been found at more than 20 times higher levels in Sjögren’s patients’ salivary glands compared to healthy controls, suggesting a possible role in the glands’ malfunction.
MicroRNAs occur in plants, animals and some viruses, and they act to quell the activities of specific genes. Ilias Alevizos, D.M.D., M.M.Sc., of the Sjögren’s Syndrome Clinic at NIDCR, wondered if ebv-miR-BART13-3p might be inactivating certain genes necessary for the salivary glands to function properly.
To find out, his team examined the RNA sequences of genes known to be involved in salivary gland function. MicroRNAs inactivate specific genes by binding to complementary, or matching, genetic sequences, interacting much like a lock and a key. By scanning genes for this type of complementarity with ebv-miR-BART13-3p, the researchers identified matches in two genes—STIM1 and AQP5. When they introduced ebv-miR-BART13-3p into cultured salivary gland cells, STIM1 and AQP5 levels plummeted, confirming the microRNA’s inhibitory effect.
AQP5 and STIM1 encode proteins that play important functions in acinar cells, the fluid-secreting cells of salivary glands. The AQP5 protein forms a channel that enables water to flow from inside the cells toward the collecting ducts. The STIM1 protein acts as a cellular calcium ion sensor, stimulating calcium entry into the cells when needed. Calcium entry triggers the flux of fluid toward the ducts and mouth, a key step in saliva production. Earlier work had revealed low levels of the STIM1 protein in salivary glands of Sjögren’s patients, but the cause of the depletion was unknown.
The researchers next analyzed biopsies of salivary gland tissue from Sjögren’s patients and compared them to those from healthy controls. They found that acinar cells with high levels of ebv-miR-BART13-3p had lower levels of STIM1, and conversely, cells with low or no ebv-miR-BART13-3p had higher levels of STIM1. This result further implicated the microRNA in tamping down the activity of the STIM1 gene, and provided a mechanism for the low STIM1 levels in Sjögren’s patients’ salivary glands that scientists had previously seen.
By showing that ebv-miR-BART13-3p can inhibit the activity of key genes in acinar cells, the findings reveal a possible new mechanism for salivary gland malfunction in Sjögren’s.
“The prevailing view has been that tissue damage in Sjögren’s arises from longstanding inflammation brought on by autoimmune attack, but there is little correlation between inflammation and salivary gland function in patients,” said Dr. Alevizos. “Our work, by showing that EBV’s ebv-miR-BART13-3p disrupts the STIM1 calcium sensing pathway, suggests a novel disease mechanism and new therapeutic strategies for combating the syndrome.”
Hepatitis delta virus: an unexpected role for a virus known to infect the liver
In the second study, Melodie L. Weller, Ph.D., a postdoctoral researcher in the lab of John A. Chiorini, Ph.D., chief of the Adeno-Associated Virus Biology Section at NIDCR, uncovered evidence of Hepatitis delta virus (HDV) in salivary glands of people with Sjögren’s syndrome. HDV is known to infect the liver, but Dr. Weller and her colleagues found low levels of replicating virus in the salivary glands of 50 percent of the Sjögren’s patients she tested.
Intrigued by HDV’s unexpected presence in patients’ salivary glands, the researchers went on to investigate a possible role for the virus in the disease. When they engineered mice to produce HDV proteins in their salivary glands, they found that the mice produced autoantibodies, antibodies that attack the mice’s own proteins, indicating the development of autoimmunity. Salivary gland tissue from these mice also showed signs of accumulation of immune cells called lymphocytes, a hallmark of Sjögren’s.
The researchers next engineered mice to produce an HDV protein abundant early in the infection cycle, mimicking an early-stage infection. These mice generated significantly less saliva than control mice, suggesting that the early stages of HDV infection have the potential to impact the flow of saliva.
The discovery of the presence of HDV in a substantial fraction of Sjögren’s patients and the finding that HDV proteins can trigger signs of the disease suggest a causal relationship between the virus and the disease. In future work, Dr. Weller aims to investigate how patients acquire HDV, and to delve into how the virus affects salivary gland function.
“We’re trying to understand how HDV works in salivary glands and what its weak points are so that we can target the virus with therapeutics,” said Dr. Weller. “In addition, we want to understand how patients become exposed to the virus because that knowledge may have a major impact on treatment as well.”
Both studies were funded by the NIDCR Intramural Research Program (Alevizos: 1ZIADE000733; Chiorini/Weller: 1ZIADE000695 and K99-DE21745).
Targeting the Ca2+ Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is Associated with Sjögren's Syndrome.Gallo A, Jang SI, Ong HL, Perez P, Tandon M, Ambudkar I, Illei G, Alevizos I. EBioMedicine. 2016 Jun 29. pii: S2352-3964(16)30298-5. doi: 10.1016/j.ebiom.2016.06.041. PMID: 27381477
Hepatitis Delta Virus Detected in Salivary Glands of Sjögren's Syndrome Patients and Recapitulates a Sjögren's Syndrome-Like Phenotype in Vivo. Weller ML, Gardener MR, Bogus ZC, Smith MA, Astorri E, Michael DG, Michael DA, Zheng C, Burbelo PD, Lai Z, Wilson PA, Swaim W, Handelman B, Afione SA, Bombardieri M, Chiorini JA. Pathog Immun. 2016 May;1(1):12-40. PMID: 27294212