Abstract
ObjectiveThe tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren’s syndrome (SS), was analyzed to identify unique miRNAs.MethodsMale NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated in additional mouse cohorts as well as in tears of SS patients versus patients with another form of dry eye disease, meibomian gland disease (MGD) using qRT-PCR. The pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis.ResultsIn comparison to tears from both healthy (male BALB/c) and additional control (female NOD) mice, initial analy1sis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS. When compared to tears from patients with MGD, miRNAs hsa-miR-203a-3p, hsa-miR-181a-5p and hsa-miR-181b-5p were also significantly increased in tears of SS patients.ConclusionsA panel of differentially expressed miRNAs were identified in tears of male NOD mice, with some preliminary validation in SS patients, including some never previously linked to SS. These may have potential utility as indicators of ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis.
Highlights
Sjögren’s Syndrome (SS) is a chronic, progressive autoimmune disease that affects ~1% of the population [1] and causes inflammation of moisture-producing glands including lacrimal glands (LG) and salivary glands (SG), leading to dry eye and dry mouth [1]
An ideal diagnostic tear biomarker for SS would be chemically stable and able to: 1) detect SS with high sensitivity and specificity [11]; 2) distinguish SS from other autoimmune and dry eye diseases; 3) be collected relatively non-invasively [11]; and 4) be processed inexpensively in a straightforward manner [11, 12]. With these characteristics in mind, we investigated a type of short non-coding RNAs called microRNA that are 18-26 nucleotides long. miRNAs circulate in the body either packaged inside secreted vesicles or bound to RNA-binding proteins and are highly stable and protected from RNAse degradation. miRNAs are responsible for transcriptional regulation of nearly 60% of all mammalian messenger RNA [13], and dysregulation of miRNAs has been implicated in diseases such as cancer and neurodegenerative disease [14, 15]
To determine the percentage of lymphocytic infiltration in mouse LG, H&E-stained images of LG sections from the mice used for the initial tear collection were acquired and analyzed
Summary
Sjögren’s Syndrome (SS) is a chronic, progressive autoimmune disease that affects ~1% of the population [1] and causes inflammation of moisture-producing glands including lacrimal glands (LG) and salivary glands (SG), leading to dry eye and dry mouth [1]. Symptoms of SS overlap with those of other autoimmune diseases such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE) and other dry eye diseases such as Meibomian gland disease (MGD), leading to delays in diagnosis or misdiagnosis [7]. It can take several years before the disease is confirmed, during which time infiltrating immune cells may further damage exocrine glands and sustain debilitating ocular and oral cavity symptoms. There is a need for more specific diagnostic tests for SS to prevent irreversible tissue and/or organ damage and to improve the health and vision-related quality of life for SS patients
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