34.1Introduction

SjšgrenÕs syndrome (SS) is an enigmatic disease of unknown etiology and elusive pathogenesis. The current understanding of the complex immune and non-immune mechanisms driving SS is covered in detail by Gottenberg and Meriette earlier in this textbook. Recognition that glandular dysfunction without overt inßammation in a signiÞcant number of patients has led to the hypothesis that glandular dryness may be caused by mechanisms independent of or in

N.P. Nikolov ( )

SjšgrenÕs Syndrome Clinic, National Institute of Dental and Craniofacial Research, Bethesda, MD, USA e-mail: nikolovn@mail.nih.gov

addition to immune-mediated glandular destruction, atrophy, or Þbrosis. Autonomic nervous system (ANS) dysfunction has emerged as one plausible mechanism underlying the sicca manifestations. In addition, advancements in our understanding of microRNAs (miRNAs) as gene expression regulators have highlighted the importance of exploring miRNAs as potential biomarkers and therapeutic targets for SS. The need for new and effective therapies for SS has led researchers to explore new approaches such as gene therapy, which could provide a local delivery mechanism of key molecules directly to the gland. Using targeted gene delivery in animal models may also be a powerful tool to investigate critical pathways in the pathogenesis of SS.

34.2Autonomic Neuropathy in Sjögren’s Syndrome

Current understanding about the etiopathogenesis of SS stems from the assumption that the immune-mediated destruction of the exocrine glands leads to their hypofunction and subsequently symptoms of dryness. However, multiple examples raise concerns with this view: (1) the degree of glandular inßammation or destruction correlates only poorly with the degree of dysfunction or symptoms of dryness; (2) many patients do not show evidence of systemic autoimmunity or inßammation; (3) traditional immunosuppressive and anti-rheumatic medications have not proven to be beneÞcial, unlike the symptomatic sialogogues, to the sicca manifestations. Thus, the existing evidence does not fully support this assumption and cannot explain the underlying pathogenic mechanisms of SS.

Exocrine glands are innervated by the autonomic nervous system (ANS), and, interestingly, dysautonomia can mimic many of the features of SS, particularly cardinal manifestations, such as xerostomia and xerophthalmia. The ANS regulates homeostasis via effects on the smooth muscles, glands, and cardiovascular system. Recent studies have focused on the fact that regulation of tear and salivary ßow involves an entire functional system that includes the mucosal surfaces, glands, afferent nerve signals sent to the midbrain (lacrimal and salivary response region), and efferent neural signals from the brain to the acinar/ductal epithelial structures in the gland. Saliva-secreting glands have both sympathetic and parasympathetic innervations with the latter being predominant. Postganglionic parasympathetic stimulation results in postsynaptic release of the neurotransmitter acetylcholine responsible for activation of G-protein-coupled muscarinic receptors on acinar cells leading to saliva secretion, while sympathetic glandular innervation is responsible for secretion of salivary proteins and glandular vascular responses.

Several systematic studies in patients with SS have found multiple clinical and subclinical abnormalities in parasympathetic and

sympathetic cholinergic, sympathetic noradrenergic, enteric, and adrenomedullary hormonal ANS components, underscoring the role of the autonomic neuropathy in the pathogenesis of the syndrome [1Ð3]. Identifying the patterns of abnormalities in the different ANS domains will help us to better understand the mechanisms involved in the initiation and tenacity of the disease. The importance of salivary gland innervation in the pathogenesis of SS is further supported by the positive results of a study utilizing a local salivary reßex (lingual nerve) electrostimulator to relieve severe xerostomia [4].

In addition, recent recognition of the Òcholinergic anti-inßammatory pathwayÓ [5] indicates the existence of a critical crosstalk between the immune and the nervous systems, supporting the need for an interdisciplinary approach to understand the mechanisms underlying dryness in SS.

34.3MicroRNAs in Sjögren’s Syndrome

Over the past decade, microRNAs (miRNAs) have become a ÒhotÓ area of interest in the research community to identify mechanism of disease, biomarkers, and therapeutic targets. MiRNAs are endogenous, small (approximately 22 nucleotides in length) non-coding RNAs that regulate gene expression posttranscriptionally [6]. MiRNAs have been shown to play important roles in numerous physiological as well as pathological processes. Their discovery dates to 1993 when lin-4, the Þrst described miRNA was shown to downregulate LIN-14 protein levels in

Caenorhabditis elegans. It was only in 2001 that the term miRNA was used in the concurrent publication of three articles [7Ð9]. The number of publications related to miRNAs has since been exponentially increasing and their role is being explored in both functional and biomarker discovery studies.

MiRNA biogenesis has been extensively studied [10]. In mammalian cells, the Þrst step involves the transcription of the pri-microRNAs by RNA polymerase II. Pri-microRNAs are

usually thousands of nucleotides (nt) long. The second step in biogenesis involves the processing of pri-miRNAs to pre-microRNAs in the nucleus by Drosha, an RNase III enzyme. The pre-microRNAs form a hairpin duplex structure and typically have a length of 70Ð100 nt. PremicroRNAs are subsequently actively transported from the nucleus to the cytoplasm where they are processed by Dicer, a cytoplasmic endonuclease, and other RNA-binding proteins into double-stranded RNAs, 19Ð24 nt long. Those double-stranded RNAs are then dissociated and get loaded to a ribonucleoprotein complex called RISC, so that they can exert their effect by binding on the target mRNAs and either degrading them or blocking their translation. The target selectivity of the miRNAs depends on the complementarities of their sequences with the sequences of the mRNA. Initially it was believed that miRNAÐmRNA binding occurs only on the 3 -UTR, but it has been shown since that binding can also occur on the 5 -UTR and the coding sequence of the mRNA [11].

Some of the speciÞc physiological functions that miRNAs have been implicated with include lymphocytic differentiation, embryonic stem cell fate, tissue morphogenesis, and organ development. Non-malignant pathologies in which miRNAs have been implicated include hepatitis infections [12], rheumatoid arthritis [13], neuropsychiatric disorders [14], and AlzheimerÕs disease [15]. Moreover, miRNA expression alterations have been identiÞed in almost all major malignancies [16Ð22].

One of the most important characteristics of miRNAs is that a single miRNA can regulate the expression of hundreds of mRNAs simultaneously, potentially exerting a much greater effect on a cellular phenotype than a single mRNA [11, 23]. This is the reason why miRNAs are also called master regulators of gene expression. For autoimmune diseases such as SjšgrenÕs syndrome, the functional importance of miRNAs is currently being explored. Their roles in SjšgrenÕs syndrome should be investigated in two major directions. First, miRNA alterations might be associated with the autoimmune characteristics of lymphocytes, i.e., increased immune activation

or loss of immune regulation. Second, miRNAs might be playing a role in the altered salivary gland function, especially in cases where inßammatory cells do not constitute the majority of the cells within the glands and there is functional salivary tissue remaining.

The use of miRNAs as biomarkers for diagnosis, prognosis, and disease activity in autoimmune diseases is currently unexplored. MiRNAs possess some characteristics that make them excellent candidates for biomarker studies. First, one miRNA can potentially capture a lot of information about the physiological status of a cell or tissue, since one miRNA controls the expression of a large number of genes. Clinically this translates into using a more limited number of miRNAs in biomarker assays reducing the complexity of measurements. Second, miRNAs can be isolated readily and reproducibly from easily obtained biological ßuids such as saliva [24], urine [25], and plasma [26] and from formalinÞxed parafÞn-embedded tissues [27]. The use of existing parafÞn-embedded samples with their associated clinical information can greatly accelerate biomarker discovery. Third, in complex diseases such as cancer, miRNAs have been shown to have the speciÞcity and sensitivity required for clinical applications. Fourth, miRNAs have a long half-life in vivo [23] and are remarkably stable and resistant to degradation by nucleases in vitro [28], thus allowing for detection in clinical specimens in which mRNA isolation is hindered by quick degradation.

In summary, miRNAs appear to be a promising tool to dissect critical mechanisms of disease, identify biomarkers for diagnosis, prognosis, and disease activity. MiRNAs also have the potential for identifying new therapeutic targets in SS and other disorders.

34.4Gene Therapy in Sjögren’s Syndrome

Current treatment strategies for SS are focused on symptomatic relief and the rationale for using available immunosuppressive and immunomodulatory therapies draws analogy from treatments

of other rheumatic diseases. This approach, however, has not been very productive and indicates the need for development of new therapies based on better understanding of the disease itself. Identifying appropriate therapeutic targets is an active area of research and one aspect of successfully targeting the identiÞed molecule(s) is the delivery method. Major salivary glands are considered a Òprime suspectÓ in the initiation and perpetuation of sicca manifestations and perhaps the extraglandular features of SS. Therefore, local delivery of key immunomodulatory proteins or other potential therapeutic molecules directly to the gland using gene therapy is a sensible treatment approach. Advantages of this approach include ease of access via retrograde cannulation of the entire salivary gland (Fig. 34.1). Depending on the choice of vector, both long-term and short-term expressions are possible and systemic complication of the drugs can be limited by treating only the salivary glands. Although several viral vectors tested in pre-clinical studies appear to be safe vehicles for gene transfer, further work will be required to develop vectors with improved transduction activity in both ductal and acinar cells with limited immune response. Incorporation of regulated expression systems and the addition of tissuespeciÞc promoters would further improve the safety proÞle of the vectors.

Despite the lack of correlation between local inÞltrate scores and salivary gland activity, several proof of concept studies with immunomodulatory proteins delivered by gene therapy in animal models have been completed and demonstrated the feasibility of this approach. One of the Þrst studies to test the local application of gene therapy to the salivary gland used a viral vector encoding the anti-inßammatory cytokine IL-10 [29]. IL-10 was administered locally to the salivary glands of NOD mice by retrograde cannulation of the salivary gland ducts or intramuscular injection in the quadriceps muscle using a recombinant adeno-associated viral type 2 (AAV2) vector. In contrast to the control or IM-treated groups, the mice treated with IL-10 locally in the salivary gland showed signiÞcantly higher salivary ßow and lower inÞltrate scores.

Similarly, positive results have also been observed using viral vectors encoding the neuropeptide vasoactive intestinal peptide (VIP) [30]. Although IL-10 has clear anti-inßammatory activity, it can promote B-cell proliferation, a concern in SjšgrenÕs patients in which 5% of the patients develop lymphomas. Similarly, while VIP did restore salivary gland activity following local expression in the salivary glands of mice, it did not reduce the focal inßammation associated with this model. These Þndings suggest that immunomodulation can have a signiÞcant role