Spontaneous and Inducible Animal Models of Sjögren’s Syndrome

1.1.1

NOD Strain Origins and Characteristics

Makino and colleagues originally developed the NOD strain in Japan during the selection of a cataract-prone strain derived from the outbred Jcl:ICR ^c

c An inbred albino mouse strain originally distributed by the Institute of Cancer Research (ICR) in the USA and distributed by CLEA, Japan (Jcl).

line of mice. During the selection of this cataract-prone strain, the NOD strain was established, through repetitive brother–sister mating, as a subline that spontaneously developed diabetes. The incidence of spontaneous diabetes in the NOD mouse is 60% to 80% in females and 20% to 30% in males. Diabetes onset typically occurs at 12–14 weeks of age in female mice and slightly later in male mice. Histological studies have shown that few immune cell infiltrates are noted in islets until approximately 3–4 weeks of age, when both male and female mice begin to demonstrate mononuclear infiltrates that surround the islet (periinsulitis). These infiltrates progress and invade the islets (insulitis) over the subsequent few weeks, such that most mice demonstrate severe insulitis by 10 weeks of age. The finding that the reduced incidence in male mice occurs in spite of similar levels of early insulitis suggests that late regulatory events control disease progression. Thus the autoimmune process in the pancreas of NOD mice includes two checkpoints: checkpoint 1, or insulitis, which is completely penetrant; and checkpoint 2, or overt diabetes, which is not completely penetrant. Autoimmune sialadenitis in NOD mice has an incidence similar to insulitis, being more common in female as compared with male NOD mice. Conversely, dacryoadenitis develops more often in male mice. Initial immune cell infiltrates in NOD submandibular glands are observed around 10 weeks of age in females, several weeks after the development of the insulitis. Importantly, decrease in exocrine secretory function follows the inflammatory process and is evident normally after 16 weeks of age with reduced salivary flow. This feature is unique of NOD mice in comparison with other animal models of SS, in which autoimmune sialadenitis is not followed by reduction of exocrine function of the salivary glands. Thus NOD mice offer the possibility to investigate not only the mechanisms leading to breach of tolerance and autoimmunity, but also to understand the events that link chronic inflammation in the target organ to reduction of exocrine function. Interestingly, the incidence of disease is highest when mice are maintained in a relatively germ-free environment but dramatically decreases when mice are maintained in conventional “dirty” housing facilities. The basis for this effect is unclear, but it has been suggested that it reflects the fine tuning of the immune system that occurs during exposure to foreign proteins and protects the individual from allergy, autoimmunity, and other diseases of immune dysregulation. In addition to the classical NOD mice, a congenic NOD strain, the NOD.H2 ^h4 , has been developed and is characterized by almost complete protection from insulitis but a high incidence of sialadenitis. In keeping with the classical NOD strain, female, but not male, NOD.H2 ^h4 mice develop lymphocytic infiltration of the salivary glands and autoantibodies against Ro and La, although this process is generally delayed compared with NOD mice. Interestingly, the NOD.H2 ^h4 strain also develops iodine-induced autoimmune thyroiditis, another organ-specific autoimmune disease which is often associated with SS in humans.

1.1.2

NOD Genetics

Multiple loci control the genetic susceptibility to diabetes in this mouse. NOD mice harbor a unique major histocompatibility complex (MHC) haplotype, termed H-2g7, that is essential and is the highest genetic contributor for disease susceptibility. This MHC haplotype does not express an I-E molecule because of a defective Eα locus. Moreover, the unique I-A molecule contains a nonaspartic acid substitution at position 57 of the β chain that substantially alters the repertoire of MHC binding peptides presented by this allele. Strikingly, this substitution is also seen in human T1D MHC susceptibility loci in the DQ β chain. Several studies that examine the MHC requirement in NOD mice for the development of insulitis and diabetes conclude that homozygosity of the H-2g7 haplotype may be necessary for diabetes development and that dominant protection may be provided by some MHC manipulations, including introducing a functional I-E or non–I-Ag7 allele but not others. The major contributor to autoimmune diseases susceptibility is the MHC class II molecule itself. Its unique structure, its ability to bind an array of low affinity peptides, and its shared structural features in humans susceptible to autoimmune disease suggests that targeting this gene product, both in terms of genetic screening and potential therapy, remains a high priority. Finally, it should be noted that multiple genes are encoded within the MHC loci, many of which have been associated with immune functions. Possibly, the high susceptibility endowed by the H-2g7 MHC may be caused, in part, by polymorphisms in other genes, such as tumor necrosis factor (TNF)-α, encoded within this chromosomal segment.

In addition to the MHC locus, many other loci contribute to disease development and are termed insulin-dependent diabetes (Idd) loci. To date, over 20 potential Idd loci have been identified, but in most cases the exact structural or regulatory elements that lie within these loci still await identification. Interestingly, different loci have been associated with the development of diabetes and sialadenitis, with the Idd3 and Idd5 intervals, on chromosomes 3 and 1 respectively, being more strongly involved in conferring susceptibility to SS-like disease. This suggests that the autoimmune process in the pancreas and in the salivary glands of NOD mice occur independently. These two NOD genetic regions, designated Aec2 and Aec1, are necessary and sufficient to recapitulate SS-like disease in nonsusceptible C57 black 6 (C57BL/6) mice. Interestingly, a bioinformatics-based approach applied to the C57BL/6.NOD- Aec1Aec2 strain demonstrated that loci-dependent aberrations in innate immune responses affecting salivary gland homeostasis precede the onset of overt disease.

Researchers have discovered some clues about the nature of some of these genetic susceptibilities. In the case of the Idd5 locus (which may encode two regulatory elements), a unique polymorphism in the ctla-4 gene was determined that affects gene splicing. Interestingly, CTLA-4 is also a candidate gene in humans susceptible to a variety of autoimmune diseases, although the structural basis for CTLA-4 dysfunction is distinct. Candidate genes have been suggested in other Idd loci as well. VAV3 polymorphisms may account for Idd18, CD101 for Idd10, and the interleukin (IL)-2 or IL-21 genes for Idd3.

Overall, differently from other mouse strains in which autoimmunity is driven by single genetic defects (ie, MRL/lpr), NOD mice represent a more reliable model of the immune dysregulation that leads to human autoimmunity and chronic inflammation in the target organs. In particular, the existence of multiple susceptibility loci in the NOD strain again highlights the inherent complexity of the autoimmune process and supports the hypothesis that multiple tolerance networks are defective and interact in this strain. In fact, the spontaneous incidence of autoimmunity in the NOD mouse strain is likely to be a consequence of the absence of protective genes, as well.

1.1.3