Identification of possible candidate genes regulating Sjögren's syndrome-associated autoimmunity: a potential role for TNFSF4in autoimmune exocrinopathy

Sjögren syndrome (SjS) is a chronic, systemic, human autoimmune disease in which an immunological attack initially against the salivary and lacrimal glands results, respectively, in dry mouth (stomatitis sicca) and dry eye (keratoconjunctivitis sicca) disease(s) [1‐3]. Despite efforts to define the genetic, environmental, and immunological bases of SjS, the underlying etiology of this disease remains ill defined. In attempts to better define the nature of SjS autoimmunity, a variety of mouse models exhibiting various aspects of SjS have been studied extensively [4]. One of the more intensively studied models of SjS is the nonobese diabetic (NOD) mouse [5‐9]. Based on disease profiling of various congenic partners and gene knockout lines of NOD, we have proposed that the development and onset of SjS-like disease in these mice can be divided into at least three distinct consecutive phases [10‐19]. In phase 1, a number of aberrant physiological and biochemical activities, thought to result from a genetically based retarded salivary gland organogenesis and increased acinar cell apoptosis, occur prior to and independent of detectable autoimmunity. In phase 2, believed to result from the glandular cell injury of phase 1, small numbers of macrophages and dendritic cells are attracted to the exocrine gland where these sentinel cells recruit T and B lymphocytes that form lymphocytic foci (LF), some of which histologically appear as germinal centers. In phase 3, the onset of clinical disease as defined by salivary and lacrimal gland secretory dysfunction occurs, possibly resulting first from the production of autoantibodies that interfere with the neural-acinar cell signaling pathways and then from progressive loss of acinar cell mass hastened by the action of effector T cells.

A genetic predisposition for development and onset of SjS-like disease in NOD mice has also been defined. First, SjS-like disease in these mice appears independent of or only weakly associated with major histocompatibility complex (MHC) class I and class II genes [10, 20], thus mimicking SjS in humans. This can be seen by the fact that the congenic strain, NOD.B10-H2 ^b , in which the NOD MHC I-A^g7Idd1 diabetes susceptibility locus was replaced by the MHC I-A ^b locus [20], continued to show SjS-like disease, including salivary and lacrimal gland dysfunction. Second, replacing Idd loci other than Idd1 (for example, Idd9, Idd10, and Idd13) resulted in the identification of Idd3 on chromosome 3 and Idd5 on chromosome 1 as critical genetic regions for development of SjS-like disease in NOD mice [10]. In a reverse approach, introducing both Idd3 and Idd5 derived from NOD mice into SjS-nonsusceptible C57BL/6 mice resulted in a severe SjS-like disease, confirming the contributions of these two genetic loci to the development and onset of SjS [21]. Furthermore, the preclinical nonimmune aspects manifested in phase 1 of the disease appeared to associate with the Idd5 locus (referred to as autoimmune exocrinopathy 2 or Aec2), whereas the immunological aspects of the disease manifested in phases 2 and 3 of the disease appeared to associate with Idd3 (referred to as Aec1). This recently generated mouse strain is referred to as C57BL/6.NOD-Aec1Aec2. While the pathophysiological and immunological aspects may not be linked solely to one or the other genetic region (as originally proposed [22]), the complete disease profile requires genes within both of these genetic loci.

For years, identification of candidate genes associated with autoimmune diseases such as T1D [23] or systemic lupus erythematosus [24] in animal models has been providing invaluable data on delineating the genetic components of these diseases, now translating to the human disease. These studies have formed a template for our current efforts to identify the SjS susceptibility loci and candidate genes underlying SjS which, in this respect, have lagged behind many other autoimmune diseases. Although our initial work defined the Aec1 and Aec2 genetic regions present in C57BL/6.NOD-Aec1Aec2 mice as being an approximately 48.5-cM centromeric region on chromosome 3 and an approximately 73.3-cM telomeric region on chromosome 1, respectively [10], the size of these regions precluded identification of candidate genes. Subsequently, we shortened Aec1 to an approximately 19.2-cM region in the first studied recombinant inbred (RI) line, C57BL/6.NOD-Aec1R01Aec2 [9]. For the present study, we generated a set of new RI lines that further demarcate the boundaries of Aec2. These new C57BL/6.NOD-Aec1Aec2R(n) RI lines identify not only a much shorter Aec2 sublocus at position 79 cM of chromosome 1, but also potential candidate SjS susceptibility genes on which to build hypothetical models that can be tested for validating possible pathogenic molecular mechanisms of SjS-like disease.

In the present study, in which the specific goal was to redefine (and narrow) the boundaries of the Aec2 genetic region on chromosome 1 known to predispose NOD and NOD-derived lines of mice to SjS, we generated a large set of new RI lines (n = 39) and examined each line for its SjS-like disease profile. Disease profiles obtained with the C57BL/6.NOD-Aec1Aec2R(n) RI lines indicate that the Aec2 genetic region of C57BL/6.NOD-Aec1Aec2 mice, postulated to regulate primarily the pathophysiological and biochemical abnormalities that subsequently result in the activation of the autoimmune attack against the submandibular and lacrimal glands [10], is a single subregion mapping to the telomeric portion of chromosome 1 located at approximately 79 ± 5 cM. However, penetrance and severity of SjS-like disease may be further influenced by genes located within a few centimorgans on the centromeric side of this region, possibly pointing to SjS-associated quantitative trait loci (QTL) genes. Although the size of the redefined Aec2 region remains relatively large for identification of individual candidate SjS susceptibility genes, the genes residing within this subregion can be grouped into four functionally clustered sets, each suspected previously of involvement in SjS susceptibility. These are (a) endogenous viruses and oncogenic genes, (b) Fas/FasL-associated apoptosis, (c) T_H17-associated activities, and (d) fatty acid, lipid, lipoprotein, and cholesterol homeostasis. However, perhaps the most obvious aspect is the fact that this redefined Aec2 region contains the QTL-Ath1 region containing some 10 genes, including tumor necrosis factor ligand superfamily member 4 (Tnfsf4 or Ox40L) and Tnfsf6 (Fasl).

Within the first set, several viral/oncogenic genes, such as Emv38 (endogenous ecotropic MuLV-38), Kras-2-rs1 (Kirsten rat sarcoma oncogene-2, related sequence-1), Xpr1 (xenotropic/polytropic retrovirus receptor-1), and Abl2 (Abelson murine leukemia viral oncogene-2), are found in this redefined Aec2 subregion. In our earlier studies with NOD mice [12], we observed that high levels of interferon-gamma (INF-γ) were present in the salivary glands of neonate mice, suggesting an important role for INF-γ in the delayed development/proliferation of acinar tissue observed in the salivary glands of neonate NOD mice. While it is logical to conclude that induction of INF-γ may be a result of short-term viral infection during the preterm and early postpartum periods, what might cause a viral outbreak at this time point remains unknown. It could be hypothesized that this occurs due to the changes in maternal hormone levels at this time. Perhaps more interesting, however, this region contains the gene Tnfsf6 encoding the proapoptotic protein FasL. FasL has numerous functions but is mainly involved in regulating immune responses, apoptosis, and retinal cell programmed death [4]. During the early phase 1 period of SjS-like disease in NOD mice, both FasL and Fas are upregulated at both the gene and protein levels, and this increased expression of Fas/FasL corresponds to the observed increase in acinar cell apoptosis within the glands [32]. However, it remains speculative whether there might be an association between endogenous/exogenous viral infection and Fas/FasL activity in the salivary and lacrimal glands.

The redefined Aec2 subregion also contains several genes involved in autoimmunity and/or tumorgenesis, the latter being one clinical manifestation of SjS that occurs in a small subset of patients. Of interest, but not thought to be directly involved in the development and onset of SjS, is the presence of genes specific to the ocular/lacrimal gland etiology (for example, Pdc [phosducin], which is a protein of the retinal photoreceptors cells [33], and Myoc [myocilin], whose product interacts with olfactemedin involved in glaucoma [34]). However, whether any of these genes are related to SjS susceptibility and lacrimal gland disease or merely influence the secondary disease phenotypes often associated with SjS remains unknown. In contrast, one genetic element in this region that has a direct association with the immunopathology of SjS is the QTL gene Cypr2 (cytokine production 2) [35]. CYPR-2 is known to regulate levels of interleukin-10 (IL-10), an important cytokine that enhances the activity of B lymphocytes, and at the same time to regulate the functions of T_H1 and T_H17 cells [36]. Our recent microarray studies indicate that Il10 is not upregulated during the development of SjS in the C57BL/6.NOD-Aec1Aec2 mouse model [32], possibly indicating a lack of immune regulation by regulatory T (T_reg) cells. If so, this lack of regulation by IL-10 would be consistent with the results of gene therapy studies in which injections of vectors expressing recombinant IL-10 reduced or suppressed clinical manifestations of SjS-like disease in both salivary and lacrimal glands of mice [37, 38].

Maintaining sufficient regulation of immune responses to prevent development of an overt autoimmunity is no doubt dependent on a physiological balance between T_H1, T_H2, T_H17, and T_reg cell interactions. This cellular interaction appears to be highly influenced by OX40L encoded by the Tnfsf4 gene and this gene is located within the redefined Aec2 region. OX40L is expressed by a number of distinct cell populations, including activated dendritic cells [39]. OX40L is capable of functioning as an inhibitor of the maturation of T_reg1 cells [40], a regulatory cell population normally producing IL-10 and INF-γ, which (in conjunction with IL-27) can inhibit the effector CD4⁺ T_H17 cells [41]. Reduced T_reg1 cell function, therefore, results in a positive feedback for the generation/activation of effector CD4⁺ T_H17 cells (Figure 7). These effector T_H17 cells produce predominantly IL-17, IL-21, and IL-22 plus factors like nitric oxide, matrix metalloproteinase, and prostaglandin E₂, each of which is shown to play an important role in the immunopathophysiology of several autoimmune diseases, including SjS [41]. Thus, we hypothesize that the presence of T_H17 cells in the salivary and lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice, as well as SjS patients [42], indicates an imbalance in the T_H17/T_reg1 ratio favoring the T_H17 population(s). A recent study indicates that retinoic acid can facilitate an increase in the numbers of Foxp3⁺ T_reg cells and simultenously inhibit the formation of effector T_H17 cells [43]. Interestingly, we have found that expression of the retinoic receptors, Rxr (retinoid × receptor) and Rar (retinoic acid receptor), is downregulated in the lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice [44]. This observation is again consistent with a potential problem in cellular homeostasis, especially at the level of macrophages, dendritic cells, and even production of the FOXP3⁺ T_reg cell populations whose differentiation and functional maturation are highly dependent on retinoic and fatty acid stimulation. As presented in Figure 7, there is a reciprocal maturation of effector CD4⁺ T_H17 and FOXP3⁺ T_reg cells dependent on the relative balance of IL-6 and transforming growth factor-beta influenced by retinoic acid. Thus, the intricacy and balance between OX40L, the retinoids, proinflammatory cytokines, and development of T_reg cells appear to impact the potential development and onset of autoimmunity, which in SjS appears to favor activation of effector T_H17 cells.

Although several factors encoded by genes in the redefined Aec2 region may be involved in secondary manifestations of SjS, OX40L is the one factor that clearly stands out as a primary candidate gene underlying not only the recognized immune dysregulation, as presented above, but also the pathophysiological aberrations associated with chromosome 1 of the C57BL/6.NOD-Aec1Aec2 SjS model. In this redefined region, a common functional cluster of lipid, lipoprotein, cholesterol, and fatty acid regulatory and processing elements is found, including Hdlq14 (high-density lipoprotein QLT-14), Hdlq5 or Apoa2 (high-density lipoprotein QTL-5), Gpa33 (glycoprotein A33), Cq1 (cholesterol QTL-1), Prdx6 (peroxiredoxin), and (of special note) Soat1 (sterol O-acyltransferase-1). Involvement of lipids and fatty acids in the pathology of SjS has become a major focus of SjS research as lipid depositions [45] and changes in lipid rafts [46] appear to influence the pathology in both salivary and lacrimal glands. Furthermore, our recent genomic microarray studies – [44, 47] (C.Q. Nguyen, S. Ashok, R.A McIndoe, J.X. She, B.H. Lee, A.B. Peck, unpublished data) – indicate that multiple genes involved in fatty acid, lipid, lipoprotein, and cholesterol homeostasis/transport are differentially expressed, corresponding with lipid deposits, dysfunctional dendritic cells, and onset of autoimmunity. As illustrated in Figure 8, various relationships between genes controlling free fatty acid, lipid, and lipoprotein homeostasis are directly or indirectly dependent on the activities of OX40L. As a consequence, imbalances in this homeostasis regulated in part by OX40/OX40L can result in widespread pathology, including inflammation and cell death. Based on differential gene expression data, there are major reductions in the levels of transcripts encoding FDFT-1 (farnesyl diphosphate farnesyl transferase-1), ABCA1 (ATP-binding cassette, subfamily A [ABC1] member 1), and the retinoic acid receptors, RRX and RAR. At the same time, increased levels of transcripts encoding the low- and high-density lipoprotein receptors, as well as SOAT-1, are observed. We hypothesize, therefore, that an imbalance occurs in the production of cholesterol and the increased level of cholesterol results in greater amounts being converted by SOAT-1 to cholesteryl esters that accumulate within the cells due to the downregulation or dysfunction of the lipid transporter. In addition, the functional activities of cells whose differentiation and maturation are dependent on the retinoids and the RXR/RAR-PPARγ (RXR/RAR-peroxisome proliferator activated receptor-gamma) signaling pathways (for example, macrophages and dendritic and FOXP3⁺ T cells) will be altered due to altered development, ultimately affecting antigen presentation and balanced production of regulatory cytokines.

Identifying gene products that are differentially expressed in the Aec2 SjS susceptibility subregion defined by the new RI lines is moving us closer to identifying specific candidate genes involved in the onset and development of SjS-like disease. Based on our current data, our focus is turning to Ox40L as an effective candidate gene for the development of SjS. The future application of genetic knockout mice and/or short interfering RNA will permit us to further our understanding of the potential role of Ox40L in SjS and, more importantly, to translate its relevancy to human SjS.