Certain autoantigens have been shown to exhibit remarkable capabilities for modulating experimental autoimmunity (1). Heat-shock proteins (HSPs), which possess such properties, are ubiquitously expressed chaperones, and also have been proven to be highly immunogenic (2). Exceptionally conserved throughout evolution, molecular mimicry between HSPs of microbial and mammalian origin has been suggested as a factor in several autoimmune and inflammatory conditions, such as rheumatoid arthritis (RA) (3) and atherosclerosis (4). Hsp60 has also been identified as the crucial component in bacillus Calmette-Guérin–mediated inhibition of type 1 diabetes mellitus (DM) in NOD mice (5). As a consequence, a peptide vaccine (DiaPep277, amino acids [aa] 437–460), based on eukaryotic Hsp60, has been developed for the treatment of type 1 DM and is currently being tested in phase II clinical trials (6). Previous characterization of the immune response induced by aa 437–460 showed that it closely mimicked the effects of whole Hsp60 regarding prevention of type 1 DM, induction of T cell proliferation, and behavior in adaptive transfer experiments and other functional analyses, whereas use of other peptides from Hsp60 has resulted in outcomes very similar to those obtained by vaccination without peptide (7, 8).
Therapeutic and preventive effects, irrespective of the specific autoimmune condition studied, have been attributed to the capacity of Hsp60 to trigger antiinflammatory and regulatory mechanisms. Through a process dependent on Toll-like receptor 2 (TLR-2), Hsp60 and aa 437–460 have been shown to alter inflammatory chemotaxis and down-regulate T cell migration in vitro (8, 9). Treg cells seem to be innately responsive to Hsp60, and to be more effective in down-regulating CD4 and CD8 effector responses after Hsp60 and aa 437–460 engagement (10). In addition, Hsp60 may regulate Th1/Th2-related transcription factors and cytokines (11). Research efforts have also expanded the focus from self-Hsp60–specific T cells to antigen-presenting cells, which may directly interact with endogenous Hsp60 through identified and unidentified cell-specific surface receptors (12).
Sjögren's syndrome (SS) is a systemic autoimmune disease, which affects ∼0.3–0.6% of the total population and is manifested by severe impairment of exocrine gland function and focal mononuclear cell infiltrates within the salivary and lacrimal glands (13, 14). Treatments used today provide merely marginal symptomatic relief (15). Previous studies have shown that anti–type 3 muscarinic acetylcholine receptor (anti-M3R) autoantibodies may potentially interfere with acinar cell innervation, assigning for the first time a defined pathogenetic role to an autoantibody in SS (16). The disease can involve organs other than the exocrine glands, and up to 5% of patients develop lymphoid malignancies. As in other rheumatic diseases, anti-Hsp60 antibodies have been found to be elevated in patients with SS (17). Nevertheless, HSPs (18) and regulatory mechanisms, such as Treg cells (19), have not been extensively investigated in SS. Importantly, however, it seems that the protective effects of HSPs are independent of their antigenic relationship with the (unfortunately often unknown) disease-causing antigen (20).
The NOD mouse is the best-characterized model of SS (21–23). It spontaneously manifests SS-like histopathologic features and hyposalivation, following a specific time course for the onset of the different SS-related disease manifestations (24). Although some genetic loci related to diabetes have been found to contribute to inflammatory changes in the exocrine glands, diabetes and SS-like disease can develop independently of each other (25). Most knowledge regarding immunostimulatory interventions in type 1 DM has been accumulated through study of the NOD strain (1). Whether potential therapeutic agents may, however, modify SS disease manifestations has not yet been addressed. Nevertheless, some SS-related findings were described in a previous report characterizing NOD mice transgenic for Hsp60 (26). Despite abundant thymic expression of Hsp60, resulting in diminished susceptibility to type 1 DM, salivary gland inflammation was found to be aggravated, and interestingly, T cell responses to Hsp60 were not abolished.
The aim of the present study was to investigate the immunomodulatory potential of Hsp60 and its peptide aa 437–460 in SS-like disease in nondiabetic NOD mice. In addition, comprehensive biomarker profiles were analyzed for systemic and local alterations related to the treatment received and treatment efficacy in preventing the onset of hyposalivation.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
The present report describes the first preventive antigen-specific intervention to successfully target experimental spontaneous SS. Although the antigenic relationships between Hsp60 and antigens specifically related to SS are unknown, our results indicate that immune responses to Hsp60 are relevant in the control of pathogenic autoimmunity. Consistent with the notion that Hsp60 and aa 437–460 treatment can alter chemotaxis of T cells in vitro (8, 9), decreased inflammation was associated with lower serum levels of chemoattractants for Th1 (IP-10) and Th2 cells (eotaxin) (30), even 14 weeks after immunization. In addition, levels of MCP-1 and MCP-3, to which monocytes and plasmacytoid dendritic cells are most responsive (30), were also reduced in serum after treatment.
Disease severity in RA, and more recently in juvenile idiopathic arthritis, has been shown to be related to the extent of Hsp60-induced propagation and activation of Treg cells (31, 32). It has been suggested that IL-10, secreted upon Hsp60 signaling through TLR-2, is a key molecule involved in this process (10). Our findings do not support the notion of increased IL-10 secretion by Treg cells in favor of immunosuppression. However, such alterations may be detectable only in specific microenvironments. Nevertheless, it should be noted that systemic overexpression of IL-10 has been shown to induce SS in mice (33). In contrast, in the present study, IL-2, which is crucial in maintaining immunologic self tolerance by promoting growth and function of Treg cells (34), was increased in saliva upon treatment with Hsp60 or aa 437–460.
Compared with IL-2, leptin exerts a reciprocal effect on effector and Treg cell populations (35). The observed modulation of leptin as a consequence of treatment therefore further supports the idea of strengthened regulatory pathways. Indeed, induction of regulatory processes might be the key to controlling pathogenic autoimmunity while avoiding long-term immunosuppression. In addition to Treg cells, IL-17 cells have received considerable attention in the study of autoimmune diseases (36). Nevertheless, our treatment regimens were not accompanied by detectable alterations in IL-17 in saliva or in serum (data not shown). In our study, induction of anti-M3R IgG1 production was strongly related to immunization with Hsp60. However, although IgG1 has previously been reported to be the anti-M3R isotype that can mediate hyposalivation (37), in our study the induction of the anti-M3R IgG1 isotype, generally considered to be an antiinflammatory isotype (38), did not seem to be related to manifestation of or protection against hyposalivation.
Prevention of hyposalivation, the other hallmark of SS, was achieved in a substantial number of mice for the duration of the experiment. Further studies are needed to determine whether NOD mice can be permanently protected against development of hyposalivation and whether such treatments might also be able to induce remission of SS. Refinement of treatment regimens may further increase the percentage of mice protected against hyposalivation, as has been shown recently in studies of diabetes using tandem repeats of aa 437–460 (39).
Interestingly, effective protection against hyposalivation was associated almost exclusively with changes in the quality of inflammation reflected by the multianalyte profiles. At the same time, we found hyposalivation to be unrelated to the quantitative degree of glandular inflammation. With focus on immune mediators, multianalyte profiles generated an overview, although it was not all-embracing, of the serum and salivary proteome. However, unlike in gene expression profiling, we could exclude distorting effects, such as RNA stability and poor correlation between messenger RNA and protein levels (40), from our analyses. Hu et al (40) used mass spectrometry to compare the salivary proteome from SS patients versus healthy controls. Not unexpectedly, of the 42 proteins reported in that study, none was included in our multianalyte profile, which shows the differential focus and strengths of the 2 methods (41, 42).
We identified low levels of VEGF-A, a key molecule mediating vascularization, as a primary predictor of prevention of hyposalivation, followed by low levels of M-CSF, which, like TPO and OSM, is an inducer of VEGF-A (43). Other molecules implicated in the process of neovascularization that were decreased in saliva in treated mice in which hyposalivation was prevented were MMP-9 and FGF-9 (44), as well as all CXCR2 ligands measured (granulocyte chemotactic protein 2, melanoma growth-stimulatory activity protein, and MIP-2) (30). These results support the notion of a strong interrelationship between pathogenic neovascularization and impaired secretory function. The importance of neovascularization in RA is recognized, and it has been explored as a target for therapeutic interventions (45). Unfortunately, the issue of pathogenic neovascularization in SS has not yet been addressed.
The salivary multianalyte profile associated with retained salivary flow in NOD mice resulted from the decrease and normalization of multiple chemokine levels. Such modulation was potentially promoted by the simultaneous decrease in levels of TNFα and IL-7, both key antagonists of Treg cell–induced immunoregulation (46). Down-regulation of CD40, IL-10, and IL-11 (47) may further suggest that Th2-associated responses were modulated by treatment. An effect of Hsp60 on B cells through innate signaling pathways has been reported (48) and may also contribute to the modulation of autoimmune diseases with a confirmed or potential B cell aspect. Nevertheless, in our study the levels of most autoantibodies remained unaffected. Similarly, we did not identify significant alterations in extraglandular disease manifestation as a consequence of the 2 treatments investigated. The development of malignancy in one mouse remains difficult to interpret, since lymphomas develop rather frequently in aged NOD mice (49). Nevertheless, antiinflammatory treatment should be critically reviewed regarding alterations induced, which might critically affect antitumor-related or host-defense–related immunity.
One other encouraging aspect of our results regards the capacity of granulocyte chemotactic protein 2, which, individually or combined with 4 other analytes, accurately predicted impaired salivary flow irrespective of treatment group and mouse strain. Validation of a biomarker-based diagnostic criterion in humans would indeed represent a significant advance in SS diagnosis and allow close followup of disease progression and the effects of therapeutic interventions.
In conclusion, our results indicated that Hsp60 vaccination may have a preventive effect in experimental SS. In addition, we found that the Hsp60 peptide aa 437–460 had, in general, the same beneficial effects as did whole Hsp60. Biomarker profiles indicated that down-regulation of inflammatory chemotaxis in parallel with strengthened regulatory and antiinflammatory mechanisms were major consequences of the treatment. Successful prevention of hyposalivation was related to a significant decrease in mediators of inflammation related to pathologic neovascularization, inflammatory chemotaxis, and cell activation. The multianalyte profile was also considerably successful in predicting treatment effectiveness. These biomarkers, in addition to the analytes indicative of normal and impaired salivary flow across the strains, should be explored further regarding their diagnostic significance in humans. In addition, the processes we identified to be involved in the onset of hyposalivation should be considered in the development of new therapeutic strategies.