Edited by: Falk Nimmerjahn, University of Erlangen-Nuremberg, Germany
Reviewed by: Abdelhadi Saoudi, Institut national de la santé et de la recherche médicale, France; Sylvie Lesage, Université de Montréal, Canada
†These authors have contributed equally to this work.
Specialty section: This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology
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Primary Sjögren’s syndrome (pSS) is characterized by a panel of autoantibodies, while it is not clear whether B cells and autoantibodies play an essential role in pathogenesis of the disease. Here, we report a novel mouse model for pSS which is induced by immunization with the Ro60_316-335 peptide containing a predominant T cell epitope. After immunization, mice developed several symptoms mimicking pSS, including a decreased secretion of tears, lymphocytic infiltration into the lacrimal glands, autoantibodies, and increased levels of inflammatory cytokines. Disease susceptibility to this novel mouse model varies among strains, where C3H/HeJ (H2-k) and C3H/HeN (H2-k) are susceptible while DBA/1 (H2-q) and C57BL/6 (H2-b) are resistant. Depletion of B cells using anti-CD20 monoclonal antibodies prevented C3H/HeN mice from development of the pSS-like disease. In addition, HLA-DRB1*0803, a pSS risk allele, was predicted to bind to the hRo60_308-328 which contains a predominant T cell epitope of human Ro60. Therefore, this study provides a novel mouse model for pSS and reveals an indispensable role of B cells in this model. Moreover, it suggests that T cell epitope within Ro60 antigen is potentially pathogenic for pSS.
Primary Sjögren’s syndrome (pSS) is an autoimmune disorder mainly targeting salivary and lacrimal glands and leading to xerostomia (dry mouth) and xerophthalmia (dry eye) (
Animal models provide a powerful tool for understanding the pathogenesis of disease. So far, several mouse models for pSS have been established and they provide some evidence for the role of autoantibodies in the disease pathogenesis (
In 2013, Jonsson et al. reported that the presymptomatic presence of anti-SSA/Ro autoantibodies shows the highest odds ratio for the risk of development of pSS, followed by anti-SSB/La autoantibodies and ANA (
In this study, we hypothesized that autoimmune response to the T cell epitope of SSA/Ro antigen contribute to the pathogenesis of pSS by producing pathogenic autoantibodies
All mice used in this study were female. C3H/HeJ, DBA/1J, and C57BL/6J mice were purchased from Shanghai SLAC laboratory Animal Co. (Shanghai, China), while C3H/HeN mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed in the animal facility with a 12-h light–dark cycle at the Xiamen University. Mice were held at specific pathogen-free conditions and fed standard mouse chow and acidified drinking water
Murine Ro60_316-335 (KARIHPFHVLIALETYRAGH) peptides used in this study were synthesized at Research Center Borstel, Germany. Mice of the age from 8 to 10 weeks were immunized at the hind footpad with 100 µg peptide emulsified (1:1) in nonionic block copolymer adjuvant Titermax (Alexis Biochemicals, Lorrach, Germany). Control mice were treated with PBS emulsified in Titermax. The mice were followed until 12 weeks after immunization.
Mouse saliva and tears were measured at week 0, 6, and 12 after immunization as described previously (
Histology of exocrine glands or kidney was evaluated by Hematoxylin and Eosin (H&E) staining with 6-μm-thick sections prepared from formalin-fixed tissues. Immunochemistry in exocrine gland specimens was performed using antibodies against murine CD3 (ab5690, Abcam) and B220 (RA3-6B, eBioscience) with 6-μm-thick paraffin sections.
An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against mRo60_316-335 peptides. The SSA peptides (10 mg/ml in 0.5 M Na2CO3 buffer; pH 9.6) were absorbed onto Costar EIA/RIA Plates (Corning Incorporated, Corning, NY, USA), washed and blocked with 3% BSA in PBS with 0.05% Tween-20 (PBS-T), incubated with the respective mouse sera (1:200), and further washed with PBS-T. Bound antibodies were detected using peroxidase conjugated goat anti-mouse IgG antibodies (Sigma, USA) and tetramethylbenzidine (Solarbio, Beijing, China). Concentrations of IFN-γ, IL-17A, IL-4, and IL-10 in mouse sera were determined by commercially available ELISA kits (Peprotech, USA) according to the manufacturer’s protocols. The detection range of the assay for IFN-γ, IL-17A, IL-4, and IL-10 was 7.5–1,000 pg/ml, 7.5–1,000 pg/ml, 40–5,000 pg/ml, and 15–2,000 pg/ml, respectively. All sera were diluted 1:3 before quantification.
Samples of total proteins extracted from lysates of salivary or lacrimal glands from healthy C57BL/6J mice were subjected a 10% SDS-PAGE gel and then separated proteins were transferred to PVDF membranes. Membranes were blocked using 5% de-fated milk in PBS-T for 2 h at room temperature. After washing, membranes were incubated with mouse sera (1:200 dilution in blocking buffer) from immunized or control mice overnight at 4°C and subsequently incubated with peroxidase conjugated goat anti-mouse IgG antibodies for 1 h at room temperature followed by visualization of bands using chemiluminescent substrate.
To deplete B cells, we injected C3H/He mice i.v. with 10 mg/kg anti-CD20 antibody 1 day before the immunization. To maintain the depletion, the same dose of anti-CD20 antibody was injected at the third, sixth, and ninth week after the immunization. Both anti-CD20 monoclonal antibody (18B12) and isotype control IgG were kind gift from Biogen (Biogen Inc., San Diego). Efficiency of depletion of the B cells was evaluated by determining B cells in the peripheral blood using FITC-conjugated anti-mouse CD19 IgG (6D5, Biolegend, USA).
The protein sequence of
All analyses were performed with GraphPad Prism statistical software (GraphPad Software Inc., version 5.01, La Jolla, CA, USA). Normality and equality of variances of the quantitative data was determined first. At
Previously, Deshmukh and colleagues reported that the predominant T cell epitope of mRo60 antigen is located within mRo60_316-335 peptide (
Immunization with the Ro60_316-335 peptide induces a primary Sjögren’s syndrome-like disease in C3H/He mice. C3H/HeJ mice were immunized with Ro60_315-336 (
Lymphocytic foci (LF), a hallmark of pSS, were observed in both lacrimal and salivary glands (Figure
In the next step, we investigated the production of autoantibodies. As expected, C3H/HeJ mice immunized with the mRo60_316-335 peptide generated autoantibodies against the antigen while corresponding control mice did not (Figure
Immunological features of the primary Sjögren’s syndrome-like disease in C3H/He mice.
Since SSA autoantibodies are also found in systemic lupus erythematosus (SLE), we examined whether immunized mice express SLE-like symptoms. No obvious difference was observed in the H&E staining of kidney sections between the mRo60_316-335 peptide immunized mice and controls. In consistence with this, no significant difference was found in the concentration of proteins in the urine of the two groups of mice (Figure S1 in Supplementary Material). Therefore, immunization with Ro60_316-335 peptide induced a pSS-like disease in both, C3H/HeJ and C3H/HeN mice.
Since genetic background plays an important role in the development of experimental autoimmune disorders, we then investigated the development of the Ro60_316-335 peptide-induced pSS-like disease in other mouse strains. We first investigated C3H/HeN mice, a strain closely related to C3H/HeJ. Six weeks after immunization, C3H/HeN mice treated with the mRo60_316-335 peptide showed a significant decrease in the production of tears as compared to control mice (Figure S2A in Supplementary Material). No significant difference was observed in the secretion of saliva between the two groups (Figure S2B in Supplementary Material). Similar to C3H/HeJ mice, C3H/HeN mice immunized with mRo60_316-335 peptide generated autoantibodies against the peptide and protein extracts from lacrimal and salivary glands (Figure S2C,D in Supplementary Material). With regard to inflammatory cell infiltration, in C3H/HeN mice, LF were almost exclusively observed in Ro60_316-335 peptide-immunized mice but not in control mice, although the frequency of mice with LF was rather low (Figures S2E,F in Supplementary Material).
We next investigated other two mouse strains carrying different MHC allele, DBA1/J and C57BL/6J mice. DBA/1J mice treated with mRo60_316-335 peptide did not show any impairment in secretion function of salivary or lacrimal glands as compared to control mice. Furthermore, neither LF nor infiltrated cells was observed in the immunized mice. Although autoantibodies against Ro60_316-335 peptide can be detected in sera of DBA/1J mice treated with the peptide, no autoantibodies against proteins from lachrymal and salivary glands were detectable. In addition, serum levels of IL-17A but not IFN-λ, IL-4, or IL-10 was increased in peptide immunized mice as compared to control mice (Figure S3 in Supplementary Material). Similar to DBA/1J mice, C57BL/6J mice were also resistant to the Ro60_316-335 peptide-induced pSS-like disease, without impairment in secretion of tears, infiltration of lymphocytes in exocrine glands or production of autoantibodies binding to proteins from exocrine gland (Figure S4 in Supplementary Material).
Table
Summary of the development of the Ro60_316-335 induced primary Sjögren’s syndrome -like disease among mouse strains.
C3H/HeJ | C3H/HeN | DBA/1J | C57BL/6J | |
---|---|---|---|---|
MHC II hyplotype | H2-d | H2-d | H2-q | H2-b |
Tears secretion | Decreased | Decreased | Not affected | Not affected |
Saliva secretion | Not affected | Not affected | Not affected | Not affected |
Lymphocytic foci | Yes | Yes | No | No |
Inflammatory cell infiltration | Yes | Yes | No | No |
anti-Ro60_316-335 IgG | Yes | Yes | Yes | Yes |
anti-exocrine gland lysate IgG | Yes | Yes | No | No |
IFN-γ | Increased | – | Not affected | Increased |
IL-17A | Increased | – | Increased | Not affected |
We next investigated the role of B cells in this novel mouse model of pSS. C3H/HeN mice were injected i.p. with anti-CD20 antibody before immunization as well as at third, sixth, and ninth weeks after immunization. We evaluated the efficiency of B cell depletion by detecting B cells in peripheral blood. As shown in Figure
B cell depletion prevents mice from Ro60_315-336-induced primary Sjögren’s syndrome-like disease. Before and after immunization with Ro60_316-335 peptide, C3H/HeN mice were injected i.p. with anti-CD20 IgG (red line) or isotype IgG (blue line). The control group (green line) represents mice immunized with PBS and adjuvants alone.
Our data suggest a pathogenic role of the predominant T cell epitope of Ro60 in the development of pSS-like but not SLE-like disease in mice. Previously, we performed a comprehensive meta-analysis and demonstrated that HLA-DRB1*0301 and HLA-DRB1*0803 are two major alleles associated with an increased risk of pSS (
Prediction of the epitopes for the primary Sjögren’s syndrome (pSS)-risk associated HLA-DRB1 alleles within the hRo60 protein. The protein sequence of
In this study, we induced a pSS-like disease by immunizing mice with a partial structure derived from the mRo60 protein, a peptide containing the predominant T cell epitope. Previously, Scofield et al. established a mouse model of pSS by repetitive immunization with Ro60 peptide containing predominant B cell epitopes (
The Ro60_316-335 peptide induced novel mouse model of pSS demonstrates that T cell epitope in this region is pathogenic in mice. Moreover, a T cell epitope in this section is predicted to bind to the pSS-risk associated HLA-DRB1*0803 allele, a HLA allele associated with pSS but not SLE (
Analysis of the sequence homology between hRo60_310-335 and human virus proteins. Sequence homologies were analyzed by BLASTP software (
As expected, different mouse strains show diverse susceptibility to these novel mouse models of pSS, which might reflect the situation in human where genetic factor contribute to the development of disease (
Despite the similarities in the pathogenesis of experimental pSS in C3H/HeJ and C3H/HeN, it should be noted that there are some fine differences in the disease phenotypes developed in both substrains. For example, C3H/HeJ mice showed a higher level of inflammatory infiltration in exocrine glands in both Ro60_316-335 peptide immunized and PBS treated groups than C3H/HeN mice. Since the only known relevant genetic difference between these closely related substrains can be referred to the expression of TLR4, which is absent in C3H/HeJ but present in C3H/HeN mice (
Notably, in this new model, the mice developed specifically pSS-like but not SLE-like symptoms. In humans, the anti-SSA/Ro60 antibodies have been detected in the presymptomatic phases of both, pSS and SLE (
Perhaps the most important finding of this study is that B cell-depletion prevents the impairment of secretion function of exocrine glands. However, this result may have two reasons. In autoimmune responses, B cells are involved in two essential processes, the presentation of antigen to T cells and producing autoantibodies. Since B cells were depleted before immunization and, thus, both processes were potentially affected, we are currently unable to delineate the individual contribution of each mechanism to the disease development. To address this issue, B cell depletion at different time points after the initiation of the immune responses will help to explore the underlying mechanism. The essential role of B cell in this novel mouse model of pSS provides some helpful evidence for clinical treatment of the human disease because it strongly argues for therapeutic approaches targeting B cells. Therefore, although the efficacy of current therapeutics targeting B cells have been proved not to be consistently effective (
It should be mentioned that our model does not cover the entire pathology of pSS. First, disease symptoms are rather mild and impairment in secretion is observed only for tear but not for salivary glands. Second, although the formation of LF as a hallmark of pSS was observed in susceptible mice after immunization, no significant difference in incidence of LF between peptide-immunized mice and controls occurred. Finally, although the incidence of LF in salivary gland was higher than that in lacrimal glands, saliva production was found not to be impaired in this setting. This result argues against a direct association between LF development and secretion function which is not consistent with findings for the pSS in humans.
In conclusion, in the current study, we have established a novel mouse model of pSS based on a single immunization with a Ro60 peptide containing a predominant T cell epitope, demonstrating that T cell epitope within SSA/Ro60 antigen is potentially pathogenic. Furthermore, our results support that B cells play an essential role in the development of pSS.
All protocols of mouse experiments were approved by the Institutional Animal Care and Use Committee of Xiamen University.
XYu was involved in conception, design and supervision of the study. JZ, QH, RH, FD, XYue, YC, RH, WZ, LW and JY were involved in the performing experiment, acquisition of data and analysis of data. JZ, JY, and RH were involved in the bioinformatics analysis. XYu, GR, ZL and FP were involved in drafting of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at