Gene therapy using IL-27 ameliorates Sjögren's syndrome-like autoimmune exocrinopathy
© Lee et al.; licensee BioMed Central Ltd. 2012
Received: 21 February 2012
Accepted: 24 July 2012
Published: 24 July 2012
Sjögren's syndrome (SjS) is a systemic autoimmune disease characterized by decreased salivary and lacrimal gland secretions, resulting in severe dry mouth and dry eyes. Recent studies have suggested that TH17 cells and its signature cytokine IL-17 are involved in the underlying pathogenic mechanisms leading to destructive inflammation and autoimmunity. In the present study, we examined whether IL-27, a natural inhibitor of TH17 activity, could down-regulate or reverse SjS in C57BL/6.NOD-Aec1Aec2 mice, a model of primary-SjS.
Recombinant serotype 2 adeno-associated viral (AAV2) vectors expressing either IL-27 (rAAV2-IL27) or LacZ (rAAV2-LacZ) were injected into 6 or 14 week-old C57BL/6.NOD-Aec1Aec2 mice. Changes in IL-27, IL-17, and IL-10 cytokine levels in peripheral blood were determined by ELISAs, while flow cytometry analyses were used to quantify cytokine-positive splenocytes. Histological assessment of salivary glands, anti-nuclear autoantibody (ANA) staining, and stimulated saliva flow rates were used to profile SjS disease severity.
Mice systemically treated with intravenous rAAV2-IL27 injections at either 6 or 14 weeks of age exhibited long-term elevated levels of serum IL-27 with concomitantly reduced levels of IL-17 compared with sera from mice injected with rAAV2-LacZ or saline out to 20 weeks post-inoculation. Most importantly, disease profiles revealed that rAAV2-IL27 treatment had little effect on lymphocytic focus (LF) scores, but resulted in structural changes in LF, lower titers of ANAs with changes in staining patterns, and a less severe clinical disease as determined by saliva flow rates.
These data support the concept that IL-27, when provided exogenously, can induce a suppressive effect on SjS development and thus may be an effective therapeutic agent for regulating TH17 pro-inflammatory activity in autoimmune diseases where the TH17 system has been shown to play an important role in their pathogenesis.
Interleukin 27 (IL-27), along with IL-12, IL-23, and IL-35, is a novel cytokine of the IL-6/IL-12 family. It is composed of two subunits: IL-12p40-related Epstein-Barr virus-induced gene 3 (Ebi3) protein and IL-12p35-related p28 protein (p28) . The orphan cytokine receptor WSX-1 (TCCR) and glycoprotein-130 (gp130) make up the heterodimeric signal transducing receptor for IL-27 . IL-27 acts on CD4+ T cells and plays a pivotal role as both a pro- and anti-inflammatory cytokine. As a pro-inflammatory cytokine, IL-27 activates T helper 1 (TH1) responses in the early phases of immunity, in which secretion of interferon-gamma (IFN-γ) is one of the key inflammatory mediators in autoimmunity. The mechanism appears to be the activation of signal transducer and activator of transcription 1 (STAT1) . As an anti-inflammatory protein, IL-27 suppresses IL-2, antagonizing IL-6 function and activating expression of suppressor of cytokine signaling (SOCS) protein(s) . In studies with WSX-/- receptor knockout mice, abnormal signal transduction of IL-27 showed hyper-production of various pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and IL-6 when challenged by Trypanosoma cruzi or T. gondii [5, 6]. Moreover, IL-27 can suppress the expression of forkhead box P3-positive (Foxp3+) regulatory T (Treg) cells and act as a negative regulator of human neutrophil function [7, 8]. Recent studies also confirmed that IL-27 has anti-tumor effects [7, 9].
IL-27 is well known for its inhibitory effects on retinoic acid-related orphan receptor gamma t (RORγt), the transcription factor for TH17 cells, by activating both T-bet, the transcription factor for TH1 cells, and the STAT1 pathway, thus inhibiting expression of IL-17A (commonly referred to as IL-17) . In addition, WSX-1-deficient mice showed greater susceptibility for experimental autoimmune encephalomyelitis (EAE) in comparison with wild-type control mice and exhibited increased levels of IL-17 . More recent reports have described the capacity of IL-27 to suppress TH17 cells by inhibiting TH17 cell differentiation, thereby reducing severity of TH17-mediated autoimmune diseases [11, 12].
Gene delivery using recombinant adeno-associated virus (rAAV)-based vectors has been shown to convey long-term gene expressions in treated hosts [13–16]. Previous studies of gene therapy using AAV have also proven its safety and ability to elicit minimal inflammatory responses in comparison with other types of gene delivery agents [17–20]. Nevertheless, to date, no study using the rAAV system has reported a role for IL-27 in Sjögren's syndrome (SjS). Thus, we examined the effects of IL-27 treatment on SjS disease of C57BL/6.NOD-Aec1Aec2 mice when delivered either at 6 weeks of age (pre-disease) or at14 weeks of age (clinical disease). Results reported here indicate that IL-27, a potent inhibitor of TH17 cell development, may be a useful reagent for treating SjS.
Materials and methods
C57BL/6.NOD-Aec1Aec2 mice were bred and maintained under specific pathogen-free conditions in the animal facility of Animal Care Services at the University of Florida (Gainesville, FL, USA). Development of the C57BL/6.NOD-Aec1Aec2 mouse and its SjS-like disease phenotype is described elsewhere . Briefly, C57BL/6.NOD-Aec1Aec2 mouse was developed by introducing two genetic regions, one on chromosome 1 (designated Aec2) and the second on chromosome 3 (designated Aec1), derived from the NOD/LtJ mouse, into the SjS-non-susceptible C57BL/6J mouse. All animals were maintained on a 12-hour light-dark schedule and provided food and acidified water ad libitum. At times indicated in the article, mice were euthanized by cervical dislocation after deep anesthetization with isoflurane, and their organs and tissues were freshly harvested for analyses. All experiments and analyses described in this article were performed with male and female mice. The breeding and the use of C57BL/6.NOD-Aec1Aec2 mice for gene therapy study as described here were approved by the University of Florida's Institutional Animal Care and Use Committee and Institution Biosafety Committee.
Recombinant AAV2-IL27 (rAAV2-IL27) vector construction
The serotype 2 adeno-associated viral vector (AAV2) was designed to contain a cytomegalovirus (CMV)/chicken-β-actin hybrid promoter, an internal ribosome entry site (IRES), and the inverted terminal repeat sequences (pTR-UF14) (Figure S1A of Additional file 1). To fully recapitulate the functionality of mouse IL-27 cytokine, a rAAV2-IL27 vector was constructed by inserting the genes encoding the two subunits of IL-27 (Ebi3 and p28) into a pTR-UF14 vector. Ebi3 and p28 were generated by polymerase chain reaction (PCR) from murine cDNA by using two pairs of primers (Ebi3: forward 5'-AAACTAGTAGGTCCTTCCCTGGGGCCAGGT-3' and reverse 5'-TTTGATATCAAGGATCCAGTCCCTCTTCAG-3' and p28: forward 5'-AAAAAGCGGCCGCATGGGCCAGGTGACAGGA-3' and reverse 5'-TTTGTCGAC TTAGGAATCCCAGGCTGAGCC-3'). Ebi3 and p28 subunits flanked by IRES component were constructed allowing the co-expression of IL-27 subunits driven by the same promoter (Figure S1B of Additional file 1). We used AAV2 vector encoding beta galactosidase (rAAV2-LacZ) as an AAV2 control vector, whose construction was previously reported .
Functional activity of IL-27 expressed from the rAAV2-IL27vector
rAAV2-IL27 and its backbone (pTR-UF14) plasmids were transfected separately into HEK 293 cells by using TransIT-293 Transfection Reagent (Mirus Bio LLC, Madison, WI, USA) in accordance with the instructions of the manufacturer. After transfection, cell pellet and culture media were collected at different time points up to 48 hours, and expression of the two subunits of IL-27 was measured. Ebi3 was determined by Western blotting by using anti-mouse Ebi3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p28 was detected by enzyme-linked immunosorbent assay (ELISA) by using a Quantikine kit for mouse IL-27 p28 (R&D Systems, Minneapolis, MN, USA). Biological activity was determined by flow cytometry. Culture supernatants from rAAV2-IL27 or empty AAV2-transfected HEK293 cells were collected (mock). C57BL/6J splenocytes were incubated with mock, IL-6/transforming growth factor-beta (IL-6/TGF-β) for optimal TH17 differentiation, and IL-6/TGF-β with supernatants of AAV2-IL27 transfected cells at 37°C for 5 days. IL-17+ cells were measured by flow cytometry.
Packaging of recombinant AAV2 (rAAV2) vector particles
To generate rAAV2 viral particles, adenoviral helper packaging plasmid pDG was used. Plates (15 cm) with less than 40% confluent HEK 293T cells were co-transfected with either pAAV2-LacZ (control vector) or rAAV2-IL27 in accordance with standardized methods . Clarified cell lysates were adjusted to a refractive index of 1.372 by the addition of CsCl and centrifuged at 38,000 revolutions per minute (rpm) for 65 hours at 20°C. Equilibrium density gradients were fractionated, and fractions with a refractive index of 1.369 to 1.375 were collected. The particle titer was quantified by real-time PCR, and the vector was stored at -80°C.
Injections of vectors
Two different age groups of C57BL/6.NOD-Aec1Aec2 mice were used to take advantage of the pre-disease and clinical disease stages of the animal  for the IL-27 gene therapy, thereby aiming to either prevent the onset of the disease or reverse the disease phenotype of SjS (6-week-old mice for pre-disease stage and 14-week-old mice for clinical disease stage, respectively). Mice were treated systemically with saline or rAAV2-LacZ or rAAV2-IL27, and there were 10 mice (five male and five female) per treatment for each disease stage. Systemic delivery was done via intravenous injection in tail veins. Mice were anesthetized with a ketamine (100 mg/mL, 1 mL/kg body weight; Fort Dodge Animal Health, Fort Dodge, IA, USA) and xylazine (20 mg/mL, 0.7 mL/kg body weight; Phoenix Scientific, St. Joseph, MO, USA) cocktail by intraperitoneal injection. Then 50 μL of vector solution containing 2 × 1010 vector genome (VG) was injected. Control groups received either the same amount of rAAV2-LacZ VG or the same volume of saline. The dosage was chosen on the basis of published studies by others for extensive study of dosage optimization [25–29]. All animals were sacrificed at 20 weeks after delivery. At the time of euthanization, systemic transgene expression was determined by using quantitative real-time PCR (Figure S2 of Additional file 2).
Intracellular cytokine staining and flow cytometry analysis
Single-cell suspensions of spleen cells were prepared from C57BL/6.NOD-Aec1Aec2 mice as described elsewhere  and at the ages designated in the text. In brief, spleens were freshly explanted, gently minced through stainless steel sieves, suspended in phosphate-buffered saline (PBS), and centrifuged (1,200 rpm for 5 minutes). Erythrocytes were lysed by a 7-minute incubation in 0.84% NH4Cl. The resulting leukocyte suspensions were washed two times in PBS, counted, and re-suspended in culture media (RPMI 1640 medium, 10% fetal bovine serum, 2 mM L-glutamine, and 0.05 mM β-mercaptoethanol) to a density of 2 × 106 cells/mL. One million cells were pipetted to individual wells of a 24-well microtiter plate pre-coated with anti-CD3 (10 μg/mL; BD Pharmingen, San Diego, CA, USA) and anti-CD28 (2 μg/mL; BD Pharmingen) antibodies for T-cell activation. Cells were incubated for 5 hours with Leukocyte Activation Cocktail containing GolgiPlug (2 μL/mL; BD Pharmingen). Collected cells were fixed and permeabilized by using a Cytofix/CytopermFixation/Permeabilization kit (BD Pharmingen). The flow cytometry acquisition for intra- and extra-cellular staining was performed following staining with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IL-27 (R&D Systems), phycoerythrin (PE)-conjugated anti-IL-17 (eBioscience, San Diego, CA, USA), and PE-Cy7-conjugated anti-mouse CD4 (Invitrogen, Carlsbad, CA, USA) for the detection of cytokines from gene-delivered mice. The cells were counted by using a BD ACCURI C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (TreeStar Inc., Ashland, OR, USA). CD3+ and CD4+ T cells were gated, and the cells were analyzed for IL-27+ or IL-17+ cells.
Cytokine level of peripheral blood
Measurement of IL-27levels in sera samples was conducted by using a Quantikine kit for mouse IL-27 p28 (R&D Systems). IL-17, and IL-10 levels are detected using mouse IL-17 and IL-10 ELISA kits (Life Technologies, Grand Island, NY, USA). All procedures were performed in accordance with the instructions of the manufacturers. Readings were carried out by using a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA).
Hematoxylin and eosin staining and histological assessment
Salivary glands, lacrimal glands, liver, lung, and kidney were surgically removed from each C57BL/6.NOD-Aec1Aec2 mouse at the time of euthanasia and placed in 10% phosphate-buffered formalin for 24 hours. Fixed tissues were embedded in paraffin and sectioned at a thickness of 5 μm. Paraffin-embedded sections were deparaffinized by immersing in xylene, followed by dehydration in ethanol. The prepared tissue sections were stained with hematoxylin and eosin (H&E) dye (Histology Tech Services, Gainesville, FL, USA). H&E-stained sections were observed under a microscope for glandular structure and leukocyte infiltration. Lymphocytic foci (LF) were enumerated by three individuals in blinded fashion. LF were defined as aggregates of more than 50 leukocytes quantified per each histological section, and adjacent sections were used for immunofluorescent staining. Stained sections were observed at ×6 magnification by using a Leica MZ8 microscope, and images were obtained with Leica application suite (version 2.4.0.R1; Leica, Wetzlar, Germany).
Tissue sections were deparaffinized, rehydrated, and blocked with hydrogen peroxide. For antigen retrieval, tissue sections were heated to 100°C under pressure with 10 mM citrate buffer for 15 minutes. The sections were incubated with rat anti-mouse B220 (BD Pharmingen, San Diego, CA, USA) or goat anti-mouse CD3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) to detect B cells and T cells, respectively, overnight at 4°C. The sections were washed two times with PBS-Tween and incubated with Texas Red-conjugated rabbit anti-rat IgG (Biomeda, Foster City, CA, USA) for B220 marker and FITC-conjugated rabbit anti-goat IgG (Sigma-Aldrich, St. Louis, MO, USA) for CD3 detection for 30 minutes at room temperature. After additional washes, the slides were treated with mounting medium with 4'-6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Stained sections were observed at ×100 magnification with a Zeiss Axiovert 200 M microscope, and images were obtained with AxioVs40 software (version 184.108.40.206; Carl Zeiss, Jena, Germany).
Detection of anti-nuclear antibodies in the sera
Anti-nuclear antibodies (ANAs) in the sera of mice were detected by using an HEp-2 ANA kit (Inova Diagnostics, Inc., San Diego, CA, USA). All procedures were performed in accordance with the instructions of the manufacturer. In brief, HEp-2-fixed substrate slides were overlaid with appropriate mouse sera diluted 1:50, 1:100, 1:200, 1:400, and 1:800. Slides were incubated for 1 hour at room temperature in a humidified chamber. After three washes for 5 minutes with PBS, the substrate slides were covered with Alexa 488-conjugated goat anti-mouse IgG (H/L) (Invitrogen) diluted 1:100 for 45 minutes at room temperature. After three washes, fluorescence was detected by fluorescence microscopy at ×200 magnification by using a Zeiss Axiovert 200 M microscope, and all images were obtained with AxioVs40 software (version 220.127.116.11; Carl Zeiss) with a constant exposure of 0.3 seconds (Carl Zeiss). In this study, three individuals in blinded fashion examined positive staining patterns.
Measurement of stimulated saliva flow
To measure stimulated saliva flow, each mouse was weighed and injected with a cocktail of isoproterenol (0.2 mg/mL) and pilocarpine (0.5 mg/mL) in 100 μL of saline by intraperitoneal injection. Saliva was collected for 10 minutes from the oral cavity of individual mice by using a micropipette starting 1 minute after injection of the secretagogue. The volume of each saliva sample was measured. Baseline saliva flow rates (SFRs) were measured a week before the gene therapy, and SFRs were measured every 4 weeks after 8 weeks of post-gene delivery periods until the end of the experiment in each group.
The data are expressed as the mean ± standard error of the mean. Statistical evaluation was determined by using one-way analysis of variance test with the GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) or chi-squared test. A P value of less than 0.05 considered statistically significant.
rAAV2-IL27 plasmid is highly efficient in producing a functional IL-27 product in vitro
Biological activity of recombinant IL-27 (rIL-27) secreted by rAAV2-IL27 was determined by using culture media supernatants collected from the rAAV2-IL27 and control by supernatants from pTR-UF14 transfected HEK293 cells or cells alone. C57BL/6J splenocytes cultured in media containing IL-6 and TGF-β supplemented with supernatants containing rIL-27 showed significant decreases in IL-17+ cells with concomitant increases in IFN-γ+ cells in comparison with media supplemented with supernatant from either pTR-UF14 or mock transfected cells (Figure 1B). Considered together, these results indicate that the heterodimeric rIL-27 protein is properly expressed by the rAAV2-IL27 vector and is biologically functional in suppressing TH17 cells in vitro.
Decreased serum IL-17 levels in mice following rAAV2-IL27treatment
Similar, yet more pronounced, results were observed in mice administered rAAV2-IL27 at 14 weeks of age, the time of adaptive immunity onset in the C57BL/6.NOD-Aec1Aec2 mice. At 8 weeks after injection, mice injected with rAAV2-IL27 showed dramatic increases in the levels of IL-27 in comparison with the rAAV2-LacZ- or saline-treated control groups (278 versus 175 and 173 pg/mL, respectively), and these levels were maintained for at least 20 weeks after injection (Figure 2B). Concomitantly, rAAV2-IL27-treated mice showed reduced levels of IL-17 at both 12 and 20 weeks after vector delivery in comparison with the rAAV2-LacZ- and saline-treated control groups (Figure 2D), indicating that decreased expression of IL-17 in the sera is correlated with the increased expression of IL-27 in the rAAV2-IL27-treated mice. In contrast to mice treated at 6 weeks of age, IL-10 was highly elevated at 8 weeks after injection in mice treated with rAAV2-IL27 in the clinical disease phase (Figure 2F). These data suggest a direct negative regulation of TH17 cells with a corresponding upregulation of IL-10 production, which could further inhibit cytokine productions from TH1, TH2, and TH17 cells. Thus, the two disease stages of SjS respond differently to IL-27 treatment.
Intravenous injection of rAAV2-IL27 negatively regulates splenic TH17 cell functions
Lymphocytic infiltrates in the exocrine glands of rAAV2-IL27-injected mice
Lymphocytic infiltration present in the salivary glands is one of the main diagnostic markers for SjS. In the C57BL/6.NOD-Aec1Aec2 mouse model, the development and onset of autoimmunity in the exocrine glands are considered the results of acinar cell apoptosis that signals an influx of leukocytes expressing pro-inflammatory cytokines. Salivary gland lymphocytic infiltrates initially are composed of T-cell clusters followed by recruitment of B lymphocytes [31, 32]. The migration of specific T-cell populations secreting IL-17 and IL-23 has been shown to directly contribute to the pathogenesis of the disease . Therefore, to determine the effect of rAAV2-IL27 treatment on lymphocytic infiltration in the salivary glands of mice injected at 6 or 14 weeks of age with rAAV2-IL27, freshly explanted, formalin-fixed, paraffin-embedded salivary gland sections were examined after euthanizaton at 20 weeks after treatment.
Quantification of lymphocytic infiltrates in the salivary glands of gene-delivered mouse groups
2.3 ± 1.5
2.8 ± 1.3
3.0 ± 2.4
1.5 ± 0.7
4.3 ± 2.2
2.0 ± 1.4
3.4 ± 2.4
2.5 ± 1.0
3.4 ± 1.3
4.5 ± 5.0
Intravenous injection of rAAV2-IL-27 treatment alters the anti-nuclear antibody profile in C57BL/6.NOD-Aec1Aec2mice
Measurement of stimulated saliva flow rates
In contrast to the above results, mice treated with either rAAV2-IL27 or rAAV2-LacZ at 14 weeks of age exhibited different responses to the treatments (Figure 6B): mice receiving the rAAV-LacZ vector again exhibited a rapid loss of SFR, whereas mice receiving the rAAV2-IL27 vector slowly showed minor improvements in their SFRs. Mice receiving saline began showing a slower SFR after about 8 weeks after treatment (or 22 weeks of age), consistent with disease in this model. These results indicate that IL-27 might impact the delayed recovery of SFRs when treatment is done prior to the onset of disease and may partially restore saliva secretion and glandular function at a later stage.
This study was undertaken to examine the effects of IL-27 gene therapy in SjS by using the C57BL/6.NOD-Aec1Aec2 mouse model of primary SjS. SjS is characterized by hyposalivation, autoantibodies in sera, and lymphocytic infiltration in the salivary glands. Previous findings also suggested a role for the IL-23/TH17/IL-17A system in both patients with SjS and animal models [33, 34]. Therefore, we hypothesized that IL-27 would have a therapeutic role in treating SjS and show potential as a therapeutic for the human disease. The results of the present study generally support our hypothesis, showing that IL-27 overexpression by the rAAV2-IL27 vector in C57BL/6.NOD-Aec1Aec2 mice increased saliva secretion and decreased ANA formation despite a lack of differences in LF scores between rAAV-IL27- and rAAV2-LacZ vector-treated mice or saline-treated controls injected at 6 or 14 weeks of age. Interestingly, rAAV2-IL27 treatment at the early clinical disease stage appeared to show a better response than treatment at a pre-disease stage.
The role of IL-27 has been studied, but the underlying mechanism of IL-27 in autoimmune disease, specifically SjS, is still unknown. Although IL-27 has pro-inflammatory functions, which are embodied in activation of IFN-γ production from TH1 cells, many recent studies are showing anti-inflammatory effects of IL-27 against microorganisms and effector T cells, including TH17 cells. Recent studies have also shown that IL-27 is involved in anti-inflammatory functions, especially interfering with TNF-α or TH17 cells (or both) in autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus [35–38]. For the potential therapeutic use of IL-27, previous in vivo and in vitro studies have shown that IL-27 can be used as an anti-tumor or anti-viral agent as well as a modulator of autoimmunity [39–43]. IL-27-expressing vectors have been shown to exhibit potent anti-tumor activity in malignancies and metastatic tumors [9, 44, 45]. Therefore, the present study, using IL-27 in a gene therapy protocol, provides evidence that supports its potential effectiveness in autoimmunity.
Our previous study, in which an adenoviral vector was used, demonstrated that overexpression of IL-17 was capable of recapitulating SjS phenotypes (lowered saliva secretion and increased autoantibodies) in normal C57BL/6J mice and, at the same time, showed differential immunological or biological responses depending on the age of mice at the time of treatment . By inducing long-term expression of IL-27 levels by using AAV2, our data showed marked reductions in IL-17 cytokine levels in blood from rAAV2-IL27-injected mice at two different phases of disease onset. Interestingly, there was delayed expression of IL-27 in rAAV2-IL27-treated mice in the innate immune phase in comparison with the expression in the adaptive immune phase. This phenomenon could be attributed to the age of the animals at the time of vector treatment. A study by Bostick and colleagues  showed that AAV9 transduction by systemic delivery could be influenced by the age of the animals. These authors found that AAVs exhibited different transduction efficiencies and that adult mice showed higher transduction efficiency than newborn mice. Furthermore, other studies have implicated the 'time lag' effect of AAV2-mediated transgene expression in various tissues, specifically myocardium [47–49]. Zincarelli and colleagues  categorized AAV2 as a low-expression and slow-onset virus along with AAV3, 4, and 5 among AAV serotypes 1 to 9 after systemic injection in mice. Our study also showed that the regulation of IL-17 could be directly attributed to the overexpression of an IL-27 transgene, but this could be the indirect systemic effect of IL-27 on other regulatory cytokines and cell types. Although serum IL-27 is elevated at 8, 12, and 20 weeks after treatment in the 14-week treated group, serum IL-17 is markedly decreased at 20 weeks after treatment. A number of factors can regulate the function of IL-17, especially TH1 cells releasing IFN-γ , which was increased in the rAAV2-IL27-treated group (Figure 3). Also, independent roles of IL-27 subunits, p28 and Ebi3, might be critical. A recent study by Stumhofer and colleagues  showed that IL-27 p28 alone binds to gp130 as a pro-inflammatory signal antagonist. Although gp130 is the subunit for IL-27 receptor with WSX-1 (IL-27Rα), it is also a subunit for IL-6 receptor, which is involved in TH17 cell differentiation. In addition, Ebi3, a cytokine subunit of IL-35, is known for its suppression of TH17 cells . Although rAAV2-IL27 vector expresses both p28 and Ebi3, the possibility of independent roles of p28 or Ebi3 could not be excluded here. Lastly, other cell types (for instance, Tr-1) can regulate TH17 cells. IL-27 has been shown to be able to stimulate Tr-1 cells, which release IL-10 through STAT1 and STAT3 signaling [53, 54]. IL-10 is a cytokine that is known for its anti-inflammatory function and the inhibitory effect on TH17 cells. However, recent studies using IL-27-overexpressing transgenic mice showed an antagonist role of IL-27 on Treg cells, another source for IL-10, through IL-2 modulation . Thus, IL-27 may have an effect on the balance of Tr-1 cells and Treg cells in the mouse or human system. Therefore, multi-factorial regulation of IL-17 might have contributed to the data in this study.
One unique feature of SjS is the formation of leukocyte aggregates in the salivary and lacrimal glands, referred to as either lymphoepithelial sialadenitis or LF [56, 57]. At times, these LF appear histologically to be germinal-like centers and may be indicative of a more pronounced and severe hypergammaglobulinemia . Although LF and LF scores are important criteria for clinical disease and depict the level of lymphocytic infiltrations of the exocrine glands, the scores often do not correlate with the severity of disease [59, 60]. Until recently, it was thought that salivary gland-infiltrating leukocytes were mostly B lymphocytes and CD4+ TH1 cells. However, we recently reported that LF contain significant numbers of CD4+ TH17 memory T cells plus IL-23-producing macrophages or dendritic cells or both . Although the present study shows no difference in mean LF scores between IL-27-treated and AAV2-LacZ vector- or saline-treated controls at the innate or adaptive immune phase, other research has suggested a poor correlation between this marker of disease and salivary gland function .
As might be anticipated, only treatment with rAAV2-IL27 at the clinical disease stage showed a stable and gradual increase of SFRs compared with baseline SFRs over the course of the experiment, whereas mice treated with rAAV2-IL27 at the pre-disease stage showed slight recovery of glandular function at 20 weeks after treatment. These data are consistent with the serum IL-17 levels, which were significantly downregulated at 20 weeks after treatment for both studied groups. Further investigations are needed to determine the underlying mechanism of how IL-27 influences SFRs, although one possible mechanism is the direct suppression of IL-17 in promoting the formation of spontaneous germinal centers , thereby dampening pathogenic autoantibody production . This concept appears to be supported in part by the altered ANA profile and significant decrease in autoantibody titers in rAAV2-IL27-treated mice.
We have provided a proof of concept that exogeneous IL-27 is a potential factor for treating SjS. Although additional research in regard to gene delivery methods and optimal disease time points are needed to further validate the use of rAAV gene transfer, the use of mouse models and systemic delivery of IL-27 provides a unique system to study treatment of autoimmunity at the clinical disease stage, which is applicable for treating patients with established disease. Our results suggest that IL-27 gene therapy could be an effective therapeutic strategy to reverse the symptoms of SjS and should be further investigated.
serotype 2 adeno-associated viral vector
experimental autoimmune excephalomyelitis
Epstein-Barr virus-induced gene 3
enzyme-linked immunosorbent assay
hematoxylin and eosin
internal ribosome entry site
polymerase chain reaction
recombinant adeno-associated virus
revolutions per minute
saliva flow rate
signal transducer and activator of transcription
transforming growth factor-beta
tumor necrosis factor-alpha
We thank Sergi Zolotuhkin (Department of Pediatrics, University of Florida College of Medicine) for generously providing the AAV2 vectors (pTR-UF14). We thank Tegan N Lavoie for helping with the flow cytometry analysis used in this study. This work was supported by the Sjögren's Syndrome Foundation (CQN) and Public Health Service (PHS) grant K99 DE018958 (CQN) from the National Institute of Dental and Craniofacial Research. ABP and CQN were supported by PHS grant 1R21AI081952 from National Institute of Allergy and Infectious Diseases.
- Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, Hibbert L, Churakova T, Travis M, Vaisberg E, Blumenschein WM, Mattson JD, Wagner JL, To W, Zurawski S, McClanahan TK, Gorman DM, Bazan JF, de Waal Malefyt R, Rennick D, Kastelein RA: IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells. Immunity. 2002, 16: 779-790. 10.1016/S1074-7613(02)00324-2.View ArticlePubMedGoogle Scholar
- Pflanz S, Hibbert L, Mattson J, Rosales R, Vaisberg E, Bazan JF, Phillips JH, McClanahan TK, de Waal Malefyt R, Kastelein RA: WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27. J Immunol. 2004, 172: 2225-2231.View ArticlePubMedGoogle Scholar
- Villarino AV, Huang E, Hunter CA: Understanding the pro- and anti-inflammatory properties of IL-27. J Immunol. 2004, 173: 715-720.View ArticlePubMedGoogle Scholar
- Batten M, Ghilardi N: The biology and therapeutic potential of interleukin 27. J Mol Med. 2007, 85: 661-672. 10.1007/s00109-007-0164-7.View ArticlePubMedGoogle Scholar
- Villarino A, Hibbert L, Lieberman L, Wilson E, Mak T, Yoshida H, Kastelein RA, Saris C, Hunter CA: The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity. 2003, 19: 645-655. 10.1016/S1074-7613(03)00300-5.View ArticlePubMedGoogle Scholar
- Hamano S, Himeno K, Miyazaki Y, Ishii K, Yamanaka A, Takeda A, Zhang M, Hisaeda H, Mak TW, Yoshimura A, Yoshida H: WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production. Immunity. 2003, 19: 657-667. 10.1016/S1074-7613(03)00298-X.View ArticlePubMedGoogle Scholar
- Li JP, Wu H, Xing W, Yang SG, Lu SH, Du WT, Yu JX, Chen F, Zhang L, Han ZC: Interleukin-27 as a negative regulator of human neutrophil function. Scand J Immunol. 2010, 72: 284-292. 10.1111/j.1365-3083.2010.02422.x.View ArticlePubMedGoogle Scholar
- Huber M, Steinwald V, Guralnik A, Brustle A, Kleemann P, Rosenplanter C, Decker T, Lohoff M: IL-27 inhibits the development of regulatory T cells via STAT3. Int Immunol. 2008, 20: 223-234.View ArticlePubMedGoogle Scholar
- Zhu S, Lee DA, Li S: IL-12 and IL-27 sequential gene therapy via intramuscular electroporation delivery for eliminating distal aggressive tumors. J Immunol. 2010, 184: 2348-2354. 10.4049/jimmunol.0902371.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang J, Yang M, Htut TM, Ouyang X, Hanidu A, Li X, Sellati R, Jiang H, Zhang S, Li H, Zhao J, Ting AT, Mayer L, Unkeless JC, Labadia ME, Hodge M, Li J, Xiong H: Epstein-Barr virus-induced gene 3 negatively regulates IL-17, IL-22 and RORgamma t. Eur J Immunol. 2008, 38: 1204-1214. 10.1002/eji.200838145.PubMed CentralView ArticlePubMedGoogle Scholar
- Batten M, Li J, Yi S, Kljavin NM, Danilenko DM, Lucas S, Lee J, de Sauvage FJ, Ghilardi N: Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nat Immunol. 2006, 7: 929-936.View ArticlePubMedGoogle Scholar
- Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, Johnson LM, Villarino AV, Huang Q, Yoshimura A, Sehy D, Saris CJ, O'Shea JJ, Hennighausen L, Ernst M, Hunter CA: Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat Immunol. 2006, 7: 937-945. 10.1038/ni1376.View ArticlePubMedGoogle Scholar
- Flotte TR, Afione SA, Conrad C, McGrath SA, Solow R, Oka H, Zeitlin PL, Guggino WB, Carter BJ: Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc Natl Acad Sci USA. 1993, 90: 10613-10617. 10.1073/pnas.90.22.10613.PubMed CentralView ArticlePubMedGoogle Scholar
- Conrad CK, Allen SS, Afione SA, Reynolds TC, Beck SE, Fee-Maki M, Barrazza-Ortiz X, Adams R, Askin FB, Carter BJ, Guggino WB, Flotte TR: Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther. 1996, 3: 658-668.PubMedGoogle Scholar
- Voutetakis A, Kok MR, Zheng C, Bossis I, Wang J, Cotrim AP, Marracino N, Goldsmith CM, Chiorini JA, Loh YP, Nieman LK, Baum BJ: Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics. Proc Natl Acad Sci USA. 2004, 101: 3053-3058. 10.1073/pnas.0400136101.PubMed CentralView ArticlePubMedGoogle Scholar
- Voutetakis A, Zheng C, Mineshiba F, Cotrim AP, Goldsmith CM, Schmidt M, Afione S, Roescher N, Metzger M, Eckhaus MA, Chiorini JA, Dunbar CE, Donahue RE, Baum BJ: Adeno-associated virus serotype 2-mediated gene transfer to the parotid glands of nonhuman primates. Hum Gene Ther. 2007, 18: 142-150. 10.1089/hum.2006.154.View ArticlePubMedGoogle Scholar
- Wagner JA, Moran ML, Messner AH, Daifuku R, Conrad CK, Reynolds T, Guggino WB, Moss RB, Carter BJ, Wine JJ, Flotte TR, Gardner P: A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis. Hum Gene Ther. 1998, 9: 889-909. 10.1089/hum.1998.9.6-889.View ArticlePubMedGoogle Scholar
- Wagner JA, Messner AH, Moran ML, Daifuku R, Kouyama K, Desch JK, Manley S, Norbash AM, Conrad CK, Friborg S, Reynolds T, Guggino WB, Moss RB, Carter BJ, Wine JJ, Flotte TR, Gardner P: Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope. 1999, 109: 266-274. 10.1097/00005537-199902000-00017.View ArticlePubMedGoogle Scholar
- Flotte TR, Brantly ML, Spencer LT, Byrne BJ, Spencer CT, Baker DJ, Humphries M: Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha 1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults. Hum Gene Ther. 2004, 15: 93-128.View ArticlePubMedGoogle Scholar
- Moss RB, Rodman D, Spencer LT, Aitken ML, Zeitlin PL, Waltz D, Milla C, Brody AS, Clancy JP, Ramsey B, Hamblett N, Heald AE: Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial. Chest. 2004, 125: 509-521. 10.1378/chest.125.2.509.View ArticlePubMedGoogle Scholar
- Cha S, Nagashima H, Brown VB, Peck AB, Humphreys-Beher MG: Two NOD Idd-associated intervals contribute synergistically to the development of autoimmune exocrinopathy (Sjögren's syndrome) on a healthy murine background. Arthritis Rheum. 2002, 46: 1390-1398. 10.1002/art.10258.View ArticlePubMedGoogle Scholar
- Kaludov N, Brown KE, Walters RW, Zabner J, Chiorini JA: Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity. J Virol. 2001, 75: 6884-6893. 10.1128/JVI.75.15.6884-6893.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Grimm D, Kern A, Rittner K, Kleinschmidt JA: Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther. 1998, 9: 2745-2760. 10.1089/hum.1998.9.18-2745.View ArticlePubMedGoogle Scholar
- Lee BH, Tudares MA, Nguyen CQ: Sjögren's syndrome: an old tale with a new twist. Arch Immunol Ther Exp. 2009, 57: 57-66. 10.1007/s00005-009-0002-4.View ArticleGoogle Scholar
- Shai E, Falk H, Honigman A, Panet A, Palmon A: Gene transfer mediated by different viral vectors following direct cannulation of mouse submandibular salivary glands. Eur J Oral Sci. 2002, 110: 254-260. 10.1034/j.1600-0722.2002.21200.x.View ArticlePubMedGoogle Scholar
- Fu H, Muenzer J, Samulski RJ, Breese G, Sifford J, Zeng X, McCarty DM: Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain. Mol Ther. 2003, 8: 911-917. 10.1016/j.ymthe.2003.08.021.View ArticlePubMedGoogle Scholar
- Yu CY, Yuan Z, Cao Z, Wang B, Qiao C, Li J, Xiao X: A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery. Gene Therapy. 2009, 16: 953-962. 10.1038/gt.2009.59.PubMed CentralView ArticlePubMedGoogle Scholar
- Mah C, Fraites TJ, Zolotukhin I, Song S, Flotte TR, Dobson J, Batich C, Byrne BJ: Improved method of recombinant AAV2 delivery for systemic targeted gene therapy. Mol Ther. 2002, 6: 106-112. 10.1006/mthe.2001.0636.View ArticlePubMedGoogle Scholar
- Adesanya MR, Redman RS, Baum BJ, O'Connell BC: Immediate inflammatory responses to adenovirus-mediated gene transfer in rat salivary glands. Hum Gene Ther. 1996, 7: 1085-1093. 10.1089/hum.1996.7.9-1085.View ArticlePubMedGoogle Scholar
- Nguyen CQ, Gao JH, Kim H, Saban DR, Cornelius JG, Peck AB: IL-4-STAT6 signal transduction-dependent induction of the clinical phase of Sjogren's syndrome-like disease of the nonobese diabetic mouse. J Immunol. 2007, 179: 382-390.PubMed CentralView ArticlePubMedGoogle Scholar
- Robinson CP, Cornelius J, Bounous DI, Yamamoto H, Humphreys-Beher MG, Peck AB: Infiltrating lymphocyte populations and cytokine production in the salivary and lacrimal glands of autoimmune NOD mice. Adv Exp Med Biol. 1998, 438: 493-497. 10.1007/978-1-4615-5359-5_68.View ArticlePubMedGoogle Scholar
- Robinson CP, Cornelius J, Bounous DE, Yamamoto H, Humphreys-Beher MG, Peck AB: Characterization of the changing lymphocyte populations and cytokine expression in the exocrine tissues of autoimmune NOD mice. Autoimmunity. 1998, 27: 29-44. 10.3109/08916939809008035.View ArticlePubMedGoogle Scholar
- Nguyen CQ, Hu MH, Li Y, Stewart C, Peck AB: Salivary gland tissue expression of interleukin-23 and interleukin-17 in Sjogren's syndrome: findings in humans and mice. Arthritis Rheum. 2008, 58: 734-743. 10.1002/art.23214.PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen CQ, Yin H, Lee BH, Carcamo WC, Chiorini JA, Peck AB: Pathogenic effect of interleukin-17A in induction of Sjogren's syndrome-like disease using adenovirus-mediated gene transfer. Arthritis Res Ther. 2010, 12: R220-10.1186/ar3207.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanida S, Yoshitomi H, Ishikawa M, Kasahara T, Murata K, Shibuya H, Ito H, Nakamura T: IL-27-producing CD14(+) cells infiltrate inflamed joints of rheumatoid arthritis and regulate inflammation and chemotactic migration. Cytokine. 2011, 55: 237-244. 10.1016/j.cyto.2011.04.020.View ArticlePubMedGoogle Scholar
- Sweeney CM, Lonergan R, Basdeo SA, Kinsella K, Dungan LS, Higgins SC, Kelly PJ, Costelloe L, Tubridy N, Mills KH, Fletcher JM: IL-27 mediates the response to IFN-beta therapy in multiple sclerosis patients by inhibiting Th17 cells. Brain Behav Immun. 2011, 25: 1170-1181. 10.1016/j.bbi.2011.03.007.View ArticlePubMedGoogle Scholar
- Murugaiyan G, Mittal A, Weiner HL: Identification of an IL-27/osteopontin axis in dendritic cells and its modulation by IFN-gamma limits IL-17-mediated autoimmune inflammation. Proc Natl Acad Sci USA. 2010, 107: 11495-11500. 10.1073/pnas.1002099107.PubMed CentralView ArticlePubMedGoogle Scholar
- Rajaiah R, Puttabyatappa M, Polumuri SK, Moudgil KD: Interleukin-27 and interferon-gamma are involved in regulation of autoimmune arthritis. J Biol Chem. 2011, 286: 2817-2825. 10.1074/jbc.M110.187013.PubMed CentralView ArticlePubMedGoogle Scholar
- Fakruddin JM, Lempicki RA, Gorelick RJ, Yang J, Adelsberger JW, Garcia-Pineres AJ, Pinto LA, Lane HC, Imamichi T: Noninfectious papilloma virus-like particles inhibit HIV-1 replication: implications for immune control of HIV-1 infection by IL-27. Blood. 2007, 109: 1841-1849. 10.1182/blood-2006-02-001578.PubMed CentralView ArticlePubMedGoogle Scholar
- Greenwell-Wild T, Vazquez N, Jin W, Rangel Z, Munson PJ, Wahl SM: Interleukin-27 inhibition of HIV-1 involves an intermediate induction of type I interferon. Blood. 2009, 114: 1864-1874. 10.1182/blood-2009-03-211540.PubMed CentralView ArticlePubMedGoogle Scholar
- Nagai H, Oniki S, Fujiwara S, Xu M, Mizoguchi I, Yoshimoto T, Nishigori C: Antitumor activities of interleukin-27 on melanoma. Endocr Metab Immune Disord Drug Targets. 2010, 10: 41-46.View ArticlePubMedGoogle Scholar
- Pan HF, Tao JH, Ye DQ: Therapeutic potential of IL-27 in systemic lupus erythematosus. Expert Opin Ther Targets. 2010, 14: 479-484. 10.1517/14728221003769911.View ArticlePubMedGoogle Scholar
- Kalliolias GD, Gordon RA, Ivashkiv LB: Suppression of TNF-alpha and IL-1 signaling identifies a mechanism of homeostatic regulation of macrophages by IL-27. J Immunol. 2010, 185: 7047-7056. 10.4049/jimmunol.1001290.PubMed CentralView ArticlePubMedGoogle Scholar
- Hu P, Hu H-D, Chen M, Peng M-L, Tang L, Tang K-F, Matsui M, Belladonna ML, Yoshimoto T, Zhang D-Z, Xiang R, Ren H: Expression of interleukins-23 and 27 leads to successful gene therapy of hepatocellular carcinoma. Mol Immunol. 2009, 46: 1654-1662. 10.1016/j.molimm.2009.02.025.View ArticlePubMedGoogle Scholar
- Ho M-Y, Leu S-JJ, Sun G-H, Tao M-H, Tang S-J, Sun K-H: IL-27 directly restrains lung tumorigenicity by suppressing cyclooxygenase-2-mediated activities. J Immunol. 2009, 183: 6217-6226. 10.4049/jimmunol.0901272.View ArticlePubMedGoogle Scholar
- Bostick B, Ghosh A, Yue Y, Long C, Duan D: Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration. Gene Therapy. 2007, 14: 1605-1609. 10.1038/sj.gt.3303029.View ArticlePubMedGoogle Scholar
- Su H, Huang Y, Takagawa J, Barcena A, Arakawa-Hoyt J, Ye J, Grossman W, Kan YW: AAV serotype-1 mediates early onset of gene expression in mouse hearts and results in better therapeutic effect. Gene Therapy. 2006, 13: 1495-1502. 10.1038/sj.gt.3302787.View ArticlePubMedGoogle Scholar
- Chu D, Sullivan CC, Weitzman MD, Du L, Wolf PL, Jamieson SW, Thistlethwaite PA: Direct comparison of efficiency and stability of gene transfer into the mammalian heart using adeno-associated virus versus adenovirus vectors. J Thorac Cardiovasc Surg. 2003, 126: 671-679. 10.1016/S0022-5223(03)00082-5.View ArticlePubMedGoogle Scholar
- Vassalli G, Bueler H, Dudler J, von Segesser LK, Kappenberger L: Adeno-associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors. Int J Cardiol. 2003, 90: 229-238. 10.1016/S0167-5273(02)00554-5.View ArticlePubMedGoogle Scholar
- Zincarelli C, Soltys S, Rengo G, Rabinowitz JE: Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008, 16: 1073-1080. 10.1038/mt.2008.76.View ArticlePubMedGoogle Scholar
- Stumhofer JS, Tait ED, Quinn WJ, Hosken N, Spudy B, Goenka R, Fielding CA, O'Hara AC, Chen Y, Jones ML, Saris CJ, Rose-John S, Cua DJ, Jones SA, Elloso MM, Grotzinger J, Cancro MP, Levin SD, Hunter CA: A role for IL-27p28 as an antagonist of gp130-mediated signaling. Nat Immunol. 2010, 11: 1119-1126. 10.1038/ni.1957.PubMed CentralView ArticlePubMedGoogle Scholar
- Niedbala W, Wei XQ, Cai B, Hueber AJ, Leung BP, McInnes IB, Liew FY: IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells. Eur J Immunol. 2007, 37: 3021-3029. 10.1002/eji.200737810.View ArticlePubMedGoogle Scholar
- Awasthi A, Carrier Y, Peron JP, Bettelli E, Kamanaka M, Flavell RA, Kuchroo VK, Oukka M, Weiner HL: A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells. Nat Immunol. 2007, 8: 1380-1389. 10.1038/ni1541.View ArticlePubMedGoogle Scholar
- Wang H, Meng R, Li Z, Yang B, Liu Y, Huang F, Zhang J, Chen H, Wu C: IL-27 induces the differentiation of Tr1-like cells from human naive CD4+ T cells via the phosphorylation of STAT1 and STAT3. Immunol Lett. 2011, 136: 21-28. 10.1016/j.imlet.2010.11.007.View ArticlePubMedGoogle Scholar
- Tait Wojno ED, Hosken N, Stumhofer JS, O'Hara AC, Mauldin E, Fang Q, Turka LA, Levin SD, Hunter CA: A role for IL-27 in limiting T regulatory cell populations. J Immunol. 2011, 187: 266-273. 10.4049/jimmunol.1004182.PubMed CentralView ArticleGoogle Scholar
- Xu KP, Katagiri S, Takeuchi T, Tsubota K: Biopsy of labial salivary glands and lacrimal glands in the diagnosis of Sjogren's syndrome. J Rheumatol. 1996, 23: 76-82.PubMedGoogle Scholar
- Agale SV, D'Costa GF, Hastak MS, Shedge RT: Primary non-Hodgkin's lymphoma of the salivary gland: a spectrum of lymphoepithelial sialadenitis, low-grade B-cell lymphoma of mucosa-associated lymphoid tissue with transformation to high-grade lymphoma. Indian J Pathol Microbiol. 2010, 53: 364-367. 10.4103/0377-4929.64351.View ArticlePubMedGoogle Scholar
- Nordmark G, Alm GV, Ronnblom L: Mechanisms of disease: primary Sjogren's syndrome and the type I interferon system. Nat Clin Pract Rheumatol. 2006, 2: 262-269. 10.1038/ncprheum0173.View ArticlePubMedGoogle Scholar
- Bombardieri M, Barone F, Pittoni V, Alessandri C, Conigliaro P, Blades MC, Priori R, McInnes IB, Valesini G, Pitzalis C: Increased circulating levels and salivary gland expression of interleukin-18 in patients with Sjogren's syndrome: relationship with autoantibody production and lymphoid organization of the periductal inflammatory infiltrate. Arthritis Res Ther. 2004, 6: R447-456. 10.1186/ar1209.PubMed CentralView ArticlePubMedGoogle Scholar
- Haga HJ: Clinical and immunological factors associated with low lacrimal and salivary flow rate in patients with primary Sjogren's syndrome. J Rheumatol. 2002, 29: 305-308.PubMedGoogle Scholar
- Nikolov NP, Alevizos I, Grisius M, Atkinson J, Cotrim A, Baum BJ, Brahim J: Uncoupling salivary gland dysfunction from inflammation in a large cohort of primary Sjogrens syndrome patients [abstract]. Arthritis Rheum. 2009, 60: 480-Google Scholar
- Hsu H-C, Yang P, Wang J, Wu Q, Myers R, Chen J, Yi J, Guentert T, Tousson A, Stanus AL, Le T-vL, Lorenz RG, Xu H, Kolls JK, Carter RH, Chaplin DD, Williams RW, Mountz JD: Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice. Nat Immunol. 2008, 9: 166-175. 10.1038/ni1552.View ArticlePubMedGoogle Scholar
- Salomonsson S, Jonsson MV, Skarstein K, Brokstad KA, Hjelmstrom P, Wahren-Herlenius M, Jonsson R: Cellular basis of ectopic germinal center formation and autoantibody production in the target organ of patients with Sjogren's syndrome. Arthritis Rheum. 2003, 48: 3187-3201. 10.1002/art.11311.View ArticlePubMedGoogle Scholar
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