Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Lab Invest. Author manuscript; available in PMC 2011 July 1.
Published in final edited form as:
PMCID: PMC3012147

IL17: potential therapeutic target in Sjögren's syndrome using adenovirus-mediated gene transfer



Sjögren's syndrome (SS) involves a chronic, progressive inflammation primarily of the salivary and lacrimal glands leading to decreased levels of saliva and tears that eventually result in dry mouth and dry eye diseases. TH17 cell populations secreting IL17A have been shown to play an important role in an increasing number of autoimmune diseases, including SS. In the present study, we investigated the function of IL17A on SS development and onset.


Adenovirus-5 vectors expressing either IL17R:Fc fusion protein or LacZ were injected via retrograde cannulation into the salivary glands of SS-susceptible (SSS) C57BL/6.NOD-Aec1Aec2 mice between 6-8 weeks of age (a pre-disease stage) or 15-17 weeks of age (a diseased stage). The mice were subsequently characterized for their SS phenotypes.


Mice cannulated with the Ad5-IL17R:Fc viral vector at either 7 or 16 weeks of age exhibited a rapid temporal, yet persistent, decrease in the levels of serum IL17 as well as the overall numbers of CD4+IL17+ T cells present in their spleens. Disease profiling indicated that these mice showed decreased lymphocytic infiltrations of their salivary glands, normalization of their ANA repertoire, and increased saliva secretion. In contrast, mice cannulated with the control Ad5-LacZ viral vector did not exhibit similar changes and progressed to the overt disease stage.


The capacity of the Ad5-IL17R:Fc blocking factor to reduce SS pathology in SSS mice, strongly suggest that IL17 is an important inflammatory cytokine in salivary gland dysfunction. Thus, therapeutic approach targeting IL17 may be an effective in preventing glandular dysfunction.

List Of Keywords: IL17, TH17 cells, IL17R, Sjögren's Syndrome, adenoviral vector, gene therapy

Sjögren's syndrome (SS) is a chronic, systemic autoimmune disease characterized most notably by development of dry eyes and dry mouth manifestations, indicative of secretory dysfunction of the lacrimal and salivary glands (1-3). Although an underlying cause of SS remains elusive, studies using the NOD/LtJ and C57BL/6.NOD-Aec1Aec2 mouse models of SS have provided evidence that this autoimmune exocrinopathy progresses through several consecutive, yet distinct phases (1-3). In the first phase, occurring between birth and 6-8 weeks (wks) of age, a series of aberrant genetic, physiological and biochemical activities associated with retarded salivary gland organogenesis and acinar cell apoptosis occur prior to initiation of detectable autoimmunity. In the second phase, occurring between 8 and 18-20 wks of age, various leukocyte populations first by antigen presenting cells, especially dendritic cells followed by T and B lymphocytes infiltrate the exocrine glands with a concomitant increase in the expression of inflammatory cytokines and production of autoantibodies. In the last phase, occurring usually after 18 wks of age, significant secretory dysfunction of the salivary and lacrimal glands occurs, most likely the result of production of pathogenic autoantibodies reactive against the muscarinic receptor type III (M3Rs) (4, 5). Previous studies have demonstrated that intervention or disruption of the biological or immunological elements identified in one or more of the three phases delays or prevents the subsequent onset of SS in these murine models (5-7).

Although these earlier studies have implicated both TH1 and TH2 cell-associated functions in the development and onset of clinical SS, recent identification of the CD4+TH17 memory cells within the lymphocytic focus (LF) of lacrimal and salivary glands of SS-susceptible (SSs) C57BL/6.NOD-Aec1Aec2 mice, as well as minor salivary glands of human SS patients, greatly expands the potential complexity in deciphering the autoimmune response underlying SS (8, 9). The TH17 cell population, while clearly a subset of CD4+ memory effector T cells, is distinct from either the TH1 or TH2 cell lineages (10-14). TH17 effector cells secrete at least one of the six cytokines belonging to the IL17 family, i.e., IL17A, IL17B, IL17C, IL17D, IL-25 and/or IL17F; however, IL17A (IL17), the signature cytokine, has received the greatest attention in studies of autoimmune diseases (15). The IL17 cytokines are potent pro-inflammatory molecules, actively involved in tissue inflammation via induction of pro-inflammatory cytokine and chemokine expressions (16). In addition, IL17 is involved in the mobilization, maturation and migration of neutrophils via the release of IL-8 at the site of injury (17). Interestingly, IL17A is known to regulate Foxp3+ TReg cells and vice versa (18).

While TH17 cells have been implicated in several autoimmune diseases (e.g., Crohn's disease (19, 20), experimental autoimmune encephalomyelitis (EAE) (21), collagen-induced arthritis CIA) (21), SS (8) and others (2, 3), this characteristic may require signaling from TH1 cells already present in the lesion (3). In any event, recent observational studies in SS patients and animal models of primary SS have identified the presence of IL17 and its activating cytokine IL-23 in the lymphocytic infiltrates of the exocrine glands, as well as higher levels of circulating IL17 in both sera and saliva (8, 9), raising the question of the importance of IL17 in SS. Thus, the goals of the present study were to determine whether blocking IL17 can directly interfere with the onset of SS-like disease by improving the pathology and clinical signs in the salivary glands and in part, identify IL17 as a potential therapeutic target in preventing the development or reversing progression of SS-like disease.

Materials & Methods


SSS C57BL/6.NOD-Aec1Aec2 mice were bred and maintained under specific pathogen-free conditions. The animals were maintained on a 12 hr light-dark schedule and provided food and acidified water ad libitum. At times indicated in the text, mice were euthanized by cervical dislocation following deep anesthetization with isoflurane, after which organs were freshly explanted for analyses. Both the breeding and use of these animals for the present studies were approved by the University of Florida's IACUC. Salivary glands of C57BL/6.NOD-Aec1Aec2 mice were cannulated with IL17 blocking vector, Ad5-IL17R:Fc using retrograde injections at either 8 wks of age (n=9) or 17 wks of age (n=12). Age- and sex-matched control mice (n=10 per age group) received the Ad5-LacZ control vector using the same protocol.

Production of Ad5-LacZ and Ad5-IL17R:Fc vectors

The recombinant adenovirus vectors used in this study were generously provided by Dr. Jay K. Kolls (Children's Hospital of Pittsburgh, Pittsburgh, PA). These vectors were constructed based on the first generation serotype 5 adenovirus (Ad5) and shown to produce their appropriate and functional products (22-24). In brief, the Ad5-IL17R:Fc vector was initially made by fusing the extracellular domain of mouse IL-17R with the murine IgG1 CH2 and CH3 domains. The functionality of the fusion protein was tested by inhibiting recombinant IL-17–induced production of IL-6 in 3T3 fibroblasts (25). The construct was expressed in vivo by transferring the fusion gene into an E1-deleted recombinant adenovirus (Ad5-IL17R:Fc) (25). To obtain sufficient viral vectors for the present studies, each recombinant vector was amplified in HEK293 cells, purified by two rounds of CsCl gradient centrifugation, then dialyzed against 100 mM Tris-HCl (pH 7.4), 10 mM MgCl2 and 10% (v/v) glycerol, as described elsewhere (26).

Retrograde salivary gland cannulation of Ad5-LacZ or Ad5-IL17R:Fc vectors

Previous studies have demonstrated that retrograde salivary gland cannulation is an effective method to direct local gene expression in the salivary glands (27-29). In brief, prior to cannulation, each mouse was anesthetized with a ketamine:xylazine mixture ((100 mg/mL, 1 mL/kg body weight; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (20 mg/mL, 0.7 mL/kg body weight; Phoenix Scientific, St. Joseph, MO)) via intramuscularly. Stretched PE-10 polyethylene tubes were inserted into each of the two openings of the salivary ducts. After securing the cannulas, the mouse received an intramuscular injection of atropine (1 mg/kg), followed 10 minutes later by a slow, steady injection of viral vector. Each salivary gland received 50 μl of vector solution containing 107 viral particles. This vector dose was chosen based on published literature in which dosage optimizations were performed extensively (30, 31). The cannulas were removed 5 minutes later to ensure successful cannulation.

Measurement of stimulated saliva flow

To measure stimulated saliva flow, individual non-anesthetized mice were weighed and given an intraperitoneal (IP) injection of 100 μl of phosphate-buffered saline (PBS) containing isoproterenol (0.02 mg/ml) and pilocarpine (0.05 mg/ml) (Sigma-Alrich, St. Louis, MO). Saliva was collected for 10 min from the oral cavity of individual mice using a micropipette starting 1 min after injection of the secretagogue. The volume of each saliva sample was measured. Prior to vector cannulation and again at each time-point designated in the text, saliva and sera were collected from each mouse. Samples were stored at -80°C until analyzed.

Determination of IL17 cytokines level

Measurement of IL17 in serum samples were performed using the mouse IL17 Bio-Plex Cytokine Assay (Bio-Rad, Hercules, CA). All procedures were performed according to the manufacturer's instructions. Readings were carried out using the Luminex 200 system (Luminex, Austin, TX). Standard curves were generated from 3.1 to 10,200 pg/ml. The lower cutoff level for detection by the software was 1 pg/ml.

Intracellular cytokine staining and flow cytometric analysis

Splenocytes were prepared as previously described (6). Cells were plated in a 24-well microtiter plate pre-coated with anti-CD3 (10μg/ml) and anti-CD28 antibodies (2 μg/ml) and incubated for 5 hrs with Leukocyte Activation Cocktail containing GolgiPlug (2 μl/ml). Collected cells were fixed and permeabilized using Cytofix/CytopermFixation/Permeabilization. Flow cytometric acquisition for intracellular staining was performed following staining with PE-Cy5-anti-mouse CD4 and PE-anti-IL17A. The cells were counted on a FACSCalibur (BD, Franklin Lakes, NJ) and analyzed by FCS Express (De Novo Software, Los Angeles, CA).


Following euthanasia, salivary glands were surgically removed from each mouse and placed in 10% phosphate-buffered formalin for 24 hrs. Fixed tissues were embedded in paraffin and sectioned at 5 μm thickness. Three non-consecutive sections separated by 100 μm and cut across the entire glands were used. Paraffin-embedded sections were de-paraffinized by immersing in xylene, followed by dehydration in ethanol. The paraffin-embedded salivary glands were prepared and stained with hematoxylin and eosin (H&E) dye. Stained sections were observed under a microscope for glandular structure and leukocyte infiltration determination. A double-blinded procedure was used to enumerate leukocytic infiltrations in the histological sections of salivary glands. In the current study, lymphocytic foci (LF) were defined as aggregates of >50 leukocytes quantified per each histological section. Calculations were based on a particular histological section with the most severe LF in the gland.

Immunofluorescent staining for B and T cells

Histological sections of salivary glands were incubated with rat anti-mouse B220 (BD Pharmingen, San Jose, CA) and goat anti-mouse CD3 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with Texas Red-conjugated rabbit anti-rat IgG (Biomeda, Foster City, CA) and FITC-conjugated rabbit anti-goat IgG (Sigma-Aldrich, St. Louis, MO). The slides were mounted with DAPI-mounting medium (Vector Laboratories, Burlingame, CA Sections were observed at 200× magnification using a Zeiss Axiovert 200M microscope.and images were obtained with AxioVs40 software (Ver., Zeiss) (Carl Zeiss, Thornwood, NY).

Immunohistochemical staining for IL17 in salivary glands

Immunohistochemical staining for IL17A were carried out as previously described (8). In brief, paraffin-embedded salivary glands were deparaffinized by immersion in xylene, followed by antigen retrieval with 10 mM citrate buffer, pH 6.0. Tissue sections were incubated overnight at 4°C with anti–IL-17 antibody (Santa Cruz Biotechnology Santa Cruz, CA). Isotype controls were done with rabbit IgG. The slides were incubated with biotinylated goat anti-rabbit IgG followed by horseradish peroxidase–conjugated avidin–biotin–peroxidase incubation using the Vectastain ABC kit. The staining was developed by using diaminobenzidine substrate (Vector Laboratories, Burlingame, CA), and counterstaining was performed with hematoxylin. Stained sections were observed at 200× magnification using a Zeiss Axiovert 200M microscope.and images were obtained with AxioVs40 software (Ver., Zeiss) (Carl Zeiss, Thornwood, NY).

Detection of antinuclear antibodies (ANA) in the sera

ANA in the sera of mice were detected using HEp-2 ANA kit (INOVA Diagnostics, Inc, San Diego, CA). All procedures were performed according to the manufacturer's instructions. In brief, HEp-2 fixed substrate slides were overlaid with appropriate mouse sera diluted 1:40, 1:80 and 1:160. Slides were incubated for 1 hr at room temperature in a humidified chamber. After three washes for five minutes with PBS, the substrate slides were covered with Alexa 488-conjugated goat anti-mouse IgG (H/L) (Invitrogen Inc, Carlsbad, CA) diluted 1:100 for 45 min at room temperature. After three washes, fluorescence was detected by fluorescence microscopy at 200× magnification using a Zeiss Axiovert 200M microscope and all images were obtained with AxioVs40 software with constant exposure of 0.3 seconds (Carl Zeiss, Thornwood, NY). In the present study, data are the results using 1:40 dilutions of sera from each experimental group.

Statistical analyses

Statistical evaluations between saliva collections were determined by using Mann-Whitney U test generated by the GraphPad InStat software (GraphPad Software, La Jolla, CA). The two-tailed p value <0.05 was considered significant.


Reduction of serum IL17 cytokine levels following transduction with Ad5-IL17R:Fc vector

Although, adenoviral vectors have been shown to elicit optimal recombinant gene expressions around day 5 post-infection which then persist for approximately 2 wks (32), the present study utilized immunohistochemistry staining against LacZ protein to demonstrate that optimal transduction efficiency was approximately 26 ± 5% at 2 wks post-infusion which decreased to 15 ± 3% by 9 wks (data not shown). To determine the efficacy of the Ad5-IL17R:Fc blocking vector to reduce the systemic levels of IL17 in C57BL/6.NOD-Aec1Aec2 mice following transduction of the salivary glands, IL17 levels were quantified in sera collected at several time points post-cannulation.

As shown in Figure 1A, C57BL/6.NOD-Aec1Aec2 mice treated with the Ad5-IL17R:Fc vector at 8 wks of age exhibited a marked temporal decrease in IL17 levels at both 3 and18 wks post-treatment compared to baseline levels at 7 wks of age. Mice receiving the control Ad5-LacZ vector showed highest IL17 levels at 7 wks of age (pre-diseased state) which gradually decreased with age; however, these decreases were not statistically significant and appeared to be consistent with normal progression of the mice (8). Similar observations were seen in C57BL/6.NOD-Aec1Aec2 mice treated with Ad5-IL17R:Fc or Ad5-LacZ vector at 17 wks of age (Figure 1B). Note, however, that by 17 wks of age, serum IL17 levels were markedly reduced in untreated mice. Nevertheless, the Ad5-IL17:Fc vector, but not the Ad5-LacZ vector, was capable of reducing the serum levels even further. These results support the functional efficacy of the Ad5-IL17:Fc viral vector in suppressing IL17 levels both transiently and stably (up to 18 wks).

Figure 1
Serum IL17 cytokine levels

Decreased numbers of IL17-producing CD4+ T cells in the spleens of Ad5-IL17R:Fc transduced mice

Mice treated with Ad5-IL17R:Fc or Ad5-LacZ at either 8 wks or 17 wks of age were euthanized at 27 and 29 wks of age, respectively. The splenocytes were isolated and examined for the number of IL17 secreting CD4+T cells. As presented in Figures 2A and 2B, spleens of C57BL/6.NOD-Aec1Aec2 mice contained about 4% ofCD4+ IL17+ T cells at 7 wks of age and this increased to over 8% by 19 wks post Ad5-LacZ treatment. This result is consistent with the natural aging of the mice (unpublished data) and is not considered a direct effect of treatment with Ad5-LacZ vector. In contrast, as shown in Figure 2C, mice treated with Ad5-IL17R:Fc at 8 wks of age showed no increased levels of CD4+IL17+ T cells at 19 wks post-treatment. A similar functional efficacy of Ad5-IL17R:Fc vector treatment was observed in mice treated at 17 wks of age (Figures 2D-F) where more than a 2-fold decrease in the number of IL17 secreting CD4+T cells was seen when examined at 29 wks of age. In contrast, only a slight decrease in the number of CD4+IL17+ T cells was observed in mice treated with the Ad5-LacZ vector. These data suggest that even though the Ad5 vector is transient and presumably restricted locally to the salivary glands, the effect on C57BL/6.NOD-Aec1Aec2 mice can be systematic and sustained longer than anticipated as evidenced by the decrease in the levels of IL17 secreting cells at 12 or 19 wks post treatment.

Figure 2
Number of splenic IL17+CD4+ cells

Reduced SS-like disease in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice following transduction with Ad5-IL17R:Fc vector

The disease profile in C57BL/6.NOD-Aec1Aec2 mice is well-characterized in that they exhibit loss of saliva secretion concomitantly with the appearance of LF in the exocrine glands and increased levels of ANAs (1). Thus, to determine the effect of blocking IL17 on the development of SS, mice treated by salivary gland cannulation with either Ad5-IL17R:Fc blocking vector or Ad5-LacZ control vector at either 8 or 17 wks of age, were examined for their SS-like disease phenotype at 18 and 12 wks after cannulation, respectively. Histological examinations of the salivary glands from mice treated with Ad5-IL17R:Fc vector at either 8 or 17 wks of age revealed a marked decrease in the number of LF at time of euthanization, i.e., 26 and 29 wks of age, respectively (Figure 3). As presented in Table 1, Figure 3A & 3D, of the C57BL/6.NOD-Aec1Aec2 mice whose salivary glands were treated with Ad5-IL17R:Fc vector at 8 wks of age (early phase treatment), 67% (6 of 9) had no detectable LF in the salivary glands, while 83% (5 of 6) mice whose salivary glands were treated with the control Ad5-LacZ vector showed a dramatic increase in the number of LF. Similarly, of the C57BL/6.NOD-Aec1Aec2 mice treated with Ad5-IL17R:Fc vector at 18 wks of age (late phase treatment), 83% (10 of 12) had no detectable LF in the salivary glands, compared to 83% (5 of 6) mice treated with the Ad5-LacZ control vector (Table 1, Figure 3G & 3J). Immunofluorescent staining revealed both B and T lymphocytes with IL17 positive cells within the salivary gland ductal and acinar cells and LF of Ad5-LacZ treated mice (Figure 3B, 3C, 3H & 3I), but not in Ad5-IL17R:Fc treated mice (Figure 3E & 3K).

Figure 3
Histological analyses of salivary glands
Table 1
Quantification of lymphocytic foci (LF) in salivary glands

Production of autoantibodies, including ANA, may be independent of the TH17-IL17 system, but it is one of the important criteria in diagnosing SS disease. Sera obtained from mice treated with the Ad5-LacZ vector at 8 wks of age in the early treatment group exhibited the expected changes in ANA staining pattern, evolving from a faint cytoplasmic/nuclear to a homogenous nuclear profile between 7 and 26 wks of age, indicative of a SS disease ANA staining profile (Figures 4A & 4B). In contrast, mice that received Ad5-IL17R:Fc vector maintained weak cytoplasmic/nuclear staining pattern between 7 and 26 wks of age, or 18 wks post-treatment (Figure 4C). As anticipated, sera collected from 16 wks old C57BL/6.NOD-Aec1Aec2 mice, i.e., one wk prior to cannulation in the late treatment group, were positive for ANA with a homogenous nuclear pattern. The ANA profile remained homogenous nuclear with higher intensity at 10 wks post treatment with Ad5-LacZ vector. Paradoxically, mice treated with Ad5-IL17R:Fc vector shifted from homogenous nuclear to normal cytoplasmic/nuclear ANA profile (Figure 4F). Therefore, blocking IL17 appears to have a significant effect on the ANA profiles in SSS C57BL/6.NOD-Aec1Aec2 mice.

Figure 4
Identification of ANA

Preventing secretory dysfunction or restoring normal saliva flow in C57BL/6.NOD-Aec1Aec2 mice following Ad5-IL17R:Fc treatment

To determine if the Ad5-IL17R:Fc vector is capable of preventing SS disease progression in C57BL/6.NOD-Aec1Aec2 mice when treated prior to development of SS (i.e., 7 wks of age), or restoring normal saliva secretion when treated at the late stage of SS development (i.e., 17 wks of age), saliva volumes were collected and measured post-cannulation. As presented in Figure 5A, mice whose salivary glands were cannulated at 7 wks of age with Ad5-IL17R:Fc vector retained normal saliva flow at 3 wk and 18 wks post-cannulation compared to baseline level at 7 wks of age. In contrast, mice that received control Ad5-LacZ vector showed the expected gradual decrease in saliva secretion over this same time intervals. Similarly, mice whose salivary glands were treated with Ad5-IL17R:Fc vector at 17 wks of age exhibited temporally progressive increases in saliva secretion over the 11 wks follow-up period (Figure 5B), suggesting a significant recovery of salivary function. This result indicates that blocking IL17 is capable of preventing development of SS when carried out prior to onset of disease, and even restoring normal salivary function when carried out at a later stage of the disease.

Figure 5
Secretory function of salivary glands


The TH17-derived IL17 (IL17A) cytokine is a potent inflammatory cytokine that has been implicated in a growing list of autoimmune diseases, e.g., multiple sclerosis, Crohn's disease, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and SS, as well as autoimmunity in animal models (3). The consequence of TH17/IL17 activation includes, in addition to the production the IL17 family of cytokines, the production of IL-21, IL-22, chemokines (MIP-2, CXCL1, CXCL2, CXCL5), and matrix metalloproteases (MMP3 and MMP13) (16) all actively involved in tissue inflammation. Interaction of the IL17 with its receptors evokes activation of CXCL8, resulting in recruitment of neutrophils to the site of injury. Thus, IL17 has emerged as an ideal therapeutic target for autoimmune disease. In the present study, we sought to examine the effect(s) of inhibiting IL17 on SS development using an adenoviral vector in a mouse model of SS. The results suggest that inhibiting IL17 at early disease stage can prevent the onset of SS development, specifically the absence of lymphocytic infiltration in the salivary glands, retention of normal ANA profiles and no loss in saliva secretion. Likewise, inhibiting IL17 at a later disease stage could rescue salivary gland function by ameliorating lymphocytic infiltrations, normalizing ANA profiles and more importantly recovering saliva secretion.

The design of the current study has taken advantage of several important observations: 1) the temporal disease profile of SSS C57BL/6.NOD-Aec1Aec2 mice is well-defined at both the genetic and pathological levels (1, 2), 2) histological examinations of salivary gland biopsies from both SS patients and C57BL/6.NOD-Aec1Aec2 mice indicate the presence of the IL-23/TH17/IL17 system within LF, while plasma IL17 levels in SS patients correlate with the disease state (8), and 3) retrograde cannulation of the salivary glands in mice via the submandibular ducts can be used to deliver viral vectors encoding recombinant proteins (27-29). Cannulations were carried out at two different ages corresponding to time-points of expected early-stage (6-8 wks of age) and later-stage (15-17 wks of age) pathogenesis. The later-stage studies were carried out based on the fact that C57BL/6.NOD-Aec1Aec2 mice still have intact glands and partial salivary flow rates. With this design, we have been able to examine the direct effect of IL17 blockage as a therapeutic target in preventing either development or onset of SS. A possible weakness in the present design to be considered is the use of the Ad5-based vector system known to express the recombinant protein for a relatively shorter defined time-span (32, 33), and therefore possibly only transient immunological functions. Interestingly, as presented in this study, the effect of the Ad5 vector using IL17R:Fc was quite stable up to 19 wks post-treatment, possibly contributed by the stability of the receptor portion which can be enhanced and prolonged due to the Fc (fragment of crystallization) protein fusion. Numerous studies have shown Fc fusion proteins extend the serum half-life of the partner protein, limit renal clearance and significantly promote protein secretion with high expression (34). Furthermore, even though adenoviral vectors are capable of inducing immunological responses (35, 36), the low-dosage treatment (107 viral particles per salivary gland) used in the present study was well-tolerated and do not elicit any observable side effects.

In addition to the longer duration or persistence of the Ad5 vector in the cannulated mice, the effect of the vectors appear to be systemic, as defined by changes observed in both sera and spleens of the Ad5-IL17R:Fc treated mice. Even though, SS targets primarily the exocrine glands specifically the salivary and lacrimal glands, the pathology can be systemic thereby affecting multiple organs. Extensive studies by Bruce Baum's laboratory have provided significant evidence into the systemic effect of the adenovirus transduction (37-40). As demonstrated by Adesanya et al. (31), retrograde salivary gland cannulation at high vector dose can injure acinar cells which likely compromise the integrity of the mucosal barrier allowing for leakage of the vector systemically. Further studies by Kagami et al. (39) and He X. et al. (41) provide evidence that ductal cannulation of salivary glands can result in systemic effects due to the secretory nature of the salivary glands which are well endowed with protein synthesis organelles and secretory machinery. As observed in this study, the systemic spread of the vector is quite expected and promising

Our studies have indicated that generation of lymphocytic foci in the salivary glands (1) requires an intricate and synchronize action between TH1, TH2 and TH1 7 cells. Study by Jonsson et al. (42) has indicated that some LF form germinal center-like structures and that the appearance of such structures correlate with a more severe disease and higher production of autoantibodies in human patients. We have shown that the initial infiltrating cells are TH1 cells producing IFN-γ which directly mediates the up-regulation of adhesion molecules, consequently recruiting inflammatory cells such as TH2 and TH17 cells to the glands. The destruction of the glands is suggested to be executed by the pathogenic potential of IL17 cytokine (Nguyen et al, unpublished data). More importantly, a recent study (24) has found that IL17 is needed to maintain the structure and formation of GC-like organization in an autoimmune animal model; therefore blocking it with Ad5-17R:Fc vector has been shown to destroy the integrity of the GC by the dissociation of B cell from CD4+ T cells within the follicles. Furthermore, Doreau et al. (43) have demonstrated that IL17 alone or in combination with BAFF (B cells activating factor) can influence the survival, proliferation and differentiation of B lymphocytes and maintain the existence of self-reactive B cells. These seminal studies clearly support our findings in which blocking the activity of IL17 will prevent the generation of LF in the glands or dissociate the existing LF due to the lack of survival or maintenance signals produced by IL17, and this dissipation of the LF ameliorates the formation of self-reactive B cell, thereby eliminating the emergence of autoreactive antibodies.

In conclusion, reduction of IL17A levels by Ad5-IL17R:Fc blocking vectors suppresses features of SS in SSS mice, demonstrating the major role this cytokine plays in the development of this autoimmune disease. How this one cytokine affects the various features of autoimmunity, and at what level or time point, will require additional studies. Nevertheless, the simple and relatively safe cannulation procedure to introduce the Ad5-IL17R:Fc vector directly into the targeted glands suggests this intervention therapy should be more thoroughly investigated. The promising aspect of the present studies is that intervention at late stage of SS can provide protection from further destruction or recovery of salivary gland function. Longer observation is needed to determine the long term effect of adenoviral vectors per se and IL17 at late stage disease. The future application of adeno-associated viral (AAV) vectors which provide a more stable and persistent factor expression could advance gene therapy application to future treatment of SS.


The authors would like to thank Dr. Jay K. Kolls and Dr. Julie Bindas (Children's Hospital of Pittsburgh) for generously providing the Ad5-LacZ and Ad5-IL17R:Fc vectors, and Dr. Phil Cohen for his critical reading of the manuscript and helpful suggestions. We greatly appreciate the assistance of Dr. Craig Meyers and Dr. Nicholas Muzyczka for the use of the microscope. This work was supported by PHS grant K99 DE018958 grant (CQN) from the NIDCR. ABP and CQN were supported by PHS grant 1R21AI081952 from NIAID. HY and JAC were supported by an NIH, NIDCR intramural research grant.

List Of Abbreviations

Sjögren's Syndrome
muscarinic receptor type III
lymphocytic focus
Sjögren's Syndrome-susceptible
T helper cells
adenoviral vector serotype 5
experimental autoimmune encephalomyelitis
collagen-induced arthritis
antinuclear antibodies
adeno-associated virus
macrophage inflammatory protein
chemokine (C-X-C motif) ligand
matrix metalloproteases
B cells activating factor
fragment of crystallization
interferon γ


Disclosure/Duality of Interest: The authors declare no conflict of interest.


1. Nguyen CQ, Cha SR, Peck AB. Sjögren's syndrome (SjS)-like disease of mice: the importance of B lymphocytes and autoantibodies. Frontiers in Bioscience. 2007;12:1767–1789. [PubMed]
2. Nguyen CQ, Peck AB. Unraveling the pathophysiology of Sjogren syndrome-associated dry eye disease. Ocul Surf. 2009;7:11–27. [PMC free article] [PubMed]
3. Lee BH, Tudares MA, Nguyen CQ. Sjogren's syndrome: an old tale with a new twist. Arch Immunol Ther Exp (Warsz) 2009;57:57–66. [PMC free article] [PubMed]
4. Nguyen KH, Brayer J, Cha S, Diggs S, Yasunari U, Hilal G, et al. Evidence for antimuscarinic acetylcholine receptor antibody-mediated secretory dysfunction in nod mice. Arthritis Rheum. 2000;43:2297–2306. [PubMed]
5. 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. [PMC free article] [PubMed]
6. Nguyen C, Cornelius J, Singson E, Killedar S, Cha S, Peck AB. Role of complement and B lymphocytes in Sjogren's syndrome-like autoimmune exocrinopathy of NOD.B10-H2b mice. Mol Immunol. 2006;43:1332–1339. [PubMed]
7. Nguyen CKH, Cornelius JG, Peck AB. Development of Sjogren's syndrome in NOD-derived autommune-prone C57BL/6.NOD-Aec1Aec2 mice is dependent on complement component-3. J Immunol. 2007;179:2318–29. [PMC free article] [PubMed]
8. 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. [PMC free article] [PubMed]
9. Sakai A, Sugawara Y, Kuroishi T, Sasano T, Sugawara S. Identification of IL-18 and Th17 cells in salivary glands of patients with Sjogren's syndrome, and amplification of IL-17-mediated secretion of inflammatory cytokines from salivary gland cells by IL-18. J Immunol. 2008;181:2898–2906. [PubMed]
10. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005;6:1123–1132. [PubMed]
11. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol. 2005;6:1133–1141. [PMC free article] [PubMed]
12. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24:179–189. [PubMed]
13. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. [PubMed]
14. Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature. 2006;441:231–234. [PubMed]
15. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006;126:1121–1133. [PubMed]
16. Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol. 2007;25:821–852. [PubMed]
17. Kastelein RA, Hunter CA, Cua DJ. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu Rev Immunol. 2007;25:221–242. [PubMed]
18. Zhou L, Lopes JE, Chong MM, Ivanov II, Min R, Victora GD, et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature. 2008;453:236–240. [PMC free article] [PubMed]
19. Duerr RH, Taylor KD, Brant SR, Rioux JD, Silverberg MS, Daly MJ, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 2006;314:1461–1463. [PMC free article] [PubMed]
20. Hue S, Ahern P, Buonocore S, Kullberg MC, Cua DJ, McKenzie BS, et al. Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med. 2006;203:2473–2483. [PMC free article] [PubMed]
21. Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature. 2003;421:744–748. [PubMed]
22. Ye P, Rodriguez FH, Kanaly S, Stocking KL, Schurr J, Schwarzenberger P, et al. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J Exp Med. 2001;194:519–527. [PMC free article] [PubMed]
23. Schwarzenberger P, La Russa V, Miller A, Ye P, Huang W, Zieske A, et al. IL-17 stimulates granulopoiesis in mice: use of an alternate, novel gene therapy-derived method for in vivo evaluation of cytokines. J Immunol. 1998;161:6383–6389. [PubMed]
24. Hsu HC, Yang P, Wang J, Wu Q, Myers R, Chen J, et al. Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice. Nat Immunol. 2008;9:166–175. [PubMed]
25. Kolls J, Peppel K, Silva M, Beutler B. Prolonged and effective blockade of tumor necrosis factor activity through adenovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1994;91:215–219. [PubMed]
26. Zheng C, Baum BJ. Evaluation of promoters for use in tissue-specific gene delivery. Methods Mol Biol. 2008;434:205–219. [PMC free article] [PubMed]
27. Kok MR, Yamano S, Lodde BM, Wang J, Couwenhoven RI, Yakar S, et al. Local adeno-associated virus-mediated interleukin 10 gene transfer has disease-modifying effects in a murine model of Sjogren's syndrome. Hum Gene Ther. 2003;14:1605–1618. [PubMed]
28. Kok MR, Voutetakis A, Yamano S, Wang J, Cotrim A, Katano H, et al. Immune responses following salivary gland administration of recombinant adeno-associated virus serotype 2 vectors. J Gene Med. 2005;7:432–441. [PubMed]
29. Lodde BM, Mineshiba F, Wang J, Cotrim AP, Afione S, Tak PP, et al. Effect of human vasoactive intestinal peptide gene transfer in a murine model of Sjogren's syndrome. Ann Rheum Dis. 2006;65:195–200. [PMC free article] [PubMed]
30. 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. [PubMed]
31. 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. [PubMed]
32. Delporte C, Redman RS, Baum BJ. Relationship between the cellular distribution of the alpha(v)beta3/5 integrins and adenoviral infection in salivary glands. Lab Invest. 1997;77:167–173. [PubMed]
33. Cotrim AP, Mineshiba F, Sugito T, Samuni Y, Baum BJ. Salivary gland gene therapy. Dent Clin North Am. 2006;50:157–173. vii. [PubMed]
34. Huang C. Receptor-Fc fusion therapeutics, traps, and MIMETIBODY technology. Curr Opin Biotechnol. 2009;20:692–699. [PubMed]
35. Crystal RG, Harvey BG, Wisnivesky JP, O'Donoghue KA, Chu KW, Maroni J, et al. Analysis of risk factors for local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions. Hum Gene Ther. 2002;13:65–100. [PubMed]
36. Harvey BG, Maroni J, O'Donoghue KA, Chu KW, Muscat JC, Pippo AL, et al. Safety of local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions. Hum Gene Ther. 2002;13:15–63. [PubMed]
37. Zheng C, Voutetakis A, Kok MR, Goldsmith CM, Smith GB, Elmore S, et al. Toxicity and biodistribution of a first-generation recombinant adenoviral vector, in the presence of hydroxychloroquine, following retroductal delivery to a single rat submandibular gland. Oral Dis. 2006;12:137–144. [PubMed]
38. O'Connell BC, Zheng C, Jacobson-Kram D, Baum BJ. Distribution and toxicity resulting from adenoviral vector administration to a single salivary gland in adult rats. J Oral Pathol Med. 2003;32:414–421. [PubMed]
39. Kagami H, O'Connell BC, Baum BJ. Evidence for the systemic delivery of a transgene product from salivary glands. Hum Gene Ther. 1996;7:2177–2184. [PubMed]
40. Wang J, Voutetakis A, Mineshiba F, Illei GG, Dang H, Yeh CK, et al. Effect of serotype 5 adenoviral and serotype 2 adeno- associated viral vector-mediated gene transfer to salivary glands on the composition of saliva. Hum Gene Ther. 2006;17:455–463. [PubMed]
41. He X, Goldsmith CM, Marmary Y, Wellner RB, Parlow AF, Nieman LK, et al. Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands. Gene Ther. 1998;5:537–541. [PubMed]
42. Jonsson MV, Skarstein K, Jonsson R, Brun JG. Serological implications of germinal center-like structures in primary Sjogren's syndrome. J Rheumatol. 2007;34:2044–2049. [PubMed]
43. Doreau A, Belot A, Bastid J, Riche B, Trescol-Biemont MC, Ranchin B, et al. Interleukin 17 acts in synergy with B cell-activating factor to influence B cell biology and the pathophysiology of systemic lupus erythematosus. Nat Immunol. 2009;10:778–785. [PubMed]