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Pathogenic effect of interleukin-17A in induction of Sjögren’s syndrome-like disease using adenovirus-mediated gene transfer
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Title: Pathogenic effect of interleukin-17A in induction of Sjögren’s syndrome-like disease using adenovirus-mediated gene transfer
Physical Description: Book
Language: English
Creator: Nguyen, Cuong Q.
Yin, Hongen
Lee, Byung Ha
Carcamo, Wendy C.
Chiorini, John A.
Peck, Ammon B.
Publisher: BioMed Central Ltd
Publication Date: 2010
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Abstract: Introduction: Sjögren’s syndrome (SS) involves a chronic, progressive inflammation primarily of the salivary and lacrimal glands leading to decreased levels of saliva and tears resulting in dry mouth and dry eye diseases. Seminal findings regarding TH17 cell populations that secrete predominantly interleukin (IL)-17A 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 IL-17A on the development and onset of SS, Methods: Adenovirus serotype 5 (Ad5) vectors expressing either IL-17A or LacZ were infused via retrograde cannulation into the salivary glands of C57BL/6J mice between 6-8 weeks of age or between 15-17 weeks of age. The mice were characterized for SS phenotypes. Results: Disease profiling indicated that SS-non-susceptible C57BL/6J mice whose salivary glands received the Ad5-IL17A vector developed a SS-like disease profile, including appearance of lymphocytic foci, increased cytokine levels, changes in antinuclear antibody profiles, and temporal loss of saliva flow. Conclusions: Induction of SS pathology by IL-17A in SS-non-susceptible mice strongly suggests that IL-17A is an important inflammatory cytokine in salivary gland dysfunction. Thus, localized anti-IL17 therapy may be effective in preventing glandular dysfunction.
Funding: Publication of this article was funded in part by the University of Florida Open-Access publishing Fund.
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Full Text
Pathogenic effect of interleukin-17A in induction of Sjogren's syndrome-like disease
using adenovirus-mediated gene transfer

Cuong Q. Nguyen ^^^'', Hongen Yin ^, Byung Ha Lee ^, Wendy C. Carcamo ^, John A. Chiorini ^
& Ammon B. Peck ^''^

^ Eli and Edythe L. Broad Institute, 7 Cambridge Center, Cambridge, MA 02142, USA
^Department of Chemical Engineering, Massachusetts Institute of Technology, 77
Massachusetts Ave, E25-545, Cambridge MA 02139, USA
^ Department of Oral Biology, University of Florida College of Dentistry, 1600 SW Archer Rd,
Gainesville, Florida 32610, USA
''Center for Orphan Autoimmune Disorders, University of Florida College of Dentistry, 1600 SW
Archer Rd, Gainesville, Florida 32610, USA
^National Institute of Dental and Craniofacial Research, NIH, 10 Center Drive MSC 1190,
Bethesda, Maryland 20892, USA
^Department of Pathology, Immunology & Laboratory Medicine, University of Florida
College of Medicine, 1600 SW Archer Rd, Gainesville, Florida 32610, USA

Running title: The role of IL-17A in Sjogren's Syndrome

Address correspondence:
Cuong Q. Nguyen, PhD
Department of Oral Biology
PO Box 100424, College of Dentistry
University of Florida, Gainesville, Florida 32610 USA
Telephone: 352-273-8865 FAX: 352-273-8829
E-mail: Nguyen@pathology.ufl.edu
1


ABSTRACT:
Introduction: Sjogren's syndrome (SS) involves a chronic, progressive inflammation primarily



of the salivary and lacrimal glands leading to decreased levels of saliva and tears resulting in



dry mouth and dry eye diseases. Seminal findings regarding Th17 cell populations that secrete



predominantly interleukin (IL)-17A 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 IL-17A on the development and onset of SS,



Methods: Adenovirus serotype 5 (Ad5) vectors expressing either IL-17A or LacZ were infused



via retrograde cannulation into the salivary glands of C57BL/6J mice between 6-8 weeks of age



or between 15-17 weeks of age. The mice were characterized forSS phenotypes.



Results: Disease profiling indicated that SS-non-susceptible C57BL/6J mice whose salivary



glands received the Ad5-IL17A vector developed a SS-like disease profile, including



appearance of lymphocytic foci, increased cytokine levels, changes in antinuclear antibody



profiles, and temporal loss of saliva flow.



Conclusions: Induction of SS pathology by IL-17A in SS-non-susceptible mice strongly



suggests that IL-17A is an important inflammatory cytokine in salivary gland dysfunction. Thus,



localized anti-IL17 therapy may be effective in preventing glandular dysfunction.
2


INTRODUCTION




Sjogren'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 the etiology of SS remains




unknown, intensive studies of an ever expanding number of animal models is beginning to




unravel the genetic, molecular and immunological basis for this disease[1]. Previous studies




have implicated critical roles for both interferon-y (IFN-y) and interleukin (IL)-4 in development




and onset of SS-like disease in NOD/LtJ and C57BL/6.NOD-/Aecf/Aec2 mice [4, 5], strongly




suggesting involvement of ThI and Th2 cell populations, respectively. While IFN-y regulates




cell-mediated immunity through activation of macrophages, NK cells and CD8'^ T cells, this




cytokine appears to predispose these SS-susceptible mice by retarding salivary gland




organogenesis, especially proliferation of acinar tissue [5]. This delay in acinar cell maturation




has been postulated to prevent expression of cellular antigens at the critical time of self-




tolerance, resulting in inefficient clonal deletion of acinar tissue-reactive T cells. In contrast to




the role of IFN-y both prior to and during development of SS, IL-4 appears to be essential during




development of adaptive immunity and subsequent onset of glandular dysfunction. Specifically,




IL-4 was shown to be necessary for proper isotypic switching, regulating B lymphocyte




synthesis of pathogenic IgGI anti-muscarinic acetylcholine type III receptor (M3R)




autoantibodies [6, 7].




Although these earlier studies have implicated both ThI and Th2 cell-associated




functions in the development and onset of clinical SS, recent identification of the CD4'^ ThI 7




memory cells within the lymphocytic focus (LF) of lacrimal and salivary glands of SS^




C57BL/6.NOD-/Aec'//Aec2 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 ThI7 cell population, while clearly a subset of CD4'^ memory effector T cells, appears to be






3


distinct from, and unrelated to, either the ThI orTH2 cell lineages [10-14]. ThI 7 effector cells



secrete at least one of the six cytokines belonging to the IL-17 family, i.e., IL-17A, IL-17B, IL-



17C, IL-17D, IL-25 and/or IL-17F; however, IL-17A, the signature cytokine, has received the



greatest attention in studies of autoimmune diseases [15]. The I LI 7 cytokines are potent pro-



inflammatory molecules, actively involved in tissue inflammation via induction of pro-



inflammatory cytokine and chemokine expressions [16]. In addition, IL-17 is involved in the



mobilization, maturation and migration of neutrophils via the release of IL-8 at the site of injury



[17]. Interestingly, IL-17A is known to regulate Foxp3+ TReg cells and vice versa [18].



While ThI7 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 ThI



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 IL-17A and its activating



cytokine IL-23 in the lymphocytic infiltrates of the exocrine glands, as well as higher levels of



circulating IL-17A in both sera and saliva [8], raising the question of the importance of IL-17 in



SS. Thus, the goals of the present study were to determine whether IL-17A can directly



influence the pathology leading to the onset of SS-like disease by administrating exogenous IL-



17A to the salivary glands at specific time points.
4


MATERIALS AND METHODS




Animals




SS non-susceptible C57BL/6J 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 lACUC and IBC. Salivary glands of mice were




cannulated with mouse IL-17A-expressing Ad5-IL17A vector using retrograde injections at either




7 weeks (wks) of age (n=11) or 16 wks of age (n=8). In addition, mice at 6 wks (n=4) and 15




wks (n=4) were randomly selected and used as pre-treated or baseline analysis. Age- and sex-




matched control C57BL/6J mice (n=10 per age group) received the Ad5-LacZ control vector




using the same protocols.




Production of Ad5-LacZ and Ad5-IL17A 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 are based on the first




generation adenovirus serotype 5 (Ad5) and shown to produce their appropriate and functional




mouse IL- 17A and LacZ products [22-24]. To obtain sufficient viral vectors for the present




studies, each recombinant vector was amplified in HEK293 cells, purified by two rounds of CsCI




gradient centrifugation, then dialyzed against 100 mM Tris-HCI (pH 7.4), 10 mM MgCb and 10%




(v/v) glycerol, as described elsewhere [25].




Retrograde salivary gland cannulation of Ad5-LacZ or Ad5-IL17A vectors




Previous studies have demonstrated that retrograde salivary gland cannulation is an effective




method to direct local gene expression in the salivary glands [26-28]. In brief, prior to




cannulation, each mouse was anesthetized with a ketamine:xylazine mixture (100 mg/mL, 1






5


mL/kg body weight; Fort Dodge Animal Health, Fort Dodge, lA) 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 |jl of vector solution containing 10'^ viral particles). The cannulas were removed 5




minutes later to ensure successful cannulation.




Measurement of saliva flow




To measure stimulated saliva flow, individual non-anesthetized mice were weighed and given an




i.p. injection of 100 |jl of PBS containing isoproterenol (0.02 mg/ml) and pilocarpine (0.05




mg/ml). 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 -80C until analyzed.




Determination of cytokines levels:




Measurements of IL-6 and IL-17A cytokine levels in sera samples were performed by an




independent contractor (Millipore, Billerica, MA) using Luminex platform.




Intracellular cytokine staining and flow cytometric analysis




Spleens were freshly explanted, gently minced through stainless steel sieves, suspended in




phosphate buffered saline (PBS) and centrifuged (1200rpm for 5 minutes). Erythrocytes were




lysed by 7 minute incubation in 0.84% NH4CI. The resulting leukocyte suspensions were




washed two times in PBS, counted and resuspended inculture media (RPMI 1640 medium, 10%




FBS, 2 mM L-glutamine, 0.05 mM |3-mercaptoethanol) at a density of 2 x 10^ cells/ml. One




million cells were pipetted to individual wells of a 24-well microtiter plate pre-coated with anti-




CD3 (10 |jg/ml) and anti-CD28 antibodies (2 |jg/ml) forT cell activation. Cells were incubated for




5 hrs with Leukocyte Activation Cocktail containing GolgiPlug (2 |jl/ml). Collected cells were




6


fixed and permeabilized using Cytofix/CytopermFixation/Permeabilization. Flow cytometric




acquisition for intracellular staining was performed following staining with PE-Cy5-conjugated




anti-mouse CD4, FITC-conjugated anti-IFN-y and PE-conjugated anti-IL-17AA. The cells were




counted on a FACSCalibur (BD, Franklin Lakes, NJ) and analyzed by FCS Express (De Novo




Software, Los Angeles, CA).




Histology




Following euthanasia, whole salivary glands containing submandibular, sublingual, and parotid




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 |jm thickness.




Paraffin-embedded sections were de-paraffinized by immersing in xylene, followed by




dehydrating in ethanol. The tissue sections 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 (lymphocytic foci) in the histological sections of salivary glands.




Lymphocytic foci (LF) were defined as aggregates of >50 leukocytes quantified per each




histological section. Calculations were based on one histological section per mouse.




Immunofluorescent staining for CD3+T cells and B220+B 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 200X magnification using a Zeiss Axiovert 200M microscope.and images




were obtained with AxioVs40 software (Ver. 4.7.1.0, Zeiss) (Carl Zeiss, Thornwood,.




Enumeration of B, T cells and total number of nuclei in the LF were performed using Mayachitra




imago software (Mayachitra, Inc, Santa Barbara, CA)




7


Immunohistochemical staining for IL17A 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




4C with anti-IL-17A 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 strepavidin incubation using the Vectastain




ABC kit. The staining was developed by using diaminobenzidine substrate (Vector Laboratories,




Burlingame, CA), and counterstaining was performed with hematoxylin. Sections were




observed at 200X magnification using a Zeiss Axiovert 200M microscope. And images were




obtained with AxioVs40 software (Ver. 4.7.1.0, Zeiss) (Carl Zeiss, Thornwood). Enumeration of




IL17A-positive cells was performed on the entire histological sections of the whole salivary




glands using Mayachitra imago software (Mayachitra, Inc, Santa Barbara, CA), although




lymphocytic infiltrations are normally seen only in the submandibular glands.













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 per 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




200X 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).




8


Negative controls are secondary antibody only and positive controls are standard serum with



nuclear speckled pattern provided with the kits. Data presented in the results are from slides



using 1:40 dilutions of sera from each experimental group.








Statistical analyses



Statistical evaluations 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.
9


RESULTS
Induction of IL-17 A and IL-6 cytokine levels in sera following transduction with Ad5-




IL17A vector




Adenoviral vectors have been reported to show peak gene expressions around day 5




post-infusion which then persist for approximately 2 wks [29]. In the current study,




immunohistochemical staining for the presence of LacZ protein in the infused salivary glands




demonstrated that optimal transduction efficiency was approximately 26 5% at 2 wks post-




infusion which decreased to 15 3% by 9 wks post-infusion. The cells within the salivary




glands positive for LacZ expression were predominantly ductal cells, as expected, and acinar




cells (data not shown), indicating the virus was capable of passing through the ducts.




To determine if transduction of salivary glands with IL-17A alters the serum cytokine




profiles, serum preparations were assessed for temporal changes in pro-inflammatory cytokine




levels. Sera of treated mice were collected at days 5 and 12 post-treatment to determine the




efficacy of the IL-17A expressing viral vectors to affect cytokine secretions. As shown in Figure




1, C57BL/6J mice treated with the Ad5-IL17A vector at 10'^ viral particles per salivary gland




exhibited a marked increase in the levels of serum IL-17A compared to baseline levels or with




C57BL/6J mice receiving the control Ad5-LacZ vector at 10'^ viral particles per salivary gland,




demonstrating the efficacy of this viral vector to produce IL-17A. In addition, Ad5-IL17A-treated




C57BL/6J mice also secreted elevated amounts of the IL-17A-related cytokine IL-6 following




cannulation. Thus, the vectors gain access into the glands and apparently secrete IL-17A in




quantities that elevate systemic levels.








Increased numbers of IL-17A-producing CD4+ T cells in the spleens of Ad5-1L17A




transduced mice
10


As mentioned previously, salivary glands were cannulated with Ad5-IL17A vector at



either 7 wks or 16 wks of age. The time points chosen are based on extensive studies of the



development and onset of disease in our C57BL/6.NOD-/Aec'//Aec2 mouse model of SS [1-3, 30,



31]. The two time points selected represent the innate and adaptive immune response phases,



respectively, in the disease model, thus they were chosen to mimic these changes in the



parental C57BL/6 mouse. Microarray analyses examined the temporal differential gene



expression of salivary and lacrimal glands of C57BL/6 mice revealed gradual change in



pathophysiological related genes from 16-20 wks of age, concomitantly, leukocyte infiltration in



the exocrine glands is often observed at these ages [32, 33]. Thus, it is important to examine



the role of IL17A in the development of SS at prior and post to any pathophysiological changes.



Mice treated with Ad5-IL17A or Ad5-LacZ at either 7 wks or 16 wks of age were



euthanized at 26 and 27 wks of age, i.e. 19 wks and 11 wks post-treatment, respectively.



Splenocytes were isolated from individual mice and examined for the number of IFN-y and IL-



17A secreting CD4+T cells. Representative data, presented in Figures 2B &C, revealed that



the number of IL-17A secreting CD4+T cells in the spleens of mice receiving the Ad5-IL17A



vector at 7 wks of age was approximately 2-fold higher than mice receiving the control Ad5-



LacZ vector, while the number of IFN-y secreting CD4+T cells was approximately half at time of



analysis. Similarly, the number of IL-17A secreting CD4+T cells in the spleens of mice receiving



the Ad5-IL17A vector at 16 wks of age was approximately 7-fold higher than mice receiving the



control Ad5-LacZ vector, while the number of IFN-y secreting CD4+T cells was less than 50% at



time of analysis (Figure 2E & F). Results of a similar analysis with untreated mice performed



one wk prior to vector cannulations are presented in Figures 2A & D. These data suggest that



even though the Ad5 vector is considered locally restricted, the effect in C57BL/6 J mice



appeared systematic. More importantly, the systemic effects of IL17A in Ad5 appears to be



correlate with the duration of gene expression after vector cannulation as evidenced by the 2-



fold increase in the levels of IL-17A secreting cells at 19 wks post-treatment in younger mice but



11


a 7-fold increase at 11 wks post-treatment in the older group. However, one cannot rule out the




possibility that different efficacies are achieved based on the status of disease development in




different ages of mice.








Induction of SS immune-pathology in C57BL/6 mice following treatment with Ad5-IL17A




vector




Lymphocyte infiltration of the salivary and/or lacrimal glands is a critical criterion for identification




of the autoimmune phase of SS in both human and animal models. Although the number of LF




present in the salivary and lacrimal glands does not often correlate directly with disease or its




severity, SS patients and NOD-derived mouse strains exhibiting SS-like disease typically have




lymphocytic infiltrates in theirsalivary glands. IL-17A appears to play a critical role in the




development of LF and has recently been found to be present within LF in both SS patients and




animal models [8]. Salivary glands of C57BL/6J mice following cannulation with Ad5-IL17A




vector were examined for the presence of infiltrating leukocytes. Salivary glands retrieved from




C57BL/6J mice treated with Ad5-LacZ vector at either 7 or 16 wks of age revealed that 10% (1




of 10) in each group had evidence of glandular infiltrations (Figure 3A, B, G, H, Table 1). This




observation is consistent with the number of healthy, untreated C57BL/6J mice expected to




have infiltration of the salivary glands [8]. In contrast, salivary glands from C57BL/6J mice




treated with Ad5-IL17A vector at 7 wks of age showed infiltrations in 91% (10 of 11) with the




mean LF per histological section numbering 4 + 1.32, while salivary glands from C57BL/6J mice




treated with Ad5-IL17A vector at 16 wks of age revealed infiltrations in 75% (6 of 8) with a mean




LF number per histological section of 2 + 0.83 (Table 1).




Besides the number of LF detected in the salivary glands of the experimental animals,




immunofluorescent staining to detect B and T cells revealed further differences in the cellular




composition of the infiltrations between mice administered Ad5-IL17A at an early or late stage.




At time of euthanasia, C57BL/6J mice treated with Ad5-IL17A vector at 7 wks of age generally




12


exhibited smaller foci containing fewer IL-17 positive cells compared to mice receiving the




vector at 16 wks of age (Figure 3 C-F & l-L). Consistent with previous observation, the smaller




foci in mice treated at 7 wks of age may have resulted from the longer duration of time after




cannulation (19 wks) reflecting the decreases in IL-17A serum levels and IL-17A- positive cell




numbers. Detailed examination of IL-17A-positive cells revealed that a majority of IL-17A cells




are present in the LF and ductal cells with smaller percentage of positive cells found in the




epithelium and acinar cells. Nevertheless, these data support the concept that formation and




maintenance of LF are due, in part, to the expression levels of IL17A in the salivary glands.








Changes in ANA profiles following instillation of the Ad5-IL-17A vector




With the appearance of B and T lymphocytes within the salivary glands of Ad5-IL17A treated




C57BL/6 mice, plus the significant changes within their splenic ThI 7 and ThI cell populations,




the presence of circulating autoantibodies, specifically ANA, detectable by staining of HEp-2




cells was examined. To identify the presence of ANA, the sera prepared from blood samples




collected from each C57BL/6J mouse both pre- and post-cannulation were tested for reactivity




on HEp-2 cells. As presented in Figure 4A, the sera collected from C57BL/6J mice at 6 wks of




age or one wk prior to vector treatment showed a general weakly diffusedcytoplasmic and




nuclear background staining of the individual target cells. However, sera collected 19 wks post-




treatment from mice treated with Ad5-IL17A vector at 7 wks of age showed no cytoplasmic




staining with course speckled staining and negative nucleoli, while Ad5-LacZ treated mice




exhibited diffused cytoplasmic staining, weak but fine speckled nucleoplasmic staining with




negative nucleoli (Figures 4B & C). Similar results were seen in C7BL/6J mice whose salivary




glands were transduced with Ad5-IL17A vector at 16 wks of age in which the pattern was




pronounced course speckled staining with no cytoplasmic staining and negative nucleoli at 29




wks of age, or 11 wks post-treatment (Figures 4D-F). Considering the functions of IL-17A, it is




interesting to see a gradual and subtle change in ANA profile from diffused cytoplasmic/nuclear




13


pattern to a distinct course nuclear speckled pattern, suggesting influence of IL-17A on the B




cells repertoire.








Induction of salivary gland dysfunction in C57BL/6J mice following cannulation with




Ad5-1 LI7A vector




To determine if the expression of exogenous IL-17A can induce salivary gland dysfunction,




saliva volumes for each mouse were measured at 1 wk prior to treatment, then at 3-5 wk




intervals post-cannulation. C57BL/6J mice that received control Ad5-LacZ vector at 7 wks of




age exhibited stable stimulated saliva volumes at 7 wks post treatment with a statistically non-




significant increase in saliva volumes at 11 wks post treatment. Nevertheless, C57BL/6J mice




whose salivary glands were cannulated at 7 wks of age with Ad5-IL17A exhibited a significant




and relatively rapid decrease in stimulated saliva volumes that was most pronounced at 7 wks




post treatment, and this observation is seen even if the saliva volumes are converted to saliva




flow rates based on weights of the mice. After 7 wks post treatment, these mice showed a slight




recovery (Figure 5A). Similar results were observed with C57BL/6J mice cannulated at 16




wks of age with Ad5-LacZ and Ad5-IL17A vectors; however, no saliva volume recovery was




observed at time of euthanization (i.e., 11 wks post-treatment) (Figure 5B). Whether a reversal




of this inhibition would occur in these older animals will require further studies. Thus, saliva




secretions of mice receiving the Ad5-IL17A vector were significantly decreased 1-2 months




post-treatment when compared to secretions of mice receiving the Ad5-LacZ vector.
14


DISCUSSION




The THl7-derived IL-17A 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]. As the Th17/IL-17A system is considered to be an important factor in




innate immunity that, in turn, regulates development of the adaptive immune response, it is not




surprising that environmental microflora trigger IL-17A responses [34]. The consequence of




Th17/IL-17A activation includes, in addition to the production the IL-17A 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 IL-17A with its receptors evokes activation of IL-8, resulting in recruitment of




neutrophils to the site of injury. However, the relationship between such early




innate/inflammatory events mediated by the Th17/IL-17A system and the role Th17 cells play in




subsequent autoimmunity remains unknown, especially in light of the multiple functions now




associated with the ThI7 cell populations. Thus, in the present study, we have attempted to




elucidate the importance of the cytokine IL-17A perse in the development of SS and whether its




function may be dependent on when it is expressed.




Results in which SS-non-susceptible C57BL/6J mice were cannulated with the Ad5-




IL17A vector revealed that increased IL-17A expression could induce several pathological




features of SS, irrespective of whether the mice received the vector at 7 or 16 wks of age, two




time points corresponding to innate and adaptive immune responses in SS-susceptible




C57BL/6.NOD-/Aec'//Aec2 mice. This was noted by decreases in saliva production compared to




control vector, elevated production of specific pro-inflammatory cytokines detected in sera,




changes in the weak cytoplasmic/nuclear ANA patterns to nuclear specked staining on HEp2




cells and increased numbers of LF and IL17A positive cells present in the salivary glands at






15


time of euthanasia. Interestingly, mice received Ad5-IL17A at 7 wks of age showed a slight



recovery of saliva secretion at 7 wks of treatment in contrast to mice received Ad5-IL17A at 16



wks of age. This observation might be supported by the differential immunological or biological



response of mice at different ages and the effect of Ad5-IL17A exerted on the mice.



Previous studies have indicated that genes placed within Ad5 vectors are generally



expressed transiently and locally restricted (i.e., 7-14 days) [29]. The present study



demonstrates that a rapid and significant increase in the levels of plasma IL-17A was affected at



12 days post-cannulation by the Ad5-IL17A transgene vector. Interestingly, this systemic



increase in IL17 cytokine levels correlated with significant increases in splenic IL-17A secreting



CD4+T cells that persisted at least 19 wks for mice treated at 7 wks of age and 11 wks for mice



treated at 16 wks of age. These observations indicated that the Ad5 vector effect was longer



than anticipated. Whether this effect might be due to an indirect secondary effect of the Ad5-



IL17 vector is unknown. In addition, the systemic increase in IL17A production by local



treatment of Ad5-IL17A presented in this study is consistent with previous studies by Bruce



Baum's laboratory [35-38]. Adesanya et al. [39] has demonstrated that acinar cells can be



punctured by retrograde salivary gland cannulation at a certain vector dosage. The injured



acinar cells which have compromised mucosal barrier integrity allow for leakage of the vector



systemically. Further studies by Kagami et al. [37] and He et al. [40] provided evidence that



ductal cannulation of salivary glands can also have systemic effects due to the secretory nature



of the salivary glands which are well endowed with protein synthesis organelles and secretory



machinery.



Nevertheless, these observations are consistent with the concept that SS develops



along specific biological processes in a sequential fashion and interference with this process



alters development of disease [1-3]. Therefore, this study clearly indicates the pathogenic



nature of IL-17A in inducing SS-like phenotypes when cannulated in the salivary glands.
16


Previous data have shown that lymphocytic infiltrates in the salivary glands secreting IL-17A



and its related cytokines are more important in local glandular destruction. Staining salivary



glands for IL-17A revealed that C57BL/6J mice receiving Ad5-IL17A vector not only expressed



significant levels of IL-17A, but that IL-17A levels correlated with recruitment of inflammatory



cells, specifically B and T cells, to the glands. This observation is important in light of the recent



study suggesting IL-17A is a critical factor in the adaptive immune response by inducing the



formation of germinal centers for the production of autoreactive antibodies [24]. Autoantibodies



represent a major component in the onset of SS, thus the changes in the ANA profiles observed



with sera of C57BL/6J mice cannulated with the Ad5-IL17A vector indicate that IL-17A affects



even the B cell compartment in SS-non-susceptible mice. The presence of LF and loss of saliva



secretion raises an interesting question about the possible role of IL-17A in B cell activation. As



BAFF is capable of inducing ThI 7 cell differentiation in addition to regulating B cell activation



[41], the possible role of BAFF and IL17A in this phenomenon needs to be better defined in SS



pathogenesis.
17


CONCLUSIONS




The capability of IL-17A to induce features of SS in SS-non-susceptible mice demonstrates the




major role this cytokine plays in the development, and possibly onset, of the autoimmune




process. How this one cytokine affects the various features of autoimmunity, and at what level




or time point, will require additional studies. More importantly, the study demonstrates that IL-




17A might be a potential therapeutic target for SS.
18


LIST OF ABBREVATIONS




SS: Sjogren's Syndrome, IL: Interleukin, Ad5: Adenovirus serotype 5, IFN-y: Interferon-y, EAE:




Experimental autoimmune encephalomyelitis, CIA: Collagen-induced arthritis, LF: Lymphocytic




Focus, ANA: Antinuclear antibodies, MIP-2: Macrophage inflammatory protein-2, CXCL1:




Chemokine (C-X-C motif) ligand, MMP: matrix metalloproteases, BAFF: B cell activating factor.
19


COMPETING INTERESTS




The authors declare no conflicts of interest.


AUTHORS' CONTRIBUTIONS
JAC produced and determined the titers of the Ad5-LacZ and Ad5-IL17A viral vectors. HY and



BL performed retrograde ductal cannulations/instillations of the vectors into the salivary glands.



CQN designed the study, performed saliva flow, flow cytometry, histology and statistical



analyses, and prepared the manuscript. WC carried out the ANA staining. ABP assisted in the



manuscript preparation. All authors read and approved the final manuscript.
21


ACKNOWLEDGEMENTS




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-IL17A 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.




Publication of this article was funded in part by the University of Florida Open-Access publishing




Fund
22


FUNDING




This work was supported by PHS grants K99DE018958 (CQN) from NIDCR, R21AI081952




(ABP) from NIAID and funds from the Sjogren's Syndrome Foundation and Center for Orphan




Autoimmune Disorders. HY and JAC were supported by an NIH, NIDCR intramural research




grant.
23


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29


FIGURE LEGENDS:
Figure 1. Rapid changes in IL-17A and IL-6 serum cytokine concentrations in C57BL/6J



mice following vector cannulations. Sera were prepared from blood collected from individual



5 wk old mice (n=4) randomly chosen 1 wk prior to vector treatment (day 0 on the graph). Mice



were allowed to acclimate for 7 days, followed by vector instillation of each salivary gland with



50 |jl of vector solution containing 10'^ viral particles of either Ad5-LacZ or Ad5-IL17A vector.



Sera were again prepared from blood collected from individual mice (n=11) at day 5 and day 12



post-treatment. Concentrations of cytokines were determined using the Luminex platform. To



ensure sufficient quantities for testing, the sera of three individual mice of each experimental



group were pooled. ND: not detected indicates levels below threshold detection.








Figure 2. Intracellular staining for IL-17A and IFN-y secreting CD4'^T cells in spleens of



Ad5-IL17A-treated mice. Splenic leukocytes prepared from C57BL/6J mice (n=4) at 6 wks of



age (one wk prior to vector treatment) and 26 wks old (19 wks post vector treatment),



considered early treatment (A-C), or splenic leukocytes prepared from C57BL/6J mice (n=4) at



15 wks of age (one wk prior to vector treatment) and 27 wks old (11 wks post vector treatment),



considered late treatment (D-F) were examined for the presence of intracellular IL-17A and IFN-



Y gated on CD4'^T cells following a 5 hr in-vitro activation with anti-CD3£ and anti-CD28 in



Leukocyte Activation Cocktail containing GolgiPlug. Flow cytometric acquisition was performed



by staining with PE-Cy5-conjugated rat anti-CD4, FITC-conjugated rat anti-IFNy and/or PE-



conjugated rat anti-IL-17A. Data was analyzed by FCS Express. Flow cytometric images shown



are from one representative analysis of 2 independent experiments that examined 2 different



mice within each experiment. Data presented as mean + SEM for n=4 per group and statistical



analyses were performed comparing the means of the Ad-LacZ and Ad-IL17A treated groups at
30


26 wks and 27 wks of early and late treatment, respectively. (*) indicates p<0.5 using Mann-




Whitney U test.








Figure 3. Histological examination of salivary glands. Salivary gland histology was




examined at 19 wks post-vector infusions of mice treated at 7 wks of age (early treatment) or at




11 wks post-vector infusions of mice treated at 16 wks of age (late treatment). Panels show




representative H&E staining of salivary gland tissue from mice receiving early treatment with




Ad5-LacZ (n=10) (A), or Ad5-IL17A (n=11) (B); fluorescent staining and enumeration of B and T




cells in Ad5-IL17A treated mice (C & D) and immunohistochemical staining and enumeration of




IL-17A-positive cells in Ad5-IL17A treated mice (E & F); H&E staining of salivary gland tissue




from mice receiving late treatment with Ad5-LacZ (n=10) (G), or Ad5-IL17 (n=8) (H); and




fluorescent staining and enumeration of B and T cells in Ad5-IL17A treated mice (I & J) and




immunohistochemical staining and enumeration of IL-17A-positive cells in Ad5-IL17A treated




mice (K & L). Black arrows indicate representative lymphocytic infiltrate.








Figure 4. Identification of the antinuclear antibodies in sera of C57BL/6J mice.




Representative patterns of cellular staining of HEp-2 cells by sera diluted at 1:40 prepared from




sera taken from C57BL/6 mice cannulated with Ad5-LacZ or Ad5-IL17A vectors at 7 wks of age




with pre-treated mice (baseline) at 6 wks of age (n=4) (A-C), and cannulated at16 wks of age




with Ad5-LacZ or Ad5-IL17A and pre-treated mice (baseline) at 15 wks of age (n=4) (D-F) with




negative control using secondary antibody only (G) and positive control with standard nuclear




speckled serum (H). Representative patterns were determined with n=4 for two baselines and




n=7 for each time point presented in the figure. Fixed HEp-2 substrate slides were incubated




with individual mouse sera diluted 1:40, 1:80 and 1:160 followed by development with FITC-




conjugated goat anti-mouse IgG. Fluorescent patterns were detected by fluorescence




microscopy at 400X magnification.




31


Figure 5. Stimulated saliva flow in treated C57BL/6J mice. One week prior to salivary gland



cannulations with either Ad5-LacZ or Ad5-IL17A vector, stimulated saliva volumes were



determined for individual mice within each of the four experimental groups: early treatment with



Ad5-LacZ (n= 10) or Ad5-I LI 7A (n= 11) at 7 wks of age (A) or late treatment with Ad5-LacZ



(n=10) or Ad5-IL17A (n=8) at 16 wks of age (B). Saliva was collected every 3-5 wks post-



treatment until the mice were euthanized. Statistical analysis was used to determine the



significance between the Ad5-LacZ and Ad5-IL17A treated mice at each time point. (NS: not



significant, p=*<0.05, p=**<0.01, p=***<0.001). Arrows indicate the initial time point of vector



cannulation.
32


Table 1: Quantification of lymphocytic foci (LF) in salivary glands
Ad5:LacZ Ad5:IL17A
NoLF LF Mean LF NoLF LF Mean LF
Early 9^ (90%)'' 1 (10%) 1 1 (9%) 10(91%) 4 + 1.32"
Late 9 (90%) 1 (10%) 1 2 (25%) 6(75%) 2 + 0.83
^ number of mice
'percentage of mice
'^ mean number of LF + SEM per histological salivary gland section
LF; lymphocytic foci, Ad5; Adenovirus serotype 5, IL; interleukin.
33


IL-17 A
5000-1



4000-
- 3000-



Q. 2000-
1000-



0
^^day 0
Figure 1
IL-6
500-1



400
- 300.


OS
Q.200.
100<



0'
^UUS^JU^_
LE^
Iday5 ^3day 12
499999999999


Early treatment
A. 6 wks old mice (Pre-treatment) B. 26 wks old mice: Ad5-LacZ
iu : 1.520.21
10'- . .
'"- '-'''. "
t ^ ii?^ -

in'^ ^tt ^jUB^.___~
PUft^^-^Pi,?:;;
B^^^^HI ^'. "-^ ?. ^ -'
m"- W^ >-^' 4.59+0.23.
C. 26 wks old mice: Ad5-IL17A
10 10 10 10
10"
10 10
Late treatment
D. 15 wks old mice (Pre-treatment) E. 27 wks old mice: Ad5-LacZ
F. 27 wks old mice: Ad5-IL17A
Figure 2


^Ad5-LacZ > ^^^^^ K^5-IL17A "J
C. Ad5:IL-17A
D.A 100i |.. g CD-'S S) 40-S 20-a. d5-IL17A
B220 CDS: Red Green Blue


CD3+ B22Q+


F. Ad5-IL17A
r-Kia/ ^i^
0 4 8 12 16 20
Percentage of cells per histological section
G. Ad5-I
Ad5-IL17A
Ts"*^ Ji f-- f' *'~

Figure 3
Ji.S4

) 10 15 20 25 30 35
Percentage of cells per histological section


Early treatment (cannulated at 7 wks old)
A. 6 wks old C57BL/6
Late treatment (cannulated at 16 wks old)
D. 15 wks old C57BL/6
B. Ad5-LacZ 26 wks
C. Ad5-L17A 26wks
E. AdS-LaczZ 29 wks
Controls
G. Negative control
H. Nuclear speckled control
F. Ad5-L17A 29 wks
!<;, '''. c" itV 1
>
z':*^ O" ft.
K ^i >
3 A
g* 9 1^ e "ci (
Figure 4


A. 7 weeks old mice cannulation
250
B. 16 weeks old mice cannulation
200
'^
"#-
-^ .
Week(s) post vector treatment
AdS-LacZ
-1 t 2 5 8
Week(s) post vector treatment

-Ad5-IL17A
Figure 5


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