17-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 TH
17/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
mice is well-defined at both the genetic and pathological levels (1
), 2) histological examinations of salivary gland biopsies from both SS patients and C57BL/6.NOD-Aec1Aec2
mice indicate the presence of the IL-23/TH
17/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
). 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
), 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
), 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
). 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 TH
2 and TH
1 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 TH
1 cells producing IFN-γ which directly mediates the up-regulation of adhesion molecules, consequently recruiting inflammatory cells such as TH
2 and TH
17 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.