SS is a chronic autoimmune disease of the exocrine glands and is characterized by an infiltration of lymphocytes and a female predominance. Although the pathogenesis of SS remains to be determined, the pathologic hallmark is an extensive lymphocytic infiltration of the salivary glands [1
]. The majority of infiltrating cells are T cells, and approximately 60% to 70% of the infiltrating T cells bear the CD4 phenotype [28
], suggesting that CD4+
T cells contribute to the pathophysiology of SS [29
T-cell subsets, Th17 cells are a unique CD4+
T-cell subset and are characterized by production of IL-17. IL-17 is a highly inflammatory cytokine with robust effects on stromal cells, resulting in the production of inflammatory cytokines and recruitment of leukocytes, and this creates a link between innate and adaptive immunity [19
]. It is well known that Th17 cells and IL-17 play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis [30
], RA [32
], multiple sclerosis [34
], inflammatory bowel disease [35
], and asthma [36
]. In regard to the study of IL-17 in SS, previous studies support the finding that IL-17 or Th17 cells or both are upregulated in the salivary glands of patients with SS [22
]. However, the pathophysiologic role of IL-17 is still undetermined.
As mentioned in the Introduction, TLRs, leading players in adaptive immunity as well as in innate immunity, have been thought to play a role in the pathogenesis of various human autoimmune inflammatory diseases. In addition, it has been reported that the expression of TLRs is upregulated in the salivary glands of patients with SS [13
]. Thus, it can be speculated that increased TLRs and IL-17/Th17 cells in salivary glands might closely interact and so contribute to the pathogenesis of SS.
There have been discordant results in regard to the role of TLR stimulation in Th17 cell differentiation in humans and mice. Like us, Aliahmadi and colleagues [37
] demonstrated that TLR2 activation promotes human Th17 polarization. Yu and colleagues [38
] showed that human plasmacytoid DCs support Th17 cell effector function in response to TLR7 ligation. In contrast, Loures and colleagues [39
] reported that TLR2 is a negative regulator of Th17 cells in mice.
In this study, we demonstrated that the expressions of TLR2, TLR4, and TLR6 were increased in the salivary glands of patients with SS in comparison with the disease controls. Moreover, not only IL-17 but IL-6 and IL-23, the major promoting factors in Th17 differentiation and amplification, were highly expressed. Using PBMCs from patients with SS, we also showed that stimulation of TLR2, TLR4, and TLR6 with specific ligands additively promoted the production of IL-17 and IL-23 in the presence of TLR2 stimulation, thus verifying that the increased TLRs and IL-17 in the salivary glands of patients with SS are biologically meaningful. Eventually, we investigated the signaling pathway by which TLR2 stimulation induces the production of IL-17 and IL-23 and we demonstrated that the IL-6, STAT3, and NF-κB pathways are implicated in TLR2-stimulated production of IL-23 and IL-17.
It is known that glandular epithelial cells appear to have the central role in the induction and perpetuation of tissue inflammatory reactions in patients with pSS [40
] and have an increased rate of apoptosis [42
]. Given the central role of glandular epithelial cells in local immune response and the immunohistochemical results presented in our study showing variably positive staining of ductal epithelial cells for IL-17 and IL-23 (Figure ), the role of epithelial cells in directing local immune responses could be more direct. Further investigations are needed to clarify the issue.
There have been a few previous reports that have tried to verify the role of Th17 cells and its associated cytokines like IL-17, IL-23, and IL-6 in patients with SS [22
]. Like our report, these reports demonstrated that the expressions of Th17-associated cytokines in salivary glands are significantly higher in patients with SS than in controls (Figures and ). However, conflicting results exist when it comes to the circulating levels of Th17-associated cytokines like IL-17, IL-23, and IL-6 in patients with SS. Like us, Katsifis and colleagues [22
] showed that the serum levels of IL-17, IL-23, and IL-6 are significantly higher in patients with SS (Figure ). The authors also showed that serum levels of IL-6 and IL-23 are positively correlated with ESR in patients with SS [22
]. In contrast, Nguyen and colleagues [23
] reported that there were no differences in the serum levels of IL-17 and IL-6 between patients with SS and controls, although the IL-6 levels in the saliva from patients with SS exhibited nearly a threefold increase over those in the saliva from controls subjects. Studies that include a large number of patients will be required to clarify this discrepancy between the different studies.
The association between the IL-17 expression and immunopathologic features has been described [22
]. One previous report showed that IL-17+
mononuclear cell infiltrations in salivary glands progressively increased with higher biopsy focus score (P
< 0.0001) and that the IL-17 mRNA expression of whole salivary gland positively correlated with ESR levels [22
]. The report also showed that TGF-β, IL-6, and IL-23, which are the requisite promoters of Th17 differentiation, were found in abundance in the salivary glands of patients with SS, thus demonstrating that the microenvironment of salivary glands in patients with SS is full of factors that are known to foster local Th17 lineage polarization [22
]. In our study, not only IL-17 but also IL-23, IL-6, IL-1β, and TNF-α, which are known to promote Th17 differentiation and amplification [19
], were highly expressed in the salivary glands of patients with pSS in comparison with the disease controls (Figures and ); similarly, another report showed that TNF-α, IL-1β, and IL-6 have consistently been detected in pSS minor salivary gland biopsy and conjunctiva samples [29
]. In addition, the expression of IL-17/IL-23 in salivary glands tended to be higher in patients with SS with higher biopsy focus score. However, in that report, like a previous report [23
], there was no direct correlation between the IL-17/IL-23 expressions and clinical profiles such as the degree of abnormal salivary flows, the presence of antinuclear antibody, rheumatoid factor, anti-Ro, anti-La, and extraglandular involvements (data not shown).
The biological relevance of our data requires comment. We examined the pathophysiologic mechanism of the TLR/IL-17 pathway in patients with SS by using PBMCs. Thus, we failed to clarify the role of epithelial cells, which are thought to be central players in the pathogenesis of pSS [40
]. However, although the ductal epithelial as well as the infiltrating mononuclear cells expressed TLRs in our results and previous reports [13
], IL-17, the main effector cytokine in Th17 cells, is produced mainly by activated CD4+
T cells. In this study, we also demonstrated that the major sources of IL-17 are CD4+
T cells by using PBMCs (Figure ). Ductal epithelial cells were variably positive for IL-17 stain, whereas IL-17 was highly expressed mainly in the infiltrating CD4+
T cells in the salivary glands of patients with SS (Figure ). Therefore, it seems that our data using PBMCs are biologically relevant, although we did not completely rule out IL-17 production by ductal epithelial cells.