Activation of IFN pathways and plasmacytoid dendritic cell recruitment in target organs of primary Sjögren's syndrome

Abstract

Gene expression analysis of target organs might help provide new insights into the pathogenesis of autoimmune diseases. We used global gene expression profiling of minor salivary glands to identify patterns of gene expression in patients with primary Sjögren's syndrome (pSS), a common and prototypic systemic autoimmune disease. Gene expression analysis allowed for differentiating most patients with pSS from controls. The expression of 23 genes in the IFN pathways, including two Toll-like receptors (TLR8 and TLR9), was significantly different between patients and controls. Furthermore, the increased expression of IFN-inducible genes, BAFF and IFN-induced transmembrane protein 1, was also demonstrated in ocular epithelial cells by quantitative RT-PCR. In vitro activation showed that these genes were effectively modulated by IFNs in salivary gland epithelial cells, the target cells of autoimmunity in pSS. The activation of IFN pathways led us to investigate whether plasmacytoid dendritic cells were recruited in salivary glands. These IFN-producing cells were detected by immunohistochemistry in all patients with pSS, whereas none was observed in controls. In conclusion, our results support the pathogenic interaction between the innate and adaptive immune system in pSS. The persistence of the IFN signature might be related to a vicious circle, in which the environment interacts with genetic factors to drive the stimulation of salivary TLRs.

Fig. 1.

Unsupervised hierarchical clustering of 120 top differentially expressed genes in patients with pSS. Expanded view of gene expression of selected differentially expressed genes in patients with pSS and controls. Expression level is shown by color: red, relatively high expression; green, low expression. The seven pSS samples clustered together and six of seven controls clustered together. Only one control sample (C10) was more closely associated with the pSS samples.

Fig. 2.

Mean ratio of 424 differentially expressed genes in patients with pSS to those in controls. Mean pSS/controls ratio of the 424 differentially expressed genes in pSS. The 23 IFN-inducible genes are in red. Twenty-one of these genes showed significantly increased expression in pSS, and two showed significantly decreased expression.

Fig. 3.

Quantitative real-time RT-PCR analysis of expression of IFN-inducible genes in salivary glands and ocular epithelial cells. (A) Quantitative RT-PCR analysis of gene expression of IFN-inducible genes and BCMA in salivary glands. Gene expression of TLR8, TLR9, IFITM1, BAFF, and BCMA was significantly increased, and SOCS3 expression was significantly decreased in patients with pSS (n = 10) compared with controls (n = 10). (B) RT-PCR analysis of expression of IFN-inducible genes in ocular epithelial cells of patients with pSS (n = 5) and controls (n = 8). BAFF and IFITM1 expression were significantly increased in patients with pSS.

Fig. 4.

In vitro IFN induction of IFITM1 in salivary gland epithelial cells. Salivary gland epithelial cells were cultured in vitro after minor salivary gland biopsy of patients with pSS (n = 4) and controls (n = 6). IFITM-1 gene expression in epithelial cells was assessed by using quantitative RT-PCR at baseline and after IFN-α (2,400 unit/ml) or IFN-γ (5 ng/ml) stimulation during 2 days. Salivary gland epithelial cells showed a strong and significant increased expression of IFITM1 mRNA after stimulation with IFN-α and IFN-γ.

Fig. 5.

Identification of pDCs in the salivary glands of patients with pSS. Immunohistochemical study of salivary glands of patients with pSS (n = 5) and controls (n = 9). The results of one patient with pSS (A, C, and E) and one control (B, D, and F) are presented. pDCs, identified as CD123 positive cells (A), were present in all of the patients with pSS. Similarly appearing plasmacytoid cells stained positively for BDCA-2, a specific marker of pDCs (C), and TLR-9 (E). No pDCs were observed in controls.