Molecular Subsetting of Interferon Pathways in Sjögren's Syndrome

Abstract

Objective: Sjögren's syndrome (SS) is an autoimmune disease that targets the salivary and lacrimal glands. While all patients demonstrate inflammatory infiltration and abnormal secretory function in the target tissues, the disease features, pathology, and clinical course can vary. Activation of distinct inflammatory pathways may drive disease heterogeneity. The purpose of this study was to investigate whether activation of the interferon (IFN) pathway correlates with key phenotypic features.

Methods: Clinical data and 1 labial salivary gland (stored frozen) were obtained from each of 82 participants (53 patients with primary SS and 29 control subjects) in the Sjögren's International Collaborative Clinical Alliance (SICCA) registry. Salivary gland lysates were immunoblotted with markers of type I or type II IFN, and patterns of IFN activity were determined by hierarchical clustering. Correlations between SS phenotypic features and IFN activity in the salivary gland were performed.

Results: A total of 58% of the SS participants had high IFN activity and differed significantly from those with low IFN activity (higher prevalence of abnormal findings on sialometry, leukopenia, hyperglobulinemia, high-titer antinuclear antibody, anti-SSA, and high focus score on labial salivary gland [LSG] biopsy). Three distinct patterns of IFN were evident: type I-predominant, type II-predominant, and type I/II mixed IFN. These groups were clinically indistinguishable except for the LSG focus score, which was highest in those with type II-predominant IFN.

Conclusion: The SS phenotype includes distinct molecular subtypes, which are segregated by the magnitude and pattern of IFN responses. Associations between IFN pathways and disease activity suggest that IFNs are relevant therapeutic targets in SS. Patients with distinct patterns of high IFN activity are clinically similar, demonstrating that IFN-targeting therapies must be selected according to the specific pathway(s) that is active in vivo in the individual patient.

Figure 1. Distinct patterns of IFN activity are evident in lysates made from LSG biopsies from SS participants

Protein lysates made from control (n=29) or SS (n=53) LSG biopsies were probed for IFN activity by Western blotting. A marker of type I IFN (IFIT3) and type II IFN (GBP1) is included. Vinculin was analyzed as a loading control.

Figure 2. Correlation of clinical features with IFN activity in LSG biopsies from SS participants

(A) IFN-induced protein expression in SS participants from Fig. 1 was quantified by densitometry and normalized to the level of vinculin expression in the same sample. The vinculin-normalized expression values were subjected to unsupervised hierarchical clustering to define patterns of IFN activity in each patient. (B) The clinical features for each individual patient are presented in line with the respective clustering data. White represents a negative value and colored blocks represent a positive value. Serologic marker values and units are as follows: WBC < 4000/µL, IgG > 1445 mg/dL and IgA > 400 mg/dL. P-values for clinical features which satisfy statistical significance between IFN low and IFN high groups are given.

Figure 3. Distinct patterns of IFN activity are evident in LSG paraffin sections from SS patients

LSG biopsies from 6 patients, each with distinct biochemically defined patterns of IFN activity (type I-predominant, type II-predominant and type I and type II equal), were stained with antibodies against IFIT3 (A, C and E) and GBP2 (B, D and F). Representative images from one patient with each pattern (A&B, type I- predominant; C&D, type II-predominant and E&F, type I/II equal) are shown. Scale bar = 100 µm.

Figure 4. CD45+ infiltrates in tissues are associated with high type II IFN activity

CD45 expression from Supplemental Figure 1 was quantified by densitometry and normalized to the level of β-actin in the same sample. Participants were divided roughly into tertiles based on normalized CD45 expression (CD45 low (n=17), CD45 intermediate (n=18) and CD45 high (n=18)). Normalized-GBP1 (A), and normalized-IFIT3 (B) expression levels were compared for each CD45 group. Error bars represent median, IQR. *** p=0.0004; Kruskal-Wallis test.