Type I and II interferon signatures in Sjogren's syndrome pathogenesis: Contributions in distinct clinical phenotypes and Sjogren's related lymphomagenesis

Abstract

Both type I and II interferons (IFNs) have been implicated in the pathogenesis of Sjogren's syndrome (SS). We aimed to explore the contribution of type I and II IFN signatures in the generation of distinct SS clinical phenotypes including lymphoma development. Peripheral blood (PB) from SS patients (n = 31), SS patients complicated by lymphoma (n = 13) and healthy controls (HC, n = 30) were subjected to real-time PCR for 3 interferon inducible genes (IFIGs) preferentially induced by type I IFN, 2 IFIGs preferentially induced by IFNγ as well as for IFNα and IFNγ genes. The same analysis was performed in minor salivary gland tissues (MSG) derived from 31 SS patients, 10 SS-lymphoma patients and 17 sicca controls (SC). In PB and MSG tissues, overexpression of both type I and type II IFIGs was observed in SS patients versus HC and SC, respectively, with a predominance of type I IFN signature in PB and a type II IFN signature in MSG tissues. In SS-lymphoma MSG tissues, lower IFNα, but higher IFNγ and type II IFIG transcripts compared to both SS and SC were observed. In receiver operating characteristic curve analysis, IFNγ/IFNα mRNA ratio in MSG tissues showed the best discrimination for lymphoma development. Discrete expression patterns of type I and II IFN signatures might be related to distinct SS clinical phenotypes. Additionally, IFNγ/IFNα mRNA ratio in diagnostic salivary gland biopsies is proposed as a novel histopathological biomarker for the prediction of in situ lymphoma development in the setting of SS.

Keywords: B cell activating factor; Lymphomagenesis; Sjogren's syndrome; Type I interferon; Type II interferon.

Fig. 1. Type I and II interferon inducible genes (IFIGs) as well as type I and II interferon (IFN) scores in peripheral blood (PB) of Sjogren's syndrome (SS) patients and healthy controls (HC)

A. Normalized relative mRNA expression of preferentially type I IFIGs in PB samples derived from 30 HC, 31 SS-non lymphoma and 13 SS-lymphoma patients. Transcript levels of the MX-1, IFIT-1 and IFI44 genes were lower in HC compared to both SS non-lymphoma (p-value: <0.0001 for all comparisons) and SS-lymphoma groups (p-values: <0.0001, 0.0004 and 0.0002, respectively). No statistically significant differences were detected between the SS subgroups. B. PB type I IFN score was found to be significantly higher in both SS subgroups compared to HC (p-values: <0.0001 for both comparisons), with no significant differences between SS subgroups. C. Normalized relative mRNA expression of the preferentially type II IFIGs GBP-1 and MIG-1 in PB from 30 HC, 31 SS-non lymphoma and 13 SS-lymphoma patients. Transcript levels of the GBP-1 and MIG-1 genes were lower in HC compared to both SS non-lymphoma (p-values: 0.001 and 0.008, respectively) and SS-lymphoma groups (p-values: 0.003 and 0.02, respectively), with no detectable differences between SS subgroups. D. PB type II IFN score was found to be significantly increased in both SS subgroups complicated or not by lymphoma compared to HC, p-values: 0.004 and 0.0005, respectively. (*p<0.05, ** p<0.01, *** p<0.001). RE: Relative expression.

Fig. 2. Type I and II IFN inducible genes (IFIGs) as well as type I and II interferon (IFN) scores in minor salivary glands (MSG) from Sjogren's patients (SS) and sicca controls (SC)

A. Normalized relative mRNA expression of the preferentially type I IFIGs MX-1, IFIT-1 and IFI44 in MSG tissues from 17 SC, 31 SS-non lymphoma and 10 SS-lymphoma patients. Transcript levels of the MX-1, IFIT-1 and IFI44 genes were lower in SC compared to both SS non-lymphoma (p-value: 0.02, 0.02 and 0.005, respectively) and SS-lymphoma groups (p- values: 0.01, 0.04 and 0.07, respectively), while no statistically significant differences were observed between SS subgroups. B. Type I IFN score was significantly increased in both SS subgroups complicated or not by lymphoma compared to SC (p-values: 0.004 and 0.0003, respectively), with no significantly detectable differences between SS subgroups. C. Normalized relative mRNA expression of the preferentially type II IFIGs GBP-1 and MIG-1 in MSGs from 17 SC, 31 SS-non lymphoma and 10 SS-lymphoma patients. Transcript levels of the GBP-1 and MIG-1 genes were significantly decreased in SC vs. both SS non-lymphoma (p-values: <0.0001 for both genes) and SS-lymphoma (p-values: 0.0002 and 0.0001, respectively); of note, significantly increased MIG-1 transcript levels were observed in SS-lymphoma compared to SS-non lymphoma group (p-value: <0.05). D. Type II IFN score was found to be significantly increased in both SS subgroups complicated or not by lymphoma compared to SC (p-values: 0.0001 and <0.0001, respectively), as well as in SS-lymphoma patients compared to the SS-non lymphoma group, p-value: 0.045. (*p<0.05, **p<0.01, ***p<0.001). RE: Relative expression.

Fig. 3. IFNγ and IFNα mRNA expression in MSG tissues

A. Normalized relative mRNA expression of IFNα and IFNγ genes in MSG tissues from 17 SC, 31 SS-non lymphoma and 10 SS-lymphoma patients. IFNα mRNA expression was significantly reduced in SS-lymphoma cases compared to both SS-non lymphoma and SC groups (p-values: 0.003 and 0.005, respectively). On the other hand, IFNγ transcript levels were significantly increased in both SS subgroups complicated or not by lymphoma compared to SC (p-values: <0.0001 and 0.007 respectively), as well as in the SS-lymphoma compared to the SS-non lymphoma group (p-value: 0.02). B. A significantly higher IFNγ/α ratio normalized relative mRNA expression was observed in MSG tissues derived from SS patients complicated by lymphoma compared to both SS-non lymphoma patients and SC (p-values: 0.004 and 0.0007, respectively). C. Area under the receiver operating characteristic (AUC-ROC) curves for gene transcripts in SS patients at the level of MSGs tissues. AUC-ROC curves for the ratio of the IFNγ/IFNα mRNA expression discriminates the SS lymphoma group (n=10) from SS-non lymphoma patients (n=31), (AUC=0.88, 95%CI: 0.7-1.0, p-value: 0.001). * p<0.05, ** p<0.01, *** p<0.001, RE: Relative expression.

Fig. 4. Immunohistochemical detection of IFNα, BDCA-2, IFNγ, CD3 and CD20 protein in MSG tissues from an SS patient without lymphoma (A, B) and an SS patient complicated by lymphoma (C-E)

A) IFN-α stained positive cells in a non lymphoma SS patient (representative cell indicated by arrow, ×400 magnification). B) Positive BDCA-2 (a specific marker for PDCs) stained cells in the same patient (representative cell indicated by arrow, ×630 magnification). C) Positive IFN-γ staining within lymphocytic infiltrates and salivary gland epithelia (×400 magnification) in representative tissue from an SS patient complicated by lymphoma. D) CD3+ T-lymphocytes and E) CD20+ B-cells in the same patient (×400 magnification).

Fig. 5. Association of IFNα mRNA transcripts with apoptotic gene mRNA expression at the level of MSG tissues

Relative expression of IFNα as well as TRAIL, p53 and Ro52 (TRIM21) was measured by real-time PCR in MSG tissues obtained from SS patients. Associations between IFNα mRNA and the apoptotic genes were tested using Spearman's non-parametric correlation test. IFNα transcript levels were positively correlated with the mRNA transcripts of the pro-apoptotic genes TRAIL and p53 (r=0.53, p-value: 0.02 and r=0.55, p-value: 0.002, respectively, panels A and B), as well as the mRNA transcripts of the Ro52 (TRIM21) (r=0.71/p=0.0002, panel C) at the level of MSG tissues. RE: Relative expression.

Fig.6. Association of type I and II IFN scores with B cell activating Factor (BAFF) mRNA expression and serum protein levels

A. Normalized relative mRNA expression of the BAFF gene at the level of peripheral blood samples from 30 HC, 31 SS-non lymphoma and 13 SS-lymphoma patients. BAFF transcript levels were significantly increased in both SS subgroups complicated or non by lymphoma compared to HC (mean±SD: 1.97±1.32 vs 1.61±0.70 vs1.09±0.36, p-values: 0.005 and 0.002, respectively), with no significantly detectable difference between SS subgroups B. Peripheral blood BAFF mRNA transcripts were positively correlated with type I IFN score in SS patients (r=0.43, p=0.0001). C.. Peripheral blood BAFF mRNA transcripts were also positively correlated with type II IFN score in SS patients (r=0.44, p=0.0002). D. BAFF protein levels in serum samples from 24 HC, 62 SS-non lymphoma and 23 SS-lymphoma patients. SS-lymphoma patients had higher BAFF serum levels compared to both SS-non lymphoma and HC (mean ±SD: 1648.00±1178.00 pg/ml versus 1052.00±793.20 pg/ml versus 781.5±147.5 pg/ml, p-values: 0.002 and <0.0001, respectively), while SS-non lymphoma patients showed also increased serum BAFF levels compared to HC (p=0.03). E-F. A relatively weak correlation was observed between BAFF serum protein levels with peripheral blood type I IFN score (r=0.30, p-value=0.04), while type II IFN score was not correlated with BAFF serum levels in SS patients. G. BAFF normalized relative mRNA expression in MSG tissues from 17 SC, 31 SS-non lymphoma and 10 SS-lymphoma patients. BAFF mRNA levels were found to be increased in both SS subgroups complicated or not by lymphoma compared to SC (p-value: 0.02, for both comparisons). H-I. BAFF mRNA transcripts in MSG tissues were found to positively correlate with both type I and II IFN MSG tissue scores (r=0.3/ p-value =0.04 and r=0.46/ p-value=0.005, respectively). *p<0.05, **p<0.01, ***p<0.001, RE: Relative expression