Abstract
Objective Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS.
Methods Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]).
Results The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-β signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity.
Conclusion Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).
Primary Sjögren syndrome (pSS) is a systemic autoimmune disease primarily affecting women, with a female to male ratio of 10:1.1 The pathophysiological process of the disease is marked by lymphocytic infiltrates in the lacrimal and salivary glands and the presence of circulating autoantibodies (anti-SSA/Ro, anti-SSB/La, and rheumatoid factor). Key elements in the pathogenesis are T cell–dependent B cell hyperactivity and type I interferon (IFN) activation.2-4 The most reported clinical symptoms of pSS encompass dryness of the eyes (keratoconjunctivitis sicca) and mouth (xerostomia), pain, and fatigue.1 Less well-known, less spontaneously reported, and less studied are symptoms of vaginal dryness, experienced by the majority of female patients with pSS.5-9 Vaginal dryness is an important factor influencing sexual function in women with pSS. We reported that 56% of women with pSS experienced sexual dysfunction, measured by the Female Sexual Function Index (FSFI), which includes 6 domains: desire, arousal, orgasm, lubrication, satisfaction, and pain. Further, patients with pSS were more frequently sexually inactive compared to age-matched healthy controls, negatively affecting general well-being and quality of life.8
Despite the considerable effect of vaginal dryness, the pathogenesis of this symptom in patients with pSS is poorly understood. Normally, transudate from the venous and lymphatic networks in the lamina propria of the vaginal epithelium and mucus produced by endocervical glandular epithelial cells keep the vaginal surface humidified and lubricated.10 In women with pSS, numbers of CD45+ cells are increased in vaginal biopsies compared to non-pSS controls.11-14 In addition, our previous study demonstrated a decrease in vascular smooth muscle cells in the vaginal biopsies of patients with pSS, indicating disturbed blood vessel homeostasis.11 Although these morphological changes may be caused by chronic inflammation of the vaginal wall in pSS, the underlying molecular pathways remain poorly understood.
We hypothesized that inflammatory pathways are activated in vaginal tissue of patients with pSS, potentially contributing to reduced lubrication by disturbing blood vessel homeostasis. To test this hypothesis, we performed immune gene profiling on vaginal tissue biopsies from premenopausal patients with pSS with vaginal dryness and non-pSS controls without an autoimmune disease. Differentially expressed genes and pathways were identified and expression levels were correlated to clinical variables, including the FSFI, Vaginal Health Index (VHI), and patient-reported vaginal dryness.
METHODS
Patients and controls. For this study, we used vaginal biopsies collected in a previous prospective exploratory case-control study. The study included 10 premenopausal female individuals with pSS who fulfilled the 2016 American College of Rheumatology/European Alliance of Associations for Rheumatology classification criteria and reported vaginal dryness and 10 premenopausal female individuals without an autoimmune disease. Detailed inclusion and exclusion criteria have been previously described.11 None of the participants used systemic corticosteroids or disease-modifying antirheumatic drugs ≤ 6 months before inclusion and participants with inflammatory or infectious gynecological diseases were excluded. The study complies with the Declaration of Helsinki and was approved by the Medical Ethics Committee of the University Medical Center Groningen (METC 2015/039). All participants gave written informed consent.
Sample collection and processing. All participants underwent a midvaginal biopsy. Punch biopsies were collected and either snap-frozen (−80 °C) or formalin-fixed and paraffin-embedded for immunohistochemistry. Immunohistochemical staining of tissue was performed for myxovirus resistance protein 1 (MxA) and CD123 protein. For details, see Supplementary Methods (available with the online version of this article). Blinded scoring of slides was done by 1 researcher (AV) and 3 pathologists (BvdV, GFHD, JB). MxA expression was scored in epithelium, endothelium, fibroblasts, and inflammatory infiltrates (when present). When staining was observed in all cell types, vaginal tissue was considered positive for MxA.
From patients with pSS, peripheral blood samples were collected in PAXgene tubes (Qiagen) and stored until use at −20 °C. Total RNA was extracted using the PAXgene blood isolation kit (Qiagen).
Patient-reported symptoms and clinical data collection. At the time of the biopsy, all participants completed a questionnaire concerning sexual function and vaginal dryness. Sexual function was measured by the 19-item FSFI. Higher scores indicate better sexual function.15 Vaginal dryness was patient-reported using a numerical rating scale (NRS), ranging from 0 (no dryness) to 10 (the worst dryness imaginable).16
Gynecological examination was performed by an experienced gynecologist by scoring the VHI. Higher scores indicate good vaginal health.
Immune gene profiling. Immune gene profiling was performed on RNA from frozen vaginal biopsies (RNeasy miniprep kit, Qiagen). Gene expression was measured using the nCounter NanoString PanCancer Immune Profiling Panel (NanoString Technologies). For details, see Supplementary Methods (available with the online version of this article). To validate Nanostrings’ immune gene profiling results, RNA was reverse transcribed to cDNA and quantitatively analyzed using an Applied Biosystems QuantStudio 6 Flex Real-Time (RT) PCR System (ThermoFisher Scientific). The relative expression (RE) was calculated based on the cycle threshold (Ct) value related to expression of the housekeeping gene GAPDH as follows: RE = 2−(Ct test gene − Ct GAPDH).
To differentiate between type I and type II IFN-dominant activation in pSS vaginal tissue, 3 IFN-related modules (M1.2, M3.4, M5.12) displaying a distinct inducibility by the different IFNs were analyzed.17 IFN scores were calculated by summing up the individual RE per gene after normalization to the non-pSS control group as follows: ∑(REsubject − meancontrol)/SDcontrol. Two separate IFN scores were calculated: (1) the IFN12 score, based on the 12 IFN-stimulated genes (ISGs) of modules M1.2, M3.4, and M5.12, and (2) the IFN3 score, based on 3 type I IFN (IFN-α)–induced genes (IFI44L, LY6E, and MX1) from module M1.2.18 An IFN score was considered positive when it was higher than the mean + 2 SD of the control values.
Statistical analyses. Differential gene expression was analyzed using nSolverTM Analysis 4.0 software provided by NanoString Technologies. Because of the exploratory character of this study and the low number of samples, we did not use P values, but genes were considered differentially expressed when their expression level was ≥ 1.5× higher or lower than the expression level of non-pSS controls. Signaling pathway scores were calculated as the first principal component of the pathway genes’ normalized expression. Pathway scores were expressed as median with IQR per pathway for patients and non-pSS controls. Pathway scores between groups were compared using the Mann-Whitney U test. A gene expression heatmap was generated by using the normalized expression data from nSolverTM into R statistical environment (R Foundation for Statistical Computing). z scores were calculated for each individual gene and subject. Hierarchical clustering was performed using Ward method (Euclidean distance). Spearman correlation coefficient (ρ) was used to measure the strength and direction of association between 2 ranked variables.
Statistical analyses were performed in SPSS statistics v28 (IBM Corp.) and figures were created in GraphPad Prism 9.1.0 (GraphPad Software) or R statistical environment (version 4.1.1). P < 0.05 was considered statistically significant.
RESULTS
Clinical characteristics. Clinical characteristics of the study participants are summarized in Table 1. We included 8 of 10 patients with pSS and 7 of 10 non-pSS controls in our analysis. Reasons for exclusion were presence of Chlamydia trachomatis (pSS; n = 1), endometriosis (control; n = 2), or low RNA yield of the biopsy (pSS and control; n = 2).
Demographical and clinical characteristics of patients and non-pSS controls.
Dysregulated molecular pathways in vaginal tissue of patients with pSS. Gene set enrichment analysis of nCounter data showed divergent pathway scores in vaginal biopsies from patients with pSS compared to non-pSS controls (Figure 1). Lower scores in pSS vaginal tissue were observed for several pathways, with a significant difference for transforming growth factor–β signaling and angiogenesis (P = 0.02 and P = 0.04, respectively). Conversely, for several pathways, higher pathway scores in pSS vaginal tissue compared to non-pSS controls were observed (Figure 1). The IFN signaling, Janus kinase and signal transducer and activator of transcription (JAK-STAT) signaling, and Notch signaling pathways showed the largest difference between groups, reaching statistical significance (P < 0.01 for all). Since IFNs play a dominant role in the immunopathogenesis and inflammation of the salivary glands, the IFN signaling pathway was analyzed in more detail. For this, we looked at transcription levels of genes stimulated by IFNs (IFN-stimulated genes [ISGs]), reflecting the activity of the IFN signaling pathway.
Pathway scores in vaginal biopsies from patients with pSS (black squares) compared to non-pSS controls (gray dots). Results are expressed as median (IQR) per pathway for patients and controls. Signaling pathways scores that were significantly different (P < 0.05) between patients with pSS and non-pSS controls are marked in bold and with an asterisk. JAK-STAT: Janus kinase and signal transducer and activator of transcription; MAPK: mitogen-activated protein kinases; NF: nuclear factor; PI3K-AKT: phosphoinositide-3-kinase–protein kinase B; pSS: primary Sjögren syndrome; TGF: transforming growth factor.
In total, 41 of 58 (71%) genes involved in IFN signaling included in the probe panel were differentially expressed in vaginal tissue of patients with pSS compared with non-pSS controls. For further analysis, the 20 ISGs with a fold change ≥ 1.5 in pSS vs non-pSS controls were selected (Supplementary Figure S1, available with the online version of this article). Hierarchical clustering of all study participants based on these ISGs showed evident clustering of patients with pSS with 1 independent cluster of 4 (50%) “IFN-high” patients (pSS10, 13, 16, and 21; Figure 2). The other 4 (50%) patients with pSS (pSS9, 11, 19, and 25) showed moderate to low ISG expression levels. The 7 non-pSS controls clustered together in a group with low ISG expression levels. All IFN-high patients were anti-SSA/Ro positive, whereas the 2 anti-SSA/Ro negative patients (pSS11 and pSS19) showed moderate to low ISG expression levels.
Heatmap showing gene expression in vaginal biopsies from patients with pSS and non-pSS controls. Hierarchical clustering (Ward method) was performed based on expression of 20 selected ISGs (≥ 1.5-fold linear fold change compared to controls including all subjects). Each column represents an individual study subject, and each row represents a gene. Depicted on top of the heatmap: the group of the studied subjects (pSS or non-pSS control), anti-SSA/B positivity of the pSS subjects, and the calculated IFN scores (IFN3, IFN12, IFN1.2, IFN3.4, and IFN5.12) of all subjects. EGR1: early growth response 1; GBP: IFN-induced guanylate-binding protein; HLA-DQA2: HLA class II histocompatibility antigen, DQ(6) alpha chain; IFI16: IFN gamma inducible protein 16; IFI27: IFN alpha inducible protein 27; IFIH1: IFN induced with helicase C domain 1; IFIT: IFN-induced protein with tetratricopeptide repeats; IFN: interferon; IRF: IFN regulatory factor; ISG: IFN-stimulated gene; MX1: MX dynamin like GTPase 1; neg: negative; OAS: 2’-5’oligoadenylate synthetase; PSMB8: proteasome subunit beta type-8; pos: positive; pSS: primary Sjögren syndrome; RSAD2: radical S-adenosyl methionine domain-containing protein 2; STAT1: signal transducer and activator of transcription 1.
In addition to autoantibody status, we determined the relationship between increased ISG expression in vaginal tissue and the clinical variables of sexual function (FSFI), patient-reported vaginal dryness (NRS), and vaginal health (VHI). Patients and non-pSS controls were ranked based on their IFN signaling pathway score in vaginal tissue to facilitate a descriptive analysis of the 3 clinical variables in relation to this score (Supplementary Table S1, available with the online version of this article). Although patients with pSS scored worse on nearly all FSFI domains, patient-reported vaginal dryness, and several VHI domains, as previously described,8,11 no clear differences were observed in clinical variables between patients with pSS with high or low IFN signaling pathway scores.
Validation of differential IFN-stimulated gene expression. To validate nCounter results, TaqMan RT-PCR analyses were performed to quantify expression of the 12 ISGs of the IFN12 score. At the individual gene level, a clear trend toward higher expression in the vaginal biopsies of patients with pSS was observed for the majority of measured ISGs (Supplementary Figure S2, available with the online version of this article). Modules M1.2 (IFN-α–induced) and M3.4 (IFN-β/γ–induced) were upregulated in 4 of 8 patients with pSS (ie, > mean + 2 SD of the controls; Figure 3). Module M5.12 (IFN-α/β/γ–induced) was upregulated in 3 of 8 patients with pSS. The 4 patients with pSS with elevated ISG transcript levels in M1.2 and M3.4 also showed an upregulated IFN3 score and IFN12 score. Thus, RT-PCR analysis shows increased IFN scores in the vaginal biopsies of patients with pSS compared to non-pSS control biopsies, indicating more IFN activity confirming the nCounter data.
IFN scores calculated from TaqMan Real-Time PCR analysis of vaginal biopsies from patients with pSS (black squares) compared to non-pSS controls (gray dots). (a) IFN3 score; (b) IFN12 score; and (c-e) IFNM1.2, IFNM3.4, and IFNM5.12 score. The dotted line represents mean + 2 × SD of controls. IFN3 score: IFI44L, LY6E, and MX1. IFNM1.2 score: CXCL10, IFI44L, IFIT3, LY6E, MX1, and SERPING1. IFNM3.4 score: IFITM1, IRF7, and STAT1. IFNM5.12 score: C1QA, IFI6, and IRF9. IFN12 score: M1.2 + M3.4 + M5.12. C1QA: complement C1q A chain; CXCL10: C-X-C motif chemokine ligand 10; IFI44L: interferon induced protein 44 like; IFI6: IFN alpha inducible protein 6; IFIT: IFN-induced protein with tetratricopeptide repeats; IFN: interferon; IRF: interferon regulatory factor; LY6E: lymphocyte antigen 6 family member E; MX1: MX dynamin like GTPase 1; pSS: primary Sjögren syndrome; SERPING1: serpin family G member 1; STAT1: signal transducer and activator of transcription 1.
Increased IFN scores and their correlation with the amount of CD45+ cells in pSS vaginal tissue. Since the increased expression of ISGs in the salivary glands of patients with pSS has been associated with the presence of lymphocytic infiltrates,19 we next investigated whether this was also the case in vaginal tissue. Immunohistochemical sections were previously stained for the pan-leucocyte marker CD45,11 and were reanalyzed by counting the number of positively stained cells per square milliliter of submucosa using QuPath.20 At the group level, there was an increase in the percentage (median 7.7 [IQR 7.1-9.0] vs 6.1 [IQR 4.0-8.5] percent positive cells, respectively) and number (median 340 [IQR 279-435] vs 197 [IQR 155-256] of positive cells/mm2, respectively) of CD45+ cells in vaginal tissue of patients with pSS compared to non-pSS controls. Even with a low number of samples, a strong correlation between the percentage of CD45+ cells and the IFN scores was observed, with the highest correlation for the IFN3 score (ρ 0.79). A moderate correlation was found between the number of CD45+ cells and the IFN scores, with the highest correlation for the IFN12 score (ρ 0.59).
Presence of MxA protein expression, but absence of plasmacytoid dendritic cells, in pSS vaginal tissue. To explore whether elevated MX1 transcript levels in vaginal tissue coincide with expression of this ISG at the protein level, we performed immunohistochemical staining on vaginal tissue sections (Figure 4). Tissue sections from 6 anti-SSA/Ro positive patients with pSS, all with highly or moderately elevated MX1 transcript levels as shown by Nanostring’s immune profiling data, showed strong positive MxA protein staining in epithelial cells, endothelial cells, fibroblasts, and infiltrates (when present). Previous work showed that these infiltrates mainly consist of T cells (CD4+ and CD8+).11 The 2 anti-SSA/Ro negative patients with pSS (pSS11 and pSS19) and the non-pSS controls, all with moderate to low MX1 transcript levels, showed only faint background staining in epithelial cells (and some endothelial cells) and were considered MxA negative. Thus, higher MX1 gene expression corresponds to higher MxA protein expression in vaginal tissue, which is related to anti-SSA/Ro positivity.
MxA immunohistochemical staining on vaginal tissue sections of patients with pSS and a non-pSS control. MxA staining of (a) an IFN-high patient (pSS16) with positive MxA staining of epithelium, endothelium, fibroblasts, and infiltrate; (b) an IFN–moderate-to-low patient with pSS (pSS9) with positive MxA staining of epithelium, endothelium, and fibroblasts; (c) an IFN–moderate-to-low patient with pSS (pSS19) negative for MxA staining; and (d) a non-pSS control (C15) negative for MxA staining. The patients pSS16 and pSS9 were both anti-SSA/Ro positive, and patient pSS19 was tested anti-SSA/Ro negative. IFN: interferon; mod: moderate; MxA: myxovirus resistance protein 1; neg: negative; pSS: primary Sjögren syndrome.
To explore whether plasmacytoid dendritic cells (pDCs) form a local source of type I IFN in vaginal tissue, tissue sections were stained for the pDC cell membrane marker CD123. However, CD123 staining was not observed in vaginal biopsies from patients with pSS or non-pSS controls. This observation was confirmed by nCounter data, showing CD123-mRNA counts below the detection limit in all samples. Apparently, an alternative local source of type I IFN (eg, epithelium) or type I IFN from the circulation is responsible for the observed increase in IFN signaling.
To investigate whether MX1 expression in circulating immune cells and vaginal tissue was correlated, we compared transcript levels of the MX1 gene in whole blood (RT-PCR) with the normalized expression of MX1 in vaginal tissue (nCounter). Despite differences in tissue type and molecular technique, we found a moderate correlation between blood and vaginal tissue MX1 expression at the group level (ρ 0.45, nonsignificant). The 2 anti-SSA/Ro negative patients with pSS showed low MX1 expression levels in both blood and vaginal tissue. These results indicate that there is some discordance in MX1 expression levels between blood and vaginal tissue, although low levels in blood are in all cases accompanied by low levels in vaginal tissue (Supplementary Figure S3, available with the online version of this article).
IFN-stimulated gene expression and its relation to angiogenesis. Since elevated IFN levels may result in endothelial dysfunction,21-23 and that the angiogenesis pathway was downregulated in vaginal tissue of patients with pSS, we hypothesized that higher ISG expression levels might be linked to endothelial dysfunction in vaginal tissue. However, we observed no clear difference in angiogenesis pathway scores between vaginal tissue-based IFN-high patients with pSS and patients with moderate to low ISG expression levels (Supplementary Table S2, available with the online version of this article). In addition, we analyzed the expression of typical endothelial cell marker genes (ie, PECAM1, CDH5, and TIE1) that were included in our probe panel, but not in the predefined angiogenesis pathway. Although lower normalized expression values were observed for these genes in patients with pSS compared to non-pSS controls, there was no clear difference between IFN-high patients with pSS and patients with moderate to low ISG expression levels (Figure 5).
ANG pathway score and transcript levels of endothelial cell markers in vaginal tissue of patients with pSS (black squares) compared to non-pSS controls (gray dots). The (a) ANG pathway score, and normalized expression values from NanoString results for (b) PECAM1, (c) CDH5, and (d) TIE1, are displayed. Horizontal lines indicate the median. ANG: angiogenesis; CDH5: cadherin 5; PECAM: platelet and endothelial cell adhesion molecule; pSS: primary Sjögren syndrome; TIE: tyrosine kinase with immunoglobulin-like and endothelial growth factor–like domains.
Thus, whereas blood vessel formation in the vaginal wall seems to be impaired in the majority of patients with pSS, our results do not indicate a direct relationship with enhanced IFN signaling.
DISCUSSION
Vaginal dryness and related sexual dysfunction are frequent symptoms reported by women with pSS, affecting general well-being and quality of life. However, very little is known about the pathophysiology of vaginal dryness in pSS, though such knowledge may be beneficial for the treatment of this common symptom. Here, we used a probe-based technology to measure transcript levels of immune response genes in vaginal tissue of premenopausal female patients with pSS reporting vaginal dryness and non-pSS controls without an autoimmune disease. We demonstrate increased IFN activity in vaginal tissue of patients with pSS. To our knowledge, this is the first study showing increased IFN activity in pSS outside the main target organs (eg, salivary glands) and peripheral blood. We found a strong positive correlation between upregulation of ISGs and the frequency of CD45+ cells in vaginal tissue, suggesting that these pathological features are linked.
IFN signaling is one of the major pathways that contributes to inflammation in salivary glands of patients with pSS.2-4,19 Increased IFN activity has been associated with more severe glandular pathology and dysfunction.24,25 Whether IFN is involved in the pathophysiology of vaginal dryness in women with pSS remains unknown. By using immune gene profiling, we found an IFN signature in vaginal tissue in 50% (4 of 8) of the patients with pSS. These IFN-high patients showed elevated transcript levels of ISGs, reflecting the activity of the IFN signaling pathway. The other 50% of patients with pSS, including 2 anti-SSA/Ro negative patients, showed moderate to low ISG transcript levels. All IFN-high patients with pSS were anti-SSA/Ro positive, confirming the strong link between activation of the IFN pathway and presence of anti-SSA/Ro autoantibodies in pSS.26 The 7 non-pSS controls clustered together in a group with low ISG expression levels.
IFNs are a large family of cytokines involved in antiviral defense mechanisms, cell growth regulation, and immune activation.27 Type I IFN (IFN-α and IFN-β) and type II IFN (IFN-γ) are the most studied IFNs. To discriminate between more type I–mediated or type II–mediated ISG expression, we used IFN-annotated modules, first described for systemic lupus erythematosus (SLE).17 The 4 patients with pSS who were assigned to the IFN-high cluster based on nCounter analysis showed elevated ISG transcripts in the IFN modules M1.2 and M3.4, indicative of type I IFN–mediated upregulation. Of these 4 patients with pSS, 3 were also M5.12 positive, indicative of the concerted action of both type I and type II IFNs in vaginal tissue of these patients. As previously described in the blood of patients with SLE and pSS, ISGs in M5.12 were not upregulated without concomitant upregulation of ISGs in M1.2 and M3.4.18,28 The increased expression of both type I–induced and type II–induced genes in the IFN-high cluster was confirmed by higher IFN3 scores (type I) and IFN12 scores (type I and type II). For the type I–induced gene MX1, gene expression results were validated at the protein level by immunohistochemistry. Although MxA staining was considered negative in all non-pSS controls, 6 of 8 patients with pSS (all anti-SSA/Ro positive) showed positive MxA staining, whereas the 2 anti-SSA/Ro negative patients did not show MxA staining in vaginal tissue, in line with gene expression results. In MxA positive tissues, both nonlymphoid cells (ie, epithelial cells, endothelial cells, fibroblasts) and lymphoid cells showed MxA expression, indicating responsiveness to type I IFN.
Next, we questioned whether upregulation of ISGs was induced by local production of type I and/or type II IFNs. Many different cell types can produce type I IFN upon activation of pattern recognition receptors. However, pDCs are specialized in type I IFN production.29-31 CD123-expressing pDCs have been observed in the salivary glands of patients with pSS, but not in control glands.32 In this study, we were not able to detect CD123-expressing pDCs in the vaginal wall. Apparently, other cells are responsible for the production of type I IFN. Since there is some evidence that the epithelial cells in the salivary glands of patients with pSS produce type I IFN,33-35 this could also be the case in vaginal tissue. The discordance between relative MX1 gene expression in whole blood and normalized MX1 gene expression in vaginal tissue supports the possibility that type I IFN is produced locally in the vaginal tissue. For type II IFNs, infiltrating T cells may be a potential source. In a previous study,11 we have shown that the CD45+ cell infiltrates largely consist of T cells, including both CD4+ and CD8+ T cells. The presence of IFN-γ–producing T cells could also explain the observed association between upregulation of ISGs and the percentage of CD45+ infiltrating cells in the submucosa of the vaginal wall.
In addition to promoting inflammation, there is evidence that increased IFN activity may also result in endothelial dysfunction. In patients with SLE, IFN alters the balance between endothelial cell apoptosis and vascular repair.21,22 Therefore, we hypothesized that higher ISG expression levels might be linked to endothelial dysfunction in vaginal tissue, leading to vaginal dryness. Endothelial cells in the vaginal wall are critically involved in the formation of transudate, which humidifies the vagina, together with mucus produced by endocervical glands. Previously we observed a decrease in the number of cells expressing caldesmon in the vaginal wall of patients with pSS, an important protein for smooth muscle contraction. This decrease presumably reflects a lower number of arterioles.11 In line with these previous findings, the current study shows reduced transcript levels of endothelial cell marker genes (ie, PECAM1, CDH5, and TIE1) in patients with pSS compared to non-pSS controls. Apparently, the damage and/or loss of blood vessels cannot be repaired, as mirrored by a lower angiogenesis pathway score in patients with pSS. Together, these results point toward a decreased number of arterioles in the vaginal wall of patients with pSS that may explain the vaginal dryness seen in these patients. We could not however demonstrate a direct link between increased IFN activity and reduced transcript levels of endothelial cell marker genes.
A main limitation of our study is the small sample size, which results in low statistical power. This may also explain why we could not find a correlation between enhanced IFN activity in vaginal tissue (ie, elevated expression of ISGs, higher IFN scores) and relevant clinical variables such as patient-reported vaginal dryness, VHI (physician-reported), and sexual functioning (FSFI). Another complicating matter is that none of these variables have been validated for pSS and the outcome of these clinical variables is influenced by multiple factors (eg, interpersonal and psychosocial factors). Also, patient-reported outcomes such as NRS vaginal dryness may not adequately reflect objective vaginal dryness. Thus, absence of a correlation does not necessarily mean that IFN is not a contributing factor to clinical symptoms and further investigation is warranted.
In conclusion, the current study provides unique insight into active immune response pathways in vaginal tissue of patients with pSS compared to non-pSS controls. We demonstrated that IFN signaling is upregulated in vaginal tissue and potentially contributes to clinical vaginal symptomatology. Unraveling the pathophysiology of vaginal dryness in pSS is critical in developing treatment strategies to ameliorate this important, common, but underreported and understudied symptom.
ACKNOWLEDGMENT
We thank Hannie Westra, Berber Doornbos, and Mirjam Mastik for their technical support.
Footnotes
This work was supported by grants from the Dutch Arthritis Society (ReumaNederland; 14-1-301) and the C&W de Boer Foundation.
HB has received unrestricted research grants, outside the submitted work, from BMS and AstraZeneca, and received consultancy fees from Argenx, BMS, Roche, Novartis, and Union Chimique Belge. FGK and GMV are coapplicants on an unrestricted grant by AstraZeneca. The remaining authors declare no conflicts of interest relevant to this article.
- Accepted for publication March 21, 2024.
- Copyright © 2024 by the Journal of Rheumatology
REFERENCES
ONLINE SUPPLEMENT
Supplementary material accompanies the online version of this article.
