The redundant role of plasmacytoid dendritic cells in Primary Sjögren's syndrome

The redundant role of plasmacytoid dendritic cells in Primary Sjögren's syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The redundant role of plasmacytoid dendritic cells in Primary Sjögren's syndrome Rongli Li, Xunyao Wu, Mu Wang, Chuiwen Deng, Yu Peng, Lidan Zhao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3534730/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective The aim of our present study is to investigate the role of plasmacytoid dendritic cells (pDCs) in the pathogenesis and type I interferon (IFN) signatures in Primary Sjögren's Syndrome (pSS) patients. Methods In the present study, we compared the percentage, activation markers, and representative cytokines secretion of pDCs derived from treatment-naive pSS and matched healthy controls (HCs) by flow cytometry. We performed pDC/B co-culture system to explore the contribution of pDC to B cell functions in pSS. Results The percentage of pDC was significantly reduced in the peripheral blood of pSS. The activation markers (CD80, CD83, and CD86) expressions, chemokine receptors, and representative cytokines production (IFN-α, IL-6, and TNF-α) of pDC were similar between pSS and HCs. Only a few pDCs infiltration were detected in the labial gland. The percentage of pDCs was negatively correlated with serum IgG, IgA, and anti-SSA autoantibody levels and resting pDCs were able to efficiently promote B cells proliferation, activation, differentiation, and antibody production in vitro . However, there was no difference between HC and pSS-derived pDCs. Finally, we found that incubation of plasma from pSS patients could significantly induce pDCs apoptosis than that from HCs and both IgG and IgA dramatically increased the apoptotic rates of pDCs. Conclusion Our data have deciphered the redundant role of pDC in the type I signature and disease development in pSS. Also, we demonstrated the decreased percentage of pDC in pSS patients might result from apoptosis induced by the excess of immunoglobulin (IgG and IgA). Primary Sjögren's syndrome Plasmacytoid dendritic cell Type I interferon B cell Hypergammaglobulinemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Primary sjögren's syndrome (pSS) is a typical autoimmune disorder characterized by focal lymphocytic infiltrations, damage, and dysfunction in salivary and lacrimal glands [1]. Lung, kidney, and nervous system involvement could also occur in severe cases [2]. Previous studies have demonstrated the type I IFN signatures in peripheral blood mononuclear cells (PBMCs), B cells, monocytes, neutrophils as well as the affected tissues [3–10]. Moreover, IFN-α inducible proteins: MDA-5, IFIT-3 [11], and B cell-activating factor (BAFF) [12] were found highly expressed in salivary glands. Type I IFN could enhance T cell and B cell responses and promote the production of autoantibodies [11, 13], implicating their important roles in the pathogenesis of pSS. Acting as a premier type I IFN producer and a key bridge between innate and adaptive immunity, pDCs also play a nonnegligible contribution to autoimmune disease development. pDCs in SLE patients can be activated through various pathways (immune complexes, neutrophil extracellular traps, mitochondrial DNA, etc .), which in turn trigger immune responses and promote the production of autoantibodies [14]. In studies of systemic sclerosis (SSc) [15], Psoriasis [16], rheumatoid arthritis [17] and autoimmune diabetes [18], pDCs could infiltrate into the target tissue and exacerbate local inflammation by releasing IFN-α and proinflammatory factors. Currently, studies on the role of pDCs in pSS are limited. Previous studies have reported the reduced frequencies of pDCs in peripheral blood [19, 20] of pSS patients. A recent study performed a transcriptional analysis of circulating pDCs and identified the aberrant activation of pDC in pSS patients [21]. Here, we systematically study the percentage, phenotype, and functions of pDCs for a better understanding of their roles in the pathogenesis and type I IFN signatures in pSS patients. Methods Patients and ethics All newly-onset pSS and SLE patients were enrolled in Peking Union Medical College Hospital (PUMCH) and met the 2016 ACR-EULAR classification[22] and the 1997 classification criteria of the American College of Rheumatology[23], respectively. Labial tissues were obtained from patients who underwent labial gland biopsy at the department of stomatology in PUMCH. Patients all signed informed consent for their residual tissues after the pathological examination. This study was approved by the Ethics Committee of PUMCH (No: K2525). PBMC Isolation and In vitro stimulation Human PBMCs were isolated with Ficoll-Paque density (DAKEWE, China) as previously described[8]. For the plasma stimulation, PBMCs from HCs were seeded into 24-well plates at a density of 1×10 6 /ml and maintained in RPMI 1640 (Gibco, A10491, USA) with 20% mixed plasma from ten pSS or HCs in the presence of 100 ng/ml IL-3 for 4 hours. For IgG and IgA stimulation, freshly isolated PBMCs from HCs were first incubated with 100 mg/ml IL-3 for 2 hours, then stimulated with human IgG (SP001, Solarbio) and human IgA (SP016, Solarbio) with indicated concentrations. IL3 is used to assist the survival of pDCs in vitro [24, 25]. In vitro purification and stimulation of pDC pDC was purified using CD304 microbeads (130-090-532, Miltenyi Biotec) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (15140122, ThermoFisher). For cytokines detection, PBMCs were stimulated with 5 µg/ml R848 (tlrl-r848, InvivoGen) or 10 µg/ml ODN-2216 (tlrl-2216, InvivoGen) in the presence of 100 ng/ml IL-3 (Peprotech, 200-03). R848 is a ligand for Toll-like receptor 7 (TLR7) and OND-2216 is a ligand for TLR9. pDC and B cell coculture B cells were purified using B cell isolation kit II (130-091-151, Miltenyi Biotec) following the manufacturer’s protocol. B cells were activated with 5 µg/mL anti-IgM (314502, BioLegend), 500 ng/mL sCD40L (310-02, Peprotech), 100 ng/ml IL-4 (200-04, Peprotech), and 50 ng/ml IL21 (200 − 21, Peprotech). For pDCs and B cells coculture, purified CD19 + B cells (5×10 5 /ml) and pDC from allogeneic HCs were added at the ratio of 10:1. The supernatants were harvested at day 7. Flow cytometry Cells were harvested and resuspended at the concentration of 5×10 6 cells/ml. Fluorochrome-conjugated monoclonal antibodies for cell surface staining were as follows: anti-human CD303, CD123, Lineage, HLA-DR, CD80, CD83, CD86, CD19, CD40, CD74, CD27, IgD, CCR2, CCR4, CCR5, CCR7, CCR10, CD38, CD138, CD24. The Intracellular Fixation & Permeabilization Buffer Set (eBioscience) was used for intracellular staining following manufacturer’s protocol. Fluorochrome-conjugated monoclonal antibodies for intracellular staining are as follows: anti-human Ki-67, TLR7, TLR9, IFN-α, TNF-α, IL-6. Detailed information about conjugated fluorescence and manufacturers for antibodies is shown in Supplementary Table 1. Apoptosis assay Cell apoptosis was detected by PE Annexin V Apoptosis Detection Kit I (559763, BD Pharmingen). Briefly, cells were washed with 1ml 1× Annexin V binding buffer after regular surface staining, then incubated with PE-conjugated Annexin V and 7-AAD for 15 minutes at room temperature, all the samples were analyzed by BD Accuri C6 flow cytometer (Becton Dickinson, USA). Immunofluorescence Freshly labial tissue was embedded in O.C.T. (Tissue-Tek) and 8µm frozen sections were prepared. Sections were immersed in 95% ethanol for 15 min, blocked in normal goat serum for 3h at room temperature, followed by incubating with 25 µg/ml mouse anti-human BDCA-2 (CD303) monoclonal antibody (MAB62991, R&D), 1:50 diluted mouse anti-human IFN-α monoclonal antibody (sc-373757, SANTA CRUZ BIOTECHNOLOGY, INC.) or 1:500 diluted rabbit anti-human EpCAM monoclonal antibody (also known as CD326, ab223582, abcam) at 4°C overnight. CoraLite594-conjugated goat anti-mouse secondary antibody (proteintech, China) or CoraLite488-conjugated goat anti-rabbit secondary antibody (proteintech, China) was incubated at a dilution of 1:500 for 1h at room temperature. Further, the slides were mounted by an antifading mounting medium with DAPI (S2110, Solarbio) and scanned by Pannoramic MIDI (3DHISTECH, Hungary). ELISA The levels of IgG, IgM, and IgA in culture supernatants were measured with Human IgG/IgM/IgA Precoated ELISA Kit (1128162, 1128182, 1128172 DAKEWE, China) respectively. The level of IFNα in plasma from pSS patients and matched HCs was determined by Human IFN-α Precoated ELISA Kit (1110012, DAKEWE, China) following the manufacturer’s instructions. Statistical analysis All the data analyses were conducted by IBM SPSS statistics (Version 25.0, IBM, Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) software. The data were first performed with the normality distribution. Student’s t-test was used for variables in a normal distribution, otherwise, the Mann-Whitney test was used. Linear regression and Pearson’s correlation were applied to explore the correlation analysis. A p -value < 0.05 was defined as statistically significant. Results The frequency and phenotype analysis of peripheral pDC in pSS patients We first analyzed the frequency of pDC in PBMCs from treatment-naïve pSS (n = 31), HCQ-treated pSS (n = 17), SLE (n = 9), and HCs (n = 29) by flow cytometry. The demographic characteristics of all the included pSS patients were demonstrated in Supplementary Table 2 and SLE patients were demonstrated in Supplementary Table 3. The gating strategy of pDCs was shown in Figure S1 a. In accordance with previous studies [20, 26], the percentage of pDCs was significantly lower in PBMCs from treatment naïve SLE and pSS patients compared with HCs. Interestingly, the reduction of pDCs rebounded in pSS patients after Hydroxychloroquine (HCQ) therapy (Fig. 1 a). However, we did not observe the hyperactivation status of pDCs in pSS as the activation markers CD80, CD83, and CD86 expressions were equal to HCs (Fig. 1 b, Fig S1 c). Detection of TLR7/9 expressions showed that unstimulated pDCs from pSS patients showed higher TLR7 but not TLR9 expressions compared with HCs (Fig. 1 c, d). However, the difference vanished after TLR7 (R848) or TLR9 ligand (ODN2216) stimulation (Fig. 1 e, f). pDCs did not contribute to peripheral and affected tissue type I IFN signature in pSS patients To determine whether pDC from pSS patients displays the enhanced capacity of IFN-α secretion, we stimulated freshly isolated PBMCs with TLR7 (R848) or TLR9 ligand (ODN-2216) and determined representative cytokines production by flow cytometry. However, upon stimulation, there was no difference in the IFN-α secretion of pDCs between HC and pSS patients (Fig. 2 a-b). The same was observed in the IL-6 and TNF-α production (Fig. 2 c-f). We next detected the infiltration of pDCs in labial glands by immunofluorescence to explore whether they contribute to tissue type I IFN signature in pSS patients. We detected the labial glands of nine pSS patients whose labial gland biopsy indicated typical focal lymphocytic infiltration. Among these cases, only two exhibited a small amount of pDC infiltration, while the remaining seven cases showed no evidence of pDC infiltration (Fig. 3 a). Recent researches highlighted the role of salivary epithelial cells in producing type I IFNs [27–29], consistently, our immunofluorescent results indicated that epithelial cells are responsible for the production of tissular IFN-α in patients with or without pDC infiltration (Fig. 3 b). We also detected the main chemokine receptors, including CCR2, CCR4, CCR5, CCR7, and CCR10 on the pDC surface showed no difference between pSS patients and HCs (Fig. 3 c, Fig S2j). pDCs promote B cell class-switching and antibody production Correlation analysis between the frequency of peripheral pDCs and clinical parameters in pSS patients showed that the percentage of pDCs was negatively correlated with serum IgG, IgA, and anti-SSA autoantibody levels (Fig S2a-i, Fig. 3 d). We, therefore, established the pDC/B cells co-culture system to explore the potential role of pDC in promoting B cell responses and antibody production in pSS patients. We found that resting or TLR-7-activated pDCs were able to efficiently promote the proliferation, activation, and differentiation into plasmablast/plasma cell of B cells (Fig. 4 a-e, Fig S3a-e). Also, we explored the effects of pDCs on antibody production and found that even resting pDCs could promote IgG, and IgA production by B cells (Fig. 4 f). However, there was no difference between HC and pSS-derived pDCs. Similarly, no difference was found in the effects of HC or pSS derived pDCs on naive or memory B cells (Fig S4). pDCs are more sensitized to pSS plasma-induced apoptosis Finally, we sought to explore the potential mechanism of reduced pDC frequency in the peripheral blood of pSS patients. Incubation of plasma from pSS patients could significantly reduce pDCs viability than that from HCs (Fig. 5 a). Considering that pSS is characterized by high autoantibodies in serum and pDC could promote antibody production as we demonstrated above, we inferred that plasma-derived IgG and IgA might be important inducers of pDC apoptosis. As we expected, both IgG and IgA dramatically increased the apoptotic rates of pDCs in a dose-dependent manner (Fig. 5 b-e). The viability of freshly isolated pDCs showed no significance between pSS and HCs (Fig S5a). Although pSS had higher serum IFN-α levels (Fig S5b), they had little effect on the pDC apoptosis (Fig S5c-e). These results indicate that hyper immunoglobulin in the serum of pSS patients contributes to the reduction of pDC in the peripheral blood (Fig. 6 ). Discussion The critical role of the type I IFN pathway in the pathogenesis of pSS has been widely addressed [30, 31]. Acting as the premier producer of IFN-a, the role of pDC in the pathogenesis of pSS still remains inclusive. In the present study, we have deciphered the redundant role of pDC in the type I signature and disease development in pSS. Consistent with previous studies, we have detected a peripheral reduction in pSS [20, 26]. We have demonstrated a negative feedback loop, in which, pDC promoted antibody production and in turn, high immunoglobulin could induce pDC apoptosis. It has been reported that IgG-complexed adenoviruses induce the apoptosis of human pDCs [32]. Hyperactivated B cells, characteristic autoantibodies (SSA, SSB), and hypergammaglobulinemia are hallmarks of pSS [33]. Previous studies have found that pDCs could efficiently promote the differentiation of B cells into plasmablasts and plasma cells through type I IFNs and IL-6 [34, 35]. Moreover, pDCs could enhance the autoreactivity of B cells via type I IFNs in a T cell-independent manner [36]. Although our data showed that, pSS-derived pDC did not display a stronger capacity for promoting plasma cell differentiation and antibody production, we gave a good explanation that the excess immunoglobulin (IgG and IgA) produced by hyperactivated B cells might result in the apoptosis and reduction of pDC in peripheral blood of pSS. In addition, Hydroxychloroquine (HCQ) effectively inhibits B cell activation [37] and a recent systematic review revealed that serum IgA in patients with pSS decreased significantly after using HCQ [38], which is consistent with our results that pDC frequency rebounded after HCQ therapy. Nonetheless, pDC is noted for its unique ability in producing type I IFNs, our data suggest that it might not be the culprit of hyperactive type I IFN signaling in pSS. Recent studies in pSS have emphasized the critical role of dysregulated epithelial cells [29, 39], especially the active IFN signaling in salivary gland epithelial cells (SEGCs) [29]. SEGCs from pSS were found to be sensitive to the stimulation of TLR agonists and produced type I IFNs as a response to the stimulation [27, 28, 40]. Type I IFNs released by SEGCs could further promote the secretion of BAFF in an NF-κB dependent way [12, 27]. These results imply that gland epithelial cells are not purely innocent victims, but can also be the trigger of type I IFN signaling in pSS. In SSc, pDCs directionally migrated to target organs and secreted IFN-α and CXCL4, thereby accelerated tissue fibrosis [15, 41]. In another autoimmune disease with fibrosis signature, IgG4-related disease, active pDCs and relevant IFN-α signaling were also observed in related pancreas, pDC also enhanced the production of IgG4 by B cells [42]. Dual role of pDCs on IFN-α production and promoting activation of pathogenic T cells in psoriasis has been revealed in psoriasis [43]. Study in RA mouse model suggested that pDC aggravated joint inflammation and bone erosion via TLR7 dependent type I IFNs [17]. Reduction of pDC in the peripheral blood as well as the concomitant infiltration and activation in related tissue were reported in SLE [44, 45]. A recent randomized controlled trial in SLE clarified that Litifilimab (anti-BDCA2 antibody, BDCA2 is an exclusive marker of pDC) is effective in disease remission [46], which indicated the feasibility of pDC purge strategy in SLE treatment. Whereas, newly published research revealed that SLE-derived pDC had senescence and inert phenotype, active keratinocytes should be responsible for the tupe I IFN signaling rather than pDC [26]. In general, pDCs contributed to the pathogenesis of autoimmune diseases through type I IFNs or interplay with other immune cells [47]. However, further studies are needed to elucidate the potential mechanism about pDC’s contribution to SLE and pSS. Our available evidence does not support an overactive phenotype (resting and activating status) of pDCs from pSS patients, particularly in the production of IFN-α. However, a previous transcriptional study reported that pDCs from patients with pSS secreted more type I IFNs after TLR7 stimulation [21]. Antonios et al. 's work that pDCs from patients with SLE or pSS did not show stronger secretion ability for inflammatory cytokines, especially IFNα [26], which is consistent with our findings. These divergent results may be caused by the difference of detection methods, further study will help to elucidate underlying mechanism. In conclusion, we demonstrated the decreased percentage of pDC in pSS patients, which might result from the excess immunoglobulin (IgG and IgA) induced apoptosis. Moreover, we showed that pDC might not be the major contributor to the hyperactivation of type I interferon signaling in pSS patients. Our research provides a good addition to pSS pathogenesis and gives implications that targeting pDC might not be a good strategy for clinical pSS treatment. Declarations Competing interests: The authors have declared no conflicts of interest. Ethics approval and consent to participate: The study was approved by the Ethics Committee of the Peking Union Medical College Hospital (No: K2525) and was conducted in accordance with the Helsinki Declaration. Informed consent of all the patients were obtained. Consent for publication: The manuscript is approved by all authors for publication, and written informed consent to publish were obtained. Funding: This study was supported by the National Natural Science Foundation of China (grant numbers: 81971545, 81971544), the National High-Level Hospital Clinical Research Funding (2022-PUMCH-C-039, 2022-PUMCH-B-013). Availability of data and material: Data of this study are available from the corresponding author upon reasonable request. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3534730","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":245413704,"identity":"b73813a8-d9d6-4d41-8b4d-7a9772678886","order_by":0,"name":"Rongli Li","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rongli","middleName":"","lastName":"Li","suffix":""},{"id":245413705,"identity":"89e351f9-db43-4402-83de-feb1173b1a4f","order_by":1,"name":"Xunyao Wu","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xunyao","middleName":"","lastName":"Wu","suffix":""},{"id":245413706,"identity":"26d1d4b6-80df-47c7-b40f-48a0a66589a1","order_by":2,"name":"Mu Wang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mu","middleName":"","lastName":"Wang","suffix":""},{"id":245413707,"identity":"ec70c454-a85f-487e-ad64-5387241e9c81","order_by":3,"name":"Chuiwen Deng","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chuiwen","middleName":"","lastName":"Deng","suffix":""},{"id":245413708,"identity":"ce8e4e54-ec37-431f-8e22-e094c6641bcf","order_by":4,"name":"Yu Peng","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Peng","suffix":""},{"id":245413709,"identity":"ad9044f6-4530-4f09-8340-7377c53e658d","order_by":5,"name":"Lidan Zhao","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lidan","middleName":"","lastName":"Zhao","suffix":""},{"id":245413710,"identity":"f55e5497-e2b2-4e71-80d5-0d1500a997de","order_by":6,"name":"Wen Zhang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zhang","suffix":""},{"id":245413711,"identity":"0b8ead19-d0b6-4d47-83ba-841b700d34a8","order_by":7,"name":"Zhou Jiaxin","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Jiaxin","suffix":""},{"id":245413712,"identity":"1b66c0de-3d3d-448b-ad3f-9c89148c8053","order_by":8,"name":"Mengtao Li","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengtao","middleName":"","lastName":"Li","suffix":""},{"id":245413713,"identity":"a07921ab-47dc-4046-b9d4-6bdf2dca6198","order_by":9,"name":"Yan Zhao","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhao","suffix":""},{"id":245413714,"identity":"661f805f-a783-4dd8-a6bf-eeb24e658da9","order_by":10,"name":"Xiaofeng Zeng","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Zeng","suffix":""},{"id":245413715,"identity":"916e4fde-3052-4f33-b529-c8c9c7de93d3","order_by":11,"name":"Yunyun Fei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBAC9gbGBhgLRB4grIXnAEwLzwGitcBYEgnEamFvbpPm3XE4cbvk22NSN2ruMPDPbiCghecgUMuZw4k7Z+elSecce8YgcYeATfYSiUAtbYcTN9zOMZPObTjMYABxIR5b5B9Ctdw8Q6wWCUaolhs8xGrhSWy2nNuWbrzhTF6ydc6xwzwSNwhpYT/+8MbbNmvZDcfPHrydU3NYjn8GAS1AwCLBwNAM0g0xg6B6IGD+wMBQR6ziUTAKRsEoGIkAAHrkRqMny6+pAAAAAElFTkSuQmCC","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yunyun","middleName":"","lastName":"Fei","suffix":""}],"badges":[],"createdAt":"2023-11-01 11:59:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3534730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3534730/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46032458,"identity":"d2200396-7b7f-4814-8f87-6eb33039c980","added_by":"auto","created_at":"2023-11-07 18:25:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFrequency and phenotype analysis of peripheral pDCs in pSS patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea:\u003c/strong\u003e The frequency of peripheral pDCs in untreated patients with pSS or SLE, pSS patients treated with HCQ and matched HC (HC: n=29; untreated pSS: n=31; pSS treated with HCQ: n=17; untreated SLE: n=9). \u003cstrong\u003eb:\u003c/strong\u003e Comparison of activation markers (Mean Fluorescence Intensity, MFI) on the surface of pDCs between pSS (n=12) and HCs (n=12). Comparison of TLR7 (n=8) and TLR9 (n=6) expression levels between pDCs from pSS and HC without stimulation \u003cstrong\u003e(c, d) \u003c/strong\u003eand activated by TLR7 agonist\u003cstrong\u003e (e)\u003c/strong\u003e or TLR9 agonist \u003cstrong\u003e(f) \u003c/strong\u003efor 6 hours with 100ng/ml IL3 (n=6). Data are shown as mean ± SEM. Unpaired two-tailed Student’s t-test was applied. *P-value \u0026lt; 0.05, **P-value \u0026lt; 0.01, ***P-value \u0026lt;0.001, **** P-value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/2539107a38ecb4285922a2b8.png"},{"id":46032459,"identity":"35e36235-2346-4b8b-9f7a-e0a5467a4688","added_by":"auto","created_at":"2023-11-07 18:25:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":396874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of characteristic cytokines in pDCs with different activation states.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea: \u003c/strong\u003eFreshly isolated PBMCs from pSS or HC were cultured in the presence of IL3, R848 (TLR7 agonist) or ODN2216 (TLR9 agonist) was added at the 6th hour, GolgiPlug was added at the 9th hour. secretion of IFN-α by pDCs was examined through intracellular staining at the 12th hour. pDCs were gated as CD303+CD123+HLA-DR+LIN- cells; \u003cstrong\u003eb:\u003c/strong\u003e Comparison of IFN-α secretion between HC (n=7) and pSS (n=7).\u003cstrong\u003e c:\u003c/strong\u003e Freshly isolated PBMCs from pSS or HC were cultured in the presence of IL3 and GolgiPlug, stimulated by R848 or ODN2216 for 12 hours, intracellular staining was used to detect the secretion of IL-6; \u003cstrong\u003ec: \u003c/strong\u003eComparison of IL-6 secretion between HC (n=7) and pSS (n=7); Representative flow cytometric graphs\u003cstrong\u003e (d)\u003c/strong\u003e and statistical graph \u003cstrong\u003e(f)\u003c/strong\u003e for TNF-α secretion (detected by the same method as IL-6). Data are shown as mean ± SEM. Unpaired two-tailed Student’s t-test was applied.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/67c40815b9a4ad0dd1dbfacb.png"},{"id":46032466,"identity":"eaccf72b-9a4e-469a-82d8-5f5fb8ea5463","added_by":"auto","created_at":"2023-11-07 18:25:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3323444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFew infiltrations of pDCs in labial gland and the clinical correlation analysis. a: \u003c/strong\u003eImmunofluorescent results of\u003cstrong\u003e \u003c/strong\u003e9 pSS patients who had labial pathological report with typical focal lymphocytic infiltrations. \u003cstrong\u003eb: \u003c/strong\u003eImmunofluorescent colocalization of epithelial cells (CD326) and IFN-α in salivary glands of pSS patients (n=2). \u003cstrong\u003ec: \u003c/strong\u003eComparison of surface chemotactic receptors (MFI) in pDCs between pSS and HCs, CCR2 (HC: n=11; pSS: n=11), CCR4 (HC: n=11; pSS: n=10), CCR5 (HC: n=12; pSS: n=12), CCR7 (HC: n=10; pSS: n=12), CCR10 (HC: n=14; pSS: n=12). \u003cstrong\u003ed:\u003c/strong\u003e Correlation analysis of pDC percentage in PBMC and serum IgG (n=30), IgA (n=29), anti-SSA IgG (n=23). Data are shown as mean ± SEM. Unpaired two-tailed Student’s t-test, Linear regression and Pearson’s correlation were performed for IgG and IgA, Spearman rank correlation were performed for anti-SSA IgG.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/a943199c4f7914ecc5a54f97.png"},{"id":46032464,"identity":"b15f1d9e-3f74-4b7a-a402-604b983101d6","added_by":"auto","created_at":"2023-11-07 18:25:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":373470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-culture of unstimulated pDCs and B cells. \u003c/strong\u003ePurified CD19+ B cells from allogeneic healthy donors cultured alone or with pDCs (without extra activation) from pSS and HC in the presence of 100ng/ml IL3 for 7 days, the ratio of pDC to B cell was 1:10. \u003cstrong\u003ea: \u003c/strong\u003eRepresentative flow cytometric graph\u003cstrong\u003e \u003c/strong\u003eof\u003cstrong\u003e \u003c/strong\u003eki-67 in B cells cultured alone or cultured with pDCs. \u003cstrong\u003eb: \u003c/strong\u003eThe effect of pDCs on B cell proliferation (n=12) and the effect comparison between HC (n=6) and pSS (n=6) derived pDC on B cells. \u003cstrong\u003ec: \u003c/strong\u003eExpression of activation markers on B cells cultured alone or cultured with pDCs. \u003cstrong\u003ed: \u003c/strong\u003eRepresentative flow cytometric graph of B cells differentiation into plasma cells/plasmablasts. \u003cstrong\u003ee: \u003c/strong\u003eThe effect of pDCs on B cell differentiation (n=12) and the effects comparison between HC (n=6) and pSS (n=6) derived pDCs on B cell differentiation. \u003cstrong\u003ef: \u003c/strong\u003eSecretion of IgG, IgA and IgM by B cells cultured alone or cultured with pDCs (n=12). Data are shown as mean ± SEM. Paired and unpaired two-tailed Student’s t-test was performed. *P-value \u0026lt; 0.05, **P-value \u0026lt; 0.01, ***P-value \u0026lt;0.001, **** P-value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/6fa79542d15d34d5720c43d9.png"},{"id":46032461,"identity":"db5049ad-63cd-4a54-85c9-f2a777f020b7","added_by":"auto","created_at":"2023-11-07 18:25:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":303691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epSS plasma, IgG and IgA down-regulate the viability of pDCs. ­\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eRepresentative flow cytometric graph and summary graph\u003cstrong\u003e \u003c/strong\u003eof pDCs viability. pDCs were stimulated by HC or pSS derived plasma (n=5) for 4 hours with 100ng/ml IL3. Representative flow cytometric graph of apoptosis detection, pDCs were stimulated by increasing concentration of IgG \u003cstrong\u003e(b)\u003c/strong\u003e or IgA \u003cstrong\u003e(c)\u003c/strong\u003e for 2 hours with 100ng/ml IL3\u003cstrong\u003e \u003c/strong\u003e(n=4). Statistical graph\u003cstrong\u003e \u003c/strong\u003eof pDCs viability when stimulated by different concentrations of human IgG\u003cstrong\u003e (d)\u003c/strong\u003e and IgA\u003cstrong\u003e (e)\u003c/strong\u003e. Unpaired two-tailed Student’s t-test was performed. *P-value \u0026lt; 0.05, **P-value \u0026lt; 0.01, ***P-value \u0026lt;0.001, **** P-value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/dca9ab106d625163e8988d97.png"},{"id":46032465,"identity":"8c6abd8e-16da-411a-95a5-4cd8c53ed52d","added_by":"auto","created_at":"2023-11-07 18:25:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":247050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe interplay between pDC and B cell contributed to the pathogenesis of primary Sjögren’s syndrome.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003epDCs promote the proliferation, activation, and differentiation into plasmablast/plasma cell of B cells, hyperactive B cells in pSS induce the apoptosis of pDC via excess IgG, IgA and autoantibodies.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/fb17c92b00f8ae7fed5ae10b.png"},{"id":50056124,"identity":"b0f7b217-e512-4aa0-abc6-44233acd19bb","added_by":"auto","created_at":"2024-01-23 17:52:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2482895,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/8d1542a3-cdc4-40d2-ab61-2b9c61744270.pdf"},{"id":46032460,"identity":"a9661e92-189d-4ece-8e05-df9da3e85314","added_by":"auto","created_at":"2023-11-07 18:25:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2155561,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-3534730/v1/a51a58a328065a6e26ca52b9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The redundant role of plasmacytoid dendritic cells in Primary Sjögren's syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrimary sj\u0026ouml;gren's syndrome (pSS) is a typical autoimmune disorder characterized by focal lymphocytic infiltrations, damage, and dysfunction in salivary and lacrimal glands [1]. Lung, kidney, and nervous system involvement could also occur in severe cases [2]. Previous studies have demonstrated the type I IFN signatures in peripheral blood mononuclear cells (PBMCs), B cells, monocytes, neutrophils as well as the affected tissues [3\u0026ndash;10]. Moreover, IFN-α inducible proteins: MDA-5, IFIT-3 [11], and B cell-activating factor (BAFF) [12] were found highly expressed in salivary glands. Type I IFN could enhance T cell and B cell responses and promote the production of autoantibodies [11, 13], implicating their important roles in the pathogenesis of pSS.\u003c/p\u003e \u003cp\u003eActing as a premier type I IFN producer and a key bridge between innate and adaptive immunity, pDCs also play a nonnegligible contribution to autoimmune disease development. pDCs in SLE patients can be activated through various pathways (immune complexes, neutrophil extracellular traps, mitochondrial DNA, \u003cem\u003eetc\u003c/em\u003e.), which in turn trigger immune responses and promote the production of autoantibodies [14]. In studies of systemic sclerosis (SSc) [15], Psoriasis [16], rheumatoid arthritis [17] and autoimmune diabetes [18], pDCs could infiltrate into the target tissue and exacerbate local inflammation by releasing IFN-α and proinflammatory factors.\u003c/p\u003e \u003cp\u003eCurrently, studies on the role of pDCs in pSS are limited. Previous studies have reported the reduced frequencies of pDCs in peripheral blood [19, 20] of pSS patients. A recent study performed a transcriptional analysis of circulating pDCs and identified the aberrant activation of pDC in pSS patients [21]. Here, we systematically study the percentage, phenotype, and functions of pDCs for a better understanding of their roles in the pathogenesis and type I IFN signatures in pSS patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and ethics\u003c/h2\u003e \u003cp\u003eAll newly-onset pSS and SLE patients were enrolled in Peking Union Medical College Hospital (PUMCH) and met the 2016 ACR-EULAR classification[22] and the 1997 classification criteria of the American College of Rheumatology[23], respectively. Labial tissues were obtained from patients who underwent labial gland biopsy at the department of stomatology in PUMCH. Patients all signed informed consent for their residual tissues after the pathological examination. This study was approved by the Ethics Committee of PUMCH (No: K2525).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePBMC Isolation and\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003estimulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHuman PBMCs were isolated with Ficoll-Paque density (DAKEWE, China) as previously described[8]. For the plasma stimulation, PBMCs from HCs were seeded into 24-well plates at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/ml and maintained in RPMI 1640 (Gibco, A10491, USA) with 20% mixed plasma from ten pSS or HCs in the presence of 100 ng/ml IL-3 for 4 hours.\u003c/p\u003e \u003cp\u003eFor IgG and IgA stimulation, freshly isolated PBMCs from HCs were first incubated with 100 mg/ml IL-3 for 2 hours, then stimulated with human IgG (SP001, Solarbio) and human IgA (SP016, Solarbio) with indicated concentrations. IL3 is used to assist the survival of pDCs in vitro [24, 25].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003epurification and stimulation of pDC\u003c/b\u003e\u003c/p\u003e \u003cp\u003epDC was purified using CD304 microbeads (130-090-532, Miltenyi Biotec) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin (15140122, ThermoFisher). For cytokines detection, PBMCs were stimulated with 5 \u0026micro;g/ml R848 (tlrl-r848, InvivoGen) or 10 \u0026micro;g/ml ODN-2216 (tlrl-2216, InvivoGen) in the presence of 100 ng/ml IL-3 (Peprotech, 200-03). R848 is a ligand for Toll-like receptor 7 (TLR7) and OND-2216 is a ligand for TLR9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003epDC and B cell coculture\u003c/h2\u003e \u003cp\u003eB cells were purified using B cell isolation kit II (130-091-151, Miltenyi Biotec) following the manufacturer\u0026rsquo;s protocol. B cells were activated with 5 \u0026micro;g/mL anti-IgM (314502, BioLegend), 500 ng/mL sCD40L (310-02, Peprotech), 100 ng/ml IL-4 (200-04, Peprotech), and 50 ng/ml IL21 (200\u0026thinsp;\u0026minus;\u0026thinsp;21, Peprotech). For pDCs and B cells coculture, purified CD19\u003csup\u003e+\u003c/sup\u003e B cells (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e/ml) and pDC from allogeneic HCs were added at the ratio of 10:1. The supernatants were harvested at day 7.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eCells were harvested and resuspended at the concentration of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml. Fluorochrome-conjugated monoclonal antibodies for cell surface staining were as follows: anti-human CD303, CD123, Lineage, HLA-DR, CD80, CD83, CD86, CD19, CD40, CD74, CD27, IgD, CCR2, CCR4, CCR5, CCR7, CCR10, CD38, CD138, CD24.\u003c/p\u003e \u003cp\u003eThe Intracellular Fixation \u0026amp; Permeabilization Buffer Set (eBioscience) was used for intracellular staining following manufacturer\u0026rsquo;s protocol. Fluorochrome-conjugated monoclonal antibodies for intracellular staining are as follows: anti-human Ki-67, TLR7, TLR9, IFN-α, TNF-α, IL-6.\u003c/p\u003e \u003cp\u003eDetailed information about conjugated fluorescence and manufacturers for antibodies is shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eCell apoptosis was detected by PE Annexin V Apoptosis Detection Kit I (559763, BD Pharmingen). Briefly, cells were washed with 1ml 1\u0026times; Annexin V binding buffer after regular surface staining, then incubated with PE-conjugated Annexin V and 7-AAD for 15 minutes at room temperature, all the samples were analyzed by BD Accuri C6 flow cytometer (Becton Dickinson, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eFreshly labial tissue was embedded in O.C.T. (Tissue-Tek) and 8\u0026micro;m frozen sections were prepared. Sections were immersed in 95% ethanol for 15 min, blocked in normal goat serum for 3h at room temperature, followed by incubating with 25 \u0026micro;g/ml mouse anti-human BDCA-2 (CD303) monoclonal antibody (MAB62991, R\u0026amp;D), 1:50 diluted mouse anti-human IFN-α monoclonal antibody (sc-373757, SANTA CRUZ BIOTECHNOLOGY, INC.) or 1:500 diluted rabbit anti-human EpCAM monoclonal antibody (also known as CD326, ab223582, abcam) at 4\u0026deg;C overnight. CoraLite594-conjugated goat anti-mouse secondary antibody (proteintech, China) or CoraLite488-conjugated goat anti-rabbit secondary antibody (proteintech, China) was incubated at a dilution of 1:500 for 1h at room temperature. Further, the slides were mounted by an antifading mounting medium with DAPI (S2110, Solarbio) and scanned by Pannoramic MIDI (3DHISTECH, Hungary).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe levels of IgG, IgM, and IgA in culture supernatants were measured with Human IgG/IgM/IgA Precoated ELISA Kit (1128162, 1128182, 1128172 DAKEWE, China) respectively. The level of IFNα in plasma from pSS patients and matched HCs was determined by Human IFN-α Precoated ELISA Kit (1110012, DAKEWE, China) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the data analyses were conducted by IBM SPSS statistics (Version 25.0, IBM, Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) software. The data were first performed with the normality distribution. Student\u0026rsquo;s t-test was used for variables in a normal distribution, otherwise, the Mann-Whitney test was used. Linear regression and Pearson\u0026rsquo;s correlation were applied to explore the correlation analysis. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was defined as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe frequency and phenotype analysis of peripheral pDC in pSS patients\u003c/h2\u003e \u003cp\u003eWe first analyzed the frequency of pDC in PBMCs from treatment-na\u0026iuml;ve pSS (n\u0026thinsp;=\u0026thinsp;31), HCQ-treated pSS (n\u0026thinsp;=\u0026thinsp;17), SLE (n\u0026thinsp;=\u0026thinsp;9), and HCs (n\u0026thinsp;=\u0026thinsp;29) by flow cytometry. The demographic characteristics of all the included pSS patients were demonstrated in Supplementary Table\u0026nbsp;2 and SLE patients were demonstrated in Supplementary Table\u0026nbsp;3. The gating strategy of pDCs was shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea. In accordance with previous studies [20, 26], the percentage of pDCs was significantly lower in PBMCs from treatment na\u0026iuml;ve SLE and pSS patients compared with HCs. Interestingly, the reduction of pDCs rebounded in pSS patients after Hydroxychloroquine (HCQ) therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). However, we did not observe the hyperactivation status of pDCs in pSS as the activation markers CD80, CD83, and CD86 expressions were equal to HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eDetection of TLR7/9 expressions showed that unstimulated pDCs from pSS patients showed higher TLR7 but not TLR9 expressions compared with HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). However, the difference vanished after TLR7 (R848) or TLR9 ligand (ODN2216) stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003e \u003cb\u003epDCs did not contribute to peripheral and affected tissue type I IFN signature in pSS patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether pDC from pSS patients displays the enhanced capacity of IFN-α secretion, we stimulated freshly isolated PBMCs with TLR7 (R848) or TLR9 ligand (ODN-2216) and determined representative cytokines production by flow cytometry. However, upon stimulation, there was no difference in the IFN-α secretion of pDCs between HC and pSS patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). The same was observed in the IL-6 and TNF-α production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f).\u003c/p\u003e \u003cp\u003eWe next detected the infiltration of pDCs in labial glands by immunofluorescence to explore whether they contribute to tissue type I IFN signature in pSS patients. We detected the labial glands of nine pSS patients whose labial gland biopsy indicated typical focal lymphocytic infiltration. Among these cases, only two exhibited a small amount of pDC infiltration, while the remaining seven cases showed no evidence of pDC infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Recent researches highlighted the role of salivary epithelial cells in producing type I IFNs [27\u0026ndash;29], consistently, our immunofluorescent results indicated that epithelial cells are responsible for the production of tissular IFN-α in patients with or without pDC infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We also detected the main chemokine receptors, including CCR2, CCR4, CCR5, CCR7, and CCR10 on the pDC surface showed no difference between pSS patients and HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, Fig S2j).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003epDCs promote B cell class-switching and antibody production\u003c/h2\u003e \u003cp\u003eCorrelation analysis between the frequency of peripheral pDCs and clinical parameters in pSS patients showed that the percentage of pDCs was negatively correlated with serum IgG, IgA, and anti-SSA autoantibody levels (Fig S2a-i, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). We, therefore, established the pDC/B cells co-culture system to explore the potential role of pDC in promoting B cell responses and antibody production in pSS patients.\u003c/p\u003e \u003cp\u003eWe found that resting or TLR-7-activated pDCs were able to efficiently promote the proliferation, activation, and differentiation into plasmablast/plasma cell of B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-e, Fig S3a-e). Also, we explored the effects of pDCs on antibody production and found that even resting pDCs could promote IgG, and IgA production by B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). However, there was no difference between HC and pSS-derived pDCs. Similarly, no difference was found in the effects of HC or pSS derived pDCs on naive or memory B cells (Fig S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003epDCs are more sensitized to pSS plasma-induced apoptosis\u003c/h2\u003e \u003cp\u003eFinally, we sought to explore the potential mechanism of reduced pDC frequency in the peripheral blood of pSS patients. Incubation of plasma from pSS patients could significantly reduce pDCs viability than that from HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Considering that pSS is characterized by high autoantibodies in serum and pDC could promote antibody production as we demonstrated above, we inferred that plasma-derived IgG and IgA might be important inducers of pDC apoptosis. As we expected, both IgG and IgA dramatically increased the apoptotic rates of pDCs in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-e). The viability of freshly isolated pDCs showed no significance between pSS and HCs (Fig S5a). Although pSS had higher serum IFN-α levels (Fig S5b), they had little effect on the pDC apoptosis (Fig S5c-e). These results indicate that hyper immunoglobulin in the serum of pSS patients contributes to the reduction of pDC in the peripheral blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe critical role of the type I IFN pathway in the pathogenesis of pSS has been widely addressed [30, 31]. Acting as the premier producer of IFN-a, the role of pDC in the pathogenesis of pSS still remains inclusive. In the present study, we have deciphered the redundant role of pDC in the type I signature and disease development in pSS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsistent with previous studies, we have detected a peripheral reduction in pSS [20, 26]. We have demonstrated a negative feedback loop, in which, pDC promoted antibody production and in turn, high immunoglobulin could induce pDC apoptosis. \u0026nbsp; It has been reported that IgG-complexed adenoviruses induce the apoptosis of human pDCs [32]. Hyperactivated B cells, characteristic autoantibodies (SSA, SSB), and hypergammaglobulinemia are hallmarks of pSS [33]. Previous studies have found that pDCs could efficiently promote the differentiation of B cells into plasmablasts and plasma cells through type I IFNs and IL-6 [34, 35]. Moreover, pDCs could enhance the autoreactivity of B cells via type I IFNs in a T cell-independent manner [36]. Although our data showed that, pSS-derived pDC did not display a stronger capacity for promoting plasma cell differentiation and antibody production, we gave a good explanation that the excess immunoglobulin (IgG and IgA) produced by hyperactivated B cells might result in the apoptosis and reduction of pDC in peripheral blood of pSS. In addition, Hydroxychloroquine (HCQ) effectively inhibits B cell activation [37] and a recent systematic review revealed that serum IgA in patients with pSS decreased significantly after using HCQ [38], which is consistent with our results that pDC frequency rebounded after HCQ therapy.\u003c/p\u003e\n\u003cp\u003eNonetheless, pDC is noted for its unique ability in producing type I IFNs, our data suggest that it might not be the culprit of hyperactive type I IFN signaling in pSS. Recent studies in pSS have emphasized the critical role of dysregulated epithelial cells [29, 39], especially the active IFN signaling in salivary gland epithelial cells (SEGCs) [29]. SEGCs from pSS were found to be sensitive to the stimulation of TLR agonists and produced type I IFNs as a response to the stimulation [27, 28, 40]. Type I IFNs released by SEGCs could further promote the secretion of BAFF in an NF-\u0026kappa;B dependent way [12, 27]. These results imply that gland epithelial cells are not purely innocent victims, but can also be the trigger of type I IFN signaling in pSS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn SSc, pDCs directionally migrated to target organs and secreted IFN-\u0026alpha; and CXCL4, thereby accelerated tissue fibrosis [15, 41]. In another autoimmune disease with fibrosis signature, IgG4-related disease, active pDCs and relevant IFN-\u0026alpha; signaling were also observed in related pancreas, pDC also enhanced the production of IgG4 by B cells [42]. Dual role of pDCs on IFN-\u0026alpha; production and promoting activation of pathogenic T cells in psoriasis has been revealed in psoriasis [43]. Study in RA mouse model suggested that pDC aggravated joint inflammation and bone erosion via TLR7 dependent type I IFNs [17]. Reduction of pDC in the peripheral blood as well as the concomitant infiltration and activation in related tissue were reported in SLE [44, 45]. A recent randomized controlled trial in SLE clarified that Litifilimab (anti-BDCA2 antibody, BDCA2 is an exclusive marker of pDC) is effective in disease remission [46], which indicated the feasibility of pDC purge strategy in SLE treatment. Whereas, newly published research revealed that SLE-derived pDC had senescence and inert phenotype, active keratinocytes should be responsible for the tupe I IFN signaling rather than pDC [26]. In general, pDCs contributed to the pathogenesis of autoimmune diseases through type I IFNs or interplay with other immune cells [47]. However, further studies are needed to elucidate the potential mechanism about pDC\u0026rsquo;s contribution to SLE and pSS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur available evidence does not support an overactive phenotype (resting and activating status) of pDCs from pSS patients, particularly in the production of IFN-\u0026alpha;. However, a previous transcriptional study reported that pDCs from patients with pSS secreted more type I IFNs after TLR7 stimulation [21]. Antonios et al. \u0026apos;s work that pDCs from patients with SLE or pSS did not show stronger secretion ability for inflammatory cytokines, especially\u0026nbsp;IFN\u0026alpha; [26], which is consistent with our findings. These divergent results may be caused by the difference of detection methods, further study will help to elucidate underlying mechanism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, we demonstrated the decreased percentage of pDC in pSS patients, which might result from the excess immunoglobulin (IgG and IgA) induced apoptosis. \u0026nbsp; Moreover, we showed that pDC might not be the major contributor to the hyperactivation of type I interferon signaling in pSS patients. Our research provides a good addition to pSS pathogenesis and gives implications that targeting pDC might not be a good strategy for clinical pSS treatment.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no conflicts of interest.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of the Peking Union Medical College Hospital (No: K2525) and was conducted in accordance with the Helsinki Declaration. Informed consent of all the patients were obtained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript is approved by all authors for publication, and written informed consent to publish were obtained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (grant numbers: 81971545, 81971544), the National High-Level Hospital Clinical Research Funding (2022-PUMCH-C-039, 2022-PUMCH-B-013). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData of this study are available from the corresponding author upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNocturne, G. and X. 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We performed pDC/B co-culture system to explore the contribution of pDC to B cell functions in pSS.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe percentage of pDC was significantly reduced in the peripheral blood of pSS. The activation markers (CD80, CD83, and CD86) expressions, chemokine receptors, and representative cytokines production (IFN-α, IL-6, and TNF-α) of pDC were similar between pSS and HCs. Only a few pDCs infiltration were detected in the labial gland. The percentage of pDCs was negatively correlated with serum IgG, IgA, and anti-SSA autoantibody levels and resting pDCs were able to efficiently promote B cells proliferation, activation, differentiation, and antibody production \u003cem\u003ein vitro\u003c/em\u003e. However, there was no difference between HC and pSS-derived pDCs. Finally, we found that incubation of plasma from pSS patients could significantly induce pDCs apoptosis than that from HCs and both IgG and IgA dramatically increased the apoptotic rates of pDCs.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data have deciphered the redundant role of pDC in the type I signature and disease development in pSS. Also, we demonstrated the decreased percentage of pDC in pSS patients might result from apoptosis induced by the excess of immunoglobulin (IgG and IgA).\u003c/p\u003e","manuscriptTitle":"The redundant role of plasmacytoid dendritic cells in Primary Sjögren's syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-07 18:25:52","doi":"10.21203/rs.3.rs-3534730/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30a446f8-ddb1-4606-ab1c-8186570185a1","owner":[],"postedDate":"November 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-23T17:44:16+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-07 18:25:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3534730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3534730","identity":"rs-3534730","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}