Expression of TLR10 in B cells Correlates With Primary Sjögren’s Syndrome Progression

Primary Sjögren’s Syndrome (pSS) is considered a B cell-mediated disease, yet the precise role of B cells in the pathogenesis is not fully understood. Toll-like receptor 10 (TLR10) is highly expressed in human B cells, indicating that TLR10 probably plays a vital role in regulating B cell function as well as B cell-related diseases. However, the biology of TLR10 in pSS was rarely researched. Here, we examined the TLR10 expression in peripheral B cell subsets isolated from both pSS patients and healthy controls (HCs) and further analyzed the correlations between TLR10 expression and disease activity. We observed that TLR10 expression in peripheral total CD19 + B cells, naïve B cells (CD19 + CD27 - IgD + ) and switched memory B cells (CD19 + CD27 + IgD - ) was signicantly increased in low-activity pSS patients as compared with HCs and high-activity pSS patients. TLR10 expression in total and switched memory B cells in pSS patients was signicantly negatively correlated with serum levels of anti-SSA antibody and B cell activating factor of TNF family (BAFF). As compared with the TLR10 low-, the TLR10 high-expressed pSS patients presented with reduced switched memory B cells. Moreover, a much lower proportion of high-activity pSS patients was observed in TLR10 high- as compared to low-expressed patients. Our study concluded that TLR10 expression in peripheral total and switched memory B is negatively correlated with pSS disease activity, suggesting that TLR10 might suppress pSS progression via inhibiting the B cell class switch recombination. These results should contribute to the diagnosis and treatment of pSS.


Introduction
Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder affecting exocrine glands of the body, preferentially lacrimal and salivary glands. The global prevalence of pSS is approximately 0.4-1%, in which around 30-40% of pSS patients will progress to at least one additional systemic autoimmune complications [1] and nearly 5% of pSS patients may develop to B cell malignancies, most commonly salivary gland mucosa-associated lymphoid tissue lymphomas [2]. Clinical treatments for pSS easily fail due to the heterogeneity of clinical phenotype in pSS patients. Moreover, plenty studies in pSS demonstrated that numerous factors seem to contribute to the progression of the pSS [3,4]. However, the mechanism by which pSS develops, although being widely researched, remains unclear.
Currently, pSS is considered a B cell-mediated disease characterized by autoantibodies and hypergammaglobulinaemia in patients [4]. Besides the production of autoantibodies, B cell can activate T cell by presenting autoantigen and secrete multiple in ammatory cytokines upon Toll Like Receptor (TLR) activation, thereby contributing to the development of pSS [5]. However, the precise role of B cells in the pathogenesis of pSS is still poorly understood. According to clinical e cacy for rituximab treatment in different trials, it was suggested that B cells at different developmental stages contribute to the broad clinical phenotypes in pSS patients, suggesting that understanding the abnormality of B cell development and differentiation is essential to uncover the pathogenesis of pSS [6][7][8].
TLRs are pattern recognition receptors that have crucial roles in the initiation of innate immunity and the activation of adaptive immunity. Many studies have proved that TLR signaling is required for human B cell activation and plays an important role in autoimmune diseases [9,10]. Among ten human TLRs (TLR1-10), TLR10 remains the least understood one because it presents in human beings, but not in the mice, and its ligand has not been identi ed yet [11]. TLR10 is expressed at the highest level in B cells, followed by plasmacytoid dendritic cells but not expressed in monocytes, natural killer cells, and T cells [12,13], indicating that TLR10 might play an important role in regulating B cell function as well as B cellrelated diseases. Recently, a few studies have reported that human TLR10 polymorphisms are associated with several diseases, including bacterial infections, cancers and autoimmune diseases [14][15][16]. Torices et al. showed that TLR10 variant rs11466657 is closely related to rheumatoid arthritis (RA) [17]. Moreover, Zhang et al. revealed that upregulated TLR10 in B cell subsets is positively correlated with disease activity in RA patients [18]. However, the biology of TLR10 in pSS is less researched.  [19]. Patients with other autoimmune or in ammatory diseases, severe renal or liver disease, or cancer were excluded. The disease activity of pSS patients was determined according to the ESSDAI. All the pSS subjects were divided into two groups: a high-activity group (ESSDAI ≥ 5), a low-activity group (ESSDAI < 5) [20]. In addition, 25 age-and sex-matched HCs were chosen for comparison. More information about age, gender and treatments are shown in Table 1.
The data are expressed as n (%), mean ± standard deviation (SD). a The age of each group is proved to be normal distribution (sample K-S test, P > 0.05). And T-test about age between each group ensured that there was no signi cant difference between them (P > 0.05). b Anti-SSA antibody, anti-SSB antibody, anti-Ro52 antibody data were lacking in a few subjects. c The patient de ned as "unknown" was someone who was on her rst visit to our hospital, could not tell which medication to use.

Specimen Collection and Laboratory Testing
For analysis of the peripheral blood, 2 mL venous blood was collected from each participant into ethylene diamine tetra acetic acid (EDTA)-containing collection tubes (Becton Dickinson). Samples were centrifuged to collect the upper serum layer, and then frozen and stored at -80°C until use. After that, the remaining sample was processed to isolate peripheral blood mononuclear cells (PBMCs) by Ficoll density-gradient centrifugation. PBMCs were used for ow cytometry analyses.

Antibodies and Flow Cytometry Analysis
The immunophenotyping of B cells was performed in the peripheral blood samples using the following uorochrome-labelled anti-human antibodies: and human Fc receptor blocking solution (cat: 422302) were purchased from Biolegend; IgD-FITC (clone IA6-2) was purchased from BD Biosciences, and 7-amino-actinomycin D (7AAD) (cat:00-6993-50) was purchased from Invitrogen. Fresh isolated PBMCs were rst blocked with human Fc blocking reagents and stained with diluted antibodies at 4°C for 15 minutes in the dark. Then, PBMCs were washed twice with cold FACS buffer (1×PBS containing 2% FBS), resuspended in 0.3 ml of FACS buffer and analyzed by ow cytometry. Approximately 300,000 ~ 500,000 events were collected per sample. The data were collected with a FACS Calibur (Beckman CytoFLEX, USA) and analyzed using the FlowJo software version 10.0.

Statistical analysis
The results are expressed as the means ± standard deviation (SD) and medians (interquartile range). Statistical comparisons were performed by Student's t-tests. Differences among the three groups were determined by the Kruskal-Wallis H nonparametric test. Correlation analyses between two parameters were performed by Spearman's correlation method. All statistical analyses were performed using the SPSS software version 20 (SPSS Inc., Chicago, Illinois, USA) and GraphPad Prism (v.8.0, CA). A P value < 0.05 was considered statistically signi cant.

TLR10 expression in B cells in pSS patients
The Human Protein Atlas showed that TLR10 mRNA mainly enriched in B cells, less in dendritic cells and monocytes, and undetectable in other peripheral blood immune cells (Sup Fig. 1). Therefore, to investigate the difference in TLR10 expression on the B cell surface between pSS patients and HCs, we isolated PBMC from the above two groups and detected by ow cytometry (Fig. 1a). Compared with the HCs, the pSS patients expressed relatively high levels of TLR10 on total CD19 + B cells' surface when determined by the mean uorescence intensity (MFI) (Fig. 1b), but there was no statistically signi cant difference between the two groups.

Correlation between TLR10 expression in B cells and pSS related autoantibodies
Anti-SSA, anti-SSB and anti-Ro52 are important clinical diagnostic indicators for pSS patients, and their concentrations are usually positively correlated with pSS progression [21][22][23].We analyzed the TLR10 expression in B cells in pSS patients according to the extractable nuclear antigen pro le results.
Interestingly, the expression of TLR10 in total CD19 + B cells from anti-SSA +++ and anti-Ro52 +++ pSS patients is signi cantly reduced as compared with the anti-SSA −/+ and anti-Ro52 +/− pSS patients respectively (Sup Fig. 2a-b), and anti-SSB +++ pSS patients show a moderate reduction in TLR10 expression as compared with anti-SSB −/+ pSS patients (Sup Fig. 2c), indicating that the expression of TLR10 in B cells might be related with the production of autoantibodies in pSS patients. Further ELISA results showed that the expression of TLR10 in total CD19 + B cells is negatively correlated with serum level of anti-SSA (r = -0.4599, P = 0.0138), anti-SSB (r = -0.4028, P = 0.0336) and ANA (r = -0.7855, P = 0.0011) in pSS patients (Fig. 2a-c). Moreover, the expression of TLR10 in CD19 + B cells was negatively correlated with BAFF (r = -0.4092, P = 0.0306) (Fig. 2d), which is important for survival and activation of B cells and presents excessive level in pSS patients [24]. These results suggested that the expression of TLR10 in CD19 + B cells might be correlated with pSS formation and/or progression.

TLR10 expression is mainly upregulated in switched memory B in pSS
Numerous studies have been reported that memory B cells, PB and plasma cells are the key subsets of B cells involved in the pathogenesis of pSS [4]. We further analyzed the expression of TLR10 in peripheral B cell subsets, including CD19 + CD24 ++ CD38 ++ transitional B cells, CD19 + IgD + CD27 − naïve B cells, CD19 + IgD + CD27 + unswitched memory B cells, CD19 + IgD − CD27 + switched memory B cells and CD19 + CD24 − CD38 ++ PB (Fig. 3a), obtained from both the pSS patients and HCs. The results showed that TLR10 expression was similar between the pSS patients and HCs in transitional B cells, naïve B cells, unswitched memory B cells and PB (Fig. 3b). Interestingly, the expression of TLR10 in switched memory B cells was signi cantly increased in pSS patients compared with the HCs (Fig. 3b). Moreover, the expression of TLR10 in switched memory B cells also correlated negatively with serum level of anti-SSA (r = -0.3953, P = 0.0373) and BAFF (r = -0.3760, P = 0.0486) in pSS patients (Fig. 3c-d). There was no signi cant correlation between TLR10 expression in switched memory B cells and serum level of anti-SSB (r = -0.2376, P = 0.2234) in pSS patients (Sup Fig. 3a). In addition, the expression of TLR10 in transitional B cells, naïve B cells, unswitched memory B cells and PB showed no obvious correlation with serum level of anti-SSA, anti-SSB and BAFF in pSS patients, respectively (Sup Fig. 3). These results further con rmed that the expression of TLR10 was increased in switched memory B cells, which might play an important role in pSS progression.

TLR10 expression in B cells is negatively correlated with pSS progression
To further clarify whether TLR10 expression in B cells correlated with pSS progression, we analyzed the changes of TLR10 expression in peripheral B subsets in the HCs, low-activity pSS patients and highactivity pSS patients evaluated by ESSDAI according to their clinical features [20]. Interestingly, the expression of TLR10 in total CD19 + B cells in low-activity pSS patients signi cantly increased compared with the HCs, while decreased in high-activity pSS patients compared with low-activity pSS patients (Fig. 4a). Consistent with above results, the expression of TLR10 in transitional B cells and PB was relatively comparable among these groups ( Fig. 4b and f). Notably, the expression of TLR10 in naïve B cells and switched memory B cells was obviously increased in low-activity pSS patients compared with the HCs (Fig. 4. c and e). With the progression of pSS, the expression of TLR10 in naïve B cells, unswitched and switched memory B cells was signi cantly decreased in high-activity pSS patients compared with low-activity pSS patients (Fig. 4c-e), indicating that TLR10 might affect pSS progression by taking part in the process of B cell activation and differentiation, especially in the phase of B cell classswitch recombination (CSR).

TLR10 suppress pSS progression might via inhibiting CSR
There were some evidences suggested that TLR10 might be able to suppress antibody responses of B cell with either T-dependent or independent antigens [25]. The upregulated expression of TLR10 in switched memory B cells indicate that TLR10 could take part in the CSR of B cells in pSS patients. Then we divided the pSS patients and HCs into TLR10 high-and low-expressed groups based on the average value of the TLR10 MFI of total CD19 + B cells, respectively, and calculated the percentage change of switched memory B cells. As shown in Fig. 5a, the percentage of switched memory B cells showed no signi cant difference between the TLR10 low-and the high-expressed group in HCs, but signi cantly decreased in the TLR10 high-as compared with the low-expressed group in pSS patients (Fig. 5a).
Moreover, the pSS progression was closely related with TLR10 expression in total CD19 + B cells (P < 0.001) ( Table 2). In addition, the proportion of high-activity patients in TLR10 low-expressed pSS patients was signi cantly higher than the TLR10 high-expressed pSS patients (76.19% vs 7.69%) (Fig. 5b).
Considering the TLR10 high-expressed pSS patients presented with reduced percentage of switched memory B cells and correspondingly lower proportion of high-activity pSS patients, it suggested that TLR10 could suppress pSS progression via inhibiting the B cell CSR in pSS patients. Discussion pSS is a systemic rheumatic autoimmune disease characterized by abnormal B cell biological function [5]. TLRs, as one kind of pattern recognition receptors, are well known for their signi cant roles in in ammation and innate immunity. Previous experiments have proved that expression of TLR7 and TLR9 in different kinds of B cells may play an important role in dysregulation of B cells in pSS [26,27]. TLR10, as the latest identi ed functional TLR in human, is mainly expressed in B cells [28]. However, few studies on TLR10 expression in pSS have been reported so far. The gene polymorphism analysis showed that TLR10 is involved in autoimmune thyroid disease [14] and rheumatoid arthritis [17]. Recently, Zhang et al reported that the expression of TLR10 in B cell subsets was increased and correlated with disease activity in RA patients [18]. These reports inspired us to detect the TLR10 expression in B cell subsets in the peripheral blood of patients with pSS. Here, we found that TLR10 was highly expressed in total CD19 + B cells, naïve B cells and switched memory B cells in low-activity pSS patients as compared with the HCs and high-activity pSS patients. TLR10 expression in CD19 + total B cells and switched memory B cells in pSS patients was signi cantly negatively correlated with anti-SSA autoantibody and BAFF production. Moreover, the TLR10 high-expressed pSS patients usually had relatively lower proportion of high-activity patients accompanied by reduced switched memory B cells as compared with the TLR10 low-expressed pSS patients.
Today, most investigators agree that there are two main CD27 + memory B cell compartments in blood, IgM + IgD + and IgM − IgD − B cells, the former is unswitched memory B cells exhibiting characteristics of marginal zone B cells, whereas the latter most likely represents class switched B cells [29,30].
Importantly, we found that TLR10 was particularly highly expressed in CD27 + IgD − switched memory B cells in pSS patients compared with the HCs, while TLR10 expression in CD27 + IgD + unswitched memory B cells in pSS patients were slightly lower than the HCs. Our results indicate that TLR10 might play an important role in B cell germinal center reaction in patients with pSS. Further studies are necessary to uncover the role and detailed mechanism of TLR10 on B cell activation and class switch response.
pSS disease activity is assessed by physician according to ESSDAI from patient's clinical manifestations [31]. Among all criterion scores, anti-SSA received the highest average weights [32]. Moreover, it has been reported that the presence and increased titers of anti-SSA, anti-SSB and rheumatoid factor serum autoantibodies are correlated with the severity in pSS patients [33]. Intriguingly, we observed that TLR10 expression in CD19 + total B cells in pSS patients was negatively correlated with anti-SSA, anti-SSB and ANA autoantibodies (Fig. 2a-c). Moreover, TLR10 expression in switched memory B cells in pSS patients was also negatively correlated with anti-SSA but not anti-SSB autoantibodies. When using anti-SSB autoantibody as the biomarker, different correlation results between TLR10 expression in CD19 + total B and switched memory B cells were achieved, but understandable because the anti-SSB shows a weaker correlation with pSS progression as compared with anti-SSA according to the ESSDAI [32]. These results suggested the expression of TLR10, especially in switched memory B cells, could be used as a diagnostic biomarker for pSS progression. Importantly, the expression of TLR10 in CD19 + total B cells and switched memory B cells in low-activity pSS patients was signi cantly increased compared with high-activity pSS patients (Fig. 5). Thus, we postulated that TLR10 could inhibit pSS progression via negatively regulating B cell function.
Although the ligand and downstream signaling pathways of TLR10 remain unclear, it has been identi ed as an immunomodulatory receptor with inhibitory properties [25,34,35]. The high expression of TLR10 in human B cells suggest that TLR10 may regulate B cell function. Hess et al. reported that TLR10 is able to suppress responses mediated by a variety of B cell co-stimulatory signals and attenuate both T cell independent and T cell dependent antibodies production in a TLR10 knock-in mouse model [25]. Therefore, we speculated that TLR10 might inhibit pSS progression via negatively regulating B cell function. In addition, the dysregulation of CSR can cause self-reactive BCRs and B cell lymphomas [36].
Considering the high expression of TLR10 in switched memory B cells in pSS patients, we speculated that TLR10 expression might affect B cell CSR in pSS patients (Fig. 3b). Notably, we further observed that the TLR10 high-expressed pSS patients presented with decreased percentage of switched memory B cells as compared with TLR10 low expression pSS patients (Fig. 5a), suggesting TLR10 suppressed the B cell CSR in pSS patients.
BAFF, an important cytokine for B cell maturation, proliferation and survival, is upregulated in salivary gland tissue and blood from pSS patients, and plays a vital role in the pathogenesis of pSS [37,38]. We observed that TLR10 expression in B cells and switched memory B cells in pSS patients also was inversely correlated with BAFF level in pSS patients' serum ( Fig. 2d and Fig. 3d). All these results further supported our hypothesis that TLR10 inhibited pSS progression by suppressing B cell function.
In summary, for the rst time, we found that the protein level of TLR10 expression in peripheral switched memory B cells was increased in pSS patients and was signi cantly negatively correlated with both anti-SSA and BAFF production. Moreover, TLR10 could inhibit pSS progression via negatively regulating B cell function. These ndings suggest that TLR10 could be used as a diagnostic biomarker of pSS progression and be a potential therapeutic target for pSS.

Declarations FUNDING
We are grateful to the patients and healthy control for their participation and cooperation in this study. All the authors read and approved the nal manuscript.

Con ict of interest
The authors have declared that no competing interests exist.

Ethics Approval and Consent of Participate
This study was performed according to the recommendations of the Declaration of Helsinki and approved by the Ethics Committee of the Seventh A liated Hospital of Sun Yat-sen University. All participants carefully read and signed the written informed consent.

Consent of Publication
The manuscript is approved by all authors for publication.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupFig.13.pdf