Niclosamide suppresses the expansion of follicular helper T cells and alleviates disease severity in two murine models of lupus via STAT3

Autoantibody production against endogenous cellular components is pathogenic feature of systemic lupus erythematosus (SLE). Follicular helper T (TFH) cells aid in B cell differentiation into autoantibody-producing plasma cells (PCs). The IL-6 and IL-21 cytokine-mediated STAT3 signaling are crucial for the differentiation to TFH cells. Niclosamide is an anti-helminthic drug used to treat parasitic infections but also exhibits a therapeutic effect on autoimmune diseases due to its potential immune regulatory effects. In this study, we examined whether niclosamide treatment could relieve lupus-like autoimmunity by modulating the differentiation of TFH cells in two murine models of lupus. 10-week-old MRL/lpr mice were orally administered with 100 mg/kg of niclosamide or with 0.5% methylcellulose (MC, vehicle) daily for 7 weeks. TLR7 agonist, resiquimod was topically applied to an ear of 8-week-old C57BL/6 mice 3 times a week for 5 weeks. And they were orally administered with 100 mg/kg of niclosamide or with 0.5% MC daily for 5 weeks. Every mouse was analyzed for lupus nephritis, proteinuria, autoantibodies, immune complex, immune cell subsets at the time of the euthanization. Niclosamide treatment greatly improved proteinuria, anti-dsDNA antibody levels, immunoglobulin subclass titers, histology of lupus nephritis, and C3 deposition in MRL/lpr and R848-induced mice. In addition, niclosamide inhibited the proportion of TFH cells and PCs in the spleens of these animals, and effectively suppressed differentiation of TFH-like cells and expression of associated genes in vitro. Niclosamide exerted therapeutic effects on murine lupus models by suppressing TFH cells and plasma cells through STAT3 inhibition.

T lymphocytes [2,3]. Although the exact pathogenesis of SLE has not yet been elucidated, various factors are known to be involved in driving SLE [1,4].
Follicular helper T (T FH ) cells are a specialized subset of CD4 + T cells required for germinal center (GC) formation and ultimately the development of memory B cells and high-affinity long-lived plasma cells (PCs) [5]. T FH cells express high levels of the chemokine receptor CXCR5, which promotes the migration of T FH cells to the B cell follicles in response to the specific ligand CXCL13 [6]. The transcription factor B cell lymphoma 6 (Bcl-6) is a master regulator of T FH cells and is critical for T FH cell differentiation [7]. However, additional transcription factors, including interferon-regulatory factor 4 (IRF4), basic leucine zipper transcriptional factor ATF-like (BATF), and Maf bZIP transcription factor (MAF), also play an important role in T FH cell differentiation [8]. B lymphocyte-induced maturation protein 1 (Blimp-1) has been found to be the most downregulated transcription factor in T FH cells, and acts as a suppressor of T FH cell differentiation [9,10]. Circulating T FH cells are significantly increased in the blood of SLE patients and correlate with disease severity. In addition, T FH cells are expanded in lupus-prone mice and lead to an increase in IL-21 production [4,11].
Signal transducer and activator of transcription 3 (STAT3) plays a key role in regulating inflammation and innate/adaptive immune responses [12]. STAT3 promotes T FH cell differentiation by positively regulating the transcription factors Bcl-6 and T cell factor 1 (TCF-1) [13,14]. STAT3 signaling is enhanced by IL-6 and IL-21, which promotes the expression of Bcl-6. Previous studies have demonstrated that STAT3 is involved in the pathogenesis of SLE; indeed, a STAT3 genetic deficiency, STAT3 inhibitors, and agents that inhibit expression of STAT3 all provide a protective effect against SLE [15][16][17][18].
Several classes of agents, such as non-steroidal antiinflammatory drugs (NSAIDs), immunosuppressants, and corticosteroids are used to treat lupus. However, these agents have a wide range of serious side effects, so their use is limited [1]. Accordingly, now, many studies are ongoing to reduce the failure of therapy by developing new agents having maximum efficacy with minimum side effects. Niclosamide is an FDA-approved anti-helminthic drug and has wide safety and few side effects, so it can be safely used for long-term period in the treatment of chronic diseases [19]. Furthermore, it is multifunctional and has also been reported by several groups to play a potential role in the treatment of various solid tumors [20,21]. In addition, it may also be effective against autoimmune diseases and renal fibrosis due to its potential anti-inflammatory effects [22,23]. Niclosamide is a potent inhibitor of STAT3 signaling [24]. However, the effect of niclosamide on T FH cells in lupus-prone mice has not yet been investigated.
In this study, we examined whether in vivo treatment with niclosamide had a therapeutic effect in lupus-prone MRL/lpr and R848-induced mice by examining proportion of T FH cells and PCs in the spleen and peripheral blood (PB). We additionally investigated the impact of niclosamide on T FH -like cells in vitro. The T FH cells were sensitive to inhibition of STAT3 signaling both in vitro and in vivo. STAT3 signaling inhibition resulted in decreased numbers of T FH cells in the spleen, which was correlated with an improvement in the lupus manifestation. Our findings demonstrate that niclosamide is a critical regulator of T FH cells, which are a therapeutic target for SLE.

Animals
MRL/lpr mice were purchased from SLC Inc. (Japan). C57BL/6 mice were purchased from OrientBio (Korea). Niclosamide (Sigma-Aldrich, St Louis, MO, USA) was resuspended in 0.5% methyl cellulose (Sigma-Aldrich, St Louis, MO, USA) for in vivo studies or in 5% DMSO for in vitro use. Female 10-week-old MRL/lpr mice received daily administration of vehicle (n = 7) or niclosamide (n = 7; 100 mg/kg) for 7 weeks by oral gavage. All mice were sacrificed at 16 weeks of age. Female 8-week-old C57BL/6 mice were treated via epicutaneous application of 50 μg of the TLR7 agonist resiquimod (R848; Sigma-Aldrich) dissolved in 10 μl of acetone, with or without 100 mg/kg of niclosamide daily for 4 weeks, or acetone alone as a control, to the right ear three times a week until euthanasia. All procedure of animal research were provided in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent experiment provided by the IACUC(Institutional Animal Care and Use Committee) in school of medicine, The Catholic University of Korea. (Approval numbers: CUMS-2018-0341-02 and 2018-0236-02).

Enzyme-linked immunosorbent assay (ELISA)
Cytokines in sera or spleen lysates were assayed using mouse IL-6 and IL-21 Duoset ELISA kits (R&D systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The serum levels of anti-double-stranded DNA (dsDNA) IgG antibodies were measured by ELISA following the manufacturer's instructions (Alpha Diagnostics, San Antonio, TX, USA). Total IgG, IgG1, IgG2a, and IgM levels in the sera of the mice were measured by ELISA following the manufacturer's instructions (Bethyl Laboratories, Montgomery, TX, USA).

Measurement of urine albumin to creatinine ratio
Urine albumin and creatinine concentrations were measured using a mouse albumin ELISA assay (Bethyl Laboratories) and a creatinine assay (R&D systems), respectively, according to the manufacturer's directions. Urine albumin excretion was expressed as the ratio of urine albumin to creatinine (ACR).

Histological assessment of the kidney
Kidney tissues were fixed with formalin and embedded in paraffin, cut into 3 μm sections, and stained with periodic acid-Schiff (PAS) stain. Kidney histological pathology was evaluated using the lupus nephritis classification system, as described [25].

Statistics analysis
Statistical analyses were performed in GraphPad Prism version 7.0 software (GraphPad, San Diego, CA, USA). Statistical significance was determined by t-tests for two groups, and by one-way ANOVA with Tukey's multiple comparisons tests for three or more groups. P < 0.05 was considered statistically significant.

Effects of niclosamide on lupus nephritis in MRL/lpr mice
To assess whether niclosamide ameliorates clinical features of LN, we orally administered 10-week-old MRL/lpr mice with niclosamide or vehicle daily for 7 weeks. While the kidney weights increased with disease progression in this spontaneous lupus model, niclosamide treatment inhibited kidney enlargement (Fig. 1a). Proteinuria, one of the major measures of LN, was reduced upon niclosamide treatment, in contrast to the vehicle (Fig. 1b). We next measured autoantibodies in these mice, which is the hallmark of SLE. Niclosamide treatment decreased serum levels of anti-dsDNA IgG (Fig. 1c), in addition to the serum levels of IgG, IgG1, and IgM (Fig. 1d). IL-6 and IL-21 are pro-inflammatory cytokine for lupus progression, and also important regulators of T FH cell generation. We found that niclosamide treatment significantly decreased the serum levels of IL-6 and IL-21 (Fig. 1e). Furthermore, MRL/lpr mice developed severe renal injury, which was characterized by diffuse glomerular, perivascular, and interstitial mononuclear cell infiltration, tubular cast deposition, increased mesangial matrix, and immune complex deposition. Niclosamide ameliorated these renal pathological features, as judged by changes in the histological scores and C3 immune complex deposition (Fig. 1f, g). These data suggest that niclosamide treatment significantly prevented LN in MRL/lpr mice.

Niclosamide inhibits CD90.2 + T cell, DNT cell, T FH cell and PC differentiation in MRL/lpr mice
To investigate the cell types related to the decreased disease severity by niclosamide in MRL/lpr mice, we analyzed the proportion of T cell and B cell subsets in spleens and PB. CD90.2 + CD4 − CD8 − double-negative T cells (DNT cells) are expanded in SLE patients, as well as massive splenomegaly and lymphadenopathy caused by hyperproliferation of DNT cells in MRL/lpr mice [26]. T FH cells, defined as CD4 + CXCR5 + PD-1 + , play a crucial role in GC formation and the production of antigen-specific memory B cells and PCs, thereby contributing to SLE disease progression [4,27]. We analyzed the proportion of CD90.2 + T cells, DNT cells, T FH cells and PCs in the spleens and PB of MRL/lpr mice. Niclosamide treatment had a significantly lower proportion of spleen CD90.2 + T cells than the vehicle treatments. The proportion of PB CD90.2 + T cells was significantly decreased in the niclosamide treatment compared with the vehicle treatment (Fig. 2a, b). Likewise, the spleen DNT cells proportion was decreased by niclosamide treatment (Fig. 2c,  d). We found that the proportion of T FH cells in both samples from these mice were significantly decreased by niclosamide treatment (Fig. 2e, f ). In addition, The proportion of Th1, Th17, and regulatory T (Treg) cells were analyzed in spleen samples using flow cytometry. The alteration of the spleen Th1 and Th17 cells proportion was not significant (Additional file 1: Figure S1a-c). However, Treg cells, characterized by immunomodulatory capabilities, were significantly increased by niclosamide treatment (Additional file 1: Figure S1d and e). The Treg/ Th17 ratio was significantly increased in the niclosamide group compared with the vehicle group in spleen (Additional file 1: Figure S1f ). In addition to, the proportion of PCs in both samples from these mice were significantly reduced by niclosamide treatment (Fig. 2g, h). These data indicate that niclosamide could suppress the number of CD90.2 + cells, DNT cells, T FH cells and PCs in the spleens and PB of MRL/lpr mice.

Niclosamide regulates major transcription factors involved in T FH cell differentiation
We next analyzed the activity of the STAT3/Bcl-6/ TCF-1 pathways in the spleens of each animal group to elucidate the molecular mechanisms regulating the effects of niclosamide treatment. Western blot analysis revealed that expression of p-STAT3/Bcl-6/TCF-1 was significantly decreased in the splenocytes of niclosamide-treated mice as compared to vehicle-treated mice (Fig. 3a). Previous studies have shown that Bcl-6, CXCR5, and Blimp-1 are important regulators of T FH cell differentiation [9,28]. Niclosamide significantly reduced Bcl-6 and CXCR5 mRNA expression, while the mRNA levels of Blimp-1, which acts as a T FH cell differentiation suppressor, was not significantly increased in spleen CD4 + T cells (Fig. 3b). In addition, niclosamide treatment significantly decreased the spleen levels of IL-6 and IL-21 (Fig. 3c). Collectively, these results indicate that niclosamide is effective in regulating T FH cell-related factors.

STAT3 is important for T FH -like cell differentiation and B cell IgG production in vitro
We further investigated the influence of niclosamide on T FH -like cell differentiation in vitro, and found that niclosamide could inhibit T FH -like cell differentiation (Fig. 4a). Niclosamide treatment reduced the protein levels of p-STAT3 and TCF-1 in cultured T FH -like cells (Fig. 4b). In addition, niclosamide inhibited mRNA expression of Bcl-6 and CXCR5, and increased Blimp-1 during the differentiation of T FH -like cells (Fig. 4c). We next examined whether T FH -like cells might play a pathogenic role in SLE by supporting the production of autoantibodies. When B cells were cultured with T FH -like cells, IgG production was significantly increased compared to CD4 + T cells. However, when B cells were cultured with niclosamide-treated T FH -like cells, IgG production markedly decreased (Fig. 4d). By enhancing antibody production in vitro, T FH -like cells thus demonstrated their capacity to promote an antibody response. Overall, these results indicate that niclosamide not only inhibits T FH -like cell differentiation, but also B cell IgG production.

Effects of niclosamide on lupus nephritis in an R848-induced mouse model
To further verify our findings in a different murine model, wild-type C57BL/6 mice received topical treatment on their right ears with the TLR-7 agonist R848 3 times weekly for 5 weeks. We orally administered 8-week-old R848-induced mice with niclosamide or vehicle daily for 5 weeks. R848-induced mice exhibited splenomegaly, while niclosamide treatment significantly reduced the spleen enlargement in these animals, although the cervical lymph node (cLN) size was not significantly different ( Fig. 5a-c). The R848 group developed proteinuria compared with the control group, but the niclosamide-treated animals showed significantly diminished proteinuria compared with the R848 group (Fig. 5d). Serum levels of anti-dsDNA IgG, IgG, and IgG2a were significantly increased in the R848-induced mice as compared to the control mice. However, niclosamide treatment significantly decreased the levels of these factors (Fig. 5e, f ). In addition, serum levels of IL-6 and IL-21 were increased following R848 stimulation, while niclosamide treatment prevented these increases (Fig. 5g). R848-induced mice also developed enlarged hypercellular glomeruli, an increase in the mesangial matrix, moderate perivascular mononuclear cell infiltration, glomerular basement membrane thickening, and immune complex deposition. Niclosamide relieved these renal pathological features, decreased histological scores, and inhibited C3 immune complex deposition (Fig. 5h, i). These results suggest that mice topically treated with R848 developed systemic autoimmunity, but this effect was significantly alleviated by niclosamide treatment.

Niclosamide inhibits CD4 + T cell, T FH cell, GC B cell, and PC differentiation in R848-induced mice
We next sought to determine whether the proportion of T cell and B cell subsets expanded in response to R848 treatment, and whether this response would be inhibited by niclosamide. CD4 + T cells, T FH cells, GC B cells, and PCs were significantly increased in the spleens of R848-induced mice (Fig. 6). However, niclosamide significantly decreased the proportion of CD4 + T cells (Fig. 6a, b). The proportion of T FH cells was also significantly decreased by niclosamide treatment (Fig. 6c,  d). Additionally, when the T cell subsets analysis was performed, Th1 cells were significantly decreased by niclosamide, while Th17 cells were not affected (Additional file 2: Figure S2). GC B cell proportion in R848induced mice were inhibited by niclosamide (Fig. 6e, f ). The proportion of PCs in these mice was significantly reduced by niclosamide treatment (Fig. 6g, h). These data suggest that R848 enhanced CD4 + T cell, T FH cell, GC B cell, and PC differentiation, but the expansion of these cells was dramatically prevented by niclosamide.

Discussion
Autoreactive antibody production is one of the hallmarks of SLE and plays a critical pathogenic role in LN [29]. T FH cells promote autoantibody-producing B cell differentiation in autoimmune diseases. Abnormal expansion of T FH cells is a common feature of lupus patients with active disease and is also found in spontaneous and induced models of murine lupus, which suggests that T FH cells affect lupus pathogenesis [30,31]. We hypothesized that STAT3-mediated regulation of T FH cells could potentially limit the pathogenicity of the disease. To address this, we used MRL/lpr mice and R848-induced mice. MRL/lpr mice spontaneously develop a severe lupus-like disease that shares several of the immunologic and clinical manifestations of human lupus [32]. Several studies have shown that T FH cells are expanded in the spleens of MRL/lpr mice [33,34]. R848 is an immune-response modifier that activates immune cells through Toll-like receptors (TLRs) 7 and 8 [35]. Epicutaneous application of R848 to C57BL/6 mice induces the development of lupus-like phenotypes, including mild LN [36]. Activation of plasmacytoid dendritic cells (pDCs) by R848 play a critical role in antigen presentation and T cell responses [37]. pDCs induce T FH cell differentiation through cytokine secretion, such as type I IFN and IL-6 [27,38].
Although the T FH cell differentiation mechanisms differ between the two mouse models, the differentiation of these cells was significantly inhibited by niclosamide, thereby alleviating the symptoms of lupus. STAT3 serves multiple biological functions, including playing roles in proliferation, differentiation, and survival through cytokine-mediated signaling in various cells. STAT3 is especially critical for T FH and Th17 cell differentiation [39,40]. Th17 cells significantly contribute to SLE pathogenesis [41], while inhibition of STAT3 delays the development of lupus pathogenesis by suppressing Th17 cell differentiation [42]. However, we found that niclosamide did not reduce Th17 cell numbers. In addition, DNT cells, which play a crucial pathological role in the development of lupus disease, are expanded in MRL/lpr mice and patients with SLE. In MRL/lpr mice, DNT cells have been found to be the most expanded T cell subset, which lead to splenomegaly and lymphadenopathy [26]. Although we suggested that splenomegaly and lymphadenopathy were not significantly reduced by niclosamide treatment, DNT cell numbers were significantly inhibited. In further studies, we will explore the mechanisms of DNT cell regulation by niclosamide.
Blimp-1 is a well-known antagonist of Bcl-6 that can directly inhibit Bcl-6 expression in T and B cells [43]. Conversely, Bcl-6 can suppress the expression of Blimp-1 [44]. Blimp-1 is a key transcription factor for regulating PC differentiation in B cell lines [45]. Genome-wide association studies have identified multiple SLE-related genes and new susceptibility loci, including Blimp-1 [46]. As mentioned previously, Bcl-6 is strongly upregulated in T FH cells [7], while conversely Blimp-1 is the most downregulated transcription factor in T FH cells [47]. Bcl-6 and Blimp-1 are mutual master transcription factors regulating T FH cell differentiation, and the presence of Bcl-6 coupled with the absence of Blimp-1 is required for T FH cell differentiation [9]. In our study, we found that Bcl-6 was significantly decreased in CD4 + T cells by niclosamide administration in MRL/lpr mice, while Blimp-1 was not significantly increased. Further, during T FH -like cell differentiation in vitro, Bcl-6 was significantly decreased and Blimp-1 was significantly increased by niclosamide treatment.
TCF-1 is crucial for T cell development, as this transcription factor promotes the formation of memory CD8 + T cells, Th2 cells, and T FH cells. TCF-1 is highly expressed and regulates the early stages process of T FH cell differentiation [13]. Xu et al. reported that TCF-1 functions as an upstream regulator of the Bcl-6 / Blimp-1 axis, critical for T FH differentiation [48]. Certain cytokines secreted by immune cells in the disease milieu, such as IL-6 and IL-21 [49,50], might induce the immediate upregulation of TCF-1 expression through STAT3 signaling. We found that niclosamide decreased serum levels of IL-6 and IL-21. Niclosamide also inhibited TCF-1 expression in the spleens of MRL/lpr mice and in T FH -like cells.
Niclosamide induce cell cycle arrest, growth inhibition and apoptosis in cancer cells by targeting multiple signaling pathways such as, Wnt/β-catenin, STAT3, mTOR, Notch signaling pathways and mitochondria metabolic pathways [51]. Han et al. demonstrated that niclosamide ethanolamine alleviated lupus nephritis in MRL/lpr mice, and regulated mitochondrial biogenesis and energy metabolism in the kidney [52]. An important feature of mitochondria is it can regulate activation, differentiation, and survival of immune cells such as, T cell, B cell, macrophage, DC in autoimmune disease [53]. However, the authors work focused primarily on the kidney and did not extensively address potential immunological mechanisms. In our study, it is necessary to further study the mechanism by which niclosamide induced mitochondria function change affects T FH cells.

Conclusion
The present study showed that niclosamide significantly alleviated SLE-like characteristics in MRL/lpr and R848induced mice models. Our data also demonstrated the regulatory function of niclosamide on T FH cells in vivo and in vitro. Targeting STAT3 signaling using niclosamide could be an effective therapy for treating LN.