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

Abstract

Background: Autoantibody production against endogenous cellular components is pathogenic feature of systemic lupus erythematosus (SLE). Follicular helper T (T_FH) 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 T_FH 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 T_FH cells in two murine models of lupus.

Methods: 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.

Results: 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 T_FH cells and PCs in the spleens of these animals, and effectively suppressed differentiation of T_FH-like cells and expression of associated genes in vitro.

Conclusions: Niclosamide exerted therapeutic effects on murine lupus models by suppressing T_FH cells and plasma cells through STAT3 inhibition.

Keywords: Follicular helper T cells; MRL/lpr; Niclosamide; R848-induced model; STAT3; Systemic lupus erythematosus.

Fig. 1

Niclosamide ameliorates disease aggravation in MRL/lpr mice. Female 10-week-old MRL/lpr mice were orally administered 0.5% methyl cellulose (vehicle, n = 7), or 100 mg/kg niclosamide (Niclosamide, n = 7) daily until they were 16-week-old. a Left, Representative photographs documenting the enlargement of kidneys. Right, kidney weights in vehicle, niclosamide groups. b Urine albumin levels normalized to creatinine. c Serum levels of anti-dsDNA IgG antibody. d Serum levels of antibody subclasses (IgG, IgG1, and IgM). e Serum levels of IL-6 and IL-21. f Left, representative photomicrographs of PAS-stained sections of kidney. Original magnification ×200 (upper), ×100 (middle, bottom). Right, histological score. g Left, representative immunofluorescent images of kidney C3 staining. Original magnification ×100 (upper), ×400 (bottom). Right, MFI of C3 deposition. Data shown as mean ± SD. t-test was performed. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Niclosamide modulates the proportion of CD90.2⁺ T cells, DNT cells, T_FH cells and PCs in the spleen and PB of MRL/lpr mice. Spleen and PB samples were collected from 16-week-old mice. a comparison of the FVD⁻ and CD90.2⁺ T-cell proportion. The gating strategies were: lymphocytes as FSC-A vs SSC-A, live cells as FVD-A vs CD90.2-A b Bar graphs show mean ± SD. c Altered proportion of DNT cells (CD4⁻ CD8⁻ gated on FVD⁻ CD90.2⁺) by niclosamide. d Bar graphs show mean ± SD. e Data show proportion of T_FH cells (CXCR5⁺ PD-1⁺ gated on CD4 +). f Bar graphs show mean ± SD. g Data show proportion of PCs (CD19⁻ CD138⁺ gated on CD19⁻). h Bar graphs show mean ± SD. t-test was performed. *P < 0.05, **P < 0.01

Fig. 3

Niclosamide treatment reduces T_FH-associated gene and cytokine levels in the spleens of MRL/lpr mice. a p-STAT3, Bcl-6, and TCF-1 expression in the spleen were analyzed by western blot. b mRNA expression levels of Bcl-6, CXCR5, and Blimp-1 in CD4⁺ T cells purified from spleens for each group of mice were measured by real-time PCR. c The levels of IL-6 and IL-21 in the spleen homogenates were detected by ELISA. Data shown as mean ± SD. t-test was performed. **P < 0.01, ***P < 0.001, ns: not significant

Fig. 4

Niclosamide inhibits T_FH-like cell differentiation and B cell IgG production in vitro. Naive CD4⁺ T cells were purified from the spleens of MRL/lpr and C57BL/6 mice. For T_FH-like cell differentiation, purified CD4⁺ T cells were activated with mouse T-activator CD3/CD28 Dynabeads, and treated with 20 ng/ml IL-6, 20 ng/ml IL-21, 10 μg/ml anti-IL-4, 10 μg/ml anti-IFN-γ, and 20 μg/ml anti-TGF-β for 4 days with or without niclosamide. a Left, T_FH-like cells (CXCR5⁺PD-1⁺, gated on CD4⁺) isolated from MRL/lpr mice were analyzed by flow cytometry. Right, the percentage of T_FH-like cells is shown. b p-STAT3 and TCF-1 expressions in T_FH-like cells isolated from MRL/lpr mice were analyzed by western blot. c mRNA expression levels of Bcl-6, CXCR5, and Blimp-1 in T_FH-like cells isolated from MRL/lpr mice were measured by real-time PCR. d T_FH-like cells and B cells isolated from C57BL/6 mice were co-cultured with or without T_FH-like cells for 3 days, and then the concentrations of IgG in the supernatants were detected by ELISA. Data shown as mean ± SD. One-way ANOVA was performed. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 5

Niclosamide ameliorates disease aggravation in R848-induced mice. Female 8-week-old C57BL/6 mice were treated with 50 μg of the TLR7 agonist R848 or control (acetone) three times weekly and were orally administered 0.5% methyl cellulose (control, R848, n = 4–6), or 100 mg/kg niclosamide (niclosamide, n = 7) daily until they were 12-week-old. a Representative photographs documenting the enlargement of spleens and cLNs. b Spleen lengths and weights in each group. c cLN lengths and weights in each group. d Urine albumin levels normalized to creatinine. e Serum levels of anti-dsDNA IgG antibody. f Serum levels of antibody subclasses (IgG, IgG2a). g Serum levels of IL-6, IL-21. h Left, representative photomicrographs of PAS-stained sections of kidney. Original magnification ×200 (upper), ×100 (bottom). Right, histological scores. i Left, representative immunofluorescent images of kidney C3 staining. Original magnification ×100 (upper), ×400 (bottom). Right, MFI of C3 deposition. Data shown as mean ± SD. One-way ANOVA was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 6

Niclosamide reduces the proportion of CD4⁺ T cells, T_FH cells, GCs, and PCs in spleens of R848-induced mice. Spleen samples were collected from 12-week-old mice. a Data show proportion of CD4⁺ T cells (CD4⁺ CD8⁻ gated on FVD⁻ CD90.2⁺). b Bar graphs show mean ± SD. c Data show proportion of T_FH cells (CXCR5⁺ PD-1⁺ gated on CD4⁺). d Bar graphs show mean ± SD. e Data show proportion of GC B cells (CD138⁻ GL7⁺ gated on CD19⁺). f Bar graphs show mean ± SD. g Data show proportion of PCs (CD19⁻ CD138⁺ gated on CD19⁻). h Bar graphs show mean ± SD. One-way ANOVA was performed. **P < 0.01