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Fludarabine Ameliorates Lupus Nephritis by Inhibiting T Cell Infiltration through STAT1 in R848-induced Mice Models
Korean J Clin Lab Sci 2024;56:207-216  
Published on September 30, 2024
Copyright © 2024 Korean Society for Clinical Laboratory Science.

Se Gwang JANG

The Rheumatism Research Center, The Catholic University, Seoul, Korea
Correspondence to: Se Gwang JANG
The Rheumatism Research Center, The Catholic University, Banpo-daero 222, Seocho-gu, Seoul 06591, Korea
E-mail: elite@catholic.ac.kr
ORCID: https://orcid.org/0000-0003-0518-5975
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease caused by both genetic and environmental factors. Fludarabine is a selective inhibitor of signal transducer and activator of transcription 1 (STAT1). Recently, STAT1 inhibitors have been considered potential treatments for SLE, due to the relationship between its pathogenesis and STAT1 pathway-mediated cytokines such as interferons. In the current study, we evaluated the therapeutic effects of fludarabine in an SLE animal model and explored its effects on T cell responses. 12-week-old C57BL/6 mice with topically administered R848 exhibited lupus-like phenotypes. Disease activity, such as proteinuria, autoantibody levels, immunoglobulin titers, the histological score, and C3 deposition, greatly improved with fludarabine treatment. In addition, fludarabine inhibited CD4+ T cells and T helper 1 (Th1) cells in the spleen and significantly decreased the differentiation of Th1 cells in vitro. These results indicate that Th1 cells play a critical role in the pathogenesis of lupus nephritis (LN). Thus, fludarabine exerted therapeutic effects on lupus animal models by suppressing Th1 cells via STAT1 inhibition. We propose that targeting STAT1 signaling using fludarabine could be an effective therapy for treating LN.
Keywords : Fludarabine, R848-induced model, STAT1, Th1 cells
INTRODUCTION

Systemic lupus erythematosus (SLE), chronic autoimmune disease, affects various organs due to autoantibodies and immune complexes deposition [1, 2], influenced by the interaction of adaptive and innate immune responses involving self-reactive T and B cells. While the precise mechanisms behind SLE remain unclear, multiple factors contribute to its development [3].

The complex mechanisms of T cell differentiation and subsets has revealed that T cells play a more critical and complex role in the pathology of autoimmune diseases [4]. The development of various types of T helper cells relies on the specific cytokine environment and additional signals from antigen-presenting cells. T helper 1 (Th1) cells play a significant role in the onset of SLE and have gained considerable attention as therapeutic targets [5]. Interferon gamma (IFNγ) is the only member of the type II IFN and has the ability to induce lymphocytes toward a proinflammatory Th1 cell phenotype [6]. Furthermore, IFNγ activates transcription factors that exacerbate lupus and is indispensable for the development of autoreactive B cells and systemic autoimmunity promoted by toll-like receptor 7 (TLR7) [7]. A better understanding of the role of Th1 cells and related cytokines may contribute to the treatment of SLE.

Signal transducer and activator of transcription (STAT) proteins are essential in facilitating various biological functions, including cell proliferation, survival, programmed cell death, and differentiation [8]. STAT1, the first STAT discovered, plays an important role in immune responses as a key mediator of cellular responses to IFN [9]. The expression of STAT1 was elevated in peripheral blood mononuclear cells (PBMCs) from SLE patients, and it correlated with the systemic lupus erythematosus disease activity index (SLEDAI) score [10]. In untreated SLE patients’ PBMCs, the expression of IFNγ response genes (STAT1, interferon-gamma receptor, integrin alpha M, S100A) was higher than that in controls [11].

Fludarabine is fluorinated purine analog, used in the treatment of a variety of hemato-oncological disorders. Fludarabine has immunosuppressive properties due to its cytotoxic potential against T lymphocytes, make it an attractive choice for both immunoregulation and chemotherapy in allogeneic transplantation for leukemia [12]. Fludarabine induces immunosuppression by inhibiting the activation of STAT1 induced by cytokines in lymphocytes, which may contribute to its immunosuppressive effect [13].

In this study, we determined whether Fludarabine had a therapeutic effect in R848-induced mice. We also examined the effect of Fludarabine on CD4+ T cells and Th1 cells in vitro.

MATERIALS AND METHODS

1. Animals and Treatment

C57BL/6 mice were purchased from OrientBio. Fludarabine (Sigma-Aldrich) was resuspended in phosphate-buffered saline for in vivo studies or in 5% dimethyl sulfoxide (DMSO) for in vitro use. 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 Fludarabine daily for 4 weeks by intraperitoneal (i.p.), or acetone alone as a control, to the right ear three times a week until euthanasia (approval numbers: CUMS-2024-0004-03).

2. Measurement of Urine Albumin to Creatinine Ratio

Urine albumin and creatinine concentrations were measured using a mouse albumin enzyme-linked immunosorbent assay (ELISA; 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).

3. Enzyme-linked Immunosorbent Assay

Serum obtained after euthanasia was separated by centrifugation at 3,000 rpm for 10 minutes at 4℃. The serum levels of anti-dsDNA immunoglobulin G (IgG) antibodies were measured by ELISA following the manufacturer’s instructions (Alpha Diagnostics). Total IgG levels in the sera of the mice were measured by ELISA following the manufacturer’s instructions (Bethyl Laboratories). IFNγ levels in spleen lysates were assayed using mouse IFNγ Duoset ELISA kits (R&D systems) according to the manufacturer’s instructions.

4. Flow Cytometry

Spleens were minced in RPMI 1640 medium and filtered through a 40-μm cell strainer to prepare single-cell suspensions. For intracellular staining, cells were stimulated with 25 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and 250 ng/mL ionomycin (Sigma-Aldrich) with monensin-containing GolgiStop (BD biosciences) for 5 hours. Cells were harvested and stained with surface eFluor780-fixable viability dye (eBioscience), Pacific Blue-anti-CD3 (Biolegend), PerCP-Cy5.5 anti-CD4 (Biolegend), PE anti-CD8a (Biolegend), PE-Cy7 IFNγ (Biolegend) antibodies. Flow cytometric analysis was performed on a LSRII Fortessa (BD biosciences), and the data were analyzed using FlowJo software (Tree Star).

5. 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. Histological analysis of pathological kidney features included assessments of inflammation, proliferation level, crescent formation, and necrosis. The glomeruli score was evaluated using scoring from 0 to 4: 0, normal; 1, mild, focal, or early proliferation; 2, moderate or definite proliferation and increased matrix; 3, diffuse, and focal or diffuse proliferation; and 4, severe diffuse proliferation with crescent/sclerosis. Perivascular infiltration was scored from 0 to 4: 0, normal; 1, occasional perivascular infiltration of mononuclear cells (MNCs); 2, several foci of MNC perivascular infiltration without necrosis; 3, multifocal MNC perivascular infiltration with/without necrosis; and 4, extensive multifocal or diffuse MNC perivascular infiltration with necrosis. The renal tubulointerstitial injury was scored from 0 to 4 for inflammation and necrosis: 0, normal; 1, occasional, focal, or small pockets of MNCs (10∼14 cells); 2, focal MNC infiltration (15∼30 cells); 3, multifocal and extensive MNC infiltration; and 4, severe MNC infiltration with extensive necrosis. Scores from 20 glomeruli were averaged to obtain a mean score for each kidney section.

6. Immunofluorescence

Kidney tissues were stained with anti-C3 (Abcam) at 4℃ overnight, followed by 2-hour incubation with secondary antibodies conjugated to Alexa488. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen). Isotype control staining was conducted via probing with rat/rabbit/mouse IgG, rather than primary antibodies. Confocal images were acquired using an LSM 800 confocal microscope (Zeiss).

7. CD4+ T Cell Proliferation

CD4+ T cells were purified from spleens of C57BL/6 mice using the CD4+ T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Purified CD4+ T cells were seeded at 1×106 cells/well and were activated with mouse T-activator CD3/CD28 DynabeadsTM (Invitrogen) and treated with or without 10 μM Fludarabine. Proliferation was assessed using the Cell Trace Violet Cell Proliferation Kit (Invitrogen) according to the manufacturer’s instructions. After 5 days, proliferation of CD4+ T cells was assessed by measuring Cell Trace Violet dye dilution on a LSRII Fortessa flow cytometer (BD biosciences), and the data were analyzed using FlowJo software (Tree Star).

8. T Helper 1 Cell Differentiation

CD4+ T cells were purified from spleens of C57BL/6 mice using the CD4+ T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. For Th1 cell differentiation, purified CD4+ T cells were seeded at 1×106 cells/well and were activated with mouse T-activator CD3/CD28 DynabeadsTM (Invitrogen) and treated with 10 ng/mL IL-2, 10 ng/mL IL-12, 5 ng/mL IFNγ, 10 μg/mL anti-IL-4 (R&D Systems) for 4 days with or without 10 μM Fludarabine.

9. Western Blot

Total protein was extracted using RIPA buffer containing Halt protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). For immunoblotting, 30 μg of protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride membrane (Bio-Rad), and probed with the following antibodies: anti-p-STAT1Y705, anti-STAT1 (Cell signaling technology), and anti-β-actin (Sigma-Aldrich). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Thermo Fisher Scientific). Reactive proteins on the membrane were visualized using SuperSignalTM West Pico Chemiluminescent substrate (Thermo Fisher Scientific), and the membrane was then exposed on an Amersham Imager 600 (GE healthcare).

10. Real-time PCR

Total RNA was collected using an RNA iso plus reagent (Takara). Up to 1∼2 µg of total RNA was converted to complementary DNA using a Transcriptor First-Strand cDNA Synthesis kit (Roche Diagnostics). A LightCycler 96 instrument (Roche) was used for PCR amplification and analysis. All reactions were performed with SYBR Green I Master Mix, according to the manufacturer’s instructions. Primers were designed using the web tool from GenScriptTM (http://www.genscript.com). Sequences are as follows (forward and reverse, respectively): beta actin, 5′- GGACTTCGAGCAAGAGATGG -3′ and 5′- TGTGTTGGGGTACAGGTCTTT -3′; T-bet, 5′- CAACAACCCCTTTGCCAAAG -3′ and 5′- TCCCCCAAGCAGTTGACAGT -3′. All mRNA expression levels were normalized to beta actin expression. Relative fold induction was calculated, following the equation 2‒(∆Cq) or 2‒(∆∆Cq), where ∆∆Cq is ∆Cq(target)‒∆Cq(beta actin), ∆Cq is Cq(stimulated)‒Cq(unstimulated), and Cq is the cycle at which the threshold is crossed. PCR product quality was monitored using post-PCR melting curve analysis.

11. Statistics

Statistical analyses were performed in GraphPad Prism version 7.0 software (GraphPad). 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.

RESULTS

1. Fludarabine Reduced the Severity of Lupus Nephritis in R848-induced Mice

To evaluate whether Fludarabine ameliorates clinical features of LN, wild-type C57BL/6 mice received topical treatment on their right ears with the TLR-7 agonist R848 3 times weekly for 4 weeks. We intraperitoneal injected 8-week-old R848-induced mice with Fludarabine daily for 4 weeks. R848-induced mice revealed splenomegaly, while Fludarabine treatment significantly decreased the spleen enlargement in these animals (Figure 1A). The R848 group developed proteinuria compared with the control group, but the Fludarabine group showed significantly diminished proteinuria compared with the R848 group (Figure 1B). Serum levels of IgG, anti-dsDNA was significantly increased in the R848 group as compared to the control group. However, Fludarabine group significantly decreased the levels of these factors (Figure 1C). In addition, serum levels of IFNγ were increased following R848 stimulation, while Fludarabine treatment prevented these increases (Figure 1D). These results indicate that mice topically treated with R848 developed systemic autoimmunity, but this effect was significantly alleviated by Fludarabine treatment.

Fig. 1. Fludarabine 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 i.p. injected 5% DMSO (control, n=4; R848, n=8), or 100 mg/kg Fludarabine (Fludarabine, n=8) daily until they were 12-week-old. (A) Left, representative photograph documenting the enlargement of spleens. Right, spleen weights in each group. (B) Urine albumin levels normalized to creatinine. (C) Serum levels of IgG, anti-dsDNA IgG antibody. (D) The levels of IFNγ in the spleen homogenates were detected by ELISA. (E) Left, p-STAT1 and STAT1 expression in the spleen were analyzed by western blot. Right, graph show the relative density of the target proteins. Data shown as mean±SD and represent two independent experiments. One-way ANOVA was performed. *P<0.05, **P<0.01.
Abbreviations: ACR, albumin creatinine ratio; Flu, fludarabine; IFNγ, interferon gamma; IgG, immunoglobulin G; i.p., intraperitoneal; STAT1, signal transducer and activator of transcription.

2. Fludarabine Reduced the Proportion of CD4+ T Cells and Th1 Cells in R848-induced Mice

To assess whether the proportions of T cell subsets expanded in response to R848 treatment, and whether this response would be inhibited by Fludarabine. CD4+ cells and Th1 cells were significantly increased in the spleens of R848-induced mice. However, Fludarabine significantly reduced the proportions of these cells (Figure 2A, 2B). These data suggest that R848 enhanced CD4+ T cells and Th1 cells expansion, but Fludarabine can modulate abnormal expansion of lupus-pathogenic T cell populations.

Fig. 2. Fludarabine reduces the populations of CD4+ T cells, Th1 cells in spleens of R848-induced mice. Spleen samples were collected from 12-week- old mice. (A) Left, data show populations of CD4+ T cells (CD4+ gated on CD3+). Right, bar graphs show mean± SD. (B) Left, data show populations of Th1 cells (IFNγ+ CD4+ gated on CD3+). Right, bar graphs show mean±SD and represent two independent experiments. One-way ANOVA was performed. *P<0.05, ***P<0.001.
Abbreviations: Flu, fludarabine; IFNγ, interferon gamma.

3. Fludarabine Prevented Renal Pathology and Immune Complex Deposition in R848-induced Mice

R848-induced mice exhibited alterations in renal tissue, including mesangial hypercellularity, signs of glomerular sclerosis, and tubular degeneration when compared to control mice. Fludarabine relieved these renal pathological features, decreased histological scores (Figure 3A). Immune complexes such as C3 are deposited in the kidneys, which causes lupus nephritis [1]. Confocal microscopic images demonstrated decreases of C3 deposition in renal tissues from Fludarabine-treated mice compared to R848-induced mice (Figure 3B).

Fig. 3. Fludarabine treatment reduces cell infiltration and C3 deposition in the kidney of R848-induced mice. (A) Left, representative photomicrographs of PAS-stained sections of kidney. Original magnification ×200. Right, histological scores. (B) Left, representative immunofluorescent images of kidney C3 staining. Original magnification ×200, ×400. Right, MFI of C3 deposition. Data shown as mean±SD and represent two independent experiments. One-way ANOVA was performed. **P< 0.01.
Abbreviation: Flu, fludarabine.

4. STAT1 Is Important for CD4+ T Cell Proliferation and Th1 Cell Differentiation in Vitro

We further investigated the influence of Fludarabine on CD4+ T cell proliferation and Th1 cell differentiation. Fludarabine inhibited the proliferation of CD4+ T cells (Figure 4A). We next examined whether Th1 cells would be inhibited by Fludarabine. Th1 cells were significantly increased in the polarizing conditions. However, Fludarabine significantly decreased the differentiation of Th1 cells (Figure 4B). Fludarabine treatment reduced the protein levels of p-STAT1in cultured Th1 cells (Figure 4C). In addition, Fludarabine inhibited the mRNA expression of T-bet, a Th1 transcription factor, during differentiation of Th1 cells (Figure 4D). Overall, these results demonstrate that Fludarabine not only inhibits CD4+ T cell proliferation, but also Th1 cell differentiation.

Fig. 4. Fludarabine inhibits CD4+ T cells proliferation and Th1 cells differentiation in vitro. Naive CD4+ T cells were purified from the spleens of C57BL/6 mice. Proliferation of Cell Trace Violet-labelled CD4+ T cells analyzed by flow cytometry 5 days after culture with or without 10 μM Fludarabine. For Th1 cells differentiation, purified CD4+ T cells were activated with mouse T-activator CD3/CD28 Dynabeads and treated with 10 ng/mL IL-2, 10 ng/mL IL-12, 5 ng/mL IFNγ, and 10 μg/mL anti-IL-4 for 4 days with or without 10 μM Fludarabine. (A) Left, representative flow cytometry histograms of Cell Trace Violet-labelled CD4+ T cells. Right, bar graphs show mean±SD. (B) Left, data show populations of Th1 cells (IFNγ+ CD4+ gated on FVD CD4+). Right, bar graphs show mean±SD. One-way ANOVA was performed. *P<0.05, **P<0.01. (C) Left, p-STAT1 and STAT1 expressions in Th1 cells isolated from C57BL/6 mice were analyzed by western blot. Right, graph show the relative density of the target proteins. (D) mRNA expression levels of T-bet in Th1 cells isolated from C57BL/6 mice were measured by real-time PCR. Represent three independent experiments.
Abbreviations: Flu, fludarabine; IFNγ, interferon gamma; STAT1, Signal transducer and activator of transcription 1; Th1, T helper 1.
DISCUSSION

In this study, Fludarabine showed effectiveness in alleviating SLE-like phenotypes, such as splenomegaly, proteinuria, infiltration of autoantibodies, kidney inflammation, and reduced proportion of Th1 cells in R848-induced mice. Furthermore, we investigated the effect of Fludarabine on Th1 cells in vitro. These cells were responsive to suppression of STAT1 signaling both in vitro and in vivo, correlating with an improvement in SLE-like phenotypes.

Lupus animal models include not only induced models but also spontaneous models such as MRL/lpr, NZB/W F1, and BXSB/Yaa. MRL/lpr mice exhibit massive lymphadenopathy, a feature not found in SLE patients. Furthermore, this model, which features expansion of double-negative T cells, differs from other spontaneous models in that the disease progresses rapidly and severely in just 2 months [14]. NZB/W F1 is a classic model used in a variety of studies, including BAFF, type 1 IFN, and lupus nephritis. NZB/W F1 mice show a phenotype most similar to lupus patients. These include lymphadenopathy, splenomegaly, and autoantibodies. Symptoms begin to appear from 5 to 6 months of age, and immune complex- mediated glomerulonephritis, which leads to renal failure, occurs at 10 to 12 months of age [15]. BXSB/Yaa mice are characterized by a severe phenotype of the disease in males, with nephritis leading to the death of BXSB/Yaa males in about 5 months and BXSB females in 14 months. Male mice develop a severe phenotype of lupus due to the Y-autoimmune accelerator (Yaa) locus, which results from a translocation from the X to the Y chromosome, leading to increased expression of at least 16 genes [16]. R848-induced mice can easily induce lupus-like phenotypes including mild LN by epicutaneous administration of R848 to C57BL/6 mice. It also has the advantage of allowing the therapeutic effect of the drug to be confirmed within a short period of time [17]. Although a variety of animal models of SLE exist, none fully represents the full clinical spectrum found in SLE patients. Therefore, it may be necessary to conduct research by selecting an animal model that suits the target mechanism or experimental conditions to be investigated.

SLE is an autoimmune disease characterized by T cell dysfunction, autoantibody production, deposition of immune complexes in tissues, and inflammatory response, resulting in damage to various organs [18]. SLE is also characterized by dysfunction of various T cell subset.

Dolff et al [19] reported that a subset of effector T cells, such as Th1, Th2 and Th17 cells, plays a pathogenic role in SLE. We showed that the number of IFNγ-expressing CD4+ T cells, which are considered Th1 cells, was significantly reduced by Fludarabine treatment. However, Th2, Th17 cells was not significantly different between R848 group (data not shown). STAT1 suppresses the activity of Th17 cells through regulation of T-bet, an inhibitory transcription factor for Th17 cells [20]. Recent studies have uncovered Th17 cell differentiation and IL-17 production played an essential role in the development of SLE. The key cytokines for Th17 cell differentiation are IL-1β, IL-6, IL-23, and TGF-β, and the specific transcription factor requires retinoid-related orphan receptor gamma (RORγt) [21]. These cytokines and transcription factors bind to naïve CD4 T cells and trigger downstream signaling, including STAT3 [22, 23]. It is well known that STAT1 and STAT3 play essentially antagonistic roles and that, when their balance is disrupted, they can switch from promoting cell survival to apoptosis and from driving inflammation to anti-inflammatory responses [24]. Further studies are required on the interaction mechanism between STAT1 and STAT3 in lupus nephritis.

Our results suggest that Fludarabine were sensitive to inhibition of CD4+ T cells and Th1 cells in the spleen, which was correlated with an aggravation in the SLE manifestation. Fludarabine could be considered as a therapeutic agent for the treatment of lupus nephritis. Fludarabine dramatically alleviated SLE-like phenotypes in R848-induced mice models. We additionally demonstrated the modulatory function of Fludarabine on Th1 cells in vivo and in vitro. Blocking STAT1 signaling using Fludarabine could be an effective therapy for LN treatment.

요 약

Fludarabine은 STAT1의 선택적 억제제이다. 다양한 연구에서 SLE의 발병기전과 인터페론(IFN)과 같은 STAT1을 매개한 사이토카인 사이의 관계로 인해 STAT1 억제제는 SLE의 잠재적 치료법으로 여겨지고 있다. 본 연구에서 우리는 R848로 유도된 동물 모델에 대한 Fludarabine의 치료 효과를 확인하고 T 세포 반응에 미치는 영향을 조사했다. 4주 동안 R848로 자극한 C57BL/6 마우스는 단백뇨, 자가항체, 신장 사구체 염증 세포 침윤, C3 침착과 같은 루푸스 질병 표현형을 나타냈지만, 약물 투여에 의해 질병 활성은 크게 개선되었다. 또한, Fludarabine은 동물 모델의 비장에서 CD4+ T 세포와 T helper 1 (Th1) 세포의 증가를 억제하고 in vitro 실험에서 Th1 세포의 분화와 관련 유전자의 발현을 효과적으로 억제했다. 이러한 결과는 Th1 세포가 루푸스 신염 발병에 중요한 역할을 한다는 것을 보여주는 결과이다. Fludarabine은 STAT1 억제를 통해 T 세포를 억제함으로써 루푸스 모델에 치료 효과를 나타냈다. 결론적으로 Fludarabine을 사용하여 STAT1 신호 전달을 억제하는 것이 루푸스에 대한 효과적인 치료법이 될 수 있다고 제안한다.

Fundings: None

Acknowledgements

None

Fundings

None

Conflict of interest

None

Author’s information (Position)

Jang SG, Research assistant professor.

Author Contributions

The article is prepared by a single author.

Ethics approval

All procedures were performed in accordance with protocols approved by the The Catholic University Animal Care and Use committee (Approval numbers: CUMS-2024-0004-03).

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