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Original research
Inhibition of receptor interacting protein kinase-1 (RIPK1) in the treatment of murine lupus
  1. Lin Peng1,
  2. Pengcheng Wang1,
  3. Xiaodong Xu1,
  4. Dacheng Chen1,
  5. Feng Xu1,
  6. Fan Yang1,
  7. Shuying Yang2,
  8. Hongguang Xia2,
  9. Zhi-Hong Liu1 and
  10. Weisong Qin1
  1. 1National Clinical Research Center for Kidney Disease, Affiliated Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, China
  2. 2Department of Biochemistry and Molecular Medical Center, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
  1. Correspondence to Dr Zhi-Hong Liu; liuzhihong{at}nju.edu.cn; Professor Weisong Qin; qinweisong{at}163.com

Abstract

Objective Systemic lupus erythematosus (SLE) is a type of autoimmune disease that involves multiple organs involved as well as cytokine dysregulation. The treatment of SLE is still challenging due to the side effects of the different drugs used. Receptor-interacting protein kinase 1 (RIPK1) is a kinase involved in T cell homeostasis and autoinflammation. Although clinical trials have shown that RIPK1 inhibition exhibits significant efficacy in different autoimmune diseases, its role in SLE remains unclear.

Methods MRL/lpr lupus-prone mice received RIPK1 inhibitor ZJU37 or vehicle intraperitoneally for 10 weeks. A BM12-induced chronic graft-versus-host-disease (cGVHD) lupus-like model was introduced in RIPK1 D138N mice or C57BL/6 mice. Nephritis, serum autoantibody levels, dysregulation of adaptive immune response and cytokines were compared in treated and untreated mice.

Results ZJU37 alleviated the clinical features of the MRL/lpr mice including nephritis and anti-dsDNA antibody production. In addition, ZJU37 treatment reduced the proportion of double-negative T cells in the spleen and the cytokines of TNFα, IFN-γ, IL-6, IL-17 and IL-1β in the serum. Moreover, RIPK1 D138N mice were able to prevent the cGVHD lupus-like model from SLE attack, manifesting as anti-dsDNA antibody production, the proliferation of germinal centre B cells, plasma cells, and T follicular helper cells as well as IgG and C3 deposits in kidneys.

Conclusion RIPK1 inhibition has a protective effect in the mouse model of SLE and can potentially become a new therapeutic target for SLE in humans.

  • Lupus Erythematosus, Systemic
  • Cytokines
  • Glucocorticoids

Data availability statement

Data are available upon reasonable request.

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WHAT IS ALREADY KNOWN ON THIS TOPIC

  • Systemic lupus erythematosus (SLE) is characterised by multiple organ involvement and cytokines dysregulation. The treatment of SLE is challenging due to the side effects and drug resistance issues with the corticosteroids and immunosuppressants used. Receptor-interacting protein kinase 1 (RIPK1 activation in patients and mice causes a kind of autoinflammatory disease. Additionally, an increasing number of studies have demonstrated the significant efficacy of RIPK1 inhibition in different autoimmune diseases, such as multiple sclerosis and ulcerative colitis.

WHAT THIS STUDY ADDS

  • This study shows that RIPK1 inhibition by its inhibitor ZJU37 or inactive mutation RIPK1 D138N has a protective effect in a mouse model of SLE. Specifically, ZJU37 treatment reduced the proliferation of double negative T cells in the spleen and the level of TNFα, IFN-γ, IL-6, IL-17, and IL-1β in the serum in MRL/lpr mice.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • This study offers RIPK1 as a new promising target for lupus management. Moreover, though further investigation is needed, a combination of ZJU37 with prednisone shows potential in mitigating SLE.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by multiple autoantibody generation and multiple systems involvement.1 2 Its treatment is still confined to immunosuppressors and sometimes monoclonal antibodies in clinics, both of which bring problems such as drug resistance, severe side effects, and high cost.3 Recently, researchers have begun testing small molecule inhibitors such as JAKs and IRAKs to treat SLE.4 5 MRL/lpr mice are the most commonly used lupus mice to research the treatment of SLE.6 Due to the spontaneous leaky mutation of the Fas gene to LPR, lymphocytes cannot normally execute programmed cell death, and autoreactive T and B cells are overactivated, which can cause splenomegaly, lymphadenopathy, increased proportion of double negative T cells (CD3+B220+CD4-CD8-, DN T cells), and secretion of a variety of inflammatory factors.6–8 Double negative T cells are in a steady state in healthy animals but expand in MRL/lpr mice due to peripheral intolerance.7 9 It was found to be a potential biomarker to predict disease activity and lupus nephritis progression in patients with SLE and MRL/lpr mice.10

Receptor-interacting protein kinase 1 (RIPK1) belongs to the family serine/threonine protein kinase, which is involved in apoptosis and necroptosis as well as inflammatory pathways.11–13 Patients with RIPK1 deficiency develop immunodeficiency, lymphopenia, and altered cytokine production.14 In addition, dominant autoinflammatory diseases caused by RIPK1 kinase activation have been found in both humans and mice.15 16 Furthermore, RIPK1 has emerged as a promising therapeutic target for the treatment of a wide range of human autoimmune and inflammatory diseases,17 such as multiple sclerosis,18–21 inflammatory bowel disease22 23 and rheumatoid arthritis.24 Although decreased expression of RIPK1 in neutrophils from patients with SLE, increasing evidence suggests RIPK1’s relationship with autoimmunity, for example, mice with RIPK1 deficiency in dendritic cells develop systemic inflammation and autoimmunity.25 26 Activation of necroptosis in the keratinocytes of patients with SLE and the podocytes of NZM2328 or MRL/lpr mice also occurs.27–29 Therefore, a better understanding of whether RIPK1 kinase inhibition can ease SLE is essential in order to illuminate the best therapeutic modalities of SLE.

Several studies have reported that RIPK1 is associated with SLE pathogenesis. For example, the mRNA of RIPK1 was significantly upregulated in the spleen and kidney of MRL/lpr mice at week 6.30 In addition, whole-genome mRNA sequencing shows a significant reduction in RIPK1 expression after prednisolone treatment with MRL/lpr mice.31 More interestingly, RIPK1 inhibitor Necrostatin-1 can alleviate lupus nephritis in MRL/lpr mice.32 In this work, we use RIPK1 kinase inhibitor and RIPK1 kinase-dead mutation mice to investigate the role of RIPK1 inhibition in SLE treatment.

In this study, the use of the RIPK1 kinase inhibitor ZJU37 from the eighth week relieved the lupus symptoms and raised the survival rate of the MRL/lpr mice. Furthermore, ZJU37 reduced the DN T cell proportion in both the peripheral blood and spleen and caused a significantly lower level of cytokines secretion in the serum. Since there are genetic differences between MRL/lpr and C57BL/6 mice, a bm12-induced chronic graft-versus-host-disease (cGVHD) lupus-like model was introduced into the study, and our results show that bm12-induced RIPK1 D138N mice had a lower autoimmune response, preventing them from SLE attacks.

Methods

Mice

Female MRL/lpr mice and C57BL/6 mice were purchased from SLAC Laboratory Animal Company (Shanghai, China). ZJU37 (patent No.: PCT/CN2019/097202) and RIPK1 D138N mice were kindly gifted from Professor Hongguang Xia from Zhejiang University. The MRL/lpr mice were divided into four groups: Vehicle, Prednisone (PNS, 2 mg/kg), ZJU37 (5 mg/kg) and combination therapy (2 mg/kg PNS+5 mg/kg ZJU37). The vehicle consisted of 2% dimethyl sulfoxide (DMSO), 30% polyethylene glycol (PEG) and 68% ddH2O. Drug administration began in the 8th week and ended in the 18th week, during which time the drugs were injected intraperitoneally once a day. Skin lesions and spleen enlargement were photographed, and body weight and spleen weight were recorded in order to calculate their ratio. The mortality every week was recorded from study start in order to plot a survival curve. Peripheral blood cell classification was carried out by an automatic peripheral blood cell analyzer (BC-2800vet, Mindray). The bm12 mice inducible model of SLE in C57BL/6 mice has been described in detail previously and herein was introduced into RIPK1 D138N and WT C57BL/6 mice. All mice were kept in a specific pathogen-free condition at Jinling Hospital Affiliated to Nanjing University School of Medicine and all experimental procedures were approved by the Ethics Committee of Jinling Hospital.

Flow cytometry

After euthanasia, the spleen of each mouse was removed and ground by the end of a syringe, and the filtrate was collected through a 70 µm sieve. After adding red blood cell lysate for 15 min and anti-Fc receptor antibody (cat#: 553141, BD Biosciences) for 30 min, the single-cell suspension was used for flow cytometric analysis in BD FACSCanto II after incubation with fluorochrome-conjugated antibodies to mouse CD3, CD4, CD8, CD19, CD21, CD23, B220, CD138, IgD, IgM, PD-1, CXCR5, GL-7, CD11b and Ly6G (all purchased from BD Biosciences). The data were analysed by Flowjo V10 software. To detect the cell number for T follicular helper cells (Tfh), plasma and germinal centre B cells (GC B cells), flow-count fluorospheres (cat#: 7547053, Beckman Coulter) were used according to the manufacturer’s instructions.

Immunofluorescence and H&E staining

The kidney tissue was embedded in an OCT agent (cat#: BL557A, Biosharp) and stored at –80℃, or fixed in a 4% formaldehyde solution for 48 hours. Frozen sections were prepared, fixed with acetone, and sealed with a blocking solution for 10 min. After washing, FITC-IgG antibody and FITC-C3 antibody were added, respectively. Next, after dark staining for 1 hour, the slides were fully washed, sealed and photographed with a fluorescence microscope (DM5000B, Leica). The scoring of IgG or C3 deposits by a pathologist was performed in a blinded manner according to the following criteria: 0=negative, 0.5=trace, 1=barely visible at high magnification, 2=moderately visible and 3=clearly visible.33 Paraffin-embedded sections were stained by H&E in an automated stainer (Autostainer XL, Leica) and each slide was scanned digitally by ScanScope CS2 at 40× magnification for evaluation. Here, the scoring of glomerular injuries by a pathologist was performed in a blinded manner according to the following criteria: 0, normal; 1, mild mesangial expansion and slight glomerular damage; 2, moderate sclerosis <50% of the glomerulus and 3, severe sclerosis >50% of the glomerulus.34

Proteinuria, serum autoantibodies and cytokine measurement

After the start of treatment, random urinary proteins were monitored by Bradford assay (Thermo Fisher Scientific) for the first 3 days of each week. The mean values were then calculated and used to plot the proteinuria curve. Mouse anti-dsDNA antibody ELISA kit (cat#: CSB-E11194e, Cusabio) and anti-nuclear antibody ELISA kit (cat#: CSB-E12912m, Cusabio) were used as per manufacturer’s protocol. Finally, serum cytokine levels were determined by anti-mIFNγ ELISA kit (cat#: MIF00-1, R&D systems) and anti-mTNFa ELISA kit (cat#: MTA00B-1, R&D systems) as manufacturer’s instructions.

Statistical analysis

All data were expressed as mean±SD, analysed by analysis of variance (ANOVA) with multiple comparison or Student’s t-test, and plotted with GraphPad Prism Software (San Diego, California). In particular, the survival curves were compared using Kaplan-Meier analysis. Statistical differences are presented with *, p<0.05; **, p<0.01 or ***, p<0.001.

Results

ZJU37 alleviated the lupus symptoms of MRL/lpr mice

MRL/lpr mice are the most commonly use animal for lupus experiments because they readily show skin lesions, splenomegaly, lymphoproliferation and organ damage.35 In our study, we tested a novel RIPK1 kinase—ZJU37 for the treatment of MRL/lpr starting from the eighth week and continuing until to the 18th week. Survival rates were evaluated and found to be statistically significantly different between groups (figure 1A). The control group demonstrated a survival rate of 52%; the PNS group exhibited a slightly better rate of 76%; the ZJU37 group had a significantly higher rate of 96.1%; and the survival rate was 100% in the PNS combined with ZJU37 treatment group. Additionally, the number of deaths was significantly lower in all treatment groups compared with the control group. These results indicate that the interventions used in this study may be effective in reducing SLE mortality rates in MRL/lpr mice.

Figure 1

ZJU37 alleviated the lupus symptoms of MRL/lpr mice. MRL/lpr mice were treated from week 8 to week 18 and the following indicators were detected in the vehicle, PNS, ZJU37 and combination groups: (A) The survival rate of the MRL/lpr mice during the 10 week treatment period (N=25 or 26). (B) Significant changes in skin lesions (black arrow). (C) Spleen weight to body weight ratio, which is a reliable indicator of splenomegaly (N=4–10). (D) Serum anti-dsDNA antibody levels (N=3–6), the experiment was repeated twice. (E) C3 and IgG deposits and scoring in the glomerulus by immunofluorescence (N=4–6). Renal tissue sections were stained with H&E (F) to assess glomerular pathological scoring (G) (N=10) (scale bar 50 μm) (original magnification: ×400). (H) Urine protein levels during the treatment by Bradford assay (N=6), the experiment was repeated twice. The data are expressed as mean±SD and the dots on the graph indicate individual mice. (A) is analysed by Kaplan-Meier method, (C–E) are analysed by one-way ANOVA with multiple comparisons, and H) is analysed by unpaired two-sided Student’s t-test. *p<0.05; **p<0.01; and ***p<0.001 indicate statistical significance compared with the vehicle group. No significance is denoted by ns. ANOVA, analysis of variance; PNS, prednisone.

Compared with the vehicle group, the ZJU37 and the combination treatment (PNS+ZJU37) also alleviated the skin lesions of the face and neck as well as spleen enlargement (figure 1B,C). Moreover, the serum level of the anti-dsDNA antibody was found to increase as weeks went by, but it was inhibited after ZJU37 usage and combination treatment (figure 1D). The combination group, PNS group and ZJU37 group had significantly less immune deposit and glomerular injury compared with the control group (figure 1E–G). The proteinuria curve was the lowest in the ZJU37 group, which was statistically different from that in the control group. Here, the combined group was the same or similar to the ZJU37 group in the sixth and eighth weeks of treatment (figure 1H). On the whole, ZJU37 relieved the general symptoms of SLE in the MRL/lpr mice.

ZJU37 reduced the proportion of DN T cells in MRL/lpr mice

T cell autoactivation and proliferation play an important role during the occurrence and progression of SLE.36 As shown in figure 2, ZJU37 reduced the number and proportion of double-negative T cells in both peripheral blood and the spleen (figure 2A, C and E) and increased the number and proportion of CD3+CD8+ T cells in the spleen (figure 2C, E). Furthermore, PNS inhibited the abnormal production of CD3+CD4-CD8-B220+double-negative T cells and reduced the consumption of functional CD3+CD8+ and CD3+CD4+ T cells in the spleen (figure 2C, E). Double-negative T cells mainly derive from CD8+T cells due to T cell receptor (TCR) complex stimulation in vivo in MRL/lpr mice and patients with SLE.37 What is more, another RIPK1 inhibitor—Nec-1 impaired T cell activation and proliferation in response to TCR stimulation in vitro,38 both of which could explain the phenomenon we observed in ZJU37 group. Besides this, the combination therapy also curbed the abnormal expansion of double-negative T cells to a new low point and reduced the proportion of CD3+CD4+T cells (figure 2C, E).

Figure 2

ZJU37 reduced the proportion of DN T cells in the peripheral blood and spleen of MRL/lpr mice. With a 10 week treatment, the peripheral blood and spleen were collected from MRL/lpr mice in the vehicle, PNS, ZJU37 and combination groups for flow cytometry analysis. A representative flow-cytometry image (A) depicting the proportion of DN T cells (CD3+CD4-CD8-B220+) in the spleen was obtained from one typical sample of each group. In the peripheral blood, the proportion (B) of B cells (CD19+), plasma cells (CD19+CD138+), CD4+T cells (CD3+CD4+CD8-) CD8+T cells (CD3+CD4 CD8+) and DN T cells (CD3+CD4-CD8-B220+) were analysed in the indicated groups (N=4–6). In the spleen, the proportion (C) and cell number (E) of the DN T cells (CD3+CD4-CD8-B220+), CD8+T cells (CD3+CD4 CD8+) and CD4+T cells (CD3+CD4+CD8-) were analysed in the indicated groups (N=4–6). Additionally, the proportion (D) and cell number (F) of the B cells (CD19+), mature B cells (IgD+IgM+), germinal centre B cells (GC B cell, CD19+GL-7+), follicular B cells (FoB cell, CD21 low CD23 high) and marginal zone B cells (MzB cell, CD21 high CD23 low) in the spleen were analysed in the indicated groups (N=4–6). The data are expressed as mean±SD. Individual mice are denoted by dots. (B–F) are analysed by two-way ANOVA with multiple comparisons, and statistical significance was defined as *p<0.05; **p<0.01; and ***p<0.001 compared with the vehicle group. No significance is denoted by ns. ANOVA, analysis of variance; PNS, prednisone.

B cell accumulation and autoantibody secretion were in the central position in the pathogenesis of SLE. MRL/lpr mice are known to have amounts of activated B lymphocytes accumulating in the peripheral immune organs.8 We investigated the efficacy of ZJU37 and combination therapy in the peripheral immune system and found that PNS normalised the changes in the B cell compartment in the spleen,39 which was indicated by an effective increase in B220+IgD+IgM+mature B cell and CD19+B220+GL-7+GC B cells (figure 2D, F). However, ZJU37 and combination treatment seemed not to affect B lymphocyte subsets (figure 2D, F). The B220+CD21 low CD23 high follicular B cells and B220+CD21 high CD23 low marginal zone B cells showed no statistical differences between all groups (figure 2D).

Collectively, these flow cytometric studies revealed that ZJU37 mainly functioned as an immune inhibitor to T lymphocytes and reversed the abnormal proportion of double-negative T cells in MRL/lpr mice.

ZJU37-reduced inflammatory cytokine secretion in the MRL/lpr mice

To explore the immune response after drug usage, peripheral blood cell classification was determined. The results showed that lymphocytes and granulocytes in the ZJU37 and PNS groups were lower in circulation and reduced to a remarkable extent in the combination group (table 1). The platelets and red blood cells showed no significant differences.

Table 1

Peripheral blood cell classification in the indicated groups

TNFα and IFN-γ are extensively secreted cell death inducers, thus making them the most important cytokines to cause fever in patients with SLE. The secretion of TNFα, IFN-γ, IL-6, IL-1β and IL-17 in the serum of the MRL/lpr mice was significantly reduced after ZJU37 treatment (figure 3A–E). Similarly, TNFα, IFN-γ, IL-6 and IL-1β were also decreased after the combination therapy (figure 3A–D).

Figure 3

ZJU37 reduced the secretion of inflammatory cytokines in MRL/lpr mice. Serum concentrations of TNF-α (A), IFN-γ (B), IL-6 (C), IL-1β (D) and IL-17 (E) were measured by ELISA kits in MRL/lpr mice in vehicle, PNS, ZJU37 and combination groups after 10-week treatment (N=5–10). The experiment was repeated twice, the data are expressed as mean±SD and the dots indicate individual mice. The results are analysed by one-way ANOVA with multiple comparisons and show statistical significance with *p<0.05; **p<0.01; and ***p<0.001 compared with the vehicle group. ANOVA, analysis of variance; PNS, prednisone.

The RIPK1 D138N inactivation mutation protected mice from cGVHD lupus-like attack

To validate the importance of RIPK1 kinase in the pathogenesis of lupus, we used bm12 mice to induce a cGVHD-like lupus model in the RIPK1 D138N mice, as seen in figure 4A. The genetic differences of bm12 mice from C57BL/6 were three amino acid mutations on major histocompatibility complex (MHC) class II, which led to bm12 CD4 T cell recognising B cells in C57BL/6 mice as a result of bm12 splenocytes transfer. In general, the bm12 inducible model of SLE in C57BL/6 mice rapidly leads to large frequencies of Tfh (CD4+PD-1+CXCR5+B220-), GC B cells (CD19+GL7+B220-) and plasma cells (CD19+CD138+B220-) followed by high levels of circulating antinuclear antibodies.

Figure 4

The RIPK1 D138N inactivation mutation prevented bm12-induced lupus symptoms. (A) This scheme describes the generation of the bm12-induced SLE model. The model was created by injecting 3×107 lymphocytes from bm12 mice into RIPK1 D138N or C57BL/6 mice via intraperitoneal injection. After a 2-week induction, the proportion (B) of T cells (CD3+) and B cells (CD19+) in the spleens of the indicated groups was determined by flow cytometry. Additionally, the proportion and cell count (C) of GC B cells (CD19+GL7+B220-), plasma cells (CD19+CD138+B220-) and Tfh cells (CD4+PD−1+CXCR5+B220-) in the spleen were examined in the indicated groups. The serum levels of anti-dsDNA antibody (D), TNF-α (E) and IFN-γ (F) were evaluated using an ELISA kit, and the experiment was repeated twice. After an 8-week induction, IgG (G) and C3 deposits (H) and scoring in the kidney were analysed by immunofluorescence tests. All groups have 4–6 mice, and individual mice are represented as dots. (B–H) are analysed by one-way ANOVA with multiple comparisons and statistically significant results are indicated as *p<0.05; **p<0.01 and ***p<0.001 compared with the bm12+C57BL/6 group. ANOVA, analysis of variance; PNS, prednisone; SLE, systemic lupus erythematosus.

Accordingly, the expansion of GC B cells, plasma cells and Tfh cells was found in bm12-induced WT C57BL/6, and the bm12-induced RIPK1 D138N mice showed less response (figure 4C). Furthermore, the serum anti-dsDNA antibody was significantly upregulated in bm12-induced C57BL/6 mice but RIPK1 D138N mutation prevented this phenomenon (figure 4D). As supporting evidence (figure 4B), the T and B lymphocyte proportions in C57BL/6 and RIPK1 D138N mice showed nearly no fluctuation, which meant a nearly normal immune state of RIPK1 D138N in contrast to C57BL/6. Additionally, cytokine detection showed a remarkably lower level of TNFα and IFN-γ in bm12-induced RIPK1 D138N mice (figure 4E,F), and the IgG and C3 deposits and their scorings in the kidney were also significantly decreased in these mice (figure 4G,H).

In summary, the cGVHD lupus-like symptoms in bm12-induced RIPK1 D138N mice were not as prominent as those of the bm12-induced C57BL/6 mice because RIPK1 D138N kinase-dead mutation had a protective effect.

Discussion

SLE is a chronic autoimmune inflammation disease, and immune intolerance causes autoimmune inflammation to occur repeatedly throughout an afflicted person’s life.2 4 Here, our data show that inhibition of RIPK1 by an inhibitor or kinase-dead mutation can significantly reduce the inflammatory response in two lupus mice models, indicating a novel treatment target in SLE. Our results suggest that inhibiting RIPK1 can improve disease manifestations for SLE, including nephritis, autoantibody production, aberrant T cell activation and inflammatory cytokine expression.

We investigated the effects of RIPK1 inhibitors on DN T cells, which further demonstrated the necessity of RIPK1 kinase in T cell development, function, proliferation and differentiation. DN T cells are aberrant T cells that express the B-cell lineage marker B220, primarily derived from CD8+T cells in patients with SLE and MRL/lpr mice.7 10 As IL-17-producing cells, they have been found to infiltrate and damage the organ tissues of patients with SLE.7 In this study, we found that ZJU37 could reduce the proportion of DN T cells and increase CD8+T cells, suggesting that ZJU37 may reverse the deviation of T cell subsets by inhibiting their necroptosis. This finding is supported by the mechanism of necroptosis of T cells in tCaspase8-/-RIPK3-/-mouse40 and the perturbation of T cell homeostasis in FADDdd-/-RIPK3-/- mouse,41 both of which suggest that the simultaneous loss of apoptosis and necroptosis in C57BL/6 mice leads to LPR disease. In addition, flow cytometric analysis of the spleen and lymph nodes in Ripk1-/-Ripk3-/-Tradd-/- mice showed a significant decrease in CD4+T cells, CD8+T cells and DN T cells,42 resulting in dramatic T lymphopenia, which directly indicates that the deletion of RIPK1 affects the development and differentiation of T cells.

In the bm12-inducible model, donor CD4+T cell-mediated responses resulted in massive host polyclonal B cell activation, expansion and immunoglobulin production.43 This chronic disease shares some similarities with SLE, including ANA, lupus-specific autoantibodies and immune complex-mediated GN.44 In this study, the RIPK1 D138N mutation significantly reduced these SLE responses. RIPK1 D138N point mutations can completely inactivate RIPK1 kinase, so we used the bm12 cGVHD model to model in vivo in RIPK1 D138N mutant mice in order to demonstrate that RIPK1 inhibition can prevent SLE.

The proliferation of GC B and plasma cells is critical to the onset and progression of SLE.45 In GCs, rapidly growing B cells undergo somatic mutations and selection before developing into memory B cells or long-lived plasma cells.46 Tfh cells also play an important role in humoral responses, assisting B cells in the formation of GCs and the production of high-affinity antibodies.47 Tfh cells, GC B cells and plasma cells proliferated greatly in the Bm12-induced lupus model, but this phenomenon was reduced in the RIPK1 D138N mutant mice, implying that RIPK1 kinase activity may play an important role in humoral immunity.

TNF-α is a multifunctional cytokine that affects both the innate and adaptive immune systems, promoting inflammation and regulating immunity. TNF-α levels in the serum and renal tissue of MRL/lpr mice have been linked to disease activity in patients with SLE and nephritis who benefited from anti-TNF-α therapy.5 48 Interestingly, our data showed that TNF-α levels dropped significantly in both the bm12-induced SLE and spontaneous MRL/lpr models after treatment. higher IFN-γ levels have been identified in lymphoid organs of diseased MRL/lpr mice, and higher IFN-γ-producing T cells have been found to be associated with autoantibody titres and proteinuria in elderly diseased mice.49 As a type of IFN-γ producing T cells, DN T cells and IFN-γ were simultaneously reduced in the treatment of RIPK 1 inhibitor and combination therapy in MRL/lpr mice.

Small molecule inhibitors, such as JAKs and IRAKs inhibitors, have emerged as a novel therapeutic approach for lupus after decades of no significant breakthroughs. In clinical settings and experimental research, combination treatment was a novel advance in SLE treatment with many advantages, including lower dosages and fewer adverse effects from inhibiting different targets.50 51 PNS has been observed to work by suppressing activated T cells and reversing the peripheral B cell compartment in a notably dose-dependent manner.39 Additionally, an intermediate dose of PNS, such as 2 mg or 2.5 mg, when administered to MRL/lpr mice, along with other medium-dose immunosuppressant medications such as FK506 and MMF, has been found to improve mouse outcomes by jointly inhibiting the TLRs family.52 Adhering to this concept, our study scheme used a combination design with PNS as a positive control and vehicle as disease baseline or negative control, and we found that the ZJU37 and PNS combined treatment better alleviated the symptoms of lupus and mutually influenced DN T cells and inflammatory response. Our add-on study design can be used as a paradigm of combined treatment strategy in exploring the function of a new-found or prospective targets on SLE. Due to the incurable nature of SLE, a combination design could better support the effect of drug treatment when compared with the positive and negative control. Specifically, combining with an intermediate dose of PNS may enhance the function of the new-target inhibitor since the immune disorder was corrected to a minor degree and the mice who received 2 mg PNS lived longer.

This study has several limitations, however. First and foremost, we must acknowledge that investigating only one dose of ZJU37 is insufficient, and that a dose-response trial must be used to determine the causal association between RIPK1 inhibition and therapeutic efficacy. Second, RIPK1 triggers apoptosis and necroptosis via kinase-dependent methods while also performing kinase-independent prosurvival and proinflammatory roles.53 RIPK1’s kinase-independent prosurvival scaffold role, although well recognised in genetic research, remains poorly understood at the biochemical level.54 As a result, the role of RIPK1’s kinase-independent function in SLE has needs to be determined.

Conclusion

The treatment of SLE poses many challenges, with no breakthroughs for decades. The three most promising treatments are3 13: immunosuppressant agents, biological agents and small-molecule inhibitors. In this study, through the use of two kinds of lupus models, we found that RIPK1 kinase can be used effectively as a therapeutic target for SLE.

Data availability statement

Data are available upon reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

All mice were maintained in a specific pathogen-free condition at Jinling Hospital Affiliated to Nanjing University School of Medicine, and all experimental procedures were approved by the Ethics Committee of Jinling Hospital. The reference number is 2021DZGKJDWLS-0012.

Acknowledgments

We thank Professor Xia Hongguang for their kind gift of the ZJU37 and RIPK1 D138N mice.

References

Footnotes

  • Contributors LP performed the experiments, analysed the data and wrote the manuscript. Z-HL designed the study, provided expert advice and revised the manuscript. PW conducted the experiments and analysed the data. XX helped to perform the flow cytometry. DZ performed the immunofluorescence staining, FX scored the immunofluorescent slides and glomerular injuries, and FY performed the H&E staining. WQ critically reviewed the paper and is the guarantor who accepted full responsibility for the submitted work. All authors were involved in drafting the article, and all authors have approved the final version for publication.

  • Funding This work was supported by the National Key Research and Development Program of China (2016YFC0904100 and 2016YFC0904103).

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer-reviewed.