Celastrol Ameliorated Lupus in Fas-Deficiency MRL/lpr Mice via Regulating in Vivo Immune Response of Lymphocytes

doi:10.21203/rs.3.rs-1339555/v1

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Celastrol Ameliorated Lupus in Fas-Deficiency MRL/lpr Mice via Regulating in Vivo Immune Response of Lymphocytes

https://doi.org/10.21203/rs.3.rs-1339555/v1

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Background

Celastrol is the bioactive constituent extracted from tripterygium wilfordii (Thunder God Vine). In present study, we explore whether celastrol would exhibit a regulatory effect on adaptive immune response in vivo in MRL/lpr mice.

Methods

In present study, we combined following approaches to determine the underlying mechanism of celastrol treatment on MRL/lpr mice, including Elisa, Luminex, and flow cytometry.

Results

Celastrol treatment significantly prevented enlargement of spleen and lymph node, alleviated renal injuries, and reduced production of ANA and anti-dsDNA antibody. Celastrol treatment also strikingly decreased serum levels of multiple cytokines and antibody subsets in MRL/lpr mice. Celastrol treatment also reduced Th1 and TNF producing cells frequencies in CD4 + T cells of MRL/lpr mice. Celastrol treatment strikingly prevented CD138 + T cells accumulation and increased apoptosis level of CD138 + T cells in MRL/lpr mice. In addition, celastrol treatment prevented plasma cells formation from B cells in MRL/lpr mice by decreasing activated and germinal center B cells frequency in B cells.

Conclusions

Celastrol treatment has a significantly therapeutic effect on MRL/lpr mice. Both alleviated accumulation of CD138 + T cells and reduced formation of plasma cells in MRL/lpr mice induced by celastrol treatment together contributed to the reduced secretion of autoantibody in celastrol-treated MRL/lpr mice.

Celastrol

T cells

B cells

Autoimmune

Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronically inflammatory autoimmune disease that is involved in multisystem injuries. It predominantly affects women, especially between puberty and menopause (Kaul et al., 2016; Dörner and Furie, 2019), and is characterized by the production of autoantibodies (Lisnevskaia et al., 2014; Zharkova et al., 2017), including anti-nuclear antibody (ANA) and double-stranded DNA (dsDNA) antibody. It has been shown that SLE has a variable incidence rate and prevalence in different regions and environments worldwide in epidemiological researches (Gergianaki et al., 2018; Stojan and Petri, 2018; Parikh et al., 2020). Despite advances of treatment in recent years, mortality in patients with SLE remains high with significant geographic variations (Kaul et al., 2016; Vukelic et al., 2018; Durcan et al., 2019). However, glucocorticoids are still applied in clinical as the first-line treatment, playing an irreplaceable role in SLE treatment, despite of recent development of therapeutic drugs such as belimumab and rituximab (Mok et al., 2013; Menon et al., 2016; Wilhelmus et al., 2016; Sciascia et al., 2017; Dammacco, 2018; Pinheiro et al., 2018). Glucocorticoids has exhibited significant therapeutic effects on SLE (Mok et al., 2013; Wilhelmus et al., 2016; Dammacco, 2018; Pinheiro et al., 2018), but chronic usage of prednisone leas to unreversible damage or has substantial side effects in patients (Thong and Olsen, 2017; Dammacco, 2018; Gatto et al., 2019; Vera and Tsang-A-Sjoe, 2020). More treatment options with fewer side effects are required to substitute glucocorticoids treatment or reduce the dose of glucocorticoids, especially for patients who have to be chronically served with glucocorticoids for SLE management.

Celastrol is the bioactive constituent extracted from tripterygium wilfordii (Thunder God Vine). Celastrol has been demonstrated to have a therapeutic effect on the experimental model of the diseases that chronic inflammatory and immunity disorders were involved in (Xu et al., 2007; Kannaiyan et al., 2011; An et al., 2020; Yan et al., 2021), such as cancer and rheumatoid arthritis. Evidences also have been provided that celastrol significantly alleviate lupus symptoms in SLE murine models (Xu et al., 2003; Li et al., 2005; Xu et al., 2007). However, the underlying mechanisms of therapeutic effects of celastrol treatment on lupus in SLE murine models were not deciphered. Neutrophil extracellular traps (NETs) have been believed to play an important role in the innate immunity fighting pathogens (Döring et al., 2017). NETs also have been reported to be involved in self-antigen exposure such as self-DNA from dying cells in autoimmune diseases (Döring et al., 2017). Recent research indicated celastrol could prevent formation of NETs in vitro that were induced by inflammatory stimuli via downregulating SYK-MEK-ERK-NF-κB signaling (Yu et al., 2015). In present study, we aim to investigate more underlying mechanisms of celastrol in treatment of SLE.

Productions of the autoantibodies in SLE make a detrimental effect on multiple tissues and organs (Kotzin, 1996; Thomas et al., 2011; You et al., 2017). It is believed that B cells are the central roles involved in adaptive immune responses, as they are specialized in production of antibodies. However, SLE is the disease that has so much variability and complexity that both T cells and B cells participate in the progress of SLE (Chesnutt et al., 1998; Nagasu et al., 2019a; Alexander et al., 2020). Recent researches found T cells, in fact, even play a more important role in the progress of lupus in SLE murine models (Alexander et al., 2020; Liu et al., 2020; Liu and Akkoyunlu, 2021). In present study, we explore whether celastrol would exhibit a regulatory effect on adaptive immune response and intervene the progression of T and B cells differentiation in vivo in Fas-deficiency MRL/lpr mice.

Mice

Female MRL/MPJ and MRL/lprmice were obtained from the Slac Laboratory (Shanghai, China). Mice were housed at 22 ± 1°C with a relative humidity of 50–60% and a 12-hour light/dark cycle.

Methods

4-week-old female MRL/MPJ and MRL/lprmice were acclimatized for one week. MRL/MPJ mice were used as controls (ddH₂O, n=9). MRL/lpr lupus mice were randomly grouped according to different treatments as follows: vehicle (ddH₂O, n=9), low dose of celastrol (CL, n=9) 2.5 mg/kg per day, high dose of celastrol (CH, n=9) 5.0 mg/kg per day, and prednisone (PNS, n=6) 2.5 mg/kg per day. Celastrol (>98%, HPLC, Batch no. DST200715-035, Supplemental Figure A) was purchased from Desite Biotechnology Co., Ltd (Chengdu, China). Oral administration of mice was performed daily from 9 to 18 weeks of age. At the 19^th-20^th week, mice were anesthetized for serum collection, and the following tissues of mice were then harvested: lymph nodes and spleen (isolated and weighed), and kidneys (for histology).

Histology

To observe renal pathologic changes in mice, paraformaldehyde-fixed kidneys were embedded in paraffin and then sectioned at 4-μm thickness. Hematoxylin and Eosin (H&E), periodic acid schiff (PAS), and Masson trichrome staining were performed on the paraffin sections. Images of kidneys were obtained and analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA)

Measurement of total IgG, anti-dsDNA IgG, and anti-nuclear antibodies in serum by ELISA

Measurement of total IgG, anti-dsDNA IgG, and anti-nuclear antibody (ANA) levels in serum of mice were performed using ELISA kit (total IgG ELISA kit, Thermo Fisher, anti-dsDNA IgG and ANA ELISA kit, Alpha Diagnostic International, San Antonio, TX, USA) according to manufacturer’s instructions. Optical density (OD) was determined at 450nm absorbance using a microplate reader.

Measurement of multiple cytokines and antibody isotypes in serum using the Luminex platform

Serum levels of multiple cytokines and antibody subtypes were measured using the Luminex assay kits (Thermo Fisher, USA). Measurements were performed according to the manufacturer’s instructions and analyzed on the Luminex™ platform.

Measurement of urine protein levels

Urine samples from individual mice were collected for 24 hours at the 16^th week. Fresh urine samples were centrifuged at 1500 rpm for 10 min and then stored at -80°C. The concentration of proteins in urine samples was determined using the Coomassie brilliant blue dye-binding assay kit and performed according to the manufacturer’s instructions (Biokits Tech. Inc, Beijing, China).

Flow cytometry

Single-cell suspensions of splenocytes were obtained by filtering through a 70um cell strainer. Splenocytes were incubated on ice with CD16/CD32 monoclonal antibody (Thermo Fisher, eBiosience) for 15 minutes, and then red blood cells were lysed using lysis buffer (BD Bioscience). The splenocytes were then fixed and permeabilized (Fixation/Permeabilization solution, BD Bioscience) before intracellular staining. Cells were stained with the following antibodies for flow cytometry analysis: anti-CD3 PE-cy7, anti-CD3 APC-cy7, anti-CD4 FITC, anti-CD8 PerCP, anti-CD19 APC-cy7, anti-CD19 PE-cy7, anti-CD138 PE, anti-CD69 APC, anti-CD25 APC, anti-Foxp3 PE, anti-IL-4 APC, anti-IFN-γ PE-cy7, anti-TNF PE, anti-CD44 PE, anti-CD62L APC, Annexin V FITC, 7-AAD PerCP, anti-CD23, anti-B220 PerCP, anti-GL-7 APC, anti-MHC-II FITC, anti-CD23 APC, anti-CD21 PE, and anti-CD69 PE. Flow cytometry data was analyzed using Flowjo software version 10.6 for PC (Tree Star).

Statistical analysis

Data from all experiments were presented as mean ± standard deviation (SD) and analyzed using the SPSS software (SPSS, Inc., Chicago, IL, USA). Comparisons between the groups were performed for statistical significance using one-way analysis of variance or Mann-Whitney U test. Differences with P values less than 0.05 were considered statistically significant.

Celastrol treatment significantly ameliorated lupus symptoms in MRL/lpr mice

Compared to MRL/MPJ mice, significant enlargement of the spleen and lymph nodes, increased levels of ANA and anti-dsDNA IgG antibodies in serum, obvious renal injuries, and elevated urine protein levels had been observed in vehicle-treated MRL/lpr mice (Figure 1). These results indicated that the murine lupus model was successfully built in MRL/lpr mice in present study. After administration of celastrol treatment (2.5mg/kg CL and 5.0mg/kg, CH), increased weight of spleen and lymph nodes, elevated levels of ANA and anti-dsDNA IgG antibodies in serum, and elevated urine protein levels in MRL/lpr mice were significantly ameliorated (Figure 1A, 1D, and 1E). Additionally, obvious renal injuries in vehicle-treated MRL/lpr mice such as hyaline deposits, interstitial and perivascular cellular inflammation infiltration, cellular crescent formation, glomerular fibrosis, glomerulosclerosis, and tubular cell necrosis were also significantly alleviated (Figure 1B and 1C) in MRL/lpr mice after celastrol treatment (2.5mg/kg CL and 5.0mg/kg, CH). These results indicated that celastrol had a significantly therapeutic effect on lupus in MRL/lpr mice.

Celastrol treatment reducesantibody secretion in MRL/lpr mice

Inflammation in vivo induced by immune complex and further activated complements results in multiple organs injuries and promotes the development of disease in SLE (Sandhu and Quan, 2017). We observed multiple antibody subsets levels in serum including total IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, and IgE were increased in MRL/lpr lupus mice compared with MRL/lpr mice (Figure 2). However, celastrol treatment exhibited obvious effects on antibody production in serum of MRL/lpr mice. Total IgG, IgG1, and IgG2b levels in serum of MRL/lpr mice with celastrol treatment (5.0mg/kg, CH) were significantly decreased, compared with vehicle-treated MRL/lpr mice (Figure 2A, 2B, and 2D).

Celastrol prevents plasma cells formation by decreasing GC and activated B cells frequency in B cells of MRL/lpr mice

Compared with MRL/MPJ mice, B cells in splenocytes of vehicle-treated MRL/lpr mice were significantly decreased (Figure 3A). We further observed follicular B cells frequency was significantly decreased but marginal B cells frequency was obviously increased in vehicle-treated MRL/lpr mice compared with those in MRL/MPJ mice. While, both MHC-II expression in B cells and activated B cells frequency in vehicle-treated MRL/lpr mice were significantly increased compared with MRL/MPJ mice (Figure 3C and 3D). But there is no significant defference of germinal center (GC) B cells frequency between vehicle-treated MRL/lpr mice and MRL/MPJ mice (Figure 3E). Importantly, plasma cells strikingly accumulated in splenocytes of MRL/lpr mice compared with MRL/MPJ mice (Figure 3F).

However, celastrol treatment (5.0mg/kg, CH) significantly increased B cells frequency in splenocytes of MRL/lpr mice (Figure 3A). Celastrol treatment (5.0mg/kg, CH) also strikingly reduced activated B cells frequency in B cells of MRL/lpr mice (Figure 3D). Moreover, GC B cells frequency was significantly decreased in celastrol-treated MRL/lpr mice (5.0mg/kg, CH) compared with vehicle-treated MRL/lpr mice (Figure 3E). Plasma cells frequencies in CD3- cells of MRL/lpr mice after oral administration of celastrol (2.5mg/kg CL and 5.0mg/kg, CH) were even obviously reduced (Figure 3F). But celastrol treatment did not exhibited significant effect on frequencies of both follicular and marginal B cells in B cells of MRL/lpr mice (Figure 3B). Celastrol treatment also failed to significantly decrease MHC-II expression in B cells of MRL/lpr mice (Figure 3C).

Celastrol suppresses in vivo inflammation response in MRL/lpr mice

In present study, we observed multiple cytokines levels including IFN-γ, IL-6, IL-12, and TNF in serum were significantly increased in MRL/lpr mice compared with MRL/MPJ mice (Figure 4A). We further observed T helper (Th) 1 cells frequency of CD4+ T cells in vehicle-treated MRL/lpr mice was significantly increased compared with that in MRL/MPJ mice (Figure 4B and 4C). However, Th2 cells frequency of CD4+ T cells was decreased in vehicle-treated MRL/lpr mice compared with MRL/MPJ mice (Figure 4B and 4C). Accordingly, ratio of Th1/Th2 in vehicle-treated MRL/lpr mice was signifcantly elevated compared with that in MRL/MPJ mice (Figure 4B and 4C). Interestingly, TNF producing cells frequency in CD4+ T cells of MRL/lpr mice was obviously reduced compared with that of MRL/MPJ mice despite of increased TNF serum level in MRL/lpr mice (Figure 4D). In addition, regular T (Treg) cells frequency in CD4+ T cells was obviously decreased in vehicle-treated MRL/lpr mice (Figure 4E).

In vivo inflammation of MRL/lpr mice was significantly suppressed by celastrol treatment. After oral administration of celastrol treatment with 2.5mg/kg (CL), serum level of TNF in MRL/lpr mice was significantly decreased compared with vehicle-treated MRL/lpr mice (Figure 4A). IFN-γ and IL-6 levels in serum of MRL/lpr mice with celastrol treatment of 5.0 mg/kg (CH) were even strikingly decreased (Figure 4A). Th1 cells frequencies in CD4+ T cells of celastrol-treated MRL/lpr mice (2.5mg/kg CL and 5.0mg/kg, CH) were significantly decreased, compared with vehicle-treated mice (Figure 4B and 4C). Th2 cells frequency in CD4+ T cells (5.0mg/kg, CH) was simultaneously reduced in MRL/lpr mice after celastrol treatment (Figure 4B and 4C). But the ratio of Th1/Th2 in MRL/lpr mice was not significantly affected by celastrol treatment (Figure 4C). In addition, TNF producing cells frequencies in CD4+ T cells of celastrol-treated MRL/lpr mice (2.5mg/kg CL and 5.0mg/kg, CH) were also strikingly decreased, compared with vehicle-treated mice (Figure 4D). But Treg cells frequency in CD4+ T cells of MRL/lpr mice was not significantly affected by celastrol treatment (Figure 4E).

Celastrol treatment reduces Tcm cells frequency in T cells of MRL/lpr mice

Compared with MRL/MPJ mice, CD3+T cells frequency was significantly increased in splencoytes of vehicle-treated MRL/lpr mice (Figure 5A). Moreover, double Negative (DN) T cells were also accumulated in CD3+ T cells of MRL/lpr mice (Figure 5B). However, CD4+ and CD8+ T cells frequencies in splenocytes of vehicle-treated MRL/lpr mice were evidently decreased compared with those in MRL/MPJ mice (Figure 5C). Despite of Fas deficiency and T cells accumulation in MRL/lpr mice, there were no difference of apoptotic and live cells frequencies in CD3+ T cells between vehicle-treated MRL/lpr mice and MRL/MPJ mice (Figure 5D). In addition, CD69+ cells frequency in CD3+ T cells of vehicle-treated MRL/lpr mice was significantly increased compared with that in MRL/MPJ mice (Figure 5E). In addition, central memory (Tcm) and effector memory (Tem) T cells frequencies in CD3+ T cells were significantly increased in vehicle-treated MRL/lpr mice compared with those in MRL/MPJ mice (Figure 5E). But CD44-CD62L- and native T (Tn) cells frequencies in CD3+ T cells of vehicle-treated MRL/lpr mice were evidently decreased (Figure 5E).

After oral administration of celastrol, we observed celastrol treatment did not significantly prevent the accumulation of T cells in MRL/lpr mice (Figure 5A). But celastrol treatment significantly reduced live cells frequency of CD3+ T cells in MRL/lpr mice (Figure 5D). Moreover, DN T cells accumulations in CD3+ T cells of MRL/lpr mice with celastrol treatment (2.5mg/kg CL and 5.0mg/kg, CH) were significantly alleviated compared with vehicle-treated MRL/lpr mice (Figure 5B). In addition, CD4+ T cells frequency in splenocytes was obviously increased in MRL/lpr mice after celastrol treatment (5.0mg/kg, CH) compared with vehicle-treated MRL/lpr mice (Figure 5C). However, celastrol treatment did not show significant effect on activated T cells frequency in MRL/lpr mice (Figure 5E). Moreover, Tcm cells frequencies in MRL/lpr mice after celastrol treatment (2.5mg/kg CL and 5.0mg/kg, CH) were significantly reduced compared with vehicle-treated MRL/lpr mice. But Tem, Tn, and CD44-CD62L- T cells frequencies in CD3+ T cells of MRL/lpr mice were not significantly affected by celastrol treatment (Figure 5F).

Celastrol treatment prevents accumulation of CD138+ T cells by promoting apoptosis of CD138+ T cells

Fas deficiency and subsequently decreased apoptosis level of CD138+ T cells results in CD138+ T cells accumulation in MRL/lpr mice (Liu et al., 2020). We had observed CD138+ T cells accumulated in CD3+ T cells of vehicle-treated MRL/lpr mice but not in MRL/MPJ mice (Figure 6A). CD138+ cells frequencies were also significantly increased in CD4+ and DN T cells of vehicle-treated MRL/lpr mice compared with MRL/MPJ mice (Figure 6B and 6C). However, CD138+ cells frequencies in CD3+ T cells and T cells subsets (DN and CD4+ T cells) were significantly decreased in MRL/lpr mice after treatment of celastrol (2.5mg/kg CL and 5.0mg/kg, CH) compared with vehicle-treated MRL/lpr mice (Figure 6A, 6B, and 6C). We had further observed apoptotic cells frequency was increased but live cells frequency was decreased in CD138+ T cells of celastrol-treated (5.0mg/kg, CH) MRL/lpr mice compared with vehicle-treated MRL/lpr mice (Figure 6D). But celastrol treatment did not exhibit significant effects on CD69+ cells frequency in CD138+ T cells of MRL/lpr mice compared with vehicle-treated MRL/lpr mice (Figure 6E).

In present study, celastrol treatment exhibited a therapeutic effect on MRL/lpr mice via significantly preventing enlargement of spleen and lymph node, alleviating renal injuries, and reducing production of ANA and anti-dsDNA antibody. Celastrol treatment also had shown significant effects on suppressing in vivo inflammation in MRL/lpr mice by strikingly decreasing serum levels of multiple cytokines and antibody subsets in MRL/lpr mice. Celastrol treatment also reduced Th1 and TNF producing cells frequencies in CD4 + T cells of MRL/lpr mice to suppress in vivo inflammation. Celastrol inhibited accumulations of DN and CD138 + T cells in MRL/lpr mice. Celastrol also significantly decreased Tcm frequency in CD3 + T cells of MRL/lpr mice. Celastrol treatment strikingly increased apoptosis level of CD138 + T cells but decreased live cells numbers of them in MRL/lpr mice. Alleviated accumulation of CD138 + T cells in MRL/lpr mice induced by celastrol treatment resulted in decreased formation of autoreactive plasma cells which subsequently contributed to the reduced secretion of autoantibody. In addition, celastrol treatment prevented plasma cells formation from B cells in MRL/lpr mice by decreasing activated and germinal B cells frequency in B cells, which further contributed to the reduced secretion of autoantibody in celastrol-treated MRL/lpr mice.

Syndecan-1/CD138 is the marker of plasma cells in lymphocytes that are believed to originate from B cells (Calame, 2001; Lu et al., 2011). CD138 + T cells, which express both CD3 and CD138, have been reported recently to be plasmablastic B-cell neoplasms as observed in clinical cases (Pan et al., 2018). However, these abnormal CD138 + cells were also observed in murine systemic lupus erythematosus (SLE) models (Seagal et al., 2003; Mohamood et al., 2008; Liu et al., 2020). Previous research has demonstrated CD138 + T cells in MRL/lpr mice significantly promote autoantibody production both in vivo and in vtro indicating CD138 + T cells are comprised of the autoreactive T cells of MRL/lpr mice (Liu et al., 2020). Moreover, CD138 expression in CD3 + T cells play a key role in the progression of lupus in MRL/lpr mice. In present study, we observed DN T cells were accumulated in MRL/lpr mice. DN T cells accumulation has been demonstrated to play an important role in promoting the progression of lupus in MRL/lpr mice (Alexander et al., 2020). Our results showed celastrol treatment significantly prevented DN T cells accumulation in MRL/lpr mice. While CD138 expression in CD3 + T cells significantly prevents the apoptosis of CD3 + T cells and significantly contributes to DN T cells accumulation in MRL/lpr mice (unpublished data in preview by Xie TH et al.). Importantly, CD138 expression in DN T cells also significantly increased FasL expression of DN T cells. However, DN T cells in MRL/lpr mice are strongly cytotoxic, overexpressing FasL, which results in autoimmune injuries of multiple tissues that express small amounts of Fas receptor (Benihoud et al., 1997; Alexander et al., 2020). CD138 expression in CD3 + T cells also significantly promotes the activation of CD3 + T cells in MRL/lpr mice. Recent research also found CD138 + T cells could strikingly contribute to formation of plasma cells from B cells of MRL/lpr mice (Liu et al., 2020; Liu and Akkoyunlu, 2021). Previous research showed majorities of CD138 + T cells were Tcm cells which were both CD44 + CD62 + cells (Liu et al., 2020). Our results showed oral administration of celastrol significantly reduced Tcm cells frequency of CD3 + T cells in MRL/lpr mice. Celastrol treatment simultaneously prevented CD138 + T cells accumulation via promoting apoptosis of CD138 + T cells in MRL/lpr mice. We also observed decreased serum levels of ANA and anti-dsDNA antibody in celastrol-treated MRL/lpr mice. Our results indicated alleviated CD138 + T cells accumulation induced by celastrol treatment contributed to the decreased autoantibody production in vivo in MRL/lpr mice. In addition, our results showed celastrol significantly reduced CD138 + cells frequency in DN T cells of MRL/lpr mice, which subsequently resulted in decreased DN T cells frequency of splenocytes in MRL/lpr mice.

Immune complex and subsequently induced increase in multiple cytokines level in serum of MRL/lpr mice promote and amplify the in vivo inflammation of MRL/lpr mice (Tshilela et al., 2016; Sandhu and Quan, 2017). In present study, our results showed celastrol significantly reduced production of antibody subsets including total IgG, IgG1, and IgG2b and decreased serum levels of multiple cytokines such as TNF, IFN-γ, and IL-6 to inhibit the progression of inflammation in vivo in MRL/lpr mice. However, more researchers believed that T cells polarization in SLE was from Th1 to Th2 cells, and IFN-α that promoted activated B cells differentiation into plasma cells, played an essential role in the progression of disease (Ehrenfeld et al., 2001; Selvaraja et al., 2019). But, in present study, our results showed Th1 cells frequency but not Th2 cells in CD4 + T cells of MRL/lpr mice was significantly increased in addition to strikingly increased serum levels of IFN-γ. Moreover, ratio of Th1/Th2 in MRL/lpr mice was simultaneously elevated compared with MRL/lpr mice. Recent years, with detailed researches, IFN-γ began to be identified as a more important role and essential in development of lupus in MRL/lpr mice (Balomenos et al., 1998; Juvet et al., 2012; Tshilela et al., 2016; Tan et al., 2019). It has been demonstrated that IFN-γ dramatically promotes proliferation and accumulation of DN T cells, and significantly increases expression of FasL on surface of DN T cells in lupus mice (Balomenos et al., 1998; Juvet et al., 2012). Previous researches also showed frequency of Th1 cells in fas-deficiency lupus mice, but not Th2, was significantly increased in vivo (Seagal et al., 2003; Tang et al., 2019). Recent researches suggested that IFN-γ and Th1 cells may be closely associated with the mechanism of lupus development and tissue injuries in MRL/lpr mice (Balomenos et al., 1998; Zhang et al., 2007; Chodisetti et al., 2020). The evidence has been also provided that IFN-γ is required for TLR7-promoted development of autoreactive B cells (Chodisetti et al., 2020). In present study, our results showed celastrol treatment could significantly prevent IFN-γ expression and reduce Th1 cells frquency in CD4 + T cells of MRL/lpr mice in addition to decrease of serum IFN-γ level. But celastrol treatment did not strikingly affect the ratio of Th1/Th2 in MRL/lpr mice.

TNF-alpha, a pro-inflammatory cytokine, however, plays a contradictory role in SLE. Increased TNF levels in serum, kidney, and skin samples of SLE patients as well as SLE murine models play an inflammatory role in SLE and promote organs injuries (Sabry et al., 2006; Aringer and Smolen, 2012). On the ther hand, TNF is also the cytokine that induces apoptosis of cytotoxic T cells via activating caspase-8 in the target cells (Nagasu et al., 2019b). However, it has been reported (Williams et al., 2009; Katz and Zandman-Goddard, 2010) that anti-TNF therapies such as TNF antibodies and soluble TNF receptors were commonly associated with the induction of autoantibodies. Some researchers even proposed that the contribution of TNF in SLE progression was limited, but increased TNF levels contributed to the deletion of DN T cells in SLE murine models resulting in the improved lupus symptoms (Nagasu et al., 2019b). In present study we observed contradictory results that although serum levels of TNF in MRL/lpr mice were obviously increased, interestingly, TNF producing cells frequency in CD4 + T cells of MRL/lpr mice was significantly decreased compared with MRL/MPJ mice as our results had shown. However, previous research has reported that the TNF blockade therapy alone could result in induction of ANA and anti-dsDNA despite the frequency and clinical characteristics of anti-TNF-induced lupus (ATIL) varied between different drugs (Williams et al., 2009). In present study, both celastrol and prednisone significantly decreased serum TNF levels and TNF producing cells frequencies in CD4 + T cells of MRL/lpr mice. But celastrol and prednisone treatment did not result in the symptoms of ATIL in MRL/lpr mice.

B cells are universally regarded as the central role in adaptive immune response. It has been believed autoreactive B cells in SLE are able to further differentiate into abnormal plasma cells secreting autoantibody after activation by self-antigen and autoreactive T cells (Laurent et al., 2017; de la Varga-Martínez et al., 2019; Jackson and Davidson, 2019; Mihaylova et al., 2020). Immature B cells are originated from stem cells in bone marrow and the diversity among BCR specificities is generated by random rearrangement of gene segments during early B-cell development (Monroe et al., 2003; Xu et al., 2020). After experiencing positive selection and pro B cells, then, pre-B cells expressed mIgM and became immature B cells (Monroe et al., 2003; Benhamou et al., 2016). Autoreactive immature B cells will suffer from apoptosis and be deleted in negative selection (Monroe et al., 2003; Xu et al., 2020). Although the mechanism that autoreactive B cells pass through negative selection in SLE is still unclear. Researchers believed Fas deficiency may result in the failure of autoreactive B cells apoptosis in SLE murine model (Akagi et al., 1998; Hancz et al., 2012). Then, MHC-II with self-antigens expressed in activated autoreactive B cells interacts with autoreactive CD4 + T cells and activate the autoreactive T cells (Chesnutt et al., 1998; Laurent et al., 2017). Activated autoreactive B cells also differentiate into plasma cells secreting autoantibody at the help of autoreactive CD4 + T cells (Chesnutt et al., 1998; Laurent et al., 2017; Liu et al., 2020). Our results showed celastrol treatment significantly reduced GC B cells frequency in B cells of MRL/lpr mice and strikingly prevented activation of B cells in MRL/lpr mice, which contributed to decrease of plasma cells formation and would further prevent the formation of autoreactive plasma cells. In celastrol-treated MRL/lpr mice, both decreased plasma cells formation and alleviated CD138 + T cells accumulation together contributed to the reduced production of autoantibody.

Celastrol has a therapeutic effect on lupus of MRL/lpr mice. Celastrol treatment has a significant effect on decreasing production of multiple cytokines and antibody subsets in serum. Celastrol treatment also reduced Th1 and TNF producing cells frequencies in CD4 + T cells of MRL/lpr mice to suppress in vivo inflammation. Celastrol treatment inhibited accumulations of DN and CD138 + T cells in MRL/lpr mice by strikingly increasing apoptosis level of CD138 + cells in CD3 + T cells, to alleviate the enlargement of spleen and lymph node. Alleviated accumulation of CD138 + T cells in MRL/lpr mice induced by celastrol treatment also contributed to decreased formation of autoreactive plasma cells and resulted in reduced autoantibody secretion in MRL/lpr mice. In addition, celastrol treatment also prevents plasma cells formation in MRL/lpr mice by decreasing activated and germinal B cells frequency in B cells. Decreased formation of plasma cells being from B cells in MRL/lpr mice induced by celastrol treatment further contributed to the decreased autoantibody secretion.

Ethics approval and consent to participate

All animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Beijing Institute of Traditional Chinese Medicine, and performed according to the institutional ethical guidelines.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conflict of interests.

Funding

This work was funded by Beijing Postdoctoral Research Foundation (ZZ2019-23) and the MiaoPu research foundation of the Beijing Institute of Traditional Chinese Medicine (MP-2020-45).

Authors' contribution

HL performed the histological examination of the kidney. PL designed the experiments and edited the manuscript. TX conceived the study, wrote the manuscript and performed the measurement of Elisa, Luminex, and Flow cytometry. XL performed data analysis in manuscript. All authors read and approved the final manuscript.

Acknowledgments

Not applicable.

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Celastrol Ameliorated Lupus in Fas-Deficiency MRL/lpr Mice via Regulating in Vivo Immune Response of Lymphocytes

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