Dexamethasone reduces autoantibody levels in MRL/lpr mice by inhibiting Tfh cell responses

Abstract Previous studies have shown that dexamethasone (Dex) reduces the levels of anti‐nuclear (ANA) and anti‐dsDNA antibodies in MRL/lpr mice (a mouse model of SLE). However, the effect of Dex on T follicular helper (Tfh) cells is less documented. Here, using the MRL/lpr mouse model, we investigated the influence of Dex on Tfh cells and potential underlying mechanisms. The data showed that the proportion of Tfh cells, identified as CD4+CXCR5+ICOS+, CD4+CXCR5+PD‐1+ or CD4+BCL‐6+ cells, markedly decreased after treatment with the Dex, in both Balb/c mice and MRL/lpr mice. Dex significantly inhibited IL‐21 expression at both the mRNA and the protein levels. Dex also significantly reduced the proportion of germinal centre B cells and decreased serum IgG, IgG2a/b and IgA levels. Moreover, a positive correlation between the proportion of Tfh cells (CD4+CXCR5+ICOS+, CD4+CXCR5+PD‐1+ or CD4+BCL‐6+) and autoantibodies was observed. Dex significantly increased the Prdm1 and Stat5b mRNA expression and decreased the Bcl‐6 and c‐Maf mRNA expression of CD4+T cells. In brief, Dex inhibited the Tfh development, which relies on many other transcription factors in addition to Bcl‐6. Our data indicate that Dex can be used as a Tfh cell inhibitor in SLE.

In MRL/lpr mice, the abnormally high expression of Tfhassociated molecules, such as ICOS, PD-1, BCL-6 and IL-21, suggests that Tfh cells may play a crucial role in the pathogenesis of SLE, which is closely connected with autoantibody production and/or lupus-like symptoms. [15][16][17][18][19] This observation is reminiscent of the significant correlation between the high proportion of circulating cells displaying a Tfh phenotype, abnormal production of autoantibodies and disease severity in SLE patients. 20,21 Recent work clearly evidenced an increased frequency of circulating Tfh cells accompanied by higher levels of serum IL-21 in SLE patients. 21, 22 Simpson et al found that the frequency of circulating Tfh cells is strongly correlated with the number of their counterparts residing in the GC. 23,24 Therefore, circulating Tfh cells in humans could serve as a biomarker, indicative of the occurrence of such a potential mechanism of GC tolerance disruption. If this hypothesis is verified, it may be an effective therapeutic target for SLE treatment through inhibiting Tfh cell and the GCs response.
At present, glucocorticoids are used as the first-line drugs for the treatment of SLE. Among them, Dex is often used to treat severe nephritis or other serious organ complications in SLE patients. [25][26][27] However, it is still unclear how Dex affects Tfh cells in SLE.
Our study aimed to evaluate the efficacy of Dex in modulating

| Mice and murine model
Six-to eight-week-old female MRL/lpr mice and Balb/c mice (from the Shanghai SLAC Laboratory Animal Co., Ltd) were all used in experiment. All 16-week-old MRL/lpr and Balb/c mice were randomized into two groups :(1) 1 mg/kg of dexamethasone injection (H42020020; China) in 100 μL normal saline; (2) 100 μL normal saline. The animals received continuously intraperitoneal injections for 4 weeks. Mice were harvested at 20 weeks old.

| Cell isolation and culture
Splenocytes were derived from 6-to 8-week-old female Balb/c mice. Total CD4 + T cells were selected by CD4 + T-cell isolation kit and stimulated with 1 μg/ml anti-CD3, 1 μg/ml anti-CD28, 10 ng/ mL IL-21 and 20 ng/mL IL-2 (all from BD Biosciences) in the presence or absence of the Dex at concentrations of 0.5,1 or 2 μg/mL. 28 At day 3, we collected the cultured cells and supernatant for the further analysis.

| Using ELISA analysis cytokine and autoantibody titre
Mice serum was collected after intervention. In the light of the manufacturer's guidelines, the total IgG, IgG2a/b, IgA (all from eBioscience), cytokine IL-21 and autoantibody (from J&l Biological) were analysed using the mouse ELISA Kit.

| Histopathology test
Kidney samples were fixed with 4% formaldehyde, dehydration as well as wax immersion, embedded in paraffin and finally cut into 5 mm sections. And then, the sections were washed in deparaffinized and dehydrated via sequential addition of xylene, 100% ethanol, 95% ethanol and distilled water and stained with H&E (all from Sigma). Finally, the slides were evaluated by an experienced pathologist blinded to the treatment protocol. HE staining was used to observe inflammatory cell infiltrates, each with a score of 0-4 (0, absent; 1, <25%; 2, 25%-50%; 3, 50%-75% and 4, >75%).

| Immunofluorescence
For the detection of glomerular IgG deposition, 6 μm frozen kidney sections were incubated with FITC-conjugated goat anti-mouse IgG (R&D) and then mounted with anti-fade mounting medium (Beyotime). Semi-quantitative analysis of glomerular IgG deposition was performed using the following scale (0-3): 0 = negative staining, 1 = barely visible at high magnification, 2 = moderately visible and 3 = clearly visible.

| Statistics
Quantitative data are expressed as means ± SD. Statistical analyses were performed using a Student's t test with SPSS 20.0 software.
Correlations were determined by Spearman's ranking. P-value less than 0.05 was considered statistically significant.

| Dex suppresses disease progression in MRL/ lpr mice
Previous research found that MRL/lpr mice exhibit splenomegaly, a significant increase of cell proliferation and IgG deposits in kidney glomeruli and high levels of serum ANA and anti-dsDNA antibodies. 28,29 Similar to a previous report, 30 our data showed that the levels of serum ANA and anti-dsDNA antibodies were significantly reduced after Dex treatment in MRL/lpr mice ( Figure 1B). To further evaluate the effects of Dex on disease progression, we determined the size of the spleen, cellular proliferation and IgG deposits in the glomeruli. As shown in Figure 1A, the splenomegaly of MRL/lpr mice was significantly lowered by Dex treatment. Histological analysis also revealed changes between the three groups. Compared with normal mice, MRL/lpr mice showed a significant increase of cell proliferation and IgG deposition in the glomeruli, which were diminished by Dex treatment ( Figure 1B,1D). Values are the mean and SD of 5 mice per group, **P < .01 and ***P < .001 were significantly decreased in Dex-treated mice. However, Dex had no effect on IgA levels ( Figure S2 2G).  Figure 3D).

| Dex suppresses Tfh cells, GC B cells and antibody secretion in MRL/lpr mice
An increasing body of evidence shows that the number of GC B cells have a directly correlated with disease activity and B-cell numbers in MRL/lpr mice. 22 Compared to Balb/c mice, we found that the frequency of GC B cells was significantly higher in MRL/lpr mice, and this frequency would decrease on treatment with Dex ( Figure 4A and Figure S1 1C-C). Previous research demonstrated a strong positive correlation between the increased number of Tfh cells and the pathogenesis and severity of autoimmune diseases, which are GCdependent. Moreover, MRL/lpr mice showed markedly higher levels of IgG, IgG subtypes and IgA than normal mice. 22 The levels of IgG, IgG2a/b and IgA were markedly decreased in MRL/lpr mice after Dex treatment ( Figure 4B).

| Dex suppresses disease progression through the inhibition of Tfh cell responses
Lupus is characterized by the overproduction of autoantibodies.
To confirm the likely mechanism whereby Dex suppresses disease progression, we analysed the correlation between the frequency of Tfh cells, identified as CD4 + CXCR5 + ICOS + , CD4 + CXCR5 + PD-1 + or CD4 + BCL-6 + cells, and serum ANA and anti-dsDNA antibody levels.
This analysis revealed a strong correlation between these biological parameters. These data indicate that Dex reduces ANA and anti-dsDNA antibody levels through the inhibition of Tfh cell responses ( Figure 5A-B).
To explore the potential mechanism underlying the biological activity of Dex, we further assayed the expression of several transcription factors. We were surprised to find a significant down-regulation in Dex-treated MRL/lpr mice were up-regulated. In contrast, there was no effect on the mRNA expression of Stat3 ( Figure 5C).

| D ISCUS I ON
SLE is an autoimmune disease that is characterized by the production of autoantibodies and multiorgan damage, especially nephritis. [30][31][32] Previous studies have shown that Dex is able to reduce ANA and anti-dsDNA antibody levels in MRL/lpr mice. 29 In keeping with these studies, our data show that the levels of serum ANA and anti-dsDNA antibodies were significantly decreased in Dex-treated MRL/lpr mice. Interestingly, we also found that disease severity, splenomegaly, significant hyperproliferation and IgG deposits in kidney glomer- Previous studies showed that the expansion of Tfh cells is closely associated with excessive production of anti-dsDNA antibodies and severe end organ damage, such as nephritis. 18,20,21 Our data showed a positive correlation between the frequency of Tfh cells and serum ANA and anti-dsDNA antibody levels.
In conclusion, we demonstrated that the levels of autoantibodies in MRL/lpr mice were reduced when Tfh cell differentiation F I G U R E 5 Potential mechanism underlying Dex effect on Tfh cell responses. Sixteen-week-old female MRL/lpr mice (36 ± 2 g) were treated with vehicle (normal saline; noted Veh) or 1 mg/kg of Dex (noted Dex) for 4 weeks. A Positive correlation between the percentages of Tfh cells (CD4 + CXCR5 + ICOS + , CD4 + CXCR5 + PD-1 + , or CD4 + BCL-6 + ) and the levels of serum anti-dsDNA antibodies in MRL/lpr mice. B Positive correlation between the percentages of Tfh cells (CD4 + CXCR5 + ICOS + , CD4 + CXCR5 + PD-1 + , or CD4 + BCL-6 + ) and the levels of serum ANA in MRL/lpr. C Relative expression of transcription factor mRNAs in CD4 + T cells from spleen of MRL/lpr mice. Bcl-6, c-Maf, Stat3, Prdm1 and Stat5b mRNA levels quantified by TaqMan PCR. Values are the mean and SD of 5 mice per group, (black represents Veh, red represents Dex ),*P < .05, **P < .01, and *** P < .001 was inhibited by Dex treatment. Few studies have addressed the effects of Dex on Tfh cells. Therefore, our study sheds new light on a previously undescribed negative effect of Dex on Tfh cell differentiation and IL-21 secretion, which are both up-regulated in SLE patients compared to healthy controls. Our next step will be to investigate the effects of Dex on other cell populations, including CD4 + T-cell subpopulations (effector memory, central memory, naïve, and if possible regulatory T cells) and CD8 + T cells, in both MRL/lpr mice and SLE patients. We will also compare the combined effects of Dex with these populations and on Tfh cells with that of other glucocorticoids, on these populations and on Tfh cells.

E TH I C A L A PPROVA L
The Ningxia Medical University Ethics Review Committee approved this study.

CO N FLI C T O F I NTE R E S T
The authors report no declarations of interest.