Prevention of Immune Nephritis by the Small Molecular Weight Immunomodulator Iguratimod in MRL/lpr Mice

<div><p>Objective</p><p>This study was performed to investigate the therapeutic effects of iguratimod in a lupus mouse model.</p><p>Methods</p><p>Female MRL/lpr mice were treated with iguratimod, vehicle solution or cyclophosphamide. Proteinuria was monitored and kidney injury was blindly scored by a renal pathologist. Serum anti-double-stranded DNA antibodies were monitored by radioimmunoassay. Kidney IgG and CD20 were stained by immunohistochemistry. Splenic lymphocyte phenotypes were analyzed by flow cytometry. BAFF, IL-17A, IL-6, and IL-21 levels in serum and splenic lymphocytes were detected by ELISA or quantitative PCR.</p><p>Results</p><p>Compared with the vehicle-treated controls, MRL/lpr mice treated with iguratimod showed less protenuria, less acute pathological lesions and no chronic changes in the kidneys. There were significant differences in glomerular injury and vasculitis scores, as well as in the semi-quantitave analysis of immune complex deposition between the two groups. Disease activity markers in sera (anti-dsDNA antibodies and immunoglobulin levels) were reduced and hypocomplementemia was attenuated. Lymphocyte expression of BAFF, IL-6, IL-17A and IL-21 was decreased. The abnormal splenic B220+ T cell and plasma cell populations in MRL/lpr mice were reduced by iguratimod treatment, with recovery of the total B cell population and inhibition of B cell infiltration of the kidney tissue. The dosage of iguratimod used in this study showed no significant cytotoxic effects <i>in vivo</i> and no overt side-effects were observed.</p><p>Conclusion</p><p>Iguratimod ameliorates immune nephritis in MRL/lpr mice via a non-antiproliferative mechanism. Our data suggest a potential therapeutic role of iguratimod in lupus.</p></div

Iguratimod affected lymphocyte subsets.

<p>(A) Gating strategy for B220+T cell and total B cell after 8-week treatment. Hexagon gates denote B cell and rectangle gates denote abnormal B220+T cell. (B) Iguratimod and Cyc both recover B cell population in spleen significantly. Data are represented as mean ±SEM. (C) Iguratimod decreases abnormal B220+T cell population of MRL/lpr mice. Data are represented as mean ±SEM. (D) Iguratimod decreases plasma cell population (CD138+) in spleen. Data are represented as mean ±SEM. (E) Representative kidney sections stained with CD20 after 20-week treatment of iguratimod or vehicle solution. B cell infiltrates in glomerulus and interstitial of kidneys from control mice. (F) Semiquantitative IOD values of all samples are analyzed. Each dot represents mean IOD value of an individual mouse. The horizontal lines represent media. Statistics are calculated by non-paired student's t test (B, C and D) or Mann-Whitney U test (F). * <i>p</i><0.05, *** <i>p</i><0.001.</p

Iguratimod attenuated proteinuria and kidney injury in MRL/lpr mice.

<p>(A) Protein concentrations of 24 h-urine from iguratimod treated and control mice. Each dot represents mean ±SEM at the time point. (B) Representative H&E-stained kidney sections from 28 weeks old mice treated with iguratimod or vehicle solution. Control mice develop crescents (arrow), glomerular sclerosis (asterisk), lymphocyte infiltrations and casts in kidneys. Fields are at 200× magnification. (C) Glomerular injury and vasculitis were scored by a renal pathologist blindly. Each dot represents average score of an individual mouse. The horizontal lines represent media. (D) Representative kidney sections stained with IgG after 20-week treatment of iguratimod or vehicle solution. The fields are at 400× magnification. (E) Semiquantitative IOD values of all samples are analyzed (right). Each dot represents mean IOD value of an individual mouse. The horizontal lines represent media. Statistics are calculated by non-paired student's t test (A) or Mann-Whitney U test (C and E). * <i>p</i><0.05, *** <i>p</i><0.001.</p

Iguratimod decreased production of BAFF, IL-6, IL-17A and IL-21.

<p>(A) Serum BAFF concentrations of the same mice above. Each dot represents an individual mouse. The horizontal bars represent mean. (B) Relative mRNA expression levels of BAFF, IL-6, IL-21 and IL-17A in splenic cells. The mice received 8-week treatment of iguratimod, cyclophosphamide or vehicle solution. Data are represented as mean ±SEM. Statistics are calculated by non-paired student's t test (A) or two-tailed Mann-Whitney U test (B). * <i>p</i><0.05, **<i>p</i><0.01, *** <i>p</i><0.001.</p

Additional file 1: of T-bet+CD11c+ B cells are critical for antichromatin immunoglobulin G production in the development of lupus

T-bet+ CD11c+ B cells are critical for antichromatin immunoglobulin G production in the development of lupus. Figure S1. The fraction of T-bet and CD11c positive population was increased in CD138+ plasma cells. Figure S2. Correlation between the percentage of T-bet+ CD11c+ CD19+ B cells and the titers of anti-ANA antibodies (A), anti-dsDNA antibodies (B) from 22 SLE patients. Figure S3. IFNγ is required for T-bet+ CD11c+ B cell differentiation and activation. 5 × 107 splenocytes from Bm12 mice or B6 mice cells were injected intraperitoneally into B6 mice (n = 5). (PDF 361 kb