Pristane-Accelerated Autoimmune Disease in (SWR X NZB) F1 Mice Leads to Prominent Tubulointerstitial Inflammation and Human Lupus Nephritis-Like Fibrosis

<div><p>Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.</p></div

Pristane treatment of SNF1 mice induces human lupus nephritis-like fibrosis.

<p>(A) Gene expression levels of <i>Tnc</i>, <i>Timp1</i>, <i>Col1a2</i>, <i>Acta2</i> in the kidneys of pristane-treated SNF1 mice and Adv-IFNα BWF1 mice (Heatmap shows expression levels in Z-scores). Masson’s Trichrome stain and Tenascin C and alpha smooth muscle actin (ASMA) immunohistochemistry (IHC) were performed on kidney tissue sections from SNF1 with or without pristane treatment (week 14) and BWF1 mice with or without Adv-IFNα injection (week 9). Diseased animals were matched for proteinuria levels. (B) Staining or immunopositive area was quantified in percentage of total tissue. Each dot represents an animal. (* p-value<0.05, **p-value<0.01, unpaired two-tailed Student’s t-test). (C) <i>TNC</i>, <i>TIMP1</i>, <i>COL1A2</i>, <i>ACTA2</i> gene expression levels in normal kidney glomeruli tissues and LN kidney glomeruli tissues from public human microarray dataset. (Heatmap shows expression levels in Z-scores). PAS and tenascin C and ASMA IHC were performed on biopsies from LN patients.</p

Serum antibody levels and kidney immune complex deposition in the pristane-SNF1 model and in the Adv-IFNα BWF1 model.

<p>(A) Titers of serum anti-nucleic acid antibodies (ANA) and anti-dsDNA antibodies (Total IgM, IgA, IgG in μg equivalents/ml) in pristane-treated, untreated age matched controls, and 36-week old SNF1 mice with spontaneous disease. (B) Frozen kidney sections from pristane-treated SNF1 mice on weeks 4, 8, 12, and 14, age match control and 36-week old SNF1 mice with spontaneous disease were stained with FITC conjugated anti-IgG Ab to detect immune complex deposition. Staining is representative of 4 mice examined in each group. (C) Frozen kidney sections from Adv-IFNα BWF1 mice on weeks 0, 3, 5, and 9 were stained with FITC conjugated anti-IgG Ab to detect immune complex deposition. Staining is representative of 4 mice examined in each group. (D) Titers of serum total IgG antibodies, anti-nucleic acid antibodies (ANA) were compared between diseased SNF1 pristane mice (14 weeks after treatment) and Adv-IFNα BWF1 mice (9 weeks after treatment). Statistical significance was assessed using unpaired two-tailed Student’s t-test (*** p<0.001, **p<0.01).</p

Pristane treatment accelerates disease in SNF1 lupus-prone mice.

<p>(A) Percentage of non-moribond mice in weeks after birth (SNF1 n = 15) or in weeks after pristane injection (pristane-SNF1 n = 15). Shown is a representative plot of 2 independent experiments. (B) Progression of proteinuria in pristane-treated, untreated age matched controls, and 36-week old SNF1 mice with spontaneous disease. Each symbol indicates an individual mouse. (C) Serum IFNα concentration, and progression of proteinuria and serum cholesterol elevation in Adv-IFNα BWF1 model and untreated age matched controls. Each symbol indicates an individual mouse. (D) Effect of CD40L and BAFF blockade on proteinuria and serum cholesterol in pristane-SNF1 mice (14 weeks after treatment) or Adv-IFNα BWF1 (9 weeks after treatment) either treated with anti-CD40L (MR1), or the isotype control (Ha 4/8) and compared with PBS treated mice. Each symbol indicates an individual mouse. This is a representative experiment of 2 independent experiments. Statistical significance was assessed using One-way Anova test.</p

Kidney transcriptional response of pristane-SNF1 model and Adv-IFNα BWF1.

<p>(A) Differentially expressed genes were identified using LIMMA package for both mouse models (with fold change difference > |2| and FDR-adjusted p-values <0.05) and using ANOVA test for the human LN kidney dataset (with fold change difference >|1.5| and FDR-adjusted p-values <0.05. Venn Diagram shows the number of differentially expressed genes common between kidneys from pristane-treated SNF1 mice compared to age matched controls, kidneys from Adv-IFNα BWF1 mice compared to age matched controls, and kidney glomeruli tissues from LN patients compared to normal human kidney glomeruli tissues. (B) Gene Set Enrichment Analysis (GSEA) was used to detect whether the upregulated (UP) and downregulated (DN) genes in the human LN were enriched in the top upregulated and top downregulated genes in the kidneys from pristane-treated SNF1 mice respectively. (C) Z-score heatmap showing expression profiles of genes in kidneys from SNF1 mice with and without pristane treatment. All genes shown were differentially expression with a fold change greater than 2 between kidneys from SNF1 at 14 week post-pristane injection vs control (FDR-adjusted p-value<0.05).</p

Innate immune sensors <i>Aim2</i> and <i>Sting</i> are upregulated in pristane-SNF1 model.

<p>(A) Gene expression changes of <i>Aim2</i> and <i>Sting</i> in the kidneys of pristane-treated SNF1 mice and Adv-IFNα BWF1 mice. Data are shown as expression fold change compared to matched control untreated mice. (B) Aim2 and Sting transcripts were detected by RNAscope in kidney tissue sections from SNF1 with or without pristane treatment (week 14) and BWF1 mice with or without Adv-IFNα injection (week 9). Diseased animals were matched for proteinuria levels. (C) Results from image quantification from RNAscope hybridization described in (B) with Aim2, Sting and Ifhi1 RNAscope staining area normalized to the mean of the staining area from the control groups. Each dot represents an animal. (* p-value<0.05, **p-value<0.01, unpaired two-tailed Student’s t-test).</p