Genetic evidence for the role of plasmacytoid dendritic cells in systemic lupus erythematosus - PubMed (original) (raw)

Genetic evidence for the role of plasmacytoid dendritic cells in systemic lupus erythematosus

Vanja Sisirak et al. J Exp Med. 2014.

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of antibodies to self-nucleic acids, immune complex deposition, and tissue inflammation such as glomerulonephritis. Innate recognition of self-DNA and -RNA and the ensuing production of cytokines such as type I interferons (IFNs) contribute to SLE development. Plasmacytoid dendritic cells (pDCs) have been proposed as a source of pathogenic IFN in SLE; however, their net contribution to the disease remains unclear. We addressed this question by reducing gene dosage of the pDC-specific transcription factor E2-2 (Tcf4), which causes a specific impairment of pDC function in otherwise normal animals. We report that global or DC-specific Tcf4 haplodeficiency ameliorated SLE-like disease caused by the overexpression of the endosomal RNA sensor Tlr7. Furthermore, Tcf4 haplodeficiency in the B6.Sle1.Sle3 multigenic model of SLE nearly abolished key disease manifestations including anti-DNA antibody production and glomerulonephritis. Tcf4-haplodeficient SLE-prone animals showed a reduction of the spontaneous germinal center reaction and its associated gene expression signature. These results provide genetic evidence that pDCs are critically involved in SLE pathogenesis and autoantibody production, confirming their potential utility as therapeutic targets in the disease.

© 2014 Sisirak et al.

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Figures

Figure 1.

Figure 1.

Tcf4 haplodeficiency ameliorates SLE-like disease in Tlr7 transgenic mice. Tlr7.Tg males, haplodeficient for Tcf4 (Tg/het) or their _Tcf4_-sufficient littermates (Tg), were analyzed. (A) Kaplan-Meier survival plot (n = 7–10). Significance was determined by log-rank test. (B) Splenic size (left) and splenic weights were determined for individual 50-wk-old transgenic animals and WT controls (right). (C) Peripheral blood cells from the indicated mice were analyzed by flow cytometry (left); thresholds of positive staining and percentages of cells within the resulting quadrants are indicated. The frequencies of the CD11c+ MHC cl. II− population (top left quadrant) in individual 30-wk-old animals were determined (right). Data were pooled from 3 independent experiments. (D) Anti-RNA IgG levels in the sera of 30–40-wk-old animals were determined by ELISA. Data were pooled from 3 independent experiments. (E) Kidney cryosections of 50–60-wk-old animals were stained for IgG (red) and DNA (blue) and analyzed by fluorescence microscopy (bars, 100 µm). Arrows show kidney glomeruli. Shown is a representative of 5 animals in each group from 2 independent experiments. Horizontal bars indicate mean. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 2.

Figure 2.

DC-specific Tcf4 haplodeficiency ameliorates disease in Tlr7 transgenic mice. Tlr7.Tg _Tcf4_flox/+ _Itgax_-Cre− males (Tg) or their _Itgax_-Cre+ littermates with DC-specific Tcf4 CKO (Tg/CKO) were analyzed along with WT controls. (A) Splenic weights were determined in individual 60-wk-old animals. (B) Frequencies of CD11c+ MHC cl. II− myeloid cells in the peripheral blood of 30–40-wk-old animals were determined by flow cytometry. Data were pooled from 3 independent experiments. (C) Peripheral blood cells from the indicated 30–40-wk-old mice were analyzed by flow cytometry, gated on CD4+ T cells, and the frequencies of activated CD44+ CD45RB− cells among CD4+ T cells were determined. Data were pooled from 4 independent experiments. (D) Levels of total IgM, IgG, and IgG subclasses in the sera of 30–40-wk-old animals were determined by ELISA (mean ± SD of 5 animals per group pooled from 2 independent experiments). (E) Anti-RNA IgG levels in the sera of 30–40-wk-old animals were determined by ELISA. Data were pooled from 3 independent experiments. (F) Fixed HEp-2 cells were incubated with sera from Tg mice, stained for IgG (red) alone or with DNA (blue), and analyzed by fluorescence microscopy (bars, 20 µm). Images are representative of 2 independent staining experiments. (G) Kidney cryosections of 60-wk-old animals were stained for IgG (red) and DNA (blue) and analyzed by fluorescence microscopy (bars, 100 µm). Arrows show kidney glomeruli. Images are representative of 6 animals in each group from 2 independent experiments. Horizontal bars indicate mean. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 3.

Figure 3.

Tcf4 haplodeficiency ameliorates immune activation in B6_.Sle1.Sle3_ mice. 30-wk-old B6_.Sle1.Sle3_ mice (Sle) or their Tcf4 haplodeficient littermates (Sle/het) were analyzed along with WT controls. (A) Splenic weights were determined in individual indicated animals. (B) Peripheral blood cells from the indicated mice were analyzed by flow cytometry, gated on CD4+ T cells, and the frequencies of activated CD44+ CD45RB− cells among CD4+ T cells were determined. Data were pooled from 3 independent experiments. (C) Sca-1 expression on gated B and T cells (left) and mean fluorescent intensities (MFIs) of Sca-1 on these cells from individual mice was determined by flow cytometry. Data were pooled from 3 independent experiments (right). (D and E) Levels of total IgM, IgG, and IgG subclasses (mean ± SD; D), anti-dsDNA, and anti-RNA IgG (E) in the sera of indicated experimental groups were measured by ELISA. Data were pooled from 2 independent experiments. (F) Fixed Hep2 cells were incubated with sera from Sle mice, stained for IgG (red) alone or with DNA (blue), and analyzed by fluorescence microscopy (bars, 20 µm). Images are representative of 2 independent staining experiments. (G) Kidney cryosections were stained for IgG (red) and DNA (blue) and analyzed by florescence microscopy (bars, 100 µm). Arrows show kidney glomeruli. Images are representative of 9 animals in each group from 3 independent experiments. Horizontal bars indicate mean. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 4.

Figure 4.

Tcf4 haplodeficiency ameliorates kidney disease in SLE-prone mice. (A) Kidney paraffin sections from Tlr7.Tg animals with or without DC-specific Tcf4 CKO (Tg/CKO) were stained with H&E and analyzed by microscopy (bars, 100 µm). Arrows show kidney glomeruli, with a representative glomerulus on the inset. Images are representative of 6 animals in each group from 2 independent experiments. (B) Mean diameter of kidney glomeruli was determined in individual 60-wk-old Tlr7.Tg animals. (C) Histopathological score of indicated disease parameters in the kidneys from individual Tlr7.Tg animals were determined using microscopy. (D) Kidney paraffin sections from B6_.Sle1.Sle3_ mice with (Sle/het) or without (Sle) Tcf4 haplodeficiency were stained with H&E and analyzed by microscopy (bars, 100 µm). Arrows show kidney glomeruli, with a representative glomerulus on the inset. Images are representative of 9 animals in each group from 2 independent experiments. (E–G) Percentage of parenchyma with interstitial inflammation in the kidneys (E), mean diameter of kidney glomeruli (F), and histopathological scores of indicated disease parameters (G) were determined in individual B6_.Sle1.Sle3_ animals. Horizontal bars indicate mean. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 5.

Figure 5.

Tcf4 haplodeficiency reduces GC signature in SLE-prone mice. (A and B) Total splenocytes from individual 30-wk-old WT (n = 2), B6_.Sle1.Sle3_ (Sle, n = 3), or B6_.Sle1.Sle3 Tcf4_+/− (Sle/het, n = 3) mice were analyzed by expression microarrays. (A) Unsupervised hierarchical clustering of the total expression profiles of each sample group. (B) Unsupervised PCA-based clustering of genes whose expression was significantly changed between groups. Shown are clusters of genes with common pattern of differential expression according to the two principal components (PC1 and PC2). No additional clusters or principal components were identified in the PCA. Data represent expression trajectories of individual genes (gray) and a mean trajectory (red) across sample groups; the number of genes in each cluster is indicated. (C) The expression of Aicda was analyzed in total splenocytes from B6_.Sle1.Sle3_ mice without (Sle) or with (Sle/het) Tcf4 haplodeficiency (top), or from Tlr7.Tg animals without (Tg) or with (Tg/CKO) DC-specific Tcf4 CKO (bottom). The expression of the IFN-inducible gene Ifi203 is shown as a control. Data represent relative expression in each sample group as determined by qRT-PCR (mean ± SD of 5 and 3 animals per group for Sle and Tg samples, respectively). *, P ≤ 0.05; **, P ≤ 0.01. (D) Spleen sections from WT, B6_.Sle1.Sle3_ (Sle), or B6_.Sle1.Sle3 Tcf4_+/− (Sle/het) mice were stained for total B cells (B220, red) and GC B cells (PNA, green). Shown is overall splenic architecture (top row: bars 100 µm) and representative GCs (bottom row: bars 20 µm). Representative of 3 spleens per genotype.

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