Fas ligand promotes an inducible TLR-dependent model of cutaneous lupus-like inflammation

Abstract

Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9-/- autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-γ+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9.

Keywords: Autoimmune diseases; Autoimmunity; Dermatology; Innate immunity; Th1 response.

Figure 1. TLR9 deficiency promotes systemic autoimmunity and B cell activation.

TLR9 ^WT, TLR9^KO, TLR7^KO, and TLR7/9^DKO Ii-TGO mice were or were not provided with Dox chow and were sublethally irradiated (400R) and injected i.v. with DO11 T cells. (A and B) Spleen weight (g) at 4 weeks and total body weight (g) at weekly time points following injection. (C) Proliferation of VPD-labeled naive DO11 T cells in sdLNs 5 days after T cell injection (n = 5 per group). (D) B220⁺ cells from the sdLNs stained for GC markers Fas and GL7. (E) Plasma cells in the bone marrow measured by ELISpot assay at 4 weeks after T cell injection (n = 6 per group). (F) Autoantibodies detected by HEp2 staining. Original magnification, ×200. Images were captured at ×2 magnification using an ImmunoSpot plate reader (CTL), and a representative well image is shown in the figure. Data are shown as mean ± SEM and are representative of 5 independent experiments with n = 20 mice per group (A, B, D, and F). ***P < 0.001; ****P < 0.0001, 1-way ANOVA with Šidák’s multiple-comparison test.

Figure 2. TLR9-deficiency promotes development of Th1 and TFH cells.

sdLN suspensions from DO11-injected TLR9^WT, TLR9^KO, TLR7^KO, and TLR7/9^DKO Ii-TGO mice at 4 weeks after T cell injection were analyzed for (A) percentages of DO11 (KJ126⁺) T cells in the CD4⁺ gate, (B) percentages of cytokine-producing cells in the KJ126⁺ gate, (C) percentages of FasL-expressing cells in the KJ126⁺ gate, and (D) percentages of TFH cells in the KJ126⁺ gate. Data are shown as mean ± SEM and are representative of 5 independent experiments with n = 15 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA with Šidák’s multiple-comparison test (A–C) and 2-tailed Student’s t test (D).

Figure 3. TLR9 deficiency results in LLSI.

(A) Clinical appearance of DO11-injected TLR9^WT or TLR9^KO Ii-TGO mice, provided or not provided with Dox chow, at 4 weeks after injection. (B and C) H&E-stained skin sections showing follicular plugging (black arrow, B), perivascular and perifollicular mononuclear infiltrate, vacuolization of basal layer (black arrowhead, C), and apoptotic KCs in the epidermis (black arrows, C). Original magnification, ×200 (B); ×400 (C).(D) Basement membrane thickening shown by PAS stain. Original magnification, ×400. (E) Mucin deposition in the dermis detected by Alcian blue stain. Original magnification, ×200. (F) TUNEL stain showing apoptotic cell death (red) counterstained with DAPI (blue). Original magnification, ×200; ×400 (inset). and (G) Ig deposition at basement membrane (white arrow) detected by FITC anti-IgG (green) and counterstained with DAPI (blue). Original magnification, ×200. Images shown are representative of 5 mice per group from 3 independent experiments.

Figure 4. DO11 T cell and myeloid cell infiltration in the skin.

Representative FACS plots and compiled data of cells isolated from the epidermis (A) or combined epidermis/dermis (B) of Dox/400R TLR9^WT (left column) or TLR9^KO (right column) recipients at 4 weeks after T cell injection. Gating summary: R1, CD45⁺ hematopoietic cells; R2, live KCs; R3, CD45⁺CD4⁺ cells; R4, CD45⁺CD11c⁺ cells; R5, CD4⁺ cells in CD45⁺ gate; R6, CD11b⁺ cells in CD45⁺ gate; R7, inflammatory monocytes in CD45⁺CD11b⁺ gate; R8, Ly6C-Ly6G intermediate cells in CD45⁺CD11b⁺ gate; R9, neutrophils in CD45⁺CD11b⁺ gate. Data are shown as mean ± SEM and are representative of 3 independent experiments with n = 6 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Student’s t test.

Figure 5. FasL-deficient DO11 T cells fail to induce skin lesions.

DO11 or DO11^gld T cell–injected Dox/400R TLR9^WT or TLR9^KO Ii-TGO recipients were evaluated 4 weeks after injection. (A) Clinical appearance (upper row) and skin histology by H&E staining. Original magnification, ×100 (middle row); ×200 magnification (bottom row). Representative images from n = 5 mice per group. (B) Spleen weights. (C) Skin-infiltrating cells, percentages of CD45⁺ cells in total skin cell suspension (left); percentages of CD11b⁺ myeloid cells, CD11b⁺Ly6C⁺ inflammatory monocytes, and CD11b⁺Ly6G⁺ neutrophils (center); and percentages of CD4⁺, CD4⁺KJ126⁺, and KJ126⁺IFN-γ⁺ T cells (right) within the CD45⁺ gate. (D) Autoantibodies detected by HEp2 staining and ANA score. Original magnification, ×200. Data are shown as mean ± SEM and are representative of 2 independent experiments with n = 8 mice per group. *P < 0.01; **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA and 2-way ANOVA with Šidák’s multiple-comparison test.

Figure 6. Gene expression in the skin of mice injected with DO11 or DO11^gld T cells.

Skin biopsies were obtained from TLR9^WT or TLR9^KO Ii-TGO mice injected with DO11 or DO11^gld T cells. (A) Heatmap showing hierarchical clustering of the 750 genes in the NanoString murine cancer immune code set (left) with data presented as log₂-transformed values on a scale of 0 (blue) to 15 (red). Genes above the threshold of mean+2SD of the background were considered for analysis. Enlarged images of gene clusters are designated by arrows (right). Small black arrow denotes the gene downregulated in group 2 compared with group 3. (B) Genes similarly expressed by groups 2 and 3 with a fold change of more than 2.5 relative to group 1 and with P > 0.05 (not significant) between groups 2 and 3. (C) Genes differentially expressed by groups 2 and 3 with fold change of more than 2.5 for group 2 relative to group 1 and P < 0.05 (significant) between groups 2 and 3. Group 1, n = 4; group 2, n = 4; and group 3, n = 3.

Figure 7. Type I IFN blockade abrogates skin disease in TLR9^KO Ii-TGO mice.

(A) Development of skin disease in TLR9^KO mice or TLR9^KO mice treated with anti–IFN-aR mAb or isotype control mAb. (B) Representative images at 5 weeks after T cell injection. (C) Spleen weights of experimental and unmanipulated (no T cells or mAb) controls. (D) Percentages of CD4⁺ and CD4⁺KJ126⁺ cells in CD45⁺ skin cell gate. (E) Representative FACS plots and compiled data showing percentages of CD11c⁺PDCA1⁺ pDCs in CD45⁺CD11b^–CD11c⁺ skin cell gate. Data are shown as mean ± SEM from 2 independent experiments and are representative of n = 6 mice per group. **P < 0.01; ***P < 0.001; ****P < 0.0001, 2-tailed Student’s t test and 2-way ANOVA with Šidák’s multiple-comparison test.

Figure 8. Gene expression in human CLE.

(A) Microarray analysis showing FasL transcript levels in healthy control skin. n =13. CLE patient skin (ACLE, n = 7; SCLE, n = 12; CCLE, n = 6) and psoriasis patient skin (n = 17). ***P < 0.001. Benjamini-Hochberg procedure was used for multiple comparisons. (B) Fold change of gene expression levels, determined by NanoString analysis, of CCLE, SCLE, and psoriasis lesional skin biopsies relative to healthy control skin biopsies. Genes above background (mean + 2SD of negative controls) with fold change of 2.0 or above and P < 0.05 in CCLE relative to healthy controls were selected and compared with genes with fold change greater than 2.5 and P < 0.05 in DO11→TLR9^KO (group 2) mice relative to control DO11→TLR9^WT (group 1) Ii-TGO mice. n = 3 each of CCLE, SCLE, psoriasis subjects, and healthy controls. Benjamini-Yekutieli procedure was used for multiple comparisons.