Interferon lambda promotes immune dysregulation and tissue inflammation in TLR7-induced lupus

Abstract

Type III IFN lambdas (IFN-λ) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, has not been determined. Here, we identify a nonredundant role for IFN-λ in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-λ protein is increased in murine lupus and IFN-λ receptor (Ifnlr1) deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys without effects on autoantibody production. Single-cell RNA sequencing in mouse spleen and human peripheral blood revealed that only mouse neutrophils and human B cells are directly responsive to this cytokine. Rather, IFN-λ activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and promote tissue inflammation. These data provide insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in this disease.

Keywords: autoimmunity; inflammation; interferon lambda; lupus; skin.

Fig. 1.

IFN-λ is elevated in murine lupus. (A) IFN gene expression in healthy donor PBMCs. Cells were stimulated with the TLR7 agonist IMQ for 4 h and gene expression was quantified by qPCR (n = 4/group). (B) Schematic diagram of the IMQ-induced murine lupus model. (C) IFN-λ2/3 protein in murine lupus serum. Cytokine concentrations were measured by ELISA in mouse serum after 5 wk of IMQ treatment (n = 8 untreated, n = 16 treated). (D) Immunofluorescent staining for pDCs in murine lupus skin. TLR7 (green) and Siglec H (red) were detected in ear skin tissue after 5 wk of IMQ treatment. Tissue was counterstained with Hoechst (blue). (E) TLR7 expression in murine lupus skin. Gene expression was measured in ear tissue after 5 wk of IMQ treatment by qPCR (n = 4 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). (F) IFN-λ2/3 production by pDCs. Mouse pDCs were isolated from splenocytes by MACS column and treated with 5 μg/mL IMQ for 24 h (n = 4 untreated, n = 5 IMQ). IFN-λ2/3 protein was measured by ELISA in culture supernatants. Optical density (OD) values were blank corrected and normalized to untreated samples. Data are represented as mean ± SEM. Statistics were calculated by nonparametric Mann–Whitney U test or one-way ANOVA with Sidak correction for multiple comparisons. *P < 0.05, **P < 0.01; ns, not significant. (Scale bars in D: 25 μm.)

Fig. 2.

IFN-λ promotes systemic immune dysregulation in murine lupus. (A) Splenomegaly in murine lupus. Spleens were collected after 5 wk of IMQ treatment and measured as percentage of total body weight (n = 4 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). (B) Leukocytosis, (C) anemia, and (D) thrombocytopenia in murine lupus. White blood cells (WBCs), hemoglobin, and platelets were quantified in peripheral blood after 5 wk of IMQ treatment (n = 3 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). (E) IFN-λ response and (F) IFN-α response in WT and Ifnlr1^−/− mice. Splenocytes were stimulated with 100 ng/mL IFN-λ2 or 20 ng/mL IFN-α for 6 h. IRF7 expression was quantified by qPCR (n = 4/group). Data are represented as mean ± SEM. Statistics were calculated by nonparametric Mann–Whitney U test or two-way ANOVA with Sidak correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Fig. 3.

IFN-λ promotes myeloid expansion and T cell activation. (A) Neutrophils, (B) monocytes, and (C) conventional dendritic cells in murine lupus. Cells were analyzed in spleen after 5 wk of IMQ treatment (n = 4 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). (D) NETs in serum and (E) skin tissue in murine lupus. NETs were detected in serum by ELISA for neutrophil elastase–DNA complexes. NETs were detected in ear skin tissue by immunofluorescent staining for citrullinated histone H3 (red) and DNA (Hoechst). (F) Representative gating strategy for T cell activation. (G) Naïve CD8⁺, (H) CD8⁺ effector memory, (I) naïve CD4, and (J) CD4⁺ effector memory cells in murine lupus. T cell subsets were identified based on surface expression of CD62L and CD44 (n = 4 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). Data are represented as mean ± SEM. Statistics were calculated by nonparametric Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (Scale bars in E: 50 μm.)

Fig. 4.

IFN-λ is not required for B cell activation or autoantibody production. (A) Plasma cells and (B) naïve B cells in murine lupus. B cells were analyzed in spleen after 5 wk of IMQ treatment (n = 4 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). (C–E) Activation status of B cells in murine lupus. Mean fluorescent intensity of activation markers on CD19⁺ CD11b⁻ B cells (n = 4 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). (F) Total IgG, (G) anti-dsDNA IgG, and (H) antinuclear antibodies in murine lupus. Antibodies were quantified in mouse serum by ELISA or immunofluorescence on HEp-2 cells after 5 wk of IMQ treatment (n = 4 WT, n = 4 Ifnlr1^−/−, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). Data are represented as mean ± SEM. Statistics were calculated by nonparametric Mann–Whitney U test. *P < 0.05; ns, not significant. (Scale bar in H: 100 μm.)

Fig. 5.

Immune cells are selectively responsive to IFN-λ. (A) UMAP clustering of WT mouse spleen cells treated with 100 ng/mL of IFN-λ2 or IFN-α for 4 h (n = 18,520 cells). (B) IFN-stimulated gene expression in untreated and IFN-treated mouse cell clusters. (C) UMAP clustering of human whole blood cells treated with 100 ng/mL of IFN-λ1 or IFN-α for 4 h (n = 19,266 cells). (D) IFN-stimulated gene expression in untreated and IFN-treated human cell clusters. Data are represented as notched boxplots (median with interquartile range; notch represents 95% confidence interval of the median). Statistics were calculated by one-way ANOVA with Tukey’s honest significant differences. *P < 0.005.

Fig. 6.

IFN-λ promotes skin inflammation. (A) H&E staining and (B) pathology scoring of ear skin in murine lupus. Tissue sections were prepared after 5 wk of IMQ treatment (n = 8 untreated, n = 8 WT + IMQ, n = 7 Ifnlr1^−/− + IMQ). (C–F) Inflammatory cytokine and chemokine expression in murine lupus skin. Gene expression was measured in ear tissue after 5 wk of IMQ treatment by qPCR (n = 8 untreated, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). Data were normalized to untreated mice. Data are represented as mean ± SEM. Statistics were calculated by one-way ANOVA with Tukey’s correction for multiple comparisons or nonparametric Mann–Whitney U test. *P < 0.05, ****P < 0.0001; ns, not significant.

Fig. 7.

IFN-λ induces chemokine expression and immune cell recruitment by keratinocytes. (A) Primary murine keratinocytes were isolated from neonate mice and treated with IFN-λ2 at the indicated concentrations (ng/mL) for 6 h. RSAD2 gene expression was measured by qPCR (n = 4/group). (B and C) HaCaT keratinocytes were treated with 20 ng/mL of IFN-λ1 or IFN-α. IFN-stimulated gene expression was measured at the indicated timepoints by qPCR (n = 4/group). (D–F) CXCL chemokine gene expression in HaCaT cells after 24 h of stimulation with IFN-λ1 or IFN-α (n = 4/group). (G) PBMC migration assay: 500,000 healthy donor PBMCs were seeded in the top chamber of a Transwell (Inset) and incubated with culture supernatants from keratinocytes treated with IFN for 48 h (n = 4/group). Cells migrated into the bottom chamber were counted after 24 h in the Transwell culture. Data are represented as mean ± SEM or median ± min/max (boxplots). Statistics were calculated by one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8.

IFN-λ promotes lupus-associated renal pathology and activates mesangial cells. (A) Immunofluorescent staining for immune complex deposition in murine lupus kidneys. Renal tissue was prepared after 5 wk of IMQ treatment. (B) IgG and (C) C3 deposition were quantified as fluorescent intensity within individual glomeruli and normalized to Hoechst staining. A total of 10 to 15 glomeruli were scored for each mouse and averaged (n = 4 untreated, n = 8 WT + IMQ, n = 7 Ifnlr1^−/− + IMQ). (D) PAS staining and (E) pathology scoring of murine lupus kidney sections (n = 8 untreated, n = 7 WT + IMQ, n = 7 Ifnlr1^−/− + IMQ). (F) IFN-stimulated gene expression in kidney tissue. Gene expression was determined by qPCR and normalized to untreated mice (n = 8 untreated, n = 16 WT + IMQ, n = 12 Ifnlr1^−/− + IMQ). (G and H) IFN-stimulated gene expression and (I–K) CXCL chemokine expression in cultured mouse Mes13 mesangial cells. Mes13 cells were treated with 20 ng/mL IFN-λ2 or IFN-α for 24 h (n = 4/group) and gene expression was determined by qPCR. Data are represented as mean ± SEM or median + min/max (boxplots). Statistics were calculated by one-way ANOVA with Tukey’s correction for multiple comparisons or nonparametric Mann–Whitney U test. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant.