Epidermal loss of JunB leads to a SLE phenotype due to hyper IL-6 signaling

Abstract

Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting various tissues. Involvement of B and T cells as well as increased cytokine levels have been associated with disease manifestation. Recently, we demonstrated that mice with epidermal loss of JunB (JunB(Deltaep)) develop a myeloproliferative syndrome (MPS) due to high levels of G-CSF which are secreted by JunB-deficient keratinocytes. In addition, we show that JunB(Deltaep) mice develop a SLE phenotype linked to increased epidermal interleukin 6 (IL-6) secretion. Intercrosses with IL-6-deficient mice could rescue the SLE phenotype. Furthermore, we show that JunB binds to the IL-6 promoter and transcriptionally suppresses IL-6. Facial skin biopsies of human SLE patients similarly revealed low JunB protein expression and high IL-6, activated Stat3, Socs-1, and Socs-3 levels within lupus lesions. Thus, keratinocyte-induced IL-6 secretion can cause SLE and systemic autoimmunity. Our results support trials to use alpha-IL-6 receptor antibody therapy for treatment of SLE.

Fig. 1.

SLE-like skin and kidney affections in JunB^Δep mice. A 3-month-old wild-type mouse with normal skin was compared to a JunB^Δep littermate control (A) with ulcerative skin lesions in the face region (B) and rescue of the skin phenotype after crossing to IL-6^−/− mice (C). In indirect immunofluorescence using α-IgG (D–F), JunB^Δep mice (E) (arrowheads) show a prominent “lupus band” in the epidermal-dermal junction, whereas in wild-type (D) and JunB^ΔepIL-6^−/− mice (F), this band is absent. (Scale bar, 50 μm.) The skin of wild-type and JunB^ΔepIL-6^−/− mice show no IL-6 expression, whereas JunB^Δep mice have high IL-6 expression levels (G–I). In the AFOG staining, the glomerular changes are characterized by mesangial hypercellularity with glomerular basement membrane thickening and luminal obstruction by hyaline immune complex deposits in the mutant (arrowheads) (K) compared to wild-type (J) and JunB^ΔepIL-6^−/− (L). (Scale bar, 50 μm.) The macroscopic kidney appearance (M) of 6-month-old wild-type mice compared to small and atrophic kidneys of mutant mice. The kidney size appears to be normal in the JunB^ΔepIL-6^−/− intercross. (Scale bar, 0.3 cm.) Note the normal width of the kidney cortex in wild-type and JunB^ΔepIL-6^−/− mice (arrowheads) compared to the mutant. Albumin ELISA shows increased protein levels in the urine of JunB^Δep mice compared to JunB^f/f controls and JunB^ΔepIL-6^−/− (N). Immunoblot analysis (O) showing increased anti-histone antibody serum levels against the individual histones H1, H2A, H2B, H3, and H4 in JunB^Δep mice: Lanes 1 and 2, 6-month-old JunB^f/f mice; lanes 3 and 4, 6-month-old JunB^Δep mice with high α-histone Ab reactivities similar to the patient in lane 7. Lanes 5 and 6, 6-month-old JunB^ΔepIL-6^−/− mice. Lane 7, SLE patient serum with high α-histone Ab reactivities. JunB^Δep mice show increased titers of α-histone Abs with cumulative age. No anti-histone antibodies can be found in JunB^ΔepIL-6^−/− mice. Line immunoassays using sera of mice were performed. JunB^Δep mice show pronounced α-SmD Abs (lanes 2 and 3), similar to a SLE patient serum (lane 6). No α-SmD antibodies are found in sera of JunB^f/f wild-type mice (lane 1) and JunB^ΔepIL-6^−/− mice (lanes 4 and 5). (P) The survival time of JunB^Δep mice was reduced compared to wild-type mice (Kaplan Meier blot).

Fig. 2.

UV irradiation experiments of JunB^f/f and JunB^Δep mice (A–D). (Scale bar, 50 μm.) JunB^f/f show no α-IgG immunofluorescence with or without UV (A and C). JunB^Δep mice show linear α-IgG-immunofluorescence at the dermo-epidermal junction (arrowheads) (B), which becomes more intense after UV-irradiation (D). Thus, UV-irradiation enhanced severeness of lupus-like skin lesions in JunB^Δep mice on immunofluorescence levels. JunB^Δep mice show dense lymphocytic infiltrates in the peripheral organs kidney, lung, and liver compared to JunB^f/f control mice. These infiltrates are mainly composed of αCD3 positive T-cells (E–J) (arrowheads). (Scale bar, 100 μm.)

Fig. 3.

Schematic overview of known transcription factor binding sites in the IL-6 promoter, indicating the location of the putative AP-1 site (filled circle) and the transcription start site (+1) (A). Arrows indicate the position of the primers used for ChIP. ChIP using an JunB Ab or IgG control Ab reveals binding of JunB to the IL-6 promoter in a region containing a conserved AP-1 binding site in keratinocytes isolated from JunB^fl/fl mice. No binding was detected in JunB^Δep keratinocytes (B). HeLa cells were either cotransfected with pCDNA4_hismax_JUNB (JunB) or pCDNA4_hismaxA (vec) together with either pIL-6 or pGL3 empty vector for control purpose. Twenty-four hours later, luciferase- and β-gal activity (expressed from a routinely cotransfected β-gal expression vector) was measured. Luciferase expression from two replicates, normalized to β-gal expression was plotted, and error bars are given, represent ± SD. Relative normalized luciferase expression was below detection limit from promoterless pGL3-basic plasmid, while intact luciferase expression from pIL-6 was abrogated by ectopic JunB expression from pCDNA4_hismax_JUNB (C).

Fig. 4.

Normal human facial skin (A) compared to SLE patient's skin (B). SLE combines systemic with cutaneous lesions such as the “butterfly” facial lesion, resembling the cardinal feature of human SLE. Compared to normal human skin (C), histology of the patient's skin (D) revealed typical vacuolar alterations of the basal cell layers, a predominant basal membrane, vasculitis of dermal blood vessels, and dermal chronic inflammatory infiltrates consisting mainly of lymphocytes. (Scale bar, 100 μm.) JunB expression is high in normal facial skin (E) when compared to the reduced JunB expression levels in SLE patient's facial skin area (F), which was quantified by automated cell acquisition and quantification software (Histoquest) (G). Immunohistochemistry of SLE patient's skin revealed high protein expression levels of IL-6 (I), IL-6R (L), and pStat3 (O) especially in the basal cell layers, compared to normal facial skin biopsies (H, K, and N, respectively). (Scale bar, 50 μm.) On the right side, the respective protein quantification results of the human patient samples depicted in (J, M, P) using Histoquest software are shown as scattergrams and bar diagrams. The relative protein expression levels compare normal (black dots/bars) versus SLE patients (green dots/bars).