The NF-κB regulator Bcl-3 modulates inflammation during contact hypersensitivity reactions in radioresistant cells

Abstract

Bcl-3 is an atypical member of the IκB family. Bcl-3 functions as a cofactor of p50/NF-κB1 or p52/NF-κB2 homodimers in nuclei, where it modulates NF-κB-regulated transcription in a context-dependent way. Bcl-3 has tumorigenic potential, is critical in host defense of pathogens, and has been reported to ameliorate or exacerbate inflammation, depending on disease model. However, cell-specific functions of Bcl-3 remain largely unknown. Here, we explored the role of Bcl-3 in a contact hypersensitivity (CHS) mouse model, which depends on the interplay between keratinocytes and immune cells. Bcl-3-deficient mice exhibited an exacerbated and prolonged CHS response to oxazolone. Increased inflammation correlated with higher production of chemokines CXCL2, CXCL9, and CXCL10, and consequently increased recruitment of neutrophils and CD8(+) T cells. BM chimera experiments indicated that the ability of Bcl-3 to reduce the CHS response depended on Bcl-3 activity in radioresistant cells. Specific ablation of Bcl-3 in keratinocytes resulted in increased production of CXCL9 and CXCL10 and sustained recruitment of specifically CD8(+) T cells. These findings identify Bcl-3 as a critical player during the later stage of the CHS reaction to limit inflammation via actions in radioresistant cells, including keratinocytes.

Keywords: Bcl-3; Chemokines; Contact hypersensitivity; Keratinocytes; NF-κB.

Figure 1

Exaggerated CHS response in Bcl-3-deficient mice. (A) WT and Bcl-3^−/− mice were sensitized on abdomens to 25 μl of 100 μg/ml oxazolone (OXA) on two consecutive days and challenged with 5 μl of 10 μg/ml OXA or solvent on ears 5 days later. Increases in ear thickness were measured over the next 4 days. Data are shown as mean ± SEM (n=14 mice/group) and are pooled from three independent experiments. (B) WT and Bcl-3^−/− mice were sensitized and challenged with DNFB and analyzed as in (A). Data are shown as mean ± SEM (n=10 mice/group) and are pooled from two independent experiments. (C) H&E-stained sections of ears from OXA-challenged and unchallenged WT and Bcl-3^−/− mice. Images were captured by light microscopy and are representative of the three independent experiments performed in (A). Original magnification 20X, scale bar 100 μm. *p < 0.05, ***p < 0.001, Student's two-tailed t-test.

Figure 2

Increased cell recruitment in Bcl3^−/− mice during CHS response. (A) WT and Bcl-3^−/− mice were sensitized to and challenged with OXA as in Figure 1A. 96 h after challenge, single cell suspensions were prepared from ear and cells stained with antibodies to CD8, Ly6G and Gr1 to identify infiltrating CD8⁺ T cells, neutrophils and neutrophils/monocytes, respectively. Data are shown as mean + SEM (n=5 mice/group) and this experiment is representative of three independent experiments. (B) Mice were treated as in (A), and frozen ear sections were prepared 48 h and 96 h after challenge, stained with biotin-conjugated anti-Gr-1 and anti-Ly6G antibodies and visualized with streptavidin-conjugated Alexa Fluor 568; formalin fixed sections were stained with biotin-conjugated anti-Ly6B antibodies (primarily stain monocytes/neutrophils) and visualized with streptavidin-conjugated HRP/with DAB substrate. Scale bar 100μm. Data are representative of ten mice/group analyzed, taken from three independent experiments. *p < 0.05, **p < 0.01, Student's two-tailed t-test.

Figure 3

Increase in IFN-γ-producing CD8⁺ T cells during CHS in Bcl-3^−/− mice. (A) WT and Bcl-3^−/− were sensitized to and challenged with OXA as in Figure 1A. 96 h after challenge, cells from skin draining lymph nodes were isolated, and analyzed for intracellular IFN-γ production after gating on CD8⁺ T cells. Representative FACS analysis is shown in the upper panels and data summarized in bottom panels. Data are shown as mean + SEM (n=4 mice/group) and this experiment is representative of two independent experiments. **p < 0.01, Student's two-tailed t-test.

Figure 4

Increased chemokine expression in Bcl-3^−/− mice during CHS response. (A and B) WT and Bcl-3^−/− were sensitized to and challenged with OXA as in Figure 1A. Ears were harvested and analyzed for relative mRNA expression for indicated genes with qPCR at 48 h (A) and 96 h (B) after challenge. Data shown as mean +SEM (n = 7-10/group) and are pooled from two experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student's two-tailed t-test.

Figure 5

Lack of Bcl-3 expression in radioresistant cells results in exaggerated CHS response. (A-C) WT or Bcl3^−/− mice were lethally irradiated and reconstituted for about 8 weeks with 2 × 10⁶ WT or Bcl3^−/− bone marrow cells, allowing for all possible combinations, as indicated. Reconstituted chimeric mice were sensitized to and challenged with OXA (or solvent) and analyzed for increased ear thickness as in Figure 1A. For ease of comparison, all panels (A-C) show the results obtained with WT recipients after adoptive transfer of WT bone marrow. In addition, (A) also shows WT mice reconstituted with Bcl3^−/− bone marrow; (B) Bcl3^−/− mice reconstituted with WT bone marrow; and (C) Bcl3^−/− mice reconstituted with Bcl3^−/− bone marrow. Data are shown as mean + SEM (n=10 mice/group (for each panel)) and are pooled from two experiments. *p < 0.05, **p < 0.01, Student's two-tailed t-test.

Figure 6

Increased expression of chemokines and CD8⁺ T-cell recruitment in mice with keratinocyte-specific ablation of Bcl-3. (A) Mice ablated of Bcl-3 in Langerhans cells (Bcl-3-Δ-LC (Bcl3^flx/flx; Langerin-Cre)) and control mice (CTR; Bcl3^flx/flx) were sensitized to and challenged with OXA and analyzed for increase in ear thickness as in Figure 1A. (B) Single cell suspensions from ears of mice treated as in (A) were analyzed by flow cytometry 96 h after challenge for expression of CD8, Ly6G and Gr1 to identify infiltrating CD8⁺ T cells, neutrophils and monocytes/neutrophils, respectively. (C) Mice ablated of Bcl-3 in keratinocytes (Bcl-3-Δ-KC [Bcl3^flx/−; K5-Cre]) and control mice (CTR; Bcl3^flx/−) mice were treated and analyzed as in (A). (D) Single cell suspensions from ears of mice treated as in (C) were analyzed 96 h after challenge for expression of CD8, Ly6G and Gr1 as in (B). (E) Relative mRNA expression for indicated genes in ears of mice as (D). (A-E) Data shown as mean + SEM ((A) n=12, (B,D) n=10-12, (C) n=14, (E) n=8 mice/group; each pooled from two independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, Student's two-tailed t-test.