Caspase-8 acts as a molecular rheostat to limit RIPK1- and MyD88-mediated dendritic cell activation

Abstract

Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death-independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8-deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type-specific manner and acts uniquely in DCs to maintain tolerance.

Figure 1. Mice with DC-specific deletion of caspase-8 exhibit systemic autoimmunity

(A–I) 8-month-old female Casp8^fl/fl (control) and Cre^CD11cCasp8^fl/fl mice (n≥10) were evaluated for systemic autoimmune disease phenotypes. (A–B) Splenomegaly. (A and C) Lymphadenopathy. (D) Number of splenocytes. (E) PAS-stained formalin-fixed kidney sections and αIgG-FITC-stained frozen kidney sections. (F) Kidney score. (G) Proteinuria. Serum levels of (H) chromatin, dsDNA- and ssDNA-reactive IgG antibodies, (I) pathogenic IgG2a antibodies and (J) cytokines and chemokines. Data are represented as mean ± SD and compared by Mann Whitney test: *, p<0.05; **, p<0.005; ***, p<0.0005. (K) Survival curve; compared using log-rank Mantel Cox test: **, p<0.005.

Figure 2. Inflammation related to DC-specific caspase-8 deficiency is independent of DC survival

Casp8^fl/fl (control) and Cre^CD11cCasp8^fl/fl (A) BMDCs (n=4) and (B–C) total splenocytes (n=3) were stimulated with superFasL ± necrostatin-1 (Nec-1) for 10 hours and stained with Annexin-V and Aqua live/dead. Total splenocytes were gated into (B) CD11c⁺ and (C) CD4/8⁺ populations for analysis. Additionally, control and Cre^CD11cCasp8^fl/fl BMDCs were stimulated with (D) etoposide for 10 hours and stained with Annexin-V and Aqua live/dead. Data are represented as the percent live compared to unstimulated. (E–F) CD4⁺ T-cells (n=3) stimulated for 72 hours with αCD3 and αCD28 ± pan-caspase inhibitor zVAD-FMK (zVAD) and Nec-1 were stained with Annexin-V and Aqua live/dead. Data are represented as the percent live compared to αCD3/28 alone. (G–H) Control and Cre^CD11cCasp8^fl/fl mice (n=4) injected with BrdU for 3 days were evaluated for percent splenic BrdU⁺ (L) CD11c⁺CD8⁻ and (M) CD11c⁺CD8⁺ conventional DCs. (I–M) Mice reconstituted with equal portions of B6.CD45.1/2 (WT) and either control or Cre^CD11cCasp8^fl/fl bone marrow (n=5). (J) Representative Lin⁻Sca-1⁺c-kit⁺ bone marrow cell percentages from 3-month-old female control, Cre^CD11cCasp8^fl/fl and WT mice. Chimeric mice were evaluated 3 months post-transfer for (K) numbers of conventional DCs, (L) distribution of WT (45.1/2) and control or Cre^CD11cCasp8^fl/fl (45.2) derived conventional DCs, (M) splenomegaly and (N) lymphadenopathy. Data are represented as mean ± SD and compared by Mann Whitney test: *, p<0.05; **, p<0.005; ***, p<0.0005.

Figure 3. RIPK3 knockout cannot reverse the consequences of DC-specific caspase-8 deletion

(A–I) 6-month-old female Casp8^fl/fl (control), Cre^CD11cCasp8^fl/fl and RIPK3^−/−Cre^CD11cCasp8^fl/fl mice (n≥5) were evaluated for systemic autoimmune disease phenotypes. (A) Splenomegaly. (B) Lymphadenopathy. (C) Number of splenocytes. (D) PAS-stained formalin-fixed kidney sections and αIgG-FITC-stained frozen kidney sections. (E) Kidney score. (F) Proteinuria. Serum was evaluated for levels of (G) chromatin, dsDNA- and ssDNA-reactive IgG antibodies, (H) pathogenic IgG2a antibodies and (I) cytokines and chemokines. Data are represented as mean ± SD and compared by Mann Whitney test. * denotes comparison between control and Cre^CD11cCasp8^fl/fl, # denotes comparison between Cre^CD11cCasp8^fl/fl and RIPK3^−/−Cre^CD11cCasp8^fl/fl. *, #:p<0.05; **, ##:p<0.005; ***, ###:p<0.0005.

Figure 4. Deletion of IRF3 exacerbates the systemic inflammation in Cre^CD11cCasp8^fl/fl mice

(A) Casp8^fl/fl (control) and Cre^CD11cCasp8^fl/fl BMDCs were stimulated with CpG, imiquimod or LPS for 6 hours and isolated nuclear lysates subjected to a multi-analyte transcription factor bead-based assay, represented as the fold change over unstimulated. (B) Control and Cre^CD11cCasp8^fl/fl BMDCs were stimulated with imiquimod and isolated total cellular lysates subjected to immunoblot analysis for total IRF3. The blot was then stripped for total IRF7 and GAPDH expression and the figures have been cropped and placed together. (C–K) 7-month-old female Casp8^fl/fl (control), Cre^CD11cCasp8^fl/flIRF3^−/−Cre^CD11cCasp8^fl/fl and IRF7^−/−Cre^CD11cCasp8^fl/fl mice (n≥5) were evaluated for systemic autoimmune disease phenotypes. (C) Splenomegaly. (D) Lymphadenopathy. (E) Number of splenocytes. (F) PAS-stained formalin-fixed kidney sections and αIgG-FITC-stained frozen kidney sections. (G) Kidney score. (H) Proteinuria. Serum was evaluated for levels of (I) chromatin, dsDNA- and ssDNA-reactive IgG antibodies, (J) pathogenic IgG2a antibodies and (K) cytokines and chemokines. Data are represented as mean ± SD and compared by Mann Whitney test. * denotes comparison between control and Cre^CD11cCasp8^fl/fl, # denotes comparison between Cre^CD11cCasp8^fl/fl and IRF3^−/−Cre^CD11cCasp8^fl/fl or IRF7^−/−Cre^CD11cCasp8^fl/fl. *, #:p<0.05; **, ##:p<0.005; ***, ###:p<0.0005.

Figure 5. Caspase-8-deficient DCs are hyper-responsive to TLR activation in a RIPK1-dependent manner

(A) GM-CSF + Flt3-L-treated BMDCs from Casp8^fl/fl (control) and Cre^CD11cCasp8^fl/fl mice were stimulated with imiquimod ± Nec-1 and/or zIETD-FMK (zIETD) and/or 1-Methyl-D-tryptophan (1-MT) for 6 hours ± ATP (5mM) and supernatants evaluated for IL-12/IL-23p40, IL-6, TNFα, and IL-1β. (B–C) 3-month-old control and Cre^CD11cCasp8^fl/fl mice (n=4) injected with imiquimod (200 µg/mouse) or PBS were evaluated 4 hours later for splenic CD11c⁺CD8⁻ conventional DC CD86, MHCII, and CD40 expression, represented as the fold change over PBS injection. Data are represented as mean ± SD and compared by Mann Whitney test: *, p<0.05; **, p<0.005; ***, p<0.0005.

Figure 6. Caspase-8 suppresses MyD88 signaling

(A–C) 3-week-old Casp8^fl/fl (control) and Cre^CD11cCasp8^fl/fl (n=4) mice treated with oral antibiotics for 8 weeks were evaluated for (A) splenomegaly and (B) lymphadenopathy. (C–K) 8-month-old female control, Cre^CD11cCasp8^fl/fl and MyD88^fl/flCre^CD11cCasp8^fl/fl mice (n≥4) were evaluated for systemic autoimmune disease phenotypes. (C) Splenomegaly. (D) Lymphadenopathy. (E) Number of splenocytes. (F) PAS-stained formalin-fixed kidney sections and αIgG-FITC-stained frozen kidney sections. (G) Kidney score. (H) Proteinuria. Serum was evaluated for levels of (I) chromatin, dsDNA- and ssDNA-reactive IgG antibodies, (J) pathogenic IgG2a antibodies and (K) cytokines and chemokines. Data are represented as mean ± SD and compared by Mann Whitney test. * denotes comparison between control and Cre^CD11cCasp8^fl/fl, # denotes comparison between Cre^CD11cCasp8^fl/fl and MyD88^fl/flCre^CD11cCasp8^fl/fl. *, #:p<0.05; **, ##:p<0.005; ***, ###:p<0.0005.

Figure 7. Caspase-8-deficient CD11c⁺CD8 conventional DCs express increased activation markers and confer a hyperactive phenotype on lymphocytes

(A–G) Splenocytes from 6-8-month-old female Casp8^fl/fl (control), Cre^CD11cCasp8^fl/flRIPK3^−/−Cre^CD11cCasp8^fl/flIRF3^−/−Cre^CD11cCasp8^fl/flIRF7^−/−Cre^CD11cCasp8^fl/fl and MyD88^fl/flCre^CD11cCasp8^fl/fl mice (n≥4) were analyzed by flow cytometry. (A) CD4⁺ and (B) CD8⁺ T-cell numbers. (C) Representative naïve (CD44⁻CD62L⁺) and activated (CD44⁺CD62L⁻) T-cell percentages of total CD4⁺ and CD8⁺ populations. (D) Total B-cell (B220⁺) numbers and subsets: follicular (FO, CD19⁺CD21/35⁺CD23⁺), marginal zone (MZ, CD19⁺CD21/35⁺CD23^low), transitional 2 (T2, B220⁺AA4.1⁺CD23⁺), transitional 1 (T1, B220⁺AA4.1⁺CD23⁻), plasmablasts (PB, CD19⁺B220^lowCD138⁺CD21/35⁻CD23⁻). (E) B-cell IgD, CD80, CD86 and PD-1 expression. (F) Conventional (CD11c⁺CD8⁻ and CD11c⁺CD8⁻) and plasmacytoid (CD11c^intermediatePDCA-1⁺B220⁺) DC numbers. (G) CD11c⁺CD8⁻ conventional DC CD80 and CD86 expression. (H) Bead-separated CD11c⁺ cells pulsed with OVA were co-cultured with OT-II/RAG^−/− CD4⁺ T-cells. Data are represented as mean ± SD and compared by Mann Whitney test. * denotes comparison between control and Cre^CD11cCasp8^fl/fl, # denotes comparison between Cre^CD11cCasp8^fl/fl and experimental knockouts. *, #:p<0.05; **, ##:p<0.005; ***, ###:p<0.0005.