Toll-like receptor-mediated down-regulation of the deubiquitinase cylindromatosis (CYLD) protects macrophages from necroptosis in wild-derived mice

Abstract

Pathogen recognition by the innate immune system initiates the production of proinflammatory cytokines but can also lead to programmed host cell death. Necroptosis, a caspase-independent cell death pathway, can contribute to the host defense against pathogens or cause damage to host tissues. Receptor-interacting protein (RIP1) is a serine/threonine kinase that integrates inflammatory and necroptotic responses. To investigate the mechanisms of RIP1-mediated activation of immune cells, we established a genetic screen on the basis of RIP1-mediated necroptosis in wild-derived MOLF/EiJ mice, which diverged from classical laboratory mice over a million years ago. When compared with C57BL/6, MOLF/EiJ macrophages were resistant to RIP1-mediated necroptosis induced by Toll-like receptors. Using a forward genetic approach in a backcross panel of mice, we identified cylindromatosis (CYLD), a deubiquitinase known to act directly on RIP1 and promote necroptosis in TNF receptor signaling, as the gene conferring the trait. We demonstrate that CYLD is required for Toll-like receptor-induced necroptosis and describe a novel mechanism by which CYLD is down-regulated at the transcriptional level in MOLF/EiJ macrophages to confer protection from necroptosis.

Keywords: Deubiquitination; Forward Genetic Mapping; Gene Regulation; Mouse Genetics; Necrosis (Necrotic Death); Toll-like Receptor (TLR); Wild-derived Mice.

FIGURE 1.

TLR-induced necroptosis in macrophages can be cell-intrinsic or require TNFR1. Shown are B6 (A), TNFR1^−/− (B), or IL1R1^−/− (C) peritoneal macrophages stimulated for 18 h with lipoteichoic acid (LTA) (2 μg/ml), IMQ (5 μg/ml), CL-097 (1 μg/ml), CpG (200 nm), polyI:C (10 μg/ml), or LPS (100 ng/ml) in the presence of zVAD (12.5 μm) with or without the RIP1 kinase inhibitor Nec-1 (30 μm). Immortalized macrophage cell lines lacking RIP1 (D) or RIP3 (E) were stimulated with TLR agonists in the presence of zVAD (Z) (100 or 50 μm, respectively) with or without Nec-1 (N). Cell viability was measured by ATP levels using the CellTiter-Glo assay (Promega). Values are a percentage of viable treated cells relative to cells in medium with vehicle (DMSO), shown as mean ± S.E. Shown is one representative experiment of at least three independent experiments. Statistical significance was determined by two-tailed Student's t test. *, p < 0.05; **, p < 0.001; ***, p < 0.0001; ns, not significant.

FIGURE 2.

MOLF peritoneal macrophages are resistant to TLR-induced necroptosis. A, B6 and MOLF peritoneal macrophages stimulated for 18 h with LPS (100 ng/ml) or IMQ (5 μg/ml) with and without zVAD (Z) (12.5 μm) and Nec-1 (N) (30 μm). B, B6, F1 (B6xMOLF), and MOLF peritoneal macrophages stimulated with IMQ and zVAD. Cell viability was measured relative to cells kept in medium with vehicle (DMSO). C, B6 and MOLF bone marrow-derived macrophages were stimulated with LPS (10 ng/ml) with and without zVAD (25 μm) and with or without Nec-1. Unstim, unstimulated. TNF production was measured by ELISA after 18 h. Shown are means for biological triplicates in a representative experiment. Error bars are mean ± S.E. (A and B) or S.D. (C). Data are representative of at least three independent experiments. Statistical significance was determined by two-tailed Student's t test. *, p < 0.05; **, p < 0.001; ***, p < 0.0001; ns, not significant.

FIGURE 3.

Resistance to necroptosis in MOLF peritoneal macrophages is linked to Cyld. A, viability of peritoneal macrophages from 67 N2 mice after stimulation with IMQ+zVAD grouped by genotype at D8Mit51 (marker within the peak of highest linkage). B, NOD2^−/− and B6 (WT) peritoneal macrophages were stimulated for 18 h with IMQ (5 μg/ml) or LPS (100 ng/ml) and zVAD (Z) (12.5 μm) ± Nec-1 (N) (30 μm). Viability was measured by CellTiter-Glo ATP assay. C, B6 and MOLF peritoneal macrophages stimulated for 8 h with IMQ (5 μg/ml), IMQ+zVAD (12.5 μm), or IMQ+zVAD+Nec-1 (30 μm). TNF production in supernatants was measured by ELISA. Unstim, unstimulated. D, B6 and MOLF peritoneal macrophages stimulated for 18 h with LPS (100 ng/ml) or IMQ (5 μg/ml) in the presence or absence of the SM164 (SM) (100 nm), zVAD (12.5 μm), and Nec-1 (30 μm). E, peritoneal macrophages from 20 F2 mice were stimulated for 4 h with LPS (100 ng/ml). CYLD mRNA levels were measured by qPCR. Samples from each mouse were sorted by genotype at D8Mit51. Error bars are mean ± S.E. (B and D) or S.D. (C). Data from two (D) or three (B and C) independent experiments were pooled, with statistical significance determined by two-tailed Student's t test. *, p < 0.05; ***, p < 0.0001; ns, not significant.

FIGURE 4.

CYLD promotes TLR-induced necroptosis. A, CYLD siRNA and control (NC) siRNA were transfected into peritoneal macrophages, followed by stimulation with LPS or IMQ and zVAD (Z). Knockdown efficiency was assessed by Western blotting for CYLD relative to GAPDH in unstimulated cells (B) and by qPCR after 4 h of LPS stimulation (C). D, RAW264.7 macrophages were infected with lentivirus containing full-length (FL) CYLD or an empty vector and stimulated with IMQ or LPS and zVAD (50 μm) ± Nec-1 (N) (30 μm). E, overexpression level of FL-CYLD measured by qPCR relative to empty vector. F, TNFR^−/− BMDM infected with lentivirus containing short hairpin RNA targeting GFP or CYLD stimulated with LPS (100 ng/ml) or polyI:C (200 μg/ml) with zVAD (25 μm). G, Western blotting for CYLD protein expression for samples in F. For all experiments, cell viability was measured at 18 h relative to cells kept in medium with vehicle (DMSO). Data shown are the mean ± S.E. pooled from six (A), two (D), or three (F) pooled, independent experiments. Statistical significance was determined by two-tailed Student's t test. *, p < 0.05; ***, p < 0.0001.

FIGURE 5.

CYLD is down-regulated by TLR stimulation in MOLF peritoneal macrophages. A–D, RNA extracted from B6 and MOLF peritoneal macrophages. A, CYLD-specific PCR and sequencing of clones obtained from 5′ rapid amplification of cDNA ends shows full-length (FL) and splice (SP) CYLD isoforms in unstimulated (UN) or 4-h LPS-stimulated (100 ng/ml) cells. B, ribonuclease protection assay using CYLD-specific (top row) and GAPDH-specific (bottom row) probes on RNA samples from macrophages stimulated for 1, 2, and 4 h with LPS (100 ng/ml). Shown is the relative amount (Rel Amt) of CYLD/GAPDH in each sample as determined by densitometry. C, quantitative RT-PCR was performed using a full-length, CYLD-specific TaqMan probe after LPS stimulation (4 h). Values are presented as relative units (RU) normalized to GAPDH. D, Northern blot on RNA samples of macrophages stimulated for 4 h with LPS (100 ng/ml) or IMQ (5 μg/ml) using a CYLD-specific probe. 28 S rRNA ethidium bromide staining was the loading control. E, B6 and MOLF peritoneal macrophages unstimulated or stimulated with LPS with zVAD (LPS+Z) for 6 h. Lysates were analyzed by Western blot analysis for CYLD protein levels with GAPDH as a loading control. Densitometry measured the fold change of the CYLD/GAPDH ratio relative to unstimulated levels within each mouse strain. F, LPS-stimulated peritoneal macrophages were cross-linked and lysed, and DNA was sheared by sonication. RNA polymerase II ChIP was performed. Quantitative real-time PCR was performed at locations in the Cyld promoter indicated in reference to the TSS. Shown is the mean ± S.E. (C and F) of pooled data from three independent experiments (C) or from one representative experiment (F) (of two independent experiments). Statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.001.

FIGURE 6.

CYLD expression and susceptibility to necroptosis are restored in MOLF BMDM. A, B6 and MOLF BMDM stimulated with LPS (100 ng/ml) or IMQ (5 μg/ml) and zVAD (Z) (25 μm) for 18 h. B, quantitative real-time PCR performed on cDNA reverse-transcribed total RNA from unstimulated (Unstim) and LPS-stimulated B6 and MOLF BMDM. The full-length (FL) CYLD/GAPDH ratio is presented as relative amounts in comparison to B6 unstimulated cells. C, BMDM infected with lentivirus containing siGFP or siCYLD hairpin stimulated with LPS (100 ng/ml) and zVAD (25 μm) for 18 h. D, relative expression of FL-CYLD as measured by qPCR in B6 and MOLF BMDM stimulated for 4 h with LPS. E, CYLD protein expression in B6 and MOLF BMDM measured by Western blot analysis in unstimulated cells. GAPDH was used as a loading control. A and C, cell viability was measured relative to cells in medium with vehicle (DMSO). Data are mean ± S.E. from one representative experiment of three independent experiments. Statistical significance was determined by Student's t test. *, p < 0.05; ***, p < 0.0001; ns, not significant.