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Figure 1. A kindred with family members bearing a <t>CARD8</t> mutation and CD-like intestinal inflammation. (A) A kindred containing 3 members bearing a CARD8 mutation. In all 3 members of the kindred, the proband (Pt), his mother (Pt M), and his maternal aunt (Pt Aunt), the mutation occurred in associa- tion with a CD-like intestinal inflammation. (B) Macroscopic examination of the colon exhibited scattered areas of superficial erythema and ulceration hav- ing a lenticular pattern especially evident in the rectosigmoid region. n = 3/group. (C–I) Biopsies from the terminal ileum and colon. (C) Index patient colon. Colitis with epithelial erosive changes and inflammation, significant crypt and goblet cell loss with regenerative changes. Features consistent with GvHD. (D) Index patient colon. Colitis with rare residual gland showing goblet cell loss, repair changes, and rare apoptotic bodies (arrow). (E) Index patient terminal ileum. Ileitis with focal erosion, villi loss, lymphocytic infiltrates, severe crypt drop-out, and repair changes. (F) Index patient terminal ileum. Chronic active ileitis with regenerative changes and poorly formed granulomas including giant cells. (G) Index patient terminal ileum. Poorly formed granuloma (arrow) with giant cells. Adjacent glands with repair/regenerative changes. (H) Aunt terminal ileum. Transmural lymphocyte infiltration with well-formed granuloma present. (I) Aunt terminal ileum. Well-formed granuloma (arrow). n = 3/group. Original magnification, ×4 (C, E, H); ×10 (F, I); ×20 (D, G). Parts G and I show higher magnification of boxed areas in F and H, respectively. (J) Anakinra therapy resulted in rapid clinical improvement marked by decreased fecal calprotec- tin levels. Data are representative of 3 independent experiments. (K) Whole exome sequencing revealed a CARD8 V44I mutation in 1 allele of chromosome 19 (see sequencing data in the text). The mutation site of V44I was present on the CARD8 T60 isoform, but not the “canonical” T48 isoform.
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Figure 1. A kindred with family members bearing a CARD8 mutation and CD-like intestinal inflammation. (A) A kindred containing 3 members bearing a CARD8 mutation. In all 3 members of the kindred, the proband (Pt), his mother (Pt M), and his maternal aunt (Pt Aunt), the mutation occurred in associa- tion with a CD-like intestinal inflammation. (B) Macroscopic examination of the colon exhibited scattered areas of superficial erythema and ulceration hav- ing a lenticular pattern especially evident in the rectosigmoid region. n = 3/group. (C–I) Biopsies from the terminal ileum and colon. (C) Index patient colon. Colitis with epithelial erosive changes and inflammation, significant crypt and goblet cell loss with regenerative changes. Features consistent with GvHD. (D) Index patient colon. Colitis with rare residual gland showing goblet cell loss, repair changes, and rare apoptotic bodies (arrow). (E) Index patient terminal ileum. Ileitis with focal erosion, villi loss, lymphocytic infiltrates, severe crypt drop-out, and repair changes. (F) Index patient terminal ileum. Chronic active ileitis with regenerative changes and poorly formed granulomas including giant cells. (G) Index patient terminal ileum. Poorly formed granuloma (arrow) with giant cells. Adjacent glands with repair/regenerative changes. (H) Aunt terminal ileum. Transmural lymphocyte infiltration with well-formed granuloma present. (I) Aunt terminal ileum. Well-formed granuloma (arrow). n = 3/group. Original magnification, ×4 (C, E, H); ×10 (F, I); ×20 (D, G). Parts G and I show higher magnification of boxed areas in F and H, respectively. (J) Anakinra therapy resulted in rapid clinical improvement marked by decreased fecal calprotec- tin levels. Data are representative of 3 independent experiments. (K) Whole exome sequencing revealed a CARD8 V44I mutation in 1 allele of chromosome 19 (see sequencing data in the text). The mutation site of V44I was present on the CARD8 T60 isoform, but not the “canonical” T48 isoform.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 1. A kindred with family members bearing a CARD8 mutation and CD-like intestinal inflammation. (A) A kindred containing 3 members bearing a CARD8 mutation. In all 3 members of the kindred, the proband (Pt), his mother (Pt M), and his maternal aunt (Pt Aunt), the mutation occurred in associa- tion with a CD-like intestinal inflammation. (B) Macroscopic examination of the colon exhibited scattered areas of superficial erythema and ulceration hav- ing a lenticular pattern especially evident in the rectosigmoid region. n = 3/group. (C–I) Biopsies from the terminal ileum and colon. (C) Index patient colon. Colitis with epithelial erosive changes and inflammation, significant crypt and goblet cell loss with regenerative changes. Features consistent with GvHD. (D) Index patient colon. Colitis with rare residual gland showing goblet cell loss, repair changes, and rare apoptotic bodies (arrow). (E) Index patient terminal ileum. Ileitis with focal erosion, villi loss, lymphocytic infiltrates, severe crypt drop-out, and repair changes. (F) Index patient terminal ileum. Chronic active ileitis with regenerative changes and poorly formed granulomas including giant cells. (G) Index patient terminal ileum. Poorly formed granuloma (arrow) with giant cells. Adjacent glands with repair/regenerative changes. (H) Aunt terminal ileum. Transmural lymphocyte infiltration with well-formed granuloma present. (I) Aunt terminal ileum. Well-formed granuloma (arrow). n = 3/group. Original magnification, ×4 (C, E, H); ×10 (F, I); ×20 (D, G). Parts G and I show higher magnification of boxed areas in F and H, respectively. (J) Anakinra therapy resulted in rapid clinical improvement marked by decreased fecal calprotec- tin levels. Data are representative of 3 independent experiments. (K) Whole exome sequencing revealed a CARD8 V44I mutation in 1 allele of chromosome 19 (see sequencing data in the text). The mutation site of V44I was present on the CARD8 T60 isoform, but not the “canonical” T48 isoform.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Sequencing

Figure 2. The CARD8 V44I mutation is associated with enhanced IL-1β production and increased NLRP3 inflammasome activation. (A) HEK293 cells were transfected with plasmids carrying human NLRP3 together with CARD8 T48 or T60 or empty vector (EV) and, 24 hours later, transfected with plas- mids carrying ASC, caspase-1. and pro–IL-1β (to allow assembly of the NLRP3 inflammasome). Twenty-four hours later, cells were stimulated with nigericin (NI) for 30 minutes, after which culture supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (B) Serum samples from the proband (with a CARD8 V44I mutation) and age- and sex-matched healthy controls (H1 and H2) were collected and subjected to IL-1 and IL-6 ELISA assays. (C) Proband and control PBMCs and monocytes were cultured for 24 or 48 hours without stimulation, after which supernatants were subjected to IL-1β ELISA assay. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (D) Proband and control mDCs were primed with LPS (100 ng/ml) for 6 hours and stimulated with ATP (5 mM) or nigericin (1.2 μM) for 30 minutes, after which culture super- natants were collected and assayed for IL-1β and IL-6 by ELISA. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (E) Western blot detection of mature IL-1β and mature caspase-1. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 2. The CARD8 V44I mutation is associated with enhanced IL-1β production and increased NLRP3 inflammasome activation. (A) HEK293 cells were transfected with plasmids carrying human NLRP3 together with CARD8 T48 or T60 or empty vector (EV) and, 24 hours later, transfected with plas- mids carrying ASC, caspase-1. and pro–IL-1β (to allow assembly of the NLRP3 inflammasome). Twenty-four hours later, cells were stimulated with nigericin (NI) for 30 minutes, after which culture supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (B) Serum samples from the proband (with a CARD8 V44I mutation) and age- and sex-matched healthy controls (H1 and H2) were collected and subjected to IL-1 and IL-6 ELISA assays. (C) Proband and control PBMCs and monocytes were cultured for 24 or 48 hours without stimulation, after which supernatants were subjected to IL-1β ELISA assay. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (D) Proband and control mDCs were primed with LPS (100 ng/ml) for 6 hours and stimulated with ATP (5 mM) or nigericin (1.2 μM) for 30 minutes, after which culture super- natants were collected and assayed for IL-1β and IL-6 by ELISA. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (E) Western blot detection of mature IL-1β and mature caspase-1. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Western Blot

Figure 3. The CARD8 mutation affects NLRP3 and AIM2 inflammasome, but not pyrin and NLRC4 inflammasome, activation. mDCs from the proband and a healthy control were primed with LPS (100 ng/ml, 6 hours) and then stimulated with ATP (5 mM, A), poly(dA:dT) (1 μg/ml, 2 hours, B), TcdB (1 μg/ml, 2 hours, C), or flagellin (1 μg/ml, 2 hours, D) to activate the NLRP3, AIM2, pyrin, or NLRC4 inflammasomes, respectively. Culture supernatants were collect- ed and subjected to IL-1β (A–D) and IL-6 (E) assay by ELISA. Primary mDCs from proband and healthy control were treated with or without LPS (100 ng/ml) for 6 hours, after which cells were harvested and subjected to RNA extraction. Quantitative reverse-transcriptase PCR (qRT-PCR) of the extracted RNA was performed to determine the expression of ASC (F), caspase-1 (G), NLRP3 (H), and pro–IL-1β (I). Data are shown as mean ± SEM. n = 3. **P < 0.01; *P < 0.05, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 3. The CARD8 mutation affects NLRP3 and AIM2 inflammasome, but not pyrin and NLRC4 inflammasome, activation. mDCs from the proband and a healthy control were primed with LPS (100 ng/ml, 6 hours) and then stimulated with ATP (5 mM, A), poly(dA:dT) (1 μg/ml, 2 hours, B), TcdB (1 μg/ml, 2 hours, C), or flagellin (1 μg/ml, 2 hours, D) to activate the NLRP3, AIM2, pyrin, or NLRC4 inflammasomes, respectively. Culture supernatants were collect- ed and subjected to IL-1β (A–D) and IL-6 (E) assay by ELISA. Primary mDCs from proband and healthy control were treated with or without LPS (100 ng/ml) for 6 hours, after which cells were harvested and subjected to RNA extraction. Quantitative reverse-transcriptase PCR (qRT-PCR) of the extracted RNA was performed to determine the expression of ASC (F), caspase-1 (G), NLRP3 (H), and pro–IL-1β (I). Data are shown as mean ± SEM. n = 3. **P < 0.01; *P < 0.05, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, RNA Extraction, Reverse Transcription, Quantitative RT-PCR, Expressing

Figure 4. CARD8 with a V44I mutation exhibits reduced binding to NLRP3 and affects NLRP3 inflammasome assembly. (A) Plasmids expressing intact CARD8, CARD8 V44I mutation, or empty vector were transfected into HEK293 cells along with a plasmid expressing NLRP3. At 48 hours after transfection, cells were harvested and cell lysates were subjected to immunoprecipitation using anti-CARD8 antibody, followed with immunoblotting. (B) mDCs from proband and healthy control were stimulated with LPS (100 ng/ml, 6 hours) or LPS plus nigericin (1.2 μM, 30 minutes). Cells were lysed, and lysates were subjected to immunoprecipitation and immunoblotting. (C) HEK293 cells were transfected with a plasmid expressing NLRP3 along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Cells were lysed at 48 hours, and lysates were subjected to immunoprecipitation using anti-Myc antibody, followed by immunoblotting. (D) HEK293 cells were transfected with NLRP3 plasmid along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or emp- ty vector. Twenty-four hours later, cells were transfected with plasmids expressing ASC, caspase-1, and pro–IL-1β to allow assembly of the NLRP3 inflam- masome. Another 24 hours later, cells were stimulated with nigericin (1.2 μM, 30 minutes). The cultural supernatants were examined for IL-1β concentra- tion by ELISA. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Dunnett’s post hoc test. (E) mDCs from proband and a healthy control were stimulated with LPS (100 ng/ml, 6 hour) plus nigericin (1.2 μM, 30 minutes). Cells were prepared for Western blot as in Methods. (F) Cell lysates shown in E were prepared for Western blot as in Methods. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 4. CARD8 with a V44I mutation exhibits reduced binding to NLRP3 and affects NLRP3 inflammasome assembly. (A) Plasmids expressing intact CARD8, CARD8 V44I mutation, or empty vector were transfected into HEK293 cells along with a plasmid expressing NLRP3. At 48 hours after transfection, cells were harvested and cell lysates were subjected to immunoprecipitation using anti-CARD8 antibody, followed with immunoblotting. (B) mDCs from proband and healthy control were stimulated with LPS (100 ng/ml, 6 hours) or LPS plus nigericin (1.2 μM, 30 minutes). Cells were lysed, and lysates were subjected to immunoprecipitation and immunoblotting. (C) HEK293 cells were transfected with a plasmid expressing NLRP3 along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Cells were lysed at 48 hours, and lysates were subjected to immunoprecipitation using anti-Myc antibody, followed by immunoblotting. (D) HEK293 cells were transfected with NLRP3 plasmid along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or emp- ty vector. Twenty-four hours later, cells were transfected with plasmids expressing ASC, caspase-1, and pro–IL-1β to allow assembly of the NLRP3 inflam- masome. Another 24 hours later, cells were stimulated with nigericin (1.2 μM, 30 minutes). The cultural supernatants were examined for IL-1β concentra- tion by ELISA. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Dunnett’s post hoc test. (E) mDCs from proband and a healthy control were stimulated with LPS (100 ng/ml, 6 hour) plus nigericin (1.2 μM, 30 minutes). Cells were prepared for Western blot as in Methods. (F) Cell lysates shown in E were prepared for Western blot as in Methods. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Binding Assay, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Figure 5. Mutant CARD8 exerts a dominant-negative effect on the NLRP3 inflammasome. (A) HEK293 cells were transfected with plasmids expressing intact and mutant CARD8 T60 alone or together (half of the amount per plasmids, to mimic the heterozygous status of the proband). After 48 hours of incubation, cell lysates were obtained and subjected to immunoprecipitation and immunoblotting to determine CARD8 binding to NLRP3. (B) HEK293 cells were transfected with NLRP3 and Flag-tagged CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti- CARD8 antibody) and immunoblotting. (C) HEK293 cells were transfected with NLRP3 and CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-NLRP3 antibody) and immunoblot- ting. (D) HEK293 cells were transfected with NLRP3 and intact CARD8 T48 or T60 alone or together with mutant CARD8 T60 plasmids. Twenty-four hours later, T cells were transfected with ASC, caspase-1, and pro–IL-1β plasmids to allow the assembly of NLRP3 inflammasome. Twenty-four hours later, cells were stimulated with nigericin (1.2 μM) for 30 minutes. Cultural supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 5. Mutant CARD8 exerts a dominant-negative effect on the NLRP3 inflammasome. (A) HEK293 cells were transfected with plasmids expressing intact and mutant CARD8 T60 alone or together (half of the amount per plasmids, to mimic the heterozygous status of the proband). After 48 hours of incubation, cell lysates were obtained and subjected to immunoprecipitation and immunoblotting to determine CARD8 binding to NLRP3. (B) HEK293 cells were transfected with NLRP3 and Flag-tagged CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti- CARD8 antibody) and immunoblotting. (C) HEK293 cells were transfected with NLRP3 and CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-NLRP3 antibody) and immunoblot- ting. (D) HEK293 cells were transfected with NLRP3 and intact CARD8 T48 or T60 alone or together with mutant CARD8 T60 plasmids. Twenty-four hours later, T cells were transfected with ASC, caspase-1, and pro–IL-1β plasmids to allow the assembly of NLRP3 inflammasome. Twenty-four hours later, cells were stimulated with nigericin (1.2 μM) for 30 minutes. Cultural supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Expressing, Incubation, Immunoprecipitation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Figure 6. Mutant CARD8 T60 disrupts interaction between T48 and NLRP3. (A) HEK293 cells were transfected with Flag-tagged CARD8 T48 and with Myc-tagged intact or mutant CARD8 T60. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (with anti-Flag antibody), fol- lowed by immunoblotting. (B) HEK293 cells were transfected with Myc-tagged CARD8 T60 along with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-Myc antibody), followed by immunoblotting. (C) HEK293 cells were transfected with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (D) HEK293 cells were transfected with Flag-tagged intact CARD8 T60 plasmid alone or together with a mutant CARD8 T60 plasmid. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (E) HEK293 cells were transfected with a Flag-tagged CARD8 T48 along with intact or mutant CARD8 T60 plasmids: after 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (F) Diagram showing that mutant CARD8 T60 exhibits increased binding with intact T60 and T48 and that this binding is thought to block interaction between intact CARD8 T60 or T48 with NLRP3. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 6. Mutant CARD8 T60 disrupts interaction between T48 and NLRP3. (A) HEK293 cells were transfected with Flag-tagged CARD8 T48 and with Myc-tagged intact or mutant CARD8 T60. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (with anti-Flag antibody), fol- lowed by immunoblotting. (B) HEK293 cells were transfected with Myc-tagged CARD8 T60 along with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-Myc antibody), followed by immunoblotting. (C) HEK293 cells were transfected with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (D) HEK293 cells were transfected with Flag-tagged intact CARD8 T60 plasmid alone or together with a mutant CARD8 T60 plasmid. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (E) HEK293 cells were transfected with a Flag-tagged CARD8 T48 along with intact or mutant CARD8 T60 plasmids: after 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (F) Diagram showing that mutant CARD8 T60 exhibits increased binding with intact T60 and T48 and that this binding is thought to block interaction between intact CARD8 T60 or T48 with NLRP3. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Blocking Assay

Figure 7. The CARD8 V44I mutation results in reduced NLRP3 serine phosphorylation as well as reduced K63 and K48 polyubiquitination. (A) HEK293 cells were transfected with human NLRP3, ASC, caspase-1, and either intact or mutant CARD8 T60 plasmids. After 48 hours, cells were stimulated with or without nigericin (1.2 μM, 30 minutes). Cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 antibody) and immuno- blotted with anti-phosphoserine antibody. (B) mDCs from the proband and a healthy control were pretreated with LPS (100 ng/ml, 6 hours) and then activated with nigericin (1.2 μM, 30 minutes). Cell lysates from these cells were subjected to immunoprecipitation and immunoblotting as described above. (C) HEK293 cells were transfected with NLRP3, ASC, and caspase-1 plasmids as well as intact or mutant CARD8 T60 plasmids together with constructs expressing K63 or K48 ubiquitin chains. Forty-eight hours later, cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 anti- body) and Western blot to examine the polyubiquitination of NLRP3. The top row image was spliced from images obtained from the same gel, but with different exposure times. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 7. The CARD8 V44I mutation results in reduced NLRP3 serine phosphorylation as well as reduced K63 and K48 polyubiquitination. (A) HEK293 cells were transfected with human NLRP3, ASC, caspase-1, and either intact or mutant CARD8 T60 plasmids. After 48 hours, cells were stimulated with or without nigericin (1.2 μM, 30 minutes). Cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 antibody) and immuno- blotted with anti-phosphoserine antibody. (B) mDCs from the proband and a healthy control were pretreated with LPS (100 ng/ml, 6 hours) and then activated with nigericin (1.2 μM, 30 minutes). Cell lysates from these cells were subjected to immunoprecipitation and immunoblotting as described above. (C) HEK293 cells were transfected with NLRP3, ASC, and caspase-1 plasmids as well as intact or mutant CARD8 T60 plasmids together with constructs expressing K63 or K48 ubiquitin chains. Forty-eight hours later, cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 anti- body) and Western blot to examine the polyubiquitination of NLRP3. The top row image was spliced from images obtained from the same gel, but with different exposure times. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Immunoprecipitation, Control, Western Blot, Construct, Expressing, Ubiquitin Proteomics

Figure 8. IL-1β antibody treatment reduced peripheral cytokine levels in proband bearing a CARD8 V44I mutation. Seven serum samples from the proband at various time points were collected and subjected to assays of IL-1β (A), IL-6 (B), and TNF-α (C) concentrations by ELISA. Data in A–C are shown as mean ± SEM. Serum samples from patients with active CD without CARD8 V44I mutations and healthy individuals were collected and subjected to assays of the concentration of IL-1β (D), IL-6 (E), and TNF-α (F) by ELISA. CD patients, n = 4; control, n = 5. Data in D–F are shown as mean ± SEM. **P < 0.01, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 8. IL-1β antibody treatment reduced peripheral cytokine levels in proband bearing a CARD8 V44I mutation. Seven serum samples from the proband at various time points were collected and subjected to assays of IL-1β (A), IL-6 (B), and TNF-α (C) concentrations by ELISA. Data in A–C are shown as mean ± SEM. Serum samples from patients with active CD without CARD8 V44I mutations and healthy individuals were collected and subjected to assays of the concentration of IL-1β (D), IL-6 (E), and TNF-α (F) by ELISA. CD patients, n = 4; control, n = 5. Data in D–F are shown as mean ± SEM. **P < 0.01, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control