Blau syndrome NOD2 mutations result in loss of NOD2 cross-regulatory function

Abstract

The studies described here provide an analysis of the pathogenesis of Blau syndrome and thereby the function of NOD2 as seen through the lens of its dysfunction resulting from Blau-associated NOD2 mutations in its nucleotide-binding domain (NBD). As such, this analysis also sheds light on the role of NOD2 risk polymorphisms in the LRR domain occurring in Crohn's disease. The main finding was that Blau NOD2 mutations precipitate a loss of canonical NOD2 signaling via RIPK2 and that this loss has two consequences: first, it results in defective NOD2 ligand (MDP)-mediated NF-κB activation and second, it disrupts NOD2-mediated cross-regulation whereby NOD2 downregulates concomitant innate (TLR) responses. Strong evidence is also presented favoring the view that NOD2-mediated cross-regulation is under mechanistic control by IRF4 and that failure to up-regulate this factor because of faulty NOD2 signaling is the proximal cause of defective cross-regulation and the latter's effect on Blau syndrome inflammation. Overall, these studies highlight the role of NOD2 as a regulatory factor and thus provide additional insight into its function in inflammatory disease. Mutations in the nucleotide binding domain of the CARD15 (NOD2) gene underlie the granulomatous inflammation characterizing Blau syndrome (BS). In studies probing the mechanism of this inflammation we show here that NOD2 plasmids expressing various Blau mutations in HEK293 cells result in reduced NOD2 activation of RIPK2 and correspondingly reduced NOD2 activation of NF-κB. These in vitro studies of NOD2 signaling were accompanied by in vivo studies showing that BS-NOD2 also exhibit defects in cross-regulation of innate responses underlying inflammation. Thus, whereas over-expressed intact NOD2 suppresses TNBS-colitis, over-expressed BS-NOD2 does not; in addition, whereas administration of NOD2 ligand (muramyl dipeptide, MDP) suppresses DSS-colitis in Wild Type (WT) mice it fails to do so in homozygous or heterozygous mice bearing a NOD2 Blau mutation. Similarly, mice bearing a Blau mutation exhibit enhanced anti-collagen antibody-induced arthritis. The basis of such cross-regulatory failure was revealed in studies showing that MDP-stimulated cells bearing BS-NOD2 exhibit a reduced capacity to signal via RIPK2 as well as a reduced capacity to up-regulate IRF4, a factor shown previously to mediate NOD2 suppression of NF-κB activation. Indeed, TLR-stimulated cells bearing a Blau mutation exhibited enhanced in vitro cytokine responses that are quieted by lentivirus transduction of IRF4. In addition, enhanced anti-collagen-induced joint inflammation in mice bearing a Blau mutation was accompanied by reduced IRF4 expression in inflamed joint tissue and IRF4 expression was reduced in MDP-stimulated cells from BS patients. Thus, inflammation characterizing Blau syndrome are caused, at least in part, by faulty canonical signaling and reduce IRF4-mediated cross-regulation.

Keywords: Blau; Crohns disease; IRF4; NFkapapB; Nod2.

Figure 1

The BS-NOD2 Mutation Causes Reduced NOD2 Self-Interaction. (A) Diagram of NOD2 structure showing main NOD2 domains and locations of nucleotide binding domain Blau mutations in the Blau constructs studied here. (B) HEK293T cells were transfected with constructs expressing Flag- or V5-tagged mouse wildtype NOD2 or Blau syndrome-associated mutations (FlagN2R314W, FlagN2R682W) or NOD2 with frameshift mutation (FlagN2FS987) with different combinations. The cells were lysed 24 hours post transfection and the cell lysates were subjected to IP using an anti-V5 antibody and Western blotting for NOD2 detection. (C) HEK293T cells were transfected with constructs expressing flag- or his-tagged mouse wild type NOD2 or NOD2 with a R314Q mutation with different combinations. The cells were lysed 24 hours post transfection and the cell lysates were subjected to IP using an anti-flag antibody and Western blotting for NOD2 detection.

Figure 2

The BS-NOD2 Mutation Causes Reduced NOD2-Induced Interaction with RIPK2, Reduced RIPK2 Activation and Reduced NF-κB Activation. (A) HEK293T cells were transfected with constructs expressing T7-tagged wildtype or BS-NOD2 mutations (R334Q or R334W) and NTAP-tagged RIPK2; 24 hours later, the cells were incubated with or without MDP (10μg/ml) for 6 hours after which the cells were lysed and subjected to pull-down assay for detecting NOD2-RIPK2 interaction and RIPK2 phosphorylation. (B) HEK293T cells were transfected with constructs expressing T7-tagged human wildtype NOD2 or with one of multiple constructs expressing various NOD2 mutations associated with Blau syndrome; in addition, the cells were transfected with NTAP-tagged RIPK2; 24 hours later, the cells were stimulated with MDP (10μg/ml) for 6 hours after which the cells were lysed and the cell lysates obtained were subjected to pull-down assay using streptavidin beads and Western blotting for detection of NOD2-RIPK2 interaction and RIPK2 phosphorylation. (C, D) HEK293T cells were transfected with constructs expressing wildtype NOD2 or one of multiple Blau syndrome constructs expressing various NOD2 mutations; in addition, the cells were transfected with constructs expressing V5-tagged RIPK2 and HA- tagged K63-only ubiquitin (C, other lysine sites mutated to alanine) or HA-tagged K48-only ubiquitin (D, other lysine sites mutated to alanine); 24 hours later, the cells were stimulated with MDP (10μg/ml) for 6 hours and then lysed; finally, the cell lysates were subjected to IP with agarose beads conjugated with anti-V5 antibody and Western blotting for detection of RIPK2 polyubiquitination. All data are representative figures from two independent experiments. (E) HEK293T cells were transfected with constructs expressing wildtype NOD2 or constructs bearing the indicated Blau mutations as well as an NF-κB reporter construct; 24 hours later, the cells were incubated with or without MDP (10μg/ml) for 6 hours and were then lysed; finally, the lysates were subjected to luciferase assay for detection of NF-κB activation. (F) HEK293T cells were transfected with constructs expressing one of multiple constructs expressing Blau syndrome NOD2 mutations, RIPK2 and NF-κB reporter; 24 hours later, the cells were incubated with or without MDP (10μg/ml) for 6 hours and were then lysed; finally, the lysates were subjected to luciferase assay for detection of NF-κB activation. Studies in (A, B) were conducted with three biological replicates for each sample. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparisons and are displayed as mean ± SEM; **p<0.01. ns, not significant.

Figure 3

Mice with transient over-expression of Blau Syndrome (BS)-associated NOD2 mutation (R314W) or BS-NOD2 transgene are not protected from induction of TNBS-colitis. (A) TNBS-colitis associated weight loss of C57BL/10 mice administered TNBS per rectum (3.5 mg/mouse) on day 0 of study and plasmids expressing intact NOD2 (NOD2-WT), Blau syndrome-associated mutation of NOD2 (NOD2-R314W) or Crohn’s disease-associated NOD2 frameshift mutation (NOD2-FS98) encapsulated in HVJ-E or the empty vector control (Vector Ctr) on day -1, day 0 and day 1 of study; mice were harvested for analysis on day 4. (B) Representative histology of colons after H&E staining of tissues from mice harvested on day 4. (C) TCR-induced production of IFN-γ, IL-17A and SAC+IFN-γ-induced IL-12p70 production by extracted MLN cells from mice harvested on day 4 determined by ELISA. (D) TNBS-colitis associated weight loss of C57BL/10 mice bearing transgenes expressing intact NOD2 (TgNOD2, n=6) or NOD2-R314W (TgBlau, n=7) and wildtype (WT, n=9) mice administered TNBS per rectum (3.5 mg/mouse); mice were harvested for analysis on day 4. (E) Representative histology of colons after H&E staining of colonic tissue from mice harvested on day 4. (F) TCR-induced production of IFN-γ and IL-17A by extracted MLN cells harvested on day 4. Data in (A, D) were analyzed using a two-way ANOVA with Dunnett’s multiple comparisons. Data in (C, F) were analyzed using a two-way ANOVA with Tukey’s multiple comparisons. Data are displayed as mean ± SEM; **p<0.01, ***p<0.001.

Figure 4

Transgenic BS-NOD2 Has a Dominant-Negative Effect which Interferes with the Cross-Regulatory Function of Endogenous NOD2 in TNBS-Colitis. (A) TNBS-colitis associated weight loss of C57BL/10 mice bearing transgenes expressing intact NOD2 (BL10 TgNOD2, n=6) or NOD2-R314W (BL10 TgR314W, n=8) and wildtype (BL10 WT, n=10) mice administered TNBS per rectum (3.0 mg/mouse); mice were harvested for analysis on day 4. Data were analyzed using a two-way ANOVA with Dunnett’s multiple comparisons and are displayed as mean ± SEM; *p<0.05; **p<0.01. (B) Representative histology of colons after H&E staining of colonic tissue from mice harvested on day 4. (C) Histology scores of mice described in (A) plus mice studied in a second identical study involving histologic study only (n=12). (D) TCR-induced production of IFN-γ and IL-17A and SAC+IFN-γ-induced IL-12p70 production by extracted MLN cells from mice harvested on day 4; IFN-γ, IL-17 and IL-12p70 production in (D) were examined by ELISA. Data in (C, D) were analyzed using a two-way ANOVA with Tukey’s multiple comparisons and are displayed as mean ± SEM; *p<0.05; **p<0.01.

Figure 5

The BS-NOD2 Transgene Exerts a Dominant Negative Effect on Cellular Responses. (A) Bone marrow derived dendritic cells (BMDC) from WT (C57BL/6) mice, or C57BL/6 mice expressing a transgene bearing a Blau (R314W) mutation (BL6TgR314W) were stimulated with the indicated doses of MDP for 24 hrs after which the cell culture supernatants were subjected to ELISA analysis of IL-12p40 and IL-6 secretion. (B) Total mRNA was extracted from PBMCs of three patients with Blau syndrome (Pt1, Pt2 and Pt3). The RNA samples were reverse transcribed to cDNA and subjected to PCR to amplify human NOD2 sequence. The amplified NOD2 sequence was subjected to sequencing. (C) Monocytes isolated from PBMCs of three patients with Blau syndrome (Pt1 and Pt2, carrying R334Q mutation in one allele; Pt3, carrying R334W mutation in one allele of NOD2) and three control individuals were stimulated with MDP (50μg/ml) or LPS (200 ng/ml) for 24 hours after which the cell culture supernatants were subjected to ELISA assay of IL-12p40 and IL-6 secretion. (D, E) Monocytes from a Blau patient and a control individual were differentiated into dendritic cells (MoDC, D) or macrophages (MoMac, E), the cells were then stimulated with MDP (50μg/ml) for 24 hours after which the cell culture supernatants subjected to ELISA assay of IL-6 and IL-12p40 secretion. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparisons and are displayed as mean ± SEM;. *p<0.05; **p<0.01; ***p<0.001.

Figure 6

NOD2 bearing a BS mutation expressed on one allele binds to intact NOD2 and exerts a dominant negative effect on intact NOD2 expressed on the other allele of BS-KI heterozygous mice. (A) BMDCs from WT mice, heterozygous Blau KI mice or homozygous Blau KI mice were stimulated with LPS (200ng/ml) only or LPS (200ng/ml) plus MDP (50ug/ml) for 6 hours, the cells were lysed, and the cell lysates were then subjected to Western blot for NOD2 detection. (B) Bone marrow derived dendritic cells (BMDCs) from WT mice or heterozygous Blau-KI mice were transfected with a plasmid expressing flag-tagged wild type mouse NOD2 as indicated; 48 hours later the cells were primed with LPS (200ng/ml) for 5 hours and then stimulated with MDP (50μg/ml) for 1 hour; the cells were then lysed and the cell lysates were subjected to IP with anti-flag antibody and then Western blot for detection of NOD2 binding forms of endogenous NOD2. (C) BMDMs from Blau KI mice or their wildtype littermates were primed with LPS (200ng/ml) for 5 hours and then MDP (50μg/ml) for 1 hours; the cells were then lysed, and the lysates obtained were subjected to IP with anti-RIPK2 antibody and Western blotting for detection of NOD2. (D) BMDMs described in (C) were incubated with MDP (20μg/ml) for various time periods after which the cells were then lysed and the lysates obtained were subjected to Western blotting for detection of IκBα phosphorylation and degradation. (E) DSS-colitis associated weight loss of C57BL/6 heterozygous (n=5) or homozygous (n=5) Blau KI mice and their wild type littermates (n=5) treated with 4% DSS in drinking water; mice were administered muramyl dipeptide (MDP, I00 μg/mouse, IP, n=5) or PBS (n=5) on days 0-5 and were harvested for analysis on day 7. (F) Colon length from mice harvested on day 7 after DSS-colitis induction. (G) Representative histology of colons after H&E staining of colonic tissue from mice harvested on day 7 after initiation of DSS-colitis induction. (H) TCR-induced production of IFN-γ and IL-17A and SAC+IFN-γ-induced IL-12p40 production by extracted MLN cells from mice harvested on day 4 were examined by ELISA. Data in (A–D) are representative of three independent experiments. Data in (E) were analyzed using a two-way ANOVA with Dunnett’s multiple comparisons and are displayed as mean ± SEM; ***p<0.001. Data in (F, H) were analyzed using a two-way ANOVA with Tukey’s multiple comparisons and are displayed as mean ± SEM; **p<0.01; ns: not significant.

Figure 7

The BS-NOD2 Mutation Causes Loss of NOD2 Cross-Regulation of TLR Responses in vitro. (A–C) BMDCs obtained from Blau-KI mice, their wildtype littermates and NOD2 KO mice were stimulated with PGN (1μg/ml, A), LPS (1μg/ml, B) or MDP (50μg/ml, C) for various time periods as indicated; the cells were then lysed and the lysates were subjected to Western blotting for detection of phosphorylation of IκBα, ERK, and IRF4. (D, E) BMDCs from Blau-KI mice and their wildtype littermates were incubated with LPS (100 ng/ml) or PGN (100 ng/ml) for 24 hours; the culture supernatants were then collected for assay of IL-6 (D) and IL-12p40 (E) by ELISA. Data in (A–C) are representative of three independent experiments. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparisons and are displayed as mean ± SEM; **p<0.01.

Figure 8

The BS-NOD2 Mutation Causes Loss of NOD2 Cross-Regulation of TLR Responses in vivo. (A, B) Blau KI mice (homozygous, n=4) and their wildtype littermates (n=4) were administered anti-collagen antibody cocktails (5mg/mouse I.P.) on day 0; the mice were then administered LPS (50ug/mouse, I.P.) on day 3 and were euthanized on day 7. (B) Representative histology of joints after H&E staining of tissues from both mice; disease severity was evaluated by arthritis score. (C) Joint tissues were harvested for RNA extraction, which were then subjected to reverse transcription and real time RT-PCR for transcription of pro-inflammatory cytokines, IL-6, IL-12p40 and TNF-𝛂. (D) Serum samples were collected from mice and concentration of IL-6, IL-12p40 and TNF-𝛂 was examined by ELISA. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparisons; *p<0.05; **p<0.01.

Figure 9

The BS-NOD2 Mutation is Associated with Reduced IRF-4 Expression and Function. (A) TNBS-colitis-induced weight loss of C57BL/10 mice administered IRF4 encapsulated in HVJ-E (n=7) or empty vector (Vector Ctr, n=4); TNBS (3.5 mg/mouse) administered per rectum on day 0, IRF4 or control vector administered on day -1, day 0 and day 1; mice harvested for analysis on day 4. (B) Blau KI mice (homozygous, n=4) and their wildtype littermates (n=4) were administered anti-collagen antibody cocktails (5mg/mouse by I.P.) on day 0 the mice were then administered LPS (50ug/mouse, by I.P.) on day 3. The mice were euthanized on day 7. Joint tissues were harvested and were homogenized in lysis buffer for protein extraction; the samples obtained were subjected to protein quantification followed by Western blotting for IRF4 detection. 1-4 represents protein samples from individual mouse. (C) mDCs from a patient with Blau syndrome or a healthy control individual were treated with or without MDP (50μg/ml) or LPS (100ng/ml) for 6 hours; the cells were then lysed, and the lysates were subjected to Western blotting for detection of IRF4 or RIPK2. (D) BMDCs from Blau-KI mice and their wildtype littermates were treated with MDP (50μg/ml, IP) for 3 or 6 hours; the cells were then lysed, and the lysates were subjected to Western blotting for detection of IRF4. (E) HEK293T cells were transfected with constructs expressing wildtype NOD2, BS-NOD2 mutations, RIPK2 and IRF4 promoter reporter; 24 hours later, the cells were stimulated with or without MDP (10μg/ml) for 6 hours and were then lysed; finally, the lysates were subjected to luciferase assay for detection of IRF4 promoter activity. Data in (A) were analyzed using a two-Two-tailed Student’s t test and are displayed as mean ± SEM; **p<0.01. Data in (E) were analyzed using a two-way ANOVA with Tukey’s multiple comparisons and are displayed as mean ± SEM; **p<0.01. Data in (B–E) are representative of two independent experiments.

Figure 10

Over-expression of IRF4 Restores Immune Suppression to TLR Stimulation in Blau-KI Cells. (A, B) BMDCs from Blau-KI mice and their wildtype littermates were incubated with lentiviral particles expressing mouse IRF4 or empty vector control as indicated for 48 hours; the cells were then stimulated with PGN (1μg/ml, A) or LPS (1μg/ml, B) for various time periods as indicated; the cells were then lysed and the lysates were subjected to Western blotting for detection of phosphorylation of IκBα, ERK and IRF4. (C, D) BMDCs as described in (A, B) after transfection with IRF4-expressing constructs were stimulated with LPS (100 ng/ml) or PGN (100 ng/ml) for 24 hours; the culture supernatants were then collected for assay of IL-6 (C) and IL-12 (D) by ELISA. Data in (C, D) were analyzed using a two-way ANOVA with Tukey’s multiple comparisons and are displayed as mean ± SEM; **p<0.01. Data in (A, B) are representative of three independent experiments.