Copper metabolism domain-containing 1 represses genes that promote inflammation and protects mice from colitis and colitis-associated cancer.

All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.

Gastroenterology 2014;-:1–12

60 61 62 63 64 65 66 67 68 1 1 2 3 4 Haiying Li, Lillienne Chan, Paulina Bartuzi, Shelby D. Melton, Axel Weber, 69 70 Shani Ben–Shlomo,5 Chen Varol,5 Megan Raetz,6 Xicheng Mao,1 Petro Starokadomskyy,1 7 1 8 1 71 Suzanne van Sommeren, Mohamad Mokadem, Heike Schneider, Reid Weisberg, 72 9 10 10 9 11 Harm-Jan Westra, Tõnu Esko, Andres Metspalu, Vinod Kumar, William A. Faubion, 73 6 2 9 4 9 Felix Yarovinsky, Marten Hofker, Cisca Wijmenga, Michael Kracht, Lude Franke, 74 Vincent Aguirre,1 Rinse K. Weersma,7 Nathan Gluck,5 Bart van de Sluis,2 and Ezra Burstein1,12 75 76 1 Department of Internal Medicine, 6Department of Immunology, and 12Department of Molecular Biology, University of Texas 77 Southwestern Medical Center, Dallas, Texas; 2Department of Pediatrics, 9Department of Genetics, and 7Department of 78 Gastroenterology and Hepatology, Section of Molecular Genetics, University of Groningen, University Medical Center 79 Groningen, Groningen, The Netherlands; 3Department of Pathology, Dallas VA Medical Center, Dallas, Texas; 4Rudolf 80 5 Buchheim Institute of Pharmacology, Justus Liebig University, Giessen, Germany; Gastroenterology Institute, Tel Aviv 10 11 81 Sourasky Medical Center, Tel Aviv, Israel; Estonian Genome Center, University of Tartu, Tartu, Estonia; Division of 82 Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota; and 8Institute of Physiological Chemistry, Hannover 83 Medical School, Hannover, Germany 84 BACKGROUND & AIMS: Activation of the transcription factor expression or function of COMMD1 could be involved in the 85 86 nuclear factor-kB (NF-kB) has been associated with the devel- pathogenesis of IBD. opment of inflammatory bowel disease (IBD). Copper meta87 bolism MURR1 domain containing 1 (COMMD1), a regulator of 88 various transport pathways, has been shown to limit NF-kB Keywords: Mouse Model; Gene Regulation; CD; UC. 89 activation. We investigated the roles of COMMD1 in the path90 ogenesis of colitis in mice and IBD in human beings. METHODS: 91 ersistent inflammation is a common maladaptive Q14 92 We created mice with a specific disruption of Commd1 in Q15 component in the pathogenesis of human diseases. A 93 myeloid cells (Mye-knockout [K/O] mice); we analyzed immune cell populations and functions and expression of genes regu- prime example of this paradigm is inflammatory bowel 94 lated by NF-kB. Sepsis was induced in Mye-K/O and wild-type disease (IBD), a chronic inflammatory process of the intes- 95 mice by cecal ligation and puncture or intraperitoneal injec- tinal tract that clinically presents as 2 phenotypic entities: 96 tion of lipopolysaccharide (LPS), colitis was induced by ulcerative colitis (UC) and Crohn’s disease (CD). This dis97 administration of dextran sodium sulfate, and colitis-associated order involves an interaction between environmental fac98 cancer was induced by administration of dextran sodium sul- tors and inherited susceptibility, and is associated with an 99 fate and azoxymethane. We measured levels of COMMD1 increased risk for colorectal cancer.1 100 messenger RNA in colon biopsy specimens from 29 patients The regulation of the inflammatory cascade is a complex 101 with IBD and 16 patients without (controls), and validated process in which the transcription factor nuclear factor-kB 102 findings in an independent cohort (17 patients with IBD and 22 (NF-kB) plays a master regulatory role.2 Consequently, this 103 controls). We searched for polymorphisms in or near COMMD1 factor also has been linked to the pathogenesis of several 104 that were associated with IBD using data from the International chronic inflammatory conditions in human beings,2 105 IBD Genetics Consortium and performed quantitative trait lo106 cus analysis. RESULTS: In comparing gene expression patterns 107 between myeloid cells from Mye-K/O and wild-type mice, we found that COMMD1 represses expression of genes induced by Abbreviations used in this paper: BMDM, bone marrow–derived myeloid 108 LPS. Mye-K/O mice had more intense inflammatory responses cells; CD, Crohn’s disease; cis-eQTL, cis-expression quantitative trait 109 COMMD1, copper metabolism MURR1 domain containing 1; DSS, to LPS and developed more severe sepsis and colitis, with locus; 110 dextran sodium sulfate; IBD, inflammatory bowel disease; IIBDGC, Intergreater mortality. More Mye-K/O mice with colitis developed national IBD Genetic Consortium; Il, interleukin; KEGG, Kyoto encyclo- 111 colon dysplasia and tumors than wild-type mice. We observed a pedia of genes and genomes; K/O, knockout; LPS, lipopolysaccharide; 112 mRNA, messenger RNA; Mye-K/O, myeloid-specific Commd1 knockout; reduced expression of COMMD1 in colon biopsy specimens and NF-kB, nuclear factor-kB; RelA, ___________________; SNP, single-nucle- 113 circulating leukocytes from patients with IBD. We associated otide polymorphism; Tnf, tumor necrosis factor; UC, ulcerative colitis; WT, 114 single-nucleotide variants near COMMD1 with reduced wild-type. 115 expression of the gene and linked them with increased risk for 116 © 2014 by the AGA Institute ulcerative colitis. CONCLUSIONS: Expression of COMMD1 0016-5085/$36.00 117 by myeloid cells has anti-inflammatory effects. Reduced http://dx.doi.org/10.1053/j.gastro.2014.04.007 118

Copper Metabolism Domain-Containing 1 Represses Genes That Promote Inflammation and Protects Mice From Colitis and Colitis-Associated Cancer

Q59

Q10

P

FLA 5.2.0 DTD YGAST59081_proof 20 May 2014 5:04 pm ce

BASIC AND TRANSLATIONAL AT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

2


119 Q16 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 Q17 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 Q18 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

Li et al

Gastroenterology Vol.

including IBD.3,4 Canonical NF-kB activity is mediated primarily by NF-kB complexes containing the RelA subunit (also known as p65) or its paralog c-Rel. Under basal conditions, these complexes are kept in the cytosol through interactions with the inhibitory IkB proteins. Their activation requires IkB degradation, an event triggered by a critical kinase complex known as IkB kinase that sits at the cross-roads of numerous signaling pathways. After signaling inputs abate, homeostatic mechanisms that restore basal NF-kB activity are essential for the physiologic function of this pathway. The induction of IkB gene expression,5 or the expression of IkB kinase inhibitory proteins, such as A20 or CYLD,6,7 participate in the timely termination of NF-kB activity. The expression of these factors is under the control of NF-kB itself, thus providing negative feedback loops in the pathway. In addition, it has been recognized that ubiquitination and proteasomal degradation of RelA is critical to terminate transcription of a variety of genes.8–13 One ligase responsible for these effects contains the scaffold protein Cul2 in association with copper metabolism MURR1 domain containing 1 (COMMD1),10,12 a prototypical member of the COMMD protein family.14 In addition to its role in NF-kB regulation,12,15 COMMD1 has been implicated in a variety of cellular processes, including copper transport,16 electrolyte balance,17–19 and hypoxia responses.20 Given these pleiotropic functions, it has remained unclear whether this factor plays a physiologically important role in the control of inflammation in vivo and whether it could play a role in chronic inflammatory diseases. Here, we report that myeloid-specific deficiency of Commd1 leads to more intense activation of lipopolysaccharide (LPS)-inducible genes and is associated with more severe inflammation. In addition, we present genetic evidence linking gene variants associated with reduced COMMD1 expression to risk for UC in human beings, highlighting the physiologic importance of this gene in immunity and IBD pathogenesis.

Materials and Methods Human Studies All procedures involving human subjects were reviewed and approved by the respective institutional review boards (at the University of Texas Southwestern Medical Center, the Mayo Clinic, and the Tel-Aviv Sourasky Medical Center). Circulating leukocytes and intestinal biopsy specimens were obtained at the time of endoscopy as part of the patients’ ongoing medical care.

Genome-Wide Association Studies and Quantitative Trait Locus Analysis Genetic association data were obtained from the 1000 genomes imputed meta-analysis from 15 genome-wide association studies of CD and/or UC conducted by the International IBD Genetic Consortium (IIBDGC).3 We conducted cis-expression quantitative trait locus (cis-eQTL) mapping on whole peripheral blood of 2 cohorts: 1240 samples from a previously published study21 and 891 samples from the Estonian Biobank,

-,

No.

-

both hybridized to Illumina HT12-v3 oligonucleotide arrays using methodology as described in detail elsewhere.22

Q19

178 179 180 181 Statistical Analysis In all graphs, the mean is presented and the error bars 182 correspond to the SEM. Statistical comparisons between mean 183 values were performed using a 1-tailed, heteroscedastic, Stu- 184 dent t test. For nonparametric variables, the chi-square test was 185 used. Survival curves were examined using the Kaplan–Meier 186 187 analysis. All other materials and methods are described in the 188 Supplementary Materials and Methods section. 189 190 191 Results 192 193 Commd1 Represses Proinflammatory Gene 194 Expression in Myeloid Cells 195 To evaluate the potential role of COMMD1 in inflam- 196 mation, and given the known embryonic lethality that re- 197 sults from complete Commd1 deficiency in mice,23 we 198 generated a tissue-specific mouse model of Commd1 defi- 199 ciency.24 First, Commd1 was selectively deleted in myeloid 200 cells (Mye-knockout [K/O]), a critical lineage in innate im- 201 munity, leading to the expected loss of Commd1 expression 202 in macrophages (Supplementary Figure 1A). Mye-K/O mice 203 were healthy and B-lymphocyte (B220þ) and T-lymphocyte 204 populations (CD3þ and CD4/CD8) were not significantly 205 different in the spleen or mesenteric lymph nodes 206 (Supplementary Figure 1B and C). Similarly, myeloid pop- 207 ulations, including granulocytes (Ly6Gþ), monocytes and 208 macrophages (CD11bþ, Ly6Cþ, and F4/80), and dendritic 209 cells (CD11chigh and CD11cintermediate) were not significantly 210 different in Mye-K/O mice (Supplementary Figure 1B–D). In 211 line with previous observations,12,25,26 Commd1 deficiency 212 did not alter substantially the phosphorylation or turnover 213 of IkB (Figure 1A), but had a profound effect on RelA Q20214 ubiquitination (Figure 1B). 215 Next, using bone marrow–derived myeloid cells 216 (BMDMs) from the Mye-K/O mice, we assessed the impact 217 of Commd1 on the LPS transcriptional response at a 218 genome-wide level. High-density microarray experiments 219 indicated that 1008 genes were regulated by LPS at least 220 3-fold in 2 independent series of experiments (Figure 1C, 221 and Supplementary Tables 1–3). In addition, the expression 222 of 225 genes was found to be regulated by Commd1 223 (Supplementary Tables 1 and 2). Notably, the vast majority 224 of Commd1-regulated genes (219 of 225) also was regulated 225 by LPS, and only few Commd1 target genes (6 of 225) were 226 outside the LPS transcriptional response (Supplementary 227 Tables 2 and 3). Hierarchical clustering of these 225 genes 228 was used to visualize the pattern of deregulated expression 229 in Commd1-deficient myeloid cells. Both early and late LPS- 230 inducible genes were affected by Commd1 deficiency 231 (Figure 1D). In most instances, the changes observed con- 232 sisted of increased gene expression, which often were noted 233 even at basal levels (Figure 1E). 234 To further understand the effect of Commd1 on gene 235 expression in myeloid cells, we performed a functional 236


-

COMMD1 and Colitis

3

296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 Figure 1. Commd1 regulates the LPS transcriptional program in myeloid cells. (A) Commd1 does not affect IkB turnover in 338 BMDMs. IkB-a phosphorylation (p-IkB-a) and degradation after LPS treatment were examined by Western blotting. *ns, 339 nonspecific band (arrow points to p-IkB-a). (B) Commd1 deficiency reduces RelA ubiquitination. Commd1-deficient mouse 340 embryo fibroblasts (K/O) or control cells (WT) were used to detect ubiquitinated RelA by ubiquitin immunoblotting of immu- 341 noprecipitated RelA. (C) Gene expression differences resulting from Commd1 deficiency substantially overlap with the LPS 342 response. BMDMs from WT and Mye-K/O mice were used in duplicate microarray analysis to ascertain genes regulated by Commd1 or by LPS (100 ng/mL) at early (3 h) or late (24 h) time points. The degree of overlapping genes is presented in Venn 343 diagram form. (D) Hierarchical cluster analysis of Commd1-regulated genes during the LPS response. Expression levels 344 normalized to basal expression in WT samples is shown as color-coded log2-transformed ratios. The gene induction pattern, 345 either early (>3-fold induction at 3 h) or late LPS response (>3-fold induction at 24 h), also is indicated. The specific effect of 346 Commd1 deficiency is noted by up arrows or down arrows. (E) The genes analyzed in panel D are reanalyzed by calculating 347 ratios between K/O and WT BMDMs, displayed in a color-coded log2-transformed manner. The direction of change is indi- 348 cated in adjacent columns by up arrows or down arrows. (F) Venn diagram of Commd1-regulated genes, indicating those Q1 349 involved in inflammatory responses or regulated by NF-kB. 350 351 analysis using the Kyoto encyclopedia of genes and genomes number of KEGG entries) (Supplementary Table 4), indi- 352 (KEGG) database. This showed that only 51 genes (23%) cating that a large number of Commd1 gene targets in 353 participate in immune regulation (as defined by any of a myeloid cells are not known inflammatory regulators 354


print & web 4C=FPO

237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295

2014


4


355 356 357 358 359 360 Q21 361 362 363 364 365 366 367 368 369 Q22 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413

Li et al


(Figure 1F). Next, we assessed the contribution of NF-kB regulation to the effects noted in Commd1-deficient myeloid cells. To that end, NF-kB regulation of genes of interest was ascertained through 2 overlapping approaches: first, using a curated database of published NF-kB gene targets (publicly available at www.NF-kB.org), and second, using chromatin immunoprecipitation seq data for RelA in LPS-stimulated BMDMs.27 This indicated that only 66 genes regulated by Commd1 (29%) (Supplementary Table 4) are known NF-kB or RelA targets, suggesting that a substantial proportion of the effects of Commd1 are either secondary or mediated through other transcriptional regulators.

Commd1 Exerts Gene-Selective Effects Among NF-kB–Regulated Targets Altogether, these findings suggest a substantial repressive role for Commd1 on the LPS response in myeloid cells and confirmed a negative regulatory role for Commd1 on selected NF-kB–regulated genes. Indeed, quantitative reverse-transcription polymerase chain reaction analysis corroborated that certain NF-kB target genes were selectively regulated (eg, Mx1, Cd86, and Sell) (Figure 2A), whereas others were relatively insensitive to Commd1 deficiency (such as Il6, Tnfaip3, and Mmp13) (Supplementary Figure 2A). These findings are in line with previous reports of gene-specific effects for COMMD1 in cancer cell lines.12 Enhanced expression of selected genes that are not regulated directly by NF-kB was confirmed as well (Figure 2B). Interestingly, in contrast to these effects, several hypoxia-induced genes were not affected by Commd1 deficiency in BMDMs (Supplementary Figure 2B), despite its reported role in hypoxia-dependent gene expression.20,23 In aggregate, the data indicate that Commd1 primarily exerts a gene-selective repressive function on the LPS response.

Myeloid Deficiency of Commd1 Leads to Exaggerated Inflammatory Responses In Vivo In view of the effects of Commd1 on LPS-induced gene expression, we used 2 models to contrast the inflammatory response of Mye-K/O mice and wild-type (WT) littermate controls. First, we used the cecal ligation and puncture model of polymicrobial sepsis. Animal survival was compromised in Mye-K/O mice compared with WT controls (Figure 3A). In contrast, cultures of peritoneal fluid showed lower bacterial counts in the Mye-K/O mice (Figure 3B), indicating that their increased mortality was not caused by greater bacterial load. Rather, after accounting for bacterial invasion (by examining animals with negative bacterial cultures), we found that Mye-K/O mice had increased cytokine expression (Figure 3C), suggesting that exaggerated proinflammatory gene expression was responsible for their increased susceptibility to sepsis. Next, animals were challenged directly with intraperitoneal LPS injection. Once more, Mye-K/O mice showed greater mortality (Figure 3D) and increased morbidity (Supplementary Figure 3A). Tissue injury, manifested by lactate dehydrogenase release in plasma, also was more

-,

No.

-

pronounced in Mye-K/O mice (Supplementary Figure 3B), which also displayed greater plasma increases of interleukin (Il)6 and tumor necrosis factor (Tnf) (Figure 3E). In contrast, plasma levels of the anti-inflammatory cytokine Il10 were comparable with WT. Tissue expression of proinflammatory genes was more pronounced in Mye-K/O mice, as exemplified by Tnf, as well as other proinflammatory genes (Figure 3F and Supplementary Figure 4). Altogether, these data indicate that Commd1 deficiency in the myeloid lineage leads to more profound acute inflammatory responses.

Reduced Expression of COMMD1 Is a Common Feature of IBD Given the findings made in the Mye-K/O mouse model, we decided to explore whether COMMD1 could play a role in the pathophysiology of inflammatory disorders in human beings, choosing IBD as a disease model. To this end, we evaluated the expression of COMMD1 in IBD patients. We found that COMMD1 messenger RNA (mRNA) levels in colonic endoscopic biopsy specimens were reduced in an Israeli cohort of patients with colitis (n ¼ 29, 18 with Crohn’s disease and 11 with ulcerative colitis) compared with unaffected controls (n ¼ 16; P ¼ .04) (Figure 4A, left). This finding was replicated in an independent patient cohort in the United States (P ¼ .04, 22 normal controls and 17 patients with IBD, 9 of whom had CD and 8 had UC) (Supplementary Figure 5). Similarly, we found that COMMD1 mRNA was reduced in circulating leukocytes from IBD patients with active disease (n ¼ 12, 9 with CD and 3 with UC) compared with normal individuals (n ¼ 14; P ¼ .001) (Figure 4A, right). Moreover, in a murine model of colitis induced by dextran sodium sulfate (DSS) administration, we also observed decreased mRNA and protein expression of Commd1 and other Commd genes in the colon (Figure 4B and C). In aggregate, the data indicate that mucosal inflammation is associated with decreased COMMD1 gene expression in clinical and experimental situations.

Genetic Variants Associated With Decreased COMMD1 Expression Are Associated With UC Risk In view of the reduced expression of COMMD1 in IBD patients, we investigated whether genetic polymorphisms of this gene might affect IBD risk in human beings. Interestingly, genome-wide association studies have linked an intergenic region near COMMD1 to the risk for various inflammatory conditions including IBD (Figure 4D),3,28–32 but the precise gene responsible for these effects remains unknown. We began by interrogating the most recent metaanalysis of genome-wide association studies for CD and UC3 by the IIBDGC, which currently is available on the Ricopili server (www.broadinstitute.org/mpg/ricopili/). These data indicated a suggestive association signal with UC for single-nucleotide polymorphisms (SNPs) located just downstream of COMMD1, including rs3943540


414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472

COMMD1 and Colitis

5

532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 analysis of the 1000Genomes–imputed IIBDGC UC and CD Q24588 meta-analyses also showed a suggestive association with 589 UC for a SNP located in the same region, rs6545924 590

Figure 2. Gene-selective effects of Commd1 on NFkB–regulated targets. BMDMs (2 mice per group, with triplicate cultures per mouse) were stimulated with LPS, and gene expression was determined by quantitative reverse-transcription polymerase chain reaction. (A) Selected NF-kB target genes are shown, and other (B) LPS-inducible genes that are not NF-kB targets also are shown.

print & web 4C=FPO

473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 Q23 531

2014


-

(P ¼ 7.89 10-5) and rs921319 (P ¼ 7.47 10-5) (Figure 4E). Interestingly, both SNPs are in linkage disequilibrium with each other (r2 ¼ 1, D’ ¼ 1). Additional


6


-,

No.

-

650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 Figure 3. Mye-K/O mice have a more severe sepsis response. (A) CLP leads to a higher mortality rate in Mye-K/O mice. Mye- Q2 684 F/F F/F K/O animals (Commd1 , LysM-Cre) were compared with WT littermate controls (Commd1 ). Survival without the need for Q3 euthanasia was plotted and the Kaplan–Meier estimate was calculated. (B) Peritoneal cultures yield lower bacterial counts in 685 Mye-K/O mice (9 mice in each group). Colony forming units (Cfu) in blood agar were determined by progressive dilution. 686 (C) Induction of proinflammatory gene expression is greater in Mye-K/O mice. Tissue RNA was analyzed by quantitative 687 reverse-transcription polymerase chain reaction (in kidney). To normalize for bacterial load, animals with no detectable bac- 688 terial counts were used for this analysis (WT, 4 mice; Mye-K/O, 6 mice). (D) Mye-K/O mice have greater mortality after LPS 689 administration. Survival was analyzed as in panel A. (E) Plasma levels of Il6, Tnf, and Il10 were determined in Mye-K/O mice 690 (n ¼ 9) and WT controls (n ¼ 10) at the time of euthanasia in the LPS model. (F) LPS induction of proinflammatory genes was determined by quantitative reverse-transcription polymerase chain reaction in tissues of Mye-K/O mice (n ¼ 10) and WT 691 692 controls (n ¼ 10). *P < .05. 693 694 695 -5 21 (P ¼ 2.27 10 ; odds ratio, 1.104; 95% confidence in- studied and 891 samples from the Estonian Biobank). 696 terval of the odds ratio, 1.06–1.15). Because the SNPs in question were not present in our eQTL 697 Epigenetic analysis in myeloid cells performed by the data set, we tested rs921320, located in very close prox- 698 Q25 ENCODE project33 showed that this genomic locus displays imity to rs921319 and a perfect proxy SNP for rs6545924 699 features of a gene regulatory region (Figure 4D and E), (r2 ¼ 0.9, D’ ¼ 1). We observed a significant eQTL effect of 700 such as histone 3 lysine 4 monomethylation and lysine 27 rs921320 on COMMD1 expression, with the same allelic 701 acetylation, both associated with active enhancers.34 In direction for the Dutch and Estonian cohorts (the P values 702 agreement with this notion, the SCAN database35 indicates for individual cohorts were .057 and .0099, respectively; 703 that there is a significant eQTL effect of rs921319 on with a weighted Z-method meta-analysis P ¼ .0033). The 704 COMMD1 gene expression. To further examine the possi- A–allele of rs921320, which is linked to the risk allele for 705 bility that these variants have an effect on COMMD1 rs6545924, was associated with reduced expression of 706 expression, we conducted cis-eQTL mapping on whole pe- COMMD1 (Figure 4F). On the other hand, we did not 707 ripheral blood of 2 cohorts (1240 samples previously observe any eQTL effects of rs921320 on the 2 other 708 print & web 4C=FPO


591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649

Li et al


-

COMMD1 and Colitis

7

768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 Figure 4. Lower COMMD1 expression is linked to IBD. (A) COMMD1 gene expression is suppressed in IBD patients. COMMD1 799 mRNA expression was determined by quantitative reverse transcription polymerase chain reaction in endoscopic colonic biopsy specimens from IBD patients with colitis (n ¼ 29, 18 with CD and 11 with UC) and normal controls (n ¼ 17). Similarly, 800 COMMD1 mRNA levels in white blood cells (WBCs) were determined in IBD patients (n ¼ 12, 9 with CD and 3 with UC) and 801 unaffected controls (n ¼ 14). (B and C) Commd gene expression at the (B) mRNA level was decreased similarly in colonic tissue 802 after DSS-induced acute colitis (7 mice in the DSS group compared with 8 mice in the water group), and (C) this was shown at 803 the protein level for Commd1 and Commd9. *P < .05, ‡P < .001, AP < .0001. (D) Polymorphisms near COMMD1 are 804 associated with UC risk. A map of the 2p15 locus, including SNPs previously implicated in inflammatory diseases, is shown. 805 The region located 3’ of the COMMD1 gene, where SNPs associated to UC were found, is marked by a red box. (E) A closer 806 view of the location of the SNPs of interest downstream of COMMD1 is shown, including chromatin modifications such as histone 3 K4 monomethylation (H3K4m1), trimethylation (H3K4m3), and histone 3 K27 acetylation (H3K27Ac). (F) cis-eQTL 807 analysis for rs921320 in 2 independent cohorts. COMMD1 gene expression (in the y axis) is shown in a box plot for all 3 808 genotypes for this SNP (CC, CA, or AA). The horizontal bar in the box is the median, the top and bottom of the box show the 809 interquartile range (Q3 - Q1); the whiskers show the quartile þ 1.5 the interquartile range. The number of individuals in each Q4 810 Q5 group is indicated (under the genotype); males and females are denoted by blue dots and red dots, respectively. 811 812 813 neighboring genes, B3GNT2 or TMEM17. Altogether, these lamina propria (Supplementary Figure 6). Furthermore, 814 findings suggest that genetic variation in the 3’ region of confocal microscopy images confirmed that resident lam- 815 COMMD1, which has regulatory effects on gene expression, ina propria macrophages (F4/80þ) are also COMMD1 816 positive, in agreement with Western blot analysis of pri- 817 is linked to risk for UC. mary myeloid cells (Figure 1A). With this in mind and in 818 view of the known contributions of both epithelial and 819 Myeloid Cell Deficiency of Commd1 Leads to myeloid cells in the pathogenesis of human and murine 820 More Severe Colitis colitis,36,37 we evaluated the role of this gene in this dis- 821 To further examine the possible involvement of ease process using both epithelial and myeloid cell– 822 COMMD1 in IBD pathogenesis, we began by assessing the specific knockout mice. 823 in situ expression of this gene in the colon. ImmunofluoFirst, we used the DSS model, which triggers colonocyte 824 rescence staining of mouse colon tissue showed staining injury and an innate immune response to luminal bacteria 825 in the epithelium as well as in mononuclear cells in the that results in acute colitis. Mye-K/O mice showed more 826


print & web 4C=FPO

709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767

2014


8

print & web 4C=FPO


827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885

Li et al


-,

No.

-

886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 Figure 5. Myeloid deficiency of Commd1 predisposes to more severe colitis. (A) Worse survival, weight loss, and disease 922 activity index (DAI) were noted in Mye-K/O mice compared with wild-type littermate controls during acute DSS-induced colitis. Q6 923 *P < .05, ‡P < .001, AP < .0001. (B) Colon length was significantly shorter in Mye-K/O mice (n ¼ 26) after acute colitis 924 compared with WT controls (n ¼ 20). Representative images are shown. (C) Worse tissue injury also was evident histologically. 925 By using a severity score, colonic sections were examined (11 Mye-K/O and 9 WT mice). Representative histologic sections are shown. (D and E) Expansion of macrophage populations in the colonic mucosa of Mye-K/O mice. In the left panel, the 926 number of CD68þ cells present in the mucosa was assessed by immunohistochemistry in a tissue microarray of DSS colitis 927 samples (Mye-K/O, 13 samples; WT, 12 samples). In the right panel, the colonic expression of the macrophage marker gene 928 Emr1 (F4/80) was determined by quantitative reverse-transcription polymerase chain reaction (Mye-K/O, 18 samples; WT, 23 929 samples). (E) Representative images of the CD68 immunohistochemistry are shown. Scale bar: 20 mm. (F) RNA extracted from 930 colonic tissue was used for quantitative reverse-transcription polymerase chain reaction analysis for the genes indicated 931 (Mye-K/O, 18 samples; WT, 23 samples). 932 933 934 severe disease than WT animals, with excess mortality, greater numbers of CD68þ myeloid cells in the colon as seen 935 more profound weight loss, and worse disease activity by immunohistochemistry (Figure 5D, left panel, and E), and 936 (Figure 5A). In agreement with this, colon shortening a consistent trend was observed at the mRNA level for the 937 (a macroscopic marker of tissue injury) and histologic macrophage-specific gene Emr1 (F4/80, Figure 5D, right 938 evaluation of the colonic mucosa both indicated more panel). In addition, Mye-K/O mice showed increased 939 damage in Mye-K/O mice (Figure 5B and C). In contrast to expression of proinflammatory genes, such as Il6 and Il1b 940 these results, Commd1 inactivation in the intestinal epithe- (Figure 5F). However, myeloid and lymphoid populations in 941 lium did not result in any appreciable alteration of DSS- the mesenteric lymph node (Supplementary Figure 8) and 942 induced colitis (Supplementary Figure 7). The more severe the spleen (not shown), were not significantly different 943 inflammatory response in Mye-K/O mice correlated with between the groups. 944 FLA 5.2.0 DTD YGAST59081_proof 20 May 2014 5:04 pm ce

-

COMMD1 and Colitis

Myeloid Cell Deficiency of Commd1 Promotes Colitis–Dysplasia Progression

9

(Figure 6D). Moreover, after 7 weeks of treatment, Mye-K/O 1004 mice had a greater propensity to develop dysplasia, high- 1005 grade dysplasia, or multifocal dysplasia as assessed histo- 1006 logically (Figure 6D). By 10 weeks, small adenomas could be 1007 visualized, but the total number of lesions was not increased 1008 in Mye-K/O mice (Figure 6E). Nevertheless, only Mye-K/O 1009 mice developed proximal colonic tumors and there was a 1010 tendency for the tumor burden to be greater in these ani- 1011 mals as a result of greater adenoma volume (Figure 6E). In 1012 colonic tissue, Mye-K/O animals showed greater NF-kB 1013 activation as evidenced by the ratio of the transcriptionally 1014 active form of RelA (phosphorylated at Ser536) to total 1015 RelA. However, STAT3 activation, another pathway known Q26 1016 to promote cancer in the context of chronic tissue 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062


Further analysis indicated that recurrent DSS administration, which mimics the chronic nature of IBD, also resulted in more severe disease in Mye-K/O mice, manifested as greater weight loss (Figure 6A) and more severe anemia (Figure 6B). Colonic tissue from these animals also produced greater amounts of several proinflammatory factors such as Tnf (Figure 6C and Supplementary Figure 9). In addition, azoxymethane followed by recurrent DSS treatments was used to examine progression to dysplasia. In this model, Mye-K/O mice also showed more severe disease, as evidenced by more severe colonic shortening, splenomegaly, and histologic distortion of the mucosal architecture

print & web 4C=FPO

945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003

2014

Figure 6. Commd1 deficiency leads to worse chronic colitis and transition to dysplasia. (A) Recurrent administration of DSS caused more weight loss in Mye-K/O mice. (B) Chronic colitis led to more severe anemia in Mye-K/O mice (5 mice per group). (C) Proinflammatory cytokine production is increased in colonic tissue of Mye-K/O mice. Colonic full-thickness tissue harvested from colitic mice was cultured overnight and the secretion of cytokines into the growth media was determined. (D) Progression to dysplasia was worse in Mye-K/O animals. Azoxymethane was administered before chronic colitis induction and dysplasia was assessed histologically at 7 weeks. Representative images are displayed. (E) Tumor number and volume in mice at 10 weeks of colitis/dysplasia induction is shown. *P < .05. (F) Greater NF-kB activation in inflamed colonic tissue of Mye-K/O mice. Activation of NF-kB/RelA and Stat3 was assessed in inflamed colonic tissue of animals treated with AOM/DSS. Western blot analysis was performed with tissue lysates and the active phosphorylated form was quantitated and normalized to expression of the respective target. HGD, high-grade dysplasia; LGD, low-grade dysplasia; MFD, multifocal dysplasia.


10

Li et al



38 1063 inflammation, was not altered in these mice (Figure 6F). 1064 Altogether, these data highlight a role for myeloid cell 1065 expression of Commd1 in chronic colonic inflammation and 1066 progression to dysplasia. 1067 1068 Discussion 1069 COMMD1 plays an essential role to restrain the activity 1070 of NF-kB, a master regulator of inflammation. Specifically, 1071 COMMD1 acts to terminate NF-kB transcriptional activation 1072 through the ubiquitin-mediated degradation of NF-kB sub1073 units. A detailed molecular mechanism by which COMMD1 1074 restrains NF-kB has been examined and reported previ1075 8,10,12,13 The studies presented here confirm the ously. 1076 negative regulatory role of this factor on the NF-kB 1077 10,12,13,15 and also underscores the gene-selective 1078 pathway, 1079 nature of these effects. Moreover, the studies in this model 1080 corroborate that key aspects of the reported mechanism are 1081 evident in the knockout mice, namely normal IkB phos1082 phorylation and turnover, but diminished ability to termi1083 nate NF-kB transcriptional activity (ie, reduced RelA/p65 Q27 1084 ubiquitination). Importantly, this study investigates the 1085 physiological relevance of these events in disease patho1086 genesis. The data indicate that COMMD1 expression in the 1087 myeloid compartment plays an important role in the reguthat diverse 1088 lation of inflammatory responses in vivo. Given 17,39–41 this functions have been reported for this gene, 1089 report fills a critical void by underscoring that COMMD1 1090 1091 participates in immune regulation. In addition, the results 1092 also suggest that COMMD1 has broader effects on proin1093 flammatory gene expression programs that might extend 1094 beyond the NF-kB pathway. Whether these effects are the 1095 result of secondary perturbations or are the result of direct 1096 regulation of transcription factor(s) other than NF-kB, 1097 should be examined further in the future. In the context of intestinal inflammation and progression 1098 to cancer, previous studies have underscored the impor1099 of epithelial and myeloid cells in these pro1100 tance 14,37,38 In particular, epithelial intrinsic NF-kB 1101 cesses. activation has been shown to be a protective response that 1102 promotes cell survival and barrier function,42 and myeloid 1103 is important in sustaining the inflam1104 cell NF-kB activation 36 The increased propensity to inflammamatory response. 1105 tion and colitis observed as a result of myeloid cell 1106 deficiency of Commd1 is in agreement with this paradigm 1107 and suggests that the immune regulatory roles of this gene 1108 Q28 are physiological. In addition, the increased progression to 1109 1110 dysplasia in Mye-K/O mice is in agreement with the role of tumor growth through various 1111 myeloid cells in promoting 43 In this regard, our model proinflammatory cytokines. 1112 displays increased production of Tnf and greater tissue 1113 activation of NF-kB, both of which have been shown to 1114 36,44 In 1115 promote cancer progression in the setting of colitis. addition, we also found no appreciable phenotype of 1116 enterocyte-specific deficiency of Commd1, which stands in 1117 contrast to other models of disrupted NF-kB activity in the 1118 36,42 This may reflect the fact that unlike other 1119 epithelium. models, cell survival genes are not regulated by COMMD1 in 1120 the transcriptome analysis presented here. Furthermore, it 1121

-,

No.

-

suggests that other reported roles of Commd1, such as the regulation of various transport mechanisms, may be more relevant in these cells. These observations in the animal model, coupled with the finding that genetic variants linked to lower COMMD1 expression also are associated with an increased risk for UC, suggest that this gene participates in the pathogenesis of this disorder in human beings. In this context, it is important to note a recent report of a risk association for COMMD7, another member of the COMMD gene family.45 Moreover, COMMD1 acts in concert with Cul2 to promote RelA ubiquitination,10,12,13 and the CUL2 gene has been linked to IBD susceptibility.46 Additional studies, including ongoing resequencing efforts, will be required to validate the genetic association seen here with greater statistical confidence. However, the aggregate of our data and published reports suggest the possibility that homeostatic mechanisms that restore NF-kB to its basal state, when decreased, may enhance the risk for IBD. Our studies also identified that inflammation can lead to suppressed expression of COMMD1, as seen in patients with colitis as well as in animal models. Our data suggest that this is likely the consequence rather than the cause of inflammation in human disease because this suppression can be induced by inflammation itself in animal studies. The decrease in COMMD1 expression probably plays a physiologic role in the initiation of an appropriate inflammatory response, but in view of the more severe inflammatory responses seen when this gene is deleted in myeloid cells, persistent COMMD1 suppression probably is maladaptive during chronic inflammation. Therefore, this work highlights the need to gain a refined understanding of the molecular events that control COMMD1 gene expression in the setting of inflammation. With this knowledge, we may begin to envision potential interventions that might restore gene expression, which could be tested for their therapeutic potential in chronic inflammatory disorders.

1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 Supplementary Material 1162 Note: To access the supplementary material accompanying 1163 this article, visit the online version of Gastroenterology at 1164 www.gastrojournal.org, and at http://dx.doi.org/10.1053/ 1165 j.gastro.2014.04.007. 1166 1167 1168 References 1169 1. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel 1170 disease. Annu Rev Immunol 2010;28:573–621. 1171 2. Hayden MS, Ghosh S. NF-kB, the first quarter-century: 1172 remarkable progress and outstanding questions. Genes 1173 Dev 2012;26:203–234. 1174 3. Jostins L, Ripke S, Weersma RK, et al. Host-microbe 1175 interactions have shaped the genetic architecture of in- 1176 flammatory bowel disease. Nature 2012;491:119–124. Q58 1177 4. Lees CW, Barrett JC, Parkes M, et al. New IBD genetics: 1178 common pathways with other diseases. Gut 2011; 1179 60:1739–1753. 1180


1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239

2014

COMMD1 and Colitis

5. Hoffmann A, Levchenko A, Scott ML, et al. The IkB-NFkB signaling module: temporal control and selective gene activation. Science 2002;298:1241–1245. 6. Kovalenko A, Chable-Bessia C, Cantarella G, et al. The tumour suppressor CYLD negatively regulates NF-kB signalling by deubiquitination. Nature 2003;424:801–805. 7. Wertz IE, O’Rourke KM, Zhou H, et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kB signalling. Nature 2004;430:694–699. 8. Natoli G, Chiocca S. Nuclear ubiquitin ligases, NF-kB degradation, and the control of inflammation. Sci Signal 2008;1:pe1. 9. Saccani S, Marazzi I, Beg AA, et al. Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor kB response. J Exp Med 2004;200:107–113. 10. Mao X, Gluck N, Li D, et al. GCN5 is a required cofactor for a ubiquitin ligase that targets NF-kB/RelA. Genes Dev 2009;23:849–861. 11. Ryo A, Suizu F, Yoshida Y, et al. Regulation of NF-kB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell 2003;12:1413–1426. 12. Maine GN, Mao X, Komarck CM, et al. COMMD1 promotes the ubiquitination of NF-kB subunits through a Cullin-containing ubiquitin ligase. EMBO J 2007;26: 436–447. 13. Geng H, Wittwer T, Dittrich-Breiholz O, et al. Phosphorylation of NF-kB p65 at Ser468 controls its COMMD1dependent ubiquitination and target gene-specific proteasomal elimination. EMBO Rep 2009;10:381–386. 14. Burstein E, Hoberg JE, Wilkinson AS, et al. COMMD proteins: a novel family of structural and functional homologs of MURR1. J Biol Chem 2005;280:22222–22232. 15. Ganesh L, Burstein E, Guha-Niyogi A, et al. The gene product Murr1 restricts HIV-1 replication in resting CD4þ lymphocytes. Nature 2003;426:853–857. 16. van de Sluis B, Rothuizen J, Pearson PL, et al. Identification of a new copper metabolism gene by positional cloning in a purebred dog population. Hum Mol Genet 2002;11:165–173. 17. Biasio W, Chang T, McIntosh CJ, et al. Identification of Murr1 as a regulator of the human d epithelial sodium channel. J Biol Chem 2004;279:5429–5434. 18. Drevillon L, Tanguy G, Hinzpeter A, et al. COMMD1mediated ubiquitination regulates CFTR trafficking. PLoS One 2011;6:e18334. 19. Smith L, Litman P, Liedtke CM. COMMD1 interacts with the COOH terminus of NKCC1 in Calu-3 airway epithelial cells to modulate NKCC1 ubiquitination. Am J Physiol Cell Physiol 2013;305:C133–C146. 20. van de Sluis B, Mao X, Zhai Y, et al. COMMD1 disrupts HIF-1a/b dimerization and inhibits human tumor cell invasion. J Clin Invest 2010;120:2119–2130. 21. Fu J, Wolfs MG, Deelen P, et al. Unraveling the regulatory mechanisms underlying tissue-dependent genetic variation of gene expression. PLoS Genet 2012;8:e1002431. 22. Fehrmann RS, Jansen RC, Veldink JH, et al. TranseQTLs reveal that independent genetic variants associated with a complex phenotype converge on

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

37.

38.

11

intermediate genes, with a major role for the HLA. PLoS Genet 2011;7:e1002197. van de Sluis B, Muller P, Duran K, et al. Increased activity of hypoxia-inducible factor 1 is associated with early embryonic lethality in Commd1 null mice. Mol Cell Biol 2007;27:4142–4156. Vonk WI, Bartuzi P, de Bie P, et al. Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation. PLoS One 2011;6:e29183. Mao X, Gluck N, Chen B, et al. Copper metabolism MURR1 domain containing 1 (COMMD1) regulates Cullin-RING ligases by preventing Cullin-associated NEDD8-dissociated (CAND1) binding. J Biol Chem 2011;286:32355–32365. Starokadomskyy P, Gluck N, Li H, et al. CCDC22 deficiency in humans blunts activation of proinflammatory NF-kB signaling. J Clin Invest 2013; 123:2244–2256. Barish GD, Yu RT, Karunasiri M, et al. Bcl-6 and NF-kB cistromes mediate opposing regulation of the innate immune response. Genes Dev 2010;24:2760–2765. Reveille JD, Sims AM, Danoy P, et al. Genome-wide association study of ankylosing spondylitis identifies non-MHC susceptibility loci. Nat Genet 2010;42: 123–127. Okada Y, Terao C, Ikari K, Kochi Y, et al. Meta-analysis identifies nine new loci associated with rheumatoid arthritis in the Japanese population. Nat Genet 2012; 44:511–516. Kenny EE, Pe’er I, Karban A, et al. A genome-wide scan of Ashkenazi Jewish Crohn’s disease suggests novel susceptibility loci. PLoS Genet 2012;8:e1002559. Anderson CA, Boucher G, Lees CW, et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat Genet 2011;43:246–252. Franke A, McGovern DP, Barrett JC, et al. Genomewide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet 2010;42:1118–1125. Consortium TEP. A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 2011;9:e1001046. Kaikkonen MU, Spann NJ, Heinz S, et al. Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol Cell 2013;51: 310–325. Nicolae DL, Gamazon E, Zhang W, et al. Trait-associated SNPs are more likely to be eQTLs: annotation to enhance discovery from GWAS. PLoS Genet 2010;6:e1000888. Greten FR, Eckmann L, Greten TF, et al. IKKb links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 2004;118:285–296. Bollrath J, Phesse TJ, von Burstin VA, et al. gp130mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitisassociated tumorigenesis. Cancer Cell 2009;15:91–102. Bollrath J, Greten FR. IKK/NF-kB and STAT3 pathways: central signalling hubs in inflammation-mediated tumour promotion and metastasis. EMBO Rep 2009;10: 1314–1319.


1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298


-

12


1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357

Li et al


39. Weiss KH, Lozoya JC, Tuma S, et al. Copper-induced translocation of the Wilson disease protein ATP7B independent of Murr1/COMMD1 and Rab7. Am J Pathol 2008;173:1783–1794. 40. Miyayama T, Hiraoka D, Kawaji F, et al. Roles of COMM-domain-containing 1 in stability and recruitment of the copper-transporting ATPase in a mouse hepatoma cell line. Biochem J 2010;429:53–61. 41. Chang T, Ke Y, Ly K, et al. COMMD1 regulates the delta epithelial sodium channel (dENaC) through trafficking and ubiquitination. Biochem Biophys Res Commun 2011; 411:506–511. 42. Nenci A, Becker C, Wullaert A, et al. Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 2007;446:557–561. 43. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010;140:883–899. 44. Popivanova BK, Kitamura K, Wu Y, et al. Blocking TNF-a in mice reduces colorectal carcinogenesis associated with chronic colitis. J Clin Invest 2008;118:560–570. 45. Kabakchiev B, Silverberg MS. Expression quantitative trait loci analysis identifies associations between genotype and gene expression in human intestine. Gastroenterology 2013;144:1488–1496. 1496 e1–3. 46. Rivas MA, Beaudoin M, Gardet A, et al. Deep resequencing of GWAS loci identifies independent rare

-,

No.

-

variants associated with inflammatory bowel disease. Nat Genet 2011;43:1066–1073.

1358 1359 1360 1361 Author names in bold designate shared co-first authorship. 1362 Received December 11, 2013. Accepted April 5, 2014. 1363 Reprint requests Q12 1364 Address requests for reprints to: Ezra Burstein, MD, University of Texas Q111365 Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas Q13 1366 75230-9151. e-mail: [email protected]; fax: (214) 648-2022. 1367 Acknowledgments 1368 The authors are grateful to Eric Fearon for kindly providing the reagents used here. The authors are also grateful to John Shelton, Christine Komarck, and 1369 Baozhi Chen for technical support for this project. 1370 1371 Conflicts of interest Q7 The authors disclose no conflicts. 1372 1373 Funding Supported by a National Institutes of Health grant (R01 DK073639 to E.B.), a Q81374 CCFA Senior Research Award (SRA 2737 to E.B.), a Cancer Prevention and 1375 Research Institute of Texas grant (CPRIT RP130409 to E.B.), the Disease Oriented Clinical Scholars’ Program at University of Texas Southwestern 1376 (E.B.), a VIDI grant (91713308 to R.K.W.) from The Netherlands Organization 1377 for Scientific Research, a start-up grant from the US–Israel Binational Science Foundation (BSF 2009339 to N.G.), a grant from the German Israeli 1378 Foundation (N.G.), and the Estonian Genome Center at the University of 1379 Q9 Tartu (T.E. and A.P.) received financing from FP7 programs (ENGAGE, 1380 OPENGENE), targeted financing from the Estonian Government (SF0180142s08), Estonian Research Roadmap through the Estonian Ministry 1381 of Education and Research, Center of Excellence in Genomics (EXCEGEN), 1382 and the University of Tartu (SP1GVARENG). 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416


-

2014

COMMD1 and Colitis

1417 Supplementary Materials and Methods 1418 Mouse Strains 1419 Mice were housed in barrier facilities and fed an irra1420 diated standard diet. All animal procedures were approved 1421 by the Care and Use Committee. The conditional Commd1 1422 allele recently was described.1 LysM-Cre knock-in mice 1423 Q29 were obtained from the Jackson Laboratory (Bar Harbor, 1424 ME). Villin-Cre mice kindly were provided by Dr Eric 1425 Fearon. 1426 1427 Sepsis Models 1428 Cecal ligation and puncture was performed as previously 1429 described.2 Overall, the surgical time was kept to fewer than 1430 15–20 minutes. After surgery, the mice were housed indi1431 vidually and monitored in a blinded manner (ie, without 1432 knowledge of the specific genotypes). This was performed 1433 every hour for the first 4–6 hours, then every 2 hours for the 1434 next 6 hours, and, finally, every 4–6 hours until the 1435 conclusion of the experiment or the need for euthanasia 1436 arose (based on morbidity criteria) (Supplementary 1437 Table 5). For the LPS model, LPS (Escherichia coli 055:B5; 1438 Q30 Sigma) was diluted in water to 0.2 mg/mL and placed in a 1439 water bath sonicator (model FS20; Fisher) for 15–30 mi1440 nutes at room temperature before use. After sonication, the 1441 LPS was filtered (0.2-mm pore) and injected intraperitone1442 ally (0.1 mg/mouse). After injection, the mice were moni1443 tored similarly for morbidity until the conclusion of the 1444 experiment (Supplementary Table 5). Bacterial cultures 1445 were performed as previously described3 and tissue RNA 1446 was extracted as noted later. Blood also was collected 1447 through orbital puncture, and plasma was separated using 1448 EDTA (5 mmol/L). 1449 1450 Acute and Chronic DSS-Induced Colitis 1451 For the acute colitis model, DSS (Fisher) was adminis1452 tered in the drinking water at a concentration of 3 g/dL 1453 (3%). Freshly prepared DSS drinking solution was replaced 1454 every 5 days. Mice were housed in individual cages and 1455 evaluated daily for their weight and the presence of diar1456 rhea or bleeding to calculate a disease activity index 1457 (Supplementary Table 6), which was based on the scoring 1458 system reported by Cooper et al.4 Evaluation for occult 1459 Q31 bleeding included Hemoccult testing (Beckman Coulter). 1460 1461 Treatment was given for up to 10 days and animals showing 1462 severe distress were euthanized at any point during the 1463 experiment. For the chronic colitis and cancer model, the animals 1464 were given azoxymethane intraperitoneally (10 mg/kg; 1465 Sigma). After 7 days, DSS was given in the drinking water 1466 over 4 days at a concentration of 2.5% followed by 10 days 1467 of regular water. This was repeated for 3 cycles and at the 1468 Q32 end of 7 of 10 weeks, the animals were euthanized, and the 1469 spleen and colon were harvested. The organ length and 1470 weight were determined and the colon was opened along 1471 1472 the mesenteric side, and tumors were counted using a disQ33 1473 secting microscope (Zeiss Stemi 2000-C). Tumor volumes 2 Q34 1474 were calculated using the following formula: (width ) (height/2). 1475

12.e1

Histology and Immunohistochemistry

1476 At the time of euthanasia, the colon was dissected and 1477 opened along the mesenteric side. The open colon was 1478 transected longitudinally with one half rolled onto itself as a 1479 Swiss roll and fixed in 4% PFA (in phosphate-buffered sa- Q351480 line). After paraffin embedding and sectioning, the tissue 1481 was stained with H&E. The second half was used for RNA 1482 extraction as described later. A histologic severity index4 1483 was used to score colonic sections from mice with acute 1484 DSS colitis (Supplementary Table 7). In chronic colitis/ 1485 dysplasia models (azoxymethane/DSS at 7 weeks), Swiss- 1486 roll sections of the colon were assessed for the presence 1487 of architectural distortion, dysplasia, multifocal dysplasia, or 1488 high-grade dysplasia according to standard clinical pathol- 1489 ogy criteria. The number of individual foci was not quanti- 1490 tated by this approach. All histopathologic analyses were 1491 performed in a blinded fashion by a gastrointestinal 1492 pathologist (S.D.M.). For CD68 staining, a tissue microarray 1493 was generated using random colonic tissue cores obtained 1494 from blocks previously prepared from mice with acute co- 1495 litis. After constructing the microarrays, 5-mm sections were 1496 subjected to CD68 staining (clone F8–11; Biolegend). Q361497 Staining was performed on a Ventana Benchmark XT 1498 Automated IHC Stainer with Mild CC1 (Cell Conditioning 1) 1499 and 32 minutes of primary antibody incubation at a 1:25 1500 dilution (in Chemmate Ab diluent). The detection was per- 1501 formed with an Ultraview Universal DAB detection Kit (cat. 1502 760–500; Ventana). The slides were counterstained with 1503 Mayer’s hematoxylin. Images then were analyzed using 1504 ImageJ software (National Institutes of Health, Bethesda, 1505 MD) and cell counts were normalized to the surface area 1506 using the surface of a microscopic field as a reference. Co- 1507 staining for COMMD15 and F4/80 (11–4801; eBioscience) 1508 was performed using frozen sections of mouse colon tissue 1509 Q37 embedded in OCT using a previously reported protocol. 1510

1511 1512 EDTA plasma was separated from blood samples by 1513 centrifugation and stored at -80 C. Full-thickness colonic 1514 tissue (1 cm of rectum) was harvested form colitic mice and 1515 cultured in a 12-well cell culture dish with 1 mL of complete 1516 Dulbecco’s modified Eagle medium culture media. The su- 1517 pernatant was collected 24 hours later, centrifuged, and 1518 stored at -80 C until measurement. Cytokine measurements 1519 were performed using Milliplex MAP beads (Millipore) and Q381520 run on a Luminex Magpix with the Luminex xPONENT 1521 software. Data analysis was performed using Milliplex 1522 1523 Analyst. 1524 1525 Cell Isolation and Cell Culture 1526 The generation and immortalization of mouse embryo 1527 fibroblasts from the Commd1 floxed mouse has been re1528 6 ported previously. These cells were maintained in high1529 glucose Dulbecco’s modified Eagle medium containing 1530 10% fetal calf serum and treated with MG-132 (20 mmol/L 1531 for 3 h) for precipitation of ubiquitinated RelA, as described 1532 later. BMDMs were generated by isolation of bone marrow 1533 cells from long bones, which were cultured in RPMI Q39 1534

Multiplex Cytokine Measurements


12.e2

Li et al


1535 supplemented with 10% fetal calf serum, antibiotics (100 1536 mg/mL penicillin G and 100 mg/mL streptomycin), and 1537 Fungizone (Fisher), in the presence of granulocyte1538 macrophage colony–stimulating factor (20 ng/mL; PeproQ40 1539 tech). Myeloid differentiation was allowed to proceed for 10 1540 days. In specific experiments, cells were treated with LPS 1541 (10 ng/mL, E coli 026:B6; Sigma). Hypoxia conditions (3% Q41 1542 oxygen) were established in HeraCell incubators. Spleno1543 cytes, thymocytes, or mesenteric lymph node cells were 1544 isolated according to standard protocols. Briefly, after 1545 removing the respective organs and placing them on a 1546 sterile dish containing RPMI, the tissue manually was strainer. After 1547 ground and strained through a 40-mm tissue C), the cell pellet centrifugation (300g for 5 minutes at 4 1548 Q42 was resuspended in 2 mL of ACK buffer (150 mmol/L 1549 þ Q43 1550 NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA, pH 7.2) to 1551 lyze red blood cells. After 3 minutes at room temperature, 1552 the ACK lysis was neutralized by adding 10 mL of fluoresQ44 1553 cence-activated cell sorter media buffer (0.01% sodium 1554 azide, 2.5% fetal calf serum in phosphate-buffered saline). 1555 This cell suspension was strained again through the tissue 1556 strainer to obtain a single-cell suspension, which was pel1557 leted by centrifugation. Gastrointestinal epithelial cells were7 1558 isolated according to a previously described method. 1559 Briefly, the entire colon, small bowel, or stomach was har1560 vested and flushed clean with 1 mmol/L dithiothreitol in 1561 Krebs–Ringer bicarbonate buffer (10 mmol/L D-glucose, 0.5 1562 mmol/L MgCl2, 4.6 mmol/L KCl, 120 mmol/L NaCl, 0.7 1563 mmol/L Na2HPO4, 1.5 mmol/L NaH2PO4, 15 mmol/L 1564 NaHCO3). The lumen was filled with Krebs–Ringer bicar1565 bonate buffer supplemented with 10 mmol/L EDTA and 1566 1 mmol/L dithiothreitol, and both ends were ligated. The mL 1567 ligated colon was placed in a 50-mL conical tube with 30 C for of Krebs–Ringer bicarbonate buffer and placed at 37 1568 20 minutes with agitation. The sealed intestinal segment 1569 1570 was palpated gently to loosen the cells, then cut on one end Q45 1571 to drain the contents into a 1.7-mL microfuge tube. The cells 1572 were collected by centrifugation at 300g for 5 minutes and 1573 rinsed once with Krebs–Ringer bicarbonate buffer. 1574 1575 Flow Cytometry 1576 Cells were resuspended in 2 mL of fluorescence1577 activated cell sorter media buffer and the cells from the 1578 mesenteric lymph node were resuspended in 600 mL. For 1579 antibody staining, 100 mL of each cell suspension was 1580 incubated on ice with the following primary antibodies: 1581 Q46 Q47 anti-B220-PE (BD Pharmingen), anti-CD3–PE (BD Phar1582 1583 mingen), anti-CD4–perCP (BD Pharmingen), anti-CD8a–APC 1584 (BD Pharmingen), anti-Ly6G–APC (eBioscience), anti1585 Ly6C–PerCP-Cy5.5 (eBioscience), anti-CD11C–APC (BD 1586 Pharmingen), anti-CD11b–PE (eBioscience), anti-F4/ 1587 80–fluorescein isothiocyanate (eBioscience), and correwere 1588 sponding isotype controls. After 30 minutes, the cells C) and pelleted by centrifugation (300g for 5 minutes at 4 1589 washed twice in 500 mL of fluorescence-activated cell sorter 1590 1591 media buffer. Flow cytometric analysis was performed using 1592 FACSCalibur (BD Biosciences, Mountain View, CA) and Q48 1593 analyzed using FlowJo software.

Protein Extraction, Immunoblotting, and Immunoprecipitation

-,

No.

-

1594 1595 Cell lysate preparation, nuclear extract preparation, 1596 immunoprecipitation, denatured immunoprecipitation, and 1597 immunoblotting were performed as previously 1598 described.5,8,9 Protein lysates from mouse tissues were 1599 prepared after snap freezing the samples in liquid nitrogen. 1600 After mincing with a razor blade, tissue pieces were sub- 1601 merged in lysis buffer (50 mmol/L Tris-Hcl, pHþ 7.5, 250 1602 mmol/L NaCl, 3 mmol/L EDTA, 3 mmol/L ethylene glycol- 1603 bis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid, 1% 1604 Triton X-100, 0.5% NP40, 10% glycerol, and 25 mmol/L Na- Q491605 pyrophosphate) supplemented with protease inhibitor tab- 1606 lets (Roche). Thereafter, tissue homogenization was per- Q501607 formed using a 2-mL Douncer and a cleared lysate 1608 (supernatant after 10,000g centrifugation) was used for 1609 further analysis. The following antibodies were used for 1610 Western blotting in this study: b-actin (A5441; Sigma), 1611 COMMD1,5 COMMD9,10 IkB-a (06–494; Upstate Bio- 1612 technologies), phosphor-IkB-a (9246; Cell Signaling), RelA 1613 (sc-372 and sc-8008; Santa Cruz Biotechnology, Santa Cruz, 1614 CA), and ubiquitin (3936; Cell Signaling). 1615 1616 1617 RNA Extraction and Quantitative Real-Time 1618 Reverse-Transcription Polymerase Chain 1619 Reaction 1620 In most cases, RNA was extracted using TRIzol (Invi- 1621 trogen) according to the manufacturer’s instructions. In the Q511622 case of colonic RNA from colitic mice, we obtained better 1623 results by first stabilizing the freshly isolated tissue in 1624 RNAlater (Qiagen), followed by RNA extraction using the 1625 RNeasy chromatography method (Qiagen), according to the 1626 manufacturer’s instructions. RNA (5 mg) was used for 1627 complementary DNA synthesis using the Superscript III 1628 strand synthesis system (Invitrogen). Real-time polymerase 1629 chain reaction was performed in an Eppendorf real-time 1630 polymerase chain reaction system. In most cases, SYBR 1631 Green–based detection (Invitrogen) was used using gene- 1632 specific primers, as noted in Supplementary Table 8. Ex- 1633 periments were performed in triplicate, data were normal- 1634 ized to housekeeping genes, and the relative abundance of 1635 transcripts was calculated by the comparative DDCt Q521636 1637 method. 1638 1639 Microarray Analysis 1640 The primary data for this analysis are available in the 1641 GEO database at NCBI (Accession GSE53368). The Whole Q531642 Mouse Genome Oligo Microarray V2 (G4846A, ID 026655; 1643 Agilent Technologies, Santa Clara, CA) used in this study 1644 contains 44,397 oligonucleotide probes covering the entire 1645 murine transcriptome. Synthesis of Cy3-labeled comple- 1646 mentary RNA was performed with the Quick Amp Labeling 1647 kit, one color (5190-0442; Agilent Technologies) according 1648 to the manufacturer’s recommendations. Complementary 1649 RNA fragmentation, hybridization, and washing steps also 1650 were performed exactly as recommended per the One-Color 1651 Microarray-Based Gene Expression Analysis Protocol V5.7. 1652


-

2014

COMMD1 and Colitis

1653 Slides were scanned on the Agilent Micro Array Scanner 1654 G2565CA (pixel resolution, 5 mm; bit depth, 20). Data 1655 extraction was performed with the Feature Extraction 1656 Software V10.7.3.1 by using the recommended default 1657 extraction protocol file GE1_107_Sep09.xml. The single1658 channel data generated by the Feature Extraction software 1659 were normalized and analyzed in Genespring GX software, 1660 version 12.0 (Agilent Technologies). Low expression values 1661 were increased to the constant value 15 (defined as the 1662 threshold) to minimize false-positive ratios. Data were log2Q54 1663 transformed and normalized to the 75th percentile of each 1664 array according to the standard procedure in Genespring 1665 GX. Only probes referring to transcripts of annotated genes 1666 were considered and Agilent probe annotation was used. In 1667 1668 cases of multiple probes per gene the value from that 1669 probe with the highest arithmetic mean intensity across all 1670 12 hybridizations was selected. Genes were considered as 1671 measurable when the arithmetic mean of (anti1672 log2–retransformed) intensity values calculated from all 12 1673 samples was greater than 100 and the mean intensity in at 1674 least 1 of 6 averaged conditions was greater than 50. En1675 tities showing technical impairments were excluded from 1676 analyses. By using these criteria, of 44,397 probes, 18,682 1677 probes corresponding to 11,441 genes were selected for 1678 further analysis. Genes were considered as regulated by 1679 LPS or by Commd1 KO according to the following criteria: 1680 the fold-change of mean values in at least 1 of 3 ratios was 1681 greater than 3-fold and regulation was consistent with the 1682 same trend in each single (nonaveraged) comparison. The Q55 1683 following ratios were calculated: Commd1 KO vs WT, LPS 3 1684 hours WT vs WT, LPS 24 hours WT vs WT. In the resulting 1685 gene set Commd1-dependent genes were defined by at 1686 least 2-fold differences of the following ratios of mean in1687 tensity values: deregulated basal expression (ratio 1688 Commd1 KO vs WT), deregulated less than 3 hours LPS 1689 treatment (Commd1 KO LPS 3 h vs WT LPS 3 h), and 1690 deregulated less than 24 hours LPS treatment (Commd1 1691 KO LPS 24 h vs WT LPS 24 h). The ratio values of the 1692 resulting COMMD1-dependent 225 genes were analyzed by 1693 hierarchical cluster analysis using MeV Multi1694 ExperimentViewer, version 4.8.1, 2011 (available: www. Q56 1695 tm4.org, and Saeed et al, 2003) with cluster settings 1696 “average” as linkage method and “Manhattan distance” as 1697 distance measure. Q57 Gene function analysis was performed using the DAVID 1698 (v6.7) software platform (maintained by NIAID and 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711

12.e3

available at http://david.abcc.ncifcrf.gov/home.jsp). Functional annotation was performed using the KEGG database (KEGG Mapper v1.6, released October 1, 2013). The regulation of genes of interest by NF-kB was ascertained by comparing each gene to 2 partially redundant data sets: a curated database of published NF-kB gene targets (publicly available and maintained at www.NF-kB.org), and, second, a chromatin immunoprecipitation seq data set of RelAregulated genes in LPS-stimulated BMDMs.11

References 1. Vonk WI, Bartuzi P, de Bie P, et al. Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation. PLoS One 2011;6:e29183. 2. Toscano MG, Ganea D, Gamero AM. Cecal ligation puncture procedure. J Vis Exp 2011;pii:2860. 3. Kirkland D, Benson A, Mirpuri J, et al. B cell-intrinsic MyD88 signaling prevents the lethal dissemination of commensal bacteria during colonic damage. Immunity 2012;36:228–238. 4. Cooper HS, Murthy SN, Shah RS, et al. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 1993;69:238–249. 5. Burstein E, Hoberg JE, Wilkinson AS, et al. COMMD proteins: a novel family of structural and functional homologs of MURR1. J Biol Chem 2005;280:22222–22232. 6. Vonk WI, de Bie P, Wichers CG, et al. The coppertransporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression. Cell Mol Life Sci 2012;69:149–163. 7. Ahnen DJ, Reed TA, Bozdech JM. Isolation and characterization of populations of mature and immature rat colonocytes. Am J Physiol 1988;254:G610–G621. 8. Burstein E, Ganesh L, Dick RD, et al. A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J 2004;23:244–254. 9. Mao X, Gluck N, Li D, et al. GCN5 is a required cofactor for a ubiquitin ligase that targets NF-kB/RelA. Genes Dev 2009;23:849–861. 10. Starokadomskyy P, Gluck N, Li H, et al. CCDC22 deficiency in humans blunts activation of proinflammatory NF-kB signaling. J Clin Invest 2013;123:2244–2256. 11. Barish GD, Yu RT, Karunasiri M, et al. Bcl-6 and NF-kB cistromes mediate opposing regulation of the innate immune response. Genes Dev 2010;24:2760–2765.


1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770

Dietary Phenethyl Isothiocyanate Protects Mice from Colitis Associated Colon Cancer.

Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation.

Human Thyroid Cancer-1 (TC-1) is a vertebrate specific oncogenic protein that protects against copper and pro-apoptotic genes in yeast.

Positive selection of primate genes that promote HIV-1 replication.

The Mbd4 DNA glycosylase protects mice from inflammation-driven colon cancer and tissue injury.

6-gingerol protects against nutritional steatohepatitis by regulating key genes related to inflammation and lipid metabolism.

Otopetrin 1 protects mice from obesity-associated metabolic dysfunction through attenuating adipose tissue inflammation.

Muscle gets stressed? p53 represses and protects.

Necrostatin-1 reduces intestinal inflammation and colitis-associated tumorigenesis in mice.

Micheliolide, a new sesquiterpene lactone that inhibits intestinal inflammation and colitis-associated cancer.

11 deficiency protects against cholesterol crystallization and hepatic inflammation in hyperlipidemic mice.

Hemidesmosome integrity protects the colon against colitis and colorectal cancer.

type 17 responses.

NPC1L1 knockout protects against colitis-associated tumorigenesis in mice.

Spred2-deficiecy Protects Mice from Polymicrobial Septic Peritonitis by Enhancing Inflammation and Bacterial Clearance.

Editorial: Steely hair, mottled mice and copper metabolism.

VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis.

Loss of Sirt1 function improves intestinal anti-bacterial defense and protects from colitis-induced colorectal cancer.

COMMD1 regulates inflammation and colitis-associated cancer progression.

Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis.

Poly(ADP-ribose) polymerase 1 inhibition protects cardiomyocytes from inflammation and apoptosis in diabetic cardiomyopathy.

Chitinase-3-like protein 1 protects skeletal muscle from TNFα-induced inflammation and insulin resistance.

c mice.

Metabolism: Inflammation keeps old mice healthy.