ORIGINAL ARTICLE
Autophagy Suppression by Appendicitis and Appendectomy Protects Against Colitis Rajkumar Cheluvappa, MBBS, PhD, Annie S. Luo, BSc, and Michael C. Grimm, MBBS, FRACP, PhD
Background: When done at a young age, appendicitis followed by appendectomy (AA) offers protection against ulcerative colitis development in later life. We developed the first ever murine AA model. Using this model, we showed earlier that previous AA ameliorated colitis. We aimed to determine whether autophagy genes contribute to the anti-colitis protection conferred by AA, and if so, to delineate the autophagy-linked genes involved in this.
Methods: Mice with 2 laparotomies each served as controls (sham–sham). Distal colons were harvested (4 AA-group colons, 4 sham–sham group colons), and RNA extracted from each. The RNA was taken through microarray analysis or reverse transcription-polymerase chain reaction validation. Gene set enrichment analysis software was used to analyze the microarray data.
Results: Out of 28 key autophagy-related genes investigated (VPS15, VPS34, FIP200, ATG03, ATG04A, ATG04B, ATG05, ATG07, ATG10, ATG12, ATG13b, ATG14, ATG16L1, BECN1, GABARAPL1, IRGM1, IRGM2, LAMP2, LC3A, LC3B, RAB7A, UVRAG, NOD2, XBP1, LRRK2, ULK1, ULK2, PTPN2), 7 have genetic associations with inflammatory bowel diseases (ATG16L1, IRGM1, NOD2, XBP1, LRRK2, ULK1, PTPN2). There was slight upregulation of IRGM1, FIP200, and ATG04A (P , 0.05), but no variations with the other 25 genes. In contrast, gene set enrichment analysis revealed that AA downregulated 74 gene sets (associated with 28 autophagy genes) while upregulating only 5 (false discovery rate ,5%; P , 0.001) gene sets. Additionally, 22 gene sets associated with the 7 autophagy + inflammatory bowel disease-associated genes were downregulated by AA, whereas only 3 were upregulated. The genes with maximum AA-induced gene set suppression were VPS15, LAMP2, LC3A, XBP1, and ULK1.
Conclusions: AA induces profound autophagy suppression in the distal colon. The AA-induced upregulation of individual genes (IRGM1, FIP200, ATG04A) could be a reflection of complex compensatory changes or the initial abnormality that led to the pronounced autophagy suppression. Autophagy suppression by AA may induce lesser antigen processing, leading to lesser cross-reactive immunity between microbes and self-antigens, and subsequent amelioration of colitis. (Inflamm Bowel Dis 2014;20:847–855) Key Words: appendicitis, appendectomy, colitis, autophagy, antigen processing
T
he vermiform appendix contains abundant lymphoid tissue and is perpetually exposed to intestinal flora. Appendicitis is the most common abdominal emergency requiring surgery.1 The highest occurrence of uncomplicated appendicitis is between 10 and 30 years.2 The complex interplay between genetic predisposition, gastrointestinal bacteria, and gut immunity in inflammatory bowel disease (IBD) (comprising of Crohn’s disease and ulcerative colitis) is yet to be deciphered. Appendicitis and appendectomy (AA) ameliorates or prevents ulcerative colitis3–5 from developing only Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.ibdjournal.org). Received for publication February 11, 2014; Accepted February 19, 2014. From the Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, Australia. Supported by National Health and Medical Research Council (NHMRC). The authors have no conflicts of interest to disclose. Reprints: Rajkumar Cheluvappa, MBBS, PhD, Inflammation and Infection Research Centre, School of Medical Sciences, Wallace Wurth Building, University of New South Wales, Gate 9, High Street, Sydney, New South Wales 2052, Australia (e-mail: [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000034 Published online 1 April 2014.
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in the patients undergoing surgery before 20 years of age.5 AA has also shown to be protective against Crohn’s disease in a few studies.4,6 However, other studies have showed a slightly elevated risk early after appendectomy, but this has been attributed to diagnostic bias (misdiagnosis of early Crohn’s disease as appendicitis) by these studies.7–10 In mice, the major cecal lymphoid patch is the equivalent of the human appendix. The development of experimental colitis was prevented by removal of the cecum in 3 murine colitis models, namely T-cell receptor-a mutants,11 dextran sulfate sodium model12 and adoptive T-cell transfer colitis model.13 The first murine model of AA was developed by our group.14 In this model, the appendiceal pathology closely resembled that of human appendicitis; and AA offered an age-, bacteria-, and antigen-dependent protection against trinitrobenzene sulfonic acid colitis.14 Autophagy is a homeostatic mechanism to recycle dispensable and damaged cell organelles. Perturbations in autophagic processes have been increasingly implicated in various disease pathogenetic processes. Three main pathways are recognized in autophagy, each differentiated by their substratedelivery mechanisms to lysosomes, their associated enzymes, their regulation, their selectivity, and their genes.15 Macroautophagy (or just autophagy, which is implicated in chronic www.ibdjournal.org |
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inflammation) and microautophagy (direct lysosomal engulfment of cytoplasmic material) are phylogenetically conserved in eukaryotes, whereas chaperone-mediated autophagy (specific and hsc70-mediated) has been observed only in mammals, to date.15 Autophagy entails16 4 major steps:
1. ULK1 kinase complex (ULK1, Atg13, Atg17) phosphorylating FIP200 and Atg13. 2. ULK1 complex translocating the Beclin 1 lipid kinase complex (Atg14, Beclin 1 and its Vps15 subunit; Vps34 PI(3)K) to the phagophore (phospoholipid-bilayered “isolation membrane”) assembly site in the endoplasmic reticulum. 3. Transfer of Agt12 to Atg10 by Atg7, followed by tetrameric complexing of Atg12 (with Atg5 and 2 molecules of Atg16L1). 4. Transfer of LC3-I to Atg3 and phosphatidylethanolamine, resulting in the reversible formation of LC3-II, which is included in the elongating phagophore. Perturbation in “normal” autophagy is pivotal to IBD pathogenesis as suggested by clearly documented IBD-related genetic associations with the autophagy genes ATG16L1,17,18 IRGM,16,18 NOD2,19,20 XBP1,21 LRRK2,22 ULK1,23 and PTPN2.24 However, gut autophagy is a complex unresolved nexus between innate immunity, antimicrobial immune responses, and endoplasmic reticulum stress responses, involving gut epitheilium, goblet cells, and Paneth cells.16 Individual gene expression differences between 2 experimental groups have been successfully elucidated by various gene expression methodologies. Unfortunately, this simplistic approach does not take into account the biological reality of cellular processes cohesively or contiguously effecting changes as groups of genes (gene sets). These changes may be minimal when individual genes per se are examined, but are glaringly conspicuous when corresponding gene sets are examined. We have previously demonstrated the utility of this approach in exploring mechanisms of immune protection in the colon.25,26 This study uses microarray analysis and gene set enrichment analysis (GSEA)27 to identify and characterize the role of autophagy genes and their associated, manifold, disparate pathways in the amelioration of colitis by AA in our murine model.
MATERIALS AND METHODS Animal Experiments Specific pathogen free Balb/c mice (Male, 5 wk) were purchased from the Animal Resource Centre, Perth, Western Australia, Australia and kept in the University of New South Wales holding and care facility in physical containment level 2 rooms. All experiments were approved and monitored by the University of New South Wales Animal Care and Ethics Committee. Mice were anesthetized with xylazine (5 mg/kg, X1251; Sigma–Aldrich, St. Louis, MO) and ketamine (100 mg/kg, K1884; Sigma–Aldrich) intraperitonealy, followed by allocation into
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2 treatment groups; the appendicitis group or the sham surgery group.14 Mice were randomized to have either appendicitis or sham operation. Appendicitis was induced by constructing an appendiceal pouch from the cecal lymphoid patch. This murine appendix was obstructed by rubber band ligation using standardized negative aspiration. Sham surgery entailed a similar procedure, but without continuous obstruction by band ligation of the cecal patch and the placement of a sterile rubber band in the abdominal cavity as a control for foreign body reaction. Although most human patients with ulcerative colitis would not have undergone any form of abdominal surgery at diagnosis, it is essential that we control for the colonic and peritoneal milieu changes induced by a simple laparotomy in our experimental AA mice group. Additionally, the small rubber ring used to encircle the main cecal follicle in the AA group needs to be controlled for too. Hence, in each sham–sham (SS) group mouse, a rubber ring is introduced in the peritoneal cavity through a small laparotomy incision. Seven days after initial surgery, appendicitis mice underwent appendectomy (AA group), whereas sham mice underwent the second sham surgery (SS group). All mice were monitored daily.
Processing of Colonic Specimens for RNA Extraction Distal colons were harvested without the recta, a 1-cm segment (from the anus) was excluded before harvesting the distal colon. Transmural distal colonic segments were chosen to minimize artifactual changes and maximize pathophysiological relevance; as not only superficial musosal tissue, but also colonic lymphoid nodules/aggregations and other tissue may “transmit” the protective immunological changes induced by AA to the distal colon. The transmural distal colonic samples were cleaned of fecal contents with normal saline and immediately transferred to TRIzol reagent (50–75 mg of tissue in 600 mL of TRIzol reagent, 15596026; Invitrogen Australia Pty Limited, Mount Waverley, Australia), snap-frozen in liquid nitrogen, and stored at 2808C until the microarray analysis. Further extraction entailed chloroform and isopropanol treatment and centrifugation followed by washing the resultant pellet with 75% ethanol, air-drying, and final reconstitution in nuclease-free H2O. Concentration and purity of RNA were determined by automated optical density evaluation (OD 260/OD 280 $ 1.8 and OD 260/OD 230 $ 1.8) using Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE). The degree of RNA degradation was analyzed by the Agilent electrophoresis bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA) with the RNA integrity number values consistently above 7.
Experimental Design of Microarray Study All experiments were designed to be compliant with minimum information about a microarray experiment standards.28,29 For Affymetrix array experiments, 4 individual test samples (for 3-d post-SS/AA time point) or 3 individual test samples (for 28-d post-SS/AA time point) were used per group (AA group versus SS group; 1 colonic sample per mouse) with each sample hybridized to an individual slide.
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Autophagy Suppression by Appendectomy
TABLE 1. Individual Gene Expression Data of 28 Important Genes Involved in Autophagy 3-Day Post-AA No.
Gene
Other Names
1
VPS15
PIK3R4
2
VPS34
PIK3C3
3
FIP200
4
ATG3
RB1CC1, LaXp180, Cc1, mammalian ortholog of ATG17 ATG03, APG3
5
ATG4A
6
ATG4B
7
ATG5
8
ATG7
9
ATG10
10
ATG12
11 12
ATG13b ATG14
13
ATG16L1
14 15
BECN1 GABARAPL1
16
IRGM1
17
IRGM2
18 19 20 21 22
LAMP2 LC3A LC3B RAB7A UVRAG
23
NOD2
Role of Product Autophagy regulation, protein trafficking, transcription elongation Autophagy regulation, protein trafficking, transcription elongation Interacts with p53 to suppress autophagy, colon cancer link
Ubiquitin conjugation, Atg8 conjugation with phosphatidylethanolamine ATG04A, APG4A, Autophagy regulation, proteolysis, protein AUTL2 transport, cysteine proteinase activity ATG04B, APG4B, Autophagy regulation, proteolysis, protein AUTL1 transport, cysteine proteinase activity ATG05, APG5L, ASP E3 ubiquitin ligase, protein lipidation, autophagosome elongation ATG07, APG7L, GSA7 Autophagy regulation, protein transport, breast cancer link, ubiquitin E1 like enzyme APG10L, PP12616 Ubiquitin-conjugating E2 enzyme, interacts with Atg7 to receive Atg12 APG12 E3 ubiquitin ligase, protein lipidation, autophagosome elongation APG13, KIAA0652 Autophagy induction alongside FIP200 KIAA0831, Barkor Endosome maturation regulator, (Beclin 1-associated autophagosome formation, triglyceride autophagy-related key metabolism regulator) APG16L Autophagosome assembly, Crohn’s disease link Beclin 1, ATG6, GT197 Key autophagic regulator ATG8L, APG8L, gec1 Promotes tubulin polymerization, protein transport, vesicle transport, cell death, cell proliferation, tumor progression IRGM, IFI1, LRG47 Small antimicrobial GTPase that regulates autophagosome formation, Crohn’s disease link Small antimicrobial GTPase that regulates autophagosome formation, Crohn’s disease link CD107B Lysosomal, phagosomal pathways MAP1LC3A Autophagic vacuole assembly MAP1LC3B Autophagic vacuole assembly RAB7 Endocytosis, phagosome formation p63 Regulates Rab7-mediated endosomal trafficking CARD15 Autophagy initiator, muramyl dipeptide responsive, recruits ATG16L1 to bacterial entry site, Crohn’s disease link
28-Day Post-AA
Reference
Fold change
P
Fold change
P
33
0.85
0.072
1.00
0.988
33
0.88
0.166
1.01
0.912
34,35
1.01
0.941
1.19
0.028a
36
1.17
0.165
0.99
0.932
37
1.18
0.168
1.20
0.026a
37
1.11
0.210
0.96
0.665
38
1.06
0.542
1.01
0.818
39
0.89
0.081
0.94
0.460
40
1.00
0.966
0.95
0.550
38
1.06
0.512
1.04
0.446
41 42,43
— —
— —
0.93 1.02
0.257 0.785
17,18
—
—
1.02
0.744
44 45
1.08 —
0.362 —
1.04 —
0.641 —
16,18
1.66
0.075
1.70
0.005a
16,18
2.17
0.095
1.56
0.053
46 16 16 47 47
1.20 1.17 1.11 1.06 1.05
0.078 0.095 0.269 0.059 0.483
0.99 0.91 0.95 1.01 0.97
0.930 0.130 0.427 0.924 0.635
19,20
1.05
0.581
1.03
0.690
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TABLE 1 (Continued) 3-Day Post-AA No.
Gene
24
XBP1
25
LRRK2
26
ULK1
27
ULK2
28
PTPN2
Role of Product
Reference
Fold change
P
Fold change
P
Transcription factor, key component of ER stress response, autophagosome maturation, secretory cell maintenance, Crohn’s disease link, ulcerative colitis link Leucine-rich repeat kinase, autophagosome maturation, Crohn’s disease link Kinase, autophagy initiation, Crohn’s disease link Autophagy initiation, nutrient assessment, protein recruitment to autophagosome Autophagosome formation regulator, responds to IFN-g and TNF-a, Crohn’s disease link, ulcerative colitis link
21
1.14
0.064
0.93
0.267
22
0.90
0.399
0.99
0.826
23
1.04
0.702
0.91
0.286
48
1.04
0.624
1.06
0.463
24
0.99
0.913
0.88
0.092
Other Names
UNC51.2, ATG1H2
28-Day Post-AA
Twenty-eight key genes involved in autophagy, namely, VPS15, VPS34, FIP200, ATG03, ATG04A, ATG04B, ATG05, ATG07, ATG10, ATG12, ATG13b, ATG14, ATG16L1, BECN1, GABARAPL1, IRGM1, IRGM2, LAMP2, LC3A, LC3B, RAB7A, UVRAG, NOD2, XBP1, LRRK2, ULK1, ULK2, and PTPN2 were examined for gene expressions levels in distal colons 3 days post-AA and 28 days post-AA. At 28 days post-AA, FIP200, ATG04A, and IRGM1 were significantly upregulated (P , 0.05). The 3 days post-AA study used 4 AA mice versus 4 SS mice. The 28 days post-AA study involved 3 AA mice versus 3 SS mice. a P , 0.05.
Affymetrix Array Process: Labeling, Hybridization, Scanning, and Normalization RNA from distal colonic tissue obtained from mice was not pooled. RNA from each mouse specimen was taken individually through the microarray process. For Affymetrix arrays, 100 ng of RNA from each sample was labeled using the Whole Transcript Sense Target Labeling Assay as described (Affymetrix). Labeled complementary RNA samples were then hybridized to Affymetrix Mouse Gene 1.0 ST Arrays (28,853 well-annotated genes) (Ramaciotti Centre for Gene Function Analysis, University of New South Wales) before being scanned using a Affymetrix GCS3000 7 G four-color Gene Array scanner with autoloader (Affymetrix). The Gene Expression Omnibus accession number for microarray data reported here, inclusive of minimum information about a microarray experiment-compliant experimental details, 28,29 is GSE23914.
Microarray Preprocessing and Filtering All noncontrol probesets from the 8 arrays were imported into Partek (Version 6.4; Partek Inc., St. Louis, MO) and then normalized using RMA.30 By principle components analysis, a batch effect was evident in principle component 1, which was removed using the batch removal tool in Partek, using default parameters. The probability of each probeset being expressed was determined using the detected above background procedure, using Affymetrix Power Tools (v1.10.2), excluding 13 probes from probeset 10338063, which had very low GC, and thus did not have matched controls. Probesets were excluded if
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none of the samples were detected above background (P ¼ 1025). To assess the degree of differential expression between AA and SS groups, a two-way analysis of variance on treatment and batch was fitted to each probeset using Partek. To correct for multiple hypothesis testing, we used the q-value/positive False Discovery Rate (FDR).31
Gene Set Enrichment Analysis We compared gene expression profiles to the c2_all collection of curated gene sets from the molecular signatures database (version 2.5).27 This collection contains gene sets that are experimentally derived and from expert curated pathway databases. A preranked file was created, containing the average difference between AA and SS for each probeset, sorted from most upregulated in SS to most downregulated. We used the na28 annotation csv file from www.affymetrix.com to determine the gene symbol for each probeset and collapsed probesets to unique genes using the default, max_probe option, resulting in 18,600 unique genes. GSEA (version 2.0)27 was run in preranked mode, using default parameters (gene set sizes between 15 and 500 leaving 1387 gene sets, 1000 permutations, images on the top 50 gene sets).
Enrichment of Autophagy-associated Gene Sets We used GSEA, developed by Mootha et al,32 which merges data from groups of gene sets previously described in the literature to detect significant expression differences. These gene set groups were Kegg Pathways (150 gene sets), micro-
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RNAs (200 gene sets), transcription factors (579 gene sets), biological processes (536 gene sets), and others (1387 gene sets). We used stringent statistical cutoffs (FDR values ,5% and P , 0.001) to delineate autophagy-associated gene sets, which were consistently altered in the distal colons of all AA mice when compared with control SS mice.
Statistics Group comparisons were analyzed using the Mann–Whitney U test with GraphPad Prism (Graphpad software, San Diego, CA). Data are expressed as mean 6 SE of mean, and the differences were considered to be significant if P , 0.05.
RESULTS Individual Distal Colonic Gene Expression of Key Autophagy-associated Genes The 28 genes chosen (Table 1) for expression-level evaluations are those with well-documented direct links with the heterogenous “autophagy system,” with or without links to IBD. Out of the 28 autophagy genes examined (VPS15, VPS34, FIP200, ATG03, ATG04A, ATG04B, ATG05, ATG07, ATG10, ATG12, ATG13b, ATG14, ATG16L1, BECN1, GABARAPL1, IRGM1, IRGM2, LAMP2, LC3A, LC3B, RAB7A, UVRAG, NOD2, XBP1, LRRK2, ULK1, ULK2, and PTPN2), 7 have human polymorphisms associated with IBD (ATG16L1, IRGM1, NOD2, XBP1, LRRK2, ULK1, and PTPN2). FIP200, ATG04A, and IRGM1 were upregulated 1.2-fold, 1.2-fold, and 1.7-fold, respectively (P , 0.05). There were no expression changes in the other 25 genes.
Enrichment of Autophagy-associated Gene Sets Using stringent statistical cutoffs for GSEA (FDR values ,5% and P , 0.001), we delineated 28 days post-AA autophagy-associated gene sets, which were also consistently altered in the distal colons of each of the AA mice when compared with that from each of the control SS mice (Table 2). GSEA analysis demonstrated that 74 gene sets associated with the 28 autophagy genes were downregulated by AA in stark contrast to only 5 upregulated gene sets.
Enrichment of Gene Sets Associated with Autophagy- and IBD-associated Genes Using stringent statistical cutoffs for GSEA (FDR values , 5% and P , 0.001), we delineated 28 days post-AA autophagy + IBD-associated gene sets which were also consistently altered in the distal colons of each of the AA mice when compared with that from each of the control SS mice (Table 3). GSEA analysis demonstrated that 22 gene sets associated with the 7 autophagy + IBD-associated genes were downregulated by AA in stark contrast to only 3 upregulated gene sets.
Autophagy Suppression by Appendectomy
TABLE 2. GSEA Analysis of 28 Important Autophagy Genes–Concise Overview* Gene VPS15 VPS34 FIP200 ATG03 ATG04A ATG04B ATG05 ATG07 ATG10 ATG12 ATG13b ATG14 ATG16L1 BECN1 GABARAPL1 IRGM1 IRGM2 LAMP2 LC3A LC3B RAB7A UVRAG NOD2 XBP1 LRRK2 ULK1 ULK2 PTPN2 TOTAL
Upregulated Gene sets in AA
Downregulated Gene sets in AA
0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 1 0 0 0 5
6 3 3 0 0 1 1 1 1 0 1 0 3 4 3 1 0 11 7 2 0 5 0 8 0 7 3 3 74
GSEA was used to analyze the differential regulation of gene sets associated with 28 key genes involved in autophagy. The gene set groups chosen for further evaluation had stringent cutoff values (FDR , 5% and P , 0.001). Although 74 gene sets associated with autophagy genes were downregulated in the AA group, only 5 were upregulated. *Table 2 is just a concise overview of the results. Please see Supplemental Digital Content 1, http://links.lww.com/IBD/A458 for a detailed version of this table, replete with statistical minutiae.
DISCUSSION As summarized earlier, using a new murine appendicitis model developed by us,14 we showed that AA protected against or prevented experimental colitis. A robust approach, GSEA, was used to delineate and enumerate genetic pathways entailed in this protection or prevention. Reverse transcription-polymerase chain reaction was used for validation of the study.25 The proximal colon would be much more likely to be affected by the inflammatory changes experimentally induced by www.ibdjournal.org |
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TABLE 3. GSEA Analysis of 7 Autophagy Genes with Genetic Links to IBD
Gene
Upregulated Gene sets in AA
ATG16L1
UPREG 0 gene sets —
IRGM1
IRGM2
NOD2 XBP1
LRRK2
ULK1
PTPN2
TOTAL
UPREG 1 gene sets ICHIBA_GRAFT_VERSUS_ HOST_DISEASE_D7_UP UPREG 1 gene sets ICHIBA_GRAFT_VERSUS_ HOST_DISEASE_D7_UP UPREG 0 gene sets — UPREG 0 gene sets —
UPREG 1 gene sets SABATES_COLORECTAL_ ADENOMA_DN UPREG 0 gene sets —
UPREG 0 gene sets —
UPREG 3 gene sets
No. of Enriched Genes
FDR q-val
Downregulated Gene sets in AA
107
0.022
DOWNREG 3 gene sets CTCTGGA,MIR-520A,MIR-525 GCACTTT,MIR-17-5P,MIR-20A,MIR-106A, MIR-1MIR-106B,MIR-20B,MIR-519D GTGCCAA,MIR-96 DOWNREG 1 gene sets BERENJENO_TRANSFORMED_BY_RHOA_UP
107
0.022
DOWNREG 0 gene sets —
0.016
DOWNREG 0 gene sets — DOWNREG 8 gene sets CHARAFE_BREAST_CANCER_LUMINAL_ VS_BASAL_UP GAGCTGG,MIR-337 GTGACGY_V$E4F1_Q6 HSIAO_HOUSEKEEPING_GENES PASQUALUCCI_LYMPHOMA_BY_GC_ STAGE_UP PENG_RAPAMYCIN_RESPONSE_DN PENG_GLUTAMINE_DEPRIVATION_DN SMITH_TERT_TARGETS_UP DOWNREG 0 gene sets —
260
DOWNREG 7 gene sets CHARAFE_BREAST_CANCER_LUMINAL_ VS_BASAL_UP DEBIASI_APOPTOSIS_BY_REOVIRUS_ INFECTION_DN DOUGLAS_BMI1_TARGETS_UP GCACTTT,MIR-17-5P,MIR-20A,MIR-106A, MIR-106B,MIR-20B,MIR-519D GRADE_COLON_VS_RECTAL_ CANCER_DN RASHI_RESPONSE_TO_IONIZING_ RADIATION_3 STEIN_ESRRA_TARGETS_RESPONSIVE_ TO_ESTROGEN_DN DOWNREG 3 gene sets RAMALHO_STEMNESS_UP V$NRF1_Q6 VANHARANTA_UTERINE_FIBROID_WITH_ 7Q_DELETION_UP DOWNREG 22 gene sets
No. of Enriched Genes
FDR q-val
130 492
0.011 0.030
266
0.047
495
0.006
350
0.036
135 468 377 262
0.006 0.043 0.032 0.027
67 82 126
0.001 0.009 0.016
—
—
350
0.036
220
0.001
491 492
0.009 0.030
39
0.026
45
0.009
41
0.032
197 158 67
0.008 0.019 0.010
GSEA was used to analyze the differential regulation of gene sets associated with 7 autophagy genes with genetic links to IBD. The gene set groups chosen for further evaluation had stringent cutoff values (FDR , 5% and P , 0.001). Although 22 gene sets associated with autophagy genes were downregulated in the AA group, only 3 were upregulated. UPREG “n” gene sets, upregulated number of gene sets pertaining to that gene; DOWNREG “n” gene sets, downregulated number of gene sets pertaining to that gene; Number of enriched genes in gene set, the number of genes in each gene set enriched by GSEA pertaining to AA mice in this study; NOM p-val, Nominal P-value statistic; FDR q-val, false discovery rate q-value statistic.
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AA, owing to the close anatomical proximity of the main mouse cecal patch (approximately human appendix) to the proximal colon. However, the novelty of our study is that the most distal regions of the large gut sustain major persistent immunological changes (protective against colitis), by manipulation at the cecum, the most proximal region of the large gut. The immunological changes sustained in T-helper cell type 1, 2, and 17 “pathways” have been published elsewhere.26 Distal colonic gene expression studies, 3 days after surgery or 28 days after surgery, reveal the various genes and gene sets that are responsible for the durable protective effect of AA against colitis.25 The gene expression data obtained from our microarray study get more distinct between the time points (days 3 and 28) and stabilize around day 28. Although observable in our overall microarray results, these are visually appraised better in the reverse transcription-polymerase chain reaction time curves (days 3, 14, and 28) of selected genes in our previous article.25 The persistence of these expression changes would be theoretically easier to demonstrate in humans than mice, as mice have an accelerated lifespan, with 1 mouse year “often equated” to 30 human years. The 28-day-extension of our study is adequate to demonstrate the chronological sustainability of these changes. For our gene expression studies, we chose 28 key autophagy genes, including 7 with previously described genetic links to IBD (Table 1), namely; ATG16L1,17,18 IRGM,16,18 NOD2,19,20 XBP1,21 LRRK2,22 ULK1,23 and PTPN2.24 GSEA analysis of the 28 autophagy genes shows that 74 gene sets were downregulated by AA versus only 5 upregulated gene sets (Table 2; see Table, Supplemental Digital Content 1, http://links.lww.com/IBD/A458). GSEA analysis of 7 autophagy + IBD-associated genes highlights 22 AA-downregulated gene sets versus only 3 upregulated gene sets (Table 3). The genes that demonstrated the maximum suppression in our GSEA analysis were VPS15, LAMP2, LC3A, XBP1, and ULK1 (Table 2), including the previously described IBD-associated genes XBP1 and ULK1 (Table 3). XBP1, a transcription factor, is a key component of the endoplasmic reticulum stress response. It has roles in the secretory cell development and maintenance. Deletion of XBP1 results in spontaneous enteritis and increased susceptibility to induced colitis in a mouse knockout study.21 In that study,21 XBP1 variantassociations with Crohn’s disease and ulcerative colitis were identified and replicated. ULK1, a ubiquitously expressed serine–threonine kinase, initiates autophagy in conjunction with ATG13 and FIP200. A genome-wide association study23 identified that the frequency of a short nucleotide polymorphism in ULK1 was significantly higher in patients with Crohn’s disease. Interestingly, ULK1 knockout mice49 displayed normal induction of autophagy (not demonstrated in the gut), hinting that autophagy may still occur through ULK2 back-up signaling or through microautophagy/chaperone-mediated autophagy. The impact of ULK1 deletion on the gut is yet to be determined.
Autophagy Suppression by Appendectomy
Although Crohn’s disease is more strongly associated with autophagy gene polymorphisms, appendectomy is more protective against ulcerative colitis (most studies) than Crohn’s disease (a proportion of studies). Our mouse model of AA is protective against trinitrobenzene sulfonic acid colitis, which simulates human ulcerative colitis. Therefore, AA-induced autophagy suppression is less likely to be relevant to Crohn’s disease-related inflammation or the exacerbation of it thereof. Although the genetic links between defective autophagy and IBD are reasonably evident, the triggering factors for autophagy dysregulation are yet to be deciphered. Although autophagy is ubiquitous and functionally diverse specialized processes like those adopted by secretory cells require it absolutely for cellular function. Variations in a single autophagy gene may therefore result in multiple manifestations of damage across different cells within the same tissue. Owing to the widespread presence of autophagy throughout human tissues, therapeutic attempts to curtail immunopathology require further elucidation of autophagy dysfunction in each cell type within the same tissue (gut mucosa). Exposure to, or inappropriate responses to gut flora, are commonly incriminated in the causality of IBD.50 In our study, suppression of autophagy by AA is profound. The increased AA-induced expression of the genes IRGM1, FIP200, and ATG04A (1.7-fold, 1.2-fold, and 1.2-fold, respectively) may be an indication of compensatory changes (positive regulatory changes), or the initial changes, which, in turn, had led to the marked autophagy suppression. Autophagy suppression by AA may induce lesser antigen processing, leading to lesser crossreactive immunity between gut flora and self-antigens and subsequent amelioration of colitis. Alternatively, the marked autophagy suppression observed in AA may reflect a controlling response to inappropriately excessive initial immune responses. AA may use these mechanisms to protect against colitis. Our study demonstrates that AA curbs autophagy, potentially contributing to suppression of autophagy-mediated immunopathology as in IBD or experimental colitis. Specific immunohistochemical analyses and “functional confirmation” using 1 or more autophagy gene knockouts would be our next logical step to pursue. Autophagy, owing to its foundational role in gut homeostatic maintenance, is an attractive therapeutic target. Elucidating the pathways involved in these changes will lead to the development of techniques to manipulate different autophagy component pathways resulting in improved IBD management.
ADDENDA 1. The raw microarray data are available in Gene Expression Omnibus. The accession number for microarray data reported here is GSE23914, and the relevant link is http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23914. 2. Specific methodologies and the reverse transcriptionpolymerase chain reaction study validation results have www.ibdjournal.org |
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previously been published.25 We selected 14 genes for confirmation of our gene expression studies. These genes broadly belonged to 4 major groups: innate immunity (SLPI, S100A8, LBP, CD68); immune mediators (IL18R1, IL33), cell migration-chemokines (CCL8, CXCL10, CCL12 or MCP5, PF4, CXCL5, CCL7 or MCP3), and cell migration receptors (FPR1, CCR5).
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21. Kaser A, Lee AH, Franke A, et al. XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell. 2008;134:743–756. 22. Liu Z, Lee J, Krummey S, et al. The kinase LRRK2 is a regulator of the transcription factor NFAT that modulates the severity of inflammatory bowel disease. Nat Immunol. 2011;12:1063–1070. 23. Henckaerts L, Cleynen I, Brinar M, et al. Genetic variation in the autophagy gene ULK1 and risk of Crohn’s disease. Inflamm Bowel Dis. 2011;17: 1392–1397. 24. Glas J, Wagner J, Seiderer J, et al. PTPN2 gene variants are associated with susceptibility to both Crohn’s disease and ulcerative colitis supporting a common genetic disease background. PLoS One. 2012;7: e33682. 25. Cheluvappa R, Luo AS, Palmer C, et al. Protective pathways against colitis mediated by appendicitis and appendectomy. Clin Exp Immunol. 2011;165:393–400. 26. Cheluvappa R, Luo AS, Grimm MC. T helper type 17 pathway suppression by appendicitis and appendectomy protects against colitis. Clin Exp Immunol. 2014;175:316–322. 27. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102:15545–15550. 28. Brazma A. Minimum information about a microarray experiment (MIAME)—successes, failures, challenges. ScientificWorldJournal. 2009;9: 420–423. 29. Brazma A, Hingamp P, Quackenbush J, et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001;29:365–371. 30. Irizarry RA, Bolstad BM, Collin F, et al. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31:e15. 31. Storey JD, Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad Sci U S A. 2003;100:9440–9445. 32. Mootha VK, Lindgren CM, Eriksson KF, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267–273. 33. Gaur NA, Hasek J, Brickner DG, et al. Vps factors are required for efficient transcription elongation in budding yeast. Genetics. 2013;193: 829–851. 34. Paun BC, Cheng Y, Leggett BA, et al. Screening for microsatellite instability identifies frequent 30 -untranslated region mutation of the RB1-inducible coiled-coil 1 gene in colon tumors. PLoS One. 2009;4:e7715. 35. Morselli E, Shen S, Ruckenstuhl C, et al. p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200. Cell Cycle. 2011;10:2763–2769. 36. Tanida I, Tanida-Miyake E, Komatsu M, et al. Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p. J Biol Chem. 2002;277:13739–13744. 37. Marino G, Uria JA, Puente XS, et al. Human autophagins, a family of cysteine proteinases potentially implicated in cell degradation by autophagy. J Biol Chem. 2003;278:3671–3678. 38. Hanada T, Noda NN, Satomi Y, et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem. 2007;282:37298–37302. 39. Mizushima N, Noda T, Yoshimori T, et al. A protein conjugation system essential for autophagy. Nature. 1998;395:395–398. 40. Hong SB, Kim BW, Kim JH, et al. Structure of the autophagic E2 enzyme Atg10. Acta Crystallogr D Biol Crystallogr. 2012;68:1409–1417. 41. Alers S, Loffler AS, Paasch F, et al. Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction. Autophagy. 2011;7: 1423–1433. 42. Kim HJ, Zhong Q, Sheng ZH, et al. Beclin-1-interacting autophagy protein Atg14L targets the SNARE-associated protein Snapin to coordinate endocytic trafficking. J Cell Sci. 2012;125:4740–4750. 43. Xiong X, Tao R, DePinho RA, et al. The autophagy-related gene 14 (Atg14) is regulated by forkhead box O transcription factors and circadian rhythms and plays a critical role in hepatic autophagy and lipid metabolism. J Biol Chem. 2012;287:39107–39114. 44. Fu LL, Cheng Y, Liu B. Beclin-1: autophagic regulator and therapeutic target in cancer. Int J Biochem Cell Biol. 2013;45:921–924.
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45. Le Grand JN, Chakrama FZ, Seguin-Py S, et al. GABARAPL1 (GEC1): original or copycat? Autophagy. 2011;7:1098–1107. 46. Eskelinen EL. Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol Aspects Med. 2006;27:495–502. 47. Liang C, Lee JS, Inn KS, et al. Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nat Cell Biol. 2008;10:776–787.
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48. Mizushima N. The role of the Atg1/ULK1 complex in autophagy regulation. Curr Opin Cell Biol. 2010;22:132–139. 49. Kundu M, Lindsten T, Yang CY, et al. Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood. 2008;112:1493–1502. 50. Strober W, Fuss IJ, Blumberg RS. The immunology of mucosal models of inflammation. Annu Rev Immunol. 2002;20:495–549.
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Autophagy Suppression by Appendicitis and Appendectomy Protects Against Colitis Rajkumar Cheluvappa, MBBS, PhD, Annie S. Luo, BSc, and Michael C. Grimm, MBBS, FRACP, PhD
Background: When done at a young age, appendicitis followed by appendectomy (AA) offers protection against ulcerative colitis development in later life. We developed the first ever murine AA model. Using this model, we showed earlier that previous AA ameliorated colitis. We aimed to determine whether autophagy genes contribute to the anti-colitis protection conferred by AA, and if so, to delineate the autophagy-linked genes involved in this.
Methods: Mice with 2 laparotomies each served as controls (sham–sham). Distal colons were harvested (4 AA-group colons, 4 sham–sham group colons), and RNA extracted from each. The RNA was taken through microarray analysis or reverse transcription-polymerase chain reaction validation. Gene set enrichment analysis software was used to analyze the microarray data.
Results: Out of 28 key autophagy-related genes investigated (VPS15, VPS34, FIP200, ATG03, ATG04A, ATG04B, ATG05, ATG07, ATG10, ATG12, ATG13b, ATG14, ATG16L1, BECN1, GABARAPL1, IRGM1, IRGM2, LAMP2, LC3A, LC3B, RAB7A, UVRAG, NOD2, XBP1, LRRK2, ULK1, ULK2, PTPN2), 7 have genetic associations with inflammatory bowel diseases (ATG16L1, IRGM1, NOD2, XBP1, LRRK2, ULK1, PTPN2). There was slight upregulation of IRGM1, FIP200, and ATG04A (P , 0.05), but no variations with the other 25 genes. In contrast, gene set enrichment analysis revealed that AA downregulated 74 gene sets (associated with 28 autophagy genes) while upregulating only 5 (false discovery rate ,5%; P , 0.001) gene sets. Additionally, 22 gene sets associated with the 7 autophagy + inflammatory bowel disease-associated genes were downregulated by AA, whereas only 3 were upregulated. The genes with maximum AA-induced gene set suppression were VPS15, LAMP2, LC3A, XBP1, and ULK1.
Conclusions: AA induces profound autophagy suppression in the distal colon. The AA-induced upregulation of individual genes (IRGM1, FIP200, ATG04A) could be a reflection of complex compensatory changes or the initial abnormality that led to the pronounced autophagy suppression. Autophagy suppression by AA may induce lesser antigen processing, leading to lesser cross-reactive immunity between microbes and self-antigens, and subsequent amelioration of colitis. (Inflamm Bowel Dis 2014;20:847–855) Key Words: appendicitis, appendectomy, colitis, autophagy, antigen processing
T
he vermiform appendix contains abundant lymphoid tissue and is perpetually exposed to intestinal flora. Appendicitis is the most common abdominal emergency requiring surgery.1 The highest occurrence of uncomplicated appendicitis is between 10 and 30 years.2 The complex interplay between genetic predisposition, gastrointestinal bacteria, and gut immunity in inflammatory bowel disease (IBD) (comprising of Crohn’s disease and ulcerative colitis) is yet to be deciphered. Appendicitis and appendectomy (AA) ameliorates or prevents ulcerative colitis3–5 from developing only Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.ibdjournal.org). Received for publication February 11, 2014; Accepted February 19, 2014. From the Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, Australia. Supported by National Health and Medical Research Council (NHMRC). The authors have no conflicts of interest to disclose. Reprints: Rajkumar Cheluvappa, MBBS, PhD, Inflammation and Infection Research Centre, School of Medical Sciences, Wallace Wurth Building, University of New South Wales, Gate 9, High Street, Sydney, New South Wales 2052, Australia (e-mail: [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000034 Published online 1 April 2014.
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in the patients undergoing surgery before 20 years of age.5 AA has also shown to be protective against Crohn’s disease in a few studies.4,6 However, other studies have showed a slightly elevated risk early after appendectomy, but this has been attributed to diagnostic bias (misdiagnosis of early Crohn’s disease as appendicitis) by these studies.7–10 In mice, the major cecal lymphoid patch is the equivalent of the human appendix. The development of experimental colitis was prevented by removal of the cecum in 3 murine colitis models, namely T-cell receptor-a mutants,11 dextran sulfate sodium model12 and adoptive T-cell transfer colitis model.13 The first murine model of AA was developed by our group.14 In this model, the appendiceal pathology closely resembled that of human appendicitis; and AA offered an age-, bacteria-, and antigen-dependent protection against trinitrobenzene sulfonic acid colitis.14 Autophagy is a homeostatic mechanism to recycle dispensable and damaged cell organelles. Perturbations in autophagic processes have been increasingly implicated in various disease pathogenetic processes. Three main pathways are recognized in autophagy, each differentiated by their substratedelivery mechanisms to lysosomes, their associated enzymes, their regulation, their selectivity, and their genes.15 Macroautophagy (or just autophagy, which is implicated in chronic www.ibdjournal.org |
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inflammation) and microautophagy (direct lysosomal engulfment of cytoplasmic material) are phylogenetically conserved in eukaryotes, whereas chaperone-mediated autophagy (specific and hsc70-mediated) has been observed only in mammals, to date.15 Autophagy entails16 4 major steps:
1. ULK1 kinase complex (ULK1, Atg13, Atg17) phosphorylating FIP200 and Atg13. 2. ULK1 complex translocating the Beclin 1 lipid kinase complex (Atg14, Beclin 1 and its Vps15 subunit; Vps34 PI(3)K) to the phagophore (phospoholipid-bilayered “isolation membrane”) assembly site in the endoplasmic reticulum. 3. Transfer of Agt12 to Atg10 by Atg7, followed by tetrameric complexing of Atg12 (with Atg5 and 2 molecules of Atg16L1). 4. Transfer of LC3-I to Atg3 and phosphatidylethanolamine, resulting in the reversible formation of LC3-II, which is included in the elongating phagophore. Perturbation in “normal” autophagy is pivotal to IBD pathogenesis as suggested by clearly documented IBD-related genetic associations with the autophagy genes ATG16L1,17,18 IRGM,16,18 NOD2,19,20 XBP1,21 LRRK2,22 ULK1,23 and PTPN2.24 However, gut autophagy is a complex unresolved nexus between innate immunity, antimicrobial immune responses, and endoplasmic reticulum stress responses, involving gut epitheilium, goblet cells, and Paneth cells.16 Individual gene expression differences between 2 experimental groups have been successfully elucidated by various gene expression methodologies. Unfortunately, this simplistic approach does not take into account the biological reality of cellular processes cohesively or contiguously effecting changes as groups of genes (gene sets). These changes may be minimal when individual genes per se are examined, but are glaringly conspicuous when corresponding gene sets are examined. We have previously demonstrated the utility of this approach in exploring mechanisms of immune protection in the colon.25,26 This study uses microarray analysis and gene set enrichment analysis (GSEA)27 to identify and characterize the role of autophagy genes and their associated, manifold, disparate pathways in the amelioration of colitis by AA in our murine model.
MATERIALS AND METHODS Animal Experiments Specific pathogen free Balb/c mice (Male, 5 wk) were purchased from the Animal Resource Centre, Perth, Western Australia, Australia and kept in the University of New South Wales holding and care facility in physical containment level 2 rooms. All experiments were approved and monitored by the University of New South Wales Animal Care and Ethics Committee. Mice were anesthetized with xylazine (5 mg/kg, X1251; Sigma–Aldrich, St. Louis, MO) and ketamine (100 mg/kg, K1884; Sigma–Aldrich) intraperitonealy, followed by allocation into
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2 treatment groups; the appendicitis group or the sham surgery group.14 Mice were randomized to have either appendicitis or sham operation. Appendicitis was induced by constructing an appendiceal pouch from the cecal lymphoid patch. This murine appendix was obstructed by rubber band ligation using standardized negative aspiration. Sham surgery entailed a similar procedure, but without continuous obstruction by band ligation of the cecal patch and the placement of a sterile rubber band in the abdominal cavity as a control for foreign body reaction. Although most human patients with ulcerative colitis would not have undergone any form of abdominal surgery at diagnosis, it is essential that we control for the colonic and peritoneal milieu changes induced by a simple laparotomy in our experimental AA mice group. Additionally, the small rubber ring used to encircle the main cecal follicle in the AA group needs to be controlled for too. Hence, in each sham–sham (SS) group mouse, a rubber ring is introduced in the peritoneal cavity through a small laparotomy incision. Seven days after initial surgery, appendicitis mice underwent appendectomy (AA group), whereas sham mice underwent the second sham surgery (SS group). All mice were monitored daily.
Processing of Colonic Specimens for RNA Extraction Distal colons were harvested without the recta, a 1-cm segment (from the anus) was excluded before harvesting the distal colon. Transmural distal colonic segments were chosen to minimize artifactual changes and maximize pathophysiological relevance; as not only superficial musosal tissue, but also colonic lymphoid nodules/aggregations and other tissue may “transmit” the protective immunological changes induced by AA to the distal colon. The transmural distal colonic samples were cleaned of fecal contents with normal saline and immediately transferred to TRIzol reagent (50–75 mg of tissue in 600 mL of TRIzol reagent, 15596026; Invitrogen Australia Pty Limited, Mount Waverley, Australia), snap-frozen in liquid nitrogen, and stored at 2808C until the microarray analysis. Further extraction entailed chloroform and isopropanol treatment and centrifugation followed by washing the resultant pellet with 75% ethanol, air-drying, and final reconstitution in nuclease-free H2O. Concentration and purity of RNA were determined by automated optical density evaluation (OD 260/OD 280 $ 1.8 and OD 260/OD 230 $ 1.8) using Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE). The degree of RNA degradation was analyzed by the Agilent electrophoresis bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA) with the RNA integrity number values consistently above 7.
Experimental Design of Microarray Study All experiments were designed to be compliant with minimum information about a microarray experiment standards.28,29 For Affymetrix array experiments, 4 individual test samples (for 3-d post-SS/AA time point) or 3 individual test samples (for 28-d post-SS/AA time point) were used per group (AA group versus SS group; 1 colonic sample per mouse) with each sample hybridized to an individual slide.
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TABLE 1. Individual Gene Expression Data of 28 Important Genes Involved in Autophagy 3-Day Post-AA No.
Gene
Other Names
1
VPS15
PIK3R4
2
VPS34
PIK3C3
3
FIP200
4
ATG3
RB1CC1, LaXp180, Cc1, mammalian ortholog of ATG17 ATG03, APG3
5
ATG4A
6
ATG4B
7
ATG5
8
ATG7
9
ATG10
10
ATG12
11 12
ATG13b ATG14
13
ATG16L1
14 15
BECN1 GABARAPL1
16
IRGM1
17
IRGM2
18 19 20 21 22
LAMP2 LC3A LC3B RAB7A UVRAG
23
NOD2
Role of Product Autophagy regulation, protein trafficking, transcription elongation Autophagy regulation, protein trafficking, transcription elongation Interacts with p53 to suppress autophagy, colon cancer link
Ubiquitin conjugation, Atg8 conjugation with phosphatidylethanolamine ATG04A, APG4A, Autophagy regulation, proteolysis, protein AUTL2 transport, cysteine proteinase activity ATG04B, APG4B, Autophagy regulation, proteolysis, protein AUTL1 transport, cysteine proteinase activity ATG05, APG5L, ASP E3 ubiquitin ligase, protein lipidation, autophagosome elongation ATG07, APG7L, GSA7 Autophagy regulation, protein transport, breast cancer link, ubiquitin E1 like enzyme APG10L, PP12616 Ubiquitin-conjugating E2 enzyme, interacts with Atg7 to receive Atg12 APG12 E3 ubiquitin ligase, protein lipidation, autophagosome elongation APG13, KIAA0652 Autophagy induction alongside FIP200 KIAA0831, Barkor Endosome maturation regulator, (Beclin 1-associated autophagosome formation, triglyceride autophagy-related key metabolism regulator) APG16L Autophagosome assembly, Crohn’s disease link Beclin 1, ATG6, GT197 Key autophagic regulator ATG8L, APG8L, gec1 Promotes tubulin polymerization, protein transport, vesicle transport, cell death, cell proliferation, tumor progression IRGM, IFI1, LRG47 Small antimicrobial GTPase that regulates autophagosome formation, Crohn’s disease link Small antimicrobial GTPase that regulates autophagosome formation, Crohn’s disease link CD107B Lysosomal, phagosomal pathways MAP1LC3A Autophagic vacuole assembly MAP1LC3B Autophagic vacuole assembly RAB7 Endocytosis, phagosome formation p63 Regulates Rab7-mediated endosomal trafficking CARD15 Autophagy initiator, muramyl dipeptide responsive, recruits ATG16L1 to bacterial entry site, Crohn’s disease link
28-Day Post-AA
Reference
Fold change
P
Fold change
P
33
0.85
0.072
1.00
0.988
33
0.88
0.166
1.01
0.912
34,35
1.01
0.941
1.19
0.028a
36
1.17
0.165
0.99
0.932
37
1.18
0.168
1.20
0.026a
37
1.11
0.210
0.96
0.665
38
1.06
0.542
1.01
0.818
39
0.89
0.081
0.94
0.460
40
1.00
0.966
0.95
0.550
38
1.06
0.512
1.04
0.446
41 42,43
— —
— —
0.93 1.02
0.257 0.785
17,18
—
—
1.02
0.744
44 45
1.08 —
0.362 —
1.04 —
0.641 —
16,18
1.66
0.075
1.70
0.005a
16,18
2.17
0.095
1.56
0.053
46 16 16 47 47
1.20 1.17 1.11 1.06 1.05
0.078 0.095 0.269 0.059 0.483
0.99 0.91 0.95 1.01 0.97
0.930 0.130 0.427 0.924 0.635
19,20
1.05
0.581
1.03
0.690
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TABLE 1 (Continued) 3-Day Post-AA No.
Gene
24
XBP1
25
LRRK2
26
ULK1
27
ULK2
28
PTPN2
Role of Product
Reference
Fold change
P
Fold change
P
Transcription factor, key component of ER stress response, autophagosome maturation, secretory cell maintenance, Crohn’s disease link, ulcerative colitis link Leucine-rich repeat kinase, autophagosome maturation, Crohn’s disease link Kinase, autophagy initiation, Crohn’s disease link Autophagy initiation, nutrient assessment, protein recruitment to autophagosome Autophagosome formation regulator, responds to IFN-g and TNF-a, Crohn’s disease link, ulcerative colitis link
21
1.14
0.064
0.93
0.267
22
0.90
0.399
0.99
0.826
23
1.04
0.702
0.91
0.286
48
1.04
0.624
1.06
0.463
24
0.99
0.913
0.88
0.092
Other Names
UNC51.2, ATG1H2
28-Day Post-AA
Twenty-eight key genes involved in autophagy, namely, VPS15, VPS34, FIP200, ATG03, ATG04A, ATG04B, ATG05, ATG07, ATG10, ATG12, ATG13b, ATG14, ATG16L1, BECN1, GABARAPL1, IRGM1, IRGM2, LAMP2, LC3A, LC3B, RAB7A, UVRAG, NOD2, XBP1, LRRK2, ULK1, ULK2, and PTPN2 were examined for gene expressions levels in distal colons 3 days post-AA and 28 days post-AA. At 28 days post-AA, FIP200, ATG04A, and IRGM1 were significantly upregulated (P , 0.05). The 3 days post-AA study used 4 AA mice versus 4 SS mice. The 28 days post-AA study involved 3 AA mice versus 3 SS mice. a P , 0.05.
Affymetrix Array Process: Labeling, Hybridization, Scanning, and Normalization RNA from distal colonic tissue obtained from mice was not pooled. RNA from each mouse specimen was taken individually through the microarray process. For Affymetrix arrays, 100 ng of RNA from each sample was labeled using the Whole Transcript Sense Target Labeling Assay as described (Affymetrix). Labeled complementary RNA samples were then hybridized to Affymetrix Mouse Gene 1.0 ST Arrays (28,853 well-annotated genes) (Ramaciotti Centre for Gene Function Analysis, University of New South Wales) before being scanned using a Affymetrix GCS3000 7 G four-color Gene Array scanner with autoloader (Affymetrix). The Gene Expression Omnibus accession number for microarray data reported here, inclusive of minimum information about a microarray experiment-compliant experimental details, 28,29 is GSE23914.
Microarray Preprocessing and Filtering All noncontrol probesets from the 8 arrays were imported into Partek (Version 6.4; Partek Inc., St. Louis, MO) and then normalized using RMA.30 By principle components analysis, a batch effect was evident in principle component 1, which was removed using the batch removal tool in Partek, using default parameters. The probability of each probeset being expressed was determined using the detected above background procedure, using Affymetrix Power Tools (v1.10.2), excluding 13 probes from probeset 10338063, which had very low GC, and thus did not have matched controls. Probesets were excluded if
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none of the samples were detected above background (P ¼ 1025). To assess the degree of differential expression between AA and SS groups, a two-way analysis of variance on treatment and batch was fitted to each probeset using Partek. To correct for multiple hypothesis testing, we used the q-value/positive False Discovery Rate (FDR).31
Gene Set Enrichment Analysis We compared gene expression profiles to the c2_all collection of curated gene sets from the molecular signatures database (version 2.5).27 This collection contains gene sets that are experimentally derived and from expert curated pathway databases. A preranked file was created, containing the average difference between AA and SS for each probeset, sorted from most upregulated in SS to most downregulated. We used the na28 annotation csv file from www.affymetrix.com to determine the gene symbol for each probeset and collapsed probesets to unique genes using the default, max_probe option, resulting in 18,600 unique genes. GSEA (version 2.0)27 was run in preranked mode, using default parameters (gene set sizes between 15 and 500 leaving 1387 gene sets, 1000 permutations, images on the top 50 gene sets).
Enrichment of Autophagy-associated Gene Sets We used GSEA, developed by Mootha et al,32 which merges data from groups of gene sets previously described in the literature to detect significant expression differences. These gene set groups were Kegg Pathways (150 gene sets), micro-
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RNAs (200 gene sets), transcription factors (579 gene sets), biological processes (536 gene sets), and others (1387 gene sets). We used stringent statistical cutoffs (FDR values ,5% and P , 0.001) to delineate autophagy-associated gene sets, which were consistently altered in the distal colons of all AA mice when compared with control SS mice.
Statistics Group comparisons were analyzed using the Mann–Whitney U test with GraphPad Prism (Graphpad software, San Diego, CA). Data are expressed as mean 6 SE of mean, and the differences were considered to be significant if P , 0.05.
RESULTS Individual Distal Colonic Gene Expression of Key Autophagy-associated Genes The 28 genes chosen (Table 1) for expression-level evaluations are those with well-documented direct links with the heterogenous “autophagy system,” with or without links to IBD. Out of the 28 autophagy genes examined (VPS15, VPS34, FIP200, ATG03, ATG04A, ATG04B, ATG05, ATG07, ATG10, ATG12, ATG13b, ATG14, ATG16L1, BECN1, GABARAPL1, IRGM1, IRGM2, LAMP2, LC3A, LC3B, RAB7A, UVRAG, NOD2, XBP1, LRRK2, ULK1, ULK2, and PTPN2), 7 have human polymorphisms associated with IBD (ATG16L1, IRGM1, NOD2, XBP1, LRRK2, ULK1, and PTPN2). FIP200, ATG04A, and IRGM1 were upregulated 1.2-fold, 1.2-fold, and 1.7-fold, respectively (P , 0.05). There were no expression changes in the other 25 genes.
Enrichment of Autophagy-associated Gene Sets Using stringent statistical cutoffs for GSEA (FDR values ,5% and P , 0.001), we delineated 28 days post-AA autophagy-associated gene sets, which were also consistently altered in the distal colons of each of the AA mice when compared with that from each of the control SS mice (Table 2). GSEA analysis demonstrated that 74 gene sets associated with the 28 autophagy genes were downregulated by AA in stark contrast to only 5 upregulated gene sets.
Enrichment of Gene Sets Associated with Autophagy- and IBD-associated Genes Using stringent statistical cutoffs for GSEA (FDR values , 5% and P , 0.001), we delineated 28 days post-AA autophagy + IBD-associated gene sets which were also consistently altered in the distal colons of each of the AA mice when compared with that from each of the control SS mice (Table 3). GSEA analysis demonstrated that 22 gene sets associated with the 7 autophagy + IBD-associated genes were downregulated by AA in stark contrast to only 3 upregulated gene sets.
Autophagy Suppression by Appendectomy
TABLE 2. GSEA Analysis of 28 Important Autophagy Genes–Concise Overview* Gene VPS15 VPS34 FIP200 ATG03 ATG04A ATG04B ATG05 ATG07 ATG10 ATG12 ATG13b ATG14 ATG16L1 BECN1 GABARAPL1 IRGM1 IRGM2 LAMP2 LC3A LC3B RAB7A UVRAG NOD2 XBP1 LRRK2 ULK1 ULK2 PTPN2 TOTAL
Upregulated Gene sets in AA
Downregulated Gene sets in AA
0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 1 0 0 0 5
6 3 3 0 0 1 1 1 1 0 1 0 3 4 3 1 0 11 7 2 0 5 0 8 0 7 3 3 74
GSEA was used to analyze the differential regulation of gene sets associated with 28 key genes involved in autophagy. The gene set groups chosen for further evaluation had stringent cutoff values (FDR , 5% and P , 0.001). Although 74 gene sets associated with autophagy genes were downregulated in the AA group, only 5 were upregulated. *Table 2 is just a concise overview of the results. Please see Supplemental Digital Content 1, http://links.lww.com/IBD/A458 for a detailed version of this table, replete with statistical minutiae.
DISCUSSION As summarized earlier, using a new murine appendicitis model developed by us,14 we showed that AA protected against or prevented experimental colitis. A robust approach, GSEA, was used to delineate and enumerate genetic pathways entailed in this protection or prevention. Reverse transcription-polymerase chain reaction was used for validation of the study.25 The proximal colon would be much more likely to be affected by the inflammatory changes experimentally induced by www.ibdjournal.org |
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TABLE 3. GSEA Analysis of 7 Autophagy Genes with Genetic Links to IBD
Gene
Upregulated Gene sets in AA
ATG16L1
UPREG 0 gene sets —
IRGM1
IRGM2
NOD2 XBP1
LRRK2
ULK1
PTPN2
TOTAL
UPREG 1 gene sets ICHIBA_GRAFT_VERSUS_ HOST_DISEASE_D7_UP UPREG 1 gene sets ICHIBA_GRAFT_VERSUS_ HOST_DISEASE_D7_UP UPREG 0 gene sets — UPREG 0 gene sets —
UPREG 1 gene sets SABATES_COLORECTAL_ ADENOMA_DN UPREG 0 gene sets —
UPREG 0 gene sets —
UPREG 3 gene sets
No. of Enriched Genes
FDR q-val
Downregulated Gene sets in AA
107
0.022
DOWNREG 3 gene sets CTCTGGA,MIR-520A,MIR-525 GCACTTT,MIR-17-5P,MIR-20A,MIR-106A, MIR-1MIR-106B,MIR-20B,MIR-519D GTGCCAA,MIR-96 DOWNREG 1 gene sets BERENJENO_TRANSFORMED_BY_RHOA_UP
107
0.022
DOWNREG 0 gene sets —
0.016
DOWNREG 0 gene sets — DOWNREG 8 gene sets CHARAFE_BREAST_CANCER_LUMINAL_ VS_BASAL_UP GAGCTGG,MIR-337 GTGACGY_V$E4F1_Q6 HSIAO_HOUSEKEEPING_GENES PASQUALUCCI_LYMPHOMA_BY_GC_ STAGE_UP PENG_RAPAMYCIN_RESPONSE_DN PENG_GLUTAMINE_DEPRIVATION_DN SMITH_TERT_TARGETS_UP DOWNREG 0 gene sets —
260
DOWNREG 7 gene sets CHARAFE_BREAST_CANCER_LUMINAL_ VS_BASAL_UP DEBIASI_APOPTOSIS_BY_REOVIRUS_ INFECTION_DN DOUGLAS_BMI1_TARGETS_UP GCACTTT,MIR-17-5P,MIR-20A,MIR-106A, MIR-106B,MIR-20B,MIR-519D GRADE_COLON_VS_RECTAL_ CANCER_DN RASHI_RESPONSE_TO_IONIZING_ RADIATION_3 STEIN_ESRRA_TARGETS_RESPONSIVE_ TO_ESTROGEN_DN DOWNREG 3 gene sets RAMALHO_STEMNESS_UP V$NRF1_Q6 VANHARANTA_UTERINE_FIBROID_WITH_ 7Q_DELETION_UP DOWNREG 22 gene sets
No. of Enriched Genes
FDR q-val
130 492
0.011 0.030
266
0.047
495
0.006
350
0.036
135 468 377 262
0.006 0.043 0.032 0.027
67 82 126
0.001 0.009 0.016
—
—
350
0.036
220
0.001
491 492
0.009 0.030
39
0.026
45
0.009
41
0.032
197 158 67
0.008 0.019 0.010
GSEA was used to analyze the differential regulation of gene sets associated with 7 autophagy genes with genetic links to IBD. The gene set groups chosen for further evaluation had stringent cutoff values (FDR , 5% and P , 0.001). Although 22 gene sets associated with autophagy genes were downregulated in the AA group, only 3 were upregulated. UPREG “n” gene sets, upregulated number of gene sets pertaining to that gene; DOWNREG “n” gene sets, downregulated number of gene sets pertaining to that gene; Number of enriched genes in gene set, the number of genes in each gene set enriched by GSEA pertaining to AA mice in this study; NOM p-val, Nominal P-value statistic; FDR q-val, false discovery rate q-value statistic.
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AA, owing to the close anatomical proximity of the main mouse cecal patch (approximately human appendix) to the proximal colon. However, the novelty of our study is that the most distal regions of the large gut sustain major persistent immunological changes (protective against colitis), by manipulation at the cecum, the most proximal region of the large gut. The immunological changes sustained in T-helper cell type 1, 2, and 17 “pathways” have been published elsewhere.26 Distal colonic gene expression studies, 3 days after surgery or 28 days after surgery, reveal the various genes and gene sets that are responsible for the durable protective effect of AA against colitis.25 The gene expression data obtained from our microarray study get more distinct between the time points (days 3 and 28) and stabilize around day 28. Although observable in our overall microarray results, these are visually appraised better in the reverse transcription-polymerase chain reaction time curves (days 3, 14, and 28) of selected genes in our previous article.25 The persistence of these expression changes would be theoretically easier to demonstrate in humans than mice, as mice have an accelerated lifespan, with 1 mouse year “often equated” to 30 human years. The 28-day-extension of our study is adequate to demonstrate the chronological sustainability of these changes. For our gene expression studies, we chose 28 key autophagy genes, including 7 with previously described genetic links to IBD (Table 1), namely; ATG16L1,17,18 IRGM,16,18 NOD2,19,20 XBP1,21 LRRK2,22 ULK1,23 and PTPN2.24 GSEA analysis of the 28 autophagy genes shows that 74 gene sets were downregulated by AA versus only 5 upregulated gene sets (Table 2; see Table, Supplemental Digital Content 1, http://links.lww.com/IBD/A458). GSEA analysis of 7 autophagy + IBD-associated genes highlights 22 AA-downregulated gene sets versus only 3 upregulated gene sets (Table 3). The genes that demonstrated the maximum suppression in our GSEA analysis were VPS15, LAMP2, LC3A, XBP1, and ULK1 (Table 2), including the previously described IBD-associated genes XBP1 and ULK1 (Table 3). XBP1, a transcription factor, is a key component of the endoplasmic reticulum stress response. It has roles in the secretory cell development and maintenance. Deletion of XBP1 results in spontaneous enteritis and increased susceptibility to induced colitis in a mouse knockout study.21 In that study,21 XBP1 variantassociations with Crohn’s disease and ulcerative colitis were identified and replicated. ULK1, a ubiquitously expressed serine–threonine kinase, initiates autophagy in conjunction with ATG13 and FIP200. A genome-wide association study23 identified that the frequency of a short nucleotide polymorphism in ULK1 was significantly higher in patients with Crohn’s disease. Interestingly, ULK1 knockout mice49 displayed normal induction of autophagy (not demonstrated in the gut), hinting that autophagy may still occur through ULK2 back-up signaling or through microautophagy/chaperone-mediated autophagy. The impact of ULK1 deletion on the gut is yet to be determined.
Autophagy Suppression by Appendectomy
Although Crohn’s disease is more strongly associated with autophagy gene polymorphisms, appendectomy is more protective against ulcerative colitis (most studies) than Crohn’s disease (a proportion of studies). Our mouse model of AA is protective against trinitrobenzene sulfonic acid colitis, which simulates human ulcerative colitis. Therefore, AA-induced autophagy suppression is less likely to be relevant to Crohn’s disease-related inflammation or the exacerbation of it thereof. Although the genetic links between defective autophagy and IBD are reasonably evident, the triggering factors for autophagy dysregulation are yet to be deciphered. Although autophagy is ubiquitous and functionally diverse specialized processes like those adopted by secretory cells require it absolutely for cellular function. Variations in a single autophagy gene may therefore result in multiple manifestations of damage across different cells within the same tissue. Owing to the widespread presence of autophagy throughout human tissues, therapeutic attempts to curtail immunopathology require further elucidation of autophagy dysfunction in each cell type within the same tissue (gut mucosa). Exposure to, or inappropriate responses to gut flora, are commonly incriminated in the causality of IBD.50 In our study, suppression of autophagy by AA is profound. The increased AA-induced expression of the genes IRGM1, FIP200, and ATG04A (1.7-fold, 1.2-fold, and 1.2-fold, respectively) may be an indication of compensatory changes (positive regulatory changes), or the initial changes, which, in turn, had led to the marked autophagy suppression. Autophagy suppression by AA may induce lesser antigen processing, leading to lesser crossreactive immunity between gut flora and self-antigens and subsequent amelioration of colitis. Alternatively, the marked autophagy suppression observed in AA may reflect a controlling response to inappropriately excessive initial immune responses. AA may use these mechanisms to protect against colitis. Our study demonstrates that AA curbs autophagy, potentially contributing to suppression of autophagy-mediated immunopathology as in IBD or experimental colitis. Specific immunohistochemical analyses and “functional confirmation” using 1 or more autophagy gene knockouts would be our next logical step to pursue. Autophagy, owing to its foundational role in gut homeostatic maintenance, is an attractive therapeutic target. Elucidating the pathways involved in these changes will lead to the development of techniques to manipulate different autophagy component pathways resulting in improved IBD management.
ADDENDA 1. The raw microarray data are available in Gene Expression Omnibus. The accession number for microarray data reported here is GSE23914, and the relevant link is http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23914. 2. Specific methodologies and the reverse transcriptionpolymerase chain reaction study validation results have www.ibdjournal.org |
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previously been published.25 We selected 14 genes for confirmation of our gene expression studies. These genes broadly belonged to 4 major groups: innate immunity (SLPI, S100A8, LBP, CD68); immune mediators (IL18R1, IL33), cell migration-chemokines (CCL8, CXCL10, CCL12 or MCP5, PF4, CXCL5, CCL7 or MCP3), and cell migration receptors (FPR1, CCR5).
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