Neonatal Colonic Inflammation Epigenetically Aggravates Epithelial Inflammatory Responses to Injury in Adult Life

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

ORIGINAL RESEARCH

59 60 61 62 63 64 65 Xiaoying S. Zhong,1 John H. Winston,1 Xiuju Luo,2 Kevin T. Kline,3 Syed Z. Nayeem,1 66 67 Yingzi Cong,4 Tor C. Savidge,5 Roderick H. Dashwood,6 Don W. Powell,1 and Qingjie Li1 68 1 3 4 Division of Gastroenterology, Department of Internal Medicine, Department of Microbiology and Immunology, The University 69 of Texas Medical Branch at Galveston, Galveston, Texas; 2Department of Laboratory Medicine, Xiangya School of Medicine, 70 Central South University, Changsha, China; 5Texas Children’s Microbiome Center, Baylor College of Medicine; 6Center for 71 Epigenetics and Disease Prevention, Texas A&M College of Medicine, Houston, Texas 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 Propranolol, a b-blocker, markedly ameliorated the inflammaSUMMARY 93 tory response and IL1b overexpression by mitigating against 94 epigenetic modifications. Adrenalectomy abrogated NI-induced This investigation showed that neonatal inflammation disease susceptibility whereas yohimbine sensitized the 95 increases susceptibility to inflammatory bowel disease by epithelium for exacerbated immune response. The macro- 96 epigenetically sensitizing the interleukin 1b promoter for phages of NI rats produced more IL1b than controls after 97 exacerbated overexpression when exposed to another exposure to lipopolysaccharide (LPS), suggesting hypersensiti- 98 episode of inflammation later in life. Propranolol might zation; incubation with LPS plus Foradil, a b2-agonist, induced Q7 99 mitigate against the inflammatory bowel disease susceptia greater IL1b expression than LPS alone. Epinephrine and 100 bility by reversing epigenetic modifications. Foradil also exacerbated LPS-induced IL1b activation in human 101 THP-1–derived macrophages, by increasing acetylated H4K12, Q8102 103 and these increases were abrogated by propranolol. BACKGROUND & AIMS: Early life adversity is considered a risk 104 factor for the development of gastrointestinal diseases, CONCLUSIONS: NI sensitizes the colon epithelium for exacer105 including inflammatory bowel disease. We hypothesized that bated IL1b activation by increasing stress hormones that 106 early life colonic inflammation causes susceptibility to aggra- induce histone hyperacetylation, allowing greater access of 107 vated overexpression of interleukin (IL)1b. nuclear factor-kB to the IL1B promoter and rendering the host 108 susceptible to aggravated immune responses. Our findings METHODS: We developed a 2-hit rat model in which neonatal suggest that b blockers have a therapeutic potential for 109 inflammation (NI) and adult inflammation (AI) were induced by inflammatory bowel disease susceptibility and establish a 110 trinitrobenzene sulfonic acid. novel paradigm whereby NI induces epigenetic susceptibility to 111 112 RESULTS: Aggravated immune responses were observed in inflammatory bowel disease. (Cell Mol Gastroenterol Hepatol 113 NI þ AI rats, including a sustained up-regulation of IL1b and 2018;-:-–-; https://doi.org/10.1016/j.jcmgh.2018.02.014) 114 other cytokines. In parallel with exacerbated loss of IkBa expression, NI þ AI rats showed hyperacetylation of histone Keywords: Early-Life Adversity; Inflammatory Bowel Disease; 115 116 H4K12 and increased RelA binding on the IL1B promoter, Epinephrine; Histone Acetylation; NF-kB.

Neonatal Colonic Inflammation Epigenetically Aggravates Epithelial Inflammatory Responses to Injury in Adult Life

Q29

Q2

Q6

accompanied by high levels of norepinephrine/epinephrine.

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

Q9

2

117 Q10 118 Q11 119 Q12 120 121 122 123 124 125 126 127 128 129 130 131 132 133 Q13 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 Q14 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175

Zhong et al

I

Cellular and Molecular Gastroenterology and Hepatology Vol.

nflammatory bowel disease (IBD) is a chronic, progressive, relapsing, and immunologically mediated disorder that often targets the young and remains a lifelong affliction. Epidemiologic studies have suggested that the incidence of IBD is increasing worldwide.1–3 Current models of human IBD posit that the inflammatory pathogenesis arises from, and is perpetuated by, interactions between host genetic and immune factors, gastrointestinal microbes, and environmental triggers.4 Accumulating clinical evidence has shown that early life infection is a risk factor for the development of pediatric and adult IBD,5–12 and gastrointestinal infection in adolescents and adults is a trigger for its onset or exacerbation.13–17 However, it is unclear how these events cause an aggravated and prolonged immune response, which is the hallmark of IBD. Early postnatal life is a uniquely vulnerable period, characterized by “epigenetic plasticity,” in which neonates are susceptible to environmental influences that induce durable-epigenetic changes that persist in the adult.18,19 It now is well recognized that adverse early life events have an important role in perinatal programming and maturation of the immune system that make the host susceptible to complex diseases,20–24 including IBD.8,10,18 However, the molecular mechanisms by which adverse early life experiences predispose to IBD remain unknown. A variety of cytokines, including interleukin (IL)1, have been implicated in the pathogenesis of IBD.25 The IL1 family of cytokines comprises 11 proteins (IL1F1–IL1F11) encoded by 11 distinct genes in human beings and mice. IL1-type cytokines are major mediators of innate immune reactions, and blockade of IL1 by the IL1 receptor antagonist has shown an essential role of IL1 in a number of human autoinflammatory diseases.26 IL1b, a proinflammatory cytokine with a wide range of systemic and local effects, has received considerable attention as a potential mediator of inflammatory cell infiltration and mucosal barrier disruption that accompanies gut inflammation.27 It can modulate the function of both immune and nonimmune cells. IL1b also appears to promote inflammation by stimulating the production of other cytokines (eg, IL6) and chemokines (eg, CXCL1, CXCL8, IL8).28–30 Stimulation with IL1b promotes the activation and effector functions of dendritic cells, macrophages, and neutrophils.31 It also induces neutrophilia and promotes neutrophil migration.32 IL1b promotes T-cell activation and survival,33 and acts in concert with other proinflammatory cytokines to promote the differentiation of CD4þ Th17 cells.34–37 Because of the potent inflammatory activity of IL1b, tight mechanisms are in place to regulate its secretion. However, our understanding of IL1b activation in the pathogenesis of IBD is limited, and it is unclear whether early life adversity, such as neonatal colonic inflammation, aggravates IL1b overexpression to exacerbate immune responses when subjected to a second inflammatory insult later in life. The present investigation sought to test the hypothesis that neonatal colonic inflammation epigenetically aggravates IL1b activation in rat colon epithelium when the host is exposed to a second episode of inflammation as an adult.

-,

No.

-

Our findings provide compelling evidence that neonatal colonic inflammation triggers aberrant increases in norepinephrine and epinephrine to enhance histone acetylation at the IL1B gene promoter. Notably, the altered chromatin status persists, facilitating nuclear factor-kB (NF-kB) recruitment and IL1b overexpression when subjected to an additional insult in adult life.

176 177 178 179 180 181 182 183 184 Materials and Methods 185 Reagents 186 Propranolol hydrochloride was purchased from Tocris 187 Bioscience (Bristol, UK). Epinephrine, norepinephrine, lipo- 188 polysaccharide (LPS, from Escherichia coli O111:B4, cat. 189 L4391), formoterol fumarate dihydrate (Foradil), phorbol Q15190 12-myristate 13-acetate (PMA), sodium butyrate, yohim- 191 bine, and 2,4,6-trinitrobenzene sulfonic acid (TNBS) were 192 from Sigma (St. Louis, MO). 193 194 195 Cell Culture THP-1 cells were purchased from ATCC (Manassas, VA) 196 and maintained in RPMI 1640 medium with 2 mmol/L 197 L-glutamine, 10% fetal bovine serum, and 0.05 mmol/L 198 2-mercaptoethanol. To induce differentiation, THP-1 cells 199 seeded at 2  105 cells/mL were incubated with 100 nmol/L 200 201 PMA for 3 days. 202 203 Animals and Procedures 204 Male Sprague Dawley rat littermates were used in the 205 preclinical studies. Five-day-old and 6-week-old Sprague 206 Dawley rats were purchased from Harlan Laboratories 207 (Houston, TX). The work was approved by the Institutional 208 Animal Care and Use Committee at The University of Texas 209 Medical Branch at Galveston. 210 Rat littermates were divided randomly into 4 groups: 211 (1) vehicle treatment in both neonatal and adult-life stages 212 (controls, Ctl); (2) sham treatment as neonates followed by 213 an inflammatory insult as adults (adult inflammation [AI]) 214 (Ctl þ AI); (3) neonatal inflammatory insult (neonatal 215 inflammation [NI]) and then sham treatment as adults; 216 and (4) NI plus AI in combination (NI þ AI) (Figure 1A). 217 Experimenters were blinded to treatment assignment. To 218 induce neonatal inflammation, TNBS (130 mg/kg, 2.86 mg 219 for a 22-g pup, dissolved in 200 mL saline containing 10% 220 ethanol) was injected intraluminally 2 cm into the colon of 221 222 223 Abbreviations used in this paper: AI, adult inflammation; ChIP, chromatin immunoprecipitation; Ctl, control; H4K12ac, _________; HDAC, 224 histone deacetylase; IBD, inflammatory bowel disease; IkB, 225 ______________; IL, interleukin; LPS, lipopolysaccharide; MPO, myelo226 peroxidase; mRNA, messenger RNA; NF-kB, nuclear factor-kB; NI, neonatal inflammation; PCR, polymerase chain reaction; PMA, phorbol 227 12-myristate 13-acetate; RelA, ___________; RNAP II, RNA polymerase 228 II; THP-1, ___________; TNBS, 2,4,6-trinitrobenzene sulfonic acid; Tnf, tumor necrosis factor. 229 © 2018 The Authors. Published by Elsevier Inc. on behalf of the AGA 230 Institute. This is an open access article under the CC BY-NC-ND 231 license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X 232 https://doi.org/10.1016/j.jcmgh.2018.02.014 233 234

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

2018

3

Q1

294 295 0.5 *# 15 296 0.4 10 297 0.3 5 298 * 0.2 0 299 -5 0.1 300 * -10 0 *# -15 Ctl Ctl+AI NI NI+AI 301 Ctl Ctl+AI NI NI+AI 302 303 304 Day 14 Day 7 Day 28 305 IL-1β β-Actin 2.5 306 2.0 # 8 * *# *# 5 2.0 307 *# * 1.5 6 4 1.5 308 3 1.0 4 * 1.0 * 309 2 2 0.5 0.5 1 310 0.0 0 0 0.0 311 Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI 312 313 3 *# 1.5 6 314 3 *# *# 315 2 * 1.0 * 4 2 * 316 * 317 1 0.5 1 2 318 0 0.0 319 0 0 Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI 320 Figure 1. Neonatal colonic inflammation induces aggravated immune responses when subjected to a secondary insult. 321 (A) Schematic presentation of the animal protocol. NS, normal saline. (B) Body weight changes of 4 groups of rats after the 322 second TNBS treatment. (C) H&E staining of the colon, from muscularis to lumen. Scale bar: 100 mm. (D) MPO activity in colon 323 mucosa/submucosa. (E) IL1b protein expression in the mucosa/submucosa dissected from the entire colon. b-Actin served as 324 the loading control. Top: representative images of Western blots. Bottom: relative optical band density ratio between IL1b and 325 b-actin. Real-time reverse-transcription PCR was performed to detect IL1b mRNA levels in the colon mucosa/submucosa 326 at (F) 1 week, (G) 2 weeks, or (H) 4 weeks after AI, as well as the mRNA accumulation of (I) Il12, (J) Tnfa, (K) interferon g (Ifng), 327 and (L) Il10 at 7 days after AI. Values are presented as means ± SEM (N ¼ 12). Two-way analysis of variance. *P < .05 vs Ctl. # 328 P < .05 vs Ctl þ AI. rRNA, ribosomal RNA. 329 330 male pups on postnatal day 10.38 The animals were kept in a first-time AI also aggravates the immune response, compa- 331 head-down position while the anus was held closed for rable with NI treatment. 332 1 minute to prevent leakage. Rats in the sham treatment For intervention experiments with propranolol, all 333 groups received 200 mL of saline. Six to 8 weeks later, 4 groups of rats were given propranolol hydrochloride 334 animals were subjected to a secondary TNBS insult (2 mg/kg dissolved in saline) by daily intraperitoneal 335 (65 mg/kg, 13 mg for a 200-g rat), as AI. Under light injection for 7 days, starting right before the AI. Control 336 anesthesia, 250 mL of TNBS in phosphate-buffered saline groups received normal saline. Animals were killed 3 hours 337 338 containing 40% ethanol was injected intrarectally via a after the last injection. catheter, advanced to 8 cm into the colon. Control rats were To ablate epinephrine/norepinephrine, adrenalectomy 339 given 250 mL of saline. One to 8 weeks after the second was performed in 6-week-old control and NI rats. Briefly, 340 TNBS treatment, animals were decapitated under anes- rats were anesthetized with 2%–3% isoflurane. The left 341 thesia, blood samples were collected for plasma prepara- flank of the rat (landmark the adrenals: caudal end of the 342 tion. For histologic examination, a full-thickness colon ribs on left lateral side of the animal) was shaved with 343 specimen located 3 cm above the anal canal was obtained, electric clippers and the fur was removed. A 1.5- to 2-cm 344 fixed in 4% paraformaldehyde in phosphate-buffered saline, dorsal incision was made over the left adrenal. The adre- 345 embedded in paraffin, sectioned, and stained with H&E. The nals, left and right, were externalized from the abdominal 346 mucosa/submucosa were dissected from the full-length cavity and a ligature was placed below each gland. 347 colon, snap-frozen in liquid nitrogen, pulverized, and The adrenals were excised using forceps, and the perito- 348 stored at -80 C for molecular studies. neal cavity was closed with Vicryl sutures, and the skin Q16 349 We also induced AI with TNBS (130 mg/kg) in 6- to with Prolene sutures. The rats were given normal saline 350 8-week-old rats followed by a second AI (65 mg/kg TNBS) in place of drinking water and they were given 2 weeks 351 6–8 weeks later (AI þ AI), to determine whether a delayed to recover before the next procedure. Sham surgery also 352 Il12/18S rRNA

Tnfa/18S rRNA

J

I

Nl

NI+AI

MPO Activity (OD655)

Ctl

Ctl+AI

G

H

K

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

Il1b/18S rRNA

L

Il10/18S rRNA

Il1b/18S rRNA

F

Fold change

E

D

C

Il1b/18S rRNA

Change of BW (%)

B

Ifng/18S rRNA

A

web 4C=FPO

235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293

Neonatal Inflammation Aggravates IL1b Overexpression

4

353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 Q17 397 398 399 400 401 402 Q18 403 404 405 406 407 408 409 410 411

Zhong et al

Cellular and Molecular Gastroenterology and Hepatology Vol.

Table 1.Primers Specific to the Human IL1B and Rat II1b Promoters Used in ChIP–Quantitative PCR Assays Species

Primer name

Primer sequence

Human IL1B-NF-kB-F

5’-TGGCCCTTCATTGTACCCAT-3’

IL1B-NF-kB-R

5’-TCGTTGTGCAGTTGATGTCC-3’

IL1B-core-F

5’-CTCAGTTTATTAGTCCCCTCCCC-3’

IL1B-core-R

5’-CTCCCTCGCTGTTTTTATGGC-3’

Rat II1b-NF-kB-F

5’-GCTCCCTCAGCTTAAGTCCA-3’

II1b-NF-kB-R

5’-CATTATTTCCCCCTGGACAA-3’

II1b-core-F

5’-ATTCCCACCAAGCTTCTTCC-3’

II1b-core-R

5’-TGGAGAGGATCCCAGATGAG-3’

F, forward; R, reverse.

was performed and served as control. All 4 groups of animals then were treated with TNBS (65 mg/kg) and killed 7 days later. To increase endogenous levels of epinephrine/ norepinephrine in animals, we treated 10-day-old rat pups with LPS-free yohimbine (2.5 mg/kg dissolved in saline) by intraperitoneal injection twice weekly for 6 weeks. The control group received saline. Four hours after the last injection, blood was collected under light anesthesia from the saphenous vein for the measurement of epinephrine levels, and the rats then were treated with TNBS (65 mg/kg) via intrarectal injection. All animals were euthanized 7 days later for tissue collection.

Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) assays were performed as described previously.38,39 Antibodies for immunoprecipitation were from the following sources: RelA (cat. 06-418; Millipore, Temecula, CA), histone H4 acetyl lysine 12 (H4K12ac, cat. 39165), histone deacetylase 3 (HDAC3; cat. 40968), and RNA polymerase II (cat. 39097; Active Motif, Carlsbad, CA). Precipitated DNA, SYBR Green Master Mix (Applied Biosystems, Foster City, CA), and primers specific to the human IL1B or rat IL1b gene promoter (Table 1) were used for real-time quantitative polymerase chain reaction (PCR). Fold differences in precipitated DNA were normalized against input.

Real-Time Reverse-Transcription PCR Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). Complementary DNA was synthesized using SuperScript III First-Strand Synthesis System (Invitrogen). IL1B and IL1b messenger RNA (mRNA) levels were quantitated using TaqMan-based quantitative PCR, as reported.38 18S ribosomal RNA served as an internal control.

Immunoblotting Western blot was performed as described previously.38,39 Primary antibodies were as follows: anti-IL1b rabbit polyclonal (cat. sc-7884), anti-IkBa rabbit polyclonal (cat. sc-371, 1:200) (Santa Cruz, Dallas, TX), and anti–b-actin

-,

No.

-

412 413 414 415 416 417 Isolation of Macrophages by Fluorescence418 Activated Cell Sorting Cell sorting was performed using a BD FACSAria IIU 419 instrument (Becton Dickinson, Franklin Lakes, NJ). Mucosa/ 420 submucosa tissue from the entire colon of each rat was 421 dissociated in 10 mL Hanks’ balanced salt solution con- 422 taining 0.1 mg/mL Liberase and 0.1 mg/mL DNase I, and Q19 423 passed through 70-micron filters. The associated macro- 424 phages were isolated by sorting CD163þ and propidium 425 iodide–negative cells (viable cells) from a 40% Percoll 426 fractionation of dissociated mucosal cells obtained from 427 4 groups of rats, euthanized 7 days after AI. This time point 428 provided the optimal isolation of mature tissue macro- 429 phages, based on pilot studies (not shown). Expression of 430 CD163, a member of the scavenger receptor cysteine-rich 431 family class B, was restricted to cells of the monocyte/ 432 macrophage lineage, and it is expressed in most sub- 433 434 populations of mature tissue macrophages. 435 436 Myeloperoxidase Assay Frozen colon mucosa/submucosa was pulverized in 437 liquid nitrogen, homogenized in 20 mmol/L phosphate 438 buffer (pH 7.4), and centrifuged at 4 C for 10 minutes. 439 Pellets were sonicated in 50 mmol/L phosphate buffer 440 (pH 6.0) containing 0.5% hexadecyl trimethyl ammonium 441 bromide and centrifuged at 4 C for 5 minutes. The super- 442 natant (100 mL) was incubated with 16 mmol/L tetramethyl 443 benzidine in 50% ethanol, 0.3 mmol/L H2O2, and 8 mmol/L 444 sodium phosphate buffer (pH 5.4) for 3 minutes. Myelo- 445 peroxidase (MPO) activity was measured by reading the 446 447 absorbance at 655 nm in a microplate reader.40 448 449 Norepinephrine and Epinephrine Assays 450 Plasma norepinephrine and epinephrine levels were 451 measured using the Noradrenaline Research Enzyme 452 Immunoassay kit and the Epinephrine Research Enzyme453 Linked Immunosorbent Assay kit (Rocky Mountain 454 Diagnostics, Colorado Springs, CO), respectively, according 455 to the manufacturer’s instructions. 456 457 Statistics 458 All data were expressed as means ± SEM. We used 1-way 459 analysis of variance followed by the Tukey post hoc analysis 460 for comparison of more than 2 means, and the Student t test 461 to compare between 2 means, and considered P < .05 to be 462 statistically significant. 463 464 Results 465 Neonatal Inflammation in the Colon Induces 466 Aggravated Immune Responses When Subjected 467 468 to a Second Inflammatory Insult as Adults We reported that neonatal colonic inflammation acti- 469 vates a1C1b gene transcription by up-regulating histone 470 mouse monoclonal antibody (cat. A5441, 1:5000; Sigma). All blots were scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Band density was determined using LI-COR Image Studio Software.

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

2018

5

530 531 4.0 0.3 532 * β-Actin 3.5 533 2.5 * * 3.0 534 * 0.2 * 535 2.0 2.5 * 536 2.0 1.5 537 1.5 0.1 1.0 538 1.0 539 0.5 0.5 540 0.0 0.0 0.0 Ctl AI+AI Ctl+AI 541 Ctl Ctl AI+AI Ctl+AI Ctl+AI AI+AI 542 Figure 2. First-time AI does not induce aggravated IL1b overexpression when the host is exposed to another episode of 543 AI later in life. Adult rats were subjected to an intrarectal injection of TNBS and received a second treatment 6 weeks later. 544 Sham controls received normal saline. Tissue was collected 7 days after the second treatment. (A) MPO activity, (B) IL1b 545 mRNA levels, and (C) IL1b protein expression were quantified in the colon mucosa/submucosa. mRNA was quantified by 546 quantitative reverse-transcription PCR and protein levels were determined by Western blot and densitometry. Data are given 547 as means ± SEM (N ¼ 8). Analysis of variance. *P < .05 vs Ctl. rRNA, ribosomal RNA. 548 549 acetylation on the a1C1b promoter, causing motility differences between Ctl þ AI, NI, and Ctl rats by day 28. 550 dysfunction in the colon.38 The latter work suggested that The mRNA levels of Il12 (Figure 1I), tumor necrosis factor 551 neonatal inflammation could induce sustained epigenetic a (Tnfa) (Figure 1J), and interferon g (Figure 1K), but not 552 changes, which might make the host susceptible to aggra- Il10 (Figure 1L), also were increased significantly in NI þ AI 553 vated immune responses when exposed to a secondary rats vs all other treatment groups. These findings strongly 554 challenge in adult life. To test this hypothesis, we performed support our hypothesis that neonatal inflammation makes 555 an experiment in vivo with 4 groups of rats (Figure 1A). the host vulnerable to aggravated immune responses when 556 Notably, TNBS-induced inflammation in adult life caused subjected to a secondary insult in adult life. 557 significant loss of body weight in both the Ctl þ AI and NI þ We next sought to investigate whether NI was a pre- 558 AI groups, when compared with control rats (n ¼ 12) requisite for the increased susceptibility later in life, or 559 (Figure 1B). There was no significant difference between NI could be substituted with a delayed first hit as an adult. Q20560 and Ctl rats. Importantly, NI þ AI rats showed a significantly Thus, colonic inflammation was first induced with TNBS in 561 greater decrease in body weight compared with Ctl þ AI 6- to 8-week-old adult rats (AI rats, saline served as con- 562 rats (P < .05). H&E staining of the colons showed severe trol),41 followed by another dose of TNBS 6 weeks later 563 local diffuse destruction, higher histologic scores, and (AI þ AI rats), and tissue was collected 7 days later. There 564 neutrophil infiltration in NI þ AI vs Ctl þ AI rats (Figure 1C). was no difference in MPO activities of colon mucosa/ 565 A significantly greater increase in MPO activity was found in submucosa for AI þ AI and Ctl þ AI rats (n ¼ 8) (Figure 2A), 566 the colon mucosa/submucosa of NI þ AI rats vs Ctl þ AI rats both being significantly greater than Ctl (P < .05). The second 567 (Figure 1D), NI rats, and Ctl rats (P < .01). Immunoblotting AI also increased IL1b mRNA (Figure 2B) and protein 568 showed a marked up-regulation of mature IL1b protein in (Figure 2C) levels in colon mucosa/submucosa of Ctl þ AI 569 colon mucosa/submucosa of Ctl þ AI and NI þ AI rats, and AI þ AI rats, respectively, but no significant differences 570 compared with Ctl rats (Figure 1E), and this was signifi- were found between Ctl þ AI and AI þ AI rats. These findings 571 cantly greater for NI þ AI vs Ctl þ AI rats. There was no suggested that a first time AI in adulthood cannot substitute 572 for NI in exacerbating immune responses when the host is 573 significant difference between NI and Ctl rats. 574 To investigate transcriptional changes, IL1b mRNA levels exposed to an inflammatory challenge later in life. 575 were examined by real-time reverse-transcription PCR in 576 the colon mucosa/submucosa of rats at 7, 14, and 28 days after AI (Figure 1F–H). One week after AI, IL1b mRNA levels Exacerbated IL1b Overexpression in the 577 were increased by 2.5- and 6-fold in Ctl þ AI and NI þ AI Colon Mucosa/Submucosa of NI þ AI Rats 578 rats, respectively, compared with Ctl rats (Figure 1F). The Is Mediated by NF-kB 579 It is well known that NF-kB activation plays an important 580 increase in NI þ AI rats was significantly greater than that in Ctl þ AI rats (P < .05). At 2 weeks, IL1b mRNA levels were role in the production of proinflammatory cytokines, such as 581 increased 1.5- and 2-fold in Ctl þ AI and NI þ AI rats, IL1b. To investigate whether NF-kB was activated in NI þ AI 582 respectively, which was statistically significant compared rats, we first assessed the levels of IkBa. The latter protein 583 with Ctl rats (Figure 1G). IL1b mRNA levels were signifi- inhibits NF-kB by masking nuclear localization signals on 584 cantly greater in NI þ AI rats vs Ctl þ AI rats. At 4 weeks NF-kB proteins, keeping them sequestered in an inactive 585 (Figure 1H), IL1b mRNA accumulation remained signifi- state in the cytoplasm,42 and thereby blocking the ability of 586 cantly increased for NI þ AI rats compared with all other NF-kB transcription factors to tether to DNA.43 We found 587 treatment groups (P < .05). There were no significant that IkBa protein levels were significantly attenuated in 588

C

IL-1β

IL-1β/β-Actin

B Il1b/18S rRNA

A MPO Activity (OD655)

471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529

Neonatal Inflammation Aggravates IL1b Overexpression

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

6

Cellular and Molecular Gastroenterology and Hepatology Vol.

-,

No.

-

D

E

Fold change (H4K12ac/Input)

G

Fold change (RNAP II/Input)

F

-419/ -303/ -410 -294

Fold change (RelA/Input)

C

Fold change (HDAC3/Input)

B

IκBα/β-Actin

648 649 TSS IκBα 650 β-Actin 651 Core promoter NFκB binding site 652 1.2 653 1.0 654 * 20 0.8 *# *# 655 0.6 15 656 0.4 657 10 0.2 658 5 * 0.0 659 Ctl Ctl+AI NI NI+AI 660 Ctl Ctl+AI NI NI+AI 661 662 663 # 10 * 1.5 664 8 665 1.0 6 666 667 4 0.5 * 668 * 2 *# 669 0.0 0 670 Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI 671 672 # 673 12 25 * 674 # 10 * 20 675 8 676 15 6 677 * 10 4 678 * 679 5 2 680 0 0 681 NI NI+AI Ctl Ctl+AI NI NI+AI Ctl Ctl+AI 682 Figure 3. Neonatal inflammation epigenetically aggravates IL1b activation through NF-kB. (A) IkBa protein expression in 683 the colon mucosa/submucosa of 4 groups of rats. Top: representative images of Western blots. Bottom: relative optical band 684 density ratio between IkBa and b-actin (N ¼ 6). *P < .05 vs Ctl. #P < .05 vs Ctl þ AI. (B) Schematic of the rat IL1b promoter with 685 2 NF-kB binding motifs. Nucleotide numbering is relative to the transcription start site (TSS). (C) ChIP–quantitative PCR 686 analysis of RelA (p65) association with the NF-kB binding sites of the rat IL1b promoter in the colon mucosa/submucosa. 687 (D) HDAC3 interaction with the NF-kB binding motifs at the rat IL1b promoter. (E) Acetylation status of H4K12ac around the NF-kB binding region of the rat IL1b promoter in colon mucosa/submucosa. (F) Acetylated histone H4K12 at the IL1b core 688 promoter region. (G) RNAP II association with the IL1b core promoter in the colon mucosa/submucosa of 4 groups of rats. 689 Chromatin was immunoprecipitated with specific antibodies, as indicated. Precipitated chromatin was quantified by real-time 690 PCR using primers specific to the NF-kB binding region, or the core promoter of the rat IL1b gene, and normalized to inputs. 691 Means ± SEM. N ¼ 3 independent experiments. Two-way analysis of variance. *P < .05 vs Ctl. #P < .05 vs Ctl þ AI. 692 693 colon mucosa/submucosa of both Ctl þ AI and NI þ AI rats were increased significantly in NI þ AI rats compared with 694 (Figure 3A). More importantly, down-regulation of IkBa Ctl þ AI. There was no significant difference in RelA binding 695 protein expression was significantly greater in NI þ AI vs between NI and Ctl groups (Figure 3C). 696 Ctl þ AI rats (P < .05), suggesting an exacerbated activation Chromatin can exist in “open” or “closed” states, Q21 697 of NF-kB in NI þ AI rats. To show a cause-and-effect rela- depending on the circumstances,44 and chromatin remod- 698 tionship between NF-kB activation and IL1b up-regulation, eling is required for the recruitment of NF-kB and subse- 699 we identified 2 NF-kB binding motifs (-419/-410 and quent activation of target genes.45 We investigated the role 700 -303/-294) in the rat IL1b promoter (Figure 3B). of HDAC3, a pivotal HDAC in the colonic epithelium,46 and 701 ChIP–quantitative PCR assays with RelA (p65) antibody H4K12ac status near the NF-kB binding region of the IL1b 702 showed low, constitutive RelA interactions on IL1b for the promoter. In colonic mucosa/submucosa samples, HDAC3 703 Ctl and NI groups (Figure 3C). These interactions were binding was significantly repressed in NI þ AI vs Ctl þ AI, 704 enhanced significantly in Ctl þ AI and NI þ AI groups, and control rats (Figure 3D). A significant repression also 705 compared with Ctl. Notably, RelA interactions with IL1b was observed in Ctl þ AI vs NI and control rats. There was 706

A

Fold change (H4K12ac/Input)

589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647

Zhong et al

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

2018

707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765

Neonatal Inflammation Aggravates IL1b Overexpression

no significant difference between Ctl and NI groups. When histone acetylation was examined for the NF-kB binding region of IL1b, significantly greater H4K12ac levels were detected in NI þ AI vs Ctl þ AI, NI, and Ctl groups (Figure 3E). Levels of H4K12ac in NI þ AI and Ctl þ AI levels were significantly higher than in Ctl and NI groups, whereas Ctl and NI groups did not differ markedly from each other. We concluded that a reciprocal relationship exists, involving increased H4K12ac and decreased HDAC3 interactions in the vicinity of the NF-kB binding region of IL1b in NI þ AI vs Ctl þ AI rats. We also examined H4K12ac and RNA polymerase II (RNAP II) recruitment near the IL1b core promoter (-141/þ42) (Figure 3B). As with the NF-kB binding region (Figure 3E), H4K12ac levels were increased significantly in the core promoter of IL1b for NI þ AI rats vs Ctl þ AI rats, NI rats, and control rats (Figure 3F). This was interpreted as evidence of increased chromatin relaxation, and greater accessibility of the preinitiation complex, on the IL1b promoter. Core promoter H4K12ac levels were significantly greater in Ctl þ AI vs NI and Ctl rats, but there was no significant difference between NI and Ctl groups. Concomitantly, RNAP II association with the IL1b core promoter was significantly augmented in NI þ AI vs Ctl þ AI, NI, and Ctl groups (Figure 3G).

Stress Hormones Play an Important Role in the Aggravated IL1b Activation in NI þ AI Rats Alterations in the brain-gut axis are considered a pillar of the modern view of irritable bowel syndrome47 and IBD pathogenesis.48 Stress responses represent a defense against real or perceived threats, which stimulate the adrenal medulla to increase plasma levels of norepinephrine and epinephrine. When blood norepinephrine and epinephrine levels were examined in the present investigation, we found that norepinephrine (Figure 4A) and epinephrine (Figure 4B) levels were increased significantly in NI þ AI rats compared with Ctl þ AI, NI, and Ctl groups, respectively. There were also increases of norepinephrine (N ¼ 30; P ¼ .068) and epinephrine (P ¼ .047) in NI vs Ctl rats (Figure 4A and B). Thus, stress hormones were implicated only in the aggravated immune responses of rats after NI þ AI treatment. Based on the findings for norepinephrine and epinephrine, we postulated that stress hormones mediate the aggravated immune responses caused by NI þ AI exposure. To test this hypothesis, we treated all 4 groups (Ctl, Ctl þ AI, NI, and NI þ AI) with propranolol hydrochloride (2 mg/kg/ day intraperitoneally), a nonselective b blocker, for 7 days, starting immediately before AI. Propranolol markedly ameliorated TNBS-induced swelling, cecal enlargement, and tissue damage in NI þ AI rats (Figure 4C). Significant improvement also was observed in Ctl þ AI rats after the treatment. The exacerbated up-regulation of IL1b mRNA in the colon mucosa/submucosa of NI þ AI rats was significantly repressed by propranolol (Figure 4D). Thus, aggravated immune responses in NI þ AI rats appear to be mediated, at least in part, by heightened levels of stress hormones. Notably, IL1b mRNA levels in Ctl þ AI remained unchanged by propranolol.

7

To investigate whether blockade of adrenoceptors by propranolol mitigates against NI-induced epigenetic modifications on the IL1b promoter, HDAC3 and H4K12ac levels were assessed, as described earlier. We found that the NI-reduced HDAC3 interactions near the NF-kB binding region of IL1b essentially were reversed by propranolol treatment in NI þ AI rats (Figure 4E). Less dramatic increases in HDAC3 binding also were noted in Ctl, Ctl þ AI, and NI groups treated with propranolol. Accordingly, propranolol reversed the histone hyperacetylation on IL1b in NI þ AI rats (Figure 4F). No significant changes were detected for H4K12ac in Ctl þ AI, NI, or Ctl rats given propranolol, compared with the corresponding vehicle controls. To verify that stress hormones mediate the disease sensitization, we depleted norepinephrine and epinephrine by adrenalectomy in Ctl and NI rats, and applied AI when the animals were 8 weeks old. Ablation of norepinephrine and epinephrine almost completely abrogated the significant increases of MPO activity (Figure 5A) and IL1b activation (Figure 5B) by NI and had little effect on control rats, suggesting that high constitutive levels of norepinephrine and epinephrine are critical to the NI-induced disease susceptibility. To further show that an increase of norepinephrine and epinephrine increases inflammatory responses after AI, we administered yohimbine, an a2 antagonist proven to increase norepinephrine and epinephrine,49 for 6 weeks starting from postnatal day 10. Serum levels of epinephrine were increased significantly by yohimbine treatment in naive rats compared with vehicle-treated rats (P < .05) (Figure 5C). Importantly, yohimbine-treated animals showed significantly higher levels of MPO activity (Figure 5D) and IL1b mRNA (Figure 5E) in the colon mucosa/submucosa after the inflammatory insult with TNBS, clearly showing sensitization in the colon.

Neonatal Inflammation Sensitizes Macrophages in the Colon Macrophages play a critical role in the initiation, maintenance, and resolution of inflammation, and they function in both innate immunity and adaptive immunity of vertebrate animals.50 We hypothesized that neonatal inflammation sensitizes macrophages in the colonic mucosa, and makes them more sensitive to inflammatory stimuli. To test this hypothesis, we isolated macrophages from the colon mucosa/submucosa by fluorescence-activated cell sorting, and analyzed IL1b mRNA expression by quantitative reverse-transcription PCR. IL1b mRNA levels were significantly greater in NI þ AI rats vs Ctl þ AI rats, NI rats, and Ctl rats (Figure 6A). No significant differences were noted for IL1b mRNA in the other treatment groups. To corroborate that NI induced macrophage sensitization in the colon, we incubated the macrophages of Ctl or NI rats with either 100 ng/mL LPS or a combination of 100 ng/mL LPS and 1 mmol/L Foradil, a selective b2-adrenergic agonist, and examined IL1b mRNA expression. Real-time PCR analyses showed that LPS induced a significantly greater increase of IL1b mRNA in the macrophages isolated from NI rats vs Ctl rats (Figure 6B), strongly supporting our hypothesis that NI sensitizes macrophages in the colon. Compared with LPS

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824

8

Cellular and Molecular Gastroenterology and Hepatology Vol.

-,

No.

-

884 885 *# *# 886 400 2.0 887 * 1.5 300 888 1.0 200 889 890 0.5 100 891 0.0 0 892 Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI 893 894 895 896 + Vehicle Prop 897 15 898 * # ** 899 + 10 900 * 901 902 5 + 903 904 0 905 Ctl Ctl+AI NI NI+AI + 906 907 908 Vehicle Prop Prop Vehicle 2.0 909 10 * # ** * 910 8 # ** 1.5 * 911 6 * 1.0 912 4 913 0.5 2 914 915 0.0 0 Ctl Ctl+AI NI NI+AI Ctl Ctl+AI NI NI+AI 916 917 Figure 4. NI induces exacerbated IL1b activation through stress hormones. (A and B) Plasma norepinephrine (NE) and 918 epinephrine (Epi) content in 4 groups of rats measured using enzyme immunoassay and enzyme-linked immunosorbent assay kits, respectively (N ¼ 30). *P < .05 vs Ctl. #P < .05 vs the other 3 groups. (C) Representative images of colons from 4 groups of 919 rats treated with vehicle or propranolol, a nonselective b-blocker. Propranolol (2 mg/kg) was dissolved in saline and given to 920 the animals by daily intraperitoneal injection. Another 4 groups of animals received saline and served as controls. (D) IL1b 921 mRNA levels in the colon mucosa/submucosa quantified by quantitative reverse-transcription PCR; 18S ribosomal RNA 922 (rRNA) served as an internal control. N ¼ 8. (E) ChIP–quantitative PCR analysis of HDAC3 binding to the NF-kB binding sites 923 at the rat IL1b promoter. (F) ChIP–quantitative PCR data of H4K12ac levels surrounding the NF-kB binding region of the 924 IL1b promoter. Data are shown as means ± SEM. N ¼ 3 independent experiments. Two-way analysis of variance. *P < .05, Q25 # 925 P < .05, **P < .05. 926 927 alone, a combination of LPS and Foradil resulted in treatment with PMA. Incubation with LPS dramatically 928 significantly greater increases of IL1b in both NI and Ctl increased IL1B mRNA expression in THP-1–derived macro- 929 rat macrophages, providing additional evidence that phages (Figure 7A), as expected. In the presence of LPS, 930 aberrant levels of stress hormones exacerbate immune epinephrine (1 mmol/L) further increased IL1B expression. 931 responses. The IL1b mRNA levels were significantly greater Surprisingly, norepinephrine (1 mmol/L) had little effect 932 in NI vs Ctl rat macrophages after the treatment of (data not shown). Propranolol treatment not only 933 LPS/Foradil (Figure 6B). completely abrogated the IL1B overexpression exacerbated 934 by epinephrine, but also significantly mitigated against 935 LPS-induced IL1B activation. Similar to epinephrine, Foradil 936 LPS-Induced IL1b Activation Is Aggravated also further increased IL1B mRNA levels in the presence 937 by Epinephrine in Human THP-1–Derived of LPS, and the increase was reversed by propranolol 938 939 Macrophages (Figure 7B). To further validate that stress hormones aggravate IL1b We next incubated THP-1 macrophages with LPS, 940 activation in inflammatory conditions, we used human epinephrine, or their combination in the presence or 941 THP-1 cells, which differentiate into macrophages after absence of propranolol for 24 hours, and performed 942

B

2.5

500

Epi (pg/ml)

NE (ng/ml)

A

Il1b/18S rRNA

Propranolol

D

NI+AI

NI

Ctl+AI

Ctl

C

F

Fold change (H4K12ac/Input)

Fold change (HDAC3/Input)

E

web 4C=FPO

825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883

Zhong et al

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

2018

9

1002 1003 * 8 # 1004 * 0.4 6 1005 0.3 1006 4 0.2 1007 0.1 2 1008 1009 0 0 Adx Sham Adx Sham Adx Sham Adx Sham 1010 Ctl+AI Nl+AI Ctl+AI Nl+AI 1011 1012 1013 5 1014 *# 400 * 0.5 *# 1015 4 0.4 300 1016 3 0.3 * 1017 200 * 2 0.2 1018 100 1 0.1 1019 1020 0 0 0 Ctl Ctl Veh Yoh Veh Yoh Ctl Veh Yoh 1021 Ctl+AI Ctl+AI 1022 Figure 5. Stress hormones play a key role in aggravating inflammatory responses in the colon. Adrenalectomy abolished 1023 aggravated increases of (A) MPO activity and (B) IL1b mRNA expression by NI in the colon mucosa/submucosa. Sham surgery 1024 was performed in control groups (N ¼ 6). *P < .05, #P < .05. Long-term administration of yohimbine (Yoh, LPS-free) by twice- 1025 weekly intraperitoneal injection not only (C) increased the serum levels of epinephrine (Epi), but also (D) exacerbated the 1026 up-regulation of MPO activity and IL1b mRNA (E) after AI (N ¼ 6). One-way analysis of variance. *P < .05 vs Ctl. #P < .05 vs Q261027 vehicle (Veh). Data are presented as the means ± SEM. Adx, adrenalectomy. 1028 1029 ChIP–quantitative PCR assays on the human IL1B promoter. epinephrine, compared with LPS alone, and the exacerbated 1030 At the NF-kB binding region, H4K12ac was increased increase was ameliorated by propranolol. These results 1031 significantly in cells treated with LPS (Figure 7C), compared suggest that epinephrine aggravates LPS-induced IL1B 1032 with control cells. Addition of epinephrine plus LPS further overexpression by enhancing histone acetylation, as in the 1033 increased H4K12ac levels surrounding the NF-kB binding preclinical rat model. 1034 motifs; this increase was mitigated significantly by pro1035 pranolol. Consistently, we found that the association of RelA Discussion 1036 with NF-kB binding motifs (-413/-404 and -297/-288) on During the perinatal period, at a time of epigenetic 1037 the human IL1B promoter was increased significantly in plasticity, environmental conditions can modulate gene 1038 LPS-treated cells vs control cells (Figure 7D). The NF-kB expression via epigenetic reprogramming.51 It is believed 1039 binding was increased further in cells treated with LPS þ that adverse early life events can interfere with the perinatal 1040 1041 1042 1043 25 4 Ctl 1044 NI 20 1045 3 1046 15 1047 2 1048 10 1049 1 5 1050 1051 0 0 1052 Veh LPS LPS+For Ctl Ctl+AI NI NI+AI 1053 1054 Figure 6. Neonatal colonic inflammation sensitizes the macrophages in the rat colon. (A) IL1b mRNA expression levels in 1055 the macrophages isolated from the colon mucosa/submucosa of 4 groups of animals by fluorescence-activated cell sorting. 1056 IL1b mRNA was quantified by quantitative reverse-transcription PCR (N ¼ 6). *P < .05 vs the other 3 groups. (B) IL1b mRNA in macrophages treated with LPS or the combination of LPS and Foradil, a b2-adrenergic agonist. Macrophages were isolated 1057 from the colon mucosa/submucosa of Ctl and NI rats by fluorescence-activated cell sorting and incubated for 24 hours in 1058 complete RPMI 1640 medium containing 100 ng/mL LPS, or 100 ng/mL LPS plus 1 mmol/L Foradil. Means ± SEM. N ¼ 3 1059 Q271060 independent experiments. Two-way analysis of variance. *P < .05.

B

Il1b/18S rRNA

#

D

E

Il1b/18S rRNA

A

B

*

Il1b/18S rRNA

Epi (pg/ml)

C

0.5

MPO Activity (OD655)

MPO Activity (OD655)

A

Il1b/18S rRNA

943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001

Neonatal Inflammation Aggravates IL1b Overexpression

*

*

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

10

Cellular and Molecular Gastroenterology and Hepatology Vol.

-,

No.

-

1120 1121 40 10 # 1122 * *# 8 1123 30 * ** 1124 * 6 20 1125 4 1126 10 ** 1127 2 1128 0 0 1129 + + + + + + 2 μg/ml LPS 2 μg/ml LPS 1130 1 uM For + + 1 uM Epi + + 1131 + 1 uM Prop + 1 uM Prop 1132 1133 1134 1135 10 1136 # 4 * *# 1137 8 3 1138 6 * 1139 * 2 4 ** 1140 ** 1 1141 2 1142 0 0 1143 + + + + + + 2 μg/ml LPS 2 μg/ml LPS 1144 1 uM Epi + + 1 uM Epi + + 1145 + + 1 uM Prop 1 uM Prop 1146 1147 Figure 7. Propranolol ameliorates the aggravated up-regulation of IL1b by epinephrine or Foradil in human THP-1 cells. 1148 THP-1 cell–derived macrophages were treated with 0.1 mg/mL LPS, 1 mmol/L Foradil (For), or their combination for 24 hours. 1149 IL1B mRNA levels were evaluated by quantitative reverse-transcription PCR and normalized to 18S ribosomal RNA (rRNA). ChIP–quantitative PCR were performed to quantify RelA binding and H4K12ac levels. (A) Propranolol (Prop) markedly 1150 repressed the IL1B mRNA up-regulation by LPS and Epi (N ¼ 3). *P < .05 vs control, #P < .05 vs LPS, **P < .05 vs LPS þ Epi. Q281151 (B) Exacerbated IL1B up-regulation by Foradil was abrogated by propranolol (N ¼ 3). *P < .05 vs control, #P < .05 vs LPS, 1152 **P < .05 vs LPS þ For. (C) H4K12ac levels at the core promoter of the human IL1B promoter. (D) ChIP–quantitative PCR 1153 evaluation of RelA association with the NF-kB binding sites on the human IL1B promoter. Means ± SEM. N ¼ 3 independent 1154 experiments. Two-way analysis of variance. *P < .05 vs Ctl, #P < .05 vs LPS, **P < .05 vs LPS þ Epi. 1155 1156 programming and maturation of the immune system, increased histone acetylation (H4K12ac) and RelA binding 1157 predisposing the host to complex diseases20–24 such as coupled to loss of HDAC3. 1158 IBD.8,10,18 A major deficiency in the field involves our Recent findings point to a critical window of early 1159 understanding of how epigenetic regulation might predis- postnatal development during which gene expression 1160 pose the host to IBD, and whether this occurs preferen- may be persistently “re-programed.”52 Adversity in the early Q221161 tially during the neonatal period. More specifically, it is stages of development can have a profound impact on 1162 unclear whether and how neonatal colonic inflammation psychological and physical health.53 Indeed, human and 1163 epigenetically interferes with the activation of IL1b, which animal studies have provided converging evidence that 1164 is highly overexpressed in patients with active IBD, and adverse early life experiences, such as prenatal exposure to 1165 animals with experimental colitis. Identification of new stress,54–56 nutritional deprivation,57 postnatal neglect and 1166 IBD mechanisms, particularly molecules that mediate the abuse,58–60 and neonatal inflammation,38,61,62 can have a 1167 exacerbation of IL1b expression, will facilitate the devel- significant long-term impact with implications for the 1168 opment of novel therapies and interventional strategies emergence of various complex diseases, including IBD. The 1169 against IBD and/or IBD susceptibility associated with underlying mechanisms remain almost as much a mystery 1170 neonatal injury. In this report, a 2-hit chemical injury today as they were a century ago.23 It is understandable 1171 model was used to investigate epigenetic mechanisms that early life adversity might cause a myriad of transient 1172 linked to aggravated IL1b overexpression in colonic alterations. However, some durable epigenetic changes 1173 epithelium of the rat. We found that neonatal inflamma- have been identified, which may underlie enduring physio- 1174 tion, followed by an additional inflammatory insult later in logical, neurologic, or pathologic outcomes. DNA methyl- 1175 adult life, caused aberrant increases in colonic MPO ation and histone modifications have been examined 1176 activity, and IL1b overexpression was linked to epigenetic after certain early life experiences, with changes noted in 1177 reprogramming of the IL1b gene transcription, via several key genes involved in the regulation of the 1178

A

D

Fold change (RelA/Input)

C

IL1B/18S rRNA

IL1B/18S rRNA

B

Fold change (H4K12ac/Input)

1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119

Zhong et al

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

2018

1179 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

Neonatal Inflammation Aggravates IL1b Overexpression

hypothalamic–pituitary–adrenal axis.63–65 A key question that remains unanswered is what mediates those epigenetic changes. We found that circulating levels of both norepinephrine and epinephrine were increased in NI rats, which is consistent with previous observations,62,66 and there was a greater increase in NI þ AI rats. Norepinephrine is a catecholamine with multiple physiological functions, including acting as a hormone and a neurotransmitter. It is best known for its role in the fight-or-flight stress response, along with epinephrine, but there is accumulating evidence for the modulation of inflammatory outcomes. In 2007, Rommelfanger and Weinshenker67 reported that norepinephrine suppresses the expression of proinflammatory molecules, such as TNF-a and IL1b, and increased the expression of anti-inflammatory molecules, such as IkB, by signaling through a1-, a2-, and b-adrenergic receptors on astrocytes and glia. This regulates the expression of inflammatory genes and nitric oxide, which are thought to contribute to neurodegenerative diseases. Another study by Takayanagi et al68 in 2012 found that norepinephrine regulates intestinal mucosal immune responses. Norepinephrine suppresses the production of interferon-g and TNF-a in murine intestinal intraepithelial lymphocytes via the b1 adrenoceptor. A major noradrenergic center of the brain is the locus coeruleus, which signals to sympathetic preganglionic cholinergic neurons in the spinal cord.69 These sympathetic nerve endings release norepinephrine, which has been found to have anti-inflammatory effects by interacting with the adrenoceptors expressed on lymphocytes and macrophages. In this way, norepinephrine has activity at both ends of the pathway: initiating signaling from the locus coeruleus and interacting with immune adrenoceptors to exert an immunomodulatory effect. In the case of IBD, it is well known that various stressors can trigger relapse, or exacerbate the inflammatory condition.70 Our findings suggest that persistently increased levels of norepinephrine and epinephrine in NI þ AI rats sensitize the IL1b promoter in intestinal macrophages, resulting in aggravated IL1b activation; significantly greater increases of IL1b also were observed in NI rat macrophages incubated with LPS or LPS plus Foradil, a b2-adrenergic agonist. This notion is strongly supported by our findings showing that propranolol, a nonselective b blocker, markedly abrogated the aberrant overexpression of IL1b in NI þ AI rats, and ameliorated against the heightened inflammatory response in the colon. Moreover, ablation of norepinephrine/ epinephrine by adrenalectomy abrogated NI-induced susceptibility, and increase of norepinephrine/epinephrine by yohimbine sensitized the colon epithelium for exacerbated immune responses. In addition, incubation of human THP-1–derived macrophages with epinephrine or Foradil, but not norepinephrine, aggravated LPS-induced IL1b upregulation; the aggravation was completely blocked by propranolol. These results provide additional evidence that epinephrine functions as a mediator of IL1b activation, and b-blockers could be beneficial to IBD patients. IL1b is produced by a wide range of cells and is a major player in immune and inflammatory processes.31 However,

11

molecular mechanisms by which epinephrine mediates or 1238 aggravates IL1b activation remain unclear. Epigenetic 1239 sensitization of the IL1b promoter by neonatal inflammation 1240 is virtually unknown. Our hypothesis was that epinephrine 1241 modulates histone modifications to remodel the chromatin 1242 state surrounding the IL1b promoter. ChIP–quantitative PCR 1243 data showed that HDAC3 association with the correspond- 1244 ing NF-kB binding region was repressed significantly in NI þ 1245 AI rats vs Ctl þ AI rats, although H4K12ac was increased 1246 markedly. Consistently, H4K12Ac and RNAP II binding at 1247 the core promoter of IL1b also were increased significantly, 1248 leading to heightened IL1b overexpression. Importantly, 1249 these epigenetic alterations were reversed by propranolol. 1250 Furthermore, LPS-induced histone acetylation on the human 1251 IL1B promoter in THP-1–derived macrophages was aggra- 1252 vated significantly by epinephrine, and this was alleviated 1253 by propranolol. Our findings show that epinephrine sensi- Q231254 tizes the rat IL1b and human IL1B gene promoters via 1255 histone hyperacetylation. 1256 Two NF-kB binding motifs exist in both human IL1B 1257 (-413/-404 and -297/-288) and rat IL1b promoters 1258 (-419/-410 and -303/-294), implicating NF-kB as a key 1259 regulator of IL1b expression in different species. We found 1260 significant down-regulation of IkBa in the colonic epithelia 1261 of NI þ AI rats, and the underlying mechanisms are 1262 currently under investigation. It is noteworthy, however, 1263 that NF-kB alone cannot tether to DNA without chromatin 1264 relaxation, which is regulated by multiple factors generating 1265 and maintaining a cell-specific chromatin landscape. 1266 Although NF-kB is able to influence the chromatin state 1267 through a variety of mechanisms, chromatin remodeling 1268 remains a key determinant for DNA access and NF-kB 1269 binding activity.71 Epigenetic modifications, especially 1270 acetylation of the lysine residues on the N-terminal tails of 1271 histone proteins, are essential to such chromatin dynamics. 1272 The lysine residue at H4K12 can be acetylated but not 1273 methylated, and is part of a “backbone” of histone modifi- Q241274 cations associated with active promoters.72 Thus, histone 1275 hyperacetylation around the NF-kB binding motifs on the 1276 IL1B/IL1b promoter becomes critical for NF-kB–mediated 1277 IL1b activation. Indeed, RelA association with the IL1b 1278 promoter was significantly greater in the colon mucosa/ 1279 submucosa of NI þ AI rats, compared with the other 1280 3 groups, which strongly correlates with the histone acety- 1281 lation levels. We also observed aggravated RelA binding to 1282 the human IL1B promoter in THP-1–derived macrophages 1283 treated with both LPS and epinephrine. These findings 1284 provide strong evidence that adrenal signaling epigenetically 1285 modulates IL1b expression. Although changes in HDAC3 and 1286 H4K12ac were seen on the IL1b promoter in response to 1287 propranolol, we cannot rule out mechanisms such as up- 1288 regulation of miRNAs that target IL1b for down-regulation, 1289 such as miR-146a, which plays a critical role in gut homeo- 1290 stasis.73 Pyrosequencing showed only subtle changes in DNA 1291 methylation on the IL1b promoter after NI (data not shown); 1292 future studies might focus on long-range genomic in- 1293 teractions impacted by altered chromatin states in IBD.74 1294 Importantly, rats given the 2-hit chemical insult protocol 1295 only as adults did not recapitulate the findings for NI þ AI, 1296

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

12

1297 1298 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

Zhong et al

Cellular and Molecular Gastroenterology and Hepatology Vol.

strongly implicating early epigenetic reprograming in the neonate as a critical regulator of the exacerbated IL1b overexpression in the colon. Adult rats exposed to NI alone were normal in appearance, and their levels of MPO activity, IL1b expression, and H4K12 acetylation at the IL1b promoter were not significantly different from controls. Thus, individuals subjected to early life adversity might not be permanently predisposed to increased susceptibility for IBD. An increase of H4K12ac in NI þ AI rats, based on ChIP–quantitative PCR assays, suggests the need to examine other epigenetic modifications that might prime the IL1B promoter for greater NF-kB access, and heightened IL1b protein expression. Comprehensive ChIP sequencing experiments are planned for the future to interrogate the epigenetic landscapes for IL1b, and other key inflammatory regulators that might be important mediators of NI responses and IBD susceptibility. In summary, neonatal colonic inflammation, but not acute adult inflammation, aggravates the expression of stress hormones, particularly epinephrine, to induce histone hyperacetylation and allow greater access of NF-kB to the IL1b promoter, resulting in IL1b overexpression in the colonic epithelium. By doing so, NI renders the rat susceptible to an aggravated immune response, a hallmark of IBD, when they are exposed to an additional inflammation later in life. Propranolol ameliorated histone hyperacetylation and the exacerbated IL1b overexpression induced by NI. Thus, in some patients, IBD susceptibility might be circumvented via a precision medicine approach, using b-blockers and possibly HDAC or histone acetyltransferase modulators75–78 to target critical points in disease pathogenesis.

9.

10.

11.

12.

13.

14.

15.

16.

17.

References 1. Loftus EV Jr. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology 2004;126:1504–1517. 2. Farrokhyar F, Swarbrick ET, Irvine EJ. A critical review of epidemiological studies in inflammatory bowel disease. Scand J Gastroenterol 2001;36:2–15. 3. Russel MG, Stockbrugger RW. Epidemiology of inflammatory bowel disease: an update. Scand J Gastroenterol 1996;31:417–427. 4. Sartor RB. Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol 2006;3:390–407. 5. Wurzelmann JI, Lyles CM, Sandler RS. Childhood infections and the risk of inflammatory bowel disease. Dig Dis Sci 1994;39:555–560. 6. Delco F, Sonnenberg A. Exposure to risk factors for ulcerative colitis occurs during an early period of life. Am J Gastroenterol 1999;94:679–684. 7. Hutfless S, Li DK, Heyman MB, Bayless TM, Abramson O, Herrinton LJ. Prenatal and perinatal characteristics associated with pediatric-onset inflammatory bowel disease. Dig Dis Sci 2012;57:2149–2156. 8. Sonntag B, Stolze B, Heinecke A, Luegering A, Heidemann J, Lebiedz P, Rijcken E, Kiesel L, Domschke W, Kucharzik T, Maaser C. Preterm birth but

18.

19.

20. 21. 22.

23.

24.

-,

No.

-

not mode of delivery is associated with an increased risk of developing inflammatory bowel disease later in life. Inflamm Bowel Dis 2007;13:1385–1390. Roberts SE, Wotton CJ, Williams JG, Griffith M, Goldacre MJ. Perinatal and early life risk factors for inflammatory bowel disease. World J Gastroenterol 2011;17:743–749. Baron S, Turck D, Leplat C, Merle V, Gower-Rousseau C, Marti R, Yzet T, Lerebours E, Dupas JL, Debeugny S, Salomez JL, Cortot A, Colombel JF. Environmental risk factors in paediatric inflammatory bowel diseases: a population based case control study. Gut 2005;54:357–363. Aspberg S, Dahlquist G, Kahan T, Källén B. Fetal and perinatal risk factors for inflammatory bowel disease. Acta Paediatr 2006;95:1001–1004. Gilat T, Hacohen D, Lilos P, Langman MJ. Childhood factors in ulcerative colitis and Crohn’s disease. An international cooperative study. Scand J Gastroenterol 1987;22:1009–1024. Mann EA, Saeed SA. Gastrointestinal infection as a trigger for inflammatory bowel disease. Curr Opin Gastroenterol 2012;28:24–29. Lidar M, Langevitz P, Shoenfeld Y. The role of infection in inflammatory bowel disease: initiation, exacerbation and protection. Isr Med Assoc J 2009;11:558–563. Kalischuk LD, Buret AG. A role for Campylobacter jejuniinduced enteritis in inflammatory bowel disease? Am J Physiol Gastrointest Liver Physiol 2010;298:G1–G9. Gradel KO, Nielsen HL, Schønheyder HC, Ejlertsen T, Kristensen B, Nielsen H. Increased short- and long-term risk of inflammatory bowel disease after salmonella or campylobacter gastroenteritis. Gastroenterology 2009; 137:495–501. Hansen R, Thomson JM, El-Omar EM, Hold GL. The role of infection in the aetiology of inflammatory bowel disease. J Gastroenterol 2010;45:266–276. Renz H, Brandtzaeg P, Hornef M. The impact of perinatal immune development on mucosal homeostasis and chronic inflammation. Nat Rev Immunol 2011;12:9–23. Schuurmans C, Kurrasch DM. Neurodevelopmental consequences of maternal distress: what do we really know? Clin Genet 2013;83:108–117. Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the hallmarks of cancer. Science 2017;357:6348. Rinaudo P, Wang E. Fetal programming and metabolic syndrome. Annu Rev Physiol 2012;74:107–130. Hatchwell E, Greally JM. The potential role of epigenomic dysregulation in complex human disease. Trends Genet 2007;23:588–595. Bale TL, Baram TZ, Brown AS, Goldstein JM, Insel TR, McCarthy MM, Nemeroff CB, Reyes TM, Simerly RB, Susser ES, Nestler EJ. Early life programming and neurodevelopmental disorders. Biol Psychiatry 2010; 68:314–319. Tulic MK, Hodder M, Forsberg A, McCarthy S, Richman T, D’Vaz N, van den Biggelaar AH, Thornton CA, Prescott SL. Differences in innate immune function between allergic and nonallergic children: new insights into immune ontogeny. J Allergy Clin Immunol 2011;127:470–478.

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 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

2018

1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473

Neonatal Inflammation Aggravates IL1b Overexpression

25. Yoshida H, Russell J, Senchenkova EY, Almeida Paula LD, Granger DN. Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis. Am J Pathol 2010;177:2774–2781. 26. Weber A, Wasiliew P, Kracht M. Interleukin-1 (IL-1) pathway. Sci Signal 2010;3:cm1. 27. Coccia M, Harrison OJ, Schiering C, Asquith MJ, Becher B, Powrie F, Maloy KJ. IL-1b mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4(þ) Th17 cells. J Exp Med 2012;209:1595–1609. 28. Witowski J, Tayama H, Ksiazek K, Wanic-Kossowska M, Bender TO, Jörres A. Human peritoneal fibroblasts are a potent source of neutrophil-targeting cytokines: a key role of IL-1beta stimulation. Lab Invest 2009;89:414–424. 29. Kwon KH, Murakami A, Hayashi R, Ohigashi H. Interleukin-1beta targets interleukin-6 in progressing dextran sulfate sodium-induced experimental colitis. Biochem Biophys Res Commun 2005;337:647–654. 30. Medzhitov R, Preston-Hurlburt P, Kopp E, Stadlen A, Chen C, Ghosh S, Janeway CA. MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol Cell 1998;2:253–258. 31. Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996;87:2095–2147. 32. Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 2009; 27:519–550. 33. Ben-Sasson SZ, Hu-Li J, Quiel J, Cauchetaux S, Ratner M, Shapira I, Dinarello CA, Paul WE. IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation. Proc Natl Acad Sci U S A 2009;106:7119–7124. 34. Sutton C, Brereton C, Keogh B, Mills KH, Lavelle EC. A crucial role for interleukin (IL)-1 in the induction of IL17-producing T cells that mediate autoimmune encephalomyelitis. Immunity 2009;31:331–341. 35. Sutton CE, Lalor SJ, Sweeney CM, Brereton CF, Lavelle EC, Mills KH. Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity. J Exp Med 2006;203:1685–1691. 36. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 2007;8:942–949. 37. Chung Y, Chang SH, Martinez GJ, Yang XO, Nurieva R, Kang HS, Ma L, Watowich SS, Jetten AM, Tian Q, Dong C. Critical regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity 2009; 30:576–587. 38. Li Q, Winston JH, Sarna SK. Developmental origins of colon smooth muscle dysfunction in IBS-like rats. Am J Physiol Gastrointest Liver Physiol 2013;305:G503–G512. 39. Li Q, Sarna SK. Nuclear myosin II regulates the assembly of preinitiation complex for ICAM-1 gene transcription. Gastroenterology 2009;137:1051–1060. 40. Choi K, Chen J, Mitra S, Sarna SK. Impaired integrity of DNA after recovery from inflammation causes persistent

41.

42. 43.

44.

45.

46.

47. 48.

49.

50.

51.

52.

53.

54.

55.

13

dysfunction of colonic smooth muscle. Gastroenterology 2011;141:1293–1301. Li Q, Sarna SK. Nitric oxide modifies chromatin to suppress ICAM-1 expression during colonic inflammation. Am J Physiol Gastrointest Liver Physiol 2012; 303:G103–G110. Jacobs MD, Harrison SC. Structure of an IkappaBalpha/ NF-kappaB complex. Cell 1998;95:749–758. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, Miyamoto S. Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. Genes Dev 1995; 9:2723–2735. Bevington SL, Cauchy P, Cockerill PN. Chromatin priming elements establish immunological memory in T cells without activating transcription: T cell memory is maintained by DNA elements which stably prime inducible genes without activating steady state transcription. Bioessays 2017;39:2. Natoli G. Control of NF-kappaB-dependent transcriptional responses by chromatin organization. Cold Spring Harb Perspect Biol 2009;1:a000224. Haberland M, Montgomery RL, Olson EN. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet 2009;10:32–42. Mayer EA, Tillisch K. The brain-gut axis in abdominal pain syndromes. Annu Rev Med 2011;62:381–396. Brzozowski B, Mazur-Bialy A, Pajdo R, Kwiecien S, Bilski J, Zwolinska-Wcislo M, Mach T, Brzozowski T. Mechanisms by which stress affects the experimental and clinical inflammatory bowel disease (IBD): role of brain-gut axis. Curr Neuropharmacol 2016; 14:892–900. Murburg MM, Villacres EC, Ko GN, Veith RC. Effects of yohimbine on human sympathetic nervous system function. J Clin Endocrinol Metab 1991;73:861–865. Navegantes KC, de Souza Gomes R, Pereira PAT, Czaikoski PG, Azevedo CHM, Monteiro MC. Immune modulation of some autoimmune diseases: the critical role of macrophages and neutrophils in the innate and adaptive immunity. J Transl Med 2017;15:36. Lucassen PJ, Naninck EF, van Goudoever JB, Fitzsimons C, Joels M, Korosi A. Perinatal programming of adult hippocampal structure and function; emerging roles of stress, nutrition and epigenetics. Trends Neurosci 2013;36:621–631. Karsten CA, Baram TZ. How does a neuron “know” to modulate its epigenetic machinery in response to earlylife environment/experience? Front Psychiatry 2013;4:89. Kundakovic M, Lim S, Gudsnuk K, Champagne FA. Sex-specific and strain-dependent effects of early life adversity on behavioral and epigenetic outcomes. Front Psychiatry 2013;4:78. Winston JH, Li Q, Sarna SK. Chronic prenatal stress epigenetically modifies spinal cord BDNF expression to induce sex-specific visceral hypersensitivity in offspring. Neurogastroenterol Motil 2014;26:715–730. Weinstock M. The long-term behavioural consequences of prenatal stress. Neurosci Biobehav Rev 2008; 32:1073–1086.

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC

1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532

14

1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591

Zhong et al

Cellular and Molecular Gastroenterology and Hepatology Vol.

56. Kinsella MT, Monk C. Impact of maternal stress, depression and anxiety on fetal neurobehavioral development. Clin Obstet Gynecol 2009;52:425–440. 57. Lumey LH, Stein AD, Susser E. Prenatal famine and adult health. Annu Rev Public Health 2011;32:237–262. 58. Horwitz AV, Widom CS, McLaughlin J, White HR. The impact of childhood abuse and neglect on adult mental health: a prospective study. J Health Soc Behav 2001; 42:184–201. 59. De Bellis MD. The psychobiology of neglect. Child Maltreat 2005;10:150–172. 60. Anda RF, Felitti VJ, Bremner JD, Walker JD, Whitfield C, Perry BD, Dube SR, Giles WH. The enduring effects of abuse and related adverse experiences in childhood. A convergence of evidence from neurobiology and epidemiology. Eur Arch Psychiatry Clin Neurosci 2006; 256:174–186. 61. MacRae M, Macrina T, Khoury A, Migliore MM, Kentner AC. Tracing the trajectory of behavioral impairments and oxidative stress in an animal model of neonatal inflammation. Neuroscience 2015;298:455–466. 62. Winston JH, Sarna SK. Developmental origins of functional dyspepsia-like gastric hypersensitivity in rats. Gastroenterology 2013;144:570–579. 63. Weaver IC, Cervoni N, Champagne FA, D’Alessio AC, Sharma S, Seckl JR, Dymov S, Szyf M, Meaney MJ. Epigenetic programming by maternal behavior. Nat Neurosci 2004;7:847–854. 64. Korosi A, Shanabrough M, McClelland S, Liu ZW, Borok E, Gao XB, Horvath TL, Baram TZ. Early-life experience reduces excitation to stress-responsive hypothalamic neurons and reprograms the expression of corticotropinreleasing hormone. J Neurosci 2010;30:703–713. 65. Murgatroyd C, Patchev AV, Wu Y, Micale V, Bockmühl Y, Fischer D, Holsboer F, Wotjak CT, Almeida OF, Spengler D. Dynamic DNA methylation programs persistent adverse effects of early-life stress. Nat Neurosci 2009;12:1559–1566. 66. Aguirre JE, Winston JH, Sarna SK. Neonatal immune challenge followed by adult immune challenge induces epigenetic-susceptibility to aggravated visceral hypersensitivity. Neurogastroenterol Motil 2017;29:9. 67. Rommelfanger KS, Weinshenker D. Norepinephrine: the redheaded stepchild of Parkinson’s disease. Biochem Pharmacol 2007;74:177–190. 68. Takayanagi Y, Osawa S, Ikuma M, Takagaki K, Zhang J, Hamaya Y, Yamada T, Sugimoto M, Furuta T, Miyajima H, Sugimoto K. Norepinephrine suppresses IFN-g and TNF-a production by murine intestinal intraepithelial lymphocytes via the b₁ adrenoceptor. J Neuroimmunol 2012;245:66–74. 69. Westlund KN, Bowker RM, Ziegler MG, Coulter JD. Noradrenergic projections to the spinal cord of the rat. Brain Res 1983;263:15–31. 70. Mawdsley JE, Rampton DS. Psychological stress in IBD: new insights into pathogenic and therapeutic implications. Gut 2005;54:1481–1491. 71. Bhatt D, Ghosh S. Regulation of the NF-kB-mediated transcription of inflammatory genes. Front Immunol 2014;5:71.

-,

No.

-

1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 Received October 9, 2017. Accepted February 1, 2018. 1622 1623 Correspondence Address correspondence to: Qingjie Li, PhD, Division of Gastroenterology, 1624 Department of Internal Medicine, The University of Texas Medical Branch at 1625 Galveston, Galveston, Texas 77555-1074. e-mail: [email protected]; fax: (409) Q3 747-1938. 1626 1627 Author contributions Xiaoying S. Zhong, John H. Winston, Yingzi Cong, Tor C. Savidge, 1628 Roderick H. Dashwood, Don W. Powell, and Qingjie Li performed the study 1629 concept and design; Xiaoying S. Zhong, John H. Winston, Xiuju Luo, Kevin T. Kline, Syed Z. Nayeem, and Qingjie Li performed the experiments; 1630 Xiaoying S. Zhong, John H. Winston, Xiuju Luo, Kevin T. Kline, Syed Z. 1631 Nayeem, Yingzi Cong, Tor C. Savidge, Roderick H. Dashwood, Don W. 1632 Powell, and Qingjie Li analyzed and interpreted the data; Xiaoying S. Zhong, Roderick H. Dashwood, and Qingjie Li drafted the manuscript; 1633 Xiaoying S. Zhong, John H. Winston, Xiuju Luo, Yingzi Cong, Tor C. 1634 Savidge, Roderick H. Dashwood, Don W. Powell, and Qingjie Li critically revised the manuscript for important intellectual content; and Yingzi Cong, 1635 Tor C. Savidge, Roderick H. Dashwood, Don W. Powell, and Qingjie Li 1636 obtained funding. 1637 Conflicts of interest 1638 Q4 The authors disclose no conflicts. 1639 Funding 1640 This work was supported in part by National Institutes of Health/National 1641 Institute of Allergy and Infectious Diseases grant R21 AI126097 and American Heart Association grant 17GRNT33460395 (Q.L.); National 1642 Institutes of Health/National Cancer Institute grants CA122959 and 1643 HHSN26100004, the John S. Dunn Foundation, and a Chancellors Research Initiative (R.H.D.); National Institutes of Health/National Institute of 1644 Diabetes and Digestive and Kidney Diseases R01 grants DK098370, 1645 DK105585, and DK112436 (Y.C.); National Institutes of Health/National Institute of Allergy and Infectious Diseases grants U011AI24290-01 and 1646 R01AI10091401 (T.C.S.); and National Institutes of Health/National Institute 1647 of Diabetes and Digestive and Kidney Diseases grants R21DK096323-01 1648 and P30 DK56338, which support the Texas Medical Center Digestive Q5 Diseases Center. 1649 1650

72. Wang Z, Zang C, Rosenfeld JA, Schones DE, Barski A, Cuddapah S, Cui K, Roh TY, Peng W, Zhang MQ, Zhao K. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet 2008; 40:897–903. 73. Runtsch MC, Round JL, O’Connell RM. MicroRNAs and the regulation of intestinal homeostasis. Front Genet 2014;5:347. 74. Dekker J, Mirny L. The 3D genome as moderator of chromosomal communication. Cell 2016;164:1110–1121. 75. Rajendran P, Williams DE, Ho E, Dashwood RH. Metabolism as a key to histone deacetylase inhibition. Crit Rev Biochem Mol Biol 2011;46:181–199. 76. Rajendran P, Delage B, Dashwood WM, Yu TW, Wuth B, Williams DE, Ho E, Dashwood RH. Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly. Mol Cancer 2011;10:68. 77. Rajendran P, Ho E, Williams DE, Dashwood RH. Dietary phytochemicals, HDAC inhibition, and DNA damage/repair defects in cancer cells. Clin Epigenetics 2011;3:4. 78. Rajendran P, Dashwood WM, Li L, Kang Y, Kim E, Johnson G, Fischer KA, Löhr CV, Williams DE, Ho E, Yamamoto M, Lieberman DA, Dashwood RH. Nrf2 status affects tumor growth, HDAC3 gene promoter associations, and the response to sulforaphane in the colon. Clin Epigenetics 2015;7:102.

FLA 5.5.0 DTD
JCMGH344 proof
26 March 2018
12:19 pm
ce DVC