Epigenetic master regulators HDAC1 and HDAC5 control pathobiont Enterobacteria colonization in ileal mucosa of Crohn's disease patients

Mélissa Chervy, Adeline Sivignon, Flavie Dambrine, Anthony Buisson, Pierre Sauvanet, Catherine Godfraind, Matthieu Allez, Lionel Le Bourhis, The Remind Group, Nicolas Barnich, Jérémy Denizot, Mélissa Chervy, Adeline Sivignon, Flavie Dambrine, Anthony Buisson, Pierre Sauvanet, Catherine Godfraind, Matthieu Allez, Lionel Le Bourhis, The Remind Group, Nicolas Barnich, Jérémy Denizot

Abstract

AIEC Adherent-Invasive Escherichia coli; BSA Bovine serum albumin; CD Crohn's disease; CEABAC10 Carcinoembryonic antigen bacterial artificial chromosome 10; CEACAM Carcinoembryonic antigen-related cell adhesion molecule; FBS Fetal bovine serum; IBD Inflammatory Bowel Disease; HAT Histone acetyltransferase; HDAC Histone deacetylase; kDa KiloDalton; SAHA Suberoylanilide Hydroxamic Acid; Scr Scramble.

Keywords: Adherent-InvasiveE. coli; Crohn’s disease; High-fat diet; Histone deacetylases; histone acetylation.

Conflict of interest statement

The authors report there are no conflict of interest to declare.

Figures

Figure 1.
Figure 1.
AIEC bacteria are preferentially associated with hyperacetylated histone H3 intestinal epithelium. a: Immunohistochemical staining of pan-H3 acetylation mark on mucosa samples from patients non-colonized by Enterobacteria (Enterobacteria -) and colonized by AIEC bacteria (AIEC +). b: Percentage of H3ac positive cells in samples of patients non-colonized by Enterobacteria (n = 8), colonized by mucosa-associated E. coli (MAEC +, n = 10) or colonized by AIEC bacteria (AIEC +, n = 9). Bars represent medians. Mann-Whitney test, *p < .05, **p < .01. c: Correlation between the percentage of H3ac positive cells and Enterobacteria load in patients non-colonized by Enterobacteria (n = 8) and colonized by AIEC bacteria (n = 9). Correlation existing between two variables was assessed by a Spearman test. H3ac: acetylated H3.
Figure 2.
Figure 2.
HDAC1 and HDAC5 oppositely control the entry of AIEC bacteria within host cell. a-b: Caco-2 cells were treated with increasing concentrations of HDAC inhibitor SAHA for 24 h before infection with the AIEC strain LF82 at MOI 100. Adhesive ability of AIEC strain LF82 was evaluated at 3 h post-infection (a) and a gentamicin protection assay was performed to evaluate invasive ability of the strain in the different conditions (b) (n = 4). c-f: Caco-2 cells transfected with control siRNA (siScr) or siRNAs directed against the different HDAC were infected with AIEC strain LF82 at MOI 100. Adherent (c,d) and invasive (e,f) bacteria were numbered at 3 h and 4 h post-infection respectively (n = 5). The results are the mean ± SD. One-way ANOVA, *p < .05, ***p < .001, ****p < .0001. MOI: multiplicity of infection, Lipof: lipofectamine, NT: untreated, Scr: scramble.
Figure 3.
Figure 3.
Class I HDAC activity prevents AIEC colonization whereas HDAC4/5 activity favors AIEC colonization in vivo. a: Experimental protocols of treatment and infection used in the study. b-e: AIEC LF82 load in feces at different days post-infection (b, d) and AIEC LF82 bacteria associated with colonic mucosa 7 days (c) or 4 days post-infection (e) were quantified on selective agar plates (n = 7 or 8). Bars represent medians. Mann-Whitney test, *p < .05, **p < .01, ***p < .001.
Figure 4.
Figure 4.
HDAC1 and HDAC5 expression is correlated with Enterobacteria load associated with ileal mucosa. HDAC1 and HDAC5 expression levels and Enterobacteria load were quantified in mucosa samples of CD patients from the REMIND cohort. a: CD patients’ ileal samples were split into two groups based on HDAC1 expression level (50% in HDAC1 low expression group and 50% in HDAC1 high expression group). The load of Enterobacteria associated with ileal mucosa in each group is plotted on the graph. The results are median. Mann-Whitney test, *p < .05. b: Correlation between HDAC1 expression level and Enterobacteria load associated with ileal mucosa. c: CD patients’ ileal samples were split into two groups based on HDAC5 expression level (50% in HDAC5 low expression group and 50% in HDAC5 high expression group). The load of Enterobacteria associated with ileal mucosa in each group is plotted on the graph. d: Correlation between HDAC5 expression level and Enterobacteria load associated with ileal mucosa. e: Immunohistochemical staining of HDAC5 on mucosa samples from patients non-colonized by Enterobacteria (Enterobact -), colonized by mucosa-associated E. coli (MAEC +) and colonized by AIEC bacteria (AIEC +). The intensity of the signal in samples was rated as following: 0- no signal, 1- low intensity signal, 2- high intensity signal, 3- very strong intensity signal (n = 9 samples/group) f: HDAC1/HDAC5 ratio expression in ileal mucosa of CD patients based on the absence of Enterobacteria associated with the mucosa (Enterobact -), the presence of mucosa-associated E. coli (MAEC +) and the presence of AIEC (AIEC +). Bars represent medians. Mann-Whitney test, *p < .05. g: Correlation between HDAC1/HDAC5 ratio expression and Enterobacteria load associated with ileal mucosa. Correlation existing between two variables was assessed by a Spearman test (one-tailed).
Figure 5.
Figure 5.
High-fat diet alters histone H3 acetylation in mice. a: Western blot targeting HDAC1, HDAC5 expression and H3ac mark in colonic mucosa of mice fed Chow or High-fat (HF) diet. b: Quantification of protein expression was performed by assessing band intensities using the Image Lab software (n = 7 mice/per group). Bars represent medians. Mann-Whitney test, *p < .05. H3ac: acetylated H3.
Figure 6.
Figure 6.
AIEC infection alters host epigenome in HF-fed mice, enhancing AIEC colonization. a: Western blot targeting HDAC1, HDAC5 and H3ac expression in colonic mucosa of Chow-fed mice uninfected or infected with AIEC strain LF82. G: HDAC1, HDAC5 and H3ac accumulation in colonic mucosa of HF-fed mice uninfected or infected with AIEC strain LF82 analyzed by western blot. b-e, h-k: Quantification of protein expression was performed by assessing band intensities using the Image Lab software (n = 7 mice/per group). Bars represent medians. Mann-Whitney test, **p < .01. f, l: Correlation between AIEC LF82 load associated with the colonic mucosa of Chow-fed mice (f) or HF-fed mice (l) and global H3 acetylation level. Correlation existing between two variables was assessed by a Spearman test. H3ac: acetylated H3.
Figure 7.
Figure 7.
HDAC1 and HDAC5 control Enterobacteria colonization in ileal mucosa of CD patients. The HDAC1/HDAC5 expression balance is crucial in the control of the interaction between Enterobacteria and intestinal epithelial cells in ileal mucosa of CD patients, a reduced HDAC1 expression associated with an increased expression of HDAC5 being conditions favoring Enterobacteria and AIEC gut colonization. Consumption of a high-fat diet leads to alterations of global H3 acetylation level and creates a specific micro-environment that allows AIEC bacteria to reach intestinal epithelium. In this environment, AIEC bacteria are able to imbalance HDAC1 and HDAC5 expressions to their advantage to colonize the gut mucosa. Targeting HDAC5 could represent an interesting approach to prevent AIEC colonization in CD patients. Ac: acetylation.

References

    1. Torres J, Mehandru S, Colombel J-F P-BL.. Crohn’s disease. The Lancet. 2017;389:1741–20. doi:10.1016/S0140-6736(16)31711-1.
    1. Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, Panaccione R, Ghosh S, Wu JCY, Chan FKL, et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. The Lancet. 2017;390(10114):2769–2778. doi:10.1016/S0140-6736(17)32448-0.
    1. DʼSouza S, Levy E, Mack D, Israel D, Lambrette P, Ghadirian P, Deslandres C, Morgan K, Seidman EG, Amre DK. Dietary patterns and risk for Crohnʼs disease in children. Inflamm Bowel Dis. 2008;14(3):367–373. doi:10.1002/ibd.20333.
    1. Chiba M, Nakane K, Komatsu M. Westernized diet is the most ubiquitous environmental factor in inflammatory bowel disease. Perm J. 2019;23(1):18–107. doi:10.7812/TPP/18-107.
    1. Lo C-H, Lochhead P, Khalili H, Song M, Tabung FK, Burke KE, Richter JM, Giovannucci EL, Chan AT, Ananthakrishnan AN. Dietary inflammatory potential and risk of Crohn’s disease and ulcerative colitis. Gastroenterology. 2020;159(3):873–883.e1. doi:10.1053/j.gastro.2020.05.011.
    1. Narula N, Wong ECL, Dehghan M, Mente A, Rangarajan S, Lanas F, Lopez-Jaramillo P, Rohatgi P, Lakshmi PVM, Varma RP, et al. Association of ultra-processed food intake with risk of inflammatory bowel disease: prospective cohort study. BMJ. 2021;374:n1554. doi:10.1136/bmj.n1554.
    1. Gill PA, Inniss S, Kumagai T, Rahman FZ, Smith AM. The role of diet and gut microbiota in regulating gastrointestinal and inflammatory disease. Front Immunol. 2022;13:866059. doi:10.3389/fimmu.2022.866059.
    1. Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B, Yantiss R, Orsi RH, Wiedmann M, McDonough P, Kim SG, et al. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum. ISME J. 2007;1(5):403–418. doi:10.1038/ismej.2007.52.
    1. Khanna S, Raffals LE. The Microbiome in Crohn’s Disease. Gastroenterol Clin North Am. 2017;46(3):481–492. doi:10.1016/j.gtc.2017.05.004.
    1. Sokol H, Brot L, Stefanescu C, Auzolle C, Barnich N, Buisson A, Fumery M, Pariente B, Le Bourhis L, Treton X, et al. Prominence of ileal mucosa-associated microbiota to predict postoperative endoscopic recurrence in Crohn’s disease. Gut. 2020;69:462–472. doi:10.1136/gutjnl-2019-318719.
    1. Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser A-L, Barnich N, Bringer M-A, Swidsinski A, Beaugerie L, Colombel J-F. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease. Gastroenterology. 2004;127:412–421. doi:10.1053/j.gastro.2004.04.061.
    1. Martinez-Medina M, Aldeguer X, Lopez-Siles M, González-Huix F, López-Oliu C, Dahbi G, Blanco JE, Blanco J, Garcia-Gil LJ, Darfeuille-Michaud A. Molecular diversity of Escherichia coli in the human gut: new ecological evidence supporting the role of adherent-invasive E. Coli (AIEC) in Crohn’s Disease Inflamm Bowel Dis. 2009;15:872–882. doi:10.1002/ibd.20860.
    1. Palmela C, Chevarin C, Xu Z, Torres J, Sevrin G, Hirten R, Barnich N, Ng SC, Colombel J-F. Adherent-invasive Escherichia coli in inflammatory bowel disease. Gut. 2018;67:574–587. doi:10.1136/gutjnl-2017-314903.
    1. Denizot J, Sivignon A, Barreau F, Darcha C, Chan HFC, Stanners CP, Hofman P, Darfeuille-Michaud A, Adherent-invasive BN. Escherichia coli induce claudin-2 expression and barrier defect in CEABAC10 mice and Crohn’s disease patients. Inflamm Bowel Dis. 2012;18:294–304. doi:10.1002/ibd.21787.
    1. Chervy M, Barnich N, Denizot J. Adherent-Invasive E. Coli: Update on the Lifestyle of a Troublemaker in Crohn’s Disease. Int J Mol Sci. 2020;21:3734.
    1. Martinez-Medina M, Denizot J, Dreux N, Robin F, Billard E, Bonnet R, Darfeuille-Michaud A, Barnich N. Western diet induces dysbiosis with increased E coli in CEABAC10 mice, alters host barrier function favouring AIEC colonisation. Gut. 2014;63:116–124. doi:10.1136/gutjnl-2012-304119.
    1. Agus A, Denizot J, Thévenot J, Martinez-Medina M, Massier S, Sauvanet P, Bernalier-Donadille A, Denis S, Hofman P, Bonnet R, et al. Western diet induces a shift in microbiota composition enhancing susceptibility to Adherent-Invasive E. Coli Infection and Intestinal Inflammation. Sci Rep. 2016;6:19032.
    1. Wehkamp J, Salzman NH, Porter E, Nuding S, Weichenthal M, Petras RE, Shen B, Schaeffeler E, Schwab M, Linzmeier R, et al. Reduced Paneth cell α-defensins in ileal Crohn’s disease. Proc Natl Acad Sci U S A. 2005;102:18129–18134. doi:10.1073/pnas.0505256102.
    1. Wehkamp J, Stange EF. Paneth’s disease. J Crohns Colitis. 2010;4:523–531. doi:10.1016/j.crohns.2010.05.010.
    1. Stappenbeck TS, McGovern DPB. Paneth cell alterations in the development and phenotype of Crohn’s disease. Gastroenterology. 2017;152(2):322–326. doi:10.1053/j.gastro.2016.10.003.
    1. Alkaissi LY, Winberg ME, Heil SDS, Haapaniemi S, Myrelid P, Stange EF, Söderholm JD, Keita ÅV. Antagonism of Adherent Invasive E. Coli LF82 with Human a-defensin 5 in the Follicle-associated Epithelium of Patients with Ileal Crohn’s Disease. Inflamm Bowel Dis. 2020;27:1116–1127.
    1. McPhee JB, Small CL, Reid-Yu SA, Brannon JR, Le Moual H, Coombes BK. Host defense peptide resistance contributes to colonization and maximal intestinal pathology by Crohn’s disease-associated adherent-Invasive Escherichia coli. Infect Immun. 2014;82(8):3383–3393. doi:10.1128/IAI.01888-14.
    1. Lin Z, Hegarty JP, Cappel JA, Yu W, Chen X, Faber P, Wang Y, Kelly AA, Poritz LS, Peterson BZ, et al. Identification of disease-associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease. Clin Genet. 2011;80(1):59–67. doi:10.1111/j.1399-0004.2010.01546.x.
    1. Ventham NT, Kennedy NA, Adams AT, Kalla R, Heath S, O’Leary KR, Drummond H, Wilson DC, Gut IG, Nimmo ER, et al. Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease. Nat Commun. 2016;7(1):13507. doi:10.1038/ncomms13507.
    1. Howell KJ, Kraiczy J, Nayak KM, Gasparetto M, Ross A, Lee C, Mak TN, Koo B-K, Kumar N, Lawley T, et al. DNA methylation and transcription patterns in intestinal epithelial cells from pediatric patients with inflammatory bowel diseases differentiate disease subtypes and associate with outcome. Gastroenterology. 2018;154(3):585–598. doi:10.1053/j.gastro.2017.10.007.
    1. Somineni HK, Venkateswaran S, Kilaru V, Marigorta UM, Mo A, Okou DT, Kellermayer R, Mondal K, Cobb D, Walters TD, et al. Blood-Derived DNA methylation signatures of Crohn’s disease and severity of intestinal inflammation. Gastroenterology. 2019;156(8):2254–2265.e3. doi:10.1053/j.gastro.2019.01.270.
    1. Moret-Tatay I, Cerrillo E, Sáez-González E, Hervás D, Iborra M, Sandoval J, Busó E, Tortosa L, Nos P, Beltrán B. Identification of epigenetic methylation signatures with clinical value in Crohnʼs disease. Clin Transl Gastroenterol. 2019;10(10):e00083. doi:10.14309/ctg.0000000000000083.
    1. Serena C, Millan M, Ejarque M, Saera-Vila A, Maymó-Masip E, Núñez-Roa C, Monfort-Ferré D, Terrón-Puig M, Bautista M, Menacho M, et al. Adipose stem cells from patients with Crohn’s disease show a distinctive DNA methylation pattern. Clin Epigenetics. 2020;12(1):53. doi:10.1186/s13148-020-00843-3.
    1. Hornschuh M, Wirthgen E, Wolfien M, Singh KP, Wolkenhauer O, Däbritz J. The role of epigenetic modifications for the pathogenesis of Crohn’s disease. Clin Epigenetics. 2021;13(1):108. doi:10.1186/s13148-021-01089-3.
    1. Seto E, Yoshida M. Erasers of histone acetylation: the histone deacetylase enzymes. Cold Spring Harb Perspect Biol. 2014;6(4):a018713. doi:10.1101/cshperspect.a018713.
    1. Park S-Y, Kim J-S. A short guide to histone deacetylases including recent progress on class II enzymes. Exp Mol Med. 2020;52(2):204–212. doi:10.1038/s12276-020-0382-4.
    1. Turgeon N, Blais M, Gagné J-M, Tardif V, Boudreau F, Perreault N, Asselin C . HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation. PLoS ONE. 2013;8(9):e73785. doi:10.1371/journal.pone.0073785.
    1. Turgeon N, Gagné JM, Blais M, Gendron F-P, Boudreau F, Asselin C. The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis. Am J Physiol-Gastrointest Liver Physiol. 2014;306(7):G594–605. doi:10.1152/ajpgi.00393.2013.
    1. Gonneaud A, Turgeon N, Jones C, Couture C, Lévesque D, Boisvert F-M, Boudreau F, Asselin C. HDAC1 and HDAC2 independently regulate common and specific intrinsic responses in murine enteroids. Sci Rep. 2019;9(1):5363. doi:10.1038/s41598-019-41842-6.
    1. Gonneaud A, Turgeon N, Boisvert F-M, Boudreau F, Asselin C. JAK-STAT pathway inhibition partially restores intestinal homeostasis in Hdac1- and Hdac2-intestinal epithelial cell-deficient mice. Cells. 2021;10(2):224. doi:10.3390/cells10020224.
    1. Alenghat T, Osborne LC, Saenz SA, Kobuley D, Ziegler CGK, Mullican SE, Choi I, Grunberg S, Sinha R, Wynosky-Dolfi M, et al. Histone deacetylase 3 coordinates commensal-bacteria-dependent intestinal homeostasis. Nature. 2013;504(7478):153–157. doi:10.1038/nature12687.
    1. Tsaprouni LG, Ito K, Powell JJ, Adcock IM, Punchard N. Differential patterns of histone acetylation in inflammatory bowel diseases. Journal of Inflammation. 2011;8(1):1. doi:10.1186/1476-9255-8-1.
    1. Friedrich M, Gerbeth L, Gerling M, Rosenthal R, Steiger K, Weidinger C, Keye J, Wu H, Schmidt F, Weichert W, et al. HDAC inhibitors promote intestinal epithelial regeneration via autocrine TGFβ1 signalling in inflammation. Mucosal Immunol. 2019;12(3):656–667. doi:10.1038/s41385-019-0135-7.
    1. Buisson A, Sokol H, Hammoudi N, Nancey S, Treton X, Nachury M, Fumery M, Hébuterne X, Rodrigues M, Hugot J-P, et al. Role of adherent and invasive Escherichia coli in Crohn’s disease: lessons from the postoperative recurrence model. Gut. pp.gutjnl-2021–325971. 2022. doi:10.1136/gutjnl-2021-325971
    1. Chan CHF, Stanners CP. Novel mouse model for carcinoembryonic antigen-based therapy. Mol Ther J Am Soc Gene Ther. 2004;9(6):775–785. doi:10.1016/j.ymthe.2004.03.009.
    1. Carvalho FA, Barnich N, Sivignon A, Darcha C, Chan CHF, Stanners CP, Darfeuille-Michaud A. Crohn’s disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM. J Exp Med. 2009;206(10):2179–2189. doi:10.1084/jem.20090741.
    1. Ngollo M, Perez K, Hammoudi N, Gorelik Y, Delord M, Auzolle C, Bottois H, Cazals-Hatem D, Bezault M, Nancey S, et al. Identification of gene expression profiles associated with an increased risk of post-operative recurrence in Crohn’s disease. J Crohns Colitis. 2022;16(8):1269–80. doi:10.1093/ecco-jcc/jjac021.
    1. Eskandarian HA, Impens F, Nahori M-A, Soubigou G, Coppée J-Y, Cossart P, Hamon MA. A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection. Science. 2013;341(6145):1238858. doi:10.1126/science.1238858.
    1. Mombelli M, Lugrin J, Rubino I, Chanson A-L, Giddey M, Calandra T, Roger T. Histone deacetylase inhibitors impair antibacterial defenses of macrophages. J Infect Dis. 2011;204(9):1367–1374. doi:10.1093/infdis/jir553.
    1. Corrêa RO, Vieira A, Sernaglia EM, Lancellotti M, Vieira AT, Avila-Campos MJ, Rodrigues HG, Vinolo MAR. Bacterial short-chain fatty acid metabolites modulate the inflammatory response against infectious bacteria. Cell Microbiol. 2017;19(7):e12720. doi:10.1111/cmi.12720.
    1. Ormsby MJ, Logan M, Johnson SA, McIntosh A, Fallata G, Papadopoulou R, Papachristou E, Hold GL, Hansen R, Ijaz UZ, et al. Inflammation associated ethanolamine facilitates infection by Crohn’s disease-linked adherent-invasive Escherichia coli. EBioMedicine. 2019;43:325–332. doi:10.1016/j.ebiom.2019.03.071.
    1. Baldelli V, Scaldaferri F, Putignani L, Del Chierico F. The role of enterobacteriaceae in gut microbiota dysbiosis in inflammatory bowel diseases. Microorganisms. 2021;9(4):697. doi:10.3390/microorganisms9040697.
    1. Li B, Zhang L, Zhu L, Cao Y, Dou Z, Yu Q. HDAC5 promotes intestinal sepsis via the Ghrelin/E2F1/NF-κB axis. FASEB J Off Publ Fed Am Soc Exp Biol. 2021;35:e21368.
    1. Parada Venegas D, De la Fuente MK, Landskron G, González MJ, Quera R, Dijkstra G, Harmsen HJM, Faber KN, Hermoso MA. Short Chain Fatty Acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases. Front Immunol. 2019;10:277. doi:10.3389/fimmu.2019.00277.
    1. Downes M, Ordentlich P, Kao H-Y, Alvarez JGA, Evans RM. Identification of a nuclear domain with deacetylase activity. Proc Natl Acad Sci U S A. 2000;97(19):10330–10335. doi:10.1073/pnas.97.19.10330.
    1. Zhang L, Deng M, Lu A, Chen Y, Chen Y, Wu C, Tan Z, Boini KM, Yang T, Zhu Q, et al. Sodium butyrate attenuates angiotensin II-induced cardiac hypertrophy by inhibiting COX2/PGE2 pathway via a HDAC5/HDAC6-dependent mechanism. J Cell Mol Med. 2019;23(12):8139–8150. doi:10.1111/jcmm.14684.
    1. Buisson A, Vazeille E, Fumery M, Pariente B, Nancey S, Seksik P, Peyrin‐Biroulet L, Allez M, Ballet N, Filippi J, et al. Faster and less invasive tools to identify patients with ileal colonization by adherent-invasive E. coli in Crohn’s disease. United European Gastroenterology Journal. 2021;9(9):1007–1018. doi:10.1002/ueg2.12161.
    1. Auzolle C, Nancey S, Tran-Minh M-L, Buisson A, Pariente B, Stefanescu C, Fumery M, Marteau P, Treton X, Hammoudi N, et al. Male gender, active smoking and previous intestinal resection are risk factors for post-operative endoscopic recurrence in Crohn’s disease: results from a prospective cohort study. Aliment Pharmacol Ther. 2018;48:924–932. doi:10.1111/apt.14944.

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