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British Journal of Pharmacology

British Journal of Pharmacology (2016) 173 2780–2792

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RESEARCH PAPER The 5A apolipoprotein A-I (apoA-I) mimetic peptide ameliorates experimental colitis by regulating monocyte infiltration Correspondence Tobias Nowacki, Department of Medicine B, University Hospital Münster, Albert Schweitzer Campus 1, Gebäude A1, D-48149 Münster, Germany. E-mail: [email protected]

Received 11 February 2016; Revised 15 June 2016; Accepted 5 July 2016

Tobias M Nowacki1, Alan T Remaley2, Dominik Bettenworth1, Michel Eisenblätter3, Thorsten Vowinkel4, Felix Becker4, Thomas Vogl5, Johannes Roth5, Uwe J Tietge6, Andreas Lügering7, Jan Heidemann1,8* and Jerzy-Roch Nofer9* 1

Department of Medicine B, University Hospital Münster, Münster, Germany, 2National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD USA, 3Translational Research Imaging Center, Department of Clinical Radiology, University Hospital Münster, Münster, Germany,

4

Department of General and Visceral Surgery, University Hospital Münster, Münster, Germany, 5Institute of Immunology, University Hospital Münster, Münster, Germany, 6Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, University of Groningen, University Medical Center

Groningen, GZ Groningen, The Netherlands, 7Medical Care Center Portal 10, Münster, Germany, 8Department of Gastroenterology, Klinikum Bielefeld, Bielefeld, Germany, and 9Center for Laboratory Medicine, University Hospital Münster, Münster, Germany

*Both authors contributed equally.

BACKGROUND AND PURPOSE New therapies for inflammatory bowel disease (IBD) are highly desirable. As apolipoprotein (apo)A-I mimetic peptides are beneficial in several animal models of inflammation, we hypothesized that they might be effective at inhibiting murine colitis.

EXPERIMENTAL APPROACH Daily injections of 5A peptide, a synthetic bihelical apoA-I mimetic dissolved in PBS, or PBS alone were administered to C57BL/6 mice fed 3% (w v-1) dextran sodium sulfate (DSS) in drinking water or healthy controls.

KEY RESULTS Daily treatment with 5A peptide potently restricted DSS-induced inflammation, as indicated by improved disease activity indices and colon histology, as well as decreased intestinal tissue myeloperoxidase levels and plasma TNFα and IL-6 concentrations. Additionally, plasma levels of monocyte chemoattractant protein-1 and the monocyte expression of adhesion-mediating molecule CD11b were down-regulated, pro-inflammatory CD11b+/Ly6chigh monocytes were decreased, and the number of intestinal monocytes was reduced in 5A peptide-treated animals as determined by intravital macrophage-related peptide-8/14-directed fluorescence-mediated tomography and post-mortem immunhistochemical F4/80 staining. Intravital fluorescence microscopy of colonic microvasculature demonstrated inhibitory effects of 5A peptide on leukocyte adhesion accompanied by reduced plasma levels of the soluble adhesion molecule sICAM-1. In vitro 5A peptide reduced monocyte adhesion and transmigration in TNFαstimulated monolayers of human intestinal microvascular endothelial cells. Increased susceptibility to DSS-induced inflammation was noted in apoA-I / mice.

CONCLUSIONS AND IMPLICATIONS The 5A peptide is effective at ameliorating murine colitis by preventing intestinal monocyte infiltration and activation. These findings point to apoA-I mimetics as a potential treatment approach for IBD.

Abbreviations BCECF-AM, 2’7’-bis-(2-carboxyethyl)-5 carboxyfluorescein acetoxymethylester; DSS, dextran sodium sulfate; HIMECs, human intestinal microvasculature endothelial cells; IBD, inflammatory bowel diseases; MCP-1, monocyte chemoattractant protein-1; MPO, myeloperoxidase; sICAM-1, soluble inter-cellular adhesion molecule 1 DOI:10.1111/bph.13556

© 2016 The British Pharmacological Society

ApoA-I mimetics in colitis

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Tables of Links TARGETS Enzymes

a

MPO

LIGANDS Other protein targets

b

TNF-α

5A Peptide

IL-6

ICAM-1

MCP-1 (CCL2)

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide a,b to PHARMACOLOGY 2015/16 ( Alexander et al., 2015a,b).

Introduction The incidence of inflammatory bowel diseases (IBD) is escalating in the industrialized world with current prevalence rates for Crohn’s disease and ulcerative colitis exceeding 200 – 250/100.000 (Burisch et al., 2013). As IBD pathogenesis is still incompletely understood, the majority of patients are treated with unspecific immunosuppressive medications, which are often limited by severe side effects, and up to 30% of all affected individuals need to undergo surgery at least once during the course of disease (Annese et al., 2016). Thus, there is an urgent need for novel anti-inflammatory therapeutic strategies in IBD that would induce and/or maintain long-term remission. HDL is a macromolecular complex in plasma formed by structural proteins such as apolipoprotein (apo)A-I and lipids that protects against atherosclerosis and cardiovascular disease by mediating reverse cholesterol transport – a process, in which HDL shuttles cholesterol from its deposits in the arterial wall to the liver for excretion (Yvan-Charvet et al., 2010). In addition, both HDL and apoA-I exhibit multiple anti-inflammatory properties, which arise from a direct interaction between HDL and various cell types (Yvan-Charvet et al., 2010; Navab et al., 2011). For instance, HDL interrupts pro-inflammatory signal transduction cascades involving sphingosine kinase and ERK activation, inhibits NF-κB signalling and reduces endothelial expression of inflammatory adhesion molecules thereby preventing monocyte recruitment to sites of inflammation (Saemann et al., 2010). Moreover, both HDL and apoA-I attenuate the T-cell contact-activation of macrophages and reduce the accompanying release of pro-inflammatory cytokines and chemokines (Norata et al., 2012). Clinical studies have revealed increased IL-6 levels as well as activated monocyte phenotype in subjects with low-HDL concentrations (Sarov-Blat et al., 2007; Zuliani et al., 2007). Conversely, administration of reconstituted HDL particles containing apoA-I to patients with peripheral vascular disease or type II diabetes reduced monocyte activation and decreased plasma levels of soluble inflammatory markers (Patel et al., 2009; Murphy et al., 2011). To better exploit the anti-inflammatory properties of HDL and apoA-I, apoA-I mimetic peptides have been developed, which do not possess sequence homology to any of the 10 individual helices of apoA-I, but exhibit similar structural properties with respect to the alternate distribution of charged, hydrophilic and hydrophobic amino acids (Anantharamaiah et al., 2007). Similar to the full-length

protein, apoA-I mimetic peptides were shown to promote reversed cholesterol transport and to elevate HDL concentrations in plasma and thereby reduce vascular lesion formation in animal models of atherosclerosis (White et al., 2014). In addition, apoA-I mimetic peptides have proved effective in a wide range of inflammatory conditions in experimental animals including murine models of lupus erythematosus, systemic sclerosis, house dust mite-induced asthma, influenza A virus infection and sepsis (Van Lenten et al., 2004; Weihrauch et al., 2007; Zhang et al., 2009; Woo et al., 2010; Nandedkar et al., 2011; Yao et al., 2011). Given the crucial role of monocyte activation and infiltration into intestine mucosa in the pathogenesis of IBD, we hypothesized that apoA-I mimetic peptides might be effective as a therapeutic approach for colitis. In this study, we showed that administration of 5A peptide, which is a synthetic bihelical apoA-I mimetic peptide exerting potent anti-inflammatory effects in vivo and in vitro (Tabet et al., 2010; Yao et al., 2011), attenuates the key manifestations of colitis and limits monocyte infiltration into the inflamed area in the dextran sodium sulfate (DSS)-induced model of colitis in mice.

Methods Animals and colitis induction Female wild-type C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Female apoA-Ideficient mice (strain B6.129P2-Apoa1tm1Unc/J) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were group-housed (max. five per cage) at the animal facility of the University Hospital of Münster in a temperaturecontrolled room at 22–24°C with 12 h light/dark cycle under pathogen-free conditions. The mice had free access to a standard rodent chow diet and tap water until reaching the desired weight (20–25 g at 6–9 weeks of age). Assessment of gut microbiota revealed comparable microbial composition in murine strains used in this study (Supporting Information Fig. S1). DSS-induced colitis represents a standardized model to study inflammatory processes in IBD and for preclinical testing of therapeutic substances, especially in terms of the involvement of the innate immune system (Kawada et al., 2007). The induction of DSS colitis was as has been described elsewhere (Wirtz et al., 2007). Briefly, mice were administered 3% (w v-1) DSS (MW ~40 000) in drinking water for up to 5days (days 0 to 5 of the experiment). Non-colitic control mice received drinking water without DSS. In parallel, mice were British Journal of Pharmacology (2016) 173 2780–2792

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injected i.p. with 5A peptide (50.0 μg day 1) dissolved in 200 μL sterile PBS or PBS only. Peptide or PBS was administered throughout the entire experiment from day 0 to euthanasia of mice. All injections were performed at the same time point of each experimental day. Animals were monitored daily throughout the entire experiment. Mice that lost greater than 20% initial weight or that became moribund (persistently hunched posture, decreased movement, labored breathing, markedly erect coat) were removed from the study and were killed. Animals were killed by CO2 asphyxia, followed by rapid cervical dislocation. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015) and were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV; permit 8.87-50.10.36.08.304) Nordrhein-Westfalen according to the German Animal Protection Law (Tierschutzgesetz).

Assessment of colitis The course of inflammation was monitored by daily assessment of weight loss and presence of blood in the stools using a guaiac paper test. At the end of the treatment, animals were killed, and colons were removed and measured. Afterwards, each colon was opened longitudinally, embedded as ‘Swiss rolls’ in optimum cutting temperature formulation (O.C.T. compound) and frozen at 80°C. For histological analysis, cryostat sections from the proximal, medial and distal colon were picked up, stained with haematoxylin and eosin and graded in a blinded fashion using a colitis score as previously described by Dieleman et al. (1998). Briefly, all sections were graded within a range from zero to three with respect to the amount of inflammation (none, slight, moderate, severe) and the extent of injury (none, mucosal, mucosal and submucosal, transmural) and within a range from zero to four with respect to the amount of crypt damage (none, basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact, entire crypt and epithelium lost). The changes were also quantified as percentage affected by the disease process: (i) 1–25%; (ii) 26–50%; (iii) 51–75%; (4) 76–100%. Each section was then scored separately by calculating the product of the grade and the percentage involved, and all numbers were summed. A mean score was then calculated from proximal, medial and distal sections that were analysed per mouse. Immunohistochemistry was performed as described previously (Kucharzik et al., 2005). Briefly, acetone fixed and frozen sections (4.0 μm) were blocked in 5.0% rat serum and incubated overnight at 4°C with diluted biotinylated primary antibodies (rat-anti-mouse F4/80 – a glycoprotein expressed by murine macrophages – or rat-anti-mouse Gr-1 – granulocyte (myeloid)-differentiation antigen Gr-1 (Ly6G) – , both 1:500 (v v-1)). Sections were washed three times in PBS and incubated with streptavidin-FITC (for F4/80) or streptavidinAlexa (for Gr-1, both 1:100 (v v-1) in PBS/BSA (0.1% w v-1) for 1 h at room temperature. Sections were then washed, and contrast staining was performed using DAPI (1:1000 v v-1). Fluorescence images were acquired using a Zeiss LSM510 confocal microscope. A number of F4/80- or Gr-1positive cells were counted per crypt/per view in three colon sections. 2782

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Tissue myeloperoxidase assay For measurements of myeloperoxidase (MPO) levels, colon samples were rinsed with PBS, blotted dry and snap-frozen for further use. Thawed samples were weighed, homogenized in ready to use sample buffer, sonicated and centrifuged (200× g, 10 min, 4°C). MPO levels in the homogenate were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions.

Plasma levels of cytokines and adhesion molecules Heparin-treated plasma samples collected from mice were assayed for IL-6, TNFα, monocyte chemoattractant protein-1 (MCP-1) and soluble inter-cellular adhesion molecule 1 (sICAM-1) using commercially available ELISA kits according to the manufacturers’ instructions.

Alarmin S100A8/S100A9 (myeloid-related protein-8/14) fluorescence-mediated tomography A polyclonal antibody, targeted for murine alarmin S100A9 (myeloid-related protein-14), was generated in rabbits and labelled with Cy5.5-NHS-ester for optical imaging approaches (Vogl et al., 2014). In brief, purified antibody was dissolved in NaHCO3 buffer (0.1 mmol·L 1, pH 8.3) and incubated with Cy5.5-NHS-ester for 1 h. The labelled antibody was purified from unbound precursors by chromatography and resolved in PBS for in vivo application. Colitic mice received the labelled antibody in an amount corresponding to 2.0 nmol Cy5.5 (80 μL antibody solution) i.v. via the tail vein 24 h prior to in vivo imaging. All in vivo imaging experiments were performed using a VisEN 2500 Beta Flurescence-mediated Tomography (FMT) device (VisEn Medical, Woburn, MAS, USA) for small animal fluorescence imaging. FMT allows for three-dimensional quantitative fluorescence mapping of dye distribution in vivo. The device was equipped with a Cy5.5-adapted filter set for excitation and emission. Animals were held under isoflurane inhalation anaesthesia for the time of examination (about 10 min for complete scan) to prevent movement artefacts and additional stress. The imaging data were screened for fluorescence uptake in the abdominal region, and the fluorescence signal was recorded and quantified for the whole abdominal region as well as for distinct segments of the bowel by manual placing of regions of interest (ROI) on the three-dimensional maps. Data were documented as total amounts of fluorescence signal in the ROI.

Intravital fluorescence microscopy The surgical preparation, intravital fluorescence microscopy and video analysis were performed as have been described in detail elsewhere (Vowinkel et al., 2004). Briefly, mice were anaesthetized using ketamine hydrochloride (150 mg·kg 1 i. p.) and xylazine (7.5 mg·kg 1 i.p.), and the right jugular vein was cannulated for the infusion of leukocyte labelling dye. Leukocytes were labelled in vivo with 100 μL of rhodamine 6G [0.1 mL rhodamine 6G (20 mg·mL 1) dissolved in 5 mL water] 5 min prior to the microscopy experiments. After laparotomy, animals were placed on the right side, and the

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proximal large bowel was exteriorized and superfused at 37°C with bicarbonate-buffered saline solution (pH 7.4). Leukocytes were observed under an inverted microscope (Nikon, Tokyo, Japan) equipped with a 75-W XBO xenon lamp. The microscopic images were received by a charge-coupled device video camera (C2400; Hamamatsu Photonics, Hamamatsu, Japan). Five randomly selected postcapillary venules (20–40 μm diameter) in each colon preparation were recorded for 1 min each. Leukocytes were classified according to their interaction with the venular wall as either free flowing or adherent (when cells remained stationary for 30 s or more), and the adherence was expressed as cell number·mm-2 venular surface.

Static adhesion and transmigration assays Human intestinal microvasculature endothelial cells (HIMECs) were isolated from surgical specimens obtained from the small intestine or colon as described previously (Heidemann et al., 2007) and cultured in growth medium (Endothelial Cell Growth Medium MV) containing FBS (10% v v-1). Experiments were performed on cultures between passages 8 and 14 to prevent replicative senescence. For static endothelial-monocyte adhesion assays, U937 cells (a human monocyte-like cell line) were labelled by incubating with 2’7’-bis-(2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethylester (BCECF-AM) (10.0 μmol·L 1) for 30 min at 37°. Labelled monocytes (2 × 106·mL 1) were added to HIMEC monolayers grown in 96-well tissue culture dishes and prestimulated for 20 h with TNFα (10ng·mL 1) in the presence or absence of 5A peptide. After co-incubation for 30 min, non-adherent cells were rinsed off with PBS, and plates were centrifuged (5 min, 500× g) and air dried. Fluorescence intensity was measured using a Fluostar (BGM Labtech, Ortenburg, Germany) fluorescence plate reader. For endothelial transmigration assays, HIMECs grown to confluence on collagen-coated Transwell® polycarbonate filter inserts (pore size 5 μm) were stimulated with TNFα with or without 5A peptide as indicated. Calcein-AM-labelled U937 cells (5 × 106) were added on top and allowed to migrate for 4 h at 37°C. Transmigrated cells (lower well) and cells remaining in the upper well were quantified by fluorescence readings (Heidemann et al., 2007). Equal volumes and counting intervals were applied. Each condition was assessed in triplicate.

Preparation of leukocytes and flow cytometry Blood was obtained from anaesthetized animals by retroorbital puncture, and leukocytes were isolated using Biocoll according to the manufacturer’s instructions. Cells (1 × 106·mL 1) were incubated in FACS buffer with anti-CD11b or anti-Ly6C antibodies (1:100 v v-1 each) for 30 min at 4°C. Cells were analysed by flow cytometry (FACSCalibur, BD Biosciences GmbH, Heidelberg, Germany).

Experimental design and statistical analysis Group sizes. All experiments were performed in control and treated groups of five animals. Repetitive experiments were performed to confirm the effects observed, or if not, all measurements could be performed in the same

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animals. Data were pooled for statistical analysis, where applicable. The number of animals included in the statistical analysis for each parameter is specified in each graph or the respective figure legend. Variations in group sizes occurred, if mice had to be excluded from the experiment due to ethical reasons (meeting predefined humane endpoints as described above). An initial control experiment evaluating the effect of the 5A peptide in non-colitic mice (receiving no DSS) was performed with animals receiving 5A peptide (50.0 μg day 1, i.p.) dissolved in sterile PBS or PBS alone (n = 5 per group). As in non-colitic mice, the peptide showed no effect on clinical indices (weight, faecal blood excretion), distribution of peripheral blood monocytes and postmortem measurements (histology, colon length, mucosal neutrophil and monocyte infiltration); the respective controls were not repeated for all parameters determined in the colitis group for animal ethics reasons.

Randomization and blinding Mice were randomly assigned to treatment and control groups. Experiments were performed in a blinded set-up with the investigator being unaware of the respective treatment both during the actual animal experiment and the post hoc analyses including biochemical assays and histological assessment.

Data and statistical analysis Data were analysed using one-way or two-way ANOVA. Homogeneity of variances was tested using the Levene test. Post hoc comparisons were conducted with Student–Newman–Keuls (S.N.K) test, if F achieved P < 0.05 and there was no significant variance in homogeneity. In case of a significant variance inhomogeneity (P < 0.05 in the Levene test), Welch’s test was used followed by Games-Howell post hoc test for multiple comparisons, if F achieved a P < 0.05. In cases of nonparametric data distribution, the Kruskal–Wallis test was performed. Pairwise comparisons between two groups were performed using Mann–Whitney rank sum test. A P-value of

The 5A apolipoprotein A-I (apoA-I) mimetic peptide ameliorates experimental colitis by regulating monocyte infiltration.

New therapies for inflammatory bowel disease (IBD) are highly desirable. As apolipoprotein (apo)A-I mimetic peptides are beneficial in several animal ...
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