Receptors in Ulcerative Colitis 2 Opposing Roles of Prostaglandin D

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Opposing Roles of Prostaglandin D2 Receptors in Ulcerative Colitis Eva M. Sturm, Balazs Radnai, Katharina Jandl, Angela Stancic, Gerald P. Parzmair, Christoph Högenauer, Patrizia This information is current as Kump, Heimo Wenzl, Wolfgang Petritsch, Thomas R. of September 25, 2021. Pieber, Rufina Schuligoi, Gunther Marsche, Nerea Ferreirós, Akos Heinemann and Rudolf Schicho J Immunol published online 13 June 2014

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 13, 2014, doi:10.4049/jimmunol.1303484 The Journal of Immunology

Opposing Roles of Prostaglandin D2 Receptors in Ulcerative Colitis

Eva M. Sturm,* Balazs Radnai,* Katharina Jandl,* Angela Stancic´,* Gerald P. Parzmair,* Christoph Ho¨genauer,† Patrizia Kump,† Heimo Wenzl,† Wolfgang Petritsch,† Thomas R. Pieber,‡ Ruﬁna Schuligoi,* Gunther Marsche,* Nerea Ferreiro´s,x Akos Heinemann,* and Rudolf Schicho*

Proresolution functions were reported for PGD2 in colitis, but the role of its two receptors, D-type prostanoid (DP) and, in particular, chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2), is less well deﬁned. We investigated DP and CRTH2 expression and function during human and murine ulcerative colitis (UC). Expression of receptors was measured

by ﬂow cytometry on peripheral blood leukocytes and by immunohistochemistry and immunoblotting in colon biopsies of patients Downloaded from with active UC and healthy individuals. Receptor involvement in UC was evaluated in a mouse model of dextran sulfate sodium colitis. DP and CRTH2 expression changed in leukocytes of patients with active UC in a differential manner. In UC patients, DP showed higher expression in neutrophils but lower in monocytes as compared with control subjects. In contrast, CRTH2 was decreased in eosinophils, NK, and CD3+ T cells but not in monocytes and CD3+/CD4+ T cells. The decrease of CRTH2 on blood eosinophils clearly correlated with disease activity. DP correlated positively with disease activity in eosinophils but inversely in

neutrophils. CRTH2 internalized upon treatment with PGD2 and 11-dehydro TXB2 in eosinophils of controls. Biopsies of UC http://www.jimmunol.org/ patients revealed an increase of CRTH2-positive cells in the colonic mucosa and high CRTH2 protein content. The CRTH2 antagonist CAY10595 improved, whereas the DP antagonist MK0524 worsened inflammation in murine colitis. DP and CRTH2 play differential roles in UC. Although expression of CRTH2 on blood leukocytes is downregulated in UC, CRTH2 is present in colon tissue, where it may contribute to inflammation, whereas DP most likely promotes anti-inflammatory actions. The Journal of Immunology, 2014, 193: 000–000.

nflammatory bowel diseases (IBD), such as ulcerative colitis their actions are cell specific and depend on activation of multiple (UC), are chronic inflammatory conditions of the intestine receptors, cross-reactivity, and coupling to different signal trans- I resulting from an inappropriate immune response (1). PGs duction pathways (6). by guest on September 25, 2021 and their receptors have been shown to play important roles in the PGs and thromboxanes are formed by cyclooxygenase (COX) pathogenesis of IBD (2). However, their actions are diverse and enzymes from arachidonic acid. COX-derived mediators are complex. Part of this complexity lies in the expression of different thought to play a beneficial role in IBD, because inhibition of receptors for each PG and in the fact that PGs are rapidly me- COX-2 by nonsteroidal anti-inflammatory drugs has been shown tabolized, a process in which the metabolite can gain new speci- to dampen healing and resolution of colitis (7). PGD2 has been ficity as a ligand (3–5). The exact task of PGs during inflammation categorized as an anti-inflammatory mediator because it caused (pro- and/or anti-inflammatory) is sometimes hard to define, as reduction of granulocyte infiltration during experimental colitis (8). Levels of PGD2 in colon biopsies of patients during remission of UC are increased (9). The ameliorating effect of PGD in ex- *Institute of Experimental and Clinical Pharmacology, Medical University of Graz, 2 8010 Graz, Austria; †Division of Gastroenterology and Hepatology, Department of perimental colitis is thought to be mediated by activation of the Internal Medicine, Medical University of Graz, 8010 Graz, Austria; ‡Division of D-type prostanoid (DP) receptor (now named DP1 receptor) (8); Endocrinology and Metabolic Diseases, Department of Internal Medicine, Medical University of Graz, 8010 Graz, Austria; and xInstitute of Clinical Pharmacology, however, the role of the other PGD2 receptor, chemoattractant re- Goethe University, 60590 Frankfurt/Main, Germany ceptor homologous molecule expressed on Th2 cells (CRTH2; Received for publication January 3, 2014. Accepted for publication May 12, 2014. now also termed DP2), in UC is still unclear. Interestingly, al- This work was supported by Austrian Science Fund Grants FWF P25633 and FWF though PGD2 is regarded as anti-inflammatory in colitis, it pro- P22771 (to R. Schicho), FWF P22521 (to A.H.), and FWF P22976 (to G.M.), and motes carcinogenesis after resolution of colitis (10). A recent Jubilaeumsfonds of the Austrian National Bank Grants 14429 (to R. Schicho), 14263 study reported increased expression of the lipocalin-type PGD syn- (to A.H.), 13487 (to R. Schuligoi), and 14446 (to E.M.S.). thase, the PGD2-producing enzyme, in UC (11). The expression Address correspondence and reprint requests to Prof. Rudolf Schicho, Medical Uni- versity of Graz, Institute of Experimental and Clinical Pharmacology, Universita¨ts- correlated with disease activity, challenging the role of PGD2 as platz 4, 8010 Graz, Austria. E-mail address: [email protected] a mediator with purely anti-inflammatory action (11). The online version of this article contains supplemental material. The therapeutic potential of the two PGD2 receptors in inflam- Abbreviations used in this article: 5-ASA, 5-aminosalicylic acid; CAI, clinical ac- matory processes, especially that of CRTH2, has been only recently tivity index; COX, cyclooxygenase; CRTH2, chemoattractant receptor homologous discovered (12). CRTH2 may not only be activated by PGD2,but molecule expressed on Th2 cells; DP, D-type prostanoid; DSS, dextran sulfate sodium; IBD, inflammatory bowel disease; LC, liquid chromatography; MPO, myelo- also by its stable metabolites and even by a thromboxane metabolite peroxidase; MS, mass spectrometry; PMNL, polymorphonuclear leukocyte; PS, (5, 13). CRTH2 has been found in leukocytes, such as human and penicillin/streptomycin; Treg, T regulatory; TXB2, thromboxane B2; UC, ulcerative mouse Th2 cells, eosinophils, basophils, and monocytes (14–16), colitis. and it selectively induces chemotaxis in these cells (3, 17, 18). Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 Human peripheral blood neutrophils lack expression of the CRTH2

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303484 2 CRTH2 AND DP IN IBD

receptor (19, 20) despite the detection of PGD2 binding sites (21), The study was approved by the Ethics Committee of the Medical University of Graz (protocol numbers: 23-002 ex 10/11 and 24-281 ex 11/ which are most likely DP receptors through which PGD2 inhibits their activity and chemotaxis (19, 22). Human monocytes and 12), and all participants provided written, informed consent. eosinophils both express DP and CRTH2 receptors and have shown Human peripheral blood cell populations chemotaxis upon activation with PGD (15, 22). 2 Citrated whole blood from patients and healthy individuals was lysed with It is known that the development of IBD is associated with dense 13 BD FACS lysing solution. Samples were washed once in PBS and resus- infiltration of leukocytes, such as neutrophils, T cells, eosinophils, pended in Ab diluent (Dako, Glostrup, Denmark). Cells were stained with and macrophages in the colon (23). Eosinophils, in particular, show FITC-conjugated CD4 Ab (1:25; Miltenyi Biotec, Bergisch Gladbach, Ger- prominent expression of DP and CRTH2 (22) and these two PGD many), PE-conjugated CD56 Ab (1:25), PerCP-conjugated CD14 Ab (1:25), 2 and PE-Cy5–conjugated CD3 Ab (1:25; all Abs from BD Biosciences, San receptors most likely cooperate in eosinophil responses such as Jose, CA) for 30 min at 4˚C. Samples were washed once in PBS, fixative 2+ Ca signaling, chemotaxis, or mobilization from bone marrow solution was added, and samples were kept on ice until analyzed on a (22, 24). Eosinophils were therefore investigated in more detail in FACSCalibur flow cytometer. Peripheral blood cell populations were identi- the current study. Although the role of eosinophils in UC is not yet fied on the basis of forward versus side scatter parameters and specific Ab clarified, there is some evidence that they may play a proinflam- binding. Eosinophils were distinguished from neutrophils in an unstained sample according to granularity (side scatter) and by their autofluorescence. matory role (25). Several reports describe that the gut mucosa of Monocytes were identified as CD14+, NK cells as CD56+/CD32, and Th cells IBD patients is densely infiltrated with eosinophils (26, 27), and as CD3+/CD4+ population. Cell populations were quantified as percentage of more recent studies show that eosinophil-selective chemokines total peripheral blood cells. Fixative solution was prepared by adding 9 ml and eosinophil peroxidase promote inflammation in human and distilled water and 30 ml FACS-Flow to 1 ml CellFix (BD Biosciences). experimental IBD (28, 29). Flow cytometric analysis of CRTH2 and DP receptor Downloaded from Because of its proinflammatory role in allergic inflammation, we expression in whole blood intended to elucidate whether CRTH2 would act similarly in human Citrated whole blood from patients with active UC or healthy individuals was and experimental UC, thus behaving differently to the anti- lysed with 13 BD FACS lysing solution to remove erythrocytes. Samples inflammatory DP. Expression of DP and CRTH2 was investi- were washed once in PBS, resuspended in fixative solution, and kept on ice gated in blood leukocytes, especially in eosinophils, and also in for 10 min. After washing with PBS, cells were blocked with Ultra-V Block (Lonza, Allendale, NJ) for 30 min at 4˚C. For flow cytometric visualization colon biopsies of human UC patients and healthy individuals. We http://www.jimmunol.org/ further wanted to assess the role of these receptors in a mouse of the CRTH2 receptor, cells were stained with Alexa Fluor 647–conjugated rat anti-human CRTH2 Ab or Alexa Fluor 647–conjugated rat IgG2a isotype model of experimental IBD by applying selective antagonists control (10 mg/ml; BD Biosciences) for 30 min at 4˚C. For flow cytometric against each receptor. We observed that the two receptors are in fact visualization of the DP receptor, cells were incubated with goat polyclonal differentially expressed in human UC and that their expression Ab against DP1 (20 mg/ml; Santa Cruz Biotechnology, Dallas, TX) or m in eosinophils correlates inversely with disease severity. Contrary normal goat IgG (20 g/ml; Sigma-Aldrich, St. Louis, MO) for 30 min at 4˚C. Cells were washed in PBS, resuspended in Ab diluent (Dako), and in- to DP, our results in the experimental model suggest that CRTH2 cubated for 30 min at 4˚C with the secondary Ab (1:2000; Alexa Fluor 647– plays a different, proinflammatory, role in IBD. conjugated rabbit anti-goat secondary Ab; Life Technologies, Carlsbad, CA). Finally, all samples were washed with PBS, fixative solution was added, and

samples were kept on ice until analyzed on a FACSCalibur flow cytometer. by guest on September 25, 2021 Materials and Methods Peripheral blood cell populations were identified on the basis of forward Patients versus side scatter parameters and specific Ab binding. CRTH2 and DP receptor expression was recorded as mean fluorescence intensity and Blood was collected from adult patients with confirmed active UC (n = 13; expressed as fold increase over isotype control signals. Representative flow mean age: 37.3 6 14.4; 10 males/3 females), from healthy control subjects plots and gating criteria are shown in Supplemental Fig. 1. (n = 12; mean age: 37.4 6 21.2; 6 males/6 females; Table I), and from adult UC patients in remission (n = 8; mean age: 38.6 6 14.2; 5 males/3 Eosinophil CD11b expression females; Table II). UC patients were recruited from the IBD clinic of the Citrated whole blood from patients or healthy individuals was stained with PE- Department of Internal Medicine at the Medical University of Graz; conjugated anti-CD11b Ab (1:25; BioLegend, San Diego, CA) and PE-Cy5– control subjects were recruited from healthy volunteers. Diagnosis of UC conjugated CD16 Ab (1:50; BD Biosciences). Samples were mixed with was established by standard clinical, endoscopic, and histologic criteria agonists and incubated for 20 min at 37˚C in a water bath. After incubation, (30). All UC patients with active disease also underwent contemporaneous fixative solution was added and samples were kept on ice for 10 min. colonoscopy to assess endoscopic disease activity. For UC patients, disease Erythrocytes were lysed with 13 BD FACS lysing solution; cells were activity was assessed by using different clinical activity scores, that is, the washed once in PBS and resuspended in fixative solution. CD11b expression clinical activity index (CAI, also termed Rachmilewitz Index) and the total on CD16-negative eosinophils was quantified by flow cytometry and ex- Mayo score, which combines clinical and endoscopic activity scores pressed as percentage of the vehicle response (in the absence of an agonist). (partial and endoscopy Mayo subscore) (31, 32). All subjects suffering from UC, except for one, were on some sort of medication (Table I). Blood CRTH2 receptor internalization assay in eosinophils was collected in Vacuette sodium citrate blood tubes (Greiner-Bio-One) and immediately processed for flow cytometric experiments. For the mea- Changes in CRTH2 surface expression in eosinophils were recorded by means surement of PGs, blood was collected in Vacuette serum tubes (Greiner-Bio- of flow cytometry, as described previously (24). Citrated whole blood was One) and frozen at 280˚C until use. mixed with agonists and stimulated for 1 h at 37˚C in a water bath. To stop For colonic tissue samples, UC patients and control subjects included in the reaction, samples were transferred onto ice for 10 min. Erythrocytes were the study were recruited from the endoscopy unit of the Department of lysed with 13 BD FACS lysing solution; cells were washed in PBS and Internal Medicine, Medical University of Graz. Colonoscopy in UC patients resuspended in Ab diluent (Dako)). Cells were stained with Alexa Fluor was performed as part of the clinical workup because of active disease 647–conjugated rat anti-human CRTH2 Ab or Alexa Fluor 647–conjugated (patients 14–18; Table I). Biopsies from control subjects were obtained rat IgG2a isotype control (10 mg/ml; BD Biosciences) for 30 min at 4˚C. from individuals undergoing colonoscopy as part of the colon cancer After washing with PBS, fixative solution was added and samples were kept screening program and from patients with diagnostic workup of occult or on ice until analyzed on a FACSCalibur flow cytometer. Eosinophils were overt gastrointestinal bleeding, with a normal colonoscopy. Subjects with detected as CD16-negative cells in a higher side scatter region. Data were significant comorbidities and individuals with an intercurrent illness as expressed as percentage of vehicle control (unstimulated sample). infections or pregnant women were excluded from the study. Control subjects who were taking nonsteroidal anti-inflammatory drugs, including Immunofluorescence microscopy of peripheral blood acetylsalicylic acid, were also excluded from the study. Biopsies were leukocytes taken from the sigmoid colon, 30 cm proximal to the anal canal, and were either immediately fixed in 10% phosphate-buffered formalin for immu- The DP and CRTH2 receptors were visualized in polymorphonuclear nohistochemistry or immediately frozen for Western blot experiments. leukocyte (PMNL) and PBMC fractions from healthy individuals by The Journal of Immunology 3

Table I. Characteristics of patients with UC

UC Ongoing Treatment, Response, and Disease Montreal Patients Mayo Score CAIa Duration of Treatment Previous Treatment Duration (y) Classification 1 9 (moderate) 4 5-ASA (PR, 2 y) AZA (NR), MTX (NR), 8E2 Corticosteroids (PR, 5 y) adalimumab (NR), infliximab (NR, I) 2 4 (mild) 2 AZA (PR, 10 y) 5-ASA (I), corticosteroids (R) 11 E3 Escherichia coli Nissle 1917 (PR, 1 y) 3 3 (mild) 2 5-ASA (PR, 3 y) Corticosteroids (R), AZA (I) 4 E3 MTX (R, 1 y) 4 5 (mild) 1 5-ASA (PR, 1.5 y) Corticosteroids (R) 12 E2 AZA (R, 6 y) 5 4 (mild) 2 — 5-ASA (PR), corticosteroids (NR), 10 E3 AZA (R), tacrolimus (R) 6 10 (moderate) 8 5-ASA (PR . 10 y) Corticosteroids (R), infliximab (I) 21 E3 Adalimumabc (PR, 5 y) Antibiotics (R, 3 d) 7 6 (moderate) 4 Corticosteroids (2 mo) 5-ASA (NR), corticosteroids (R), 2E2 AZA (I), adalimumab (NR) 8 10 (moderate) 5 5-ASA (NR, 4 mo) Corticosteroids (NR) 2 E3

AZA (NR, 4 mo) Downloaded from Tacrolimus (PR, 4 mo) 9 10 (moderate) 8 5-ASA (PR . 10 y) Corticosteroids (R) 16 E2 10 8 (moderate) 11 5-ASA (R, 1.5 y) — 2 E3 11 9 (moderate) 6 5-ASA (NR, 0.5 y) Corticosteroids (R) 1 E2 12 10 (moderate) 12 5-ASA (PR, 7 d) — 0 E3 13 9 (moderate) 6 5-ASA (PR, 1 y) — 2 E3 14b 12 (severe) 9 5-ASA (NR, 1 mo) — 4 E3 Corticosteroids (NR, 14 d) http://www.jimmunol.org/ Antibiotics (NR, 3 d) 15b 9 (moderate) 5 5-ASA (PR, 2 y) Corticosteroids (R) 2 E2 AZA (PR, 2 y) 16b 6 (moderate) 4 Corticosteroids (PR, 2 mo) AZA (I), inﬂiximab (NR) 2 E2 17b 7 (moderate) 6 5-ASA (PR, 21 d) — 0 E2 Corticosteroids (R, 21 d) 18b 5 (mild) 3 5-ASA (R . 10 y) Corticosteroids (R) 14 E2 aCAI is also termed Rachmilewitz index. bSamples 14–18 were used for Western blots only. cIndication for therapy: ankylosing spondylitis. by guest on September 25, 2021 AZA, azathioprine; I, intolerance; MTX, methotrexate; NR, no response; PR, partial response; R, response.

5 immunofluorescence staining. PMNL and PBMC (33 10 ) were fixed Membranes were blocked in TBST buffer (1 mM CaCl2, 136 mM NaCl, with 3.7% formaldehyde on ice for 10 min prior to the staining. 2.5 mM KCl, 25 mM Tris-HCl, 0.1% [v/v] Tween 20) containing 5% For visualization of CRTH2, cells were washed once in PBS and blocked milk powder. Furthermore, membranes were incubated with rabbit anti- with Ultra-V Block (Thermo Fisher Scientific, Waltham, MA) for 30 min at CRTH2 (1:1000; Acris Antibodies), rabbit anti-DP1 (1:1000; Cayman 4˚C. Thereafter, cells were washed once with PBS, resuspended in Ab diluent Chemicals, Ann Arbor, MI), and mouse anti–b-actin Abs (Sigma-Aldrich) (Dako), and incubated with rabbit polyclonal Ab against CRTH2 (10 mg/ml; overnight at 4˚C. Membranes were washed and subsequently immuno- Acris Antibodies, Herford, Germany) or normal rabbit IgG (10 mg/ml; Santa blotted with HRP-conjugated Abs (1:4000; Jackson ImmunoResearch Cruz Biotechnology) for 1 h at 4˚C. After washing with PBS, cells were Laboratories, West Grove, PA) for 1 h at room temperature. ECL Western resuspended in Ab diluent (Dako) and incubated for 1 h at 4˚C with the blotting substrate (Thermo Fisher Scientific) was used to visualize pro- secondary Ab (1:1000; Cy3-conjugated donkey anti-rabbit secondary Ab). tein bands, and blots were quantified with ImageJ software (National For visualization of the DP receptor, cells were washed once in PBS and Institutes of Health, Bethesda, MD). Protein expression of DP and CRTH2 blocked with 2% donkey serum for 2 h at 4˚C. After washing with PBS, cells was normalized to their respective actin levels. were resuspended in Ab diluent (Dako) and incubated with goat polyclonal Ab against DP (20 mg/ml; Santa Cruz Biotechnology) or normal goat IgG (20 Induction of dextran sulfate sodium colitis and tissue treatment mg/ml; Sigma-Aldrich) for 1 h at 4˚C. Cells were washed in PBS, resuspended in Ab diluent (Dako), and incubated for 1 h at 4˚C with the secondary C57BL/6 (males, 5–9 wk old, 20–26 g) were obtained from Charles Ab (1:2000; Cy5-conjugated donkey anti-goat secondary Ab; Life Technol- River (Sulzfeld, Germany) and kept in house for 2 wk prior to ex- ogies). All samples were finally washed twice with PBS, resuspended in periments. Mice were housed in plastic sawdust floor cages at constant fixative solution, and cytospun on microscopy slides. After being mounted temperature (22˚C) and a 12:12-h light–dark cycle with free access to with Vectashield/DAPI medium (Vector Laboratories, Burlingname, CA), the standard laboratory chow and tap water. Animals were matched by age samples were analyzed on an Olympus IX70 fluorescence microscope and body weight. Colitis was induced in mice by adding 3% (w/v) of (Olympus, Vienna, Austria) and an Olympus UPlanApo-603/1.20 lens using dextran sulfate sodium (DSS salt; reagent grade; 36–50,000 Da; MP an Olympus DP50-CU digital camera and Olympus Cell^P software. Biomedicals Europe) to the drinking water (tap water), whereas control animals received tap water only. Mice were kept on DSS over a 7-d Western blots period. Body weights were measured daily, and the DSS-containing drinking water was daily monitored to insure that the different treat- Proteins were normalized to wet weight by giving an appropriate volume (100 ment groups would consume comparable amounts of DSS. Scoring of ml/20 mg colon) of extraction buffer (50 mmol/l Tris, 10 mmol/l EDTA, 1% inflammation was carried out, as previously published, in a blinded Triton X, and protease inhibitor mixture; Roche Diagnostics, Vienna, Austria). fashion (33). Following macroscopic scoring, segments of the distal Samples were homogenized accurately, and lysates were diluted in equal colon were stapled flat onto cardboard with the mucosal side up and amounts of reducing sample buffer (15% glycerol, 5% SDS, 5% 2-ME, and fixed for 24 h in 10% neutral-buffered formalin. Tissue was then bromphenol blue in Tris-HCl [pH 6.8]) and incubated for 5 min at 95˚C. dehydrated and embedded in paraffin, and standard H&E staining was Proteins were resolved by SDS-PAGE (Life Technologies) and transferred performed on 5-mm–thick sections. Experimental procedures were to a polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA). approved by the Austrian Federal Ministry of Science and Research 4 CRTH2 AND DP IN IBD

(protocol number: BMWF-66.010/0146-II/3b/2012) and performed in four times for 10 min with HBSS/HEPES/PS, followed by four washes accordance with international guidelines. with HBSS/EDTA/PS. Afterward, samples were rinsed in complete RPMI 1640 medium for 5 min and then transferred into complete RPMI 1640 Isolation and ﬂow cytometric analysis of lamina propria with 100 U/ml collagenase type 2 (Life Technologies) for 1 h at 37˚C. leukocytes After collagenase digestion, the cell suspension was passed through a 40-mm cell strainer and centrifuged at 400 3 g for 7 min. The pellet was The colon was removed, rinsed in HBSS, weighed, cut into small pieces, and washed with PBS and centrifuged, and cells were prepared for ﬂow transferredintoa50-mlfalcontubecontainingHBSS,HEPES,and cytometry. Leukocytes were stained with Alexa Fluor 647–conjugated penicillin/streptomycin (PS). Samples were incubated at 37˚C and washed CD11b Ab (1:100), PE-conjugated Siglec F Ab (1:100), Alexa Fluor 488– Downloaded from

FIGURE 1. Expression of DP and CRTH2 on peripheral blood leukocytes. DP and CRTH2 http://www.jimmunol.org/ receptor expression were evaluated in peripheral blood leukocytes of patients with active UC and healthy (control) subjects. (A) Differences in DP expression between UC patients and control subjects were observed for neutrophils and monocytes, whereas receptor expression in the other leukocyte populations did not differ between the two cohorts. (B) Differences in CRTH2 expression between UC patients and by guest on September 25, 2021 control subjects were observed for eosinophils, NK cells, and CD3+ T cells, whereas expression on monocytes and CD3+/CD4+ T cells did not differ between the two cohorts. Changes in DP and CRTH2 receptor expression were recorded as mean fluorescence intensity, and data were expressed as fold increase of fluorescence over isotype control. Student t test; n = 8–13. The p values ,0.05 were considered significant. (C) Comparison of CRTH2 expression on eosinophils between UC patients on 5-ASA treatment (n = 10) and UC patients in remission also on 5-ASA treatment (n = 8; Student t test). Remis- sion patients highly expressed CRTH2 similar to control subjects, indicating that 5-ASA treatment was not responsible for the decrease in CRTH2 expression seen in eosinophils from UC patients (see B). The Journal of Immunology 5 conjugated F4/80 Ab (1:20; all Abs from BD Biosciences), and PerCP- and mouse blood. Sample analysis was performed using LC–electrospray Cy5.5–conjugated Gr1 Ab (1:100; eBioscience, Vienna, Austria) for ionization–tandem MS. The LC–tandem MS system consisted of a hybrid 30 min at 4˚C. Samples were washed once in PBS, fixative solution was triple quadrupole–ion trap QTrap 5500 mass spectrometer (AB Sciex, added, and samples were kept on ice until analyzed on a FACSCalibur flow Darmstadt, Germany) equipped with a Turbo-V-source operating in cytometer. Data are normalized to colon weight and expressed as per- negative electrospray ionization mode, an Agilent 1200 binary pump centage of total cells. Representative flow plots and gating criteria are and degasser (Agilent, Waldbron, Germany), and a HTC Pal autosampler shown in Supplemental Fig. 2. (Chromtech, Idstein, Germany). Serum samples were spiked with the isotopically labeled internal standards and twice extracted using ethyl Immunohistochemistry of colon tissue acetate. After evaporation of the organic extracts and reconstitution in mobile phase, the chromatographic separation of the analytes was carried Paraffin-embedded sections of human colon from UC patients and controls out in a Synergi Hydro-RP column (150 3 2 mm I.D., 4 mm; Phenom- were cut (10 mm) and deparaffinized, microwaved for 2 3 5-min cycles in enex, Aschaffenburg, Germany) under gradient conditions (300 ml/min) 10 mM citrate buffer, and then processed by ABC method (Vectastain using water/formic acid (100:0.0025, v/v) and acetonitrile/formic acid ABC kit; Vector Laboratories), according to the manufacturer’s protocol. (100:0.0025, v/v) as mobile phases. Sample run time was 16 min, and Sections were incubated with rabbit anti-CRTH2 (1:200; Acris Antibodies) injection volume of samples, 45 ml. Retention times of TXB2, PGE2, and (34) or rabbit anti-DP1 (1:100; Cayman Chemicals, Ann Arbor, MI) (9) PGD2 were 8.0, 8.7, and 9.2 min, respectively. visualized with 3-39-diaminobenzidine and counterstained with hematox- The mass spectrometer was operated in the negative ion mode with an ylin. The specificity of the Abs was tested by omitting the primary Ab and electrospray voltage of 24500 V at 450˚C. Multiple reaction monitoring by incubating with the respective blocking peptide provided by the man- was used for quantification. The mass transitions used were m/z 351.1→m/z ufacturers (see Supplemental Fig. 3). Images were taken with a high- 315.0 for PGE2 and PGD2, m/z 369.1→m/z 169.1 for TXB2, m/z 355.1→m/z resolution digital camera (Olympus DP 50), processed, and analyzed by 2 2 275.1 for [ H4]-PGE2 and [ H4]-PGD2,andm/z 373.1→m/z 173.1 for CellÂ imaging software (Olympus, Vienna, Austria). Only contrast and 2 [ H4]-TXB2, all with a dwell time of 50 ms. brightness of images were adjusted. Both quadrupoles were working at unit resolution. Quantitation was Downloaded from performed with Analyst Software V1.5 (Applied Biosystems, Darmstadt, Myeloperoxidase activity Germany) using the internal standard method. Ratios of analyte peak area and internal standard peak area (y-axis) were plotted against concentration Myeloperoxidase (MPO) activity represents an index of neutrophil accu- (x-axis), and calibration curves for each analyte were calculated by least mulation in the tissue and correlates with the severity of the colitis. MPO square regression using 1/concentration2 weighting. activity has been performed, as described previously (33). Statistical analysis

Liquid chromatography–mass spectrometry http://www.jimmunol.org/ Data were analyzed either by Student t test or one-way ANOVA, followed Liquid chromatography (LC)–mass spectrometry (MS)/MS was employed by Tukey’s post hoc test, using GraphPad Prism (GraphPad Software). The to detect PGD2, PGE2, and thromboxane B2 (TXB2) in serum of human p values ,0.05 were considered signiﬁcant. by guest on September 25, 2021

FIGURE 2. Correlation between UC scores and CRTH2/DP receptor expression in peripheral blood leukocytes. In eosinophils, indices of disease activity in UC patients (Mayo score and CAI) correlate inversely with CRTH2 (A and B) but positively with DP expression (C and D), whereas, in neutrophils, DP expression correlates inversely with the Mayo score (E)andCAI(F). The x-axes in graphs depict fold increase of receptor expression (fluorescence) over isotype control. Linear regression; r2, goodness of fit; n = 7–10. The p values ,0.05 were considered significant. 6 CRTH2 AND DP IN IBD

Results with Mayo scores and CAI (Fig. 2A, 2B) as opposed to DP ex- Peripheral blood leukocytes show differential expression of DP pression, which correlated positively with the scores (Fig. 2C, and CRTH2 in UC 2D). In contrast, DP expression showed inverse correlation with the disease scores on neutrophils (Fig. 2E, 2F). We first investigated the surface expression of the two PGD2 receptors in peripheral blood leukocytes using flow cytometry. Effect of PGD2 ligands on CD11b expression and CRTH2 The DP receptor expression was increased in neutrophils but de- internalization in blood eosinophils + creased in CD14 monocytes of UC patients in comparison with The b2 integrin CD11b is rapidly activated and upregulated on the + healthy individuals (controls), whereas in NK cells, CD3 T cells, leukocyte membrane upon activation of the cells. To investigate the + + and CD3 /CD4 T cells, expression was similar between the two inflammatory activity of peripheral blood eosinophils, expression of cohorts (Fig. 1A). Although the means of the DP receptor ex- CD11b was evaluated in the presence of PGD2 and the selective pression in eosinophils did not differ significantly between con- CRTH2 agonist DK-PGD2 (Fig. 4). Although eosinophils from trols and UC patients, the expression was increased in correlation healthy controls showed increased expression of CD11b against ris- to disease activity in UC (see Fig. 2C, 2D). Contrary to the DP ing concentrations of both ligands, hardly any increase in CD11b receptor, expression of CRTH2 was downregulated in eosinophils, expression was seen in UC patients (Fig. 4A). Because agonist- + + NK cells, and CD3 T cells, whereas in monocytes and CD3 / induced stimulation of G protein–coupled receptors can lead to re- + CD4 T cells, no significant differences in receptor expression ceptor internalization, we tested whether the low expression of were seen between UC patients and healthy controls (Fig. 1B). CRTH2 on eosinophils in UC patients could have been caused by CRTH2 was practically undetectable in neutrophils from both

internalization. Peripheral blood eosinophils isolated from healthy Downloaded from cohorts (Fig. 1B). Immunofluorescence stainings of DP and subjects were incubated with agonists for 60 min, and CRTH2 ex- CRTH2 on PMNLs and PBMCs from healthy individuals are pression on the cell surface was recorded thereafter by flow cytom- shown in Fig. 3. DP receptor expression on PMNLs and PBMCs etry. The internalization assay showed that CRTH2 is profoundly appeared generally moderate in comparison with the expression of internalized in the presence of PGD2, DK-PGD2,andtheCRTH2- the CRTH2 receptor. See Supplemental Fig. 3 for isotype control. activating metabolite 11-dehydro TXB2 (Fig. 4B). To document that Because nearly all patients were on 5-aminosalicylic acid (5-ASA) specific agonist binding with PGD2 or the CRTH2-activating me- http://www.jimmunol.org/ treatment, we compared eosinophils from active UC patients on tabolite 11-dehydro TXB2 is required for CRTH2 internalization, we 5-ASA therapy (Table I) with remission patients on 5-ASA therapy tested another important chemokine involved in UC, that is, eotaxin (Table II) to investigate whether the decreased expression of (29), in the internalization assay. Eotaxin did not lead to internali- CRTH2 could have been due to effects of the drug. CRTH2 ex- zation of CRTH2 (Fig. 4B). In a previous study, we have already pression was increased in UC patients in remission as compared demonstrated that the DP receptor does not internalize in human with patients with acute UC (Fig. 1C), excluding the possibility that eosinophils in the presence of DP agonists (24). 5-ASA is responsible for the decrease in CRTH2 in active UC). Increased expression of CRTH2 and DP in colon biopsies from DP and CRTH2 receptor expression correlate with the activity UC patients versus control subjects by guest on September 25, 2021 of UC Immunohistochemistry revealed increased infiltration of CRTH2- We also analyzed the correlations of DP/CRTH2 expression for positive cells into the lamina propria of the colonic mucosa in bi- eosinophils and neutrophils with the clinical disease scores of UC opsies of UC patients as compared with control subjects (Fig. 5A). patients. CRTH2 expression on eosinophils correlated inversely CRTH2-positive staining was also observed in epithelial cells of the colon (Fig. 5A). To show the Ab’s specificity, preabsorption experiments with a blocking peptide were carried out (Supplemental Fig. 3). In previous studies, Western blots for CRTH2 revealed two bands (∼41 and 55 kDa) (14, 34). The 41-kDa band represents the predicted molecular mass of CRTH2, whereas the 55-kDa band represents the glycosylated form of CRTH2, as previously shown (14). Evaluation of Western blots from colon biopsies of UC patients showed a strong and significant increase of CRTH2 protein at 55 kDa as well as for total CRTH2 protein (41- and 55-kDa bands together) when compared with healthy subjects (Fig. 5A), suggesting a shift of native CRTH2 to its glycosylated form in UC. Immunohistochemical staining with a DP Ab in sections of colon biopsies showed moderate expression in lamina propria and epithelial cells from UC patients and controls (preabsorption control shown in Supplemental Fig. 3). FIGURE 3. Immunofluorescence for CRTH2 and DP in polymorphonu- Similar to CRTH2, Western blots detected two bands for DP, a strong clear (PMNL) and mononuclear (PBMC) leukocytes. Stainings were per- band at ∼41 kDa and a weak band at 55 kDa (34). Density of the formed in PMNLs and PBMCs from healthy donors. DAPI was used to 55-kDa band was increased in samples from UC patients, whereas counterstain cell nuclei. Immunofluorescence images demonstrate that there was no change in the band density at 41 kDa (Fig. 5B). CRTH2 is highly expressed on the cell surface of peripheral blood eosinophils (PMNL; upper left image, cell with bilobed nucleus), but The increased expression of CRTH2 in colon biopsies from UC hardly expressed on neutrophils (PMNL; upper left image, cell with patients correlates with disease activity polymorph nucleus). A moderate to high CRTH2 expression is found on We correlated disease activity of the UC patients with the CRTH2 the cell surface of mononuclear cells (PBMCs). Immunofluorescence staining of DP displays a low to moderate expression on peripheral blood protein expression measured in the Western blots and found that both eosinophils and neutrophils (PMNLs) and a moderate surface expression Mayo score and CAI strongly correlated with the level of 41-kDa on mononuclear cells (PBMCs). Calibration bar, 20 mm. The respective CRTH2 protein (Fig. 6A, 6B). We also performed a histologic score isotype controls for CRTH2 (rabbit IgG) and DP (goat IgG) are shown in of CRTH2-positive cells in the lamina propria of colon biopsies Supplemental Fig. 3. from UC patients and compared severely affected mucosa from UC The Journal of Immunology 7

Table II. Characteristics of UC patients in remission

UC Patient Mayo Partial Ongoing Treatment, Response, Disease Montreal in Remission Score Mayo Score CAIa and Duration of Treatment Previous Treatment Duration (y) Classification 1 0 0 0 5-ASA (R, 5 y) Corticosteroids (R) 12 E3 2 0 0 3 5-ASA (R . 10 y) AZA (I) 20 E3 Corticosteroids (R, 2 y) Infliximab (I) Adalimumab (I) MTX (I) 3 2 1 0 5-ASA (PR, 7 y) Corticosteroids (R) 18 E2 Adalimumab (R, 4 y) AZA (I) 4 1 0 1 5-ASA (R, 0.5 y) None 1 E1 5 b 0 0 5-ASA (R, 1 y) Corticosteroids (R) 1 E1 6 b 0 0 5-ASA (R . 10 y) Corticosteroids (R) 17 E2 AZA (PR) 7 b 0 0 5-ASA (PR, 7 y) 8 E2 Infliximab (R, 1 y) — AZA (PR, 6 y) 8 0 1 1 5-ASA (PR . 10 y) Corticosteroids (R) 20 E3 AZA (R, 6 y) aCAI is also termed Rachmilewitz index. Downloaded from bOnly partial Mayo score available (no colonoscopy performed). AZA, azathioprine; I, intolerance; MTX, methotrexate; PR, partial response; R, response. patients that exhibited a Mayo score consistent with moderate- treatment of DSS colitic mice with the selective CRTH2 an- severe disease activity with mucosa from UC patients that exhibited tagonist CAY10595 (5 mg/kg; s.c.) (35) reduced weight loss, a Mayo score consistent with mild activity. We observed a signifi- inflammation score, and MPO activity, indicating proin- http://www.jimmunol.org/ cant increase in CRTH2-positive cells in the biopsy sections of UC flammatory actions of CRTH2 in this mouse model (Fig. 7A). patients with a moderate-severe disease (Fig. 6E, 6F). H&E stainings of histologic sections (10 mm) show reduced mucosal damage and less infiltration of immunocytes into the The selective CRTH2 antagonist CAY10595 improved, whereas submucosa of the mouse colon after treatment with CAY10595 the DP antagonist MK0524 worsened DSS-induced colitis in (Fig. 7A). As most populations of mouse leukocytes express mice CRTH2 (5, 16–18), we were interested whether CAY10595 may In an attempt to clarify the functional role of DP and CRTH2 in alter leukocyte influx into the lamina propria of the mouse colon in UC, we used a mouse model of DSS-induced colitis and treated the DSS colitis. Treatment with CAY10595 significantly lowered

mice with antagonists against each of the PGD2 receptors. Daily the lymphocyte (comprising T, B, and NK cells) and neutrophil by guest on September 25, 2021

FIGURE 4. CD11b expression and CRTH2internalizationinhumanpe- ripheral blood eosinophils. (A) CD11b expression was evaluated in peripheral blood eosinophils in response to the

CRTH2 ligands PGD2 and DK-PGD2. UC patients showed less CD11b up- regulation than healthy individuals (controls). Changes in CD11b surface expression were recorded as percentage of the vehicle control, and data are shown as means 6 SEM, n =6.*p , 0.05 versus controls, ANOVA. (B)

PGD2,DK-PGD2, and 11-dehydro TXB2 induced internalization of CRTH2. Assays were performed in citrated whole blood from healthy individuals by means of ﬂow cytometry. CRTH2 surface expression in CD16-negative eosinophils was expressed as percentage of the vehicle control (absolute ethanol only), and data are shown as means, n = 6–12, ANOVA. *p , 0.05, ***p , 0.001 versus vehicle. 8 CRTH2 AND DP IN IBD Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Immunohistochemistry and Western blots of colon biopsies from UC patients and control subjects. Immunohistochemical staining was performed for CRTH2 and DP in colon biopsies of UC patients and healthy (control) subjects. (A) Brown diaminobenzidine staining of CRTH2 was visible in lamina propria cells and epithelial cells of colon sections (scale bar, 50 mm). The density of lamina propria cells was strongly increased in UC patients. Western blots revealed significant increase in CRTH2 55-kDa protein in biopsies from UC patients. (B) With DP immunohistochemistry, some diaminobenzidine-stained cells were visible in lamina propria; however, epithelial cells only lightly stained in the colon (scale bar, 50 mm). In contrast to the CRTH2 staining, there was no prominent increase in DP-positive lamina propria cells. Density of the 55-kDa band of DP was stronger in UC patients, whereas, for the 41-kDa band, no increase was noticed. UC values were set at 100%. Other data are relative to these values (n = 5. Student t test, *p , 0.05. n.s., not significant). population, thus corroborating the findings of reduced MPO activity Discussion (Fig. 7A). Contrary to the CRTH2 antagonist, daily treatment with IBD can be described as a multifactorial disease with an uncon- the DP antagonist MK0524 (1 mg/kg; s.c.) worsened colitis and trolled immune response causing inflammation in genetically pre- increased MPO activity in the colon, confirming the anti-inflam- disposed individuals; however, much of the etiology of IBD still matory action for DP in colitis (Fig. 7B) (8). remains unresolved (23). Despite significant progress in the pharmacotherapy of IBD, such as with the introduction of anti- Increase of PG levels in serum from human UC patients and TNF agents, side effects of drugs and treatment failure are still mice with DSS colitis major drawbacks (36, 37). In the current study, we focused on the Levels of the PGs PGD2,PGE2,andTXB2 were measured in role of the two PGD2 receptors in human and experimental UC serum from UC patients by LC-MS and were found increased and provide several lines of evidence that the CRTH2 receptor as compared with healthy controls (Fig. 8A). PGE2 and PGD2 may play a different role than the anti-inflammatory DP receptor were also measured in sera of mice with DSS colitis and were and that blockade of CRTH2 in fact improves inflammation in found to have increased during experimental UC. The CRTH2 mice with DSS colitis. The results of the current study raise the antagonist CAY10595 decreased PGE2 levels at a daily dose of possibility that proinflammatory CRTH2 receptors could represent 5 mg/kg s.c. in mice with DSS colitis but had no effect on PGD2 a new drug target for IBD. It should be noted that the study may levels (Fig. 8B). have certain limitations, as only a low patient sample number was The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. CRTH2 in colon biopsies of UC patients correlates with disease activity. Levels of CRTH2 protein, as measured by Western blot (41-kDa and total CRTH2), were correlated with UC disease activity (Mayo score and CAI) by linear regression (r2, goodness of fit; n =5.p , 0.05). (A and B) The increase in CRTH2 levels (41 kDa) correlated significantly with the activity scores. (C and D) Total levels of CRTH2 (41 and 55 kDa) also correlated positively with the activity scores, although not significantly. (E and F) Counting of CRTH2-positive cells in sections of colon biopsies from UC patients (3 different biopsies from 3 UC patients consistent with Mayo score severe-moderate and from 3 UC patients consistent with Mayo score mild; Student t test) revealed a significant increase in CRTH2-positive cells in severely affected mucosa in comparison with mildly affected mucosa. investigated and only UC patients that were already on medica- CRTH2 is to facilitate chemotaxis (12), the high presence of tion, except for one, were included in the study. CRTH2 in the colon of UC patients may reflect increased che- First, we demonstrated that DP and CRTH2 expression is reg- motaxis of CRTH2-positive leukocytes to the inflamed colon ulated in a differential manner in peripheral blood leukocytes, indi- tissue. cating that the two receptors may follow different tasks in UC. The low expression of CRTH2 on peripheral blood leukocytes Whereas during UC CRTH2 demonstrates low surface expression seemed contradictory to the strong influx of CRTH2-positive cells on eosinophils and other types of leukocytes in the blood, DP is into the colon of UC patients. Interestingly, however, recent studies upregulated on neutrophils, but not on eosinophils as compared demonstrated that IBD patients had significant decreases in T with healthy subjects. Expression of CRTH2, however, is restored regulatory (Treg)/Th17 ratios and Treg frequency in peripheral on eosinophils of UC patients in remission. In contrast, we could blood but increased expression of Foxp3, IL-17a, and higher also show that a high amount of CRTH2-positive leukocytes in- amounts of Tregs in the colon mucosa, suggesting a disparity filtrated the colon in UC patients and was largely present in the between inflammatory activity of leukocytes in the blood and the lamina propria of the colonic mucosa. Because the major task of inflamed tissue during IBD (38, 39). We therefore studied CRTH2 10 CRTH2 AND DP IN IBD Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. Effects of CRTH2 and DP receptor antagonists in DSS-induced colitis. (A) The CRTH2 antagonist CAY10595 (5 mg/kg; s.c.) lowered inflammation scores, MPO activity, and weight loss in C57BL/6 mice with DSS colitis. n = 8–12; means 6 SEM. ANOVA, Tukey’s post hoc, *p , 0.05. Histological images (H&E staining) show representative colon sections (5 mm) from control mice, mice with DSS colitis (+vehicle treatment), and DSS mice treated with CAY10595 (scale bar, 200 mm). MPO assay demonstrates relative change to DSS + vehicle (100%). Treatment with CAY10595 reduced infiltration of lymphocytes and neutrophils into the lamina propria of the colon (n = 4; Student t test). (B) Effect of daily treatment with DP antagonist MK0524 (1 mg/kg s.c.) on severity of DSS colitis. The DP antagonist worsened inflammation score and increased MPO activity, thereby showing opposite effects to the CRTH2 antagonist (n = 6–8; means 6 SEM. ANOVA, Tukey’s post hoc). MPO assays show relative change to DSS + vehicle (100%). The p values ,0.05 were considered significant. n.s., not significant. expression in more detail in eosinophils, a cell type that colocal- of UC patients, which confirms our observations (40). Sixty-two izes both receptors (22) and promotes inflammation in IBD (25). percent of UC patients in that study were on no treatment, indi- In accordance with the low expression of CRTH2 on blood eosin- cating that medication most likely does not interfere with CD11b ophils, activation of CRTH2 with PGD2 and its specific ligand expression on leukocytes in our UC patients. In our study, we DK-PGD2 induced little expression of CD11b in eosinophils from could rule out that 5-ASA treatment resulted in different CRTH2 UC patients as compared with eosinophils from healthy controls, receptor expression, because UC patients in remission taking 5- indicating low inflammatory activity of blood eosinophils from ASA were similar to controls. It is also unlikely that the low ex- UC patients. A different study also described low expression of pression of CRTH2 in UC patients is due to some immature form CD11b, CD18, and the chemokine receptor CCR3 in eosinophils of leukocytes because CRTH2 is largely present in eosinophils and The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 8. Increased levels of PGs in serum from UC patients and mice with DSS colitis. (A) PGD2, PGE2, and TXB2 were measured by LC-MS in by guest on September 25, 2021 serum from UC patients and healthy (control) subjects and were found increased in the UC cohort. n = 9–13; Student t test. (B) An increase in PGE2 and PGD2 levels was also observed in mice with DSS colitis. The CRTH2 antagonist CAY10595 (5 mg/kg s.c.) decreased PGE2 levels, whereas PGD2 remained unaltered after treatment with the antagonist. DSS + vehicle was set at 100%. Data of groups denote relative change to DSS + vehicle group. n = 8–15. ANOVA, Tukey’s post hoc, p values ,0.05 were considered significant. precursor eosinophils of human bone marrow (22). Other groups high DP but low CRTH2 expression in severe cases of UC. This described that eosinophils from IBD patients are activated because suggests that CRTH2 receptors are widely internalized in severe they noticed higher eosinophil peroxidase baseline release (41). cases. Whereas the DP receptor correlates positively with activity We conducted additional experiments to investigate whether the scores in eosinophils, it correlates inversely with activity scores in low expression of CRTH2 may have been caused by receptor in- neutrophils, indicating low expression on neutrophils in severe ternalization due to long-term activation and exposure to CRTH2 cases. DP has previously been linked with the amelioration of ligands, such as PGD2 and other metabolites. We have previously experimental colitis by inhibition of granulocyte—mostly neutrophil— described internalization for CRTH2 in HEK293 cells (24). In- influx to the colon following application of the DP agonist BW-245C deed, presence of CRTH2 ligands promoted internalization of (8). The opposing correlation of DP and CRTH2 on eosinophils CRTH2 in assays with human peripheral blood eosinophils. No- and its functional significance are not clear at the moment, but DP tably, internalization was not observed for DP in human eosino- and CRTH2 are coexpressed in many leukocytes, and down- or up- phils (24). Internalization of CRTH2 could therefore occur in the regulation of one receptor may modify the other’s signaling behavior, serum of UC patients in which higher PGD2 levels are present as recently demonstrated for DP and/or CRTH2-overexpressing than in healthy subjects. Although PGD2 has been shown to be HEK293 cells (24). rapidly degraded in plasma, the generated PGD2 metabolites, such In contrast to peripheral blood leukocytes, we found high ex- 12 12 as D -PGD2 and D -PGJ2, are known to act as stable CRTH2 pression of CRTH2 protein and influx of CRTH2-positive cells into agonists (42). Additionally, a stable metabolite of TXB2, 11-dehydro the lamina propria of the inflamed colon, a finding also described by TXB2, which is a major product of TXB2 in plasma (43), also acti- others (45). This study also used UC patients that were on standard vates CRTH2 and causes internalization in eosinophils (Fig. 4B) (44). medication for UC (45). Additionally, we noticed high expression We found highly increased levels of TXB2 in sera from UC patients, of CRTH2 in epithelial cells. The CRTH2 cell influx to the colon raising the possibility that CRTH2 internalization may not only be in UC is in line with a recent study that described increased levels driven by PGD2 but also by metabolites like 11-dehydro TXB2 of the PGD2-producing enzyme lipocalin-type PGD synthase in in vivo. the colon of UC patients and in mice with DSS colitis (11), sug- Expression of DP and CRTH2 in blood eosinophils clearly cor- gesting that a prominent PGD2–CRTH2 axis may contribute to related with the clinical and endoscopic activity scores displaying inflammation during UC. We therefore investigated the functional 12 CRTH2 AND DP IN IBD role of DP and CRTH2 in UC and applied selective antagonists 6. Milatovic, D., T. J. Montine, and M. Aschner. 2011. Prostanoid signaling: dual role for prostaglandin E2 in neurotoxicity. Neurotoxicology 32: 312–319. against each of the receptors in a model of experimental colitis. 7. Wallace, J. L. 2001. Prostaglandin biology in inflammatory bowel disease. Whereas the DP antagonist MK0524 produced an exacerbation of Gastroenterol. Clin. North Am. 30: 971–980. colitis, confirming the anti-inflammatory role of DP from previous 8. Ajuebor, M. N., A. Singh, and J. L. Wallace. 2000. Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis. work (8), the CRTH2 antagonist CAY10595 improved disease Am. J. Physiol. Gastrointest. Liver Physiol. 279: G238–G244. severity and lowered MPO activity and PGE2 levels, suggesting 9. Vong, L., J. G. Ferraz, R. Panaccione, P. L. Beck, and J. L. Wallace. 2010. A pro- a different, that is, proinflammatory, role for CRTH2 in experi- resolution mediator, prostaglandin D(2), is specifically up-regulated in individuals in long-term remission from ulcerative colitis. Proc. Natl. Acad. Sci. USA mental UC. CRTH2 may therefore exert its proinflammatory role 107: 12023–12027. in the colon itself because much higher levels of CRTH2 and 10. Zamuner, S. R., A. W. Bak, P. R. Devchand, and J. L. Wallace. 2005. Predis- CRTH2-positive cells were found in biopsies from UC patients position to colorectal cancer in rats with resolved colitis: role of cyclooxygenase- 2-derived prostaglandin d2. Am. J. Pathol. 167: 1293–1300. than in healthy individuals. The CRTH2 antagonist was also able 11. Hokari, R., C. Kurihara, N. Nagata, K. Aritake, Y. Okada, C. Watanabe, to reduce infiltration of lymphocytes and neutrophils into the S. Komoto, M. Nakamura, A. Kawaguchi, S. Nagao, et al. 2011. Increased expression of lipocalin-type-prostaglandin D synthase in ulcerative colitis and lamina propria. It should be noted that, unlike in humans, CRTH2 exacerbating role in murine colitis. Am. J. Physiol. Gastrointest. Liver Physiol. is expressed in mouse Th1 cells and neutrophils next to other 300: G401–G408. leukocytes (16, 46). Thus, a direct inhibitory effect of the drug on 12. Ulven, T., and E. Kostenis. 2006. Targeting the prostaglandin D2 receptors DP and CRTH2 for treatment of inflammation. Curr. Top. Med. Chem. 6: 1427– the migration of these cells seems possible. 1444. Serum samples of UC patients revealed a small increase in 13. Schuligoi, R., M. Sedej, M. Waldhoer, A. Vukoja, E. M. Sturm, I. T. Lippe, B. A. Peskar, and A. Heinemann. 2009. Prostaglandin H induces the migration PGD2 but more prominent increases in TXB2 and PGE2,aspre- 2 of human eosinophils through the chemoattractant receptor homologous mole- viously described (47). Similarly, PGD2 and PGE2 levels were cule of Th2 cells, CRTH2. J. Leukoc. Biol. 85: 136–145. Downloaded from elevated in serum of mice with DSS colitis. Although the CRTH2 14. Nagata, K., K. Tanaka, K. Ogawa, K. Kemmotsu, T. Imai, O. Yoshie, H. Abe, antagonist lowered PGE levels, it showed no effect on PGD . K. Tada, M. Nakamura, K. Sugamura, and S. Takano. 1999. Selective expression of 2 2 a novel surface molecule by human Th2 cells in vivo. J. Immunol. 162: 1278–1286. The decrease in PGE2, however, may have contributed to im- 15. Gosset, P., F. Bureau, V. Angeli, M. Pichavant, C. Faveeuw, A. B. Tonnel, and provement of colitis. PGE2 is widely seen as a dual lipid mediator F. Trottein. 2003. Prostaglandin D2 affects the maturation of human monocyte- with pro- as well as anti-inflammatory effects. PGE can actually derived dendritic cells: consequence on the polarization of naive Th cells. J. 2 Immunol. 170: 4943–4952. act, depending on its level, as a proinflammatory mediator in IBD (48). 16. Spik, I., C. Breńuchon, V. Ange´li, D. Staumont, S. Fleury, M. Capron, http://www.jimmunol.org/ F. Trottein, and D. Dombrowicz. 2005. Activation of the prostaglandin D re- High levels of PGE2 may worsen IBD by shifting the IL-12/IL-23 2 ceptor DP2/CRTH2 increases allergic inflammation in mouse. J. Immunol. 174: balance in dendritic cells in favor of IL-23 (48), whereas a certain 3703–3708. level might be necessary to protect the epithelium from damage 17. Gervais, F. G., R. P. Cruz, A. Chateauneuf, S. Gale, N. Sawyer, F. Nantel, K. M. Metters, and G. P. O’Neill. 2001. Selective modulation of chemokinesis, via EP4 receptors (49, 50). PGE2 may also impair wound healing degranulation, and apoptosis in eosinophils through the PGD2 receptors CRTH2 in IBD (51). and DP. J. Allergy Clin. Immunol. 108: 982–988. In conclusion, CRTH2 displays expression levels contrary to 18. Royer, J. F., P. Schratl, S. Lorenz, E. Kostenis, T. Ulven, R. Schuligoi, DP in blood leukocytes during UC. Whereas CRTH2 decreases in B. A. Peskar, and A. Heinemann. 2007. A novel antagonist of CRTH2 blocks eosinophil release from bone marrow, chemotaxis and respiratory burst. Allergy several peripheral blood leukocyte populations, expression of DP 62: 1401–1409. increases in neutrophils. 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In light of the clinically already available CRTH2 receptor 22. Schratl, P., J. F. Royer, E. Kostenis, T. Ulven, E. M. Sturm, M. Waldhoer, antagonists, blocking of CRTH2 in UC may provide a worthwhile G. Hoefler, R. Schuligoi, I. T. Lippe, B. A. Peskar, and A. Heinemann. 2007. The therapeutic approach. role of the prostaglandin D2 receptor, DP, in eosinophil trafficking. J. Immunol. 179: 4792–4799. 23. Strober, W., I. Fuss, and P. Mannon. 2007. The fundamental basis of inflam- Acknowledgments matory bowel disease. J. Clin. Invest. 117: 514–521. 24.Sedej,M.,R.Schro¨der, K. Bell, W. Platzer, A. Vukoja, E. Kostenis, A. Heinemann, We thank Veronika Pommer for excellent technical support. and M. Waldhoer. 2012. D-type prostanoid receptor enhances the signaling of chemoattractant receptor-homologous molecule expressed on T(H)2 cells. J. Allergy Clin. Immunol. 129: 492-500.e1-9. Disclosures 25. Wedemeyer, J., and K. Vosskuhl. 2008. 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Gating strategy and flow cytometric staining pattern of whole blood samples. (A) Granulocytes, monocytes and lymphocytes were identified on the basis of forward scatter (FSC) versus side scatter (SSC) parameters. (B) Granulocyte region: eosinophils were distinguished from neutrophils by their autofluorescence detected in the FL-1/Fl-2 channels. (C) CD14+ cells in the monocyte region were considered pure monocytes. (D) T helper cells were distinguished from other lymphocytes as CD3+/CD4+ double positive cells in the lymphocyte region. (E) NK-cells were identified as CD56+/CD3- cells in the lymphocyte region. Citrated whole blood from UC patients and healthy controls was lysed with 1x BD FACS Lysing solution. Samples were stained with FITC-conjugated CD4 Ab (1:25; Miltenyi Biotec), PE-conjugated CD56 Ab (1:25; BD Pharmingen), PerCP-conjugated CD14 Ab (1:25; Beckton Dickinson) and PE-Cy5-conjugated CD3 Ab (1:25; BD Pharmingen) for 30 minutes at 4°C. Samples were washed once in PBS, fixative solution was added and samples were kept on ice until analyzed on a FACSCalibur flow cytometer. For population staining, a master mix of all antibodies was added to lysed whole blood samples. In CRTH2/DP staining experiments cell populations were stained in separate samples.

Supplemental material S2

A B C A

D E

Gating strategy and flow cytometric staining pattern of lamina propria leukocytes. (A) Lamina propria leukocytes were identified on the basis of forward scatter (FSC) versus side scatter (SSC) parameters. (B) CD11b positive cells were selected. (C) Macrophages, monocytes and granulocytes were identified via their differential expression of F4/80 and Gr-1. (D) Lymphocytes were selected via forward/side scatter appearance. (E) The granulocyte-population was further gated on the expression of Siglec F. Eosinophils were gated as Siglec F+ cells and neutrophils as Siglec F – cells. Lamina propria leukocytes from vehicle- and CAY10595- treated animals were isolated and stained with Alexa Fluor 647-conjugated CD11b Ab (1:100; BD Pharmingen), PE-conjugated Siglec F Ab (1:100; BD Pharmingen), PerCP- Cy5.5 conjugated Gr1 Ab (1:100, eBioscience) and Alexa Fluor 488 conjugated F4/80 Ab (1:20; BD Pharmingen) for 30 minutes at 4°C. Samples were washed once in PBS, fixative solution was added and samples were kept on ice until analyzed on a FACSCalibur flow cytometer.

Supplemental material S3

B A

C D

E F

G H

Immunohistochemistry in human colon biopsies and immunofluorescence in polymorphonuclear and mononuclear leukocytes. (A) Rabbit CRTH2 antibody (1:200; Acris; SP4548P) and (C) rabbit DP1 antibody (1:100; Cayman Chemicals) were preincubated with 30 µg of the respective blocking peptide (B, D) overnight at 4°C and stainings were performed as described in the Methods section. Scale bars in B and D:50 µm. Isotype control experiments for CRTH2 (rabbit IgG) and DP (goat IgG) were performed in polymorphonuclear leukocytes (E, G) and peripheral blood mononuclear cells (F, H) from healthy donors to demonstrate the specificity of CRTH2 and DP staining shown in Fig. 2 (main manuscript). No staining was observed with the respective IgG. DAPI was used to counterstain cell nuclei. Calibration bar in E: 20 µm.