HLA-DRhi and CCR9 Define a Pro-Inflammatory Monocyte Subset in IBD

Abstract

OBJECTIVES:It has been demonstrated that circulating monocytes relocate to the intestinal mucosa during intestinal inflammation, but the phenotype and inflammatory mechanisms of these monocytes remain poorly understood. Here, we have investigated blood monocytes expressing high levels of HLA-DR and CCR9 in patients with inflammatory bowel disease (IBD).METHODS:Fifty-one patients with mild to severe ulcerative colitis (UC; n=31; UC-DAI 3-12) or Crohn's disease (CD; n=20; Harvey-Bradshaw indices (HBI) 2-16) were included together with 14 controls, during IBD therapy for four consecutive weeks. The frequency of CD14 ⁺ HLA-DR^hi monocytes was monitored weekly in peripheral blood, using flow cytometry. The surface phenotype and cytokine profile of these monocytes were established using flow cytometry and real-time PCR. Clinical parameters were assessed weekly in all patients.RESULTS:The frequency of circulating CD14 ⁺ HLA-DR^hi monocytes was significantly higher in IBD patients with moderate to severe disease compared with healthy controls (P<0.001). During treatment with corticosteroids and granulocyte/monocyte apheresis, the proportion of circulating CD14⁺ HLA-DR^hi monocytes was significantly reduced. CD14⁺ HLA-DR^hi monocytes produced high levels of inflammatory mediators, such as tumor necrosis factor (TNF)-α, and expressed the gut-homing receptor CCR9. Furthermore, we found that the CCR9 ligand, CCL25/TECK, was expressed at high levels in the colonic mucosa in IBD patients with active disease.CONCLUSIONS:CD14 ⁺ HLA-DR^hi blood monocytes were increased in patients with active IBD. These monocytes exhibit a pro-inflammatory, gut-homing phenotype with regard to their TNF-α production and expression of CCR9. Our results suggest that these monocytes are important in mediating intestinal inflammation, and provide potential therapeutic targets in IBD.

Citation: Clinical and Translational Gastroenterology (2012) 3, e29; doi:10.1038/ctg.2012.23

& 2012 the American College of Gastroenterology All rights reserved 2155-384X/12 http://www.nature.com/ctg

Web End =www.nature.com/ctg

Ludvig Linton1, Mats Karlsson2, Jeanette Grundstrm1, Eric Hjalmarsson2, Annelie Lindberg2, Emma Lindh1, Hans Glise1, Ragnar Befrits3, Izabella Janczewska4, Per Karln2, Ola Winqvist1 and Michael Eberhardson3

OBJECTIVES: It has been demonstrated that circulating monocytes relocate to the intestinal mucosa during intestinal inammation, but the phenotype and inammatory mechanisms of these monocytes remain poorly understood. Here, we have investigated blood monocytes expressing high levels of HLA-DR and CCR9 in patients with inammatory bowel disease (IBD). METHODS: Fifty-one patients with mild to severe ulcerative colitis (UC; n 31; UC-DAI 312) or Crohns disease (CD; n 20;

HarveyBradshaw indices (HBI) 216) were included together with 14 controls, during IBD therapy for four consecutive weeks. The frequency of CD14 HLA-DRhi monocytes was monitored weekly in peripheral blood, using ow cytometry. The surface phenotype and cytokine prole of these monocytes were established using ow cytometry and real-time PCR. Clinical parameters were assessed weekly in all patients.

RESULTS: The frequency of circulating CD14HLA-DRhi monocytes was signicantly higher in IBD patients with moderate to severe disease compared with healthy controls (Po0.001). During treatment with corticosteroids and granulocyte/monocyte apheresis, the proportion of circulating CD14 HLA-DRhi monocytes was signicantly reduced. CD14 HLA-DRhi monocytes produced high levels of inammatory mediators, such as tumor necrosis factor (TNF)-a, and expressed the gut-homing receptor

CCR9. Furthermore, we found that the CCR9 ligand, CCL25/TECK, was expressed at high levels in the colonic mucosa in IBD patients with active disease.

CONCLUSIONS: CD14 HLA-DRhi blood monocytes were increased in patients with active IBD. These monocytes exhibit a proinammatory, gut-homing phenotype with regard to their TNF-a production and expression of CCR9. Our results suggest that these monocytes are important in mediating intestinal inammation, and provide potential therapeutic targets in IBD.

Clinical and Translational Gastroenterology (2012) 3, e29; doi:http://dx.doi.org/10.1038/ctg.2012.23

Web End =10.1038/ctg.2012.23 ; published online 20 December 2012 Subject Category: Inammatory Bowel Disease

inammation in genetically susceptible individuals, whereas the chronic state might be maintained by adaptive elements.5

Monocytes are bone marrow-derived leukocytes of the myeloid lineage that migrate to the tissue and differentiate into macrophages or DCs. Increased turnover rates and elevated levels of circulating monocytes have been demonstrated in IBD.6,7

Furthermore, monocytes have the ability to migrate to the inamed mucosa and mediate inammation, but the phenotype of these monocytes as well as the mechanisms underlying this relocation remains to be elucidated.810 Currently, two main human monocyte subpopulations have been characterized. The CD14CD16 cells have been shown to produce the regulatory cytokine IL-10 and are most commonly referred to as classical monocytes. The CD14loCD16 subset is characterized by production of pro-inammatory cytokines as well as high surface expression of inammatory markers, such as CD43.1113 How

ever, a larger degree of heterogeneity among human myeloid cell populations with regard to their surface antigen expression as well as their functionality has lately been observed.14

1Department of Medicine, Translational Immunology Unit, Karolinska Institutet, Stockholm, Sweden; 2Department of Clinical Research and Education, Karolinska Institutet, Sodersjukhuset, Stockholm, Sweden; 3Department of Gastroenterology and Hepatology, Karolinska University Hospital Solna, Stockholm, Sweden and

4Department of Internal Medicine and Pathology, Karolinska Institutet, Danderyd University Hospital, Stockholm, Sweden

Correspondence: L Linton, MSc, Department of Medicine, Translational Immunology Unit, Karolinska Institutet, SE-171 76 Stockholm, Sweden. E-mail: mailto:[email protected]

Web End [email protected] Received 27 June 2012; revised 27 September 2012; accepted 1 November 2012

HLA-DRhi and CCR9 Dene a Pro-Inammatory Monocyte Subset in IBD

INTRODUCTION

Crohns disease (CD) and ulcerative colitis (UC) (inamma-tory bowel diseases, IBDs) are chronic, inammatory disorders of the gastrointestinal tract resulting from a disrupted balance between the mucosal immune system and commensal ora. To date, the immunological pathophysiology behind IBD remains poorly understood. Traditionally, adaptive immunity was believed to have an important role in the onset of IBD. Studies in patients and animal models have shown that CD is driven by T-helper 1 signaling with interleukin (IL)-12 and interferon-g production, whereas UC is characterized by T-helper 2 responses and IL-13.1

However, the Th1/Th2 paradigm has been questioned over the past decade.2 Since the discovery of the NOD2/CARD15 susceptibility locus that encodes a pattern recognition receptor mainly expressed on dendritic cells (DCs) and monocytes, the focus of IBD research has shifted toward innate immunity.3,4 Currently, innate mechanisms are believed to be responsible for the onset of acute mucosal

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Reports have established that myeloid-derived suppressor cells, characterized by their lack of expression of classical myeloid markers such as CD14 and HLA-DR, have the ability to signicantly suppress antigen-specic T-cell responses in cancer patients.15,16 It has also been shown that patients with

IBD display elevated levels of functionally suppressive HLADR myeloid-derived cells, reecting the need for immuno-suppression in the state of disease.17

It has been demonstrated that monocyte HLA-DR expression has an important role in conditions characterized by immune responses against bacterial agents.18,19 Although the CD14CD16 subset has been reported to express HLADR, the specic contribution of CD14HLA-DRhi monocytes to intestinal inammation has not been studied. As it is well established that induction of colitis in human as well as in animal models requires the presence of bacteria, we set out to study CD14HLA-DRhi monocytes in patients with chronic intestinal inammation.20

MATERIALS AND METHODS

Patients. In total, 51 IBD patients (UC 31 and CD 20)

were included in this study (Table 1). The patients were monitored during treatment with corticosteroids (n 16), the

antitumor necrosis factor (TNF)-a antibodies iniximab or adalimumab (Remicade or Humira; n 17), or granulocyte/

monocyte apheresis (GMA; Adacolumn; n 18). Four to six

biopsies from affected rectum and sigmoid colon were collected together with blood samples before the start of treatment, followed by analysis of intestinal and blood specimens after 12 weeks and 4 weeks into treatment. Patients were clinically assessed using the UC-DAI (UC) and HarveyBradshaw (HBI; CD) indices. For glucocorticoid- and anti-TNF-treated patients, the response was evaluated at week 5, and GMA-treated patients were assessed at week 11 post treatment, owing to the delayed response observed in this treatment group. Clinical

remission was dened as o3 for UC-DAI and o5 for HBI.8,21

Fourteen controls without IBD were included in the study. All patients were enrolled through formal consent, and the study was approved by the regional ethics committee.

Leukocyte isolation and activation. For ow cytometry studies, peripheral blood mononuclear cells (PBMCs) were obtained from heparinized whole blood by incubation in hypotonic buffer (160 mM NH4Cl, 10 mM Tris-HCl, pH 7.4).

For PCR and CCL25 depletion experiments, PBMCs were obtained from anticoagulated healthy donor buffy coats by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Amersham, UK). For PCR experiments, CD14 monocytes were negatively isolated using Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes were subsequently activated with LPS (lipopolysaccharide; Sigma, St Louis, MO, USA) (200 ng/ml/ 106 cells) for 2 h (TNF-a PCR) or 6 h (PCR array) in RPMI medium (Thermo Scientic Hyclone, Waltham, MA, USA)

supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% PEST (penicillinstreptomycin).

Flow cytometry. PBMCs were stained for ow cytometry analysis or sorting using combinations of the antibody conjugates described in Table 2. All stainings were carried out according to the instructions of the manufacturer of the respective antibody conjugate. Isotype- and uorochrome-matched control antibodies were used to dene chemokine receptor expression in Figures 5 and 6. Flow cytometry analyses and sorting experiments were carried out using a FACSAria cytometer and data were analyzed using FACSDiva software (BD Biosciences, San Jose, CA, USA).

PCR experiments. For TNF-a PCR experiments, RNA isolation was performed using TRIZOL reagent (Invitrogen,

Carlsbad, CA, USA). For the CCL25 experiment, intestinal

Table 1 Patient demography

Gender Male 32

Female 19 Age mean 37.9 Diagnosis Ulcerative colitis 31

Crohns disease 20 Extension Extensive 25

Left-sided 19 Proctitis 5 Ileocekal 2 Intervention Corticosteroidsa,b 16

Anti-TNF-ac,d 17 GMA apheresisd,e 18

Azathioprine Yes 21 No 30

Abbreviations: GMA, granulocyte/monocyte apheresis; TNF, tumor necrosis factor.

aFifteen patients were introduced to 2045 mg prednisone followed by tapering of 5 mg weekly.

bOne patient received topical corticosteroids for ulcerative proctosigmoiditis.

cAnti-TNF-a treatment was administered either as infusions of 5 mg/kg iniximab week 0, 2, and 6 or subcutaneous injections of 80 mg Adalimumab week 0 followed by 40 mg every other week.

dSome patients were receiving baseline corticosteroid medication.

eIn the GMA apheresis group, each patient received a total of 58 Adacolumn leukocytapheresis sessions 12 times weekly.

Table 2 Flow cytometry antibodies used in the study

Marker Conjugate Clone Manufacturer

CD4 Pacic Blue RPA-T4 BDCD14 FITC RMO52 Beckman CD16 PE-Cy7 3G8 BDHLA-DR APC-Cy7 L243 BDCCR1 Alexa Fluor 647 TG4 BioLegend CCR2 PerCP-Cy5.5 TG5 BioLegend CCR3 PE 5E8 BioLegend CCR4 PerCP-Cy5.5 1G1 BDCCR5 PE HM-CCR5 BioLegend CCR6 PerCP-Cy5.5 11A9 BDCCR7 PerCP-Cy5.5 TG8 BioLegend CCR9 APC 112509 R&D Systems CCR10 PE 314305 R&D Systems CXCR1 APC 8F1 BioLegend CXCR2 PE 5E8 BioLegend CXCR3 PerCP-Cy5.5 TG1 BioLegend CXCR4 APC 12G5 R&D Systems CXCR5 PerCP-Cy5.5 TG2 BioLegend CXCR6 PE 56811 R&D Systems CXCR7 PE 8F11-M16 BioLegend CX3CR1 APC 2A9-1 BioLegend XCR1 PE polyclonal R&D Systems ChemR23 APC 84939 R&D Systems

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biopsies were collected through exible sigmoidoscopy from UC patients and immediately submerged in RNAlater (Ambion, Austin, TX, USA). RNA was subsequently isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers protocol. For the TNF-a and CCL25 experiments, 100 ng RNA per sample was included in a reverse transcriptase reaction using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). For PCR array analyses, RNA was isolated from sorted CD14HLADRhi and CD14HLA-DRlo populations using the RNeasy Mini Kit (Qiagen). For each of the analyzed populations, equal amounts of RNA from three independent donors were pooled, and complementary DNA was synthesized using RT2 First Strand Kit (SABiosciences, Germantown, MD, USA) from 150 ng of RNA. Subsequently, complementary DNA was put into a RT2 qPCR Master Mix (SABiosciences) reaction and loaded onto a Human Inammatory Response and Autoimmunity 96-well PCR array plate according to the instructions of the manufacturer (SABiosciences). TNF-a

PCR experiments were performed on an iCyclerIQ Optical System using 2 IQ SYBR Green supermix and iCycler IQ

Optical System Software v3.1 (Bio-Rad) for data retrieval. The CCL25 PCR was performed on a CFX96 PCR system (Bio-Rad) using Go-Taq hotstart polymerase (Promega, Madison, WI, USA). In the TNF-a and CCL25 experiments, expression levels were normalized to RNA polymerase II using the 2 DDCt method. Primers used were TNF-a forward (50-CTCTCTCCCCTGGAAAGGAC-30), TNF-a reverse (50-

GCCAGAGGGCTGATTAGAGA-30), CCL25 forward (50-CC ACACCCAAGGTGTCTTTGA-30), CCL25 reverse (50-GAGC ACAGCCCACCCAAT-30), RPII forward (50-GCACCACGTC CAATGACAT-30), RPII reverse (50-GTGCGGCTGCTTCCAT AA-30), and CCL25 Taqman probe (50-FAM-ACTGCTGCC TGGCCTACCACTACCC-TAMRA-30 (Cybergene, Huddinge, Sweden). CCL25 primer sequences were adopted from Eksteen et al.22 For CCL25 PCR array analyses, expression levels were normalized to the arithmetic mean expression of the B2M, HPRT1, RPL13A, GAPDH, and ACTB housekeeping genes, using the 2 DDCt method.

CCL25 depletion assay. Biotinylated CCL25 (Almac Sciences, Craigavon, UK) was bound to a solid support consisting of a streptavidinsepharose matrix (GE Health-care). PBMC from six healthy donors was perfused through the device, and CCR9 expression was analyzed before and after using ow cytometry.

Statistical analyses. All group analyses were carried out using two-tailed-dependent Students t-test (Figures 4 and 6) or two-tailed-independent Students t-test (Figures 2, 3, and 6). Regression analyses were performed using ordinal regression test for nonparametric data (Figures 2b and c). All calculations were carried out in GraphPad Prism v5 software (GraphPad Software, San Diego, CA, USA). Values of Pr0.05 were regarded as signicant, and depicted as follows: Pr0.05*, Po0.01**, and Pr0.001***. In all gures, bars represent means.d.

Ethical considerations. The study was approved by the Stockholm Regional Ethics Review Board in Stockholm, Sweden

(http://www.epn.se

Web End =http://www.epn.se). The ethical approval applies to all centers from which patients were recruited (South Hospital, Stockholm, Sweden; Karolinska Hospital, Stockholm, Sweden; and Danderyd Hospital, Stockholm, Sweden). All patients were enrolled through formal written consent.

RESULTS

The frequency of CD14HLA-DRhi monocytes is increased in active UC and CD. To investigate the role of CD14HLA-DRhi monocytes in IBD patients, we used ow cytometry to identify the population in peripheral blood (Figure 1). To rule out any possibility that the CD14HLADRhi population is in fact a mixed population mainly composed of CD16-expressing monocytes, we investigated the relative expression of CD16 and HLA-DR as well as side and forward scatter appearance in the CD14HLA-DRhi, CD14loCD16, and CD14CD16 populations. This analysis revealed the HLA-DRhi subset to be clearly distinguished from the other established monocyte population with regard to its CD16 and HLA-DR expression pattern (data not shown).

When analyzing blood from patients and controls, we found that active inammation in the colon correlated to a signicantly higher frequency of HLA-DRhi monocytes compared with the control group (Figure 2a, P 0.006). In

addition, a correlation was observed with disease activity and the prevalence of HLA-DRhi monocytes in UC (UC-DAI) (Figure 2b; P 0.0137, r2 0.072) and CD (HBI) (Figure 2c;

P 0.0182, r2 0.1898).

CD14HLA-DRhi monocytes are potential therapeutic targets and markers of inammation in colitis. Next, we investigated whether the CD14HLA-DRhi population was affected by conventional IBD therapy. Patients with active intestinal inammation who received either corticosteroids or anti-TNF-a antibodies (iniximab or adalimumab) were monitored for 5 consecutive weeks. A patient group treated with GMA was included for comparison, considering the

Figure 1 Flow cytometry gating strategies. Representative ow cytometry plots showing the gating strategies used throughout the study for the CD14CD16 (yellow; lower left), CD14loCD16 (purple; lower middle), and CD14HLA-DRhi (red; lower right) monocyte populations. The presented plots are from one representative inammatory bowel disease patient.

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Figure 2 CD14HLA-DRhi monocytes are increased and correlate to disease activity in patients with ulcerative colitis (UC) and Crohns disease (CD). (a) Frequency of CD14HLA-DRhi monocytes in peripheral blood of inammatory bowel disease patients compared with controls, as determined with ow cytometry. Bars represent mean valuess.d. from controls (n 11) and patients (n 31) with active UC (n 20; UC-DAI 612) or CD (n 11; HarveyBradshaw (HBI) 816) (P 0.006). (b) Regression

analysis of CD14HLA-DRhi monocytes and clinical disease activity in patients with UC (P 0.0137; r2 0.072). Data represent measurements (n 84) from 28 unique

patients at different time points during treatment. (c) Regression analysis of CD14HLA-DRhi monocytes and clinical disease activity in patients with CD (P 0.0182;

r2 0.190). Data represent measurements (n 29) from 11 unique patients at different time points during treatment. Axes represent percentage CD14HLA-DRhi of its

parent CD14 monocyte population.

selective removal of monocytes associated with Adacolumn.23 When these treatment regimens were plotted separately, the patient group receiving GMA therapy accounted for the most prominent decrease (Figure 3a). The monocyte population was also attenuated after only 1 week of therapy among corticosteroid patients. The suppression was maintained, reaching levels well below those of healthy control patients at week 4 (Figure 3b, Po0.05). The decreased population of CD14HLA-DRhi during treatment was not inuenced by the diagnosis UC or CD, extension of the disease in the colon, concomitant azathioprine treatment, or gender (data not shown). The treatment groups were too small to allow for any comparison between responders and nonresponders. However, adding the patients of the GMA-and corticosteroid-treated groups together, the HLA-DRhi

population was signicantly decreased after 4 weeks of treatment (16.951.809.680.75; Po0.001) in those who achieved remission. This was not observed among the non-remission patients (15.361.6611.851.73; not signi-cant). Interestingly, biological therapy with antibodies against TNF-a did not signicantly affect the proportion of CD14

HLA-DRhi monocytes (Figure 3c). Among these patients, CD14HLA-DRhi never reached the reference level observed in the controls.

CD14HLA-DRhi monocytes produce high levels of inammatory mediators. With the purpose of investigating the capacity of CD14HLA-DRhi monocytes to produce inammatory mediators, monocytes from healthy blood donors were cultured in the presence of LPS. The HLA-DRhi

population and its CD14HLA-DRlo counterpart were subsequently sorted using ow cytometry (Figure 4a). The two cell populations were investigated with regard to the production of the pro-inammatory cytokine TNF-a. Interestingly, the CD14HLA-DRhi population produced 500-fold increased levels of TNF-a transcripts upon LPS stimulation compared with the CD14HLA-DRlo cells (Figure 4b). Furthermore, PCR array analyses were carried out on sorted

CD14HLA-DRhi monocytes from three independent donors after activation with LPS in order to establish the distinctive phenotype of the population. In accordance with our hypothesis, several gene transcripts described as being involved in monocyte-mediated immune responses were upregulated in the CD14HLA-DRhi monocytes. Increased gene expression was mainly found among chemotactic cytokines and genes involved in the humoral immune response (Figure 4c; Supplementary Table). The most prominent fold-change difference between the CD14HLADRhi and the CD14HLA-DRlo monocytes was observed for the chemotactic cytokine CCL4. The transcript with the most apparent downregulation among HLA-DRhi monocytes was the HDAC4 gene that encodes a histone deacetylase that functions as a transcriptional repressor.24 Together, these data show that CD14HLA-DRhi monocytes have strong pro-inammatory potential.

CD14HLA-DRhi monocytes express the gut-homing chemokine receptor CCR9. Next, we studied the surface expression of various chemokine receptors on CD14 HLA-DRhi monocytes in relation to the CD14CD16 and CD14loCD16 subsets. Although we could observe signicant overlap between many of these markers in terms of their expression in the respective subsets, the CD14HLA-DRhi population was clearly distinguished by its expression of CCR7 and CCR9 (Figure 5; Supplementary Figure 1). CCR7 is mainly described as a lymph-node-homing receptor for DCs and T-helper cells, and has previously not been reported to be expressed on circulating monocytes.25 CCR9 has been shown to be important in lymphocyte homing to the gut through interactions with its ligand CCL25, expressed in the intestinal mucosa.26,27 Although CCR9CCL25 interactions have been well characterized in T-helper cells, their role in monocytes is unclear. The general CD14 monocyte population exhibited clearly higher CCR9 expression compared with CD3 T lymphocytes, which has been described as the main CCR9-expressing cell type (Figure 6a).28

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Figure 3 CD14HLA-DRhi monocyte levels are decreased during inammatory bowel disease (IBD) therapy. CD14HLA-DRhi monocyte levels in IBD patients during treatment with (a) granulocyte/monocyte apheresis (GMA) apheresis (n 18), (b) corticosteroids (n 16), or (c) anti-tumor necrosis factor (TNF)-a biological therapy

(n 14). Control patient reference levels (n 11) are included in all graphs. Error bars represent group mean valuess.d. Axes represent percentage CD14HLA-DRhi of

its parent CD14 monocyte population. NS, not signicant.

When comparing CCR9 expression in CD14HLA-DRhi with CD14loCD16 and CD14CD16 monocytes, the HLA-DRhi subset displayed markedly increased levels (Figure 5; Supplementary Figure 1). In contrast, the expression of CCR2, a chemokine receptor responsible for general monocyte migration, was not increased on the HLA-DRhi

monocytes, indicating that gut-homing phenotype constitutes a specic feature of the monocytes, rather than reecting general immunological activation (Figure 5; Supplementary Figure 1).29 In conclusion, pro-inammatory CD14HLA-DRhi monocytes express high levels of the gut-homing chemokine receptor CCR9, which directs them to the site of mucosal inammation.

CCR9 expression on CD14 monocytes is signicantly increased during active intestinal inammation. After establishing that CCR9 is indeed expressed on human monocytes, we investigated monocytic CCR9 expression levels during active colonic inammation. In patients with active IBD, we observed signicantly increased CCR9 expression levels on CD14 monocytes compared with healthy controls (Figure 6a; Po0.01). The same pattern was observed on CD3 lymphocytes, although the levels were generally lower (Po0.01).

We also analyzed colonic mucosal tissue biopsies with real-time PCR and found that the CCR9 ligand, CCL25/TECK, was

expressed in the colon (Figure 6b). To establish a functional interaction between CCL25 and CCR9, we carried out depletion experiments by perfusing peripheral blood cells through a solid support containing CCL25-coated sepharose beads. The frequency of CCR9-positive CD14HLA-DRhi monocytes was signicantly reduced after encounter with the CCL25-coated sepharose beads, showing that CCR9 on CD14HLA-DRhi monocytes could bind CCL25 and be removed from the blood (Figure 6c; Po0.05). These results indicate that CCR9 expressed on monocytes may functionally interact with colonic CCL25/TECK and has a role in human colonic inammation.

DISCUSSION

In this study, we identify CCR9-expressing CD14HLA-DRhi blood monocytes as an important factor in intestinal inammation. The expression of HLA-DR on monocytes is vital to the inammatory response and has been shown to determine the efcacy of antigen presentation to T-helper cells.30,31

Monocytes with high expression of HLA-DR have also been shown to inltrate the joints of patients with rheumatoid arthritis, an inammatory disease successfully treated with TNF-a antibodies.32 In addition, carrying the class II

HLA-DRB1*0103 allele correlates with an increased risk for developing UC.33

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Figure 4 CD14HLA-DRhi monocytes produce high levels of inammatory mediators in response to lipopolysaccharide (LPS). (a) Representative ow cytometry plots depicting CD14HLA-DRhi and CD14HLA-DRlo purity after ow cytometry sorting. (b) PCR analysis of tumor necrosis factor (TNF)-a in CD14HLA-DRhi monocytes after

LPS activation for 2 h (n 4, P 0.0047). Graph shows analysis of duplicate samples using the 2 DDCt method and RNA polymerase II as housekeeping gene. (c) Target

transcripts from PCR array analysis of CD14HLA-DRhi monocytes from three independent healthy donors after LPS activation for 6 h, grouped into functional categories. (d) The 20 target transcripts that represented the strongest up- and downregulation in PCR array analyses of CD14HLA-DRhi monocytes following LPS stimulation for 6 h. Fold changes range from 347.3 to 10.9 and 10.3 to 232.3, respectively. Axes in (c) and (d) represent transcript fold changes in the CD14HLA-DRhi subset using

CD14HLA-DRlo as a reference population, and the arithmetic mean of the B2M, HPRT1, RPL13A, GAPDH, and ACTB transcript levels as housekeeping genes using the 2 DDCt method. Panels (c) and (d) represent data from equal amounts of pooled input RNA isolated from three independent healthy blood donors. Error bars represent group mean valuess.d. mRNA, messenger RNA.

Several studies have suggested that monocytes per se are targeted by conventional IBD therapy.6,34,35 Our results

suggest that specic downregulation of the HLA-DRhi

subpopulation may be an important mechanism behind resolution of the inammation. In this study, patients treated with GMA were added for comparison, as Adacolumn is the only treatment specically targeting circulating monocytes. These cells are removed through Fcg receptor binding to the cellulose acetate beads in the column, leaving circulating T cells unaffected.23 Corticosteroid therapy mediates a decrease in the number of circulating CD14HLA-DRhi monocytes that is comparable to GMA (Figure 3b). Surprisingly, patients subjected to biological treatment did not display a decrease of pro-inammatory monocytes (Figure 3c). We speculate that by removing TNF-a, one of the main products of these monocytes, autocrine feedback mechanisms leading to cellular activation might be induced. The observation underscores the difference in mode of action between anti-TNF-a antibodies and corticosteroids, and should be further investigated, as anti-TNF-a failure may partly depend on the monocytes production of additional pro-inammatory chemokines, cytokines, and integrin receptors counterbalancing TNF-a suppression.

Classically, leukocyte populations have been dened through their capability to produce inammatory mediators such as cytokines and chemokines. In order to gain a functional understanding of how CD14HLA-DRhi monocytes mediate inammation, we investigated their pro-inammatory potential at the messenger RNA level compared with their HLA-DRlo-expressing counterpart. In this context, the HLADRhi subset produced markedly elevated levels of gene transcripts associated with activation and pro-inammatory phenotype. The population displayed a 500-fold increase of TNF-a transcript levels, which establishes the HLA-DRhi

subset as one of the most important producers of this cytokine. Other genes were also investigated by PCR array analysis, revealing the highest upregulation in CCL4, CCL3, and IL-1b, all cytokines previously described as being involved in the recruitment of inammatory cells to the intestinal mucosa in IBD (Figure 4d).3640

The most prominent difference was observed in the CCL4/ MIP-1b gene, with upregulated transcript levels of more than 300-fold. The inammatory role of CCL4 was reported by Bystry et al.,36 who demonstrated that activated T-helper cells efciently migrate toward a CCL4 tissue gradient through CCR5 interaction. Thus, our nding that

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CD14+HLA-DRhi CD14loCD16+ CD14+CD16- CD3+

CCR1

CCR2

CCR3

CCR4

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CXCR1 CX3CR1

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CXCR1 CX3CR1

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CXCR1 CX3CR1

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CXCR1 CX3CR1

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CXCR2

Figure 5 The relative chemokine receptor expression in the CD14HLA-DRhi, CD14loCD16, and CD14CD16 monocyte populations, as well as CD3 lymphocytes, as determined by ow cytometry. Each monocyte subset is represented by one spider web chart. Data points indicate isotype control-subtracted expression for each chemokine receptor. Expression levels are dened as median uorescence intensity channel numbers (MFC). Data presented are mean values from ve healthy control patients.

CCL4 transcripts were produced in high levels by CD14 HLA-DRhi monocytes supports their inammatory potential.41 The transcript with the most apparent down-regulation in HLA-DRhi monocytes was the HDAC4 gene that encodes a histone deacetylase that functions as a transcriptional repressor.24 Together, these data indicate that CD14HLA-DRhi is a transcriptionally active subset that readily expresses genes important for mediating mucosal immune responses.

On the surface level, CD14HLA-DRhi monocytes only partially express CD16, suggesting that the population constitutes a separate subset that is not included in its entirety when dening pro-inammatory monocytes through their expression of CD14loCD16 (Figure 1). The population is further dened by its expression of CCR7 and the gut-homing receptor CCR9, which clearly distinguishes HLA-DRhi monocytes from the CD14CD16 and CD14loCD16 subsets (Figure 5; Supplementary Figure 1).

In the context of intestinal inammation, interactions between CCR9-expressing T cells and CCL25 (TECK) expressed in the gut epithelium have been implicated as an important mechanism for recruiting circulating lymphocytes to the intestinal mucosa.26 However, whether this mechanism also applies to the extensive inltration of blood monocytes to the intestinal mucosa observed during inammation has never been studied.42 It was recently shown that CCR9-expressing monocytes are increased in the peripheral blood of patients with rheumatoid arthritis. In accordance with the results from this study, we show that the CD14CD16 and CD14loCD16 subsets express similar levels of CCR9.43 Those levels were notably superseded by the CCR9 expression on CD14HLA-DRhi monocytes (Figure 5; Supplementary Figure 1). Interestingly, this expression was considerably higher than that observed on T cells, which are considered to be the main CCR9-carrying cell type (Figure 6a).28 In contrast, the expression of CCR2, another chemokine receptor important for monocyte relocation in several disease groups, was not increased on HLA-DRhi

monocytes (Figure 5; Supplementary Figure 1).29 This

suggests that the specic increase in CCR9 expression among CD14HLA-DRhi may reect a gut-specic pheno-type, rather than a generally activated subset. CCL25, the ligand for CCR9, was found to be expressed in mucosal tissue by QT-PCR analysis, which is supported by other reports identifying CCL25 in the colon in mice (Figure 6b).27,44

Interestingly, colonic CCL25 messenger RNA levels were consistently higher in pretreatment samples (UC-DAI 78, HBI 9; n 3) compared with samples from the same patients

Figure 6 The CCR9CCL25 axis has a role in monocytes contributing to chronic intestinal inammation. (a) CCR9 expression levels in CD14 monocytes and CD3 lymphocytes are signicantly increased in peripheral blood in inammatory bowel disease (IBD) patients with active intestinal inammation (n 5) compared with healthy controls (HC) (n 5), as determined by ow

cytometry. Expression levels are dened as median uorescence intensity channel numbers (MFC). Error bars represent group mean valuess.d. (b) The graph shows CCL25 messenger RNA transcript levels from colonic biopsy specimens from IBD patients (n 3) before and after 4 weeks of corticosteroid therapy. Graph

shows analysis of duplicate samples using the 2 DDCt method and RNA polymerase II as housekeeping gene. (c) Depletion of peripheral blood CCR9-expressing CD14 HLA-DRhi monocytes from IBD patients using CCL25-coated

beads (n 5; P 0.011).

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Pro-Inammatory Monocytes in IBD Linton et al.

8

after 4 weeks of corticosteroid therapy (UC-DAI 12, HBI 2) (Figure 6b).

Our data also show that CCR9 expression levels on circulating blood monocytes are signicantly increased in patients with active colonic inammation (Figure 6a). In conclusion, these data suggest that monocytes in general and CD14HLA-DRhi in particular possess the ability to relocate to the intestinal mucosa through CCR9CCL25 interactions, particularly in patients with active disease.

T cells have been shown to acquire their CCR9 expression through retinoic acid-dependent imprinting by mesenteric lymph node DCs.45 The issue of whether CCR9 expression on monocytes is acquired through similar mechanisms seems controversial, especially as blood monocytes are not known to trafc lymph nodes to the same extent as T cells. Here, we show that CD14HLA-DRhi monocytes are dened by their high expression of CCR7, a marker mainly described as a lymph-node-homing receptor for DCs and T-helper cells (Figure 5; Supplementary Figure 1).25 Therefore, it is tempting to speculate that CCR7-expressing CD14HLA-DRhi monocytes trafc the lymph nodes to a higher extent than has previously been known, and that CCR9 imprinting in these monocytes may occur through mechanisms similar to those reported in T cells. Being beyond the scope of this study, the mechanisms behind CCR9 induction in monocytes, as well as the functional role of their CCR7 expression, need to be addressed in the future.

In this study, we have shown that CD14HLA-DRhi blood monocytes are increased in patients with active intestinal inammation. This subset is distinguished by its capability to produce pro-inammatory cytokines and its expression of CCR9 that may direct the monocytes toward CCL25 gradients produced in the inamed colon during IBD. In summary, these ndings indicate that CD14HLA-DRhi blood monocytes have an important role in IBD and that future studies evaluating these monocytes as specic targets for IBD therapy are highly indicated.

CONFLICT OF INTEREST

Guarantor of the article: Michael Eberhardson, MD, PhD. Specic author contributions: Study design: ME and OW; data collection: LL, EH, and JG; data analysis and interpretation: LL, MK, OW, EL, and ME; manuscript drafting: LL, EL, and ME; critical revision of the manuscript: OW, PK, and HG; statistical analysis: MK and LL; obtained funding: OW, HG, and ME; and study supervision and patient inclusion: AL, PK, IJ, and RB.

Financial support: The investigators have received nancial support from the Swedish Medical Society, Stockholm, Sweden; ALF-support, Stockholm, Sweden; Immune Therapy Holdings AB, Stockholm, Sweden; Schering-Plough AB, Stockholm, Sweden; and Otsuka Pharma Scandinavia AB, Stockholm, Sweden. The study was designed and performed independent of any nancial source.

Potential competing interests: None.

Acknowledgements. We gratefully acknowledge Martina Jones and Petra Jones for valuable input and excellent technical assistance.

Study Highlights

WHAT IS CURRENT KNOWLEDGE

| Monocytes are involved in mucosal inammation.| Monocyte HLA-DR levels correlate with inammatory

activity in allergy and rheumatoid arthritis.| CCR9CCL25 interactions are important for the

recruitment of activated lymphocytes to the inamed

intestine.

WHAT IS NEW HERE

| CD14HLA-DRhi monocytes are upregulated in active IBD

and decrease during therapy.| CD14HLA-DRhi monocytes produce high levels of

inammatory mediators.| CD14HLA-DRhi monocytes express functional CCR9

protein.| The CCR9 ligand, CCL25, is expressed in the colonic

mucosa of IBD patients.

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