The expression of dendritic cell subsets in severe chronic rhinosinusitis with nasal polyps is altered

Accepted Manuscript Title: The expression of dendritic cell subsets in severe chronic rhinosinusitis with nasal polyps is altered Author: Rog´erio Pezato Claudina A. P´erez-Novo Gabriele Holtappels Natalie De Ruyck Koen Van Crombruggen Geert De Vos Claus Bachert Lara Derycke PII: DOI: Reference:

S0171-2985(14)00096-5 http://dx.doi.org/doi:10.1016/j.imbio.2014.05.004 IMBIO 51148

To appear in: Received date: Revised date: Accepted date:

25-10-2013 9-5-2014 27-5-2014

Please cite this article as: Pezato, R., P´erez-Novo, C.A., Holtappels, G., De Ruyck, N., Van Crombruggen, K., De Vos, G., Bachert, C., Derycke, L.,The expression of dendritic cell subsets in severe chronic rhinosinusitis with nasal polyps is altered, Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The expression of dendritic cell subsets in severe

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chronic rhinosinusitis with nasal polyps is altered

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Rogério Pezato1,2*, Claudina A. Pérez-Novo1*, Gabriele Holtappels1, Natalie De Ruyck1, Koen Van

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Crombruggen1, Geert De Vos3, Claus Bachert1, Lara Derycke1

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Department of Otolaryngology-Head and Neck Surgery, Federal University of São Paulo, Brazil 2

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Department of Otorhinolaryngology, AZ ST Lucas Hospital, Ghent, Belgium

* These authors equally contributed to this work

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The work was conducted at University of Ghent, Belgium.

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Running title: pDCs are dampened in severe CRSwNP

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Upper Airway Research Laboratory (URL), Department of Otorhinolaryngology, Ghent University Hospital, Ghent University, Belgium

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Corresponding author: Rogério Pezato, Rua dos Otonis, 700- piso superior, Vila Clementino, São

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Paulo, Brazil, CEP 04025002. E-mail: [email protected], phone: 55 11 55752552

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Key words: airway, antigen-presenting cell, dendritic cells, nasal polyps, sinusitis

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List of abbreviations: Chronic rhinosinusitis with nasal polyps (CRSwNP), Chronic rhinosinusitis

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without nasal polyps (CRSsNP), dendritic cells (DCs), plasmacytoid dendritic cells (pDCs),

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myeloid dendritic cells (mDCs). indoleamine 2,3-dioxygenase (IDO), eosinophil cationic protein

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(ECP), T helper (Th), toll-like receptor (TLR), transforming growth factor (TGF), interferon (IFN),

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interleukin (IL), staphylococcus enterotoxins (SE),

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The authors declare no conflict of interest 1 Page 1 of 29

ABSTRACT

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Background

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Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized as a Th2-driven disease.

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Activated dendritic cells (DCs) are the main T-cell activators; their role in the chronic inflammatory

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process of nasal polyposis is still unclear.

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Methods

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The regulation of DC subsets was analyzed in nasal polyp tissue from CRSwNP patients and

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compared to inferior turbinate tissue from healthy subjects. Tissue localization and expression of

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both plasmacytoid and myeloid DCs were assayed by means of immunohistochemistry and flow

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cytometry. Plasmacytoid DCs were also assayed by PCR, and tissue homogenates were assayed for

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various inflammatory markers.

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Results

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The number of plasmacytoid (pDCs) and myeloid (mDCs) dendritic cells was significantly

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increased in nasal polyp tissue when compared to non-inflamed nasal mucosa. The number of

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pDCs, but not mDCs, was down-regulated in more severe cases (nasal polyps with asthma) and

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varied with the cytokine milieu. The amount of pDCs was significantly decreased in IL5+IFN-

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nasal polyp tissue compared to tissues with high IFNγ levels (IL5+IFNγ+). Furthermore, levels of

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indoleamine 2,3-dioxygenase were increased in nasal polyp compared to inferior turbinate tissue

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and correlated negatively with the number of pDCs.

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Conclusions

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There is an altered balance of pDC and mDC numbers in nasal polyp tissue. pDCs seem to be more

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susceptible to an inflammatory cytokine milieu and may play a crucial role in disease severity.

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INTRODUCTION

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Chronic rhinosinusitis with nasal polyps (CRSwNP) is mainly characterized by increased eosinophil

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infiltration and high levels of eosinophil-related mediators, such as IL-5 and eosinophil cationic

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protein (ECP). However, altered production of other pro-inflammatory molecules, including

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chemokines, IgE, and lipid metabolites, actively contributes to the inflammatory pattern of this

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disease (Huang et al., 2001). Furthermore, the inflammatory reaction observed in these patients is

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mainly orchestrated by a T helper-2 (Th2) response, although other patterns have been recently

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described (Bachert et al., 2010).

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Human dendritic cells (DCs) are among the most potent antigen-presenting cells (APC) in the

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airways, and play a crucial role in the link between “innate” (i.e., pathogen recognition) and

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“adaptive” (i.e., regulation of the type of T cell-mediated response) immune responses (Adema,

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2009; Schroeder et al. 2005). They are divided into two major populations, myeloid DCs (mDCs)

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and plasmacytoid DCs (pDCs), which can be distinguished by their level of expression and by the

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type of specific cell surface markers expressed (Adema, 2009). One of the main features of these

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cells is that they can follow several pathways of differentiation and maturation. This leads to high

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functional plasticity according to the immune environment, allowing the cells to counter-regulate

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specific innate and IgE-dependent immune responses and, hence, influence the type of T-cell

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response (Adema, 2009). For example, pDCs express toll-like receptor 9 (TLR9) molecules through

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which CpG DNA favors Th1 responses. However, they also possess IgE receptors, which are

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implicated in allergen presentation and induction of Th2 responses (Schroeder et al. 2005).

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Imbalance in DC subsets has been linked to severe airway diseases. Asthmatic patients exhibit a

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significantly higher number of pDCs as compared with normal subjects (Matsuda et al., 2002). In

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the nasal mucosa, however, some discrepancies have been found regarding the balance of these

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cells. While the number of DCs was increased and showed a more mature phenotype in patients 3 Page 3 of 29

with perennial allergic rhinitis compared with control subjects (Klein Jan et al., 2006), patients with

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rhinitis had a lower number of pDCs in the nasal epithelia as compared with non-inflamed mucosa

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from healthy subjects (Hartmann et al. 2006).

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Besides the cytokine milieu, other mucosal factors may influence DC functions, such as the enzyme

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indoleamine 2,3-dioxygenase (IDO). IDO catalyzes the degradation of tryptophan and, hence,

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influences cell proliferation (von Bubnoff et al., 2012). In the last decade, it has emerged as an

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important negative regulator of the immune system, mainly due to its effect on DCs. Expression of

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IDO within chronic rhinosinusitis tissue has been observed in epithelium and leukocytes, and IDO

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levels are associated with clinical parameters when analyzed in patients with CRSsNP (CRS

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without nasal polyps), CRSwNP, and antrochoanal polyps (Honkanen et al., 2011; Sekigawa et al.,

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2009).

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The extent, to which DCs and mucosal factors contribute to the development of CRSwNP, and how

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they are needed to maintain the chronic Th2-inflammatory process, remains unknown. The present

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study sought to analyze the expression of the different DC subsets in nasal polyp tissue and non-

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inflamed nasal mucosa and examine the relationship between the inflammatory milieu and the DC

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subset balance.

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MATERIAL AND METHODS

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Patients

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Patients were recruited from the Department of Otorhinolaryngology of Ghent University Hospital,

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Belgium. Inferior turbinate samples from patients without sinus disease undergoing septoplasty

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were collected to serve as controls (control, n=17; mean age, 36 (16-66) years; 6 female/11 male).

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Samples from patients with adult nasal polyposis (CRSwNP, n=50; mean age, 53 (20-79) years; 14

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female/36 male) were obtained during functional endoscopic sinus surgery procedures.

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The diagnosis of sinus disease was based on history, clinical examination, nasal endoscopy, and

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computed tomography of the paranasal cavities, in accordance with current EPOS guidelines

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(Fokkens et al., 2012). All subjects underwent a skin prick test to common inhalant allergens; 5 out

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of 17 patients in the control group and 20 out of 50 in the CRSwNP group were positive. Diagnosis

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of asthma was obtained from the Department of Pulmonology at Ghent University Hospital. In the

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CRSwNP group, 20 out of 50 patients were asthmatic, versus none in the control group. The

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occurrence of aspirin intolerance was based on patient history, and 3 patients were reported as

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intolerant. All patients stopped oral and nasal corticosteroids at least 1 month before the operation.

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The study was approved by the local Ethics Committee of Ghent University Hospital, Belgium, and

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written informed consent was obtained from each patient before specimen collection.

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Measurement of inflammatory markers in tissue homogenates

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Cytokine measurements were performed on tissue homogenates as previously described (Derycke et

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al., 2012) and were assayed by using the Luminex xMAP suspension array technology in a Bio-Plex

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200 system (BioRad, MI, USA). For this purpose, commercially available kits for IL-5, IL-6, IL-10,

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IL-17 were purchased from R&D Systems (Minneapolis, MN, USA) and for IL-12 and IFN-α from

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Invitrogen (Carlsbad, CA, USA). IFN-γ, IDO, and TGF-β1 concentrations were determined with 5 Page 5 of 29

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commercially available ELISA kits (IFN-γ and TGF-β1 from R&D Systems; IDO from USCN Life

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Science, Wuhan, China). Total IgE and specific IgE to staphylococcal enterotoxins (SE-IgE), as

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well as eosinophil cationic protein (ECP), were measured by the UNICAP system according to

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manufacturer guidelines (Phadia, Uppsala, Sweden).

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Immunohistochemical staining

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Frozen nasal tissue sections (7 µm) were dried overnight at room temperature and stored at -20°C.

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Before immunostaining, frozen sections were fixed in acetone for 10 minutes and rinsed and

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incubated with 0.3% H2O2 in TBS for 10 minutes. Then, sections were incubated with mouse

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monoclonal antibodies (mAb) against human blood dendritic cell antigen-1 (BDCA1) and blood

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dendritic cell antigen-2 (BDCA2), obtained from Miltenyi (Bergisch Gladbach, Germany). Signal

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detection was performed with the LSAB+ kit using 3-amino-9-ethylcarbazole (AEC) as substrate

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(DAKO, Glostrup, Denmark). Positive cells in 10 fields of view were counted, and a semi-

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quantitative score of 0 to 3, according to the number of positive cells (0 = no positive cells; 1= 0-20

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positive cells; 2 = 20-100 positive cells; 3 = more than 100 positive cells), was then assigned to

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each slide.

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RNA isolation and qPCR analysis

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Quantitative real-time PCR was used to quantify mRNA levels of BDCA2 (CLEC4C gene) with

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SybrGreen with primers for CLEC4C (SABiosciences, USA). Isolation of RNA, cDNA synthesis

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and qPCR analysis was performed as previously described (Van Bruaene et al., 2009).

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Quantification of DC by flow cytometry

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Preparation of single-cell tissue suspensions

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Fresh human nasal mucosa (9 specimens from controls and 17 specimens from NP) was transferred

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to a GentleMACs tube (Miltenyi Biotec) with 10 mL of RPMI 1640 culture medium containing 2

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mM L-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin and 2% fetal bovine serum (FBS),

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all purchased from Invitrogen, and single-cell suspensions were prepared as previously described

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(Derycke et al., 2012).

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Flow cytometry staining

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Single cells were first stained with LIVE/DEAD® Fixable Near-IR dye (Invitrogen) (1µL/106

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cells). Then, each cell pellet was washed/dissolved in FACS buffer (1X PBS, 2% FBS, 0.05%

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sodium azide) and stained with the following conjugated antibodies: Lineage1-FITC (CD3, CD14,

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CD16, CD19, CD20, and CD56); HLADR-PerCp; CD11c-APC; CD123-PE (BD Bioscience, New

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Jersey, USA); CD45-PeCy7 (Beckman Coulter, Brea, CA, USA); and BDCA1-APC, BDCA3-PE or

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BDCA2-PE or BDCA2-APC (Miltenyi Biotec). The cells were analyzed in a Canto II flow

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cytometer using the FACS Diva software (BD Bioscience).

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Statistics

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The data generated in the study were analyzed using SPSS version 18 (IBM Corporation, NY,

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USA). The Mann–Whitney U test was applied to evaluate the statistical differences between patient

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groups. Correlations between two variables were calculated with Spearman’s rank correlation

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coefficient. P-values of less than 0.05 were regarded as significant.

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RESULTS

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Levels of inflammatory markers in nasal tissue

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Concentrations of IL-5 and ECP were statistically higher in the CRSwNP group when compared

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with control tissue (p<0.01 for both markers), as shown in table 1. There were no significant

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between-group differences in levels of IFN-α, IFN-γ, IL-6, IL-10, IL-17, IL-12, or TGF-β1. Total

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IgE and SE-IgE were also significantly increased in the CRSwNP group (p<0.01 and p=0.01

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respectively).

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Localization and quantification of DCs in nasal tissue by immunohistochemistry

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Positive staining for DCs was observed in mucosal specimens from both control and CRSwNP

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patients. DCs were found scattered throughout the nasal mucosa, but preferentially concentrated

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along the basement membrane, as shown in figure 1. The following markers were used to identify

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DC subsets: BDCA1 (mDC1) (figure 1a,c) and BDCA2 (pDC) (figure 1b,d). Semi-quantitative

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scoring showed that nasal polyp sections had increased numbers of the pDC and mDC1 subtypes as

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compared with healthy mucosa (Table 2).

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Analysis of BDCA2 mRNA (pDC marker)

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To confirm the pDC results obtained by immunohistochemistry, we analyzed the transcript levels of

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BDCA2 by real-time PCR. Amplification data indicated that the normalized relative expression

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units (NREU) of BDCA2 mRNA levels were significantly higher in CRSwNP (n=6, 1.95 NREU;

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IQR 0.47-4.93) than in controls (n=7, 0.22 NREU; IQR 0.08-0.75) (p=0.04).

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Analysis of dendritic cell subsets by flow cytometry

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Quantification of tissue DC subsets by flow cytometry was performed in a subgroup of the

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CRSwNP (n=17) and control (n=8) patients. For all samples, 300,000 events were analyzed and the 8 Page 8 of 29

gating strategy was as follows: mDC1 were gated as Lin1-, HLADR+, CD11c+, BDCA1+,

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BDCA3-, CD123- and BDCA2-, while mDC2 were BDCA3+ and BDCA1-. Furthermore, pDC

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could be recognized as Lin1-, HLADR+, CD11c-, BDCA2+ and CD123+ positive (figure 2a).

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Results are presented as percentage of total living cells, which remained equal after processing

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healthy or diseased nasal mucosa samples.

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The total number of mDCs was significantly increased in CRSwNP (1.13%; IQR 0.89-0.14) when

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compared to control nasal tissue (0.45%; IQR 0.29-0.69) (p < 0.01) (figure 2b). Individually, both

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mDC1 and mDC2 subsets were also increased in nasal polyp samples (mDC1: 0.68%, IQR 0.41-

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0.73; mDC2: 0.20%, IQR 0.18-0.28) compared to control tissue (mDC1: 0.20%, IQR 0.11-0.31;

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mDC2: 0.10%, IQR 0.05-0.16), p<0.01 and p=0.01 respectively (figure 2c-d). The same pattern was

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found for pDCs in nasal polyp tissue (0.24%, IQR 0.18-0.33) versus control nasal tissue (0.08%,

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IQR 0.03-0.12), with p<0.01 (figure 2e). Correcting our data for the total increase in immune cells

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(CD45+) in CRSwNP (figure 3a), where the relative values of mDC and pDC were calculated, did

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not alter the previously obtained results (figure 3b-c).

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DC number and occurrence of asthma

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The number of pDCs was decreased in asthmatic CRSwNP patients (0.21%, IQR 0.15-0.30) when

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compared with non-asthmatics (0.28%, IQR 0.21-0.41) (p=0.05). Interestingly, in the asthmatic

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CRSwNP group, the 3 patients with aspirin intolerance had the lowest number of pDCs (0.14%,

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IQR 0.07-0.16), as shown in figure 4a. This was not the case for mDCs, numbers of which were

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similar in non-asthmatic and asthmatic patients (figure 4b). The ratio of total mDCs to pDCs was

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downregulated in non-asthmatic CRSwNP patients (3.52, IQR 2.94-5.53) as compared with healthy

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subjects (7.73, IQR 4.49-11.13) (p<0.05) and with asthmatic CRSwNP patients (7.05, IQR 3.46-

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9.19) (figure 4c).

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Influence of the cytokine milieu and inflammatory markers on DC subsets

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Within the CRSwNP group, we were able to identify 2 major subgroups of patients: the IL-5+

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(n=11) and the IL-5+/IFNγ+ group (n=5). The number of pDCs in the IL-5+ group (0.22%, IQR

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0.19-0.30) was significantly lower as compared with the IL-5+/IFNγ+ group (0.37%, IQR 0.26-0.48)

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(p=0.04) (figure 5a). Conversely, there was no significant difference in number of mDCs between

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the subgroups (IL-5+ 0.7%, IQR 0.41-0.76; IL-5+/IFN-γ+ 0.68%, IQR 0.33-0.90), as shown in figure

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5b. Furthermore, all IL-5+/IFN+ patients were non-asthmatic, and the number of pDCs in these

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subjects correlated positively with their levels of IFN (r = 0.5, p<0.05).

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Levels of IDO and DC balance

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Levels of IDO were significantly increased in the CRSwNP group (1895 IU/ml, IQR 1407-4221) as

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compared with the control group (836 IU/ml, IQR 475-1483) (p<0.01) (figure 6a). Spearman’s rank

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correlation analysis showed that IDO levels correlated negatively with the number of pDCs in the

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CRSwNP patient group (r = -0.53, p=0.03).

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Occurrence of asthma did not statistically influence IDO levels within the CRSwNP group,

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although the median concentration in asthmatic subjects (3448 IU/ml, IQR 1635-4665) was more

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than twofold higher than the one observed in the non-asthmatic subjects (1626 IU/ml, IQR 980-

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3810) (figure 6b). No statistical difference were observed between IL5+ (2027 IU/ml, IQR 1383-

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4311) and IL5+IFN-+ (1626 IU/ml, IQR 1346-4057) CRSwNP patients, as shown in figure 6c.

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DISCUSSION

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Although the best control comparator for nasal polyp tissue should be healthy nasal mucosa from

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the middle meatus, the procedure for biopsy collection from this area entails injury to healthy

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structures such as the uncinate process and ethmoidal bulla, rendering it infeasible from an ethical

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standpoint. It has been demonstrated that the inflammatory patterns found in the inferior turbinate

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do not differ from those seen in the middle turbinate among patients in the early stage of chronic

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sinusitis (Van Bruaene et al., 2012). As usual in the academic literature (Groot Kormelink et al.,

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2012; Van Crombruggen et al., 2012; Yukitatsu et al., 2013), we chose to use inferior turbinate

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specimens as controls for comparison with nasal polyp tissue in this study.

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In the present study, we demonstrated that the number of both plasmacytoid (pDCs) and myeloid

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(mDCs) dendritic cells was significantly increased in chronic rhinosinusitis with nasal polyp tissue

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when compared to normal nasal mucosa. Of interest, only the expression of pDCs varied with

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disease severity and cytokine milieu, showing a remarkable decrease in asthmatic patients and in

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those with no IFNγ production in the nasal tissue. We also found that levels of IDO (a crucial

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regulator of DC function) were significantly upregulated in nasal polyp mucosa and correlated

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inversely with the number of pDCs.

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Although it is known that DCs play an essential role in the pathophysiology of airway diseases,

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knowledge about these cells in the upper airways is quite limited.

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Our results confirm a previous report showing that presence of DCs is increased in CRSwNP

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(Ayers et al. 2011), and partially confirm the findings of Kirsche et al., 2010, showing an imbalance

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in the mDC/pDC ratio in CRSwNP when comparing the relative increase of pDCs and mDCs,

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taking into account the CD45+ cells only. However, discrepancies between the Kirsche et al. study

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and ours (the authors claimed this imbalance in the mDC/pDC ratio was only due to decrease of 11 Page 11 of 29

mDCs) may be partially explained by the fact that the former did not consider the variation in pDCs

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according to the severity of inflammatory process in CRSwNP at the moment of analysis; by the

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use of different approaches to identify DC subsets by flow cytometry; and/or by differences in the

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clinical characteristics of the patients (Derycke., 2010).

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Chronic rhinosinusitis with nasal polyps is mainly characterized by increased local production of

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IL-5, total IgE, and ECP, and it has been considered a Th2-driven disease. However, we recently

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demonstrated that nasal polyps can be classified on the basis of cytokine expression patterns. This

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classification includes nasal polyp with strong Th2 profile (IL-5+ only) or nasal polyp with mixed

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Th profile (IL-5+IFNγ+ or IL-5+IL-17+) (Bachert et al., 2010). These subgroups were retrospectively

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found in our patient population, with the exception of the IL-5+IL-17+ profile, which is generally

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very rare (data not shown). Furthermore, we observed that the number of pDCs was significantly

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decreased in IL-5+ nasal polyps compared to IL-5+/IFNγ+ nasal polyps. Moreover, the number of

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pDCs correlated positively with IFNγ. However, the percentage of mDCs remained similar in both

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nasal polyp groups. IFN is known to be able to modulate DC, affects receptor molecules important

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in antigen presentation, and plays a role in the production of IL-12 (a Th1 cytokine) (Billiau and

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Matthys, 2009).

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There are different hypotheses about the role of each DC subset in the activation of T cells:

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activated mDCs are able to activate Th1 (IL-12) or Th2 cells (TSLP, IL-4) (Rissoan et al. 1999;-

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Macatonia et al., 1995; Soumelis et al., 2002), whereas activated pDCs can induce T regulatory

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cells via the production of IFNα or Th2 cells via IL-3. While the latter hypothesis supports a pro-

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inflammatory role of pDCs, other data suggest that pDCs act as anti-inflammatory cells in an

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allergic airway inflammation model (de Heer et al, 2004; Kool et al., 2009). This confirms the idea

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that DCs are not doomed to a fixed profile and may play a role in complex cross-talking that

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involves countless regulatory and counter-regulatory mechanisms. This, in turn, will allow DCs to

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interact with different T cells, regardless of the subset. Furthermore, stimulated pDCs are able to 12 Page 12 of 29

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cross-activate immature mDCs (Cantisani et al., 2011), and Th2 cells can attract monocytes which

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can differentiate into inflammatory dendritic cells, also known as Th2-promoting DCs (Alonso et

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al., 2011).

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In our study, the local presence of IFNγ (a Th1 cytokine) correlated positively with pDCs; in the

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severe cases where IFNγ expression is lost, however, pDC could no longer dampen the severe

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inflammation. Our findings are in line with those reported by Kool et al., 2009, who showed that

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massive recruitment of pDCs after allergen challenge was accompanied by a suppression of

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eosinophilic airway inflammation. Importantly, these authors also noticed that such pDC

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mobilization was needed to attenuate the inflammatory reaction, as removal of these cells restored

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airway eosinophilia and Th2 cytokine levels.

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The impact of the cytokine milieu and of the mucosal environment on DCs is now the object of

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research. In a Th1 milieu, pDCs are more able to stimulate T cells than in a Th2 milieu (Bratke et

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al., 2011). Not only T-cell cytokines, but also mucosal immunoglobulins such as IgA and IgE play

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an important role in the maintenance of CRSwNP. IgE could directly influence T-cell

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differentiation by means of alterations in APC-cytokine production (for instance, by IL-12

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suppression). Human DCs express the high-affinity IgE receptor FcεRI, but also Fcγ receptors, such

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as FcγRIII. Blink and Fu, 2010, demonstrated that IgE has regulatory effects on DCs through

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FcγRIII, which could be especially important in the development and maintenance of allergic

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disease or asthma. Patients with allergic asthma or atopy exhibit increased expression of FcεRI on

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pDCs, which downregulates the innate receptor TLR9 in these cells (Schroeder et al., 2005). In

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CRSwNP, a high local IgE production is detected independent of atopic status (Zhang et al., 2011),

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which could result in blockade of the innate signaling pathway via TLR9 and low production of

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IFN-α. More recently, it was demonstrated that pDCs from asthmatic patients were dysfunctional

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and secreted less innate IFNs in response to rhinovirus exposure, thus contributing to asthma

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exacerbations (Pritchard et al., 2012). In our study we found pDC decreased in the more severe

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groups (NP + asthma, or aspirin sensitive group) where it is found a high concentration of IgE

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drived by Th2 cells and we found also low production of IFN, creating a parallel with what is

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found in the lower airway mucosa of asthmatic patients, suggesting a dysfunctional

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immunosuppressive role of pDCs in a Th2-drived disease in the upper and lower airway.

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Finally, indoleamine 2,3-dioxygenase (IDO), a key enzyme that catalyzes the initial and rate-

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limiting step in the degradation of tryptophan, is widely expressed in DCs, macrophages,

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eosinophils, fibroblasts and endothelial cells (Ball et al., 2007). IDO is weakly expressed under

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homeostatic conditions, but is found at increased levels during chronic inflammatory diseases

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induced by inflammatory mediators such as IFNs and IL-6 (Huang et al. 2010). In this study,

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concentrations of IDO were increased in the CRSwNP group as compared with the control group,

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and correlated inversely with the number of pDCs; this confirms recent findings reported by

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Honkanen et al. [9]. Moreover, it has been suggested that IDO plays an important role in Th1/Th2

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regulation, namely by suppressing Th1 and promoting Th2 responses (Xu et al., 2008a; Xu et al.,

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2008b). IFN- induces an IDO-mediated immunosuppressive function in pDCs, which is attributed

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to degradation of tryptophan, and suppresses T cell responses by this mechanism (Chen, 2011).

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However, it is still unclear whether IDO has additional and distinct functions for regulating the

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phenotype and function of pDCs. IDO-expressing DCs are also implicated in promoting Th2 bias in

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an attempt to activate local suppression. In this sample we measured IDO from tissue homogenates

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and IDO activity could be attributed to many different inflammatory cells, but the results found is

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still interesting once DCs are the main cells to express IDO. We found the highest levels of IDO,

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the lowest number of pDCs, and low levels of IFN- in the most severe cases (asthmatic and

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aspirin-sensitive patients), this fact could be partially explained by the fact that asthmatic and

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aspirin-sensitive patients have a severe Th2-drived inflammatory process, consequently lower IFN

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production, lower immunosuppressive function by pDCs, increase of Th2 response creating a

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positive feedback. Further research is required to elucidate the roles of the microenvironment and of

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IDO in nasal polyposis.

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In conclusion, this study demonstrated an increased number of both mDCs (absolute percentage)

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and pDCs (absolute and relative percentage) in nasal polyp tissue as compared with healthy, non-

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inflamed nasal mucosa. Interestingly, pDCs, but not mDCs, were downregulated in patients with a

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more severe Th2 profile, suggesting an important role of the cytokine and pro-inflammatory milieu

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in the functional response of this DC subtype in upper airway disease.

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ACKNOWLEDGEMENTS

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This work was supported by grants to Claus Bachert from the Flemish Scientific Research Board

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(FWO Nr. A12/5-HB-KH3 and G.0436.04), from the Global Allergy and Asthma European

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Network (GA²LEN), and from the Interuniversity Attraction Poles programme (IUAP), Belgian

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State - Belgian Science Policy P6/35; by grants to Rogério Pezato from the European Union

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(Erasmus 17) and from the São Paulo State Military Police Health Department, São Paulo, Brazil;

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by a grant to Claudina Pérez-Novo from the Flemish Research Board, FWO Post-doctoral mandate

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(Nr. FWO08-PDO-117); and by a grant to Lara Derycke from the Ghent University Faculty of

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Medicine. We thank Melissa Dullaers for the fruitful discussions.

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36. Yukitatsu, Y., Hata, M., Yamanegi, K., et al., 2013. Decreased expression of VE-cadherin

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FIGURE LEGENDS

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Figure 1. Representative photomicrographs (original magnification 200×) of immunostaining for

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BDCA1 and BDCA2 in inferior turbinate (control) nasal tissue (a,b) and nasal polyp tissue (c,d)

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respectively.

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Figure 2. Flow cytometry analysis of the different DC subsets in nasal tissue: a) gating strategy

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used to identify DCs (Lin1-HLADR+) from CD45+ fraction, mDC1 (BDCA1+), mDC2 (BDCA3+)

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and pDCs (BDCA2+ or CD123+); b-e) quantification of data expressed in box-whisker plots.

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Significance (p) values after Mann–Whitney U represented as follows: * p< 0.05, ** p < 0.01 and

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*** p < 0.005.

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Figure 3. Comparison of relative amount of DC subsets using the percentage of DC subsets

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taking into account the total number of CD45+ cells (number of DC cells, respecting the

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subsets/number of total cells positive for CD45): a) Percentage of CD45+ cells, number of CD45-

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positive cells/number of living cells, b) relative amount of pDCs, c) relative amount of mDCs.

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Significance (p) values after Mann–Whitney U represented as follows: * p< 0.05, ** p < 0.01.

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Figure 4. The balance of DC subsets differs depending on the presence of comorbidities.

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Number of pDCs (a) and mDCs (b) as analyzed by flow cytometry in nasal polyp tissue from

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asthmatic (n=9) and non-asthmatic (n=8) patients. The filled dots represent aspirin-intolerant

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subjects (n=3). Box-whisker plots represent the ratio of mDCs to pDCs in control and nasal polyp

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tissue (c). Significance (p) values after Mann–Whitney U represented as follows: * p< 0.05, ** p <

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0.01 and *** p < 0.005.

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Figure 5. Balance of DC subsets in different tissue inflammatory milieu

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Post-hoc analysis data showing the number of pDCs (a) and mDCs (b) in IL5+ and/or IL5+/IFNγ+

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CRSwNP patients. Significance (p) values after Mann–Whitney U represented as follows: * p<

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0.05, ** p < 0.01 and *** p < 0.005.

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Figure 6. IDO levels in CRSwNP are increased as compared with control nasal mucosa.

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Concentration of IDO in tissue homogenates from (a) CRSwNP patients and healthy (control)

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subjects; (b) non-asthmatic and asthmatic patients; and (c) IL5+ and/or IL5+/IFN+ CRSwNP

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patients. Significance (p) values after Mann–Whitney U represented as follows: * p< 0.05, ** p <

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0.01 and *** p < 0.005. IU, international units.

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Table 1. Concentrations of cytokines and mediators in nasal tissue homogenates (median and IQR) Mediators

Mann-Whitney test

CRSwNP

286 (119–804)

6,315 (2,821–12,833)

p < 0.01

Total IgE (kU/L)

11.7 (5.3–37.7)

244 (106–682)

p < 0.01

SE-IgE (kUA/L)

BDL

2.6 (1.8–3.5)

IFNγ (pg/mL)

BDL

42.9 (42.9–93.9)

NS

TGF-β1 (pg/mL)

15,070 (12,674–22,534)

10,944 (6,302–17,465)

NS

IL-5 (pg/mL)

BDL

190 (30–752)

p < 0.01

IL-17 (pg/mL)

BDL

IL-6 (pg/mL)

280 (146–888)

IL-10 (pg/mL)

11.02 (11–15.2)

IFNα (pg/mL)

75.2 (75.2–109.8)

IL-12 (pg/mL)

519 (275–843)

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ECP (µg/L)

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p = 0.01

NS

129 (74–453)

NS

12.5 (11–28.1)

NS

75.2 (75.2–258.3)

NS

379 (215–535)

NS

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CRSwNP, chronic rhinosinusitis with nasal polyposis. BDL, below detection limit. NS, not statistically significant.

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Table 2. Semiquantitative median score* for the number of DCs from each subset in healthy

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(controls) and inflamed (CRSwNP) nasal mucosa.

pDC (BDCA2+)

CRSwNP

Mann-Whitney test

0.75 (IQR 0.5-1.25)

2 (IQR 1.0-3.0)

p = 0.01

0 (IQR 0-0.5)

1.5 (IQR 0.37-3.0)

p = 0.02

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mDC1 (BDCA1+)

Control

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*Median values calculated after a score of 0-3 was assigned to each slide (0 = no positive cells,

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1= 0-20 positive cells, 2 = 20-100 positive cells, 3 = more than 100 positive cells)

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Figure 5

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Figure 6

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