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Original article
Tissue-infiltrating neutrophils represent the main source of IL-23 in the colon of patients with IBD
  1. Egle Kvedaraite1,2,
  2. Magda Lourda1,2,
  3. Maja Ideström3,
  4. Puran Chen1,
  5. Selma Olsson-Åkefeldt2,
  6. Marianne Forkel1,
  7. Désirée Gavhed2,
  8. Ulrik Lindforss4,
  9. Jenny Mjösberg1,
  10. Jan-Inge Henter2,
  11. Mattias Svensson1
  1. 1Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
  2. 2Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
  3. 3Paediatric Gastroenterology, Hepatology and Nutrition Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
  4. 4Department of Clinical Science, Gastromedical Center, Intervention and Technology (CLINTEC), Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
  1. Correspondence to Dr Mattias Svensson, Department of Medicine, Center for Infectious Medicine, F59, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden; mattias.svensson{at}ki.se

Abstract

Objective In IBD, interleukin-23 (IL-23) and its receptor (IL-23R) are implicated in disease initiation and progression. Novel insight into which cells produce IL-23 at the site of inflammation at an early stage of IBD will promote the development of new tools for diagnosis, treatment and patient monitoring. We examined the cellular source of IL-23 in colon tissue of untreated newly diagnosed paediatric patients with IBD.

Design Colon tissues from IBD and non-IBD patients were analysed by quantitative real-time PCR (qPCR), immunofluorescence confocal microscopy and flow cytometry after appropriate sample preparation. Blood samples from IBD and non-IBD patients and healthy controls were analysed using flow cytometry and qPCR.

Results We discovered that tissue-infiltrating neutrophils were the main source of IL-23 in the colon of paediatric patients with IBD, while IL-23+ human leucocyte antigen-DR+ or IL-23+CD14+ cells were scarce or non-detectable, respectively. The colonic IL-23+ neutrophils expressed C-X-C motif (CXC)R1 and CXCR2, receptors for the CXC ligand 8 (CXCL8) chemokine family, and a corresponding CXCR1+CXCR2+IL-23+subpopulation of neutrophils was also identified in the blood of both patients with IBD and healthy individuals. However, CXCL8-family chemokines were only elevated in colon tissue from patients with IBD.

Conclusions This study provides the first evidence of CXCR1+CXCR2+IL-23-producing neutrophils that infiltrate and accumulate in inflamed colon tissue of patients with IBD. Thus, this novel source of IL-23 may play a key role in disease progression and will be important to take into consideration in the development of future strategies to monitor, treat and prevent IBD.

  • GUT INFLAMMATION
  • GUT IMMUNOLOGY
  • IBD BASIC RESEARCH
  • IMAGE ANALYSIS
  • INFLAMMATORY CELLS
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Significance of this study

What is already known on this subject?

  • There are unmet needs for diagnosis, treatment and monitoring of IBD.

  • Interleukin-23 (IL-23) and its receptor are implicated in disease progression.

  • IBD is associated with intestinal inflammation-mediated recruitment of neutrophils.

  • Several neutrophil-associated proteins are detected in patients with IBD and commonly used as biomarkers of IBD.

What are the new findings?

  • Identification of IL-23+ neutrophils.

  • Accumulation of IL-23+ neutrophils in colon tissue biopsies from patients with IBD.

  • IL-23-producing neutrophils are positive for the C-X-C motif ligand 8 (CXCL8) chemokine family receptors, CXCR1 and CXCR2.

  • Higher numbers of extra-epithelial CXCL8-producing cells in IBD colon tissue.

How might it impact on clinical practice in the foreseeable future?

  • Examining colon tissue from untreated newly diagnosed paediatric patients, we point out new potential targets to monitor and treat IBD at an early stage of disease.

Introduction

IBD, including Crohn's disease (CD) and UC, are chronic immune-mediated disorders of the GI tract. Although the cause(s) of IBD is not known, both environmental and genetic cues have been suggested to play a role in disease initiation and progression, and to aggravate tissue pathology.1 Initiation and maintenance of the mucosal inflammatory process have been linked to abnormalities of innate immunity in association with defects in epithelial cell barrier functions.2

Interleukin (IL)-23 and its receptor (IL-23R) promote the development and maintenance of tissue inflammation that is associated with the induction of a pathogenic T-helper 17 (Th17) population.3 The IL-23-Th17 pathway has been implicated in chronic disorders ranging from psoriasis and rheumatoid arthritis to IBD,4 and pharmacological approaches interfering with IL-23 and IL-17A are in clinical use.5 For example, anti-IL23 antibody therapy has proven useful in managing refractory,6 and moderate to severe CD,7 as well as in the treatment of anti-tumour necrosis factor (TNF) antibody-induced psoriasiform skin lesions in patients with IBD.8 In addition to Th17 cells, many innate immune cells such as natural killer T, gamma-delta (γδ)T and retinoic-acid receptor-related orphan receptor (ROR) γt innate lymphoid cells respond to IL-23 and contribute to resistance to infection as well as inflammation. IL-23-induced local tissue inflammation is mainly mediated by type 17 signature cytokines such as IL-17A, IL-22 and colony-stimulating-factor-2.3 The importance of IL-23 in IBD pathogenesis has been well established in various animal models,9–12 and IL-23 has been implicated as essential for manifestation of chronic intestinal inflammation, likely via the activation of memory T cells, which are stimulated to produce pro-inflammatory IL-17A and IL-6.13 In addition to the human IL-23R,14 evidence from genome-wide association studies (GWAS) has indicated several IBD susceptibility loci that contain genes that encode proteins involved in cytokine and chemokine signalling, for example, signal transducer and activator of transcription (STAT)1, STAT3, STAT4, chemokine C-C motif (CC)R6, IL-12R, janus kinase (JAK)2, as well as genes that encode cytokines, for example, IL-2, IL-21, interferon (IFN)-γ, IL-10 and IL-27.15–17 Furthermore, IL23 mRNA and IL-17A protein are found at higher levels in the inflamed intestinal tissue of adult patients with IBD compared with controls.18 Despite reports suggesting that macrophages and dendritic cells (DCs) are the predominant source of IL-23 in adults with IBD,18–20 the cellular source of IL-23 in patients with IBD at an early stage of the disease and at the site of inflammation remains ill defined.

Neutrophils, polynuclear myeloid cells that make-up the largest proportion of blood leucocytes, are recruited to inflamed tissue to exert antimicrobial activities. A key component in neutrophil recruitment is the C-X-C motif ligand 8 (CXCL8) chemokine family, which acts on the CXCR1 and CXCR2 receptors, and in addition to CXCL8 includes the chemokines CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 and CXCL7.21 At the local site of inflammation, neutrophils are a source of pro-inflammatory cytokines, recently shown to include the production of IL-17A in response to IL-23 and IL-6.22 Moreover, neutrophils likely play an important role in the pathogenesis of IBD,23 and neutrophil-associated proteins, such as calprotectin and lactoferrin, are detected in patients with IBD and commonly used as biomarkers of IBD, both for diagnosis and monitoring purposes.24 Whether neutrophils also contribute to IL-23-mediated inflammation in IBD by being a source of IL-23 has not yet been reported.

Here, we used tissue biopsies collected from untreated paediatric patients undergoing colonoscopy for suspected IBD to investigate the cellular source of IL-23 at the local site of inflammation and at an early stage of disease. We show that tissue-infiltrating neutrophils expressing CXCR1 and CXCR2, receptors for the CXCL8 chemokine family,21 represent the main source of IL-23 in the colon of patients with IBD. Moreover, circulating IL-23-producing CXCR1+CXCR2+ neutrophils were also identified in blood samples collected from patients with IBD as well as from non-IBD patients and adult healthy donors.

Patients and methods

Patients and samples

Paediatric patients were recruited at the Astrid Lindgren Children's Hospital, Stockholm, Sweden, after signed informed consents were obtained. Newly diagnosed and untreated patients that had no other inflammatory or infectious disease were included. Post colonoscopies, 16 of the 26 patients included were confirmed patients with IBD and 10 were confirmed non-IBD patients. The non-IBD individuals were considered as controls. The demographic data, together with the physician global assessment (PGA), are provided in table 1. The PGA was used to classify disease activity, based on medical history, physical examination, laboratory findings and endoscopic examination.25

Table 1

Demographic data and disease activity of paediatric patients

Upon diagnostic colonoscopy, intestinal biopsies were collected for research purposes from five areas (biopsy I from ileum, II from caecum, IV from flexura coli dextra, VI from flexura coli sinistra and IX from upper colon sigmoideum). The biopsies were snap frozen in liquid nitrogen or enzymatically digested. Frozen biopsies were stored at −80°C until cryosectioning for immunohistochemistry, immunofluorescence or quantitative real-time PCR (q-PCR) analyses. For the analyses of frozen tissue, we focused on samples from caecum and flexura coli sinistra (II and VI). Enzymatically digested tissue biopsies from caecum (II), flexura coli dextra (IV), flexura coli sinistra (VI) and upper colon sigmoideum (IX) were pooled and then processed for surface labelling and flow cytometry analysis.

Colon tissue was also collected from adult patients with IBD with chronic disease undergoing resection surgery at the Karolinska University Hospital after signing informed consents. Blood samples from healthy volunteers were received from the Department of Clinical Immunology and Transfusion Medicine at the Karolinska University Hospital.

Antibodies and reagents

A detailed description of the antibodies and reagents used in this study is provided in online supplementary file—text 1.

Tissue immunostaining, microscopy and image analyses

Eight-micrometre tissue sections were immunostained with specific antibodies and isotype matched control antibodies followed by microscopic image analyses as described in online supplementary file—text 1.

Isolation of neutrophils, peripheral blood mononuclear cells and monocytes

Whole blood was collected in blood collection tubes containing sodium heparin or EDTA. Neutrophils were isolated from HetaSep (Stemcell Technologies, Vancouver, Canada) treated blood using the EasySep Human Neutrophil Enrichment Kit (Stemcell Technologies) according to the manufacturer's instructions. Peripheral blood mononuclear cells (PBMCs) were isolated after separation of the blood components with Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) and the monocytes were negatively selected using the EasySep Human Monocyte Enrichment Kit (Stemcell Technologies) according to the manufacturer's protocol.

Isolation of single cells from colon biopsies

A minimum of two tissue biopsies per patient were enzymatically digested in cell culture medium (see ‘Antibodies and reagents’, supplementary file—text 1) using a magnetic stirrer at 37°C for 40 min. Cell suspension was then filtered through a 40 μm cell strainer. Isolated cells were routinely >90% viable, as determined by flow cytometry.

Quantitative PCR

Total mRNA was extracted and purified from tissue and blood samples as described in online supplementary file—text 1.

Flow cytometry

In total, 200 μL of whole blood or isolated colon tissue cells were stained for flow cytometry analysis as previously described26 and in online supplementary file—text 1.

Statistical analysis

The data were analysed with the GraphPad Prism V.6 software (GraphPad, San Diego, USA). Statistically significant differences were determined using one-way analysis of variance applying Kruskal–Wallis test with Dunn's multiple comparison tests or the Mann–Whitney test. Pearson's correlation test was used for the correlation analysis. Differences between groups were considered to be statistically significant at p<0.05. Data are presented as mean±SD or as whisker plots.

Results

Neutrophils in colon tissue of patients with IBD are positive for IL-23

To increase our knowledge on IL-23-promoting inflammation at an early stage of IBD, clinical data, blood and tissue biopsies were collected from untreated paediatric patients undergoing colonoscopy for suspected IBD. qPCR analysis of IL-23p19 mRNA in tissue biopsies showed higher relative expression of IL-23 mRNA in IBD versus non-IBD patients (figure 1A). Further analysis revealed the highest IL-23 mRNA expression levels in patients with severe IBD (figure 1A), whereas no differences were detected between patients with CD and UC (data not shown). Next, immunohistochemical analyses of IBD colon biopsy sections revealed IL-23-positive staining in cells with polynuclear morphology, indicative of granulocytes (figure 1B). To identity the cellular source of IL-23 in IBD colon tissue, whole biopsy sections from paediatric patients were analysed using a four-colour immunofluorescence staining protocol, including staining for IL-23, human leucocyte antigen (HLA)-DR, neutrophil-elastase and the nuclear stain 4′,6-diamidino-2-phenylindole, followed by confocal microscopy analysis. Maximum intensity projection of the images showed that neutrophil-elastase+ cells were IL-23+, whereas HLA-DR+ cells were essentially IL-23 (figure 1C, D). Moreover, 3D computerised segmentation projections of lamina propria indicated that the IL-23 signal was located predominantly inside neutrophils (figure 1E). The 3D reconstructed images also demonstrated that the brightest IL-23 staining was found in neutrophil-elastase+ cells rather than in HLA-DR+ cells (see online supplementary file—video 1). Minimal background staining was detected using an isotype matched control antibody (figure 1F). Notably, the presence of neutrophil-elastase+IL-23+ cells and the absence of HLA-DR+IL-23+ cells were also evident in resected colon tissue from adult patients with IBD (figure 1G).

Figure 1

Interleukin-23 (IL-23) mRNA expression and immunofluorescence analyses of colon tissue from patients with IBD. (A) IL-23 mRNA quantification in colon tissue biopsies from paediatric patients with IBD (n=16) and non-IBD paediatric patients (n=10), comparing IBD versus non-IBD patients (left) and the IL-23 mRNA expression levels in relation to disease activity (right). (B) Immunohistochemical analysis for IL-23 (brown) detection in IBD colon biopsies. White arrowheads indicate polymorphonuclear cells. (C) A representative mean intensity immunofluorescence projection of a confocal image of the whole biopsy section (left) with higher magnification in Q1 (right, 4′,6-diamidino-2-phenylindole (DAPI) excluded). White dashed lines indicate epithelial linings; Q2 and Q3 define areas with neutrophil-elastase+ and human leucocyte antigen (HLA)-DR+ cells presented in (C) and (D), respectively. (D) Higher magnification of the Q2 region selected in (C). (E) Higher magnification of the Q3 region selected in C (left, DAPI excluded) and a 3D projection in Q4 (right, DAPI and HLA-DR excluded). (F) A 3D projection showing elastase in blue and an isotype matched control antibody for IL-23 in green (DAPI and HLA-DR excluded). (G) A mean intensity projection of a confocal image of resected colon tissue from an adult patient with IBD, higher magnification in Q5. The bottom and top of the boxes in A indicates the 25th and 75th percentile, the horizontal line indicates the median value and the error bars indicate the lower and higher values. The statistically significant differences were determined using Mann–Whitney test (IBD vs non-IBD) or one-way analysis of variance applying Kruskal–Wallis with Dunn's multiple comparison tests (comparing IBD3/IBD2/IBD1/non-IBD). *p<0.05, **p<0.01.

The maximum intensity projections of the whole tissue section images of colon tissue from IBD (figure 2A; upper panels) and non-IBD (figure 2A; lower panels) revealed significantly more neutrophils per square millimetre in IBD compared with non-IBD patient tissues (p=0.0013, figure 2B). Almost all (>95%) neutrophils found in both IBD and non-IBD patients’ colon tissue sections were positive for IL-23 (figure 2A Q1 and Q2 and data not shown). Although more IL-23-positive neutrophils per square millimetre were found in patients with IBD compared with the non-IBD patients (p=0.0013, figure 2C), the mean fluorescent intensity (MFI) per neutrophil was comparable between the two groups (data not shown). To gain further knowledge on neutrophil infiltration and IL-23-producing capacities in IBD colon tissue compared with non-IBD colon tissue, single cells were isolated from tissue biopsies within 1 h after collection and analysed with flow cytometry. The identification of neutrophils was based on a combination of the markers CD15, CD16 and CD66b. Confirming our results obtained from tissue sections using confocal microscopy, the flow cytometry analyses demonstrated that colon tissue from paediatric patients with IBD contained infiltrating neutrophils (CD14CD16+CD15+CD66b+) positive for IL-23, CXCR1 and CXCR2 (figure 2D), whereas CD14+ cells were IL-23 (figure 2D). The neutrophils and monocytes comprised approximately one-third each of the total cells in the R2 gate, and most of the neutrophils were IL-23+ (figure 2D). In addition to viable neutrophils and monocytes, the R2 gate also contained viable CD14CD16 cells (figure 2D), half of which were HLA-DR+ (data not shown), most likely representing tissue macrophages and/or DCs. Although 40–50% of the CD14CD16HLADR+ cells were IL-23+, their relative IL-23 MFI was approximately fivefold lower compared with that of neutrophils, which is in line with the confocal microscopy analysis (see online supplementary file—video 1). Taken together, neutrophils with a relatively high IL-23 MFI accounted for >85% of the IL-23+ cells in colon tissue biopsies from all patients with IBD analysed. Additionally, neutrophils were largely absent from non-IBD colon tissue (figure 2E).

Figure 2

Levels of interleukin-23 (IL-23)-producing neutrophils in colon biopsies of paediatric IBD compared with non-IBD patients. (A) Representative mean intensity projections of confocal images of the whole tissue sections from IBD (upper row) and non-IBD (lower row) patients are shown. The dashed line indicates biopsy section area. Q1 and Q2 are representative areas of images presented without nuclear stain (middle) demonstrating elastase (blue) and IL-23 (green) staining at a higher magnification (right). (B and C) The mean (±SD) values of numbers of neutrophil-elastase+ cells (B) and neutrophil-elastase+IL-23+ cells (C) in the whole biopsy sections analysed (IBD, n=10; and non-IBD, n=5). (D and E) Representative flow cytometry analyses of cells isolated from colon biopsies of IBD (D) and non-IBD patients (E). Populations of cells were identified as positive relative to the isotype control for each sample. In (D), black areas denote antibody-stained cells and grey areas isotype-stained cells. The statistically significant differences were determined using Mann–Whitney test. *p<0.05, **p<0.01.

Colon tissue from patients with IBD has increased levels of CXCR1- and CXCR2-binding chemokines that belong to the CXCL8 family

Chemokines and their receptors, such as CXCR1 and CXCR2,27 ,28 play an important role in attracting neutrophils into tissue upon inflammation. Since an accumulation of IL-23+ neutrophils expressing CXCR1 and CXCR2 was observed in IBD colon tissue (figure 2D), we investigated the relative expression levels of CXCR1 and CXCR2 mRNA in IBD versus non-IBD patients. The analysis showed that both the CXCR1 and CXCR2 relative mRNA expression was higher in patients with IBD, particularly in patients with severe IBD (figure 3A, B). CD or UC had similarly high CXCR1 and CXCR2 mRNA expression levels (data not shown). We further hypothesised that the ligands for these receptors, namely CXCL8 and other CXCL8-familly chemokines,21 might mediate this attraction. As illustrated in figure 3C, higher expression of CXCL8 mRNA was detected in the colon tissue of paediatric patients with IBD compared with non-IBD patients (p<0.01). This was not due to an altered epithelium composition, as the relative level of E-cadherin, which identifies adherence junctions in the gut epithelium, was similar between IBD and non-IBD patients (figure 3D). Furthermore, the CXCL8 mRNA level positively correlated with both CXCR1 and CXCR2 mRNA levels in colon tissue, with the strongest correlation between CXCL8 and CXCR2 mRNA levels (figure 3E). mRNA expression analyses of additional CXCL8-family chemokines that also bind CXCR1 and/or CXCR2 revealed upregulated expression of CXCL2, CXCL3 and CXCL6, but unaltered expression of CXCL5 or CXCL7 (data not shown), indicating that the IBD intestinal environment has a strong neutrophil attractant capability.

Figure 3

Analyses of the C-X-C motif ligand 8 (CXCL8)-CXCR1/CXCR2 chemotactic axis and the inflammatory environment in colon tissue from patients with IBD. (A–D) CXCR1 (A), CXCR2 (B), CXCL8 (C) and E-cadherin (D) mRNA quantification in colon tissue biopsies from paediatric patients with IBD (n=16) and non-IBD patients (n=10), comparing IBD versus non-IBD patients (left) and mRNA expression levels in relation to disease activity (right). The bottom and top of the boxes indicate the 25th and 75th percentile, the horizontal line indicates the median value and the error bars indicate the lower and higher value in (A–D). (E) Correlation graphs of mRNA expression levels of CXCR1 versus CXCL8 (left), and CXCR2 versus CXCL8 (right). (F) Representative mean intensity projections of confocal images (CXCL8 and E-cadherin) of colon biopsy sections from IBD (left) and non-IBD (right) patients (nuclear stain excluded). White arrowheads point to CXCL8+E-cadherin cells in colon lamina propria. (G and H) Mean (±SD) CXCL8 signal intensity inside the E-cadherin+ area (G) and mean values (±SD) of numbers of CXCL8+ cells outside the E-cadherin+ area (H). (I) Representative mean intensity projections of confocal images (CXCL8, neutrophil-elastase and human leucocyte antigen (HLA)-DR) of colon biopsy sections from one IBD (left) and one non-IBD patient (right). White arrowhead points to a CXCL8+ cell close to a neutrophil. Error bars indicate SD in (G) and (H). The statistically significant differences were determined using Mann–Whitney test (IBD vs non-IBD) and one-way analysis of variance applying Kruskal–Wallis with Dunn's multiple comparison test (comparing IBD3/IBD2/IBD1/non-IBD). *p<0.05, **p<0.01. The Pearson's correlation test was used to determine the correlation coefficient rp.

CXCL8, in particular, is a potent and well-established human neutrophil chemoattractant expressed by epithelial cells in the gut.29 We therefore investigated the spatial distribution of CXCL8 in the IBD colon tissue. As expected, a spatial overlap was observed between CXCL8 and E-cadherin in both IBD and non-IBD colon tissue sections (figure 3F). The results revealed that the mean MFI of CXCL8 in E-cadherin+ areas of the tissue section was comparable between the IBD and non-IBD patients (figure 3G). When maximum intensity projections of the images were examined in more detail, increased numbers of extra-epithelial E-cadherin cells positive for CXCL8 were observed in the lamina propria of patients with IBD compared with non-IBD patients (figure 3H). Furthermore, we observed neutrophil-elastase+ neutrophils closely interacting with extra-epithelial CXCL8+ cells in the IBD (figure 3I) but not in the non-IBD colon tissue (figure 3H).

A subpopulation of IL-23+CXCR1+CXCR2+ neutrophils exists in blood of both patients with IBD and healthy controls

We next turned our attention to the blood to investigate the existence of circulating IL-23+ neutrophils. Flow cytometry analyses of whole blood from patients with IBD revealed the existence of a subpopulation of neutrophils (CD15+CD66bbright) that expressed IL-23, CXCR1 and CXCR2 (figure 4A), corresponding to the IL-23+ neutrophils in IBD tissue. In contrast, CD15+CD66bdim neutrophils expressed only TNF and CXCR1 and CD15CD66b myeloid cells expressed TNF alone (figure 4A). Using an anti-human neutrophil-elastase antibody in the flow cytometry analyses, we verified that both the CD15+CD66bbright and CD15+CD66bdim populations were positive for neutrophil-elastase (figure 4B). IL-23-producing CXCR1+CXCR2+ neutrophils were also identified in blood samples collected from non-IBD patients and adult healthy donors at similar frequencies (data not shown), indicating that circulating IL-23+ neutrophils are not uniquely present in patients with IBD. Isolation of blood neutrophils (figure 4C, D) and PBMCs (figure 4E) from healthy donors confirmed the presence of IL-23+ cells predominantly among neutrophils. While comparative qPCR analyses revealed similar levels of IL-23 mRNA in neutrophils and monocytes, relatively higher levels of CXCR1 and CXCR2 were observed in neutrophils as compared to monocytes (figure 4F). Furthermore, similar expression levels of CXCR1 and CXCR2 mRNA were detected in peripheral blood neutrophils from IBD, non-IBD and healthy controls (data not shown). These results, thus, show that circulating IL-23+ neutrophils are conditioned to respond to the CXCL8-family cytokines produced in IBD colon tissue.

Figure 4

Interleukin-23 (IL-23) production by blood neutrophils. (A and B) Representative flow cytometry analyses of whole blood from patients with IBD are shown. In (A), the expression of IL-23, tumour necrosis factor (TNF), C-X-C motif (CXC)R1 and CXCR2 in the CD15+CD66bbright (R3), CD15+CD66bdim (R4) and CD15CD66b (R5) populations are shown. Black areas denote antibody-stained cells, and grey areas denote isotype-stained cells. In (B), panels to the left illustrate the expression of neutrophil-elastase in the CD15+CD66bbright cells in the R3 gate defined in (A), and panels to the right illustrate the expression and of neutrophil-elastase in the CD15+CD66bdim cells defined by the R4 gate in (A). Populations of cells were identified as neutrophil-elastase positive relative to the isotype control as indicated (quadrants). (C–E) Representative light microscopy image of control individual's isolated neutrophils stained with H&E (C, black arrowhead points towards a possible eosinophil), and representative immunofluorescence images of healthy adults’ isolated neutrophils (D) and peripheral blood mononuclear cells (PBMCs) (E) evaluated for IL-23-producing capacity. (F) IL-23, CXCR1 and CXCR2 mRNA quantification in blood neutrophils and monocytes isolated from control individuals (n=4). The bottom and top of the boxes in (F) indicate the 25th and 75th percentile, the horizontal line indicates the median value and the error bars indicate the lower and higher value in the population. The statistically significant differences were determined using Mann–Whitney test. *p<0.05.

Activation of the STAT3 signalling pathway in IBD colon tissue

As studies have shown that IL-23 contributes to pathogenic T cell responses, including enhanced IL-17A production, we searched for support of actual IL-23R signalling in situ, likely caused by IL-23 release from neutrophils. For this purpose, we immunostained and analysed the IBD colon tissue sections for pSTAT3, which is a component of the signalling pathway downstream of the IL-23R.30 This analysis identified pSTAT3+ cells situated in close association with neutrophils (figure 5A). Immunostainings of IBD colon tissue also revealed the presence of CD3+ as well as CD3 cells that were positive for pSTAT3 (figure 5B). In further support of IL-23-mediated activation and possible T cell stimulation in tissue, qPCR analyses of IL-17A, a cytokine that is regulated by the STAT3 signalling pathway,31 revealed elevated IL-17A mRNA expression levels in colon tissue of paediatric patients with IBD compared with non-IBD patients, with the highest levels detected in the most severely sick patients with IBD (figure 5C).

Figure 5

Activation of signal transducer and activator of transcription 3 (STAT3) in colon tissue from children with IBD. (A) Whole tissue sections from IBD colon biopsies were stained with 4′,6-diamidino-2-phenylindole (DAPI) and antibodies against neutrophil-elastase and phosphorylated STAT3 (pSTAT3). Boxed area in the image (left) shows staining of neutrophil-elastase and pSTAT3 presented at a higher magnification (right). (B) Whole tissue sections from IBD colon were stained with DAPI and antibodies against CD3 and pSTAT3. White arrows indicate CD3+pSTAT3+ cells and white arrowheads indicate CD3pSTAT3+ cells. (C) Relative mRNA levels of interleukin (IL)-17A in colon tissue biopsies from IBD (n=16) and non-IBD (n=10) patients. The bottom and top of the boxes in (C) indicate the 25th and 75th percentile, the horizontal line indicates the median value and the error bars indicate the lower and higher value in the population. The statistically significant differences were determined using Mann–Whitney test (IBD vs non-IBD) or one-way analysis of variance applying Kruskal–Wallis with Dunn's multiple comparison tests (comparing IBD3/IBD2/IBD1/non-IBD). **p<0.01, ***p<0.001.

Discussion

Studying colon tissue from untreated newly diagnosed paediatric patients with IBD represents a unique opportunity to monitor disease mechanisms at an early stage, with low risk of introducing bias from treatments and concurrent diseases, which is often the case in adult IBD studies. Notably, our studies on colon tissue from newly diagnosed untreated paediatric patients showed that IL-23 is predominantly produced by neutrophils and not by HLA-DR+ or CD14+ cells, previously described to be the main source of IL-23 in IBD.18–20 To our knowledge, this is the first time that neutrophils are described as IL-23-producing cells.

The increased numbers of neutrophil-elastase+ cells in the lamina propria of colon tissue from untreated newly diagnosed paediatric patients with IBD is in agreement with the main features of both UC and CD, including robust inflammatory responses associated with the recruitment of neutrophils.32 Although the primary function of neutrophils in the gut is to kill invading pathogens, neutrophils can also contribute to exacerbated inflammatory responses.33 However, the relative contribution of neutrophils in IBD pathogenesis remains controversial. Some studies using different experimental animal models directly targeting neutrophils describe a beneficial role,34–36 and in line with this, neutrophils have also been reported to contribute to mucosal healing by releasing mediators necessary for the resolution of inflammation.37 ,38 Yet, others report pathological contributions from neutrophils in experimental colitis39 ,40 or fail to demonstrate their role in the pathogenesis.41 ,42 Therefore, it is clear that the role of neutrophils in regulating intestinal inflammation depends on the conditions and models used, highlighting the necessity of studying clinically relevant samples to better understand human IBD.

It is notable that IL-23+HLA-DR+ cells were relatively few in colon tissue from paediatric patients with IBD despite the general understanding, based mainly on murine models and in vitro observations, that antigen-presenting cells provide the main source of IL-23 in the gut.18–20 Despite the fact that IL-23+HLA-DR+ cells could be identified in IBD colonic tissue, it is noteworthy that IL-23+ neutrophils surpassed the IL-23+HLA-DR+ cells both in frequency and IL-23 levels. The fact that only few human studies have been performed on tissue biopsies and that neutrophils are often discarded during the process of purification of PBMCs may explain why the neutrophils were not previously identified as IL-23 positive in tissue.

The finding that IL-23-producing neutrophils are recruited to the site of inflammation in IBD, likely via the CXCL8-CXCR1/CXCR2 axis, significantly increases our understanding of human IBD disease mechanisms. Among circulating neutrophils we also identified a subpopulation of CD15+CD66bbright neutrophils that expressed IL-23, CXCR1 and CXCR2 corresponding to the IL-23+ neutrophils identified in IBD tissue. Furthermore, circulating CD15+CD66bdim neutrophils did not express IL-23 or CXCR2, revealing phenotypic and functional heterogeneity among circulating human neutrophils. As most neutrophils detected in colon tissue of patients with IBD were IL-23 positive, this may reflect a selective recruitment of neutrophils expressing both CXCR1 and CXCR2 to colonic IBD tissue. Indeed, our results demonstrate that both CXCR1 and CXCR2 mRNA levels were increased in IBD colon tissue, as well as the levels of the potent neutrophil chemoattractant, CXCL8. Furthermore, colon tissue neutrophils identified using flow cytometry had relatively higher surface expression of CXCR2 compared with CXCR1, while the neutrophils in circulation had similar surface expression of both CXCR1 and CXCR2. In line with this, the mRNA expression of CXCR1 and CXCR2 in peripheral blood neutrophils from IBD, non-IBD and healthy controls were similar. Whether the colon tissue microenvironment influences CXCR1 and CXCR2 expression in neutrophils differently and contributes to distinct neutrophil migratory behaviour needs to be further investigated. However, CXCR1 binds only CXCL8 and CXCL1, with high affinity, while CXCR2 binds, in addition to CXCL8, the chemokines CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 and CXCL7.21 ,43 We observed elevated expression of both CXCL8 and CXCL3 mRNA in IBD colon tissue, and this is in line with a previous report by Willson et al.44 In addition, we found increased expression levels of CXCL2 and CXCL6 mRNA, while CXCL5 and CXCL7 mRNA were not significantly altered in colonic tissue of IBD compared with non-IBD patients. CXCL7 mRNA levels were similar between our IBD (11 CD, 4 UC, 1 unclassified) and non-IBD patients, which is in line with previous reports suggesting increased CXCL7 levels only in patients with UC but not in patients with CD.45 Taken together, the altered chemokine expression pattern may not be general for tissue inflammation but rather reflect disease and/or tissue-specific characteristics, as analyses of oral tissue from patients with chronic periodontal disease revealed increased levels of CXCL5, but not CXCL2 (unpublished observation). Notably, synergistic effects of CXCL6 and CXCL8 on neutrophil recruitment have been reported,42 and it is plausible that several of the CXCL8-family members act in concert to support neutrophil chemotaxis in IBD. The relative contribution of distinct chemokines to neutrophil activation and recruitment to human intestinal tissue and whether selective recruitment of CXCR2+ neutrophils occurs, however, needs to be investigated in more detail. Nevertheless, the phenotype of circulating IL-23+ neutrophils positive for both CXCR1 and CXCR2 supports the importance of the CXCL8-family chemokines in neutrophil recruitment and IL-23-mediated tissue pathology not only in IBD but also in other chronic inflammatory diseases. Notably, several registered clinical trials involving drugs targeting CXCL8, CXCR1 and CXCR2 in inflammatory diseases are ongoing or have recently been completed.21

The recruitment of CXCR1+CXCR2+IL-23+neutrophils into inflamed tissue followed by IL-23 release may have a major impact on IL-23-mediated responses involved in peripheral tissue inflammation and regeneration. Our findings indicate IL-23-mediated stimulation by intestinal neutrophils, as STAT3 was phosphorylated in cells close to IL-23+ neutrophils in colon tissue. As evident from the identification of cells double positive for pSTAT3 and CD3, target cells may include αβ T, natural killer T or γδ T cells, which all could respond and contribute to local IL-17A production.31 In line with this, we observed increased IL-17A mRNA expression in colon tissue of patients with IBD, with the highest levels observed in patients with severe disease. IL-23R mutations influence IL-17A production,46 and this may be reflected in our results demonstrating that increased IL-17A expression is mainly associated with a high disease severity score; however, the patients included in this study have not been genotyped. Neutrophil-derived IL-23 may also act in an autocrine fashion stimulating IL-17A production by neutrophils themselves.22 ,41 Our findings suggest a novel role of neutrophils in IL-23/IL-17-mediated development and maintenance of tissue inflammation relevant in resistance to pathogens,47 ,48 as well as in chronic inflammatory conditions and related immunopathology.23 Although we observed increased IL-17A mRNA levels in IBD colon, IL-22, which is also induced by the IL-23R/STAT3 signalling pathway, remained unaltered (data not shown). IL-22 can be produced by several cell types,49 including neutrophils themselves,50 to promote innate immunity of tissue-specific cells, protect tissue from damage or enhance tissue regeneration.51–53 IL-22-induced epithelial activation and survival via STAT3 signalling favour mucosal healing,54 while administration of neutralising anti-IL-22 antibodies in murine models of IBD leads to more extensive epithelial destruction and inflammation in the colon.55 The fact that the levels of IL-22 mRNA were similar in all paediatric patients analysed may reflect a relatively high constitutive expression of IL-22 in colon tissue. Another possibility, although speculative, is that a failure to enhance expression of IL-22 and take advantage of its tissue protective mechanisms may be part of the pathogenic process in paediatric IBD. From these examples, it is apparent that neutrophils can act as double-edged swords that, on the one hand, contribute to intestinal homeostasis and elimination of unwanted pathogens and, on the other hand, participate in harmful inflammatory processes by exacerbating the inflammation via the release of toxic granule contents and pro-inflammatory molecules. Thus, this novel source of IL-23 may play a significant role in pathways associated with both tissue inflammation and homeostasis. Recently, it was also suggested that murine epithelial cells can produce IL-23 leading to restricted mucosal damage;56 however, we did not detect specific IL-23 staining in human epithelial cells lining the colon.

Increased understanding of the principles and mechanisms of neutrophil recruitment, release of IL-23 and activation of target cells locally is, we believe, essential for the management of human IBD. Taken together, our results also highlight the importance of neutrophils in tissue inflammation and pathology, providing strong incentives to initiate new lines of research and to generate new treatments related to these novel findings.

Acknowledgments

The authors thank all patients, parents and unrelated volunteers for participating in this study, all clinical staff involved in collection of biopsies and blood samples and Dr A. Kirby for critical review of the manuscript.

References

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Footnotes

  • Contributors EK and ML contributed to the study design, performed the experiments, analysed the data, interpreted the results and prepared the figures. MI and UL cared for patients with IBD, provided blood samples, tissue biopsies and clinical data for IBD and non-IBD patients. PC contributed to imaging acquisition and analyses. MF performed experiments. SOÅ and DG contributed to the study design and to interpretation of the results. JM contributed to the study design, performed experiments and provided feedback on the results. MI and JIH supervised the clinical study and contributed to interpretation of the results. JIH and MS initiated the study. MS supervised the study, contributed to the study design and analysis and interpretation of the results and prepared the figures. EK, ML and MS wrote the manuscript and all authors participated in editing and finalising the manuscript, which was approved by all authors.

  • Funding The work in the authors’ laboratory was supported by grants from the Swedish Research Council, the Karolinska Institutet, Stockholm County Council (ALF-project). This study was partly performed at the Live Cell Imaging unit, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Centre for Biosciences and the Jonasson donation to the School of Technology and Health, Kungliga Tekniska Högskolan, Huddinge, Sweden.

  • Competing interests None declared.

  • Ethics approval The collection of patient data, colon tissue and blood samples was approved by the Ethical Review Board at the Karolinska Institutet, Stockholm, Sweden, and the investigations were conducted according to the Helsinki Declaration.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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