Expression of neutrophil gelatinase-associated lipocalin (NGAL) in the gut in Crohn’s disease

Expression of neutrophil gelatinase-associated lipocalin (NGAL) in the gut in Crohn’s disease The antimicrobial glycoprotein neutrophil gelatinase-associated lipocalin (NGAL) is strongly expressed in several infectious, inflammatory and malignant disorders, among these inflammatory bowel disease (IBD). Fecal and serum NGAL is elevated during active IBD and we have recently shown that fecal NGAL is a novel biomarker for IBD with a test performance comparable to the established fecal biomarker calprotectin. This study examines expression of NGAL in the healthy gut and in Crohn’s disease (CD), with emphasis on the previously unexplored small intestine. Pinch biopsies were taken from active and inactive CD in jejunum, ileum and colon and from the same sites in healthy controls. Microarray gene expression showed that the NGAL gene, LCN2, was the second most upregulated among 1820 differentially expressed genes in terminal ileum comparing active CD and controls (FC 5.86, p = 0.027). Based on immunohistochemistry and in situ hybridization findings, this upregulation most likely represented increased expression in epithelial cells. Double immunofluorescence showed NGAL expression in 49% (range 19–70) of Paneth cells (PCs) in control ileum with no change during inflammation. In healthy jejunum, the NGAL expression in PCs was weak to none but markedly increased during active CD. We further found NGAL also in metaplastic PCs in colon. Finally, we show for the first time that NGAL is expressed in enteroendocrine cells in small intestine as well as in colon. . . . . Keywords LCN2 Inflammatory bowel disease Crohn’sdisease Paneth cells Enteroendocrine cells Introduction of the lipocalin superfamily known to bind and transport small hydrophobic molecules. NGAL was originally shown to be a Neutrophil gelatinase-associated lipocalin (NGAL, also component of specific granules in human neutrophils known as lipocalin 2 or siderocalin, gene symbol LCN2) (Kjeldsen et al. 2000) and has been identified with cell-type- was first discovered in 1996. This 24 kDa glycoprotein is part specific expression in several tissues like bronchus, stomach, small intestine, pancreas, kidney, prostate gland and thymus (Friedl et al. 1999). * Arne Kristian Sandvik NGAL is a potent bacteriostatic agent, acting by binding bac- arne.sandvik@ntnu.no terial catecholate-type ferric siderophores (Flo et al. 2004;Goetz et al. 2002; Miethke and Skerra 2010). However, NGAL is also Centre of Molecular Inflammation Research, NTNU, Norwegian present in several aseptic inflammatory disorders, as well as in University of Science and Technology, Trondheim, Norway various cancer types. Bao et al. found evidence for catechol as an Department of Clinical and Molecular Medicine, NTNU, Norwegian endogenous siderophore and demonstrated that the catechol- University of Science and Technology, 7489 Trondheim, Norway NGAL complex plays a role in trafficking iron in non- Department of Gastroenterology, St Olav’s University Hospital, infectious tissue (Bao et al. 2010). NGAL has also been sug- Trondheim, Norway gested to act as a growth and differentiation factor Clinic of Medicine, St Olav’s University Hospital, (Chakraborty et al. 2012; Devireddy et al. 2005;Schmidt-Ott et Trondheim, Norway al. 2007) and as an adipokine (Wang et al. 2007; Yan et al. 2007). Department of Pathology, St Olav’s University Hospital, It has further been suggested to have chemotactic properties, to Trondheim, Norway act as an acute phase protein and to stabilize the proteolytic Department of Infectious Diseases, St Olav’s University Hospital, enzyme MMP-9 (Chakraborty et al. 2012). A recent study Trondheim, Norway Cell Tissue Res suggested a role as an appetite regulating hormone (Mosialou et Materials and methods al. 2017). NGAL can be induced by various inflammatory fac- tors, among these toll-like receptor 3 and 5 and cytokines like Samples TNF, IL1β, IL22 and IL17 (Nielsen et al. 1996;Ostviketal. 2013; Singh et al. 2016; Stallhofer et al. 2015). NGAL has been Patients with known CD and healthy controls coming for an found to be a promising biomarker for acute kidney injury ileo-colonoscopy or enteroscopy at the Department of (Mishra et al. 2005). Gastroenterology and Hepatology, St. Olav’s University The present study is relevant to the ongoing exploration of Hospital, Trondheim, Norway, were included in the study af- NGAL as a biomarker for gastrointestinal disease, in particu- ter written informed consent. The study was approved by the lar the two forms of inflammatory bowel disease (IBD), ulcer- Regional Committee for Medical and Health Research Ethics, ative colitis (UC) and Crohn’s disease (CD). While UC is approval nos. 5.2007.910 and 2013/212/REKmidt. characterized by inflammation of the colonic mucosa, CD is Pinch biopsies were obtained during the ileo-colonoscopy a deeper, transmural inflammation that can affect all parts of from the terminal ileum or from the neo-terminal ileum in the gastrointestinal tract, with the predilection area being the patients having undergone ileocecal resection. Two of the bi- terminal ileum. Nielsen et al. and our group have shown that opsies from each patient were formalin-fixed for IHC and ISH the gene encoding NGAL, LCN2, is one of the most upregu- and two were snap-frozen for gene expression analysis. A lated genes in colonic biopsies from patients with UC or CD gastrointestinal pathologist (ESR) classified the biopsies into compared to healthy controls (Nielsen et al. 1996; Ostvik et al. normal, chronic active, or chronic inactive inflammation and 2013). Immunohistochemistry (IHC) shows strong expression samples with discrepancy between endoscopic and histologic of NGAL in the human colonic epithelial cells and in mucosal diagnoses were excluded. The patients with chronic inactive granulocytes during active inflammation (Ostvik et al. 2013). inflammation all previously had verified CD of the terminal NGAL is increased in serum and feces during active IBD and ileum. Patients coming for an endoscopy for other reasons several studies have explored its role as a disease biomarker (de than IBD and with normal endoscopic and histological find- Bruyn et al. 2015; de Bruyn et al. 2014; Janas et al. 2014;Magro ings served as controls. For comparison of PCs between prox- et al. 2017; Stallhofer et al. 2015; Yesil et al. 2013). Our group imal and distal small intestine and for comparison between found fecal NGAL to be massively increased in active IBD, with active jejunal CD and controls, biopsies from the jejunum a test performance comparable to the established fecal biomarker were obtained by enteroscopy. Moreover, for comparison with calprotectin (Thorsvik et al. 2017). Moreover, we observed that metaplastic PCs of the colon, biopsies from patients with ac- NGAL is easily detectable in feces from most healthy controls, tive colonic IBD were obtained from a previously described despite weak to no expression of NGAL in the colonic mucosa. biobank (Granlund et al. 2013). Most likely, this NGAL derives from the small intestine, where its expression in health and disease has not been studied. Thus, in Microarray our ongoing studies of NGAL as a biomarker for IBD, we found it important to characterize NGAL expression also in the small Biopsies from six individuals of each of the categories active intestine, in particular the ileum, which is a predilection area for CD, inactive CD and healthy controls were analyzed for gene CD. To this end, we investigated the expression of NGAL in expression using microarray. Patient characteristics are given healthy individuals and in patients with small intestinal CD using in Table 1. Microarray gene expression analysis followed gene expression analysis, IHC and in situ hybridization (ISH). standard protocols. In brief, cRNA was prepared with The small intestinal epithelium consists of various cell types Ambion’s Illumina® TotalPrep™ RNA Amplification Kit with different functions (Peterson and Artis 2014). In the pres- (Thermo Fisher Scientific, cat. no. AMIL1791), using ent study, we show NGAL expression in Paneth cells (PCs) and 300 ng total RNA as input material. For each sample, the enteroendocrine cells (EECs). PCs are located in the crypt bot- biotin-labeled cRNA concentrations were determined toms of the small intestine. These are pyramidally shaped co- (NanoDrop, Thermo Fisher Scientific) and 750 ng hybridized lumnar cells rich in granules intermingled between LGR5- to HumanHT-12 Expression BeadChips, scanned on a positive stem cells in the crypts of Lieberkühn (Bevins and Illumina HiScan instrument (Illumina Inc., CA, USA) and Salzman 2011). PCs are rare in the healthy colon but are com- processed in GenomeStudio (version 2011.1). monly seen as metaplastic PCs in IBD patients. The antimicro- GenomeStudio output was read and processed using the bial substances defensin-5, defensin-6 and lysozyme are com- lumi R-package (Du et al. 2008) and probe annotations were mon markers for PCs. EECs typically have an apical cytoplas- added from the IlluminaHumanv4.db R-package (Dunning et mic process with microvilli that extend towards the luminal al. 2015). The gene level data values were filtered, log2 trans- surface. Various markers exist for EECs, among these formed and quantile normalized to create the expression chromogranin A, which is considered a general EEC marker values used for statistical modeling. Differential expression (Gunawardene et al. 2011). was modeled using a linear model and fitted using the limma Cell Tissue Res Table 1 Characteristics of Controls CD, active CD, inactive subjects enrolled in microarray analysis of ileum. TNFα tumor Number of subjects 6 6 6 necrosis factor alpha, MAb monoclonal antibody Average age, years (range) 41 (26–57) 34 (21–47) 35 (18–62) Treatment 5-aminosalicylic acid – 01 Anti-TNFα MAb – 12 Azathioprine – 11 Corticosteroids – 41 None 6 2 3 R-package (Ritchie et al. 2015). Significance was considered Quantification of NGAL in Paneth cells on genes with a false discovery adjusted (Benjamini- and enteroendocrine cells Hochberg method) p value below 0.05. IHC of NGAL was performed on biopsies from a total of 67 individuals (24 active CD, 14 inactive CD, 29 controls). In some Immunohistochemistry and double samples, especially in the inflamed biopsies, it could be difficult immunofluorescence to distinguish between PC and EEC expression using IHC. Double fluorescence was thus used for comparison of expression Formalin-fixed, paraffin-embedded (FFPE) biopsies were cut of NGAL in the two cell types between patient groups. To com- into 4 μm thick sections and mounted on glass slides. The pare expression of NGAL in PCs between active ileal CD and sections were pretreated with permeabilization, quenching of controls, five biopsies in each group were double-stained for endogenous peroxidases and antigen retrieval. Primary anti- NGAL and the PC specific Defensin-6 (DEFA6). The number bodies for human NGAL (Abcam, cat. no. ab41105 and of dual positive cells and the total number of DEFA6-positive AntiBodyShop, cat. no. ABS 062-14B), DEFA6 (Sigma, cat. cells (89–214 cells per biopsy in active CD and 62–133 in con- no. HPA019462) and CgA (Immunostar, cat.no. 20086) were trols) were counted in all biopsies and the percentages of NGAL used with an incubation time of 1 h at room temperature or expressing PCs were calculated. To examine NGAL-expression overnight at 4 °C. Secondary antibody was rabbit/mouse in EECs, five biopsies from healthy ileum, five from healthy EnVision-HRP and visualization was made with DAB colon and four from active CD (two colon, two ileum) were (Dako, Glostrup, Denmark, cat. no. K5007) before double-stained with NGAL and chromogranin A (CgA). The counterstaining with hematoxylin. Double immunofluores- number of dual positive cells and the total number of CgA- cence staining was performed using the MaxDouble™ positive cells (17–40 cells per biopsy in colon and 35–80 in M488&R650 ImmunoFluorescence Double Staining Kit for ileum) were counted in all biopsies and the percentage of human tissue (cat. no. DSMR-H3, MaxVision Biosciences NGAL expressing EECs was calculated. Inc., WA, USA) according to the manufacturer’sinstructions, For comparison of NGAL expression between proximal and with antigen retrieval in boiling Tris-EDTA buffer (pH 9.0), distal small intestine, the ileal controls were compared to four followed by incubation with the primary antibodies at 4 °C controls from jejunum (61–136 DEFA6-positive cells per biop- and DAPI as nuclear counterstaining. Omitting the primary sy) using the same method as described above. For comparison antibody (IF and IHC) and matching isotype immunoglobu- of LCN2 mRNA expression between proximal and distal small lins (IHC only) were used as negative controls. intestine, ISH was used and the number of LCN2-positive PCs was quantified in five biopsies from jejunum and eight from terminal ileum. We used score 0 for no staining, 1 for staining In situ hybridization of ≤ 2 PCs per crypt and 2 for > 3 per crypt. Serial sections with staining for the PC-specific DEFA6 were used to confirm the ISH was performed using an RNAscope 2.5 HD Reagent Kit presence of these cells in the biopsies. (Brown or Duplex) for FFPE tissue (Advanced Cell For identification of PC metaplasia, colonic biopsies with Diagnostics (ACD), Hayward, CA, USA) and probes for known active IBD-inflammation were stained with the PC- LCN2, CHGA and DEFA6 according to the manufacturer’s specific DEFA6. Biopsies from five patients with confirmed instructions. All probes were gene- and species-specific. All PC metaplasia were double-stained for NGAL and DEFA6 samples were tested with positive and negative control probes and quantified as described above (15–38 DEFA6-positive supplied by the manufacturer. cells per biopsy). Cell Tissue Res Fig. 2 IHC of NGAL and ISH of LCN2 in biopsies from colon (a–d), terminal ileum (e–h)and jejunum (i–l). a IHC of control colon. Weak to no staining of the epithelium. b ISH of control colon with no signal. c IHC of active CD in colon. Prominent staining of both granulocytes, goblet cells and enterocytes. d ISH of active CD in colon with intense signal in the epithelium. e, f IHC (e)and ISH(f) of control ileum. Marked expression in PCs (arrows) and EECs (arrowhead). Weak to no staining of the epithelium. g IHC of active CD in terminal ileum with markedly distorted architecture and complete loss of villi. Marked staining of granulocytes, the epithelium and scattered PCs and EECs. h ISH of active CD in terminal ileum. Expression in epithelium, PCs and EECs. i, j IHC (i)and ISH (j) of control jejunum with no expression. k IHC of active CD in jejunum with staining of granulocytes, epithelium, PCs and EECs. l ISH of active CD in jejunum with expression in the epithelium, PCs and EECs. Scale bar 200 μm(50 μm in inserts) Statistics A Mann-Whitney test was used in the comparison of NGAL Fig. 1 Scatter plot of LCN2 mRNA levels in microarray gene expression expression in PCs and EECs between active CD and controls analysis of biopsies from controls, inactive CD and active CD in terminal ileum. *p <0.05 and in the comparison of NGAL/LCN2 expression in PCs between ileum and jejunum. A p value < 0.05 (two-sided) was considered statistically significant. Individual values are given as median (range). Calculations were performed using Image capture and processing Prism 5 (GraphPad Software, San Diego, CA, USA). Chromogenic images were acquired by using a Nikon E400 microscope and NIS-Elements BR imaging software (Nikon Instruments, Melville, NY, USA). Immunofluorescence im- Results ages were captured using a Leica SP8 inverted microscope (LeicaMicrosystems, Mannheim, Germany) and further LCN2 mRNA overexpression in ileum processed using ImageJ (Wayne Rasband, National Institutes of Health, USA). Color adjustments were made Microarray gene expression analysis of pinch biopsies from by Lightroom (Adobe, San Jose, CA, USA). InkScape 0.92 terminal ileum showed that LCN2 was the second most up- was used for subsequent image orientation and cropping. regulated gene among all 1820 differentially expressed genes Table 2 List of the 15 most Gene symbol Description FC p value upregulated among 1820 significantly differentially MUC1 Mucin-1 8.57 0.001 expressed genes in inflamed ileal biopsies vs healthy controls. N =6 LCN2 Neutrophil gelatinase-associated lipocalin 5.86 0.027 in each group. FC fold change vs MMP3 Stromelysin-1 4.50 0.022 control CLCA4 Calcium-activated chloride channel regulator 4 4.20 0.050 CXCL9 C-X-C motif chemokine 9 3.71 0.039 NOS2 Nitric oxide synthase, inducible 3.68 0.015 IGFBP5 Insulin-like growth factor-binding protein 5 3.58 0.005 TIMP1 Metalloproteinase inhibitor 1 3.53 0.006 IFITM3 Interferon-induced transmembrane protein 3 3.43 0.002 IFITM2 Interferon-induced transmembrane protein 2 3.25 0.003 ITLN1 Intelectin-1 3.23 0.020 IL8 Interleukin-8 3.23 0.030 S100A9 Protein S100-A9 3.20 0.050 MXRA5 Matrix-remodeling-associated protein 5 3.18 0.001 GPX2 Glutathione peroxidase 2 3.16 0.016 Cell Tissue Res Cell Tissue Res Fig. 3 NGAL expression in PCs. Double immunofluorescence. Red: NGAL. Green: DEFA6. Yellow: overlapping signals. a Colon. Overlap in a subset of metaplastic PCs in inflamed colon. NGAL expression can also be seen in granulocytes, EECs and in the inflamed epithelium. b Terminal ileum. Overlap in a subset of PCs. NGAL also in EECs. c No overlap in control jejunum. d Overlap in a subset of PCs in inflamed jejunum. NGAL also in EECs. Scale bar 50 μm between active CD and controls (fold change (FC) 5.86, p = colons. We now also noted expression in EECs and metaplas- 0.027). Figure 1 shows a scatter plot of LCN2 expression in tic PCs (Fig. 2a, c). ISH confirmed the increased LCN2 the three patient groups. Only MUC1 was more overexpressed mRNA expression of the inflamed epithelium (Figs. 2b, d), (FC 8.57, p = 0.001). When comparing inactive CD with con- whereas there was no expression in granulocytes. The latter is trols, there were no differentially expressed genes. The proin- probably due to prepacking of NGAL in the granulocytes in flammatory cytokine IL8 was significantly upregulated (FC the bone marrow with LCN2 mRNA being downregulated in 3.23, p = 0.03) confirming the inflammatory status of the bi- peripheral cells, as shown earlier (Nielsen et al. 1996). opsies from patients with active CD. The 15 most upregulated In healthy terminal ileum, there was prominent NGAL/LCN2 genes between active CD and controls are listed in Table 2. expression in a subset of PCs and EECs, with weak to no ex- The whole gene list with the differentially expressed genes is pression in epithelial goblet cells or enterocytes (Fig. 2e, f). available in Array Express, GSE number E-MTAB-6593. Inflamed terminal ileum showed expression in granulocytes (IHC only) and epithelial cells, both in crypts and villi. NGAL-expression in colon, ileum and jejunum Expression was particularly prominent in biopsies having mark- edly distorted architecture and loss of villi (Fig. 2g, h). We also Figure 2 shows IHC and ISH of NGAL/LCN2 in the three noticed a more patchy appearance of the expression in the in- examined segments of colon, ileum and jejunum in active CD flamed ileal epithelium compared to the inflamed colonic epithe- and controls. The results for IHC and ISH for NGAL/LCN2 in lium, where expression appeared more continuous. inactive CD were identical to controls (data not shown). In healthy jejunum, there was weak to no NGAL/LCN2 In the colon, consistent with our previous findings (Ostvik expression in the epithelium and the EECs. There was low et al. 2013), IHC showed intense staining of NGAL in expression in very few PCs (Fig. 2i, j). In inflamed jejunum, granulocytes, goblet cells and enterocytes of the colonic epi- however, there was expression in granulocytes (IHC only), thelium in active CD with weak to no staining in the control epithelial cells, EECs and PCs (Figs. 2k, l and 3d). Cell Tissue Res NGAL expression in distinct cell types Paneth cells In active CD of the colon, we now, in addition to more general epithelial NGAL expression, also noticed prominent expression in granules in scattered crypt bottom cells. Since PC metaplasia is a common finding in the colon during active IBD inflammation, we performed double immunofluorescence staining of NGAL and the PC-specific DEFA6, which confirmed NGAL expression in 56 (40–73) % of metaplastic PCs (Fig. 3a). PC metaplasia can persist for a period after amelioration of inflammation and NGAL staining was positive alsoinPCmetaplasiaininactive CD (data not shown). Using double immunofluorescence staining, we further assessed the co-localization of NGAL and DEFA6 in ileal PCs. There was a clear overlap between DEFA6 and NGAL, with NGAL expression in 33 (26–58) % of the DEFA6- positive cells in inflamed ileum and 49 (19–70) % in control ileum (Fig. 3b). This difference was not significant (p =0.35). There was considerable variance in the intensity and the num- ber of NGAL-stained PCs between individuals, both in con- trols and active CD. NGAL expression was most prominent in PC granules but there was also staining in the cytoplasm of some cells. When comparing NGAL expression in the PCs in biopsies from healthy terminal ileum and jejunum (Fig. 3b, c), we found significantly higher expression of NGAL in the PCs in terminal ileum than in the jejunum (p =0.02). Enteroendocrine cells In all segments, we noticed staining of cells with a location and morphology consistent with being EECs. Double immu- nofluorescence staining with CgA confirmed NGAL expres- sion in 63 (49–85) % of CgA-positive EECs in the colon (Fig. 4a) and 53 (45–75) % in the ileum (Fig. 4b), with no difference between controls and active CD (data not shown). The expression in EECs was confirmed with two different Fig. 4 NGAL expression in EECs. Double immunofluorescence. Green: antibodies against NGAL. The mouse monoclonal antibody NGAL. Red: CgA. Yellow: overlapping signals. a Control colon. used in the double immunofluorescence staining (ABS-062- Overlapping signals in the majority of EECs. b Control ileum. NGAL expression in the majority of EECs. NGAL expression also in PCs. Scale 14B) showed NGAL in a higher proportion of the EECs than bar 50 μm the rabbit polyclonal antibody (ab41105), probably due to lower sensitivity of the latter. than seen for the NGAL protein. Co-localization was con- In situ hybridization confirming firmed by duplex ISH with the CgA gene, CHGA (Fig. 5b). immunohistochemistry results The LCN2 ISH signals in PCs were comparable to the NGAL Discussion expression pattern seen by IHC (Fig. 5a). ISH confirmed the difference between LCN2 expression in PCs between healthy We have shown here that LCN2 is the second most upregulat- terminal ileum and jejunum (p = 0.02). ISH also showed LCN2 ed gene in the mucosa of the terminal ileum in active CD expression in EECs, although with generally weaker signals compared to controls, with a 5.86-fold increase. This suggests Cell Tissue Res microarray analysis of ileal CD derives from upregulation of this gene and the NGAL protein in epithelial cells other than PCs. One previous study reported NGAL staining of a subset of intestinal PCs in normal individuals (Friedl et al. 1999). The present study is, however, the first to examine NGAL’sex- pression and regulation in the small intestine in health and disease. PCs have been suggested to play an important part in the pathogenesis of CD, as several of the genes associated with IBD can be related to these cells. CD risk alleles associated with ATG16L1 and NOD2 can lead to defects in packaging and secretion of antimicrobial peptides in PCs. It has been shown that patients with active CD of the small intestine have a decreased number of PCs, as gene expression of alpha- defensins is reduced during active disease (Perminow et al. 2010; Simms et al. 2008; Wehkamp et al. 2005). Interestingly and in contrast to ileal PCs, the NGAL expres- sion in jejunal PCs is clearly increased during CD inflamma- tion. This discrepancy may be explained by the lower bacterial load in the healthy jejunum compared to the terminal/ neoterminal ileum with potentially less baseline stimuli for NGAL induction. The localization of NGAL in the granules along with other antimicrobial peptides suggests a controlled release. The main physiological and pathobiological role of PC NGAL may then be a bacteriostatic effect, with its expres- sion fluctuating due to variable induction by microbial stimuli. This is in contrast to the defensins, lysozyme, Pla2g2a and MMP7, whose expression (in mice) is similar in germ-free and conventional mice (Ouellette 2010). While PCs are found in the healthy small intestine, these cells are very scarce in the normal colon. However, metaplastic PCs are commonly seen in IBD, both CD and UC. The knowledge of PC’s role in the colon is limited but like in the small intestine, they pro- duce antimicrobial peptides such as lysozyme and defensins and as shown in this study also NGAL. It is also likely that PCs have other role(s), such as providing a niche for neighboring stem cells by secreting Wingless/Int (Wnt) and other factors (Clevers and Bevins 2013). Fig. 5 Duplex ISH. a Duplex ISH of control terminal ileum showing We moreover show, for the first time, NGAL expression in overlapping signals in a subset of PCs. Red: DEFA6.Green: LCN2. b Duplex ISH of inflamed terminal ileum demonstrating expression of EECs of the intestine. This cell-type comprises about 1% of LCN2 mRNA in EECs. Red: CHGA.Green: LCN2. Scale bar 50 μm the total epithelial cell population and plays an important role in the regulation of appetite and digestive responses through that NGAL has a major role in the inflammatory process in the secretion of biogenic amines and peptides. Although the pre- ileum, which is a predilection area of CD. In our morpholog- cise role of NGAL in EECs is unknown, recent data show key ical analyses of ileum, we found that NGAL is expressed in two-way interactions between EECs and the mucosal immune PCs but with no significant difference between active CD and system (Harrison et al. 2013; Worthington 2015). A complete- controls. NGAL expression in epithelial cells is, however, ly different role that is consistent with known physiological clearly increased during inflammation. Although we found functions of EECs is also possible, such as suggested in a marked staining of granulocytes in inflamed biopsies, there recent work that proposed NGAL as an appetite suppressing is no LNC2 mRNA in the granulocytes. Bearing in mind the hormone in mice (Mosialou et al. 2017). limitations of IHC and ISH as quantitative methods, it is most In conclusion, we have shown that NGAL is strongly up- regulated in active CD of the small intestine, likely due to likely that the massive overexpression of LCN2 mRNA in our Cell Tissue Res Du P, Kibbe WA, Lin SM (2008) lumi: a pipeline for processing Illumina increased expression in intestinal epithelial cells other than microarray. Bioinformatics (Oxford, England) 24:1547–1548 PCs. This upregulation underlines the putative role of Dunning M, Lynch A, Eldridge M (2015) illuminaHumanv4. db: NGAL as a fecal biomarker of inflammation in the small in- Illumina HumanHT12v4 annotation data (chip illuminaHumanv4). testine. Moreover, we demonstrated the presence of NGAL in R package version 1: Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S, PCs and EECs in both healthy and inflamed intestine where its Aderem A (2004) Lipocalin 2 mediates an innate immune response to role may be both as an antimicrobial peptide and as a regula- bacterial infection by sequestrating iron. Nature 432:917–921 tory substance related to the physiological function of EECs. Friedl A, Stoesz SP, Buckley P, Gould MN (1999) Neutrophil gelatinase- associated lipocalin in normal and neoplastic human tissues. Cell Acknowledgements We want to thank the staff of the Department of type-specific pattern of expression. Histochem J 31:433–441 Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Gastroenterology and Hepatology at St Olav’s University Hospital, Trondheim, for including research subjects. We thank Bjørn Munkvold, Strong RK (2002) The neutrophil lipocalin NGAL is a bacteriostatic Berit Doseth and Wahida Afroz for technical assistance. agent that interferes with siderophore-mediated iron acquisition. The confocal imaging analyses were performed in collaboration with Mol Cell 10:1033–1043 Dr. Bjørnar Sporsheim at Cellular & Molecular Imaging Core Facility Granlund A, Flatberg A, Ostvik AE, Drozdov I, Gustafsson BI, Kidd M, (CMIC), the Norwegian University of Science and Technology (NTNU). BeisvagV,TorpSH, Waldum HL,Martinsen TC,Damas JK, Espevik T, Sandvik AK (2013) Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demon- Funding information The microarray gene expression and bioinformat- strates lack of major differences between Crohn’s disease and ulcer- ics analysis were provided by the Genomics Core Facility (GCF), NTNU. ative colitis. PLoS One 8:e56818 GCF is funded by the Faculty of Medicine and Health Sciences at NTNU Gunawardene AR, Corfe BM, Staton CA (2011) Classification and func- and Central Norway Regional Health Authority. tions of enteroendocrine cells of the lower gastrointestinal tract. Int J The study was supported by grants from the Liaison Committee be- Exp Pathol 92:219–231 tween the Central Norway Regional Health Authority, NTNU and the Harrison E, Lal S, McLaughlin JT (2013) Enteroendocrine cells in gas- Research Council of Norway. trointestinal pathophysiology. Curr Opin Pharmacol 13:941–945 Janas RM, Ochocinska A, Snitko R, Dudka D, Kierkus J, Teisseyre M, Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// Najberg E (2014) Neutrophil gelatinase-associated lipocalin in blood in children with inflammatory bowel disease. J creativecommons.org/licenses/by/4.0/), which permits unrestricted use, Gastroenterol Hepatol 29:1883–1889 distribution and reproduction in any medium, provided you give appro- Kjeldsen L, Cowland JB, Borregaard N (2000) Human neutrophil priate credit to the original author(s) and the source, provide a link to the gelatinase-associated lipocalin and homologous proteins in rat and Creative Commons license and indicate if changes were made. mouse. Biochim Biophys Acta 1482:272–283 Magro F, Lopes S, Coelho R, Cotter J, Dias de Castro F, Tavares de Sousa H, Salgado M, Andrade P, Vieira AI, Figueiredo P, Caldeira P, Sousa A, Duarte MA, Avila F, Silva J, Moleiro J, Mendes S, Giestas S, Ministro P, Sousa P, Goncalves R, Goncalves B, Oliveira A, Chagas References C, Torres J, Dias CC, Lopes J, Borralho P, Afonso J, Geboes K, Carneiro F (2017) Accuracy of faecal calprotectin and neutrophil gelatinase B-associated lipocalin in evaluating subclinical inflam- Bao G, Clifton M, Hoette TM, Mori K, Deng SX, Qiu A, Viltard M, mation in UlceRaTIVE colitis-the ACERTIVE study. Journal of Williams D, Paragas N, Leete T, Kulkarni R, Li X, Lee B, Crohn's & colitis 11:435–444 Kalandadze A, Ratner AJ, Pizarro JC, Schmidt-Ott KM, Landry Miethke M, Skerra A (2010) Neutrophil gelatinase-associated lipocalin DW, Raymond KN, Strong RK, Barasch J (2010) Iron traffics in expresses antimicrobial activity by interfering with L- circulation bound to a siderocalin (Ngal)-catechol complex. Nat norepinephrine-mediated bacterial iron acquisition. Antimicrob Chem Biol 6:602–609 Agents Chemother 54:1580–1589 Bevins CL, Salzman NH (2011) Paneth cells, antimicrobial peptides and Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, Ruff SM, maintenance of intestinal homeostasis. Nat Rev Microbiol 9:356–368 Zahedi K, Shao M, Bean J (2005) Neutrophil gelatinase-associated de Bruyn M, Arijs I, Wollants WJ, Machiels K, Van Steen K, Van Assche lipocalin (NGAL) as a biomarker for acute renal injury after cardiac G, Ferrante M, Rutgeerts P, Vermeire S, Opdenakker G (2014) surgery. Lancet 365:1231–1238 Neutrophil gelatinase B-associated lipocalin and matrix Mosialou I, Shikhel S, Liu JM, Maurizi A, Luo N, He Z, Huang Y, Zong metalloproteinase-9 complex as a surrogate serum marker of muco- H, Friedman RA, Barasch J, Lanzano P, Deng L, Leibel RL, Rubin sal healing in ulcerative colitis. Inflamm Bowel Dis 20:1198–1207 M, Nicholas T, Chung W, Zeltser LM, Williams KW, Pessin JE, de Bruyn M, Arijs I, De Hertogh G, Ferrante M, Van Assche G, Rutgeerts Kousteni S (2017) MC4R-dependent suppression of appetite by P, Vermeire S, Opdenakker G (2015) Serum neutrophil gelatinase B- bone-derived lipocalin 2. Nature 543:385–390 associated Lipocalin and matrix Metalloproteinase-9 complex as a Nielsen BS, Borregaard N, Bundgaard JR, Timshel S, Sehested M, surrogate marker for mucosal healing in patients with Crohn's dis- Kjeldsen L (1996) Induction of NGAL synthesis in epithelial cells ease. J Crohn's Colitis 9:1079–1087 of human colorectal neoplasia and inflammatory bowel diseases. Chakraborty S, Kaur S, Guha S, Batra SK (2012) The multifaceted roles Gut 38:414–420 of neutrophil gelatinase associated lipocalin (NGAL) in inflamma- Ostvik AE, Granlund AV, Torp SH, Flatberg A, Beisvag V, Waldum HL, tion and cancer. Biochim Biophys Acta 1826:129–169 Flo TH, Espevik T, Damas JK, Sandvik AK (2013) Expression of Clevers HC, Bevins CL (2013) Paneth cells: maestros of the small intes- toll-like receptor-3 is enhanced in active inflammatory bowel dis- tinal crypts. Annu Rev Physiol 75:289–311 ease and mediates the excessive release of lipocalin 2. Clin Exp Immunol 173:502–511 Devireddy LR, Gazin C, Zhu X, Green MR (2005) A cell-surface recep- tor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Ouellette AJ (2010) Paneth cells and innate mucosal immunity. Curr Opin Cell 123:1293–1305 Gastroenterol 26:547–553 Cell Tissue Res Perminow G, Beisner J, Koslowski M, Lyckander LG, Stange E, Vatn MH, Thorsvik S, Damas JK, Granlund AV, Flo TH, Bergh K, Ostvik AE, Sandvik AK (2017) Fecal neutrophil gelatinase-associated lipocalin Wehkamp J (2010) Defective paneth cell-mediated host defense in pe- diatric ileal Crohn’s disease. Am J Gastroenterol 105:452–459 as a biomarker for inflammatory bowel disease. J Gastroenterol Peterson LW, Artis D (2014) Intestinal epithelial cells: regulators of barrier Hepatol 32:128–135 function and immune homeostasis. Nat Rev Immunol 14:141–153 Wang Y, Lam KS, Kraegen EW, Sweeney G, Zhang J, Tso AW, Chow W- Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015) S, Wat NM, Xu JY, Hoo RL (2007) Lipocalin-2 is an inflammatory Limma powers differential expression analyses for RNA- marker closely associated with obesity, insulin resistance, and hy- sequencing and microarray studies. Nucleic Acids Res 43:e47–e47 perglycemia in humans. Clin Chem 53:34–41 Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Wehkamp J, Salzman NH, Porter E, Nuding S, Weichenthal M, Petras Barasch J (2007) Dual action of neutrophil gelatinase-associated RE, Shen B, Schaeffeler E, Schwab M, Linzmeier R, Feathers RW, lipocalin. J Am Soc Nephrol 18:407–413 Chu H, Lima H Jr, Fellermann K, Ganz T, Stange EF, Bevins CL Simms LA, Doecke JD, Walsh MD, Huang N, Fowler EV, Radford-Smith (2005) Reduced Paneth cell alpha-defensins in ileal Crohn’s disease. GL (2008) Reduced α-defensin expression is associated with in- Proc Natl Acad Sci U S A 102:18129–18134 flammation and not NOD2 mutation status in ileal Crohn’s disease. Worthington JJ (2015) The intestinal immunoendocrine axis: novel cross-talk Gut 57:903–910 between enteroendocrine cells and the immune system during infection Singh V, San Yeoh B, Chassaing B, Zhang B, Saha P, Xiao X, Awasthi D, and inflammatory disease. Biochem Soc Trans 43:727–733 Shashidharamurthy R, Dikshit M, Gewirtz A (2016) Microbiota- YanQ-W,YangQ,ModyN,GrahamTE, HsuC-H,Xu Z, inducible innate immune siderophore binding protein lipocalin 2 is Houstis NE, Kahn BB, Rosen ED (2007) The adipokine critical for intestinal homeostasis. CMGH Cellular and Molecular lipocalin 2 is regulated by obesity and promotes insulin re- Gastroenterology and Hepatology sistance. Diabetes 56:2533–2540 Stallhofer J, Friedrich M, Konrad-Zerna A, Wetzke M, Lohse P, Glas J, Yesil A, Gonen C, Senates E, Paker N, Gokden Y, Kochan K, Erdem ED, Tillack-Schreiber C, Schnitzler F, Beigel F, Brand S (2015) Gunduz F (2013) Relationship between neutrophil gelatinase- Lipocalin-2 is a disease activity marker in inflammatory bowel dis- associated lipocalin (NGAL) levels and inflammatory bowel disease ease regulated by IL-17A, IL-22, and TNF-alpha and modulated by type and activity. Dig Dis Sci 58:2587–2593 IL23R genotype status. Inflammatory bowel diseases http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell and Tissue Research Springer Journals

Expression of neutrophil gelatinase-associated lipocalin (NGAL) in the gut in Crohn’s disease

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Springer Berlin Heidelberg
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Copyright © 2018 by The Author(s)
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Biomedicine; Human Genetics; Proteomics; Molecular Medicine
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0302-766X
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10.1007/s00441-018-2860-8
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Abstract

The antimicrobial glycoprotein neutrophil gelatinase-associated lipocalin (NGAL) is strongly expressed in several infectious, inflammatory and malignant disorders, among these inflammatory bowel disease (IBD). Fecal and serum NGAL is elevated during active IBD and we have recently shown that fecal NGAL is a novel biomarker for IBD with a test performance comparable to the established fecal biomarker calprotectin. This study examines expression of NGAL in the healthy gut and in Crohn’s disease (CD), with emphasis on the previously unexplored small intestine. Pinch biopsies were taken from active and inactive CD in jejunum, ileum and colon and from the same sites in healthy controls. Microarray gene expression showed that the NGAL gene, LCN2, was the second most upregulated among 1820 differentially expressed genes in terminal ileum comparing active CD and controls (FC 5.86, p = 0.027). Based on immunohistochemistry and in situ hybridization findings, this upregulation most likely represented increased expression in epithelial cells. Double immunofluorescence showed NGAL expression in 49% (range 19–70) of Paneth cells (PCs) in control ileum with no change during inflammation. In healthy jejunum, the NGAL expression in PCs was weak to none but markedly increased during active CD. We further found NGAL also in metaplastic PCs in colon. Finally, we show for the first time that NGAL is expressed in enteroendocrine cells in small intestine as well as in colon. . . . . Keywords LCN2 Inflammatory bowel disease Crohn’sdisease Paneth cells Enteroendocrine cells Introduction of the lipocalin superfamily known to bind and transport small hydrophobic molecules. NGAL was originally shown to be a Neutrophil gelatinase-associated lipocalin (NGAL, also component of specific granules in human neutrophils known as lipocalin 2 or siderocalin, gene symbol LCN2) (Kjeldsen et al. 2000) and has been identified with cell-type- was first discovered in 1996. This 24 kDa glycoprotein is part specific expression in several tissues like bronchus, stomach, small intestine, pancreas, kidney, prostate gland and thymus (Friedl et al. 1999). * Arne Kristian Sandvik NGAL is a potent bacteriostatic agent, acting by binding bac- arne.sandvik@ntnu.no terial catecholate-type ferric siderophores (Flo et al. 2004;Goetz et al. 2002; Miethke and Skerra 2010). However, NGAL is also Centre of Molecular Inflammation Research, NTNU, Norwegian present in several aseptic inflammatory disorders, as well as in University of Science and Technology, Trondheim, Norway various cancer types. Bao et al. found evidence for catechol as an Department of Clinical and Molecular Medicine, NTNU, Norwegian endogenous siderophore and demonstrated that the catechol- University of Science and Technology, 7489 Trondheim, Norway NGAL complex plays a role in trafficking iron in non- Department of Gastroenterology, St Olav’s University Hospital, infectious tissue (Bao et al. 2010). NGAL has also been sug- Trondheim, Norway gested to act as a growth and differentiation factor Clinic of Medicine, St Olav’s University Hospital, (Chakraborty et al. 2012; Devireddy et al. 2005;Schmidt-Ott et Trondheim, Norway al. 2007) and as an adipokine (Wang et al. 2007; Yan et al. 2007). Department of Pathology, St Olav’s University Hospital, It has further been suggested to have chemotactic properties, to Trondheim, Norway act as an acute phase protein and to stabilize the proteolytic Department of Infectious Diseases, St Olav’s University Hospital, enzyme MMP-9 (Chakraborty et al. 2012). A recent study Trondheim, Norway Cell Tissue Res suggested a role as an appetite regulating hormone (Mosialou et Materials and methods al. 2017). NGAL can be induced by various inflammatory fac- tors, among these toll-like receptor 3 and 5 and cytokines like Samples TNF, IL1β, IL22 and IL17 (Nielsen et al. 1996;Ostviketal. 2013; Singh et al. 2016; Stallhofer et al. 2015). NGAL has been Patients with known CD and healthy controls coming for an found to be a promising biomarker for acute kidney injury ileo-colonoscopy or enteroscopy at the Department of (Mishra et al. 2005). Gastroenterology and Hepatology, St. Olav’s University The present study is relevant to the ongoing exploration of Hospital, Trondheim, Norway, were included in the study af- NGAL as a biomarker for gastrointestinal disease, in particu- ter written informed consent. The study was approved by the lar the two forms of inflammatory bowel disease (IBD), ulcer- Regional Committee for Medical and Health Research Ethics, ative colitis (UC) and Crohn’s disease (CD). While UC is approval nos. 5.2007.910 and 2013/212/REKmidt. characterized by inflammation of the colonic mucosa, CD is Pinch biopsies were obtained during the ileo-colonoscopy a deeper, transmural inflammation that can affect all parts of from the terminal ileum or from the neo-terminal ileum in the gastrointestinal tract, with the predilection area being the patients having undergone ileocecal resection. Two of the bi- terminal ileum. Nielsen et al. and our group have shown that opsies from each patient were formalin-fixed for IHC and ISH the gene encoding NGAL, LCN2, is one of the most upregu- and two were snap-frozen for gene expression analysis. A lated genes in colonic biopsies from patients with UC or CD gastrointestinal pathologist (ESR) classified the biopsies into compared to healthy controls (Nielsen et al. 1996; Ostvik et al. normal, chronic active, or chronic inactive inflammation and 2013). Immunohistochemistry (IHC) shows strong expression samples with discrepancy between endoscopic and histologic of NGAL in the human colonic epithelial cells and in mucosal diagnoses were excluded. The patients with chronic inactive granulocytes during active inflammation (Ostvik et al. 2013). inflammation all previously had verified CD of the terminal NGAL is increased in serum and feces during active IBD and ileum. Patients coming for an endoscopy for other reasons several studies have explored its role as a disease biomarker (de than IBD and with normal endoscopic and histological find- Bruyn et al. 2015; de Bruyn et al. 2014; Janas et al. 2014;Magro ings served as controls. For comparison of PCs between prox- et al. 2017; Stallhofer et al. 2015; Yesil et al. 2013). Our group imal and distal small intestine and for comparison between found fecal NGAL to be massively increased in active IBD, with active jejunal CD and controls, biopsies from the jejunum a test performance comparable to the established fecal biomarker were obtained by enteroscopy. Moreover, for comparison with calprotectin (Thorsvik et al. 2017). Moreover, we observed that metaplastic PCs of the colon, biopsies from patients with ac- NGAL is easily detectable in feces from most healthy controls, tive colonic IBD were obtained from a previously described despite weak to no expression of NGAL in the colonic mucosa. biobank (Granlund et al. 2013). Most likely, this NGAL derives from the small intestine, where its expression in health and disease has not been studied. Thus, in Microarray our ongoing studies of NGAL as a biomarker for IBD, we found it important to characterize NGAL expression also in the small Biopsies from six individuals of each of the categories active intestine, in particular the ileum, which is a predilection area for CD, inactive CD and healthy controls were analyzed for gene CD. To this end, we investigated the expression of NGAL in expression using microarray. Patient characteristics are given healthy individuals and in patients with small intestinal CD using in Table 1. Microarray gene expression analysis followed gene expression analysis, IHC and in situ hybridization (ISH). standard protocols. In brief, cRNA was prepared with The small intestinal epithelium consists of various cell types Ambion’s Illumina® TotalPrep™ RNA Amplification Kit with different functions (Peterson and Artis 2014). In the pres- (Thermo Fisher Scientific, cat. no. AMIL1791), using ent study, we show NGAL expression in Paneth cells (PCs) and 300 ng total RNA as input material. For each sample, the enteroendocrine cells (EECs). PCs are located in the crypt bot- biotin-labeled cRNA concentrations were determined toms of the small intestine. These are pyramidally shaped co- (NanoDrop, Thermo Fisher Scientific) and 750 ng hybridized lumnar cells rich in granules intermingled between LGR5- to HumanHT-12 Expression BeadChips, scanned on a positive stem cells in the crypts of Lieberkühn (Bevins and Illumina HiScan instrument (Illumina Inc., CA, USA) and Salzman 2011). PCs are rare in the healthy colon but are com- processed in GenomeStudio (version 2011.1). monly seen as metaplastic PCs in IBD patients. The antimicro- GenomeStudio output was read and processed using the bial substances defensin-5, defensin-6 and lysozyme are com- lumi R-package (Du et al. 2008) and probe annotations were mon markers for PCs. EECs typically have an apical cytoplas- added from the IlluminaHumanv4.db R-package (Dunning et mic process with microvilli that extend towards the luminal al. 2015). The gene level data values were filtered, log2 trans- surface. Various markers exist for EECs, among these formed and quantile normalized to create the expression chromogranin A, which is considered a general EEC marker values used for statistical modeling. Differential expression (Gunawardene et al. 2011). was modeled using a linear model and fitted using the limma Cell Tissue Res Table 1 Characteristics of Controls CD, active CD, inactive subjects enrolled in microarray analysis of ileum. TNFα tumor Number of subjects 6 6 6 necrosis factor alpha, MAb monoclonal antibody Average age, years (range) 41 (26–57) 34 (21–47) 35 (18–62) Treatment 5-aminosalicylic acid – 01 Anti-TNFα MAb – 12 Azathioprine – 11 Corticosteroids – 41 None 6 2 3 R-package (Ritchie et al. 2015). Significance was considered Quantification of NGAL in Paneth cells on genes with a false discovery adjusted (Benjamini- and enteroendocrine cells Hochberg method) p value below 0.05. IHC of NGAL was performed on biopsies from a total of 67 individuals (24 active CD, 14 inactive CD, 29 controls). In some Immunohistochemistry and double samples, especially in the inflamed biopsies, it could be difficult immunofluorescence to distinguish between PC and EEC expression using IHC. Double fluorescence was thus used for comparison of expression Formalin-fixed, paraffin-embedded (FFPE) biopsies were cut of NGAL in the two cell types between patient groups. To com- into 4 μm thick sections and mounted on glass slides. The pare expression of NGAL in PCs between active ileal CD and sections were pretreated with permeabilization, quenching of controls, five biopsies in each group were double-stained for endogenous peroxidases and antigen retrieval. Primary anti- NGAL and the PC specific Defensin-6 (DEFA6). The number bodies for human NGAL (Abcam, cat. no. ab41105 and of dual positive cells and the total number of DEFA6-positive AntiBodyShop, cat. no. ABS 062-14B), DEFA6 (Sigma, cat. cells (89–214 cells per biopsy in active CD and 62–133 in con- no. HPA019462) and CgA (Immunostar, cat.no. 20086) were trols) were counted in all biopsies and the percentages of NGAL used with an incubation time of 1 h at room temperature or expressing PCs were calculated. To examine NGAL-expression overnight at 4 °C. Secondary antibody was rabbit/mouse in EECs, five biopsies from healthy ileum, five from healthy EnVision-HRP and visualization was made with DAB colon and four from active CD (two colon, two ileum) were (Dako, Glostrup, Denmark, cat. no. K5007) before double-stained with NGAL and chromogranin A (CgA). The counterstaining with hematoxylin. Double immunofluores- number of dual positive cells and the total number of CgA- cence staining was performed using the MaxDouble™ positive cells (17–40 cells per biopsy in colon and 35–80 in M488&R650 ImmunoFluorescence Double Staining Kit for ileum) were counted in all biopsies and the percentage of human tissue (cat. no. DSMR-H3, MaxVision Biosciences NGAL expressing EECs was calculated. Inc., WA, USA) according to the manufacturer’sinstructions, For comparison of NGAL expression between proximal and with antigen retrieval in boiling Tris-EDTA buffer (pH 9.0), distal small intestine, the ileal controls were compared to four followed by incubation with the primary antibodies at 4 °C controls from jejunum (61–136 DEFA6-positive cells per biop- and DAPI as nuclear counterstaining. Omitting the primary sy) using the same method as described above. For comparison antibody (IF and IHC) and matching isotype immunoglobu- of LCN2 mRNA expression between proximal and distal small lins (IHC only) were used as negative controls. intestine, ISH was used and the number of LCN2-positive PCs was quantified in five biopsies from jejunum and eight from terminal ileum. We used score 0 for no staining, 1 for staining In situ hybridization of ≤ 2 PCs per crypt and 2 for > 3 per crypt. Serial sections with staining for the PC-specific DEFA6 were used to confirm the ISH was performed using an RNAscope 2.5 HD Reagent Kit presence of these cells in the biopsies. (Brown or Duplex) for FFPE tissue (Advanced Cell For identification of PC metaplasia, colonic biopsies with Diagnostics (ACD), Hayward, CA, USA) and probes for known active IBD-inflammation were stained with the PC- LCN2, CHGA and DEFA6 according to the manufacturer’s specific DEFA6. Biopsies from five patients with confirmed instructions. All probes were gene- and species-specific. All PC metaplasia were double-stained for NGAL and DEFA6 samples were tested with positive and negative control probes and quantified as described above (15–38 DEFA6-positive supplied by the manufacturer. cells per biopsy). Cell Tissue Res Fig. 2 IHC of NGAL and ISH of LCN2 in biopsies from colon (a–d), terminal ileum (e–h)and jejunum (i–l). a IHC of control colon. Weak to no staining of the epithelium. b ISH of control colon with no signal. c IHC of active CD in colon. Prominent staining of both granulocytes, goblet cells and enterocytes. d ISH of active CD in colon with intense signal in the epithelium. e, f IHC (e)and ISH(f) of control ileum. Marked expression in PCs (arrows) and EECs (arrowhead). Weak to no staining of the epithelium. g IHC of active CD in terminal ileum with markedly distorted architecture and complete loss of villi. Marked staining of granulocytes, the epithelium and scattered PCs and EECs. h ISH of active CD in terminal ileum. Expression in epithelium, PCs and EECs. i, j IHC (i)and ISH (j) of control jejunum with no expression. k IHC of active CD in jejunum with staining of granulocytes, epithelium, PCs and EECs. l ISH of active CD in jejunum with expression in the epithelium, PCs and EECs. Scale bar 200 μm(50 μm in inserts) Statistics A Mann-Whitney test was used in the comparison of NGAL Fig. 1 Scatter plot of LCN2 mRNA levels in microarray gene expression expression in PCs and EECs between active CD and controls analysis of biopsies from controls, inactive CD and active CD in terminal ileum. *p <0.05 and in the comparison of NGAL/LCN2 expression in PCs between ileum and jejunum. A p value < 0.05 (two-sided) was considered statistically significant. Individual values are given as median (range). Calculations were performed using Image capture and processing Prism 5 (GraphPad Software, San Diego, CA, USA). Chromogenic images were acquired by using a Nikon E400 microscope and NIS-Elements BR imaging software (Nikon Instruments, Melville, NY, USA). Immunofluorescence im- Results ages were captured using a Leica SP8 inverted microscope (LeicaMicrosystems, Mannheim, Germany) and further LCN2 mRNA overexpression in ileum processed using ImageJ (Wayne Rasband, National Institutes of Health, USA). Color adjustments were made Microarray gene expression analysis of pinch biopsies from by Lightroom (Adobe, San Jose, CA, USA). InkScape 0.92 terminal ileum showed that LCN2 was the second most up- was used for subsequent image orientation and cropping. regulated gene among all 1820 differentially expressed genes Table 2 List of the 15 most Gene symbol Description FC p value upregulated among 1820 significantly differentially MUC1 Mucin-1 8.57 0.001 expressed genes in inflamed ileal biopsies vs healthy controls. N =6 LCN2 Neutrophil gelatinase-associated lipocalin 5.86 0.027 in each group. FC fold change vs MMP3 Stromelysin-1 4.50 0.022 control CLCA4 Calcium-activated chloride channel regulator 4 4.20 0.050 CXCL9 C-X-C motif chemokine 9 3.71 0.039 NOS2 Nitric oxide synthase, inducible 3.68 0.015 IGFBP5 Insulin-like growth factor-binding protein 5 3.58 0.005 TIMP1 Metalloproteinase inhibitor 1 3.53 0.006 IFITM3 Interferon-induced transmembrane protein 3 3.43 0.002 IFITM2 Interferon-induced transmembrane protein 2 3.25 0.003 ITLN1 Intelectin-1 3.23 0.020 IL8 Interleukin-8 3.23 0.030 S100A9 Protein S100-A9 3.20 0.050 MXRA5 Matrix-remodeling-associated protein 5 3.18 0.001 GPX2 Glutathione peroxidase 2 3.16 0.016 Cell Tissue Res Cell Tissue Res Fig. 3 NGAL expression in PCs. Double immunofluorescence. Red: NGAL. Green: DEFA6. Yellow: overlapping signals. a Colon. Overlap in a subset of metaplastic PCs in inflamed colon. NGAL expression can also be seen in granulocytes, EECs and in the inflamed epithelium. b Terminal ileum. Overlap in a subset of PCs. NGAL also in EECs. c No overlap in control jejunum. d Overlap in a subset of PCs in inflamed jejunum. NGAL also in EECs. Scale bar 50 μm between active CD and controls (fold change (FC) 5.86, p = colons. We now also noted expression in EECs and metaplas- 0.027). Figure 1 shows a scatter plot of LCN2 expression in tic PCs (Fig. 2a, c). ISH confirmed the increased LCN2 the three patient groups. Only MUC1 was more overexpressed mRNA expression of the inflamed epithelium (Figs. 2b, d), (FC 8.57, p = 0.001). When comparing inactive CD with con- whereas there was no expression in granulocytes. The latter is trols, there were no differentially expressed genes. The proin- probably due to prepacking of NGAL in the granulocytes in flammatory cytokine IL8 was significantly upregulated (FC the bone marrow with LCN2 mRNA being downregulated in 3.23, p = 0.03) confirming the inflammatory status of the bi- peripheral cells, as shown earlier (Nielsen et al. 1996). opsies from patients with active CD. The 15 most upregulated In healthy terminal ileum, there was prominent NGAL/LCN2 genes between active CD and controls are listed in Table 2. expression in a subset of PCs and EECs, with weak to no ex- The whole gene list with the differentially expressed genes is pression in epithelial goblet cells or enterocytes (Fig. 2e, f). available in Array Express, GSE number E-MTAB-6593. Inflamed terminal ileum showed expression in granulocytes (IHC only) and epithelial cells, both in crypts and villi. NGAL-expression in colon, ileum and jejunum Expression was particularly prominent in biopsies having mark- edly distorted architecture and loss of villi (Fig. 2g, h). We also Figure 2 shows IHC and ISH of NGAL/LCN2 in the three noticed a more patchy appearance of the expression in the in- examined segments of colon, ileum and jejunum in active CD flamed ileal epithelium compared to the inflamed colonic epithe- and controls. The results for IHC and ISH for NGAL/LCN2 in lium, where expression appeared more continuous. inactive CD were identical to controls (data not shown). In healthy jejunum, there was weak to no NGAL/LCN2 In the colon, consistent with our previous findings (Ostvik expression in the epithelium and the EECs. There was low et al. 2013), IHC showed intense staining of NGAL in expression in very few PCs (Fig. 2i, j). In inflamed jejunum, granulocytes, goblet cells and enterocytes of the colonic epi- however, there was expression in granulocytes (IHC only), thelium in active CD with weak to no staining in the control epithelial cells, EECs and PCs (Figs. 2k, l and 3d). Cell Tissue Res NGAL expression in distinct cell types Paneth cells In active CD of the colon, we now, in addition to more general epithelial NGAL expression, also noticed prominent expression in granules in scattered crypt bottom cells. Since PC metaplasia is a common finding in the colon during active IBD inflammation, we performed double immunofluorescence staining of NGAL and the PC-specific DEFA6, which confirmed NGAL expression in 56 (40–73) % of metaplastic PCs (Fig. 3a). PC metaplasia can persist for a period after amelioration of inflammation and NGAL staining was positive alsoinPCmetaplasiaininactive CD (data not shown). Using double immunofluorescence staining, we further assessed the co-localization of NGAL and DEFA6 in ileal PCs. There was a clear overlap between DEFA6 and NGAL, with NGAL expression in 33 (26–58) % of the DEFA6- positive cells in inflamed ileum and 49 (19–70) % in control ileum (Fig. 3b). This difference was not significant (p =0.35). There was considerable variance in the intensity and the num- ber of NGAL-stained PCs between individuals, both in con- trols and active CD. NGAL expression was most prominent in PC granules but there was also staining in the cytoplasm of some cells. When comparing NGAL expression in the PCs in biopsies from healthy terminal ileum and jejunum (Fig. 3b, c), we found significantly higher expression of NGAL in the PCs in terminal ileum than in the jejunum (p =0.02). Enteroendocrine cells In all segments, we noticed staining of cells with a location and morphology consistent with being EECs. Double immu- nofluorescence staining with CgA confirmed NGAL expres- sion in 63 (49–85) % of CgA-positive EECs in the colon (Fig. 4a) and 53 (45–75) % in the ileum (Fig. 4b), with no difference between controls and active CD (data not shown). The expression in EECs was confirmed with two different Fig. 4 NGAL expression in EECs. Double immunofluorescence. Green: antibodies against NGAL. The mouse monoclonal antibody NGAL. Red: CgA. Yellow: overlapping signals. a Control colon. used in the double immunofluorescence staining (ABS-062- Overlapping signals in the majority of EECs. b Control ileum. NGAL expression in the majority of EECs. NGAL expression also in PCs. Scale 14B) showed NGAL in a higher proportion of the EECs than bar 50 μm the rabbit polyclonal antibody (ab41105), probably due to lower sensitivity of the latter. than seen for the NGAL protein. Co-localization was con- In situ hybridization confirming firmed by duplex ISH with the CgA gene, CHGA (Fig. 5b). immunohistochemistry results The LCN2 ISH signals in PCs were comparable to the NGAL Discussion expression pattern seen by IHC (Fig. 5a). ISH confirmed the difference between LCN2 expression in PCs between healthy We have shown here that LCN2 is the second most upregulat- terminal ileum and jejunum (p = 0.02). ISH also showed LCN2 ed gene in the mucosa of the terminal ileum in active CD expression in EECs, although with generally weaker signals compared to controls, with a 5.86-fold increase. This suggests Cell Tissue Res microarray analysis of ileal CD derives from upregulation of this gene and the NGAL protein in epithelial cells other than PCs. One previous study reported NGAL staining of a subset of intestinal PCs in normal individuals (Friedl et al. 1999). The present study is, however, the first to examine NGAL’sex- pression and regulation in the small intestine in health and disease. PCs have been suggested to play an important part in the pathogenesis of CD, as several of the genes associated with IBD can be related to these cells. CD risk alleles associated with ATG16L1 and NOD2 can lead to defects in packaging and secretion of antimicrobial peptides in PCs. It has been shown that patients with active CD of the small intestine have a decreased number of PCs, as gene expression of alpha- defensins is reduced during active disease (Perminow et al. 2010; Simms et al. 2008; Wehkamp et al. 2005). Interestingly and in contrast to ileal PCs, the NGAL expres- sion in jejunal PCs is clearly increased during CD inflamma- tion. This discrepancy may be explained by the lower bacterial load in the healthy jejunum compared to the terminal/ neoterminal ileum with potentially less baseline stimuli for NGAL induction. The localization of NGAL in the granules along with other antimicrobial peptides suggests a controlled release. The main physiological and pathobiological role of PC NGAL may then be a bacteriostatic effect, with its expres- sion fluctuating due to variable induction by microbial stimuli. This is in contrast to the defensins, lysozyme, Pla2g2a and MMP7, whose expression (in mice) is similar in germ-free and conventional mice (Ouellette 2010). While PCs are found in the healthy small intestine, these cells are very scarce in the normal colon. However, metaplastic PCs are commonly seen in IBD, both CD and UC. The knowledge of PC’s role in the colon is limited but like in the small intestine, they pro- duce antimicrobial peptides such as lysozyme and defensins and as shown in this study also NGAL. It is also likely that PCs have other role(s), such as providing a niche for neighboring stem cells by secreting Wingless/Int (Wnt) and other factors (Clevers and Bevins 2013). Fig. 5 Duplex ISH. a Duplex ISH of control terminal ileum showing We moreover show, for the first time, NGAL expression in overlapping signals in a subset of PCs. Red: DEFA6.Green: LCN2. b Duplex ISH of inflamed terminal ileum demonstrating expression of EECs of the intestine. This cell-type comprises about 1% of LCN2 mRNA in EECs. Red: CHGA.Green: LCN2. Scale bar 50 μm the total epithelial cell population and plays an important role in the regulation of appetite and digestive responses through that NGAL has a major role in the inflammatory process in the secretion of biogenic amines and peptides. Although the pre- ileum, which is a predilection area of CD. In our morpholog- cise role of NGAL in EECs is unknown, recent data show key ical analyses of ileum, we found that NGAL is expressed in two-way interactions between EECs and the mucosal immune PCs but with no significant difference between active CD and system (Harrison et al. 2013; Worthington 2015). A complete- controls. NGAL expression in epithelial cells is, however, ly different role that is consistent with known physiological clearly increased during inflammation. Although we found functions of EECs is also possible, such as suggested in a marked staining of granulocytes in inflamed biopsies, there recent work that proposed NGAL as an appetite suppressing is no LNC2 mRNA in the granulocytes. Bearing in mind the hormone in mice (Mosialou et al. 2017). limitations of IHC and ISH as quantitative methods, it is most In conclusion, we have shown that NGAL is strongly up- regulated in active CD of the small intestine, likely due to likely that the massive overexpression of LCN2 mRNA in our Cell Tissue Res Du P, Kibbe WA, Lin SM (2008) lumi: a pipeline for processing Illumina increased expression in intestinal epithelial cells other than microarray. Bioinformatics (Oxford, England) 24:1547–1548 PCs. This upregulation underlines the putative role of Dunning M, Lynch A, Eldridge M (2015) illuminaHumanv4. db: NGAL as a fecal biomarker of inflammation in the small in- Illumina HumanHT12v4 annotation data (chip illuminaHumanv4). testine. Moreover, we demonstrated the presence of NGAL in R package version 1: Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S, PCs and EECs in both healthy and inflamed intestine where its Aderem A (2004) Lipocalin 2 mediates an innate immune response to role may be both as an antimicrobial peptide and as a regula- bacterial infection by sequestrating iron. Nature 432:917–921 tory substance related to the physiological function of EECs. Friedl A, Stoesz SP, Buckley P, Gould MN (1999) Neutrophil gelatinase- associated lipocalin in normal and neoplastic human tissues. Cell Acknowledgements We want to thank the staff of the Department of type-specific pattern of expression. Histochem J 31:433–441 Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Gastroenterology and Hepatology at St Olav’s University Hospital, Trondheim, for including research subjects. We thank Bjørn Munkvold, Strong RK (2002) The neutrophil lipocalin NGAL is a bacteriostatic Berit Doseth and Wahida Afroz for technical assistance. agent that interferes with siderophore-mediated iron acquisition. The confocal imaging analyses were performed in collaboration with Mol Cell 10:1033–1043 Dr. Bjørnar Sporsheim at Cellular & Molecular Imaging Core Facility Granlund A, Flatberg A, Ostvik AE, Drozdov I, Gustafsson BI, Kidd M, (CMIC), the Norwegian University of Science and Technology (NTNU). BeisvagV,TorpSH, Waldum HL,Martinsen TC,Damas JK, Espevik T, Sandvik AK (2013) Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demon- Funding information The microarray gene expression and bioinformat- strates lack of major differences between Crohn’s disease and ulcer- ics analysis were provided by the Genomics Core Facility (GCF), NTNU. ative colitis. PLoS One 8:e56818 GCF is funded by the Faculty of Medicine and Health Sciences at NTNU Gunawardene AR, Corfe BM, Staton CA (2011) Classification and func- and Central Norway Regional Health Authority. tions of enteroendocrine cells of the lower gastrointestinal tract. Int J The study was supported by grants from the Liaison Committee be- Exp Pathol 92:219–231 tween the Central Norway Regional Health Authority, NTNU and the Harrison E, Lal S, McLaughlin JT (2013) Enteroendocrine cells in gas- Research Council of Norway. trointestinal pathophysiology. Curr Opin Pharmacol 13:941–945 Janas RM, Ochocinska A, Snitko R, Dudka D, Kierkus J, Teisseyre M, Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// Najberg E (2014) Neutrophil gelatinase-associated lipocalin in blood in children with inflammatory bowel disease. J creativecommons.org/licenses/by/4.0/), which permits unrestricted use, Gastroenterol Hepatol 29:1883–1889 distribution and reproduction in any medium, provided you give appro- Kjeldsen L, Cowland JB, Borregaard N (2000) Human neutrophil priate credit to the original author(s) and the source, provide a link to the gelatinase-associated lipocalin and homologous proteins in rat and Creative Commons license and indicate if changes were made. mouse. Biochim Biophys Acta 1482:272–283 Magro F, Lopes S, Coelho R, Cotter J, Dias de Castro F, Tavares de Sousa H, Salgado M, Andrade P, Vieira AI, Figueiredo P, Caldeira P, Sousa A, Duarte MA, Avila F, Silva J, Moleiro J, Mendes S, Giestas S, Ministro P, Sousa P, Goncalves R, Goncalves B, Oliveira A, Chagas References C, Torres J, Dias CC, Lopes J, Borralho P, Afonso J, Geboes K, Carneiro F (2017) Accuracy of faecal calprotectin and neutrophil gelatinase B-associated lipocalin in evaluating subclinical inflam- Bao G, Clifton M, Hoette TM, Mori K, Deng SX, Qiu A, Viltard M, mation in UlceRaTIVE colitis-the ACERTIVE study. Journal of Williams D, Paragas N, Leete T, Kulkarni R, Li X, Lee B, Crohn's & colitis 11:435–444 Kalandadze A, Ratner AJ, Pizarro JC, Schmidt-Ott KM, Landry Miethke M, Skerra A (2010) Neutrophil gelatinase-associated lipocalin DW, Raymond KN, Strong RK, Barasch J (2010) Iron traffics in expresses antimicrobial activity by interfering with L- circulation bound to a siderocalin (Ngal)-catechol complex. Nat norepinephrine-mediated bacterial iron acquisition. Antimicrob Chem Biol 6:602–609 Agents Chemother 54:1580–1589 Bevins CL, Salzman NH (2011) Paneth cells, antimicrobial peptides and Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, Ruff SM, maintenance of intestinal homeostasis. Nat Rev Microbiol 9:356–368 Zahedi K, Shao M, Bean J (2005) Neutrophil gelatinase-associated de Bruyn M, Arijs I, Wollants WJ, Machiels K, Van Steen K, Van Assche lipocalin (NGAL) as a biomarker for acute renal injury after cardiac G, Ferrante M, Rutgeerts P, Vermeire S, Opdenakker G (2014) surgery. Lancet 365:1231–1238 Neutrophil gelatinase B-associated lipocalin and matrix Mosialou I, Shikhel S, Liu JM, Maurizi A, Luo N, He Z, Huang Y, Zong metalloproteinase-9 complex as a surrogate serum marker of muco- H, Friedman RA, Barasch J, Lanzano P, Deng L, Leibel RL, Rubin sal healing in ulcerative colitis. Inflamm Bowel Dis 20:1198–1207 M, Nicholas T, Chung W, Zeltser LM, Williams KW, Pessin JE, de Bruyn M, Arijs I, De Hertogh G, Ferrante M, Van Assche G, Rutgeerts Kousteni S (2017) MC4R-dependent suppression of appetite by P, Vermeire S, Opdenakker G (2015) Serum neutrophil gelatinase B- bone-derived lipocalin 2. Nature 543:385–390 associated Lipocalin and matrix Metalloproteinase-9 complex as a Nielsen BS, Borregaard N, Bundgaard JR, Timshel S, Sehested M, surrogate marker for mucosal healing in patients with Crohn's dis- Kjeldsen L (1996) Induction of NGAL synthesis in epithelial cells ease. J Crohn's Colitis 9:1079–1087 of human colorectal neoplasia and inflammatory bowel diseases. Chakraborty S, Kaur S, Guha S, Batra SK (2012) The multifaceted roles Gut 38:414–420 of neutrophil gelatinase associated lipocalin (NGAL) in inflamma- Ostvik AE, Granlund AV, Torp SH, Flatberg A, Beisvag V, Waldum HL, tion and cancer. Biochim Biophys Acta 1826:129–169 Flo TH, Espevik T, Damas JK, Sandvik AK (2013) Expression of Clevers HC, Bevins CL (2013) Paneth cells: maestros of the small intes- toll-like receptor-3 is enhanced in active inflammatory bowel dis- tinal crypts. Annu Rev Physiol 75:289–311 ease and mediates the excessive release of lipocalin 2. Clin Exp Immunol 173:502–511 Devireddy LR, Gazin C, Zhu X, Green MR (2005) A cell-surface recep- tor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Ouellette AJ (2010) Paneth cells and innate mucosal immunity. Curr Opin Cell 123:1293–1305 Gastroenterol 26:547–553 Cell Tissue Res Perminow G, Beisner J, Koslowski M, Lyckander LG, Stange E, Vatn MH, Thorsvik S, Damas JK, Granlund AV, Flo TH, Bergh K, Ostvik AE, Sandvik AK (2017) Fecal neutrophil gelatinase-associated lipocalin Wehkamp J (2010) Defective paneth cell-mediated host defense in pe- diatric ileal Crohn’s disease. Am J Gastroenterol 105:452–459 as a biomarker for inflammatory bowel disease. J Gastroenterol Peterson LW, Artis D (2014) Intestinal epithelial cells: regulators of barrier Hepatol 32:128–135 function and immune homeostasis. Nat Rev Immunol 14:141–153 Wang Y, Lam KS, Kraegen EW, Sweeney G, Zhang J, Tso AW, Chow W- Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015) S, Wat NM, Xu JY, Hoo RL (2007) Lipocalin-2 is an inflammatory Limma powers differential expression analyses for RNA- marker closely associated with obesity, insulin resistance, and hy- sequencing and microarray studies. Nucleic Acids Res 43:e47–e47 perglycemia in humans. Clin Chem 53:34–41 Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Wehkamp J, Salzman NH, Porter E, Nuding S, Weichenthal M, Petras Barasch J (2007) Dual action of neutrophil gelatinase-associated RE, Shen B, Schaeffeler E, Schwab M, Linzmeier R, Feathers RW, lipocalin. J Am Soc Nephrol 18:407–413 Chu H, Lima H Jr, Fellermann K, Ganz T, Stange EF, Bevins CL Simms LA, Doecke JD, Walsh MD, Huang N, Fowler EV, Radford-Smith (2005) Reduced Paneth cell alpha-defensins in ileal Crohn’s disease. GL (2008) Reduced α-defensin expression is associated with in- Proc Natl Acad Sci U S A 102:18129–18134 flammation and not NOD2 mutation status in ileal Crohn’s disease. Worthington JJ (2015) The intestinal immunoendocrine axis: novel cross-talk Gut 57:903–910 between enteroendocrine cells and the immune system during infection Singh V, San Yeoh B, Chassaing B, Zhang B, Saha P, Xiao X, Awasthi D, and inflammatory disease. Biochem Soc Trans 43:727–733 Shashidharamurthy R, Dikshit M, Gewirtz A (2016) Microbiota- YanQ-W,YangQ,ModyN,GrahamTE, HsuC-H,Xu Z, inducible innate immune siderophore binding protein lipocalin 2 is Houstis NE, Kahn BB, Rosen ED (2007) The adipokine critical for intestinal homeostasis. CMGH Cellular and Molecular lipocalin 2 is regulated by obesity and promotes insulin re- Gastroenterology and Hepatology sistance. Diabetes 56:2533–2540 Stallhofer J, Friedrich M, Konrad-Zerna A, Wetzke M, Lohse P, Glas J, Yesil A, Gonen C, Senates E, Paker N, Gokden Y, Kochan K, Erdem ED, Tillack-Schreiber C, Schnitzler F, Beigel F, Brand S (2015) Gunduz F (2013) Relationship between neutrophil gelatinase- Lipocalin-2 is a disease activity marker in inflammatory bowel dis- associated lipocalin (NGAL) levels and inflammatory bowel disease ease regulated by IL-17A, IL-22, and TNF-alpha and modulated by type and activity. Dig Dis Sci 58:2587–2593 IL23R genotype status. Inflammatory bowel diseases

Journal

Cell and Tissue ResearchSpringer Journals

Published: Jun 5, 2018

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