Increased IL-13 expression is independently associated with neo-osteogenesis in patients with chronic rhinosinusitis
2017; Elsevier BV; Volume: 140; Issue: 5 Linguagem: Inglês
10.1016/j.jaci.2017.05.021
ISSN1097-6825
AutoresSakiko Oue, Mahnaz Ramezanpour, S. Paramasivan, Dijana Miljkovic, Clare Cooksley, Ahmed Bassiouni, Judy Ou, Alkis J. Psaltis, Peter‐John Wormald, Sarah Vreugde,
Tópico(s)Osteomyelitis and Bone Disorders Research
ResumoChronic rhinosinusitis (CRS) is a multifactorial inflammatory disease of the paranasal sinus mucosa and might also involve underlying bone, inducing new bone formation. Neo-osteogenesis has been thought to be a possible contributing factor to the pathophysiology of CRS, especially in the recalcitrant group of patients. It can be recognized as areas of increased thickness of the sinus bone, with the overlying sinus lining being irregular on computed tomographic (CT) scans.1Videler W. Gerogalas C. Menger D. Freling N. Van Drunen C. Fokkens W. Osteitic bone in recalcitrant chronic sinusitis.Rhinology. 2011; 49: 139-147PubMed Google Scholar, 2Georgalas C. Videler W. Freling N. Fokkens W. Global Osteitis Scoring Scale and chronic rhinosinusitis: a marker of revision surgery.Clin Otolaryngol. 2010; 35: 455-461Crossref PubMed Scopus (31) Google Scholar Associations between the inflammatory process and neo-osteogenesis in patients with CRS have been previously established3Perloff J. Gannon F.B. Montone W. Orlandi K. Kennedy R. Bone involvement in sinusitis: an apparent pathway for the spread of disease.Laryngoscope. 2000; 110: 2095-2099Crossref PubMed Scopus (110) Google Scholar; however, no studies have specifically used pathological bone samples with associated overlying mucosa to determine gene expression changes. Moreover, the effect of confounding variables, such as polyp status and asthma, in determining differential gene expression has also not been addressed systematically. By using histology, quantitative PCR, immunohistochemistry, and in vitro bone mineralization assays, this study aimed to identify tissue genetic and cellular changes associated with neo-osteogenesis in the context of CRS. Details of the materials and methods used in this study are found in the Methods section in this article's Online Repository at www.jacionline.org. Sinonasal bone and mucosa samples were harvested from patients with CRS with and without CT scan–evident neo-osteogenesis (19 CRSwNeO+ and 19 CRSwNeO−, respectively) and 9 control patients. Details of patients' demographics are summarized in Table E1 in this article's Online Repository at www.jacionline.org. The CRSwNeO+ group had more patients undergoing revision surgery (P = .018), higher average number of previous surgeries (P = .018), worse disease evidenced by a greater average Lund-Mackay score (P = .006), and greater average Global Osteitis Scoring Scale (GOSS; P < .001).2Georgalas C. Videler W. Freling N. Fokkens W. Global Osteitis Scoring Scale and chronic rhinosinusitis: a marker of revision surgery.Clin Otolaryngol. 2010; 35: 455-461Crossref PubMed Scopus (31) Google Scholar Relative mRNA expression of 46 genes involved in bone remodeling and chronic inflammation was determined by using a microfluidic quantitative RT-PCR assay by normalizing to 2 housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH] and hypoxanthine phosphoribosyltransferase 1 [HPRT1]) and to control subjects without CRS. Genes examined are listed in Tables E2 and E3 in this article's Online Repository at www.jacionline.org. Upregulation (of >2 fold) was found for IL13 (284.97-fold, P = .02), CCL13 (107.54-fold, P = .002), colony-stimulating factor 3 (CSF3; 10.68-fold, P = .02), integrin αM (ITGAM; 5.7-fold, P = .005), TNFA (3.2-fold, P = .02), and alkaline phosphatase liver/bone/kidney (ALPL; 2.2-fold, P = .007) in CRSwNeO+ patients compared with control subjects (Fig 1 and see Table E3). Further bivariate models showed that expression of IL13, TNFA, CSF3, ALPL, and COL5A1 was affected independently by the neo-osteogenesis state (P < .05), whereas expression of CCL13 and ITGAM was primarily affected by the asthma status (P < .05). In addition, a significant positive correlation was found between GOSS and IL13 mRNA expression (Pearson product-moment correlation = 0.56, P = .0003; see Fig E1 in this article's Online Repository at www.jacionline.org). Next, we determined IL-13 localization by using immunohistochemical analysis on tissue sections from representative patient groups. IL-13 expression was more abundant and mainly localized in immune cells in CRSwNeO+ compared with CRSwNeO−. IL-13 was also expressed in osteoblasts lining the bone (Fig 2, A-D). An Alizarin Red assay was performed on primary osteoblasts isolated from patients with CRS to determine the IL-13–related osteoblast mineralization (see Fig E2 in this article's Online Repository at www.jacionline.org). There was a significant positive correlation between IL-13 dose and observed calcium-rich mineralization (Pearson product-moment correlation = 0.586; 95% CI, 0.128-0.838; P = .017). Denaturation of IL-13 before osteoblast stimulation saw mineralization levels return to baseline (Fig 2, E and F). We then used Hematoxylin and Eosin and Sirius Red staining on paraffin-embedded tissue sections to determine eosinophil/neutrophil cell numbers and basement membrane thickness in mucosal tissue overlying the bone and in relation to neo-osteogenesis (see Fig E3 in this article's Online Repository at www.jacionline.org). Forty-five patients were assessed, including 30 patients with CRS (neo-osteogenic and non–neo-osteogenic bone from 15 CRSwNeO+ and 15 matched CRSwNeO− patients) and 15 control subjects (demographic information is shown in Table E4 and results are shown in Fig E4 and Table E5 in this article's Online Repository at www.jacionline.org). There were significantly more eosinophils and neutrophils in CRSwNeO+ and CRSwNeO− tissue compared with numbers in control tissue (P < .05). Comparing neo-osteogenic with non–neo-osteogenic areas from matched CRSwNeO+ or CRSwNeO− patients, similar numbers of eosinophils and neutrophils were observed. Similar levels of basement membrane thickness were observed among all patient groups. These histologic results were surprising because previous reports have associated tissue eosinophilia with neo-osteogenesis in patients with CRS.4Snidvongs K. McLachlan R. Chin D. Pratt E. Sacks R. Earls P. et al.Osteitic bone: a surrogate marker of eosinophilia in chronic rhinosinusitis.Rhinology. 2012; 50: 299-305PubMed Google Scholar They suggest that the activation status rather than an increase in eosinophils could contribute to neo-osteogenesis in patients with CRS. Further research is warranted to define the complex interplay of cytokines and their role in eosinophil cell recruitment and activation in patients with CRS. Bone remodeling is a complex physiologic process, with balanced osteoclast and osteoblast activities tightly regulated. In the context of severe ongoing mucosal inflammation, the balance of osteoblastic bone formation and osteoclastic resorption can be disrupted, resulting in neo-osteogenesis.3Perloff J. Gannon F.B. Montone W. Orlandi K. Kennedy R. Bone involvement in sinusitis: an apparent pathway for the spread of disease.Laryngoscope. 2000; 110: 2095-2099Crossref PubMed Scopus (110) Google Scholar Several local inflammatory mediators have been identified as being involved in the bone-remodeling process, including IL-1, IL-6, and TNF.1Videler W. Gerogalas C. Menger D. Freling N. Van Drunen C. Fokkens W. Osteitic bone in recalcitrant chronic sinusitis.Rhinology. 2011; 49: 139-147PubMed Google Scholar, 5Tuszynska A. Krzeski A. Postula M. Wyczalkowska-Tomasik A. Gornicka B. Pykalo R. Inflammatory cytokines gene expression in bone tissue from patients with chronic rhinosinusitis—a preliminary study.Rhinology. 2010; 48: 415-419PubMed Google Scholar In this study expression of 5 genes (TNFA, CSF3, IL13, COL5A1, and ALPL) was significantly upregulated in CRSwNeO+ patients compared with the control group and independently associated with neo-osteogenesis. ALPL is thought to be involved in bony matrix mineralization, and COL5A1 is expressed by osteoblasts and upregulated during mammalian osteogenesis.6Kahai S. Vary C. Gao Y. Seth A. Collagen, type V, alpha1 (COL5A1) is regulated by TBF-beta in osteoblasts.Matrix Biol. 2004; 23: 445-455Crossref PubMed Scopus (48) Google Scholar In contrast to TNFA and CSF3, which promote osteoclastogenesis, TH2 cytokines, such as IL-4 and IL-13, suppress bone resorption by inhibiting COX-2–dependent prostaglandin synthesis in osteoblasts7Onoe Y. Miyaura C. Kaminakayashiki T. Nagai Y. Noguchi K. Chen Q. et al.IL-13 and IL-4 inhibit bone resorption by suppressing cyclooxygenase-2-dependent prostaglandin synthesis in osteoblasts.J Immunol. 1996; 156: 758-764PubMed Google Scholar and osteoclastic bone resorption.8Palmqvist P. Lundberg P. Persson E. Johansson A. Lundgren I. Lie A. et al.Inhibition of hormones and cytokine-stimulated osteoclastogenesis and boe resorption by interleukin-4 and interleukin-13 is associated with increased osteoprotegrin and decreased RANKL and RANK in a STAT-6 dependent pathway.J Biol Chem. 2006; 281: 2414-2429Crossref PubMed Scopus (106) Google Scholar Our results indicate a potential causative role for IL-13 in neo-osteogenesis in patients with CRS evidenced by (1) a significant manifest increase in mRNA and protein expression of IL-13 in CRSwNeO+ patients, (2) our bivariate permutational ANOVA models indicating IL13 mRNA expression levels to independently affect the neo-osteogenesis state, (3) a significant positive correlation between GOSS and IL-13 expression, and (4) a significant positive correlation between IL-13 protein levels and observed mineralization by osteoblasts in vitro. These findings provide a potential pathophysiologic link between increased IL-13 expression found among patients with CRS and the subsequent neo-osteogenesis observed in certain CRS subsets. Further investigation into the specific mechanistic actions of IL-13 on bone remodeling in patients with CRS, as well as its source within sinonasal tissue, might provide further insights into the pathogenesis of neo-osteogenesis in patients with CRS. With IL-13–targeted biological agents currently in different stages of clinical development,9Lam K. Kern R.C. Luong A. Is there a future for biologics in the management of chronic rhinosinusitis?.Int Forum Allergy Rhinol. 2016; 6: 935-942Crossref PubMed Scopus (25) Google Scholar it will be interesting to determine the effect they might have on halting or reversing new bone formation in these patients. The study was approved by the local Human Research Ethics Committee, and written informed consent was obtained from all participants for tissue collection and use of clinical information. Patients recruited to the study included those who were undergoing endoscopic sinus surgery for CRS and patients undergoing endoscopic transsphenoidal pituitary tumor removal who had no clinical or radiologic evidence of CRS as the control group. Clinical information collected from patients included preoperative symptoms, age, allergies, medications, and medical comorbidities. In patients with CRS, the presence of nasal polyps was documented at the time of surgery, and Lund-Mackay scoresE1Lund L. Staging in rhinosinusitis.Rhinology. 1993; 107: 183-184Google Scholar and GOSSE2Georgolas C. Videler W. Freling N. Fokkens W. Global Osteitis Scoring Scale and chronic rhinosinusitis: a marker of revision surgery.Clin Otolaryngol. 2010; 35: 455-461Crossref PubMed Scopus (85) Google Scholar were graded from preoperative sinus CT scans by 2 assessors. Clinically significant neo-osteogenesis was defined by a GOSS of 5 or greater. Patients with CRS were divided into patients with CT scan–evident neo-osteogenesis (CRSwNeO+ group) and patients without CT scan–evident neo-osteogenesis (CRSwNeO− group). Patients who had been receiving oral steroids or antibiotics 1 month before the procedure, those who were immunocompromised, or those less than 18 years of age were excluded from the study. Patients with a history or symptoms of CRS or with endoscopic or radiologic evidence of sinus disease were excluded from the control cohort. During endoscopic sinus surgery, bone and overlying mucosal tissue samples were harvested from the sinonasal cavity. In patients with neo-osteogenesis (CRSwNeO+ patients), separate samples were taken from areas corresponding to radiologic regions of neo-osteogenesis, as well as from areas with no CT scan evidence of neo-osteogenesis. Harvested tissue was placed in Dulbecco modified Eagle medium (DMEM; Gibco, Invitrogen, Melbourne, Victoria, Australia) for transport to the laboratory. For the gene expression study, samples were placed in RNAlater solution (Ambion, Life Technologies, Mulgrave, Victoria, Australia) overnight at 4°C before storage at −80°C. For the histology and immunohistochemistry study, samples were placed in 10% neutral buffered formalin for more than 24 hours, routinely decalcified in formic acid solution, and embedded in paraffin. Samples for flow cytometry were kept in DMEM at 4°C to be analyzed within 24 hours of collection. Approximately 100 mg of sample tissue was weighed and homogenized by using a TissueRuptor (Qiagen, Hilden, Germany). Total RNA was extracted by using an RNeasy Lipid Tissue Mini Kit (Qiagen) per the manufacturer's protocol. An on-column DNase treatment was performed by using an RNase-Free DNase Set (Qiagen). After RNA extraction, quantification was performed by using a NanoDrop 1000 Spectrophometer (Thermo Scientific, Waltham, Mass), and RNA integrity was confirmed with an Experion RNA StdSens Kit with an Electrophoresis Station (Bio-Rad Laboratories, Hercules, Calif) per the manufacturer's protocol. Only RNA samples verified as having an RNA integrity number of 7 or greater were used. cDNA was prepared with a Quantitect Reverse Transcription Kit (Qiagen) and a MyCycler Thermal Cycler (Bio-Rad Laboratories), according to the manufacturer's protocol. Forty-eight TaqMan gene expression assays (Life Technologies, Grand Island, NY) were selected and guided by results of an initial pilot Human Osteogenesis RT2 Profiler Array (Qiagen), and samples with the cDNA and TaqMan gene expression assays were prepared according to the protocol of Fluidigm Gene Expression Specific Target Amplification (Fluidigm, South San Francisco, Calif). cDNA was preamplified for 14 cycles before performing 35 cycles of 48 × 48 Dynamic Array Integrated Fluidic Circuit (Fluidigm). Data were analyzed with Fluidigm Real-Time PCR Analysis Software v4.1.2. ΔCt values were calculated in relation to 2 housekeeping genes (GAPDH and HPRT1), and 2−ΔCt values were calculated according to the ΔCt method by Schmittgen and Livak.E4Ramezanpour M. Moraitis S. Smith J. Wormald P. Vreugde S. Th17 cytokines disrupt the airway mucosal barrier in chronic rhinosinusitis.Mediators Inflamm. 2016; 2016: 1-7Crossref Scopus (53) Google Scholar Fold changes were calculated as ratios of the 2−ΔCt values in relation to the control group. Paraffin-embedded tissue samples were cut at a thickness of 4 μm and stained with hematoxylin and eosin or Sirius Red or processed for immunohistochemistry. Slides were scanned by using digital whole-slide imaging technology (NanoZoomer 2.0-HT, Hamamatsu, Japan) and viewed with NDP.view2software (Hamamatsu). Digital files were deidentified, allowing blind analysis. Tissue eosinophils and neutrophils were identified histologically and quantified according to a protocol detailed previously.E4Ramezanpour M. Moraitis S. Smith J. Wormald P. Vreugde S. Th17 cytokines disrupt the airway mucosal barrier in chronic rhinosinusitis.Mediators Inflamm. 2016; 2016: 1-7Crossref Scopus (53) Google Scholar, E5Bassiouni A. Ou J. Rajiv S. Cantero D. Vreugde S. Wormald P. Subepithelial inflammatory load and basement membrane thickening in refractory chronic rhinosinusitis with nasal polyposis: a histopathological study.Int Forum Allergy Rhinol. 2016; 6: 248-255Crossref PubMed Scopus (30) Google Scholar In short, eosinophil and neutrophil numbers were determined by averaging cell numbers per high-power field (hpf; 0.035 mm2) randomly selected immediately below the epithelium overlying the bone from at least 10 hpfs per slide (see Fig E1). Corresponding Sirius Red–stained sections from the same cohort of patients were analyzed for basement membrane thickness at 2 separate points per hpf per patient then averaged. Thickness was measured by using the linear measurement function within NDP.view2 software. Slides were deparaffinized and rehydrated. Antigen retrieval was induced at 100°C for 10 minutes in 10 mmol/L sodium citrate buffer, pH 6. Slides were then blocked in 25% normal horse serum blocking buffer for 10 minutes and incubated with primary antibodies to IL-13 (rabbit polyclonal [ab9576] at 1:200 dilution; Abcam, Cambridge, United Kingdom) overnight at 4°C. Rabbit serum IgG I5006 (Sigma, St Louis, Mo) was used as a negative control. Specific binding was detected with the Vectastain Universal Quick kit #PK-7800 and DAB Substrate kit #SK-4100 (Vector Laboratories, Burlingame, Calif). Slides were observed with a Nikon Eclipse90I microscope equipped with NIS-Elements AR3.2 software. Bone and mucosa from collected tissue were dissociated under sterile conditions by using a scalpel to harvest primary human osteoblasts from patients with CRS. Isolated bone was washed twice with PBS before placement in 6-well plates with α-MEM medium (M4526, Sigma) supplemented by 1:100 l-glutamine, 10% FBS, 1:100 ascorbic acid 2-phosphate, and 1:100 penicillin streptomycin (Gibco, Life Technologies). Bone was then placed in an incubator at 37°C for 2 to 3 weeks with medium changes every 2 to 3 days until confluent. Primary osteoblasts were seeded in 24-well tissue-culture plates at a density of 2 × 105 grown in DMEM until confluent. Duplicate wells were stimulated with IL-13 recombinant human protein at 10, 100, and 1000 ng/mL (Gibco, Life Technology) in DMEM comprising 10 mmol/L β-glycerophosphate disodium salt hydrate (G9422, Sigma-Aldrich) and 10−8 mol/L dexamethasone (D4902, Sigma-Aldrich) for 21 days. L-Ascorbic acid-2 phosphate (100 μmol/L) was used as a positive control (Sigma-Aldrich), and DMEM and DMEM plus 1000 ng/mL heat-inactivated IL-13 was used as a negative control. Fresh medium with treatments (IL-13) or negative controls were added weekly. After 21 days, cells were washed in PBS and fixed with 10% buffered formalin at room temperature for 15 minutes. Cells were then stained with 2% Alizarin Red (122777, Sigma-Aldrich), pH 4.1, at room temperature for 20 minutes, followed by a final wash with distilled water until all unbound stain was removed. For quantitative analysis, Alizarin Red dye was eluted in 10% (wt/vol) cetylpyridinium chloride (PHR1226, Sigma-Aldrich) in 10 mmol/L phosphate buffer, pH 7.0, for 30 minutes, followed by measuring the OD of the solubilized stain at 570 nm with a microplate fluorometer (FLUOstar Optima; BMG Labtech, Ortenberg, Germany). Paired parametric t tests were used for comparison between CT scan–evident areas of neo-osteogenesis and no CT scan–evident areas of neo-osteogenesis from the CRSwNeO+ group by using a PCR array, histology, and flow cytometry. One-way ANOVA was used for comparison of 3 sample groups in PCR array and histology. These analyses were performed with GraphPad Prism software (version 6; GraphPad software, La Jolla, Calif). Data from Fluidigm Real-Time qPCR Analysis Software v4.1.2 (Fluidigm) were analyzed by using R software (R Foundation for Statistical Computing, Vienna, Austria) on R studio version 0.99.489. A nonparametric permutational ANOVA, as implemented in the 'lmPerm' R package,E6Wheeler B. lmPerm:Permutational tests for linear models.2010Google Scholar was used for comparing 3 groups (CRSwNeO+, CRSwNeO−, and control groups), and Games-Howell yes was used for post hoc analysis. Genes with a nonsignificant differential expression between the comparison groups or with a down-fold or up-fold change of less than 2 were not considered for further analysis. Bivariate permutational ANOVA models were then used to explore the effect of confounding factors in determination of gene expression levels. The Pearson product-moment correlation coefficient was determined by using R software to find correlations of IL-13 dosages with OD values in the Alizarin assay. Statistical significance was defined as a P value of less than .05.Fig E2Representative images from the Alizarin Red mineralization assay. Red staining indicates calcium deposition by osteoblasts. A-D, When compared with control values (Fig E2, A), increasing doses of IL-13 (Fig E2, B, 10 ng/mL; Fig E2, C, 100 ng/mL; Fig E2, D, 1000 ng/mL) stimulated higher deposition by osteoblasts. E, Denatured IL-13 did not have any additional effect over the control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Micrograph of a representative CRSwNeO− patient, showing multiple bone fragments and selection of multiple hpfs measuring 0.035 mm2 beneath the epithelial layer. The inset shows an eosinophil (dashed red arrow points to an eosinophil with a 2-lobed nucleus and intense red eosin-positive staining) and 2 neutrophils (full arrows point to neutrophils harboring a multiple [2-5]–lobed nucleus and neutral pink staining).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Comparison between eosinophil (A) and neutrophil (B) cell counts and basement membrane thickness (C) on histology in samples without neo-osteogenesis in CRSwNeO+ patients (CRSwNeO+ [non-neo-osteogenic area]) and in samples with neo-osteogenesis in the same CRSwNeO+ patients (CRSwNeO+ [neo-osteogenic area]) compared with patients who do not have evidence of neo-osteogenesis (CRSwNeO−) and control subjects without CRS. *P < .05. ns, Nonsignificant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Demographics of patients in the gene expression studyControl subjects (n = 9)CRSwNeO− patients (n = 19)CRSwNeO+ patients (n = 19)Mean age (y)62.2 (51-78)54.4 (25-78)60.9 (31-82)Sex (male/female)7/211/813/6Asthma (%)111142Aspirin sensitive0516Revision surgery05895∗Statistically significant compared with the CRSwNeO− group (P < .05, Fisher exact test or t test as appropriate).Average no. of previous operations01.33.3∗Statistically significant compared with the CRSwNeO− group (P < .05, Fisher exact test or t test as appropriate).Average LM score010.114.3†Statistically significant compared with the CRSwNeO− group (P < .01, Fisher exact test or t test as appropriate).Average GOSSNA1.019.3‡Statistically significant compared with the CRSwNeO− group (P < .001, Fisher exact test or t test as appropriate).CRS with nasal polypsNA5358CRSwNeO+, CRS with neo-osteogenesis evident on CT scan; CRSwNeO−, CRS without neo-osteogenesis evident on CT scan; LM, Lund-Mackay; NA, not assessed.∗ Statistically significant compared with the CRSwNeO− group (P < .05, Fisher exact test or t test as appropriate).† Statistically significant compared with the CRSwNeO− group (P < .01, Fisher exact test or t test as appropriate).‡ Statistically significant compared with the CRSwNeO− group (P < .001, Fisher exact test or t test as appropriate). Open table in a new tab Table E2Genes examined on Fluidigm real-time PCR with its significance to neo-osteogenesis status (bivariate analysis) and associated fold changesGenesDescriptionCRSwNeO+ statusFold change: CRSwNeO− group over control groupFold change: CRSwNeO+ group over control groupFold change: CRSwNeO+ group over CRSwNeO− groupACVRIActivin A receptor, type 1NS———AHSGAlpha-2-HS-glycoproteinNS———ANXA5Annexin A5‡P < .1.0.920.81∗P < .05.0.88BMP3Bone morphogenetic protein 3NS———BMP5Bone morphogenetic protein 5NS———BMPR1ABone morphogenetic protein receptor 1ANS———CALCRCalcitonin receptorNS———CCL5Chemokine (C-C motif) ligand 5NS———CCL11Chemokine (C-C motif) ligand 11CNS———COL1A1Collagen type 1 alpha 1‡P < .1.1.12.82.4COL3A1Collagen type 3 alpha 1NS———COMPCartilage oligometric matrix proteinNS———CSF2Colony-stimulating factor 2NS———CTSKCathepsin KNS———EGFREpidermal growth factor receptorNS———EPXEosinophil peroxidaseNS———FGFR1Fibroblast growth factor receptor 1NS———FN1Fibronectin 1∗P < .05.0.50‡P < .1.0.951.88∗P < .05.GLI1Glabrous1‡P < .1.0.651.18∗P < .05.1.83IGF1Insulin-like growth factor 1NS———IGF1RInsulin-like growth factor 1 receptorNS———IHHIndian hedgehogNS———IL4IL-4NS———IL5IL-5NS———IL6IL-6NS———IL11IL-11NS———ITGB1Integrin beta 1‡P < .1.———LPOLactoperoxidaseNS———MMP2Matrix metalloproteinase 2‡P < .1.0.811.662.05MPOMyeloperoxidaseNS———NOGNogginNS———RUNX2Runt-related transcription factor 2NS———SEPRINH1Serpin peptidase inhibitor, clade A, member 1†P < .01.0.82‡P < .1.1.51‡P < .1.1.84†P < .01.SMAD3SMAD family member 3NS———SP7Sp7 transcription factorNS———SPP1Secreted phosphoprotein 1NS———TGFB1TGF-β1NS———TGFBR2TGF-β receptor 2†P < .01.0.931.48‡P < .1.1.56‡P < .1.TNFSF11TNF (ligand) superfamily, member 11‡P < .1.0.971.611.66GAPDHGlyceraldehyde-3-phosphate dehydrogenaseHousekeeping genesHPRT1Hypoxanthine phosphoribosyltransferase 1NS, P > .1.—, Further evaluation was performed because of gene expression not affected by neo-osteogenesis.CRSwNeO+, CRS with neo-osteogenesis; CRSwNeO−, CRS without neo-osteogenesis.∗ P < .05.† P < .01.‡ P < .1. Open table in a new tab Table E3Significant findings from Fluidigm real-time PCR with significance to neo-osteogenesis status and associated fold changesGenesDescriptionCRSwNeO+ statusFold change: CRSwNeO− group over control groupFold change: CRSwNeO+ group over control groupFold change: CRSwNeO+ group over CRSwNeO− groupALPLAlkaline phosphatase liver/bone/kidney†P < .01.1.002.22†P < .01.2.34†P < .01.CCL13Chemokine (C-C motif) ligand 13∗P < .05.55.60∗P < .05.107.54†P < .01.1.93COL5A1Collagen type 5 alpha 1∗P < .05.0.861.872.17∗P < .05.CSF3Colony-stimulating factor 3∗P < .05.2.6410.68∗P < .05.4.04‡P < .1.IL13IL-13∗P < .05.50.94∗P < .05.284.97∗P < .05.5.59‡P < .1.ITGAMIntegrin alpha M†P < .01.2.425.70†P < .01.2.35‡P < .1.TNFATNF-α†P < .01.1.093.20∗P < .05.2.95∗P < .05.GAPDHGlyceraldehyde-3-phosphate dehydrogenaseHousekeeping genesHPRT1Hypoxanthine phosphoribosyltransferase 1CRSwNeO+, CRS with neo-osteogenesis; CRSwNeO−, CRS without neo-osteogenesis.∗ P < .05.† P < .01.‡ P < .1. Open table in a new tab Table E4Demographics of patients in histologic studyControl subjects (n = 15)CRSwNeO− patients (n = 15)CRSwNeO+ patients (n = 15)Mean age (y)5652.855.3Sex (male/female)8/710/511/4Asthma (%)06747Aspirin sensitive (%)02027Average no. of previous operations01.932.4Average LM score013.813.2Average SNOT-22 scoreNA50.752.8CRS with nasal polyps (%)07387CRSwNeO+, CRS with neo-osteogenesis evident on CT scan; CRSwNeO−, CRS without neo-osteogenesis evident on CT scan; LM, Lund-Mackay; NA, data not assessed; SNOT-22, Sino-Nasal Outcome Test. Open table in a new tab Table E5Inflammatory cells and basement membrane thickness from histologic studyControl subjects (n = 15)CRSwNeO− patients (n = 15)CRSwNeO+ patients (n = 15)Neo-osteogenic area on CT scanNon-neo-osteogenic area on CT scanEosinophil/0.035 mm20.0722.946.277.06Neutrophil/0.035 mm20.0170.240.340.25Average BM thickness (μm)5.115.375.105.15BM, Basement membrane; CRSwNeO+, CRS with neo-osteogenesis evident on CT scan; CRSwNeO−, CRS without neo-osteogenesis evident on CT scan. Open table in a new tab CRSwNeO+, CRS with neo-osteogenesis evident on CT scan; CRSwNeO−, CRS without neo-osteogenesis evident on CT scan; LM, Lund-Mackay; NA, not assessed. NS, P > .1. —, Further evaluation was performed because of gene expression not affected by neo-osteogenesis. CRSwNeO+, CRS with neo-osteogenesis; CRSwNeO−, CRS without neo-osteogenesis. CRSwNeO+, CRS with neo-osteogenesis; CRSwNeO−, CRS without neo-osteogenesis. CRSwNeO+, CRS with neo-osteogenesis evident on CT scan; CRSwNeO−, CRS without neo-osteogenesis evident on CT scan; LM, Lund-Mackay; NA, data not assessed; SNOT-22, Sino-Nasal Outcome Test. BM, Basement membrane; CRSwNeO+, CRS with neo-osteogenesis evident on CT scan; CRSwNeO−, CRS without neo-osteogenesis evident on CT scan.
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