Otolaryngology http://oto.sagepub.com/ -- Head and Neck Surgery
Immunomodulatory Effect of Mesenchymal Stem Cells on T Lymphocyte and Cytokine Expression in Nasal Polyps Kyu-Sup Cho, Yong-Wan Kim, Myoung Joo Kang, Hee-Young Park, Sung-Lyong Hong and Hwan-Jung Roh Otolaryngology -- Head and Neck Surgery published online 13 March 2014 DOI: 10.1177/0194599814525751 The online version of this article can be found at: http://oto.sagepub.com/content/early/2014/03/06/0194599814525751
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Original Research
Immunomodulatory Effect of Mesenchymal Stem Cells on T Lymphocyte and Cytokine Expression in Nasal Polyps
Otolaryngology– Head and Neck Surgery 1–9 Ó American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0194599814525751 http://otojournal.org
Kyu-Sup Cho, MD, PhD1*, Yong-Wan Kim, MD, PhD2*, Myoung Joo Kang, MD3, Hee-Young Park1, Sung-Lyong Hong, MD1, and Hwan-Jung Roh, MD, PhD4
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Keywords immunosuppression, adipose tissue, mesenchymal stem cells, nasal polyps, T lymphocytes, cytokines
Abstract Objectives. Adipose tissue–derived stem cells (ASCs) have been reported to have immunomodulatory effects in various inflammatory diseases, including asthma and allergic rhinitis, through the induction of T cell anergy. Nasal polyps (NPs) are a chronic inflammatory disease in the nose and paranasal sinus characterized histologically by the infiltration of inflammatory cells, such as eosinophils or lymphocytes. This study was performed to investigate whether ASCs have immunomodulatory effects on T lymphocyte and cytokine expression in eosinophilic NPs. Study Design. Basic science experimental study. Setting. University tertiary care facility. Subjects and Methods. NP specimens were obtained from 20 patients with chronic rhinosinusitis and eosinophilic NPs. ASCs were isolated and cultured from the abdominal fat of 15 subjects undergoing intra-abdominal surgery. Infiltrating cells (1 3 106) were isolated from NP tissue and cocultured with 1 3 105 ASCs. To determine whether ASCs affect infiltrating T lymphocyte and cytokine expression in eosinophilic NP, T lymphocyte subsets and cytokine expression were analyzed before and after ASC treatment. Results. ASC treatment significantly decreased the proportions of CD41 and CD81 T cells. After ASC treatment, Th2 cytokine (interleukin [IL]–4 and IL-5) levels decreased significantly. In contrast, levels of Th1 (interferon-g and IL-2) and regulatory cytokines (transforming growth factor–b and IL-10) increased significantly after ASC treatment. Conclusions. ASCs have immunomodulatory effects in the eosinophilic inflammation of NPs, characterized by downregulation of activated T lymphocytes and a Th2 immune response. These effects would be expected, over time, to significantly contribute to the control of eosinophilic inflammation and, possibly, growth of eosinophilic NPs.
Received September 4, 2013; revised January 23, 2014; accepted February 6, 2014.
Introduction Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesenchymal lineages, such as bone,1-3 cartilage,1,2 adipose tissue,1,2 and muscle.1,4 Because of their capacity for differentiation, MSCs have emerged as a promising source for therapeutic applications in tissue engineering and regenerative medicine.5,6 In addition to their multilineage potential, MSCs derived from adipose tissue (ASCs) may share with other MSCs the unique ability to suppress immune responses and modulate inflammation.7 MSCs can inhibit natural killer cell function,8,9 modulate dendritic cell maturation,10 and suppress the allogeneic T cell response8 by altering the cytokine secretion profile of dendritic cells and T cells induced by an allogeneic immune reaction. 1 Department of Otorhinolaryngology and Biomedical Research Institute, Pusan National University School of Medicine, Pusan National University Hospital, Busan, South Korea 2 Department of Otorhinolaryngology, Inje University Haeundae Paik Hospital, Busan, South Korea 3 Department of Hemato-Oncology, Inje University Haeundae Paik Hospital, Busan, South Korea 4 Department of Otorhinolaryngology and Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, South Korea * These authors contributed equally to this article.
Corresponding Author: Hwan-Jung Roh, MD, PhD, Department of Otorhinolaryngology, Pusan National University Yangsan Hospital, Beom-eo li, Mul-geum eup, Yang-san si, Gyeongsangnam-do 626-770, South Korea Email: [email protected]
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Figure 1. Histologic findings in nasal polyps (NPs). (A) Eosinophilic NPs contained an abundance of eosinophils. (B) Noneosinophilic NPs showed a predominant accumulation of lymphocytes and neutrophils with fewer eosinophils (hematoxylin and eosin, 4003).
Nasal polyps (NPs) are a chronic inflammatory disease of the mucous membranes in the nose and paranasal sinuses, and its frequent recurrence is clinically challenging for clinicians. An NP is characterized by an edematous mass of hyperplastic epithelium and lamina propria prolapsing into the nose, leading to nasal obstruction, hypersecretion, loss of the sense of smell, and reduced quality of life.11 Histopathologically, NPs are characterized by the infiltration of inflammatory cells, such as eosinophils, neutrophils, and lymphocytes.12 Most NPs contain abundant eosinophils, but some show only a few eosinophils. Recent reports have suggested immunohistologic features of eosinophilic NPs that differ from those of noneosinophilic NPs.12,13 Eosinophilic NPs are characterized generally by Th2-skewed eosinophilic inflammation, with high interleukin (IL)–4 and IL-5 concentrations, but noneosinophilic NPs have a lower degree of total inflammatory cell infiltration and a higher frequency of neutrophil infiltration than eosinophilic NPs.14-18 Although several recent studies have shown that MSCs inhibit eosinophilic inflammation and have immunomodulatory effects in allergic rhinitis and an asthma mouse model,19-21 no immunomodulatory effect of MSCs on NPs showing predominantly eosinophilic infiltration has been reported to date. Thus, we hypothesized that MSCs would be capable of modulating inflammatory cell and cytokine production in eosinophilic NP. The purpose of this study was to evaluate the effects of ASCs on the T lymphocytes and cytokines that modulate eosinophilic inflammation in NP.
Subjects and Methods Demographics and Clinical Characteristics After the purpose and methods of the work were explained to each participant, all provided written informed consent. When child participants were involved, written informed consent was obtained from their parents. This study was approved by the Institutional Review Board of Pusan National University Hospital (H-1209-006-010). Eleven male and 9 female patients, ranging in age from 12 to 61 years (mean, 35.2 years), who had chronic rhinosinusitis (CRS) with eosinophilic NPs and underwent endoscopic sinus surgery were studied. CRS with NPs was defined on
the basis of the current American guidelines.22 Patients with obvious NPs on endoscopic examination and positive results on computed tomographic scans of the paranasal sinus, and for whom control of the symptoms with medical therapy had failed, were included. Patients with histories of endoscopic sinus surgery, antrochoanal polyps, allergic fungal sinusitis, immunodeficiency disorders, aspirin-exacerbated respiratory disease, and cystic fibrosis were excluded. The control group included 11 patients with nasal septal deviation without CRS. None of the patients had an acute infection of the upper respiratory tract, and none had received oral or topical corticosteroids, nonsteroidal antiinflammatory drugs, macrolide antibiotics, or antihistamines in the 4 weeks before endoscopic sinus surgery. Only patients without allergies were included, to exclude any confounding influence of type I allergies. During surgery under general anesthesia, NP tissues were collected and mucosa samples were taken from the inferior turbinate in the control group. The polyp tissues and mucosa were divided into 2 parts. Half of the tissue was fixed in 10% formalin and embedded in paraffin wax for histologic examination, and the other half was used to isolate infiltrating cells for analysis of T lymphocyte subsets and cytokine profiles.
Histologic Analysis Sections (4 mm) were obtained from the paraffin wax– embedded specimens and stained with hematoxylin and eosin. The sections were randomly indicated and observed by 1 pathologist under a light microscope at 4003 magnification. NPs were classified as eosinophilic or noneosinophilic according to the predominant inflammatory cells. In eosinophilic NPs, it was confirmed that eosinophils constituted .75% of the inflammatory cells infiltrating in the polyp tissue (Figure 1).
Isolation and Culture of ASCs Adipose tissue was obtained, after obtaining informed patient consent, from 15 subjects undergoing conventional and laparoscopic intra-abdominal surgeries, 7 men and 8 women aged 45 to 55 years (mean, 47.5 years).
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To isolate human ASCs, adipose tissues were washed extensively with phosphate-buffered saline (PBS), and tissues were digested with 0.075% collagenase type I (Sigma, St Louis, Missouri) at 37°C for 30 minutes. Enzyme activity was neutralized with a-modified Eagle’s medium containing 10% fetal bovine serum, and the sample was centrifuged (1200g, 10 minutes) to obtain a pellet. The pellet was suspended and filtered through a 100-mm nylon mesh to remove cellular debris and incubated overnight at 37°C in 5% CO2/air in control medium (a-modified Eagle’s medium, 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin). After incubation, the plates were washed extensively with PBS to remove residual nonadherent red blood cells. The resulting cell population was maintained at 37°C in 5% CO2/air in control medium. Then, 1 week later, when the monolayer of adherent cells had reached confluence, cells were trypsinized (0.05% trypsin–ethylenediaminetetraacetic acid; Sigma), resuspended in a-modified Eagle’s medium containing 10% fetal bovine serum, and subcultured at a concentration of 2000 cells/cm2, with the medium changed twice per week.
Immunophenotypic Analysis of ASCs Flow cytometric analysis was used to characterize the phenotypes of the ASCs. At least 50,000 cells (in 100-mL PBS, 0.5% bovine serum albumin, 2 mmol/L ethylenediaminetetraacetic acid) were incubated with fluorescein isothiocyanate–labeled monoclonal antibodies against human CD90, CD44, CD73, CD45, CD31, and HLA-DR (BD Biosciences Clontech, Palo Alto, California). After washing, labeled cells were analyzed by flow cytometry using a FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences, San Diego, California).
Multilineage Differentiation of ASCs ASCs were analyzed for their capacity to differentiate toward the adipogenic, osteogenic, and chondrogenic lineages, as described previously.23 For adipogenic and osteogenic differentiation, cells were seeded in 6-well plates at a density of 20,000 cells/cm2 and treated for 3 weeks with adipogenic and osteogenic medium. Adipogenic and osteogenic differentiation was assessed using oil red O staining as an indicator of intracellular lipid accumulation and alizarin red S staining for extracellular matrix calcification, respectively. Chondrogenic differentiation was induced using the micromass culture technique. Briefly, 10 mL of a concentrated MSC suspension (3 3 105 cells/mL) were plated in the center of each well and treated for 3 weeks with chondrogenic medium. Chondrogenesis was confirmed by immunohistochemistry.
Isolation of Infiltrating Cells from Polyp Tissue and CoCulture with ASCs Infiltrating cells were isolated from polyp tissue using the previously published method of Zhang et al.24 Briefly, tissue samples were aseptically cut into small fragments and
incubated with 100 U/mL collagenase type IV (Sigma) and 100 mg/mL DNase I (R&D Systems, Minneapolis, Minnesota) for 60 min at 37°C in a shaking water bath. Fragments were disrupted by grinding and passage through a cell strainer (BD Falcon, Bedford, Massachusetts). Isolated cells were then washed with PBS. Isolated cells (1 3 106) from polyp tissues were cocultured with 1 3 105 ASCs in 12-well tissue culture plates (Corning Inc, Corning, New York) in the presence of 10 mg/mL phytohemagglutinin (PHA; Sigma). After 24 hours, cells were harvested for flow cytometric analysis, and supernatants were collected for cytokine measurement.
Analysis of T-Lymphocyte Subsets Isolated cells were washed with FACS buffer (PBS containing 1% fetal calf serum and 0.05% NaN3). Next, cells were incubated with purified human immunoglobulin G (Sigma) for 15 minutes at 4°C to block nonspecific binding of antibodies to the Fc receptor and then stained with phycoerythrinconjugated antihuman CD4 (L3T4; eBioscience, San Diego, California) and fluorescein isothiocyanate–conjugated antihuman CD8 (RPA-T8; e-Bioscience) for 30 minutes at 4°C. Fluorescein isothiocyanate–conjugated mouse immunoglobulin G1 and phycoerythrin-conjugated mouse immunoglobulin G2a antibodies (eBioscience) were used as isotype controls. After staining, cells were washed with FACS buffer and analyzed using a FACSCalibur flow cytometer.
Detection of Cytokine Expression After 24 hours of co-culture, IL-2, IL-4, IL-5, IL-10, IL-13, tumor necrosis factor–a, interferon (IFN)-g, and transforming growth factor (TGF)–b levels in the supernatant were determined by sandwich enzyme-linked immunosorbent assay (eBioscience). Briefly, microtiter plates (Costar, Cambridge, Massachusetts) were coated with the capture antibody at 4°C overnight. After blocking with 2% bovine serum albumin in PBS for 1 hour, undiluted supernatant was added and incubated for 2 hours at room temperature. Subsequently, the cytokine levels were detected with a biotin-conjugated detecting antibody and developed with horseradish peroxidase–conjugated streptavidin and tetramethylbenzidine as a substrate (Endogen, Rockford, Illinois). Optical densities were measured at 450 nm using an enzyme-linked immunosorbent assay plate reader (Molecular Devices, Sunnyvale, California).
Statistical Analysis All experiments were repeated a minimum of 3 times. Data are expressed as medians and interquartile ranges (IQRs). SPSS for Windows version 20.0 (SPSS, Inc, Chicago, Illinois) was used to perform all statistical analysis. Wilcoxon’s signed-rank tests were used to evaluate differences before and after PHA stimulation and before and after ASC treatment. Differences between patients with eosinophilic NPs and controls were evaluated using MannWhitney U tests. P values \ .05 were considered to indicate statistical significance.
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Figure 2. Characteristics of adipose tissue–derived stem cells (ASCs). ASCs show characteristics of mesenchymal stem cells in the immunophenotypic analysis (A), fibroblast-like morphology (B), adipogenesis (C), osteogenesis (D), and chondrogenesis (E) (original magnification 2003).
Results Isolation, Immunophenotypic Analysis, and Multilineage Differentiation of ASCs ASCs were positive for CD90, CD44, and CD73 and negative for CD45, CD31, and HLA-DR (Figure 2A). At an initial plating density of 1 3 106 cells/cm2, adipose tissue– derived fibroblastoid cells formed a monolayer 4 to 5 days after initial plating. These putative ASCs were of serpiginous or fibroblast-like morphology, similar to previously reported ASCs and bone marrow–derived MSCs (Figure 2B). This morphology was maintained during passage in monolayer culture. Adipogenic differentiation was demonstrated by the accumulation of neutral lipid vacuoles by oil red O staining. A significant fraction of the cells contained multiple, intracellular lipid-filled droplets that stained with oil red O (Figure 2C). Osteogenic differentiation was confirmed by the deposition of alizarin red S–stained mineralized matrix. Calcification appeared as red regions within the cell monolayer (Figure 2D). Chondrogenic differentiation was confirmed by the formation of a sphere in micromass culture (Figure 2E).
Infiltrating T Lymphocyte Subsets Infiltrating T lymphocyte populations in eosinophilic NPs were compared with those from control mucosa. The numbers of CD41 and CD81 T cells were significantly higher in eosinophilic NPs compared with control mucosa before PHA stimulation and after ASC treatment. To determine whether ASCs affected T lymphocyte infiltrating NP tissue, T cell populations were determined after PHA stimulation and ASC treatment. Both CD41 and CD81 T cells were detected in polyp tissue, and the percentages of CD41 and CD81 T cells were increased significantly by PHA stimulation from 3.2% (IQR, 2.3%-4.3%) and 6.4% (IQR, 5.1%7.9%) to 11.8% (IQR, 10.2%-13.1%) and 18.6% (IQR, 16.3%-20.4%) (P = .001 and P = .003, respectively). ASC treatment also significantly decreased the percentages of CD41 and CD81 T cells to 3.5% (IQR, 1.3%-5.8%) and 7.5% (IQR, 5.3%-9.6%) (P \ .001 and P = .009, respectively; Table 1, Figure 3). However, in the control group, there were no significant differences in CD41 or CD81 T cells after ASC treatment, although the percentages of CD41 and CD81 T cells were increased significantly by PHA stimulation from 1.2% (IQR, 0.1%-2.5%) and 1.8% (IQR, 0.2%-3.4%) and to 6.3% (IQR, 4.8%-8.0%) and 7.4%
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Table 1. T Lymphocyte and Cytokine Expressions in Eosinophilic NPs. NPs 1 PHA
NPs CD41 T cells CD81 T cells IL-2 IL-4 IL-5 IL-10 IL-13 TNF-a IFN-g TGF-b
3.2 (2.3-4.3) 6.4 (5.1-7.9) 6.3 (4.4-10.2) 4.8 (4.4-6.4) 18.0 (16.4-18.5) 64.1 (20.7-67.8) 337.8 (276.8-362.7) 36.6 (26.3-59.6) 32.2 (31.2-33.8) 109.7 (106.7-125.9)
11.8 18.6 15.9 16.6 33.2 180.1 370.4 189.5 566.5 119.1
(10.2-13.1) (16.3-20.4) (12.5-18.8) (13.8-20.1) (28.1-38.4) (126.5-192.6) (306.9-481.2) (158.4-233.3) (463.0-691.7) (104.4-150.2)
NPs 1 PHA 1 ASCs 3.5 7.5 22.1 12.1 27.4 236.5 355.7 176.4 831.0 145.3
(1.3-5.8) (5.3-9.6) (17.3-27.1) (9.5-16.8) (20.6-32.6) (201.2-257.4) (298.3-464.1) (123.1-197.5) (628.8-940.9) (121.6-180.0)
Pa
Pb
.001 .003 .015 .021 .005 .019 .078 .001 \.001 .521
\.001 .009 .003 .031 .028 .042 .067 .225 .001 .012
Abbreviations: ASC, adipose tissue–derived stem cells; IL, interleukin; IFN, interferon; NP, nasal polyp; PHA, phytohemagglutinin; TGF, transforming growth factor; TNF, tumor necrosis factor. a NPs versus NPs plus PHA. b NPs plus PHA versus NPs plus PHA plus ASCs.
Figure 3. Analysis of infiltrating T lymphocyte subsets in nasal polyps (NPs) and control mucosa. The R2 and R3 regions indicate the percentages of CD41 and CD81 T cells relative to the total numbers of infiltrating cells in eosinophilic NPs (A) and control mucosa (B). The percentages of CD41 and CD81 T cells were decreased significantly by adipose tissue–derived stem cell (ASC) treatment in eosinophilic NPs (C), but not in the control mucosa (D). PHA, phytohemagglutinin.
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Table 2. T Lymphocyte and Cytokine Expressions in Control Nasal Mucosa. Control CD41 T cell CD81 T cell IL-2 IL-4 IL-5 IL-10 IL-13 TNF-a IFN-g TGF-b
1.2 1.8 2.1 1.7 4.5 11.8 5.1 4.7 3.2 11.0
(0.1-2.5) (0.2-3.4) (1.5-2.5) (1.4-2.6) (3.4-6.1) (5.1-13.2) (3.5-9.2) (2.5-6.1) (2.8-4.0) (10.7-13.4)
Control 1 PHA
Control 1 PHA 1 ASCs
Pa
Pb
6.3 (4.8-8.0) 7.4 (4.9-9.5) 3.1 (2.4-3.1) 3.1 (2.3-3.9) 5.2 (4.7-7.1) 24.1 (12.4-26.9) 14.1 (8.5-18.5) 8.5 (5.9-10.7) 5.8 (4.8-7.1) 23.5 (18.6-27.4)
5.1 (3.0-7.9) 6.3 (4.9-8.1) 3.7 (3.2-5.4) 2.9 (2.4-4.3) 5.9 (5.1-7.8) 29.8 (11.5-42.4) 11.9 (10.1-13.9) 7.2 (6.5-10.4) 6.7 (5.2-8.6) 23.5 (18.6-28.7)
.001 .003 .032 .254 .452 .087 .584 .685 .016 .008
.078 .067 .748 .542 .345 .138 .686 .756 .893 .893
Abbreviations: ASC, adipose tissue–derived stem cells; IL, interleukin; IFN, interferon; PHA, phytohemagglutinin; TGF, transforming growth factor; TNF, tumor necrosis factor. a Control versus control plus PHA. b Control plus PHA versus control plus PHA plus ASCs.
(IQR, 4.9%-9.5%) (P = .001 and P = .003, respectively; Table 2, Figure 3).
Analysis of Cytokine Expression Significantly higher levels of all cytokines were found in patients with eosinophilic NPs than in controls before PHA stimulation and after ASCs treatment. To determine whether ASCs affected cytokine expression in NP tissue, cytokines were analyzed after PHA stimulation and ASC treatment. NPs showed a mixed Th1 and Th2 cytokine profile, and significantly higher expression was observed after PHA stimulation in IL-2 (6.3 pg/mL [IQR, 4.4-10.2 pg/mL] vs 15.9 pg/ mL [IQR, 12.5-18.8 pg/mL], P = .015), IL-4 (4.8 pg/mL [IQR, 4.4-6.4 pg/mL] vs 16.6 pg/mL [IQR, 13.8-20.1 pg/ mL], P = .021), IL-5 (18.0 pg/mL [IQR, 16.4-18.5 pg/mL] vs 33.2 pg/mL [IQR, 28.1-38.4 pg/mL], P = .005), IL-10 (64.1 pg/mL [IQR, 20.7-67.8 pg/mL] vs 180.1 pg/mL [IQR, 126.5-192.6 pg/mL], P = .019), tumor necrosis factor-a (36.6 pg/mL [IQR, 26.3-59.6 pg/mL] vs 189.5 pg/mL [IQR, 158.4-233.3 pg/mL], P = .001), and IFN-g (32.2 pg/mL [IQR, 31.2-33.8 pg/mL] vs 566.5 pg/mL [IQR, 463.0-691.7 pg/mL], P \ .001). After ASC treatment, Th2 cytokine (IL4 and IL-5) levels were decreased significantly to 12.1 pg/ mL (IQR, 9.5-16.8 pg/mL) and 27.4 pg/mL (IQR, 20.6-32.6 pg/mL) (P = .031 and P = .028, respectively). In contrast, Th1 cytokine (IL-2 and IFN-g) levels were increased significantly to 22.1 pg/mL (IQR, 17.3-27.1 pg/mL) and 831.0 pg/mL (IQR, 628.8-940.9 pg/mL) (P = .003 and P = .001, respectively). Moreover, after ASC treatment, regulatory cytokine (IL-10 and TGF-b) levels were increased significantly to 236.5 pg/mL (IQR, 201.2-257.4 pg/mL) and 145.3 pg/mL (IQR, 121.6-180.0 pg/mL) (P = .042 and P = .012, respectively). There was no significant difference in IL-13 or tumor necrosis factor-a levels before and after ASC treatment (Table 1, Figure 4). In the control group, after PHA stimulation, cytokine levels were increased significantly in IL-2 (2.1 pg/mL [IQR, 1.5-2.5 pg/mL] vs 3.1 pg/mL [IQR, 2.4-3.1 pg/mL],
P = .032), IFN-g (3.2 pg/mL [IQR, 2.8-4.0 pg/mL] vs 5.8 pg/mL [IQR, 4.8-7.1 pg/mL], P = .016), and TGF-b (11.0 pg/mL [IQR, 10.7-13.4 pg/mL] vs 23.5 pg/mL [IQR, 18.627.4 pg/mL], P = .008]. However, there was no significant difference in the level of any cytokine before and after ASC treatment (Table 2).
Discussion MSCs have been reported to have anti-inflammatory effects in experimental models of acute pulmonary and renal injury.25,26 MSCs have also been shown to cause attenuation of experimentally induced autoimmune encephalomyelitis through the induction of T cell anergy.27 More recently, ASCs were shown to ameliorate experimental airway allergic diseases, such as allergic rhinitis and asthma, by shifting to a Th1-biased from a Th2-biased immune response.19-21 Histologically, eosinophilic NPs have been thought to be characterized by predominantly eosinophilic infiltration, similar to allergic rhinitis and asthma.11 This study was performed to assess whether ASCs possess immunomodulatory properties in eosinophilic NPs, a common chronic inflammatory disease of the upper airways. Among MSCs, ASCs were used because of their abundance, relatively easy harvesting, and high proliferation potential. To our knowledge, this is the first reported study to investigate the effects of ASCs on T lymphocytes and cytokine expression in NPs. NPs are a chronic inflammatory disease in the nose and paranasal sinuses and are commonly associated with CRS. NPs involve a complex inflammatory process regulated by various chemical mediators and cytokines produced by inflammatory cells in the sinonasal mucosa.11,14 However, eosinophilic and noneosinophilic NPs display distinct features on the basis of histopathology and the expression of inflammatory mediators. Eosinophilic NPs have been characterized by Th2-skewed eosinophilic inflammation with defective functions in regulatory T (Treg) cells, and infiltration of natural killer T cells, whereas noneosinophilic NPs showed a predominant Th1 situation with adequate Treg
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Figure 4. Cytokine expression in nasal polyps. After adipose tissue–derived stem cell (ASC) treatment, interleukin (IL)–4 and IL-5 levels were decreased significantly. In contrast, IL-2, IL-10, interferon (IFN)–g, and transforming growth factor (TGF)–b levels were increased significantly after ASC treatment.
cell function.17 Investigations of subsets of lymphocytes in eosinophilic NPs indicate that T lymphocytes predominate over B cells and that there are more CD81 T cells than CD41 T cells.16 Consistently, our study showed the accumulation of CD41 and CD81 T cells, including this distinctive pattern of increased CD81 T cells in eosinophilic NPs. These data suggest that activated T cells, both the CD41 and CD81 subtypes, contribute to the pathogenesis of NPs. Moreover, this is consistent with evidence that NPs involve a mixed profile of Th1 and Th2 cytokines because Th1 cytokines may be secreted by the increased CD 81 T cell population in eosinophilic NPs.12,15
The present study showed immunomodulatory effects of ASCs on T lymphocytes and cytokine expression in eosinophilic NPs. In terms of eosinophilic inflammation, Th1 and Th2 cytokines are mutually antagonistic, and selective suppression of Th2 cytokines may be crucial for protection against eosinophilic inflammation.15,20 ASC treatment significantly reduced the percentage of CD41 and CD81 T cells in eosinophilic NPs. In addition to decreased T cell infiltration, ASC treatment significantly decreased Th2 cytokines (IL-4 and IL-5) and increased Th1 (IFN-g and IL-2) and regulatory (TGF-b and IL-10) cytokines in eosinophilic NPs. These data are consistent with previous reports that
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ASCs attenuate allergic airway inflammation in asthma and allergic rhinitis.19-21 Taken together with inhibition of eosinophilic inflammation by ASCs, these findings indicate that ASCs have immunomodulatory effects in eosinophilic NP, one of which is down-regulating Th2 cytokines and upregulating Th1 and regulatory cytokines. However, there was no significant difference in the number of T lymphocytes or cytokine expression before and after ASC treatment in control mucosa because T lymphocytes and cytokine expression were much lower than in polyp tissue. Despite the general consensus that MSCs inhibit T cell proliferation, little is known about the exact molecular mechanism responsible for this effect. Cell-cell contact and soluble factors, such as TGF-b1 and prostaglandin E2, are thought to be responsible for the effect.28,29 In part, T cell suppression by MSCs appears to depend on cross-talk between T cells and MSCs, leading to the production of IFN-g with indoleamine 2,3-dioxygenase activity, which in turn inhibits the proliferation of activated T and/or natural killer T cells.30 Further studies are required to fully characterize the immunomodulatory mechanism of ASCs in eosinophilic inflammation, whether by direct contact of ASCs with T lymphocytes or by secretion of crucial mediators of ASCs in eosinophilic NPs. Recently, a number of studies have shown that enhanced Th17 response existed not only in CRS without NPs with neutrophil-biased inflammation but also in CRS with eosinophilic and noneosinophilic NPs.17,31 It has been reported that an impaired balance of Th17/Treg (increased Th17 proportion and decreased Treg proportion) may play an important role in the development of NPs.32 Moreover, glucocorticoid treatment induced immunomodulation in NP via up-regulation of Treg cells and down-regulation of the Th2 and Th17 response.33 Taken together with these findings, ASCs may have downregulating effects of Th17 response in eosinophilic NPs. However, the exact role of Th17 response in the immunomodulatory mechanism of ASCs needs further investigation. This study had a number of limitations inherent to an in vitro study. Because the T lymphocyte numbers and cytokine expression tended to be low in polyp tissue and resting T lymphocytes isolated from NPs do not proliferate without PHA, T lymphocyte activation and proliferation were induced by PHA. Therefore, there was no significant difference before and after ASC treatment in the absence of PHA (data not shown). Because in vitro studies do not always reflect the in vivo situation, the immunomodulatory effect of ASCs in patients with NPs should be evaluated further.
Conclusions This study showed that ASC treatment has an immunomodulatory effect in eosinophilic inflammation associated with eosinophilic NPs, which is characterized by down-regulation of activated T lymphocytes and a Th2 immune response. These effects would be expected, over time, to contribute significantly to the control of eosinophilic inflammation and, possibly, the growth of eosinophilic NPs.
Author Contributions Kyu-Sup Cho, drafting and revising the article; Yong-Wan Kim, drafting the article; Myoung Joo Kang, analysis and interpretation of data; Hee-Young Park, acquisition of data; Sung-Lyong Hong, acquisition of data; Hwan-Jung Roh, conception and design.
Disclosures Competing interests: None. Sponsorships: None. Funding source: This work (data collection and analysis) was supported by a 2013 research grant from Inje University.
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14. Bachert C, Gevaert P, Holtappels G, van Cauwenberge P. Mediators in nasal polyposis. Curr Allergy Asthma Rep. 2002; 2:481-487. 15. Otto BA, Wenzel SE. The role of cytokines in chronic rhinosinusitis with nasal polyps. Curr Opin Otolaryngol Head Neck Surg. 2008;16:270-274. 16. Kirtsreesakul V. Update on nasal polyps: etiopathogenesis. J Med Assoc Thai. 2005;88:1966-1972. 17. Cao PP, Li HB, Wang BF, et al. Distinct immunopathologic characteristics of various types of chronic rhinosinusitis in adult Chinese. J Allergy Clin Immunol. 2009;124:478-484. 18. Van Crombruggen K, Zhang N, Gevaert P, Tomassen P, Bachert C. Pathogenesis of chronic rhinosinusitis: inflammation. J Allergy Clin Immunol. 2011;128:728-732. 19. Park HK, Cho KS, Park HY, et al. Adipose-derived stromal cells inhibit allergic airway inflammation in mice. Stem Cells Dev. 2010;19:1811-1818. 20. Cho KS, Park HK, Park HY, et al. IFATS collection: immunomodulatory effects of adipose tissue-derived stem cells in an allergic rhinitis mouse model. Stem Cells. 2009;27:259-265. 21. Sun YQ, Deng MX, He J, et al. Human pluripotent stem cellderived mesenchymal stem cells prevent allergic airway inflammation in mice. Stem Cells. 2012;30:2692-2699. 22. Meltzer EO, Hamilos DL, Hadley JA, et al. Rhinosinusitis: Establishing definitions for clinical research and patient care. Otolaryngol Head Neck Surg. 2004;131:S1-S62. 23. Caterson EJ, Nesti LJ, Danielson KG, Tuan RS. Human marrow-derived mesenchymal progenitor cells: isolation, culture expansion, and analysis of differentiation. Mol Biotechnol. 2002;20:245-256. 24. Zhang N, Gevaert P, van Zele T, et al. An update on the impact of Staphylococcus aureus enterotoxins in chronic sinusitis with nasal polyposis. Rhinology. 2005;43:162-168.
25. Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A. 2003;100:8407-8411. 26. Lange C, Togel F, Ittrich H, et al. Administrated mesenchymal stem cells enhance recovery from ischemia/reperfusioninduced acute renal failure in rats. Kidney Int. 2005;68:16131617. 27. Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood. 2005;106:1755-1761. 28. Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells. 2006;24:74-85. 29. Rasmusson I, Ringden O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res. 2005;305:33-41. 30. Krampera M, Cosmi L, Angeli R, et al. Role for interferongamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells. 2006;24:386-398. 31. Jiang XD, Li GY, Li L, Dong Z, Zhu DD. The characterization of IL-17A expression in patients with chronic rhinosinusitis with nasal polyps. Am J Rhinol Allergy. 2011;25:171-175. 32. Shen Y, Tang XY, Yang YC, et al. Impaired balance of Th17/ Treg in patients with nasal polyposis. Scand J Immunol. 2011; 74:176-185. 33. Li CW, Zhang KK, Li TY, et al. Expression profiles of regulatory and helper T-cell-associated genes in nasal polyposis. Allergy. 2012;67:732-740.
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Immunomodulatory Effect of Mesenchymal Stem Cells on T Lymphocyte and Cytokine Expression in Nasal Polyps Kyu-Sup Cho, Yong-Wan Kim, Myoung Joo Kang, Hee-Young Park, Sung-Lyong Hong and Hwan-Jung Roh Otolaryngology -- Head and Neck Surgery published online 13 March 2014 DOI: 10.1177/0194599814525751 The online version of this article can be found at: http://oto.sagepub.com/content/early/2014/03/06/0194599814525751
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Original Research
Immunomodulatory Effect of Mesenchymal Stem Cells on T Lymphocyte and Cytokine Expression in Nasal Polyps
Otolaryngology– Head and Neck Surgery 1–9 Ó American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0194599814525751 http://otojournal.org
Kyu-Sup Cho, MD, PhD1*, Yong-Wan Kim, MD, PhD2*, Myoung Joo Kang, MD3, Hee-Young Park1, Sung-Lyong Hong, MD1, and Hwan-Jung Roh, MD, PhD4
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Keywords immunosuppression, adipose tissue, mesenchymal stem cells, nasal polyps, T lymphocytes, cytokines
Abstract Objectives. Adipose tissue–derived stem cells (ASCs) have been reported to have immunomodulatory effects in various inflammatory diseases, including asthma and allergic rhinitis, through the induction of T cell anergy. Nasal polyps (NPs) are a chronic inflammatory disease in the nose and paranasal sinus characterized histologically by the infiltration of inflammatory cells, such as eosinophils or lymphocytes. This study was performed to investigate whether ASCs have immunomodulatory effects on T lymphocyte and cytokine expression in eosinophilic NPs. Study Design. Basic science experimental study. Setting. University tertiary care facility. Subjects and Methods. NP specimens were obtained from 20 patients with chronic rhinosinusitis and eosinophilic NPs. ASCs were isolated and cultured from the abdominal fat of 15 subjects undergoing intra-abdominal surgery. Infiltrating cells (1 3 106) were isolated from NP tissue and cocultured with 1 3 105 ASCs. To determine whether ASCs affect infiltrating T lymphocyte and cytokine expression in eosinophilic NP, T lymphocyte subsets and cytokine expression were analyzed before and after ASC treatment. Results. ASC treatment significantly decreased the proportions of CD41 and CD81 T cells. After ASC treatment, Th2 cytokine (interleukin [IL]–4 and IL-5) levels decreased significantly. In contrast, levels of Th1 (interferon-g and IL-2) and regulatory cytokines (transforming growth factor–b and IL-10) increased significantly after ASC treatment. Conclusions. ASCs have immunomodulatory effects in the eosinophilic inflammation of NPs, characterized by downregulation of activated T lymphocytes and a Th2 immune response. These effects would be expected, over time, to significantly contribute to the control of eosinophilic inflammation and, possibly, growth of eosinophilic NPs.
Received September 4, 2013; revised January 23, 2014; accepted February 6, 2014.
Introduction Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesenchymal lineages, such as bone,1-3 cartilage,1,2 adipose tissue,1,2 and muscle.1,4 Because of their capacity for differentiation, MSCs have emerged as a promising source for therapeutic applications in tissue engineering and regenerative medicine.5,6 In addition to their multilineage potential, MSCs derived from adipose tissue (ASCs) may share with other MSCs the unique ability to suppress immune responses and modulate inflammation.7 MSCs can inhibit natural killer cell function,8,9 modulate dendritic cell maturation,10 and suppress the allogeneic T cell response8 by altering the cytokine secretion profile of dendritic cells and T cells induced by an allogeneic immune reaction. 1 Department of Otorhinolaryngology and Biomedical Research Institute, Pusan National University School of Medicine, Pusan National University Hospital, Busan, South Korea 2 Department of Otorhinolaryngology, Inje University Haeundae Paik Hospital, Busan, South Korea 3 Department of Hemato-Oncology, Inje University Haeundae Paik Hospital, Busan, South Korea 4 Department of Otorhinolaryngology and Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, South Korea * These authors contributed equally to this article.
Corresponding Author: Hwan-Jung Roh, MD, PhD, Department of Otorhinolaryngology, Pusan National University Yangsan Hospital, Beom-eo li, Mul-geum eup, Yang-san si, Gyeongsangnam-do 626-770, South Korea Email: [email protected]
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Figure 1. Histologic findings in nasal polyps (NPs). (A) Eosinophilic NPs contained an abundance of eosinophils. (B) Noneosinophilic NPs showed a predominant accumulation of lymphocytes and neutrophils with fewer eosinophils (hematoxylin and eosin, 4003).
Nasal polyps (NPs) are a chronic inflammatory disease of the mucous membranes in the nose and paranasal sinuses, and its frequent recurrence is clinically challenging for clinicians. An NP is characterized by an edematous mass of hyperplastic epithelium and lamina propria prolapsing into the nose, leading to nasal obstruction, hypersecretion, loss of the sense of smell, and reduced quality of life.11 Histopathologically, NPs are characterized by the infiltration of inflammatory cells, such as eosinophils, neutrophils, and lymphocytes.12 Most NPs contain abundant eosinophils, but some show only a few eosinophils. Recent reports have suggested immunohistologic features of eosinophilic NPs that differ from those of noneosinophilic NPs.12,13 Eosinophilic NPs are characterized generally by Th2-skewed eosinophilic inflammation, with high interleukin (IL)–4 and IL-5 concentrations, but noneosinophilic NPs have a lower degree of total inflammatory cell infiltration and a higher frequency of neutrophil infiltration than eosinophilic NPs.14-18 Although several recent studies have shown that MSCs inhibit eosinophilic inflammation and have immunomodulatory effects in allergic rhinitis and an asthma mouse model,19-21 no immunomodulatory effect of MSCs on NPs showing predominantly eosinophilic infiltration has been reported to date. Thus, we hypothesized that MSCs would be capable of modulating inflammatory cell and cytokine production in eosinophilic NP. The purpose of this study was to evaluate the effects of ASCs on the T lymphocytes and cytokines that modulate eosinophilic inflammation in NP.
Subjects and Methods Demographics and Clinical Characteristics After the purpose and methods of the work were explained to each participant, all provided written informed consent. When child participants were involved, written informed consent was obtained from their parents. This study was approved by the Institutional Review Board of Pusan National University Hospital (H-1209-006-010). Eleven male and 9 female patients, ranging in age from 12 to 61 years (mean, 35.2 years), who had chronic rhinosinusitis (CRS) with eosinophilic NPs and underwent endoscopic sinus surgery were studied. CRS with NPs was defined on
the basis of the current American guidelines.22 Patients with obvious NPs on endoscopic examination and positive results on computed tomographic scans of the paranasal sinus, and for whom control of the symptoms with medical therapy had failed, were included. Patients with histories of endoscopic sinus surgery, antrochoanal polyps, allergic fungal sinusitis, immunodeficiency disorders, aspirin-exacerbated respiratory disease, and cystic fibrosis were excluded. The control group included 11 patients with nasal septal deviation without CRS. None of the patients had an acute infection of the upper respiratory tract, and none had received oral or topical corticosteroids, nonsteroidal antiinflammatory drugs, macrolide antibiotics, or antihistamines in the 4 weeks before endoscopic sinus surgery. Only patients without allergies were included, to exclude any confounding influence of type I allergies. During surgery under general anesthesia, NP tissues were collected and mucosa samples were taken from the inferior turbinate in the control group. The polyp tissues and mucosa were divided into 2 parts. Half of the tissue was fixed in 10% formalin and embedded in paraffin wax for histologic examination, and the other half was used to isolate infiltrating cells for analysis of T lymphocyte subsets and cytokine profiles.
Histologic Analysis Sections (4 mm) were obtained from the paraffin wax– embedded specimens and stained with hematoxylin and eosin. The sections were randomly indicated and observed by 1 pathologist under a light microscope at 4003 magnification. NPs were classified as eosinophilic or noneosinophilic according to the predominant inflammatory cells. In eosinophilic NPs, it was confirmed that eosinophils constituted .75% of the inflammatory cells infiltrating in the polyp tissue (Figure 1).
Isolation and Culture of ASCs Adipose tissue was obtained, after obtaining informed patient consent, from 15 subjects undergoing conventional and laparoscopic intra-abdominal surgeries, 7 men and 8 women aged 45 to 55 years (mean, 47.5 years).
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To isolate human ASCs, adipose tissues were washed extensively with phosphate-buffered saline (PBS), and tissues were digested with 0.075% collagenase type I (Sigma, St Louis, Missouri) at 37°C for 30 minutes. Enzyme activity was neutralized with a-modified Eagle’s medium containing 10% fetal bovine serum, and the sample was centrifuged (1200g, 10 minutes) to obtain a pellet. The pellet was suspended and filtered through a 100-mm nylon mesh to remove cellular debris and incubated overnight at 37°C in 5% CO2/air in control medium (a-modified Eagle’s medium, 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin). After incubation, the plates were washed extensively with PBS to remove residual nonadherent red blood cells. The resulting cell population was maintained at 37°C in 5% CO2/air in control medium. Then, 1 week later, when the monolayer of adherent cells had reached confluence, cells were trypsinized (0.05% trypsin–ethylenediaminetetraacetic acid; Sigma), resuspended in a-modified Eagle’s medium containing 10% fetal bovine serum, and subcultured at a concentration of 2000 cells/cm2, with the medium changed twice per week.
Immunophenotypic Analysis of ASCs Flow cytometric analysis was used to characterize the phenotypes of the ASCs. At least 50,000 cells (in 100-mL PBS, 0.5% bovine serum albumin, 2 mmol/L ethylenediaminetetraacetic acid) were incubated with fluorescein isothiocyanate–labeled monoclonal antibodies against human CD90, CD44, CD73, CD45, CD31, and HLA-DR (BD Biosciences Clontech, Palo Alto, California). After washing, labeled cells were analyzed by flow cytometry using a FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences, San Diego, California).
Multilineage Differentiation of ASCs ASCs were analyzed for their capacity to differentiate toward the adipogenic, osteogenic, and chondrogenic lineages, as described previously.23 For adipogenic and osteogenic differentiation, cells were seeded in 6-well plates at a density of 20,000 cells/cm2 and treated for 3 weeks with adipogenic and osteogenic medium. Adipogenic and osteogenic differentiation was assessed using oil red O staining as an indicator of intracellular lipid accumulation and alizarin red S staining for extracellular matrix calcification, respectively. Chondrogenic differentiation was induced using the micromass culture technique. Briefly, 10 mL of a concentrated MSC suspension (3 3 105 cells/mL) were plated in the center of each well and treated for 3 weeks with chondrogenic medium. Chondrogenesis was confirmed by immunohistochemistry.
Isolation of Infiltrating Cells from Polyp Tissue and CoCulture with ASCs Infiltrating cells were isolated from polyp tissue using the previously published method of Zhang et al.24 Briefly, tissue samples were aseptically cut into small fragments and
incubated with 100 U/mL collagenase type IV (Sigma) and 100 mg/mL DNase I (R&D Systems, Minneapolis, Minnesota) for 60 min at 37°C in a shaking water bath. Fragments were disrupted by grinding and passage through a cell strainer (BD Falcon, Bedford, Massachusetts). Isolated cells were then washed with PBS. Isolated cells (1 3 106) from polyp tissues were cocultured with 1 3 105 ASCs in 12-well tissue culture plates (Corning Inc, Corning, New York) in the presence of 10 mg/mL phytohemagglutinin (PHA; Sigma). After 24 hours, cells were harvested for flow cytometric analysis, and supernatants were collected for cytokine measurement.
Analysis of T-Lymphocyte Subsets Isolated cells were washed with FACS buffer (PBS containing 1% fetal calf serum and 0.05% NaN3). Next, cells were incubated with purified human immunoglobulin G (Sigma) for 15 minutes at 4°C to block nonspecific binding of antibodies to the Fc receptor and then stained with phycoerythrinconjugated antihuman CD4 (L3T4; eBioscience, San Diego, California) and fluorescein isothiocyanate–conjugated antihuman CD8 (RPA-T8; e-Bioscience) for 30 minutes at 4°C. Fluorescein isothiocyanate–conjugated mouse immunoglobulin G1 and phycoerythrin-conjugated mouse immunoglobulin G2a antibodies (eBioscience) were used as isotype controls. After staining, cells were washed with FACS buffer and analyzed using a FACSCalibur flow cytometer.
Detection of Cytokine Expression After 24 hours of co-culture, IL-2, IL-4, IL-5, IL-10, IL-13, tumor necrosis factor–a, interferon (IFN)-g, and transforming growth factor (TGF)–b levels in the supernatant were determined by sandwich enzyme-linked immunosorbent assay (eBioscience). Briefly, microtiter plates (Costar, Cambridge, Massachusetts) were coated with the capture antibody at 4°C overnight. After blocking with 2% bovine serum albumin in PBS for 1 hour, undiluted supernatant was added and incubated for 2 hours at room temperature. Subsequently, the cytokine levels were detected with a biotin-conjugated detecting antibody and developed with horseradish peroxidase–conjugated streptavidin and tetramethylbenzidine as a substrate (Endogen, Rockford, Illinois). Optical densities were measured at 450 nm using an enzyme-linked immunosorbent assay plate reader (Molecular Devices, Sunnyvale, California).
Statistical Analysis All experiments were repeated a minimum of 3 times. Data are expressed as medians and interquartile ranges (IQRs). SPSS for Windows version 20.0 (SPSS, Inc, Chicago, Illinois) was used to perform all statistical analysis. Wilcoxon’s signed-rank tests were used to evaluate differences before and after PHA stimulation and before and after ASC treatment. Differences between patients with eosinophilic NPs and controls were evaluated using MannWhitney U tests. P values \ .05 were considered to indicate statistical significance.
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Figure 2. Characteristics of adipose tissue–derived stem cells (ASCs). ASCs show characteristics of mesenchymal stem cells in the immunophenotypic analysis (A), fibroblast-like morphology (B), adipogenesis (C), osteogenesis (D), and chondrogenesis (E) (original magnification 2003).
Results Isolation, Immunophenotypic Analysis, and Multilineage Differentiation of ASCs ASCs were positive for CD90, CD44, and CD73 and negative for CD45, CD31, and HLA-DR (Figure 2A). At an initial plating density of 1 3 106 cells/cm2, adipose tissue– derived fibroblastoid cells formed a monolayer 4 to 5 days after initial plating. These putative ASCs were of serpiginous or fibroblast-like morphology, similar to previously reported ASCs and bone marrow–derived MSCs (Figure 2B). This morphology was maintained during passage in monolayer culture. Adipogenic differentiation was demonstrated by the accumulation of neutral lipid vacuoles by oil red O staining. A significant fraction of the cells contained multiple, intracellular lipid-filled droplets that stained with oil red O (Figure 2C). Osteogenic differentiation was confirmed by the deposition of alizarin red S–stained mineralized matrix. Calcification appeared as red regions within the cell monolayer (Figure 2D). Chondrogenic differentiation was confirmed by the formation of a sphere in micromass culture (Figure 2E).
Infiltrating T Lymphocyte Subsets Infiltrating T lymphocyte populations in eosinophilic NPs were compared with those from control mucosa. The numbers of CD41 and CD81 T cells were significantly higher in eosinophilic NPs compared with control mucosa before PHA stimulation and after ASC treatment. To determine whether ASCs affected T lymphocyte infiltrating NP tissue, T cell populations were determined after PHA stimulation and ASC treatment. Both CD41 and CD81 T cells were detected in polyp tissue, and the percentages of CD41 and CD81 T cells were increased significantly by PHA stimulation from 3.2% (IQR, 2.3%-4.3%) and 6.4% (IQR, 5.1%7.9%) to 11.8% (IQR, 10.2%-13.1%) and 18.6% (IQR, 16.3%-20.4%) (P = .001 and P = .003, respectively). ASC treatment also significantly decreased the percentages of CD41 and CD81 T cells to 3.5% (IQR, 1.3%-5.8%) and 7.5% (IQR, 5.3%-9.6%) (P \ .001 and P = .009, respectively; Table 1, Figure 3). However, in the control group, there were no significant differences in CD41 or CD81 T cells after ASC treatment, although the percentages of CD41 and CD81 T cells were increased significantly by PHA stimulation from 1.2% (IQR, 0.1%-2.5%) and 1.8% (IQR, 0.2%-3.4%) and to 6.3% (IQR, 4.8%-8.0%) and 7.4%
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Table 1. T Lymphocyte and Cytokine Expressions in Eosinophilic NPs. NPs 1 PHA
NPs CD41 T cells CD81 T cells IL-2 IL-4 IL-5 IL-10 IL-13 TNF-a IFN-g TGF-b
3.2 (2.3-4.3) 6.4 (5.1-7.9) 6.3 (4.4-10.2) 4.8 (4.4-6.4) 18.0 (16.4-18.5) 64.1 (20.7-67.8) 337.8 (276.8-362.7) 36.6 (26.3-59.6) 32.2 (31.2-33.8) 109.7 (106.7-125.9)
11.8 18.6 15.9 16.6 33.2 180.1 370.4 189.5 566.5 119.1
(10.2-13.1) (16.3-20.4) (12.5-18.8) (13.8-20.1) (28.1-38.4) (126.5-192.6) (306.9-481.2) (158.4-233.3) (463.0-691.7) (104.4-150.2)
NPs 1 PHA 1 ASCs 3.5 7.5 22.1 12.1 27.4 236.5 355.7 176.4 831.0 145.3
(1.3-5.8) (5.3-9.6) (17.3-27.1) (9.5-16.8) (20.6-32.6) (201.2-257.4) (298.3-464.1) (123.1-197.5) (628.8-940.9) (121.6-180.0)
Pa
Pb
.001 .003 .015 .021 .005 .019 .078 .001 \.001 .521
\.001 .009 .003 .031 .028 .042 .067 .225 .001 .012
Abbreviations: ASC, adipose tissue–derived stem cells; IL, interleukin; IFN, interferon; NP, nasal polyp; PHA, phytohemagglutinin; TGF, transforming growth factor; TNF, tumor necrosis factor. a NPs versus NPs plus PHA. b NPs plus PHA versus NPs plus PHA plus ASCs.
Figure 3. Analysis of infiltrating T lymphocyte subsets in nasal polyps (NPs) and control mucosa. The R2 and R3 regions indicate the percentages of CD41 and CD81 T cells relative to the total numbers of infiltrating cells in eosinophilic NPs (A) and control mucosa (B). The percentages of CD41 and CD81 T cells were decreased significantly by adipose tissue–derived stem cell (ASC) treatment in eosinophilic NPs (C), but not in the control mucosa (D). PHA, phytohemagglutinin.
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Table 2. T Lymphocyte and Cytokine Expressions in Control Nasal Mucosa. Control CD41 T cell CD81 T cell IL-2 IL-4 IL-5 IL-10 IL-13 TNF-a IFN-g TGF-b
1.2 1.8 2.1 1.7 4.5 11.8 5.1 4.7 3.2 11.0
(0.1-2.5) (0.2-3.4) (1.5-2.5) (1.4-2.6) (3.4-6.1) (5.1-13.2) (3.5-9.2) (2.5-6.1) (2.8-4.0) (10.7-13.4)
Control 1 PHA
Control 1 PHA 1 ASCs
Pa
Pb
6.3 (4.8-8.0) 7.4 (4.9-9.5) 3.1 (2.4-3.1) 3.1 (2.3-3.9) 5.2 (4.7-7.1) 24.1 (12.4-26.9) 14.1 (8.5-18.5) 8.5 (5.9-10.7) 5.8 (4.8-7.1) 23.5 (18.6-27.4)
5.1 (3.0-7.9) 6.3 (4.9-8.1) 3.7 (3.2-5.4) 2.9 (2.4-4.3) 5.9 (5.1-7.8) 29.8 (11.5-42.4) 11.9 (10.1-13.9) 7.2 (6.5-10.4) 6.7 (5.2-8.6) 23.5 (18.6-28.7)
.001 .003 .032 .254 .452 .087 .584 .685 .016 .008
.078 .067 .748 .542 .345 .138 .686 .756 .893 .893
Abbreviations: ASC, adipose tissue–derived stem cells; IL, interleukin; IFN, interferon; PHA, phytohemagglutinin; TGF, transforming growth factor; TNF, tumor necrosis factor. a Control versus control plus PHA. b Control plus PHA versus control plus PHA plus ASCs.
(IQR, 4.9%-9.5%) (P = .001 and P = .003, respectively; Table 2, Figure 3).
Analysis of Cytokine Expression Significantly higher levels of all cytokines were found in patients with eosinophilic NPs than in controls before PHA stimulation and after ASCs treatment. To determine whether ASCs affected cytokine expression in NP tissue, cytokines were analyzed after PHA stimulation and ASC treatment. NPs showed a mixed Th1 and Th2 cytokine profile, and significantly higher expression was observed after PHA stimulation in IL-2 (6.3 pg/mL [IQR, 4.4-10.2 pg/mL] vs 15.9 pg/ mL [IQR, 12.5-18.8 pg/mL], P = .015), IL-4 (4.8 pg/mL [IQR, 4.4-6.4 pg/mL] vs 16.6 pg/mL [IQR, 13.8-20.1 pg/ mL], P = .021), IL-5 (18.0 pg/mL [IQR, 16.4-18.5 pg/mL] vs 33.2 pg/mL [IQR, 28.1-38.4 pg/mL], P = .005), IL-10 (64.1 pg/mL [IQR, 20.7-67.8 pg/mL] vs 180.1 pg/mL [IQR, 126.5-192.6 pg/mL], P = .019), tumor necrosis factor-a (36.6 pg/mL [IQR, 26.3-59.6 pg/mL] vs 189.5 pg/mL [IQR, 158.4-233.3 pg/mL], P = .001), and IFN-g (32.2 pg/mL [IQR, 31.2-33.8 pg/mL] vs 566.5 pg/mL [IQR, 463.0-691.7 pg/mL], P \ .001). After ASC treatment, Th2 cytokine (IL4 and IL-5) levels were decreased significantly to 12.1 pg/ mL (IQR, 9.5-16.8 pg/mL) and 27.4 pg/mL (IQR, 20.6-32.6 pg/mL) (P = .031 and P = .028, respectively). In contrast, Th1 cytokine (IL-2 and IFN-g) levels were increased significantly to 22.1 pg/mL (IQR, 17.3-27.1 pg/mL) and 831.0 pg/mL (IQR, 628.8-940.9 pg/mL) (P = .003 and P = .001, respectively). Moreover, after ASC treatment, regulatory cytokine (IL-10 and TGF-b) levels were increased significantly to 236.5 pg/mL (IQR, 201.2-257.4 pg/mL) and 145.3 pg/mL (IQR, 121.6-180.0 pg/mL) (P = .042 and P = .012, respectively). There was no significant difference in IL-13 or tumor necrosis factor-a levels before and after ASC treatment (Table 1, Figure 4). In the control group, after PHA stimulation, cytokine levels were increased significantly in IL-2 (2.1 pg/mL [IQR, 1.5-2.5 pg/mL] vs 3.1 pg/mL [IQR, 2.4-3.1 pg/mL],
P = .032), IFN-g (3.2 pg/mL [IQR, 2.8-4.0 pg/mL] vs 5.8 pg/mL [IQR, 4.8-7.1 pg/mL], P = .016), and TGF-b (11.0 pg/mL [IQR, 10.7-13.4 pg/mL] vs 23.5 pg/mL [IQR, 18.627.4 pg/mL], P = .008]. However, there was no significant difference in the level of any cytokine before and after ASC treatment (Table 2).
Discussion MSCs have been reported to have anti-inflammatory effects in experimental models of acute pulmonary and renal injury.25,26 MSCs have also been shown to cause attenuation of experimentally induced autoimmune encephalomyelitis through the induction of T cell anergy.27 More recently, ASCs were shown to ameliorate experimental airway allergic diseases, such as allergic rhinitis and asthma, by shifting to a Th1-biased from a Th2-biased immune response.19-21 Histologically, eosinophilic NPs have been thought to be characterized by predominantly eosinophilic infiltration, similar to allergic rhinitis and asthma.11 This study was performed to assess whether ASCs possess immunomodulatory properties in eosinophilic NPs, a common chronic inflammatory disease of the upper airways. Among MSCs, ASCs were used because of their abundance, relatively easy harvesting, and high proliferation potential. To our knowledge, this is the first reported study to investigate the effects of ASCs on T lymphocytes and cytokine expression in NPs. NPs are a chronic inflammatory disease in the nose and paranasal sinuses and are commonly associated with CRS. NPs involve a complex inflammatory process regulated by various chemical mediators and cytokines produced by inflammatory cells in the sinonasal mucosa.11,14 However, eosinophilic and noneosinophilic NPs display distinct features on the basis of histopathology and the expression of inflammatory mediators. Eosinophilic NPs have been characterized by Th2-skewed eosinophilic inflammation with defective functions in regulatory T (Treg) cells, and infiltration of natural killer T cells, whereas noneosinophilic NPs showed a predominant Th1 situation with adequate Treg
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Figure 4. Cytokine expression in nasal polyps. After adipose tissue–derived stem cell (ASC) treatment, interleukin (IL)–4 and IL-5 levels were decreased significantly. In contrast, IL-2, IL-10, interferon (IFN)–g, and transforming growth factor (TGF)–b levels were increased significantly after ASC treatment.
cell function.17 Investigations of subsets of lymphocytes in eosinophilic NPs indicate that T lymphocytes predominate over B cells and that there are more CD81 T cells than CD41 T cells.16 Consistently, our study showed the accumulation of CD41 and CD81 T cells, including this distinctive pattern of increased CD81 T cells in eosinophilic NPs. These data suggest that activated T cells, both the CD41 and CD81 subtypes, contribute to the pathogenesis of NPs. Moreover, this is consistent with evidence that NPs involve a mixed profile of Th1 and Th2 cytokines because Th1 cytokines may be secreted by the increased CD 81 T cell population in eosinophilic NPs.12,15
The present study showed immunomodulatory effects of ASCs on T lymphocytes and cytokine expression in eosinophilic NPs. In terms of eosinophilic inflammation, Th1 and Th2 cytokines are mutually antagonistic, and selective suppression of Th2 cytokines may be crucial for protection against eosinophilic inflammation.15,20 ASC treatment significantly reduced the percentage of CD41 and CD81 T cells in eosinophilic NPs. In addition to decreased T cell infiltration, ASC treatment significantly decreased Th2 cytokines (IL-4 and IL-5) and increased Th1 (IFN-g and IL-2) and regulatory (TGF-b and IL-10) cytokines in eosinophilic NPs. These data are consistent with previous reports that
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Otolaryngology–Head and Neck Surgery
ASCs attenuate allergic airway inflammation in asthma and allergic rhinitis.19-21 Taken together with inhibition of eosinophilic inflammation by ASCs, these findings indicate that ASCs have immunomodulatory effects in eosinophilic NP, one of which is down-regulating Th2 cytokines and upregulating Th1 and regulatory cytokines. However, there was no significant difference in the number of T lymphocytes or cytokine expression before and after ASC treatment in control mucosa because T lymphocytes and cytokine expression were much lower than in polyp tissue. Despite the general consensus that MSCs inhibit T cell proliferation, little is known about the exact molecular mechanism responsible for this effect. Cell-cell contact and soluble factors, such as TGF-b1 and prostaglandin E2, are thought to be responsible for the effect.28,29 In part, T cell suppression by MSCs appears to depend on cross-talk between T cells and MSCs, leading to the production of IFN-g with indoleamine 2,3-dioxygenase activity, which in turn inhibits the proliferation of activated T and/or natural killer T cells.30 Further studies are required to fully characterize the immunomodulatory mechanism of ASCs in eosinophilic inflammation, whether by direct contact of ASCs with T lymphocytes or by secretion of crucial mediators of ASCs in eosinophilic NPs. Recently, a number of studies have shown that enhanced Th17 response existed not only in CRS without NPs with neutrophil-biased inflammation but also in CRS with eosinophilic and noneosinophilic NPs.17,31 It has been reported that an impaired balance of Th17/Treg (increased Th17 proportion and decreased Treg proportion) may play an important role in the development of NPs.32 Moreover, glucocorticoid treatment induced immunomodulation in NP via up-regulation of Treg cells and down-regulation of the Th2 and Th17 response.33 Taken together with these findings, ASCs may have downregulating effects of Th17 response in eosinophilic NPs. However, the exact role of Th17 response in the immunomodulatory mechanism of ASCs needs further investigation. This study had a number of limitations inherent to an in vitro study. Because the T lymphocyte numbers and cytokine expression tended to be low in polyp tissue and resting T lymphocytes isolated from NPs do not proliferate without PHA, T lymphocyte activation and proliferation were induced by PHA. Therefore, there was no significant difference before and after ASC treatment in the absence of PHA (data not shown). Because in vitro studies do not always reflect the in vivo situation, the immunomodulatory effect of ASCs in patients with NPs should be evaluated further.
Conclusions This study showed that ASC treatment has an immunomodulatory effect in eosinophilic inflammation associated with eosinophilic NPs, which is characterized by down-regulation of activated T lymphocytes and a Th2 immune response. These effects would be expected, over time, to contribute significantly to the control of eosinophilic inflammation and, possibly, the growth of eosinophilic NPs.
Author Contributions Kyu-Sup Cho, drafting and revising the article; Yong-Wan Kim, drafting the article; Myoung Joo Kang, analysis and interpretation of data; Hee-Young Park, acquisition of data; Sung-Lyong Hong, acquisition of data; Hwan-Jung Roh, conception and design.
Disclosures Competing interests: None. Sponsorships: None. Funding source: This work (data collection and analysis) was supported by a 2013 research grant from Inje University.
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