Nasal Polyp-Derived Mesenchymal Stromal Cells Exhibit Lack of Immune-Associated Molecules and High Levels of Stem/Progenitor Cells Markers.

Mesenchymal stromal cells (MSCs) are considered adult progenitor stem cells and have been studied in a multitude of tissues. In this context, the microenvironment of nasal polyp tissue has several inflammatory cells, but their stroma compartment remains little elucidated. Hence, we isolated MSCs from nasal polyps Polyp-MSCs (PO-MSCs) and compared their molecular features and gene expression pattern with bone marrow-derived MSCs (BM-MSCs). Initially, both PO-MSCs and BM-MSCs were isolated, cultivated, and submitted to morphologic, differentiation, phenotypic, immunosuppressive, and gene expression assays. Compared to BM-MSCs, PO-MSCs showed normal morphology and similar osteogenic/adipogenic differentiation potential, but their immunophenotyping showed lack of immune-associated molecules (e.g., CD117, HLA-DR, PDL-1, and PDL-2), which was linked with less immunoregulatory abilities such as (i) inhibition of lymphocytes proliferation and (ii) regulatory T cell expansion. Furthermore, we detected in the PO-MSCs a distinct gene expression profile in comparison with BM-MSCs. PO-MSC expressed higher levels of progenitor stem cells specific markers (e.g., CD133 and ABCB1), while BM-MSCs showed elevated expression of cytokines and growth factors (e.g., FGF10, KDR, and GDF6). The gene ontology analysis showed that the differentially modulated genes in PO-MSC were related with matrix remodeling process and hexose and glucose transport. For BM-MSCs, the highly expressed genes were associated with behavior, angiogenesis, blood vessel morphogenesis, cell-cell signaling, and regulation of response to external stimulus. Thus, these results suggest that PO-MSCs, while sharing similar aspects with BM-MSCs, express a different profile of molecules, which presumably can be implicated in the development of nasal polyp tissue.

TGF-beta signaling and collagen deposition in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. OBJECTIVE: The objective was to analyze the presence of TGF-beta isoforms, receptors, intracellular signaling, and collagen deposition in chronic rhinosinusitis. METHODS: Sinonasal mucosal samples obtained from chronic rhinosinusitis with nasal polyps (CRSwNP; n = 13), chronic rhinosinusitis without nasal polyps (CRSsNP; n = 13), and controls (n = 10) were analyzed for TGF-beta isoforms 1 and 2 by means of ELISA and IHC, and for TGF-beta R1, 2, and 3 by RT-PCR and IHC. As downstream proteins, phospho-Smad 2 (pSmad 2) and collagen were analyzed by performing immunostaining and picrosirius red staining, respectively. RESULTS: TGF-beta 1 and 2 protein concentrations, TGF-beta receptor (R) I and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly higher in CRSsNP versus controls. In CRSwNP, TGF-beta 1 protein concentration, TGF-beta RII and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly lower versus controls. Only TGF-beta 2 protein was found higher in CRSwNP versus controls. CONCLUSION: A high TGF-beta 1 protein expression, increased TGF-beta RI expression, and a high number of pSmad 2-positive cells all indicate an enhanced TGF-beta signaling in CRSsNP, whereas a low TGF-beta 1 protein concentration, a decreased expression of TGF-beta RII, and a low number of pSmad 2-positive cells in CRSwNP indicate a low level of TGF-beta signaling in CRSwNP. These findings are compatible with the remodeling patterns observed, reflected by a lack of collagen in CRSwNP, and excessive collagen production with thickening of the collagen fibers in the extracellular matrix in CRSsNP.

T-cell regulation in chronic paranasal sinus disease.

BACKGROUND: Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by a T(H)2-skewed eosinophilic inflammation, whereas chronic rhinosinusitis without nasal polyps (CRSsNP) represents a predominant T(H)1 milieu. OBJECTIVE: We aimed to study the direct tissue expression of transcription factors for T-cell subpopulations, including T regulatory cells, in relation to the cytokine expression patterns in the different disease subgroups. METHODS: The expression of forkhead box P3 (FOXP3), T-box transcription factor (T-bet), GATA-3, retinoid acid-related orphan receptor C (RORc), the suppressive cytokines TGF-beta1 and IL-10, and T(H)1/ T(H)2/ T(H)17 cytokines (IFN-gamma, IL-4, IL-5, IL-13, IL-17) were analyzed by means of RT-PCR in 13 CRSsNP, 16 CRSwNP, and 10 control samples. Additional protein measurements were performed for TGF-beta1 and IFN-gamma. RESULTS: In CRSwNP, we observed a significantly lower FOXP3 mRNA and TGF-beta1 protein expression, but a significantly higher T-bet, GATA-3, IL-5, and IL-13 mRNA expression compared with controls, whereas RORc was not significantly different compared with controls. In CRSsNP, FOXP3, T-bet, GATA-3, and RORc expression was not significantly different from controls, whereas TGF-beta1 mRNA, IFN-gamma mRNA, and protein were significantly higher in CRSsNP compared with controls. For IL-17, no significant differences were noted among all groups. CONCLUSION: We demonstrate for the first time a decreased FOXP3 expression accompanied by an upregulation of T-bet and GATA-3 and a downregulation of TGF-beta1 in CRSwNP versus controls and CRSsNP.

Eicosanoid metabolism and eosinophilic inflammation in nasal polyp patients with immune response to Staphylococcus aureus enterotoxins.

BACKGROUND: Staphylococcus aureus-derived enterotoxins (SEs) have been implicated in the pathogenesis of airway inflammatory diseases, especially nasal polyposis. However, the exact role of these molecules in the regulation of eicosanoid synthesis in this pathology remains unexplored. We studied the possible impact of SE-induced immune responses on the eicosanoid production in nasal polyp (NP) patients. METHODS: Tissue sample homogenates from NP patients, with (NP-SEs[+]) and without detectable IgE-antibodies to SEs (NP-SEs[-]; ImmunoCap system), were assayed for IL-5, myeloperoxidase, leukotriene CJD4/E4 (LTC4/D4/E4), LTB4, lipoxin A4, total IgE, and eosinophil cationic protein. RESULTS: Inflammatory makers, eicosanoids, and total IgE were significantly increased in NP-SEs(+) compared with NP-SEs(-) tissues, with the exception of myeloperoxidase, which was similar in both groups. Eicosanoid concentrations correlated to IL-5 and eosinophil cationic protein; however, only cys-leukotriene levels correlated with IgE-antibodies to SEs, independently of allergy and asthma. CONCLUSION: Eicosanoid synthesis is up-regulated in polyp tissue of patients with immune response to SEs and seems to be related to the inflammatory reaction induced by these enterotoxins.

Expression of eicosanoid receptors subtypes and eosinophilic inflammation: implication on chronic rhinosinusitis.

BACKGROUND: Eicosanoid receptors are G-protein-coupled receptors playing an important immunomodulatory role in airway diseases. However, there is little information on the expression of these receptors and their link with eosinophilic inflammation in paranasal sinus diseases. We aimed with this study to investigate the tissue expression of leukotrienes and prostaglandin E2 receptors in chronic rhinosinusitis patients and the link of this regulation with eosinophilic inflammation. METHODS: Samples were prepared from nasal tissue of patients with chronic rhinosinusitis without nasal polyps (CRS, n = 11), with nasal polyps (CRS-NP, n = 13) and healthy subjects (Controls, n = 6). mRNA expression of CysLT1, CysLT2, BLT1, BLT2, E-prostanoid receptors (EP1, EP2, EP3, EP4) and sol-IL-5Ralpha was determined by real-time PCR. Concentrations of PGE2, LTC4/D4/E4, LTB4 and sol-IL-5Ralpha were determined by ELISA and of ECP by ImmunoCap. Protein expression and tissue localization of eicosanoid receptors and activated eosinophils were evaluated by immunohistochemistry. RESULTS: CysLT1 mRNA expression was significantly increased in CRS-NP compared to CRS and controls, and CRS compared to controls, whereas CysLT2 mRNA was enhanced in both CRS groups without differences between them. Levels of both receptors correlated to the number of activated eosinophils, sol-IL-5Ralpha, ECP and LTC4/D4/E4 concentrations in the disease groups. PGE2 protein concentrations and prostanoid receptors EP1 and EP3 were down-regulated in the CRS-NP tissue vs. CRS and controls, whereas EP2 and EP4 expression was enhanced in CRS and CRS-NP patients vs. controls. No differences in BLT receptors were observed between patients and controls. CONCLUSION: CyLTs receptors are up-regulated in nasal polyp tissue and their expression correlate with eosinophilic inflammation supporting previous results. Eicosanoid receptors mRNA pattern observed suggests that down-regulation of EP1 and EP3 in CRS-NP and up-regulation EP2 and EP4 in CRS and CRS-NP groups may have some role in the development of the diseases and their regulation may not be directly linked to eosinophil activation but involve post-transcriptional events mainly related to other inflammatory cell sources.

Prostaglandin, leukotriene, and lipoxin balance in chronic rhinosinusitis with and without nasal polyposis.

BACKGROUND: Upper airway diseases and especially the aspirin hypersensitivity syndrome have been linked to changes in the arachidonic acid cascade; however, the specificity of these changes and their relation to inflammatory reactions in these diseases still remain controversial. OBJECTIVE: We aimed to study the tissue eicosanoid production in 3 subgroups of patients with chronic rhinosinusitis (CRS) and control subjects and to correlate it with the severity of inflammation and clinical manifestation of aspirin sensitivity. METHODS: Samples were prepared from sinonasal tissue of patients with CRS with (CRS-NP group, n = 13) and without nasal polyposis (CRS group, n = 11), sinonasal tissue of patients with nasal polyposis and aspirin sensitivity (CRS-ASNP group, n = 13), and normal nasal mucosa from healthy subjects (NM group, n = 8). Real-time PCR was applied for mRNA quantification of COX-2, 5-lipoxygenase, leukotriene C 4 synthase, and 15-lipoxygenase. Enzyme immunoassays were used to measure IL-5, eosinophil cationic protein, and eicosanoid (leukotriene [LT] C 4 , LTD 4 , and LTE 4 ; lipoxin A 4 ; and prostaglandin E 2 [PGE 2 ]) concentrations. RESULTS: COX-2 mRNA and PGE 2 concentrations were similar in the CRS and NM groups but significantly decreased in nasal polyp tissue, especially in the CRS-ASNP group. LTC 4 synthase, 5-lipoxygenase mRNA, LTC 4 , LTD 4 , and LTE 4 concentrations increased with disease severity among the patient groups. 15-Lipoxygenase and lipoxin A 4 concentrations were increased in all CRS groups compared with in the NM group but were significantly downregulated in the CRS-ASNP group when compared with the CRS-NP group. IL-5 and eosinophil cationic protein were increased in both groups of nasal polyp tissue compared with in the NM and CRS groups and correlated directly with LTC 4 , LTD 4 , and LTE 4 concentrations and inversely with PGE 2 concentrations. CONCLUSION: Changes of tissue eicosanoid metabolism do occur in CRS, even in the absence of clinical aspirin sensitivity, and these changes appear to be related to the severity of eosinophilic inflammation.