IL-17A mediates pyroptosis via the ERK pathway and contributes to steroid resistance in CRSwNP.

BACKGROUND: Pyroptosis is closely related to inflammation. However, the molecular mechanisms and pathologic contributions of pyroptotic epithelial cell are not yet fully understood. OBJECTIVE: This study aimed to explore the function and molecular mechanisms of IL-17A on human nasal epithelial cell (hNEC) pyroptosis. METHODS: The expression of pyroptosis-related biomarkers and IL-17A was assessed in sinonasal mucosa from control individuals, patients with chronic rhinosinusitis without nasal polyps, and patients with chronic rhinosinusitis with nasal polyps (CRSwNP) by using quantitative RT-PCR. Their localization was analyzed via immunohistochemistry and immunofluorescence. The ultrastructural characteristics of IL-17A-induced pyroptosis in hNECs were visualized by using electron microscopy. IL-17A functional assays were performed on hNECs and airway epithelial cell lines. Cytokine levels were quantified via ELISA. The signaling pathways involved in IL-17A-induced pyroptosis were studied via unbiased RNA sequencing and Western blotting. RESULTS: The expression of IL-17A and the pyroptotic biomarkers NOD-like receptor family, pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D, and IL-1ß was increased in nasal mucosa from patients with CRSwNP compared with in those with chronic rhinosinusitis without nasal polyps and the control subjects. IL-17A was positively correlated and colocalized with the pyroptotic biomarkers. IL-17A treatment induced pyroptosis in the hNECs and cell lines analyzed, primarily through the extracellular signal-regulated kinase (ERK)-NLRP3/caspase-1 signaling pathway, and increased IL-1ß and IL-18 secretion in hNECs. Moreover, IL-17A-induced pyroptosis contributed to steroid resistance by affecting glucocorticoid receptor-α and glucocorticoid receptor-ß expression, and the inhibition of pyroptotic proteins partially abolished IL-17A-induced steroid resistance in hNECs. CONCLUSION: Elevated IL-17A level promotes pyroptosis in hNECs through the ERK-NLRP3/caspase-1 signaling pathway and contributes to glucocorticoid resistance by affecting glucocorticoid receptor homeostasis in patients with CRSwNP.

Evidence for the Presence of Long-Lived Plasma Cells in Nasal Polyps.

PURPOSE: Plasma cells and immunoglobulins (Igs) play a pivotal role in the induction and maintenance of chronic inflammation in nasal polyps. During secondary immune responses, plasma cell survival and Ig production are regulated by the local environment. The purpose of the present study was to investigate the presence of long-lived plasma cells (LLPCs) and specific survival niches for LLPCs in human nasal polyps. METHODS: Nasal mucosal samples were cultured with an air-liquid interface system and the Ig levels in culture supernatants were analyzed by enzyme-linked immunosorbent assay. The characteristics of LLPCs in nasal polyps were determined by immunohistochemistry and immunofluorescence. The expression of neurotrophins as well as their receptors was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and Western blotting. RESULTS: The numbers of CD138⁺ total plasma cells and BCL2⁺ plasma cells were increased in both eosinophilic and non-eosinophilic nasal polyps compared with those in normal tissues. The production of IgG, IgA, and IgE was detected in culture supernatants even after a 32-day culture of nasal polyps. Although the total numbers of plasma cells were decreased in nasal polyps after culture, the numbers of BCL2⁺ plasma cells remained stable. The expression of nerve growth factor (NGF) as well as tropomyosin receptor kinase (Trk) A, a high-affinity receptor for NGF, was upregulated in both eosinophilic and non-eosinophilic nasal polyps. In addition, BCL2⁺ plasma cell numbers were positively correlated with NGF and TrkA mRNA expression in nasal mucosal tissues. Polyp plasma cells had the expression of TrkA. CONCLUSIONS: Human nasal polyps harbor a population of LLPCs and NGF may be involved in their prolonged survival. LLPCs may be a novel therapeutic target for suppressing the local Ig production in nasal polyps.

miR-186 inhibits proliferation, migration, and epithelial-mesenchymal transition in breast cancer cells by targeting Twist1.

OBJECTIVE: Breast cancer (BC) is the most prevalent malignancy in women worldwide. Our study aimed to investigate the expression and biological effect of miR-186 in BC. METHODS: Expression of miR-186 was determined by quantitative reverse transcription PCR. Kaplan-Meier curves were calculated for the survival data analysis. Functional assays were performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and wound healing assay. Protein expression was analyzed by Western blot. RESULTS: miR-186 was downregulated in BC tissues and cells. Downregulation of miR-186 was associated with tumor metastasis and a poor overall survival in patients with BC. Overexpression of miR-186 inhibited BC cells proliferation, migration, and epithelial-mesenchymal transition process; while suppression of miR-186 exhibited an opposite effects on BC cells. In addition, Twist1 was identified as a direct target of miR-186 in BC and restoration of Twist1 attenuated the biological effect of miR-186 on BC cells. CONCLUSION: Our findings suggest that miR-186 functions as a tumor suppressor by targeting Twist1 in BC. miR-186 may serve as a novel biomarker in BC diagnosis or a new therapeutic target in BC treatment.

Ectopic lymphoid tissues support local immunoglobulin production in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: The contribution of ectopic lymphoid tissues (eLTs) to local immunoglobulin hyperproduction in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) is unclear. OBJECTIVE: We sought to explore the cellular basis, formation mechanisms, and function of eLTs in patients with CRSwNP. METHODS: We graded lymphoid aggregations in sinonasal mucosa and histologically studied their structures. The expression of lymphorganogenic factors and molecules required for immunoglobulin production was measured by using real-time PCR, and their localization was analyzed by means of immunohistochemistry and immunofluorescence. The phenotype of follicular helper T cells was analyzed by performing flow cytometry. Immunoglobulin levels were quantified by using the Bio-Plex assay or ImmunoCAP system. Nasal tissue explants were challenged ex vivo with Dermatophagoides pteronyssinus group 1 (Der p 1), and the expression of Iε-Cµ and Iε-Cγ circle transcripts was detected by using seminested PCR. RESULTS: Increased formation of eLTs with germinal center-like structures was discovered in patients with eosinophilic (20.69%) and noneosinophilic (17.31%) CRSwNP compared with that in patients with chronic rhinosinusitis without nasal polyps (5.66%) and control subjects (3.70%). The presence of eLTs was associated with increased expression of lymphorganogenic and inflammatory chemokines and cytokines, as well as their receptors. The expression of molecules required for immunoglobulin production, generation of follicular helper T cells, and production of IgE in eosinophilic polyps and IgG and IgA in both eosinophilic and noneosinophilic polyps were predominantly upregulated in patients with eLTs. After Der p 1 challenge ex vivo, Iε-Cµ transcript was detected only in eosinophilic polyps with eLTs but not in polyps without eLTs and noneosinophilic polyps. CONCLUSION: eLTs might support local immunoglobulin production and therefore significantly contribute to the development of CRSwNP.

P-mTOR Expression and Implication in Breast Carcinoma: A Systematic Review and Meta-Analysis.

OBJECTIVE: Phosphorylated mammalian target of rapamycin (p-mTOR) is a promising prognostic marker in many types of cancer. However, its survival benefit in patients with breast carcinoma remains unknown. The aim of the present study was to assess the relationship between p-mTOR expression and prognosis in breast carcinoma based on a systematic review and meta-analysis. MATERIALS AND METHODS: Electronic databases (including Pubmed, Embase, ISI web of science, and Cochrane Library) were searched up to November 24, 2015. The outcome measures were hazard ratios (HRs) with 95% confidence interval (CI) for the association between the prognosis of breast carcinoma patients and p-mTOR expression. Primary end points were disease-free survival (DFS), overall survival (OS), and recurrence-free survival (RFS). Statistical analysis was performed with STATA 12.0. RESULTS: Nine cohort studies including 3051 patients met full eligibility criteria. The pooled HRs (95% CI) for OS, DFS, and RFS were 0.84 (0.27-2.63), 0.71 (0.40-1.23), and 0.48 (0.20-1.18), respectively. CONCLUSIONS: Our findings suggested that p-mTOR overexpression was not significantly related to prognosis in breast carcinoma regarding OS and disease recurrence. Prospective studies are warranted to examine the association between p-mTOR expression and survival outcomes in breast carcinoma.

Nasal IL-4(+)CXCR5(+)CD4(+) T follicular helper cell counts correlate with local IgE production in eosinophilic nasal polyps.

BACKGROUND: Locally produced IgE contributes to the initiation and development of eosinophilic inflammation in eosinophilic nasal polyps independent of systemic atopy. However, whether CXCR5(+)CD4(+) T follicular helper (TFH) cells are involved in local IgE production at mucosal sites remains unexplored. OBJECTIVE: We sought to explore the presence, phenotype, and function of CXCR5(+)CD4(+) TFH cells in eosinophilic nasal polyp tissues compared with noneosinophilic nasal polyp and control normal nasal tissues. METHODS: TFH cell-surface phenotypes and subsets and B-cell subsets in nasal tissues and peripheral blood were studied by means of flow cytometry. Immunohistochemistry was used to detect the tissue location of TFH cells. Sorted nasal TFH cells and CXCR5(-) T cells were cultured with autologous naive B cells purified from blood. RESULTS: Nasal TFH cells expressed inducible costimulator, programmed cell death protein 1, and the transcription factor B-cell lymphoma 6 (Bcl-6) at an intermediate level when compared with bona fide TFH cells in tonsils and circulating TFH cells. Although counts of total TFH cells and IL-21(+), IFN-γ(+), and IL-17(+) TFH cells were increased in both eosinophilic and noneosinophilic nasal polyp tissues compared with those in normal nasal tissues, IL-4(+) TFH cell counts were only increased in eosinophilic polyp tissues. IL-4 and IL-21 were involved in polyp TFH cell-induced IgE production from naive B cells, and nasal IL-4(+) TFH cell counts correlated highly with local IgE levels in vivo. IL-4(+)Bcl-6(+)CD4(+) TFH cells were identified in ectopic lymphoid structures in eosinophilic nasal polyps. TFH cells also positively correlated with germinal center B cells and plasma cells in nasal tissues. CONCLUSION: Nasal IL-4(+) TFH cells might be involved in local IgE production in eosinophilic nasal polyps.

Disease-specific T-helper cell polarizing function of lesional dendritic cells in different types of chronic rhinosinusitis with nasal polyps.

RATIONALE: Although eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps (CRSwNP) exhibit distinct T-helper (Th) responses, the underlying mechanisms remain unclear. OBJECTIVES: To clarify the phenotypes and Th-cell polarizing functions of dendritic cells (DCs) in different types of CRSwNP. METHODS: DC subsets, their surface phenotypes, and Th-cell subsets were studied by means of immunohistochemistry and flow cytometry. The sorted lesional DCs were activated or cultured with autologous naive CD4(+) T cells, and cytokine production was determined by ELISA. Thymic stromal lymphopoietin and osteopontin expression were detected by means of reverse-transcriptase polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Although elevated local Th1 and Th17 cells were noted in both eosinophilic and noneosinophilic CRSwNP, increased Th2 cells were found only in eosinophilic CRSwNP. Increased numbers of myeloid DCs, plasmacytoid DCs, and their activated subsets were found in both types of CRSwNP, but only myeloid DCs and plasmacytoid DCs from eosinophilic CRSwNP demonstrated an up-regulation of OX40 ligand (OX40L) and programmed death ligand 1(PD-L1) expression. Lesional DCs from both types of CRSwNP produced enhanced levels of IL-12, IL-6, and transforming growth factor-ß, and induced increased Th1 and Th17 responses; in contrast, only DCs from eosinophilic CRSwNP induced obviously enhanced Th2 responses, when cocultured with naive CD4(+) T cells. Blockade of OX40L and PD-L1 on lesional DCs from eosinophilic CRSwNP suppressed Th2 responses, but promoted Th1 responses in DC-T cell coculture. CONCLUSIONS: Distinct subsets of lesional DCs were found in eosinophilic and noneosinophilic CRSwNP, where OX40L/PD-L1(+) lesional DCs in eosinophilic CRSwNP could prime Th2 cells, whereas the low OX40L/PD-L1-expressing lesional DCs in noneosinophilic CRSwNP primarily induced Th1/Th17 cells.

MicroRNA in chronic rhinosinusitis and allergic rhinitis.

Inflammatory upper airway diseases, particularly chronic rhinosinusitis (CRS) and allergic rhinitis (AR), have a high worldwide prevalence. CRS and AR involve sustained and exaggerated inflammation that is associated with marked changes in gene and protein expression under tight regulation. A novel group of gene expression regulators is a class of short single-stranded RNA molecules termed microRNAs (miRNAs). miRNAs can cause gene silencing through degradation of target mRNAs or inhibition of translation. Dysregulated expression of miRNAs has been shown in various human diseases, such as cancer, inflammatory skin and bowel diseases, rheumatoid arthritis, and asthma. Although studies of miRNAs in inflammatory upper airway diseases are relatively new and few, emerging evidence implicates an involvement of miRNAs in shaping the inflammation pattern in upper airways. The purpose of this review is to provide an overview on our current understanding of miRNA expression and function in CRS and AR, and to underscore the potential for clinical usage of miRNAs in CRS and AR.

Clara cell 10-kDa protein inhibits T(H)17 responses through modulating dendritic cells in the setting of allergic rhinitis.

BACKGROUND: T(H)17 responses have recently been implicated to play a role in allergic airway diseases, but their local expression in the setting of allergic rhinitis (AR) and their regulation in allergic airway diseases remain unclear. OBJECTIVE: We sought to investigate the regulatory role of Clara cell 10-kDa protein (CC10), an endogenous regulator of airway inflammation, on T(H)17 responses in the setting of AR. METHODS: Wild-type and homozygous CC10-null mice were used to establish an ovalbumin (OVA)-induced AR model. Human recombinant CC10 was given during sensitization or challenge. T(H)17 responses in human subjects and mice were examined by using flow cytometry, quantitative RT-PCR assay, immunohistochemistry, and ELISA. The direct effect of CC10 on T(H)17 cells and CD11c(+) dendritic cells (DCs) was studied by means of cell culture. Adoptive transfer was used to examine the influence of CC10-conditioned DCs on airway inflammation. The regulatory effect of CC10 on the expression of the CCL20 gene was tested by using the BEAS-2B cell line. RESULTS: Compared with those of control subjects, T(H)17 responses were enhanced in the nasal mucosa of patients with AR. CC10-null mice with AR showed enhanced T(H)17 responses, and CC10 treatment significantly decreased T(H)17 responses. CC10 had no direct effect on in vitro T(H)17 cell differentiation. CC10 could significantly decrease the expression of OX40 ligand, IL-23, and IL-6 but enhance CD86 and TGF-ß expression in DCs. Importantly, CC10 was able to inhibit T(H)17 cell polarization in the presence of OVA-pulsed DCs. CC10 pretreatment inhibited T(H)17 responses elicited by adoptive transfer of OVA-pulsed DCs. Furthermore, CC10 decreased the expression of CCL20 in BEAS-2B cells induced by inflammatory cytokines. CONCLUSION: T(H)17 responses are enhanced in patients with AR, and CC10 inhibits T(H)17 responses through modulation of the function of DCs.

[The binding property of different regions in Duffy-binding-like domain alpha of Plasmodium falciparum erythrocyte membrane protein 1 to heparin].

OBJECTIVE: To clone and express Plasmodium falciparum erythrocyte membrane protein 1 DBLalpha (PfEMP1-DBLalpha) and three fragments genes, and screen the strongest affinity sequence with the red blood cell surface receptors-heparin or heparin sulfate in the structure of PfEMP1-DBLalpha. METHODS: The sequence of PfEMP1-DBLalpha1245 was optimized according to the characteristics of E. coli codon, synthesized, and divided into three fragments (DBLaA, DBLalphaB, and DBLalphaC) by PCR. Full-length gene and three gene fragments were subcloned into PGEX-4T-1 vector, and transformed into E. coli BL21 and then induced with IPTG for expression. The recombinant protein was purified from bacterial lysates using glutathione-Sepharose 4B. Heparin affinity test and glycosaminoglycan (GAG) inhibition test were used to analyze the affinity between recombinant protein and heparin. RESULTS: Four recombinant proteins(DBLalpha1245, DBLalphaA, DBLalphaB, and DBLalphaC) were expressed as solubility and the relative molecular weight (M(r) 73 600, M(r) 41 600, M(r) 42 500, and M(r) 41 500) were conformed to the prediction size. Heparin affinity test and GAG inhibition test showed that the four recombinant proteins were binded to the heparin-Sepharose, but not for the GST control. DBLalphaC (Q285-Y415) had the strongest affinity to heparin. CONCLUSION: The strongest affinity sequence with heparin or heparin sulfate in the structure of PfEMP1-DBLalpha is Q285-Y415, which plays a role in binding of Plasmodium falciparum infected red blood cells to the peripheral red blood cells.

Expression of microRNA machinery proteins in different types of chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: Dysregulation of microRNAs (miRNAs) has recently been shown in chronic rhinosinusitis (CRS), the biogenesis and function of which are modulated by miRNA machinery proteins. The expression of these proteins in inflammatory airway diseases is unclear. The aim of this study was to investigate the expression of miRNA machinery components in CRS. STUDY DESIGN: Case-control experimental study. METHODS: The mRNA expression levels of miRNA machinery components including Drosha, Dicer, protein activator of the interferon-induced protein kinase (PACT), human immunodeficiency virus transactivating response RNA-binding protein, fragile X mental retardation protein, and argonaute 2/eukaryotic translation initiation factor 2C, 2 in nasal biopsies from control, CRS without nasal polyps (CRSsNP), eosinophilic, and noneosinophilic CRS with nasal polyps (CRSwNP) subjects were determined by quantitative reverse transcription polymerase chain reaction. Immunohistochemical staining was employed to examine the protein expression of PACT and the cellular source of PACT. RESULTS: Among the tested components, only PACT mRNA expression was found to be altered in CRS, the levels of which were upregulated in CRSwNP as compared with control. In comparison with control and CRSsNP, PACT protein expression was also significantly upregulated in CRSwNP, with a further increase in eosinophilic CRSwNP. PACT was mainly expressed in CD138(+) plasma cells. A higher percentage of PACT-positive plasma cells in total plasma cells was detected in eosinophilic CRSwNP than in noneosinophilic CRSwNP. PACT protein expression correlated with disease severity and eosinophil infiltration. CONCLUSIONS: PACT may be associated with the plasma cell function and eosinophilic inflammation in CRSwNP. However, further experimentation is needed to clarify the functions of PACT.

Overexpression of miR-125b, a novel regulator of innate immunity, in eosinophilic chronic rhinosinusitis with nasal polyps.

RATIONALE: Eosinophilic chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) represents a hard-to-treat subtype of CRS. OBJECTIVES: To determine the pattern of expression and biologic role of microRNAs (miRNAs) in CRS, particularly in eosinophilic CRSwNP. METHODS: Global miRNA expression in sinonasal mucosa from controls, CRS without nasal polyps (CRSsNP), and patients with eosinophilic CRSwNP was compared using miRNA microarrays. MiR-125b expression was detected by means of quantitative reverse-transcriptase polymerase chain reaction. The cellular localization of miR-125b was determined by in situ hybridization. MiR-125b functional assays were performed on airway epithelial cells and mice. MiR-125b expression regulation was studied by tissue and cell culture. MEASUREMENTS AND MAIN RESULTS: CRSsNP and eosinophilic CRSwNP exhibited distinct miRNA expression profiles. MiR-125b was specifically up-regulated in eosinophilic CRSwNP. MiR-125b was mainly expressed by sinonasal and bronchial epithelial cells. EIF4E-binding protein 1 (4E-BP1) was identified as a direct target of miR-125b. MiR-125b mimic or inhibitor enhanced or decreased IFN-α/ß production elicited by dsRNA in vitro or in vivo, respectively. 4E-BP1 expression was decreased, whereas IFN regulatory factor-7 and IFN-ß expression was increased, in eosinophilic CRSwNP. IFN-ß mRNA levels positively correlated with IL-5 mRNA levels and eosinophil infiltration in sinonasal mucosa. IFN-ß stimulated B cell-activating factor of the tumor necrosis factor family production in airway epithelial cells. miR-125b could be induced by lipopolysaccharide, dsRNA, and IL-10. CONCLUSIONS: The up-regulated expression of miR-125b may enhance type I IFN expression through suppressing 4E-BP1 protein expression in airway epithelial cells, which potentially contributes to mucosal eosinophilia in eosinophilic CRSwNP.