Role of LINC00240 on T-helper 9 differentiation in allergic rhinitis through influencing DNMT1-dependent methylation of PU.1.

BACKGROUND: Allergic rhinitis (AR) is a common allergic disease with increasing prevalence globally. However, the molecular mechanism underlying AR pathogenesis remains largely undefined. METHODS: Peripheral blood and nasal mucosa samples obtained from patients with AR (n = 22), and ovalbumin-induced AR mouse model (n = 8 per group) were prepared for subsequent detection. qRT-PCR and western blot were used to detect the expression of LINC00240, miR-155-5p, PU.1 and other key molecules. ELISA assay and flow cytometry were employed to evaluate the secretion of IL-9 and T-helper 9 (Th9) cell ratio, respectively. Bioinformatics analysis, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and luciferase reporter assays were employed to further elucidate the regulatory network of LINC00240/miR-155-5p/DNMT1. The methylation of PU.1 promoter was assessed by methylation-specific PCR (MSP). This signaling axis was further validated in the mouse model of AR. RESULTS: LINC00240 was downregulated, while miR-155-5p and PU.1 were upregulated in the peripheral blood and nasal mucosa of AR patients, as well as in AR mice. This was accompanied with the increased ratio of Th9 cells and elevated IL-9 secretion. Mechanistically, LINC00240 served as a miR-155-5p sponge, and DNMT1 was a target of miR-155-5p. In addition, DNMT1 mediated the methylation of PU.1 promoter. In vivo studies verified that LINC00240 mitigated AR progression, possibly via miR-155-5p/DNMT1/PU.1-dependent Th9 differentiation. CONCLUSION: The involvement of LINC00240 in AR pathogenesis is closely associated with Th9 differentiation through modulating DNMT1-dependent methylation of PU.1 by sponging miR-155-5p.

IRF4 transcriptionally activate HOTAIRM1, which in turn regulates IRF4 expression, thereby affecting Th9 cell differentiation and involved in allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is an inflammatory reaction caused by irritation of nasal mucosa by external allergens, which seriously affects the life of patients. Here, we aimed to investigate the effect and mechanism of long non-coding RNA HOX antisense intergenic RNA myeloid 1 (lncRNA HOTAIRM1) on AR development. METHODS: The nasal mucosa samples were collected from AR patients and AR model mice (induced by ovalbumin). T helper type 9 (Th9) cells were examined by flow cytometry. Fluorescence in situ hybridization was conducted to examine the localization of HOTAIRM1 in CD4+ T cells. Dual-luciferase reporter assay or RNA immunoprecipitation was conducted to examine the bond between HOTAIRM1 and miR-148a-3p, miR-148a-3p, and interferon regulatory factor 4 (IRF4). Chromatin Immunoprecipitation assay was conducted to detect the interaction between IRF4 and HOTAIRM1 promoter. RESULTS: HOTAIRM1, interleukin-9 (IL-9), and IRF4 were highly expressed in the AR model. The ratio of Th9 cells was increased in AR mice and overexpressing HOTAIRM1 further promoted Th9 cell differentiation, while the effect was reversed after overexpression of miR-148a-3p. Besides, in vivo experiments showed that interfering with HOTAIRM1 reduced the number of sneezing and rubbing movements, reduced immunoglobulin E (IgE) and IL-9 levels, as well as Th9 cells. HOTAIRM1 was expressed in the cytoplasm and the interactions between HOTAIRM1 and miR-148a-3p, miR-148a-3p and IRF4, were confirmed. Furthermore, IRF4 bound to the HOTAIRM1 promoter and promoted its transcriptional activation. CONCLUSION: HOTAIRM1 was highly expressed in the AR model. Besides, IRF4 activated HOTAIRM1 transcription, and HOTAIRM1, in turn, up-regulated IRF4 expression through competitively binding to miR-148a-3p with IRF4, thereby affecting Th9 cell differentiation and participating in the occurrence and development of AR. Our results suggested that interference with HOTAIRM1 might become a treatment for AR.

Circ_0067835 regulates allergic inflammatory response in type-2 innate lymphoid cells in allergic rhinitis (AR) via miR-155/GATA3.

Allergic rhinitis (AR) is a familiar respiratory allergic inflammatory disease with higher incidence. The pathogenesis of AR is particularly complex. Therefore, a lot of work is acquired to excavate deep mechanisms, thereby providing effective strategies for AR diagnose and treatment. AR mice model was induced by recombinant murine IL-33 (0.05 µg/µl) on days 1, 3, and 5. The lentiviral vectors carrying si-circ_0067835, miR-155 mimic, si-NC or miR-NC were injected into AR mice. Thus, mice were divided into control, AR, AR + si-NC, AR + si-circ_0067835, AR + si-circ_0067835 + miR-NC, and AR + si-circ_0067835 + miR-155 mimic groups. qRT-PCR experiment was used to measure the expression of circ_0067835 and miR-155. Behavioral test result was quantified to assess AR mice model. Hematoxylin and eosin (HE) staining was performed to analyze histopathological changes. Helper T cell 2 (Th2) cytokines (IL-4, IL-5, IL-9 and IL-13) and percentage of type-2 innate lymphoid cells (ILC2s) in nasal mucosa tissues in AR mice model were evaluated needing western blot, ELISA, and flow cytometry. Besides, the targeting relationship between circ_0067835 and miR-155, or between miR-155 and GATA3, was investigated via luciferase report assay. Circ_0067835 expression levels were raised in the nasal mucosa tissues of AR mice. Inhibiting circ_0067835 could reduce Type2 cytokines and ILC2s levels in AR mice model. Furthermore, circ_0067835 targeted and positively regulated miR-155 expression, and GATA3 was a downstream target of miR-155 and adjusted by circ_0067835/miR-155 axis. In addition, silencing circ_0067835 inhibited cytokines and ILC2s levels by down-regulating miR-155. Circ_0067835 effectively inhibited AR response in ILC2s through participation of miR-155/GATA3 axis.

Iloprost inhibits acute allergic nasal inflammation by GATA3 -ILC2 pathway in mice.

BACKGROUND: Acute allergic nasal inflammation is very common in the clinical allergic diseases, Prostaglandin I2 (PGI2) has been found to effective in combating inflammation. Iloprost, as an analog of PGI2, whose role and mechanisms in the acute allergic nasal inflammation remains unclear. It's necessary to elucidate the efficacy and potential mechanism of Iloprost in acute allergic nasal inflammation. METHODS: 36 female mice were randomly divided into DMSO group, IL 33 group, Iloprost group and IL 33+Iloprost intervention group. Mice were stimulated with IL 33 to construct an acute allergic nasal inflammation model. Hematoxylin and eosin (HE) and periodic acid Schiff reagent (PAS) staining, flow cytometry, Real time PCR and Enzyme linked immunosorbent assay (ELISA) was used to identify the role of Iloprost in acute allergic nasal inflammation. The comparison between multiplied groups was analyzed by ANOVA, and the Bonferroni method was used for further comparison of two groups. RESULTS: Compared with IL 33 group, the inflammatory cell infiltration around the trachea and blood vessels of the lung tissue in the IL 33+ Iloprost group were reduced; goblet cell hyperplasia was observed in airway mucosa of IL 33 group, and the mucus secretion increased; the percentage of EOS and ILC2s in the BALF and lung single cell suspensions in IL 33+ Iloprost group were statistically lower than that of IL 33 group (p < 0.05); The mRNA expression levels of IL 5, IL 13, ST2 and GATA3 in the lung tissue of IL 33 group were higher than those in DMSO group (p < 0.05). After intervention with Iloprost, the mRNA expression levels of IL 5, IL 13, GATA3 and ST2 were lower than those in IL 33 group (p < 0.05) CONCLUSION: Iloprost may potentially inhibit the proliferation and activation of innate lymphoid cells 2 in mice with acute allergic inflammation, which maybe an effective option for the treatment of acute allergic inflammation related diseases.

Involvement of T-Helper 9 Activation in a Mouse Model of Allergic Rhinitis.

BACKGROUND We aimed to investigate the role of T-Helper (TH) 9 cells in the pathogenesis of allergic rhinitis (AR) in mice. MATERIAL AND METHODS An AR model was produced in BALB/c mice, and the viral encoding interleukin (IL)-9 silencing sequence was used to reduce IL-9 expression. The experiment was divided into a control group, an AR group, an IL-9 shRNA+AR group, and a vector+AR group. Hematoxylin and eosin (H&E) staining was used to detect pathological changes. The cytokine expression was detected by ELISA method. Cellular typing was detected by flow cytometry. RESULTS Cells in the control group were regularly arranged, with clear layers and no congestion, edema, or necrosis observable. By contrast, in the AR model group and the vector treatment group, nasal mucosa showed clear hyperemia and edema in upper tissues and infiltration of inflammatory cells, which were ameliorated by IL-9 silencing. Compared with the control group, interferon-γ (IFN-γ) was significantly down-regulated, while IL-4, IL-17, and IL-9 were significantly elevated in the AR model group. TH1 cells in nasal mucosa, lymph, nasal lavage, spleen, and peripheral blood were significantly reduced, while TH2, TH9, TH17, and Treg cells were significantly elevated in the AR group compared with the control group. Importantly, all these changes in AR model were ameliorated by IL-9 silencing. CONCLUSIONS AR is related to the changes of cytokines in TH1, TH2, TH9, TH17, and Treg, which are improved by IL-9 silencing. Activation of TH9 cells is involved in the pathogenesis of AR.

MicroRNA-let-7e regulates the progression and development of allergic rhinitis by targeting suppressor of cytokine signaling 4 and activating Janus kinase 1/signal transducer and activator of transcription 3 pathway.

The present study aimed to explore the microRNA-let-7e (miR-let-7e) expression in allergic rhinitis (AR), and to investigate the underlying molecular mechanisms. miR-let-7e expression in the nasal mucosa of mice and patients with AR were detected. The expression levels of three inflammatory factors, including histamine, immunoglobulin E and tumor necrosis factor-α (TNF-α), in the blood of AR mice and in interleukin (IL)-13-stimulated nasal epithelial cells (NECs) were also measured. Furthermore, the target gene of miR-let-7e was predicted and validated using a luciferase reporter assay. The expression levels of Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) were detected. The results demonstrated that miR-let-7e was downregulated in patients and mice with AR compared with the controls. In addition, the expression levels of inflammatory factors were higher in the blood of mice with AR compared with the control group, while miR-let-7e overexpression inhibited these levels in AR mice and IL-13-stimulated NECs. Furthermore, suppressor of cytokine signaling 4 (SOCS4) was revealed as a potential target gene of miR-let-7e and was negatively regulated by miR-let-7e. Overexpression of SOCS4 abrogated the anti-inflammatory activity of miR-let-7e overexpression. Finally, miR-let-7e overexpression activated the JAK1/STAT3 signaling pathway. In conclusion, miR-let-7e may serve an important role in the progression and development of AR, while overexpression of miR-let-7e had an anti-inflammatory effect by targeting SOCS4, which may be achieved by activation of the JAK1/STAT3 signaling pathway.