Microbiota regulates the TLR7 signaling pathway against respiratory tract influenza A virus infection.

Although intestinal flora are crucial in maintaining immune homeostasis of the intestine, the role of intestinal flora in immune responses at other mucosal surfaces remains less clear. Here, we show that intestinal flora composition critically regulates the toll-like receptor 7 (TLR7) signaling pathway following respiratory influenza virus infection. TLR7 ligands rescued the immune impairment in antibiotic-treated mice. Intact microbiota provided signals leading to the expression of mRNA for TLR7, MyD88, IRAK4, TRAF6, and NF-κB at steady state. Significant changes in the composition of culturable commensal bacteria reduced the expression levels of components of the TLR7 signaling pathway. Our results reveal the importance of intestinal flora in regulating immunity in the respiratory mucosa through the upregulation of the TLR7 signaling pathway for the proper activation of inflammasomes.

Immunologic mechanism of Patchouli alcohol anti-H1N1 influenza virus may through regulation of the RLH signal pathway in vitro.

Patchouli alcohol (PA) is a kind of methanol extracted from traditional Chinese medicine Pogostemonis Herba. Our research aimed to observe the anti-influenza virus role of PA in vitro. 16HBE (human respiratory epithelial cell) was infected by H1N1 (A/FM1/1/47) to set the cell model. Then the 16HBE was co-cultivated with three kinds of immune cells: dendritic cells, macrophages, and monocytes, PA (the concentration is 10 µg/mL) was added as a treatment intervention for 24 h. The immune cells and the supernate were collected for RT-PCR and ELISA detection related to RLH (RIG-1-like helicases) pathway. Results showed that the IL-4 and IFN-γ in supernate were increased after H1N1 infection, and the PA treatment suppressed the expression of cytokines and the mRNA of RLH pathway. PA anti-influenza virus may through regulate the RLH singal pathway.

Study on the anti-H1N1 virus effects of quercetin and oseltamivir and their mechanism related to TLR7 pathway.

The antivirus effect of quercetin and oseltamivir on the Toll-like receptor 7 (TLR7) signaling pathway was observed when dendritic cells and macrophages were infected with H1N1. Leukomonocytes were obtained from umbilical cord blood and harvested after stimulation by recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) and recombinant human Interleukin 4 (rhIL-4). Virus-infected cell model was established by human bronchial epithelial cells (16HBE) infected with H1N1. After immunological cells and virus-infected cells were co-cultured, quercetin and oseltamivir were also added into the medium as a treatment intervention. Then the immunological cells were collected for Real Time PCR (RT-PCR) and Western blot to determine the expression levels of genes related to TLR7 pathway. Viral infection led to cell death and increased the gene expression levels of TLR7 signal pathway. Quercetin and oseltamivir increased cell viability and reduced the expression levels of TLR7 signal pathway.

Dysbiosis of gut microbiota induced the disorder of helper T cells in influenza virus-infected mice.

It is widely understood that commensal microbiota contributes to the maintenance of intestinal homeostasis through dynamic interactions with a body's immunity. And the immune regulation is important for the influenza vaccine's effectiveness after body injection, however, the mechanism between commensal microbiota and vaccine's effectiveness remains unknown. The impact that individual bacteria species have on the balance of the systemic immune system beyond the local intestinal mucosal tissues also remains less clear, and the related mechanism is still unknown. In this study, through the administration of various antibiotics, we examined the balance of helper T cell subsets in mice after inoculating them with the influenza virus and then, attempted to imitate the clinical practice in which patients are always prescribed with an antibiotic treatment in flu season. The data indicates that the mice in each group present differential immune responses in terms of the makeup of helper T cell subsets, although the Th17 cell activity seems to not be involved in the systemic immune modulation in the mice that are susceptible to the intervention of antibiotic. Th1, Th2, and anti-inflammatory regulatory T cells have been implicated in the contribution to the systemic immune response influenced by the antibiotic-induced dysbiosis. Thus we believe that the normal intestinal flora could maintain the immune balance and inhibit the inflammatory responses, which may be useful for clinical application to take intestinal flora into consideration when influenza vaccination was used.

Andrographolide as an anti-H1N1 drug and the mechanism related to retinoic acid-inducible gene-I-like receptors signaling pathway.

OBJECTIVE: To observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1. METHODS: Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR). RESULTS: The optimal concentration of AD for anti-virus effect was 250 µg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-ß promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05). CONCLUSIONS: The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

The inducible effect of LBP on maturation of dendritic cells and the related immune signaling pathways in hepatocellular carcinoma (HCC).

OBJECTIVE: To investigate the effect of LBP on differentiation and maturation of healthy human peripheral blood-derived dendritic cells cultured in different tumor microenvironment in vitro, and discuss the molecular and immunological mechanisms of LBP in treatment of tumor. METHODS: In this study, we procured the peripheral blood-derived dendritic cells precursor cell by the Density gradient centrifugation method, and used the tumor-cell supernatant to prepare conditioned medium. The GM-CSF and IL-4 induced DCs precursor cell differentiation to DCs, the TNF-α promoted the immature DCs developed to mature DCs. In this way, we detected the influence of LBP on the expressions of surface molecules of DCs cultured in different environments, and especially on the role of related-immunity and NF-κB activity. RESULTS: In LBP-treated group, the molecular phenotype of DCs, its capacity to stimulate allogeneic lymphocyte proliferation, and the levels of IL-12p70 and IFN-γ secretion were higher than the untreated group (p < 0.05), with statistical significance. Meanwhile the expression of NF-κB of the DCs in the medium treated by the LBP was higher than the untreated group (p < 0.05), also with statistical significance. Between the two different tumor microenvironment groups, the cell nucleus protein NF-κB expression is obviously different, the hepG2.2.15 group higher than the hepG2 group. CONCLUSION: LBP could increase the expression of the phenotype of DCs, the secretion of IL-12p70 and IFN-γ in MLR, and enhance the NF-κB expression, especially in the virus-related group, suggesting LBP plays the anti-tumor role stronger in the virus-related environment and this phenomenon correlates with the NF-κB signaling pathway.

Carboxymethylpachymaran enhances immunologic function of dendritic cells cultured in two kinds of hepatoma carcinoma cell line's supernatant via nuclear factor κ B/Rel pathway.

OBJECTIVE: To study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs. METHODS: DCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis. RESULTS: The proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P <0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P <0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P <0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P <0.05). However, there was no significant difference between the two CM groups (P >0.05). CONCLUSIONS: Two kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.

The suppression effects of desacetyluvaricin on hepatocellular carcinoma and its possible mechanism.

OBJECTIVE: To investigate the anticancer effects of desacetyluvaricin (DES) on hepatocellular carcinoma (HCC) in vitro, and to study its mechanism. MATERIALS AND METHODS: Using DES and cisplatin (DDP) to intervene the cell lines of hepatocarcinoma G2.2.15 (HepG2.2.15) and HepG2, by detecting the expression of HBxAg by immunofluorescence method, the cell cycle and apoptosis by flow cytometry method (FCM), and expression of NF-κB protein by ELISA. RESULTS: DES and DDP showed to suppress proliferation of HepG2.2.15 and HepG2; they increase the S-phase cells and decrease G2/M phase cells. DES and DDP both could promote the apoptosis and reduce the expression of NF-κB on the cell line. DES and DDP both can suppress the expression of HbxAg in HepG2.2.15. There were no statistical differences of the above results between these two drugs (P > 0.05). CONCLUSIONS: DES possesses anticancer effect on hepatocarcinoma. The possible mechanism might be due to promotion the apoptosis of the cancer cells, and downregulate the expression of HBx andNF-κB protein. DES is a kind of natural products, Because of the lighter clinical side effects; our observations suggest that DES has the potential to be explored as an effective anticancer agent for HCC.