Allan S. Y. Lau

Induction of Proinflammatory Cytokines in Primary Human Macrophages by Influenza A Virus (H5N1) Is Selectively Regulated by IFN Regulatory Factor 3 and p38 MAPK

The hyperinduction of proinflammatory cytokines and chemokines such as TNF-α, IFN-β, and CCL2/MCP-1 in primary human macrophages and respiratory epithelial cells by the highly pathogenic avian influenza H5N1 is believed to contribute to the unusual severity of human H5N1 disease. Here we show that TNF-α, IFN-β, and IFN-λ1 are the key mediators directly induced by the H5N1 virus in primary human macrophages. In comparison with human influenza (H1N1), the H5N1 virus more strongly activated IFN regulatory factor 3 (IRF3). IRF3 knockdown and p38 kinase inhibition separately and in combination led to a substantial reduction of IFN-β, IFN-λ1, and MCP-1 but only to a partial reduction of TNF-α. IRF3 translocation was independent of p38 kinase activity, indicating that IRF3 and p38 kinase are distinct pathways leading to cytokine production by H5N1 virus. We conclude that IRF3 and p38 kinase separately and predominantly contribute to H5N1-mediated induction of IFN-β, IFN-λ1, and MCP-1 but only partly control TNF-α induction. A more precise identification of the differences in the regulation of TNF-α and IFN-β could provide novel targets for the design of therapeutic strategies for severe human H5N1 influenza and also for treating other causes of acute respiratory distress syndrome.

MHC expression kinetics and immunogenicity of mesenchymal stromal cells after short-term IFN-γ challenge

OBJECTIVE Under the influence of interferon-gamma (IFN-gamma), mesenchymal stromal cells (MSCs) are conditional antigen-presenting cells, which have immunosuppressive potential. Apart from IFN-gamma upregulation of major histocompatibility complexes class I and II (MHC-I and MHC-II) expression, the underlying kinetics and mechanisms have not been described previously. This information is helpful to delineate how human MSCs can be modulated by IFN-gamma in different clinical scenarios. MATERIALS AND METHODS Here, we demonstrated that IFN-gamma-treated MSCs underwent classical signal transduction pathway via phosphorylation of signal transducers and activators of transcription-1, activation of interferon regulatory factor-1, and class II transactivator comparable to that of primary human blood macrophages. RESULTS IFN-gamma markedly induced expression of MHC-I instantly, while its effects on MHC-II were less dramatic and delayed up to 4 days. This is due to a slower intracellular transport of the MHC-II antigen to the membrane surface. More important is that MSCs showed a reduction in their proliferation by 50% without evidence of cell death after prolonged IFN-gamma treatment for 8 days. High-dose IFN-gamma-treated MSCs (500 U/mL) could initiate T-cell activation as indicated by expression of CD25 and proliferation of allogeneic T cells. CONCLUSIONS The summative IFN-gamma effects will adversely affect the immunoprivilege status and lifespan of MSCs.

Senkyunolides reduce hydrogen peroxide-induced oxidative damage in human liver HepG2 cells via induction of heme oxygenase-1

Rhizoma Chuanxiong is widely used as folk medicine to treat the diseases caused by oxidative stress and inflammation. To delineate the underlying molecular mechanisms, we recently found that Rhizoma Chuanxiong extract significantly induced heme oxygenase-1 (HO-1), an enzyme that degrades intracellular heme into three bioactive products: biliverdin, carbon monoxide and free iron. The anti-inflammatory, antiapoptotic and antiproliferative actions of these products highlight HO-1 as a key endogenous antioxidant and cytoprotective gene. This study was designed to further characterize HO-1 induction of Rhizoma Chuanxiong through bioactivity-guided fractionation. All isolated fractions were assayed for HO-1 induction in human HepG2 cell line at mRNA and protein levels. Based on chromatographic profiling, nuclear magnetic resonance (NMR) and mass spectrometric analysis, the active compounds were identified as senkyunolide-H and its stereoisomer senkyunolide-I. Both senkyunolide isomers inhibited the formation of reactive oxygen species and lipid peroxidation and enhanced the cellular resistance to hydrogen peroxide-induced oxidative damage. Notably, heme oxygenase inhibitor tin protoporphyrin IX (SnPP) significantly suppressed the antioxidant activity of senkyunolide stereoisomers. Thus, this study demonstrated that senkyunolide-H and -I attenuated oxidative damage via activation of HO-1 pathway.

Interleukin-17A differentially modulates BCG induction of cytokine production in human blood macrophages

The pathogenesis of Mtb depends in part on cytokine cross‐regulation between macrophages and T cells in host immunity. Th17 cells produce IL‐17A to induce granuloma formation and to restrict mycobacterial dissemination. IL‐17A also mediates cytokine responses induced by proinflammatory cytokines such as TNF‐α. Our previous results showed that BCG induces IL‐6, IL‐10, and TNF‐α via activity of protein kinases, including dsRNA‐activated serine/threonine protein kinase and glycogen synthase kinase‐3 in primary human monocytes. Therefore, we investigated whether IL‐17A, upon its induction by BCG, plays an additional role to aid the production of downstream proinflammatory cytokines in macrophages. Here, we showed that IL‐17A enhanced IL‐6 mRNA and protein levels inducible by BCG in a time‐ and dose‐dependent manner, whereas it had no effect on IL‐10 and TNF‐α production. We also demonstrated that IL‐17A activated the phosphorylation of ERK1/2 triggered by BCG. With the use of a specific chemical inhibitor of a MAPK/ERK‐activating kinase (MEK1/2), we confirmed the correlation between the enhanced ERK1/2 activation and augmented IL‐6 production. Additionally, we revealed that IL‐17A acts in concert with BCG‐induced TNF‐α to enhance the level of IL‐6 synthesis. Taken together, our results suggest a significant role of IL‐17A to serve as a modulator of cytokine expression in innate immune response during mycobacterial infection.

A role for interleukin‐17A in modulating intracellular survival of Mycobacterium bovis bacillus Calmette–Guérin in murine macrophages

Interleukin 17A IL‐17A is a crucial immunomodulator in various chronic immunological diseases including rheumatoid arthritis and inflammatory bowel disease. The cytokine has also been demonstrated to control the pathogenesis of the Mycobacterium tuberculosis by dysregulating production of cytokines and chemokines and promoting granuloma formation. Whether IL‐17A regulates innate defence mechanisms of macrophages in response to mycobacterial infection remains to be elucidated. In the current report, we investigated the effects of IL‐17A on modulating the intracellular survival of Mycobacterium bovis bacillus Calmette–Guérin (BCG) in RAW264.7 murine macrophages. We observed that IL‐17A pre‐treatment for 24 hr was able to synergistically enhance BCG‐induced nitric oxide (NO) production and inducible nitric oxide synthase expression in dose‐ and time‐dependent manners. We further delineated the mechanisms involved in this synergistic reaction. IL‐17A was found to specifically enhanced BCG‐induced phosphorylation of Jun N‐terminal kinase (JNK), but not of extracellular signal‐regulated kinase 1/2 and p38 mitogen‐activated protein kinase. By using a specific JNK inhibitor (SP600125), we found that the production of NO in BCG‐infected macrophages was significantly suppressed. Taken together, we confirmed the involvement of the JNK pathway in IL‐17A‐enhanced NO production in BCG‐infected macrophages. We further demonstrated that IL‐17A significantly enhanced the clearance of intracellular BCG by macrophages through an NO‐dependent killing mechanism. In conclusion, our study revealed an anti‐mycobacterial role of IL‐17A through priming the macrophages to produce NO in response to mycobacterial infection.

Antiviral effect of forsythoside A from Forsythia suspensa (Thunb.) Vahl fruit against influenza A virus through reduction of viral M1 protein

ETHNOPHARMACOLOGICAL RELEVANCE Yinqiaosan is a classical traditional Chinese medicine formula, which has been used to treat respiratory diseases since ancient China. It consists of nine herbs and among them, Forsythia suspensa (Thunb.) Vahl fruit is one of the major herbal components. Despite the long history of Yinqiaosan, the active compounds and the mechanisms of action of this formula remain elusive. AIM OF THE STUDY The present study aimed to examine the suppressive effect of Yinqiaosan on influenza virus and to identify the active components in the formula targeting influenza. MATERIALS AND METHODS Anti-influenza virus effect of Yinqiaosan was assessed by tissue culture infective dose assay, and was also tested in an in vivo mouse model. Active compound from the formula was identified with a bioactivity-guided fractionation scheme. The potential mode of action of the compound was further investigated by identifying the host cell signaling pathways and viral protein production using in vitro cell culture models. RESULTS Our results showed that forsythoside A from Forsythia suspensa (Thunb.) Vahl fruit, a major herbal component in Yinqiaosan, reduced the viral titers of different influenza virus subtypes in cell cultures and increased the survival rate of the mice in an in vivo influenza virus infection model. Further experiments on the mode of action of forsythoside A showed that it reduced the influenza M1 protein, which in turn intervened the budding process of the newly formed virions and eventually limited the virus spread. CONCLUSION Results of our present study provides scientific evidence to support to the application of a traditional herbal formula. We also identify novel candidate compound for future drug development against influenza virus.