Glycyrrhizin inhibits highly pathogenic H5N1 influenza A virus-induced pro-inflammatory cytokine and chemokine expression in human macrophages

Abstract

Hypercytokinaemia is thought to contribute to highly pathogenic H5N1 influenza A virus disease. Glycyrrhizin is known to exert immunomodulatory and anti-inflammatory effects and therefore a candidate drug for the control of H5N1-induced pro-inflammatory gene expression. Here, the effects of an approved parenteral glycyrrhizin preparation were investigated on H5N1 virus replication, H5N1-induced pro-inflammatory responses, and H5N1-induced apoptosis in human monocyte-derived macrophages. Glycyrrhizin 100 μg/ml, a therapeutically achievable concentration, impaired H5N1-induced production of CXCL10, interleukin 6, and CCL5 and inhibited H5N1-induced apoptosis but did not interfere with H5N1 replication. Global inhibition of immune responses may result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8(+) T-lymphocytes. Notably, glycyrrhizin concentrations that inhibited H5N1-induced pro-inflammatory gene expression did not affect cytolytic activity of natural killer cells. Since H5N1-induced hypercytokinaemia is considered to play an important role within H5N1 pathogenesis, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

Fig. 1

Influence of glycyrrhizin on H5N1-replication in human monocyte-derived macrophages (MDMs). a Representative pictures showing staining of nucleuses by DAPI (blue), H5N1 nucleoprotein (NP) (green), or merge of both stainings in H5N1 (MOI 2)-infected MDMs without treatment, with glycyrrhizin 200 μg/ml, or with ribavirin 40 μg/ml 24-h post-infection. Mock-infected cells did not show NP staining (not shown). b Fraction of H5N1 nucleoprotein-expressing cells in H5N1 (MOI 2)-infected MDMs without treatment, with glycyrrhizin (gly) 200 μg/ml, or with ribavirin (ribav) 40 μg/ml 24-h post-infection. No H5N1 nucleoprotein staining was detected in Mock-infected cells. *P < 0.05 relative to virus control. c Virus titres in H5N1 (MOI 2)-infected MDMs without treatment, with glycyrrhizin (gly) 200 μg/ml, or with ribavirin (ribav) 40 μg/ml 24-h post-infection. No virus titre was detected in mock-infected cells

Fig. 2

Influence of glycyrrhizin on H5N1-induced caspase activation in monocyte-derived macrophages (MDMs). Caspase activation was measured in non-infected (MOCK) or H5N1 A/Thailand/1(Kan-1)/04 (MOI 2)-infected glycyrrhizin-treated or non-treated MDMs 24-h post-infection. Glycyrrhizin treatment did not affect caspase activation in mock cells (not shown). *P < 0.05 relative to non-treated virus control

Fig. 3

Influence of glycyrrhizin on H5N1-induced cytokine expression in monocyte-derived macrophages (MDMs). a–c Excretion of cytokines by non-infected MDMs (Mock) or H5N1 A/Thailand/1(Kan-1)/04 (MOI 2)-infected MDMs with or without glycyrrhizin (100 μg/ml) treatment 24-h post-infection determined by ELISA. Glycyrrhizin treatment did not affect cytokine excretion in mock cells (not shown). d Relative expression of cytokines at the mRNA level detected via real-time PCR. Glycyrrhizin treatment did not affect cytokine expression in mock cells (not shown). *P < 0.05 relative to non-treated virus control

Fig. 4

Influence of glycyrrhizin on natural killer (NK) cell cytolytic activity. A 4 h cytotoxicity assay against 5,000 K562 target cells was performed at indicated effector cell/target cell (E/T) ratios using glycyrrhizin (100 μg/ml)-, ribavirin (20 μg/ml)-treated, or non-treated (control) IL-2-activated NK cells. *P < 0.05 relative to non-treated virus control