Immunomodulatory and anti-viral effects of statins in influenza H5N1 virus infection of human alveolar epithelial cells and peripheral blood–derived macrophages

Poster Session: Novel TherapeuticsBackground: Highly pathogenic avian influenza (HPAI) H5N1 virus panzootic in poultry continues to spread. It causes zoonotic human disease with a high (> 60%) fatality rate and continues to pose a pandemic threat. Based on clinical, animal, and in vitro cell studies, we and others have suggested that differences in viral replication competence, tissue tropism, and cytokine dysregulation between H5N1 and low pathogenic viruses may contribute to disease pathogenesis. Statins as HMG-CoA inhibitors act to reduce cholesterol and have been demonstrated to have anti-inflammatory and immune-modulatory activities. However, there is controversy about the benefits of statin use on influenza infection in mice and humans. In this study, we aimed to evaluate the effects of statin treatment in influenza infection using physiologically relevant in vitro models—human alveolar epithelial cells (AECs) and peripheral blood–derived macrophages (PBDMs). Materials and Methods: Primary human AECs and PBDMs were infected with HPAI H5N1 (A/HK/483/97) and seasonal H1N1 (A/HK/54/98) viruses in the presence or absence of statin (simvastatin and sevastatin) treatment. Virus replication was monitored by measuring infectious viral particles in cell culture supernatants using TCID50. Immuno-modulatory effects of statins were examined by measuring the mRNA and protein expression of cytokines and chemokines using qPCR and ELISA. In order to understand the intervention of statins and influenza infection, the gene expression profile of selected members of the sterol-biosynthesis pathway in influenza virus–infected AECs and PBDMs were also monitored. The responses of a variety of cytokine treatments on the genes of the sterol-biosynthesis pathway were investigated in AECs. Furthermore, the intracellular free cholesterol level was also examined by enzymatic assay in AECs infected with influenza virus. Results: We demonstrated that both simvastatin and mevastatin exhibited a dose-dependent inhibition of influenza virus replication for both HPAI H5N1 and seasonal H1N1 viruses in human AECs and PBDMs. The observed inhibitory effect of simvastatin and mevastatin occurred below the non-specific toxic effects to cells, which were measured by MTT assay. Treatment of simvastatin and mevastatin significantly suppressed H5N1 virus–induced pro-inflammatory cytokines such as TNF-α in PBDMs and chemokines, including IP-10 and MCP-1 secretion in both AECs and PBDMs at 24 hours post-infection. We further showed that human AECs and PBDMs infected with both HPAI H5N1 and seasonal H1N1 viruses had significant down-regulation of sterol pathway gene expression at 24 hours post-infection. AECs and PBDMs treated with IFN-γ or IFN-β but not IL-1β, TNF, or IL-6, showed down-regulation of sterol pathway gene expression. In addition, we found that the free cholesterol level was significantly reduced at 24 and 48 h post-H5N1 virus infection in AECs and in IFN-β–treated AECs. These results further support a specific modulation of the sterol metabolic pathway upon influenza virus infection. Conclusions: Taken together, the controversy about the beneficial effects of statin use in influenza infection and our data suggest that statins possess both the antiviral and immune-regulatory effects in H5N1-infected in vitro cell models. We also demonstrated a highly specific response of AECs and PBDMs through a coordinated negative regulation of multiple sterol pathway members upon influenza virus infection or treatment of interferon. Identification of a reduction in sterol pathway gene expression and cholesterol levels with IFN treatment in human AECs offers new insights on the host-mediated antiviral responses through the sterol metabolism pathway and opens new therapeutic options for human influenza disease.published_or_final_versio

Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis.published_or_final_versio

Tropism and innate host responses of a novel avian influenza A H7N9 virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract

BACKGROUND: Since March, 2013, an avian-origin influenza A H7N9 virus has caused severe pneumonia in China. The aim of this study was to investigate the pathogenesis of this new virus in human beings. METHODS: We obtained ex-vivo cultures of the human bronchus, lung, nasopharynx, and tonsil and in-vitro cultures of primary human alveolar epithelial cells and peripheral blood monocyte-derived macrophages. We compared virus tropism and induction of proinflammatory cytokine responses of two human influenza A H7N9 virus isolates, A/Shanghai/1/2013 and A/Shanghai/2/2013; a highly pathogenic avian influenza H5N1 virus; the highly pathogenic avian influenza H7N7 virus that infected human beings in the Netherlands in 2003; the 2009 pandemic influenza H1N1 virus, and a low pathogenic duck H7N9 virus that was genetically different to the human disease causing A H7N9 viruses. FINDINGS: Both human H7N9 viruses replicated efficiently in human bronchus and lung ex-vivo cultures, whereas duck/H7N9 virus failed to replicate in either. Both human A H7N9 viruses infected both ciliated and non-ciliated human bronchial epithelial cells and replicated to higher titres than did H5N1 (p<0·0001 to 0·0046) and A/Shanghai/1/2013 replicated to higher titres than did H7N7 (p=0·0002-0·01). Both human A H7N9 viruses predominantly infected type II alveolar epithelial cells and alveolar macrophages in the human lung and replicated to higher titres than did H5N1 (p<0·0001 to 0·0078); A/Shanghai/1/2013 replicated to higher titres than did H1N1 (p=0·0052-0·05) and H7N7 (p=0·0031-0·0151). Human H7N9 viruses were less potent inducers of proinflammatory cytokines compared with H5N1 virus. INTERPRETATION: Collectively, the results suggest that the novel H7N9 viruses are better adapted to infect and replicate in the human conducting and lower airways than are other avian influenza viruses, including H5N1, and pose an important pandemic threat.postprin

The neck-region polymorphism of DC-SIGNR in peri-centenarian from Han Chinese Population

<p>Abstract</p> <p>Background</p> <p>DC-SIGNR (also called CD209L) has been extensively studied on its role in host genetic predisposition to viral infection. In particular, variable number tandem repeat (VNTR) of the neck-region of DC-SIGNR is highly polymorphic and the polymorphism has been investigated for genetic predisposition to various infectious diseases, though conflicting results had been reported. As infection is a major cause of human death and a mechanism of natural selection, we hypothesized that VNTR polymorphism of DC-SIGNR might have an effect on human life span.</p> <p>Methods</p> <p>Here we collected 361 peri-centenarian individuals (age ≥94 for female and age ≥90 for male) and 342 geographically matched controls (age 22-53, mean 35.0 ± 12.0) from Han Chinese. The VNTR polymorphism of the neck region was determined by PCR and genotype was called by separating the PCR products in agarose gel.</p> <p>Results</p> <p>A total of 11 genotypes and 5 alleles were found in our population. The genotype distribution, allele frequencies and homozygote proportion did not show a significant difference between peri-centenarian and control group. As gender differences in lifespan are ubiquitously observed throughout the animal kingdom, we then stratified the samples by gender. There was more 6/7 genotypes in female peri-centenarian group than that in female control group, at a marginal level of significance (5.56 vs. 1.28%, p = 0.041). The difference was not significant after correction by Bonferroni method. It suggests a possible differential effect of DC-SIGNR VNTR genotypes between sexes. Further studies are warranted to confirm our preliminary findings and investigate the mechanisms of the underlying functions.</p> <p>Conclusions</p> <p>Our study indicated that there was absence of association between the neck region polymorphism of DC-SIGNR and longevity in Han Chinese population. But the question of whether the DC-SIGNR could affect longevity in a gender-specific pattern remains open.</p

Diosgenin, a Steroidal Saponin, Inhibits Migration and Invasion of Human Prostate Cancer PC-3 Cells by Reducing Matrix Metalloproteinases Expression

BACKGROUND: Diosgenin, a steroidal saponin obtained from fenugreek (Trigonella foenum graecum), was found to exert anti-carcinogenic properties, such as inhibiting proliferation and inducing apoptosis in a variety of tumor cells. However, the effect of diosgenin on cancer metastasis remains unclear. The aim of the study is to examine the effect of diosgenin on migration and invasion in human prostate cancer PC-3 cells. METHODS AND PRINCIPAL FINDINGS: Diosgenin inhibited proliferation of PC-3 cells in a dose-dependent manner. When treated with non-toxic doses of diosgenin, cell migration and invasion were markedly suppressed by in vitro wound healing assay and Boyden chamber invasion assay, respectively. Furthermore, diosgenin reduced the activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatin zymography assay. The mRNA level of MMP-2, -9, -7 and extracellular inducer of matrix metalloproteinase (EMMPRIN) were also suppressed while tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased by diosgenin. In addition, diosgenin abolished the expression of vascular endothelial growth factor (VEGF) in PC-3 cells and tube formation of endothelial cells. Our immunoblotting assays indicated that diosgenin potently suppressed the phosphorylation of phosphatidylinositide-3 kinase (PI3K), Akt, extracellular signal regulating kinase (ERK) and c-Jun N-terminal kinase (JNK). In addition, diosgenin significantly decreased the nuclear level of nuclear factor kappa B (NF-κB), suggesting that diosgenin inhibited NF-κB activity. CONCLUSION/SIGNIFICANCE: The results suggested that diosgenin inhibited migration and invasion of PC-3 cells by reducing MMPs expression. It also inhibited ERK, JNK and PI3K/Akt signaling pathways as well as NF-κB activity. These findings reveal new therapeutic potential for diosgenin in anti-metastatic therapy

Epidermal growth factor receptor dimerization status determines skin toxicity to HER-kinase targeted therapies

Skin toxicity, a common drug-related adverse event observed in cancer patients treated with epidermal growth factor receptor (EGFR)-directed therapies is rarely seen with therapies targeting HER2. This study reports the significance of the EGFR and HER2 dimerization status in skin with regard to these dermatologic side effects. We demonstrate the differential effect of HER-directed therapies on the ligand driven activation status of EGFR, HER2 and MAPK in normal human epidermal keratinocytes. EGFR-directed therapies, such as gefitinib and cetuximab, inhibited ligand-induced activation of EGFR and MAPK in human keratinocytes. Pertuzumab, an antibody interfering with functional HER2 heterodimerization, failed to block ligand-induced HER signaling in primary keratinocytes. Using a novel proximity-based dimerization assay (eTag™) we show that EGFR homodimers are the predominant HER dimer pair in normal primary kertinocytes and in normal skin tissue from 16 patients with solid malignancies. The presence of [p]EGFR and [p]MAPK, but the absence of [p]HER2, demonstrates productive signaling via EGFR but not HER2 in human skin. These data illustrate the importance of the EGFR dimerization partner in human skin and suggests that inhibition of EGFR homodimer signaling rather than EGFR/HER2 heterodimer signaling maybe the key molecular event determining dermatologic toxicity discrepancies observed between EGFR-targeted versus HER2-targeted therapies