Pandemic H1N1 2009 influenza A virus induces weak cytokine responses in human macrophages and dendritic cells and is highly sensitive to the antiviral actions of interferons

Abstract

In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-alpha/beta]) and type III (IFN-lambda1 to -lambda3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-alpha) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-alpha and IFN-beta inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-lambda3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-lambda3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.

FIG. 1.

Infectivity and replication of the pandemic 2009 influenza A (H1N1) virus in primary human DCs. (A) To study the infectivity of the pandemic 2009 (H1N1) influenza A virus, human monocyte-derived DCs from four blood donors were infected separately with different doses (starting from 1 HA unit/ml) of A/Finland/553/2009 strain of the virus (H1/09) for 18 h. The expression of viral glycoproteins was detected by flow cytometry using cross-reacting H1N1 glycoprotein-specific rabbit anti-H1N1 antibodies. The proportion of infected cells was analyzed, and the multiplicity of infection (MOI) was determined as 0.5 in the dilution where 50% of cells were infected. (B) To study the kinetics of virus replication, DCs were infected with a pandemic A/Finland/553/2009 virus (H1/09) and seasonal influenza viruses A/Brisbane/59/07 (H1/07), A/New Caledonia/20/99 (H1/99), A/WSN/33 (H1/33), and A/Udorn/72 (H3/72) at MOI of 5. After 1, 3, 8, 24, or 48 h of infection, the cells from different donors were harvested and pooled, and total cellular RNA samples for quantitative RT-PCR were prepared. Due to significant cell death, 48-h samples were not available for the cells infected with seasonal viruses, and thus the 48-h time point is missing. The expression of viral M1 RNA was measured by qRT-PCR, and the values were normalized to 18S rRNA and presented as relative copy numbers of M1 RNA.

FIG. 2.

The pandemic 2009 and seasonal influenza A (H1N1) viruses induce weak cytokine gene expression in DCs. (A) DCs were infected with influenza viruses A/Finland/553/2009 (H1/09), A/Brisbane/59/07 (H1/07), A/New Caledonia/20/99 (H1/99), A/WSN/33 (H1/33), and A/Udorn/72 (H3/72) at MOI of 5. Cells from different blood donors were collected at 1 h to 48 h after infection and pooled, and cellular RNA was isolated. Virus-induced cytokine expression was analyzed by quantitative RT-PCR with primers and probes for IFN-λ1, IFN-λ2/3, IFN-β, IFN-α1, CXCL10, and TNF-α. The values were normalized to 18S rRNA and presented as relative gene expression in relation to the RNA sample obtained from uninfected control cells. (B) Protein levels of IFN-λ1, IFN-β, IFN-α, CXCL10, and TNF-α measured by ELISA from the supernatants of DCs after 24 h of infection with different influenza A viruses at decreasing doses (MOI of 5, 1, 0.2, 0.04, and 0.008, respectively). The data are presented as mean values with standard deviations calculated from the results from 3 different experiments, each having cells from 2 to 4 different donors. c, control supernatant of cells without stimulation.

FIG. 3.

Comparison of pandemic influenza A (H1N1) virus-induced cytokine responses in monocyte-derived DCs and macrophages. Monocyte-derived human DCs and macrophages and MDCK cells were infected with decreasing doses (representing MOI of 10, 2, 0.4, 0.08, or 0.016 in DCs) of A/Finland/553/2009 virus. Cells were collected at 20 h after infection and pooled, cellular RNA was isolated, and qRT-PCR was carried out. (A) Virus replication in DCs, macrophages, and MDCK cells as measured by quantifying influenza virus M1 gene expression by qRT-PCR. The values were normalized to 18S rRNA and presented as relative copy numbers. (B) Virus dose-dependent expression of IFN-λ1, IFN-λ2/3, IFN-β, IFN-α1, CXCL10, and TNF-α mRNAs in DCs and macrophages as analyzed by qRT-PCR. The values were normalized to 18S rRNA, and the fold inductions were calculated from the uninfected control cells.

FIG. 4.

Pandemic 2009 (H1N1) influenza A virus is highly sensitive to the antiviral activity of IFNs. Monocyte-derived human DCs and macrophages from four different donors were primed with different doses of IFN-α (1, 10, or 100 IU/ml), IFN-β (1, 10, or 100 IU/ml), IFN-λ1 (1, 10, or 100 ng/ml), or IFN-λ3 (1, 10, or 100 ng/ml) for 16 h, followed by infection with pandemic A/Finland/553/2009 (H1/09) and seasonal A/Brisbane/59/07 (H1/07) influenza virus at MOI of 2 for 18 h. The effect of IFNs on the replication of the viruses and MxA gene expression was analyzed at the protein level by FACS in DCs (A) and at the mRNA level by qRT-PCR in macrophages (B). (A) Expression of cell surface influenza A virus glycoproteins in DCs after priming the cells with different doses of IFN-α, IFN-β, IFN-λ1, or IFN-λ3. To control the effectiveness of IFN priming, induction of MxA protein was measured in a sample of cells prior to the infection. The expression of viral glycoproteins and MxA protein was detected by FACS, and the results are expressed as the means of fluorescence intensities. (B) Expression of the influenza A virus M1 gene in IFN-α-, IFN-β-, IFN-λ1-, or IFN-λ3-primed, virus-infected macrophages. Cellular MxA gene expression was analyzed in macrophages obtained before virus infection. The values were normalized to 18S rRNA and presented as relative MxA gene expression in relation to the RNA sample obtained from untreated (no IFN stimulation) and uninfected control cells or as relative copy number of M1 RNA.