Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1

Abstract

Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.

Figure 1

Human host gene expression profiles following HPAI H5N1 infection and apocynin treatment. Genes that were statistically upregulated (shown as red circles) or downregulated (shown as blue circles) were assessed in pairwise comparisons as indicated. Data are plotted as the mean FPKM of each gene obtained from HD or HA against a Log2 fold change compared to ND as (A) ND vs. HD or (B) ND vs. HA, or compared to HD as (C) HD vs. HA. (D–H) The number and overlap of genes in each pairwise comparison is illustrated by Venn diagrams. ND, uninfected cells treated with 1% DMSO vehicle control; HD and HA, cells infected with H5N1 at a MOI of 2 for 24 hours in the presence of 1% DMSO or 1 mM apocynin, respectively. Up; upregulated. Down; downregulated. FPKM; fragments per kilobase of exon per million fragments mapped. RNA samples were obtained from ATII cells isolated from the lung tissue of three donors. The full list of transcripts identified in the HiSeq analysis, as well as differentially regulated transcripts in the three experimental groups is presented in Table S1.

Figure 2

Hiseq analysis of H5N1 influenza virus gene expression and validation of differential gene expression identified with HiSeq using qRT-PCR. The same RNA samples used for HiSeq analysis were also subjected to qRT-PCR analysis of (A) IL6, (B) IFNB1, (C) CXCL10, (D) CCL5, (E) SOCS1 and (F) SOCS3 mRNA expression in three experimental groups of ATII cells (ND, HD and HA). Fold-changes following qRT-PCR analysis were calculated using 2^−ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; ^#p < 0.05, ^##p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. ^#p < 0.05, compared with HD.

Figure 3

Upregulation of CEACAM1 in influenza virus-infected cells. (A) HiSeq analysis showed elevated transcription of CEACAM1 in H5N1-infected ATII cells (HD) when compared to uninfected cells (ND). ***p < 0.01, compared with ND. (B) The transcription level of a related CEACAM family member, CEACAM5, was not altered following H5N1 infection. (C) A representative Western blot of three individual experiments and protein band density analysis of endogenous CEACAM1 protein expression in A549 cells following infection with PR8 virus at MOIs of 2, 5 and 10 at 24 and 48 hpi. (D) A representative Western blot of three individual experiments and protein band density analysis of endogenous CEACAM1 protein expression in primary human ATII cells infected with PR8 or HPAI H5N1 virus at various MOIs and time points as indicated. *p < 0.05, **p < 0.01, ***p < 0.001, compared with PR8 at the corresponding time point. ^##p < 0.01, compared between 24 and 48 hpi. All samples on protein blots were run and cropped from the same gel for accurate standardization. Full blots are provided in the supplementary data file.

Figure 4

siRNA-mediated silencing of CEACAM1 in ATII and A549 cells. (A) SYBR Green qRT-PCR analysis of siRNA-mediated silencing of endogenous CEACAM1-4L, -4S, -3L and -3S in ATII cells compared to cells transfected with siNeg controls. Data are expressed as fold-change (2^−ΔΔCt method) normalized to β-actin and compared to mock-transfected cells that have a fold-change of 1 (dotted line). *p < 0.05, **p < 0.01. qRT-PCR was performed in duplicate and ATII cells were cultured from three donors. (B) A representative Western blot of three individual experiments and protein band density analysis of siCEACAM1-mediated knockdown of endogenous CEACAM1 in ATII and A549 cells transfected with siCEACAM1 or siNeg control or in mock-transfected cells. *p < 0.05, **p < 0.01, compared to siNeg control. Experiments were performed using ATII cells from three donors and three different passages of A549 cells. All samples on protein blots were run and cropped from the same gel for accurate standardization. Full blots are provided in the supplementary data file.

Figure 5

Knockdown of CEACAM1 inhibits H5N1 replication and H5N1-induced inflammation. ATII cells (A) and A549 cells (B) transfected with 15 pmol siCEACAM1 showed reduced cytokine and chemokine production in response to H5N1 infection as analyzed by qRT-PCR and compared to siNeg-transfected control cells. Plaque assay of H5N1 virus titers in ATII (C) and A549 (D) cells that were either mock-transfected or transfected with siCEACAM1 or siNeg. *p < 0.05 and **p < 0.01, compared with mock-transfected cells, ^#p < 0.05 and ^##p < 0.01, ^##p < 0.001, compared with siNeg-transfected cells. Experiments were performed using ATII cells from three donors and three different passages of A549 cells.