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Paragraph: We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . 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. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ). In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3

Question: What mediates the anti-apoptosis of neutrophils?
Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3