Expression of inflammatory cytokines in DF-1 and MDCK cells infected by H9N2 and H5N1 AIVs
mRNA transcripts for inflammatory cytokines including IL-1β, IL-6 and TNF-α increased significantly in DF-1 and MDCK cells infected by H5N1 and H9N2 AIVs. In DF-1 cells, IL-1β, IL-6 and TNF-α increased significantly from 6 h after infection with both AIVs (p < 0.01), but greater expression levels were observed in response to H5N1 than H9N2 infection (Fig. 1A-C). This is consistent with the expression trend of inflammatory cytokines in DF-1 cells in our previous study [5]. In MDCK cells, IL-1β expression increased significantly from 6 h with H9N2 and H5N1 infection (p < 0.01), with greater expression in response to H9N2 AIV than H5N1 (Fig. 1D). IL-6 expression increased significantly from 3 to 24 h following both H9N2 and H5N1 infection (p < 0.05). Between 3 and 12h post-infection, H5N1 AIV elicited significantly greater IL-6 expression than H9N2 infection (Fig. 1E). TNF-α expression increased significantly from 3 to 24 h after infection with H9N2 AIV (p < 0.01), and from 3 to 12 h after infection with H5N1 (p < 0.01). Similar to other tested cytokines, H5N1 infection caused a greater change in TNF-α expression than H9N2 infection (Fig. 1F).
Expression of PTX3, Del-1 and GDF-15 in DF-1 and MDCK cells infected by H9N2 and H5N1 AIVs
Upon H9N2 and H5N1 AIV infection in DF-1 cells, PTX3, Del-1 and GDF-15 expression levels increased significantly. In DF-1 and MDCK cells PTX3 expression levels peaked at 12 h after infection by H9N2 AIV (p < 0.01), while expression levels in response to H5N1 infection steadily increased until study endpoint (p < 0.05; Fig. 2A, D). In response to H9N2 AIV infection, Del-1 expression increased and remained stable from 3 to 12 h (p < 0.05), peaking at 24 h in both cell lines (p < 0.01). H5N1 infection increased significantly Del-1 expression levels, which remained relatively stable from 3 to 24 h (p < 0.01). However, the elicited change in expression was not as robust as that observed in the H9N2 infection group (Fig. 2B, E). GDF-15 analysis revealed that expression levels gradually increased over 24 h in DF-1 cells irrespective of the AIV used for infection (p < 0.01). In contrast to DF-1 cells, MDCK cells exhibited different GDF-15 expression dynamics in response to both H9N2 and H5N1 AIVs infection. Specifically, GDF-15 slowly peaked at 12 h in MDCK cells, after which expression levels subsequently declined (p < 0.01; Fig. 2C, F). Altogether, these findings show that H9N2 and H5N1 AIVs infection in DF-1 cells and MDCK cells led to increased PTX3, Del-1 and GDF-15, and that the change in expression due to H9N2 is greater than that due to H5N1.
Dynamic expression of inflammatory cytokines in H9N2 AIV-infected DF-1 and MDCK cells overexpressing PTX3, Del-1 and GDF-15 alone
To determine the effects of PTX3, Del-1 and GDF-15 on cytokine expression, DF-1 and MDCK cells with and without overexpression of these proteins were infected by H9N2 AIV. Cells infected by H9N2 AIV served as the control. In DF-1 cells infected with H9N2 AIV, PTX3 overexpression resulted in significantly decreased IL-1β, IL-6 and TNF-α expression at 24 h compared to the control (p < 0.01; Fig. 3A-C). In similarly infected MDCK cells, PTX3 overexpression resulted in significantly decreased IL-1β expression at 6 (p < 0.05) and 24 h (p < 0.01; Fig. 3D), IL-6 expression after 12 h (p < 0.01; Fig. 3E), and TNF-α expression at 6 h compared to the control (p < 0.01; Fig. 3F).
Del-1 overexpression in H9N2-infected DF-1 cells caused significantly reduced expression of IL-1β at 12 h and 24 h (p < 0.01; Fig. 3G) and IL-6 at 6 and 12 h (p < 0.01; Fig. 3H) compared to the control. Unlike IL-1β and IL-6, TNF-α expression was uninhibited under these conditions (Fig. 3I). In MDCK cells infected with H9N2 AIV, Del-1 overexpression significantly reduced IL-1β and TNF-α expression from 3 to 24 h (p < 0.05; Fig. 3J, L), whereas IL-6 expression decreased at 3 (p < 0.01) and 12 h (p < 0.05; Fig. 3K).
H9N2 AIV infection in GDF-15-overexpressing DF-1 cells, resulted in significantly decreased IL-1β expression at 12 h (p < 0.01; Fig. 3M), whereas IL-6 and TNF-α expression were uninhibited (Fig. 3N, O). In GDF-15-overexpressing MDCK cells, H9N2 AIV infection significantly decreased IL-1β expression from 6 to 24 h (p < 0.01; Fig. 3P), while IL-6 and TNF-α expression significantly decreased from 3 to 24 h (p < 0.05, respectively; Fig. 3Q, R).
Dynamic expression of inflammatory cytokines in H9N2 AIV-infected DF-1 and MDCK cells combined overexpressing PTX3, Del-1 and GDF-15
Similar to the above, DF-1 and MDCK cells with and without combined overexpression of PTX3, Del-1 and GDF-15 were infected by H9N2 AIV. Cells infected by H9N2 AIV served as the control. Combined overexpression of PTX3 and Del-1 in H9N2 AIV-infected DF-1 cells resulted in significantly decreased IL-1β expression between 6 and 24 h compared to the control (p < 0.01; Fig. 4A), whereas IL-6 expression was only decreased significantly at 6 h (p < 0.05; Fig. 4B), TNF-α expression was only significantly reduced at 24 h (p < 0.05; Fig. 4C). In similarly transfected MDCK cells, H9N2 AIV infection caused decreased IL-1β expression at 24 h compared to the control (p < 0.01; Fig. 4D). Additionally, IL-6 expression decreased at 12 h (p < 0.01; Fig. 4E) and TNF-α expression reduced at 3 and 6 h post- H9N2 AIV infection (p < 0.01; Fig. 4F).
In DF-1 cells with combined PTX3 and GDF-15 overexpression, H9N2 AIV infection resulted in a significant reduction in IL-1β and TNF-α expression at 12 and 24 h (p < 0.01; Fig. 4G, I), IL-6 expression was only decreased at 24 h (p < 0.01; Fig. 4H). In MDCK cells, PTX3 and GDF-15 co-expression followed by H9N2 AIV infection resulted in a significant reduction in IL-1β and TNF-α from 3 to 24 h (p < 0.05; Fig. 4J, L), and IL-6 at 12 h (p < 0.05; Fig. 4K).
qPCR analysis of H9N2 AIV infected Del-1 and GDF-15-expressing DF-1 cells revealed that IL-1β expression significantly decreased from 3 to 24 h compared with the control (p < 0.01; Fig. 4M), IL-6 expression from 6 to 24 h (p < 0.01; Fig. 4N) and TNF-α expression at 12 and 24 h (p < 0.01; Fig. 4O). Similar findings for IL-1β were observed in MDCK cells (p < 0.01; Fig. 4P). However, IL-6 expression decreased significantly at 3 (p < 0.01), 6 (p < 0.05) and 24 h (p < 0.01; Fig. 4Q) and TNF-α decreased at 3 and 6 h in this cell line (p < 0.01; Fig. 4R).
Combined PTX3, Del-1 and GDF-15 overexpression prior to H9N2 AIV infection in DF-1 cells attenuated IL-1β and IL-6 expression after 3 h compared to control (p < 0.0105; Fig. 5A, B) and reduced TNF-α after 6 h (p < 0.01; Fig. 5C). Similar experiments in MDCK cells showed significant inhibition of IL-1β, IL-6 and TNF-α expression levels after 6 h of H9N2 AIV infection (p < 0.01; Fig. 5D-F).
Expression profile of the inflammatory cytokine in PTX3-, Del-1-, and GDF-15- overexpressing DF-1 and MDCK infected with H5N1 AIV
To determine if the inflammatory responses observed were unique to H9N2 or shared with other type A influenza viruses, we performed the same experiments using H5N1 AIV. IL-1β, IL-6, and TNF-α expression decreased significantly from 6 to 24 h in PTX3-overexpressing DF-1 cells infected with H5N1 AIV compared to control (p < 0.05; Fig. 6A-C). Similar experiments in MDCK cells showed that IL-1β expression decreased significantly at 6 and 24 h (p < 0.01; Fig. 6D), IL-6 decreased from 3 to 24 h (p < 0.01; Fig. 6E), and TNF-α decreased at 6 and 12 h (p < 0.01; Fig. 6F).
In Del-1-overexpressing DF-1 cells infected with H5N1 AIV, IL-1β and IL-6 expression decreased significantly at 6 and 24 h compared with the control (p < 0.01; Fig. 6G, H), whereas TNF-α decreased at 3 and 6 h (p < 0.01; Fig. 6I). In MDCK cells, IL-1β and TNF-α decreased significantly from 6 to 24 h compared with the control (p < 0.05; Fig. 6J, L), while IL-6 expression decreased from 3 to 24 h (p < 0.01; Fig. 6K).
H5N1 AIV infection in GDF-15 overexpressing DF-1 cells led to a reduction in IL-1β at 12 and 24 h compared to the control (p < 0.01; Fig. 6M), while IL-6 decreased from 6 to 24 h (p < 0.05; Fig. 6N), TNF-α decreased at 12 h (p < 0.01; Fig. 6O) relative to the control. When performed in MDCK cells, we observed that IL-1β and IL-6 expression decreased significantly at 3 and 6 h compared to the control (p < 0.05; Fig. 6P, Q), TNF-α was decreased significantly at 6 and 12 h (p < 0.01; Fig. 6R).
H5N1 AIV infection in DF-1 cells with paired co-expression of PTX3, Del-1 and GDF-15, the expression of IL-1β, IL-6 and TNF-α decreased significantly from 6 to 24 h compared to the control (p < 0.01). In MDCK cells, expression levels for all three cytokines decreased significantly from 6 to 24 h compared to the control (p < 0.01; Fig. 7).
Simultaneous overexpression of PTX3, Del-1 and GDF-15 in DF-1 cells significantly attenuated H5N1 AIV-induced expression of IL-1β, IL-6 and TNF-α after 6 h compared to the control (p < 0.01; Fig. 8A-C). When repeated in MDCK cells, IL-1β and IL-6 expression levels decreased significantly after 6 h (Fig. 8D, E), while TNF-α decreased after 3 h (p < 0.01; Fig. 8F).