Table_3_Overexpression of Chicken IRF7 Increased Viral Replication and Programmed Cell Death to the Avian Influenza Virus Infection Through TGF-Beta/FoxO Signaling Axis in DF-1.XLSX

During mammalian viral infections, interferon regulatory factor 7 (IRF7) partners with IRF3 to regulate the type I interferon response. In chickens, however, it is still unclear how IRF7 functions in the host innate immune response, especially given that IRF3 is absent. To further elucidate the functional role of chicken IRF7 during avian influenza virus (AIV) infection, we generated inducible IRF7 overexpression DF-1 cell lines and performed in vitro infection using low pathogenic AIVs (LPAIVs). Overexpression of IRF7 resulted in higher viral replication of H6N2 and H10N7 LPAIVs compared to empty vector control cells regardless of IRF7 expression level. In addition, a high rate of induced cell death was observed due to elevated level of IRF7 upon viral infection. RNA-seq and subsequent transcriptome analysis of IRF7 overexpression and control cells discovered candidate genes possibly controlled by chicken IRF7. Functional annotation revealed potential pathways modulated by IRF7 such as TGF-beta signaling pathway, FoxO signaling pathway and cell structural integrity related pathways. Next, we analyzed the host response alteration due to the IRF7 overexpression and additionally discovered the possible connection of chicken IRF7 and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 could modulate a wide range of cellular processes in the host innate immune response thus meticulous control of IRF7 expression is crucial to the host in response to AIV infection.</p

Validation of differentially expressed genes by quantitative reverse transcriptase PCR (qRT-PCR).

<p>Expression level of mRNAs were confirmed by qRT-PCR in the poly(I:C) induced overexpressed (left graph), knockdown (right graph) cell lines and their controls. (A) EIF2AK2; (B) LY6E; (C) MX1; (D) WIF1; (E) CLDN5; (F) ITGA8; (G) G0S2; (H) DCLRE1C. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> <0.001, N.S.: not significant. Error bars indicate the SEM of triplicate analyses.</p

Image_1_Overexpression of Chicken IRF7 Increased Viral Replication and Programmed Cell Death to the Avian Influenza Virus Infection Through TGF-Beta/FoxO Signaling Axis in DF-1.TIF

During mammalian viral infections, interferon regulatory factor 7 (IRF7) partners with IRF3 to regulate the type I interferon response. In chickens, however, it is still unclear how IRF7 functions in the host innate immune response, especially given that IRF3 is absent. To further elucidate the functional role of chicken IRF7 during avian influenza virus (AIV) infection, we generated inducible IRF7 overexpression DF-1 cell lines and performed in vitro infection using low pathogenic AIVs (LPAIVs). Overexpression of IRF7 resulted in higher viral replication of H6N2 and H10N7 LPAIVs compared to empty vector control cells regardless of IRF7 expression level. In addition, a high rate of induced cell death was observed due to elevated level of IRF7 upon viral infection. RNA-seq and subsequent transcriptome analysis of IRF7 overexpression and control cells discovered candidate genes possibly controlled by chicken IRF7. Functional annotation revealed potential pathways modulated by IRF7 such as TGF-beta signaling pathway, FoxO signaling pathway and cell structural integrity related pathways. Next, we analyzed the host response alteration due to the IRF7 overexpression and additionally discovered the possible connection of chicken IRF7 and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 could modulate a wide range of cellular processes in the host innate immune response thus meticulous control of IRF7 expression is crucial to the host in response to AIV infection.</p

Functional Analysis of Chicken IRF7 in Response to dsRNA Analog Poly(I:C) by Integrating Overexpression and Knockdown

<div><p>In order to develop novel strategies to protect against increasingly virulent bird-linked pathogens, a better understanding of the avian antiviral response mechanism is essential. Type I interferons (IFNs) are recognized as the first line of defense in a host’s antiviral response; and it has been suggested that IRF7, a member of the IFN regulatory factor (IRF) family, plays an important role in modulating the immune response to avian influenza virus infection in chickens. The objective of this study was to identify candidate genes and pathways associated with IRF7 regulation at the transcriptome level as a first step towards elucidating the underlying cellular mechanisms of IRF7 modulation in the chicken antiviral response. <i>IRF7</i> overexpression and knockdown DF-1 cell lines were established and stimulated by various pathogen-associated molecular patterns. Significant <i>IRF7</i> and type I IFN expression changes were observed in both the <i>IRF7</i> overexpression cell line and the <i>IRF7 </i>knockdown cell line upon exposure to the double stranded RNA (dsRNA) analog poly(I:C). Using RNA-seq based transcriptome analysis, we identified potential novel genes that IRF7 may help regulate as part of the host immune response to dsRNA; potential biomarkers and therapeutic targets revealed as a result of this study warrant further investigation. Based on our results, we suggest that IRF7 may have conserved functional activity in the avian antiviral response, and plays a crucial role in type I IFN regulation.</p></div

Expression of <i>IFNB</i> and <i>TLR3</i> in the overexpression and knockdown cell lines.

<p>Relative expression levels of <i>IFNB</i> (A, C) and <i>TLR3</i> (B, D) were confirmed by qRT-PCR in the mock or poly(I:C) induced overexpression or knockdown cell lines and their respective controls. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> <0.001, N.S.: not significant. Error bars indicate the SEM of triplicate analyses.</p

Expression changes of <i>IRF7</i> and <i>IFNA</i> in response to different pathogen associated molecular patterns (PAMPs) stimulated cell lines.

<p>(A, B) <i>IRF7</i> overexpression (CMV-IRF7) or (C, D) knockdown (KD) DF-1 cell lines and their respective control lines at 6h post stimulation by PAMPs. Mock: media (negative control); PGN: peptidoglycan; poly(I:C): polyinosinic-polycytidylic acid; LPS: lipopolysaccharide. * <i>P</i> < 0.05, ** <i>P</i> < 0.01. Error bars indicate the SEM of triplicate analyses.</p

Table_2_Overexpression of Chicken IRF7 Increased Viral Replication and Programmed Cell Death to the Avian Influenza Virus Infection Through TGF-Beta/FoxO Signaling Axis in DF-1.XLSX

During mammalian viral infections, interferon regulatory factor 7 (IRF7) partners with IRF3 to regulate the type I interferon response. In chickens, however, it is still unclear how IRF7 functions in the host innate immune response, especially given that IRF3 is absent. To further elucidate the functional role of chicken IRF7 during avian influenza virus (AIV) infection, we generated inducible IRF7 overexpression DF-1 cell lines and performed in vitro infection using low pathogenic AIVs (LPAIVs). Overexpression of IRF7 resulted in higher viral replication of H6N2 and H10N7 LPAIVs compared to empty vector control cells regardless of IRF7 expression level. In addition, a high rate of induced cell death was observed due to elevated level of IRF7 upon viral infection. RNA-seq and subsequent transcriptome analysis of IRF7 overexpression and control cells discovered candidate genes possibly controlled by chicken IRF7. Functional annotation revealed potential pathways modulated by IRF7 such as TGF-beta signaling pathway, FoxO signaling pathway and cell structural integrity related pathways. Next, we analyzed the host response alteration due to the IRF7 overexpression and additionally discovered the possible connection of chicken IRF7 and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 could modulate a wide range of cellular processes in the host innate immune response thus meticulous control of IRF7 expression is crucial to the host in response to AIV infection.</p

Differential expression analyses of RNA-seq samples.

<p>Differentially expressed genes (DEGs) between cell lines and treatments in the (A) overexpression and (D) knockdown data set. (B, E) Venn diagrams of overlapped DEGs between cell lines upon poly(I:C) expression. (C, F) Venn diagrams of overlapped DEGs between treatments in effect of IRF7 expression changes.</p

Establishment of <i>IRF7</i> overexpression and knockdown DF-1 cell lines.

<p>(A) Multiple sequence alignment of IRF7 sequences in exon 8 nucleotide position 169 to 221. Molecular cloned IRF7 clone (accession) and the NCBI reference sequence (NM_205372) were aligned against the reference chicken genome (galGal4). (B) Schematic diagram of overexpression (up) and knockdown (down) PiggyBac transposon expression vectors. (C) Related quantitation of IRF7 in control (Vector) and overexpression (CMV-IRF7) cell lines. (D) Knockdown efficiency of each siRNA for IRF7 in the overexpressed cell line. siRNA non-specific (NS) to the chicken genome was used as a control. (E) Knockdown efficiency of IRF7 in the knockdown cell line (KD) as compared to control cells (NS). * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> <0.001. Error bars indicate the SEM of triplicate analyses.</p

Image_2_Overexpression of Chicken IRF7 Increased Viral Replication and Programmed Cell Death to the Avian Influenza Virus Infection Through TGF-Beta/FoxO Signaling Axis in DF-1.TIF

During mammalian viral infections, interferon regulatory factor 7 (IRF7) partners with IRF3 to regulate the type I interferon response. In chickens, however, it is still unclear how IRF7 functions in the host innate immune response, especially given that IRF3 is absent. To further elucidate the functional role of chicken IRF7 during avian influenza virus (AIV) infection, we generated inducible IRF7 overexpression DF-1 cell lines and performed in vitro infection using low pathogenic AIVs (LPAIVs). Overexpression of IRF7 resulted in higher viral replication of H6N2 and H10N7 LPAIVs compared to empty vector control cells regardless of IRF7 expression level. In addition, a high rate of induced cell death was observed due to elevated level of IRF7 upon viral infection. RNA-seq and subsequent transcriptome analysis of IRF7 overexpression and control cells discovered candidate genes possibly controlled by chicken IRF7. Functional annotation revealed potential pathways modulated by IRF7 such as TGF-beta signaling pathway, FoxO signaling pathway and cell structural integrity related pathways. Next, we analyzed the host response alteration due to the IRF7 overexpression and additionally discovered the possible connection of chicken IRF7 and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 could modulate a wide range of cellular processes in the host innate immune response thus meticulous control of IRF7 expression is crucial to the host in response to AIV infection.</p