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A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice.

Boon AC, Williams RW, Sinasac DS, Webby RJ - BMC Genomics (2014)

Bottom Line: A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic.Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26).Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13.

View Article: PubMed Central - PubMed

Affiliation: Departments of Internal Medicine, Division of Infectious Diseases, Molecular Microbiology and Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA. jboon@dom.wustl.edu.

ABSTRACT

Background: Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.

Results: Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.

Conclusion: A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

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Increased concentration of CCL2, TNFα, and IFN-α in lung homogenates of A/Hong Kong/213/03 H5N1 virus infected DBA/2J mice. (A) DBA/2J (D2) and C57BL/6J (B6) were inoculated with 104 EID50 of HK213 virus (+) or mock-inoculated (-) in 30 μl PBS. Forty-eight hours post inoculation the lungs of the inoculated animals were collected, homogenized in sterile PBS, and stored at -80°C. The concentration of CCL2, TNFα, and IFN-α in these homogenates was quantified by ELISA. The average cytokine concentration (pg/ml ± standard error of the mean) of CCL2, TNFα and IFN-α is shown for mock-infected (n =4, single experiment), C57BL/6 (n =13 from 4 different experiments), and DBA/2J (n =15 from four different experiments). *** is P <0.0001. (B) Significant association between CCL2, TNF-α, and IFN-α concentration and lung virus titer 48 hours post inoculation with 104 EID50 of HK213 virus in B6, D2 and BXD recombinant inbred mouse strains (P < 0.0001 for all three cytokines).
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Fig1: Increased concentration of CCL2, TNFα, and IFN-α in lung homogenates of A/Hong Kong/213/03 H5N1 virus infected DBA/2J mice. (A) DBA/2J (D2) and C57BL/6J (B6) were inoculated with 104 EID50 of HK213 virus (+) or mock-inoculated (-) in 30 μl PBS. Forty-eight hours post inoculation the lungs of the inoculated animals were collected, homogenized in sterile PBS, and stored at -80°C. The concentration of CCL2, TNFα, and IFN-α in these homogenates was quantified by ELISA. The average cytokine concentration (pg/ml ± standard error of the mean) of CCL2, TNFα and IFN-α is shown for mock-infected (n =4, single experiment), C57BL/6 (n =13 from 4 different experiments), and DBA/2J (n =15 from four different experiments). *** is P <0.0001. (B) Significant association between CCL2, TNF-α, and IFN-α concentration and lung virus titer 48 hours post inoculation with 104 EID50 of HK213 virus in B6, D2 and BXD recombinant inbred mouse strains (P < 0.0001 for all three cytokines).

Mentions: Genetically diverse mice vary greatly in response to influenza virus infection and we think this is the result of higher virus replication in the first 24–72 h after inoculation. The increase in viral titers in the lungs of susceptible strains, such as D2, leads to the increased production of pro-inflammatory cytokines compared to resistant strains, such as B6 [11, 13]. To understand the genetic and molecular basis for this difference in host response and early viral load, we quantified inflammatory host response 48 hours after inoculation with 104 EID50 of an H5N1 virus (HK213). This time point coincided with peak viral replication [13] and the earliest point at which we can reliably measure the production of IFN-α (Additional file 1: Figure S1). To quantify the inflammatory response after HK213 infection we measured titers of CCL2, TNFα and IFN-α in whole lung homogenates. Compared to mock-infected animals the concentration of CCL2, TNFα, and IFN-α increased significantly (P <0.0001, Figure 1) in both parental strains after inoculation. More importantly, levels of CCL2, TNFα, and IFN-α were significantly higher in D2 compared to B6 (P <0.001 for all three cytokines), confirming our previous studies suggesting that early cytokine levels correlate with virus titer.To evaluate this further we quantified the lung virus titer as well as the CCL2, TNFα, and IFN-α concentration in 12 different BXD RI mouse strains (Figure 1B) 48 hours post inoculation. Correlation analysis revealed a highly significant association between lung virus titer and TNF-α (r =0.96, P < 0.0001), CCL2 (r =0.86, P < 0.0001), and IFN-α concentration (r =0.88, P <0.0001) at 48 hours post inoculation.Figure 1


A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice.

Boon AC, Williams RW, Sinasac DS, Webby RJ - BMC Genomics (2014)

Increased concentration of CCL2, TNFα, and IFN-α in lung homogenates of A/Hong Kong/213/03 H5N1 virus infected DBA/2J mice. (A) DBA/2J (D2) and C57BL/6J (B6) were inoculated with 104 EID50 of HK213 virus (+) or mock-inoculated (-) in 30 μl PBS. Forty-eight hours post inoculation the lungs of the inoculated animals were collected, homogenized in sterile PBS, and stored at -80°C. The concentration of CCL2, TNFα, and IFN-α in these homogenates was quantified by ELISA. The average cytokine concentration (pg/ml ± standard error of the mean) of CCL2, TNFα and IFN-α is shown for mock-infected (n =4, single experiment), C57BL/6 (n =13 from 4 different experiments), and DBA/2J (n =15 from four different experiments). *** is P <0.0001. (B) Significant association between CCL2, TNF-α, and IFN-α concentration and lung virus titer 48 hours post inoculation with 104 EID50 of HK213 virus in B6, D2 and BXD recombinant inbred mouse strains (P < 0.0001 for all three cytokines).
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Fig1: Increased concentration of CCL2, TNFα, and IFN-α in lung homogenates of A/Hong Kong/213/03 H5N1 virus infected DBA/2J mice. (A) DBA/2J (D2) and C57BL/6J (B6) were inoculated with 104 EID50 of HK213 virus (+) or mock-inoculated (-) in 30 μl PBS. Forty-eight hours post inoculation the lungs of the inoculated animals were collected, homogenized in sterile PBS, and stored at -80°C. The concentration of CCL2, TNFα, and IFN-α in these homogenates was quantified by ELISA. The average cytokine concentration (pg/ml ± standard error of the mean) of CCL2, TNFα and IFN-α is shown for mock-infected (n =4, single experiment), C57BL/6 (n =13 from 4 different experiments), and DBA/2J (n =15 from four different experiments). *** is P <0.0001. (B) Significant association between CCL2, TNF-α, and IFN-α concentration and lung virus titer 48 hours post inoculation with 104 EID50 of HK213 virus in B6, D2 and BXD recombinant inbred mouse strains (P < 0.0001 for all three cytokines).
Mentions: Genetically diverse mice vary greatly in response to influenza virus infection and we think this is the result of higher virus replication in the first 24–72 h after inoculation. The increase in viral titers in the lungs of susceptible strains, such as D2, leads to the increased production of pro-inflammatory cytokines compared to resistant strains, such as B6 [11, 13]. To understand the genetic and molecular basis for this difference in host response and early viral load, we quantified inflammatory host response 48 hours after inoculation with 104 EID50 of an H5N1 virus (HK213). This time point coincided with peak viral replication [13] and the earliest point at which we can reliably measure the production of IFN-α (Additional file 1: Figure S1). To quantify the inflammatory response after HK213 infection we measured titers of CCL2, TNFα and IFN-α in whole lung homogenates. Compared to mock-infected animals the concentration of CCL2, TNFα, and IFN-α increased significantly (P <0.0001, Figure 1) in both parental strains after inoculation. More importantly, levels of CCL2, TNFα, and IFN-α were significantly higher in D2 compared to B6 (P <0.001 for all three cytokines), confirming our previous studies suggesting that early cytokine levels correlate with virus titer.To evaluate this further we quantified the lung virus titer as well as the CCL2, TNFα, and IFN-α concentration in 12 different BXD RI mouse strains (Figure 1B) 48 hours post inoculation. Correlation analysis revealed a highly significant association between lung virus titer and TNF-α (r =0.96, P < 0.0001), CCL2 (r =0.86, P < 0.0001), and IFN-α concentration (r =0.88, P <0.0001) at 48 hours post inoculation.Figure 1

Bottom Line: A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic.Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26).Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13.

View Article: PubMed Central - PubMed

Affiliation: Departments of Internal Medicine, Division of Infectious Diseases, Molecular Microbiology and Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA. jboon@dom.wustl.edu.

ABSTRACT

Background: Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.

Results: Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.

Conclusion: A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

Show MeSH
Related in: MedlinePlus