Limits...
RSV-Induced H3K4 Demethylase KDM5B Leads to Regulation of Dendritic Cell-Derived Innate Cytokines and Exacerbates Pathogenesis In Vivo.

Ptaschinski C, Mukherjee S, Moore ML, Albert M, Helin K, Kunkel SL, Lukacs NW - PLoS Pathog. (2015)

Bottom Line: In vivo, infection of Kdm5bfl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs.Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells.These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5bfl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5bfl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5bfl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

No MeSH data available.


Related in: MedlinePlus

Inhibiting KDM5B in human monocyte-derived DCs leads to increased cytokine production.(A) DCs were grown from blood monocytes and infected with RSV. RNA was isolated and KDM5B expression was determined by qPCR. n = 6 samples from three pooled donors. (B) DCs were treated with DMSO or 2,4-PDCA for 24 hours, and were then infected with RSV for 24 hours. Cytokine levels were measured by qPCR. n = 3 samples/group and is representative of three independent experiments. (C) DMSO or 2,4-PDCA-treated BMDCs were infected with RSV for 24 hours. A ChIP assay was performed to determine the H3K4me3 status at the promoter regions of the IFNB, TNF and IL6 genes. n = 4 samples/group. (D) DCs were treated with DMSO or 2,4-PDCA and infected with RSV. Autologous CD4+ T cells were cultured with the DCs for 48 hours. mRNA was extracted and cytokines levels measured by qPCR. n = 3 samples/group and data are representative of two independent experiments. p<0.05,**p<0.01 ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4470918&req=5

ppat.1004978.g003: Inhibiting KDM5B in human monocyte-derived DCs leads to increased cytokine production.(A) DCs were grown from blood monocytes and infected with RSV. RNA was isolated and KDM5B expression was determined by qPCR. n = 6 samples from three pooled donors. (B) DCs were treated with DMSO or 2,4-PDCA for 24 hours, and were then infected with RSV for 24 hours. Cytokine levels were measured by qPCR. n = 3 samples/group and is representative of three independent experiments. (C) DMSO or 2,4-PDCA-treated BMDCs were infected with RSV for 24 hours. A ChIP assay was performed to determine the H3K4me3 status at the promoter regions of the IFNB, TNF and IL6 genes. n = 4 samples/group. (D) DCs were treated with DMSO or 2,4-PDCA and infected with RSV. Autologous CD4+ T cells were cultured with the DCs for 48 hours. mRNA was extracted and cytokines levels measured by qPCR. n = 3 samples/group and data are representative of two independent experiments. p<0.05,**p<0.01 ***p<0.001.

Mentions: The above data show that inhibiting KDM5B function leads to increased innate cytokine production after RSV infection. To determine the role of KDM5B in human cells, human monocyte-derived DCs (MoDCs) cultured from peripheral blood monocytes were used. As shown in Fig 3A, KDM5B is significantly upregulated in MoDCs at 12 and 24 hours following RSV infection, and although the degree of upregulation is less pronounced than in mouse DCs, the kinetics are very similar (Fig 3A). To assess the role of KDM5B in human DCs, the function of KDM5B was inhibited by treating the cells with 2,4-PDCA for 24 hours, followed by RSV infection. Similar to observations in mouse cells treated with 2,4-PDCA, inhibiting KDM5B in human MoDCs led to increased production of IFNB, TNF and IL6 compared to RSV alone or DMSO control (Fig 3B). To determine whether the presence of the inhibitor affected the H3K4me3 status of these genes, a ChIP analysis for H3K4 was performed and examined the promoter regions of specific innate cytokine genes. Interestingly, MoDCs exhibited a slight increase in promoter methylation following incubation with the inhibitor alone, which was further increased when the cells were infected with RSV (Fig 3C). Similar to observations in mouse DCs, no change in methylation at the IL10 an IL12 promoters was observed (S4 Fig). Finally, infected MoDCs that had been treated with DMSO or 2,4-PDCA were cultured with autologous CD4+ T cells in the presence of RSV to assess the APC function of the MoDCs (Fig 3D). While the T cells co-cultured with 2,4-PDCA-treated DCs had similar levels of IFN-γ production, the Th2 cytokines IL-5 and IL-13 were significantly decreased. These data suggest that inhibiting KDM5B function in human MoDCs results in regulation of Th2 cytokine production, supporting the hypothesis that RSV drives an altered immune phenotype that relies on epigenetic regulation of DC.


RSV-Induced H3K4 Demethylase KDM5B Leads to Regulation of Dendritic Cell-Derived Innate Cytokines and Exacerbates Pathogenesis In Vivo.

Ptaschinski C, Mukherjee S, Moore ML, Albert M, Helin K, Kunkel SL, Lukacs NW - PLoS Pathog. (2015)

Inhibiting KDM5B in human monocyte-derived DCs leads to increased cytokine production.(A) DCs were grown from blood monocytes and infected with RSV. RNA was isolated and KDM5B expression was determined by qPCR. n = 6 samples from three pooled donors. (B) DCs were treated with DMSO or 2,4-PDCA for 24 hours, and were then infected with RSV for 24 hours. Cytokine levels were measured by qPCR. n = 3 samples/group and is representative of three independent experiments. (C) DMSO or 2,4-PDCA-treated BMDCs were infected with RSV for 24 hours. A ChIP assay was performed to determine the H3K4me3 status at the promoter regions of the IFNB, TNF and IL6 genes. n = 4 samples/group. (D) DCs were treated with DMSO or 2,4-PDCA and infected with RSV. Autologous CD4+ T cells were cultured with the DCs for 48 hours. mRNA was extracted and cytokines levels measured by qPCR. n = 3 samples/group and data are representative of two independent experiments. p<0.05,**p<0.01 ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470918&req=5

ppat.1004978.g003: Inhibiting KDM5B in human monocyte-derived DCs leads to increased cytokine production.(A) DCs were grown from blood monocytes and infected with RSV. RNA was isolated and KDM5B expression was determined by qPCR. n = 6 samples from three pooled donors. (B) DCs were treated with DMSO or 2,4-PDCA for 24 hours, and were then infected with RSV for 24 hours. Cytokine levels were measured by qPCR. n = 3 samples/group and is representative of three independent experiments. (C) DMSO or 2,4-PDCA-treated BMDCs were infected with RSV for 24 hours. A ChIP assay was performed to determine the H3K4me3 status at the promoter regions of the IFNB, TNF and IL6 genes. n = 4 samples/group. (D) DCs were treated with DMSO or 2,4-PDCA and infected with RSV. Autologous CD4+ T cells were cultured with the DCs for 48 hours. mRNA was extracted and cytokines levels measured by qPCR. n = 3 samples/group and data are representative of two independent experiments. p<0.05,**p<0.01 ***p<0.001.
Mentions: The above data show that inhibiting KDM5B function leads to increased innate cytokine production after RSV infection. To determine the role of KDM5B in human cells, human monocyte-derived DCs (MoDCs) cultured from peripheral blood monocytes were used. As shown in Fig 3A, KDM5B is significantly upregulated in MoDCs at 12 and 24 hours following RSV infection, and although the degree of upregulation is less pronounced than in mouse DCs, the kinetics are very similar (Fig 3A). To assess the role of KDM5B in human DCs, the function of KDM5B was inhibited by treating the cells with 2,4-PDCA for 24 hours, followed by RSV infection. Similar to observations in mouse cells treated with 2,4-PDCA, inhibiting KDM5B in human MoDCs led to increased production of IFNB, TNF and IL6 compared to RSV alone or DMSO control (Fig 3B). To determine whether the presence of the inhibitor affected the H3K4me3 status of these genes, a ChIP analysis for H3K4 was performed and examined the promoter regions of specific innate cytokine genes. Interestingly, MoDCs exhibited a slight increase in promoter methylation following incubation with the inhibitor alone, which was further increased when the cells were infected with RSV (Fig 3C). Similar to observations in mouse DCs, no change in methylation at the IL10 an IL12 promoters was observed (S4 Fig). Finally, infected MoDCs that had been treated with DMSO or 2,4-PDCA were cultured with autologous CD4+ T cells in the presence of RSV to assess the APC function of the MoDCs (Fig 3D). While the T cells co-cultured with 2,4-PDCA-treated DCs had similar levels of IFN-γ production, the Th2 cytokines IL-5 and IL-13 were significantly decreased. These data suggest that inhibiting KDM5B function in human MoDCs results in regulation of Th2 cytokine production, supporting the hypothesis that RSV drives an altered immune phenotype that relies on epigenetic regulation of DC.

Bottom Line: In vivo, infection of Kdm5bfl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs.Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells.These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5bfl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5bfl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5bfl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

No MeSH data available.


Related in: MedlinePlus