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

siRNA knockdown of Kdm5b leads to increased cytokine and chemokine gene expression.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control siRNA (1 μM). Total RNA was extracted from the cells and gene expression levels measured by qPCR. n = 6 from two combined experiments. (B) Transcript levels of Ifnb, Tnf and Il6 were measured from Kdm5b-siRNA transfected BMDCs and were compared to scrambled siRNA control following infection with RSV. n = 3 samples/group and is representative of two independent experiments. (C) BMDCs were treated with 1μM 2,4-PDCA or 0.1% DMSO for 24 hours and subsequently infected with RSV for 24 hours. Transcript levels were measured by qPCR. n = 4 samples/group and is representative of two independent experiments. (D) 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. Data are representative of three different experiments, with each sample run in duplicate. n = 4 samples/group. *p<0.05, **p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004978.g002: siRNA knockdown of Kdm5b leads to increased cytokine and chemokine gene expression.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control siRNA (1 μM). Total RNA was extracted from the cells and gene expression levels measured by qPCR. n = 6 from two combined experiments. (B) Transcript levels of Ifnb, Tnf and Il6 were measured from Kdm5b-siRNA transfected BMDCs and were compared to scrambled siRNA control following infection with RSV. n = 3 samples/group and is representative of two independent experiments. (C) BMDCs were treated with 1μM 2,4-PDCA or 0.1% DMSO for 24 hours and subsequently infected with RSV for 24 hours. Transcript levels were measured by qPCR. n = 4 samples/group and is representative of two independent experiments. (D) 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. Data are representative of three different experiments, with each sample run in duplicate. n = 4 samples/group. *p<0.05, **p<0.01, ***p<0.001.

Mentions: To determine whether KDM5B affects DC function, specific siRNA was used to knock down Kdm5b resulting in >70% reduction in expression levels (Fig 2A). Previous reports have indicated that RSV, unlike many viruses, is a poor inducer of type I IFN, including IFN-β [9,10]. BMDCs infected with RSV produced low levels of IFN-β at both 4 and 24 hours, whereas H1N1 virus produced very high levels (S2 Fig). We therefore hypothesized that the increase in KDM5B in BMDCs contributed to the suppression of type I IFN production and that knocking down Kdm5b expression would result in increased IFN-β. Following in vitro treatment of BMDCs with Kdm5b-specific siRNA or with a scrambled siRNA control, significantly increased expression levels of Ifnb, as well as the pro-inflammatory cytokines Tnfa and Il6 were observed in in vitro RSV-infected cells compared to sham-infected BMDCs (Fig 2B). To determine whether APC function was affected by Kdm5b siRNA or inhibitor treatment, MHC-II expression on the cell surface of BMDCs was measured, as well as expression of the co-stimulatory molecules CD80 and CD86. No differences in any maturation markers were noticed in treated cells compared to controls (S3 Fig). Furthermore, when a chemical inhibitor, 2,4-pyridinedicarboxylic acid (2,4-PDCA), was used to block the function of KDM5B [24,25] prior to RSV infection, significantly higher levels of Ifnb, Tnfa and Il6 transcripts compared to controls were observed (Fig 2C). While this inhibitor also interacts with other KDM family members, it has the highest specificity for KDM5B. Thus, two independent approaches to block KDM5B function demonstrated an altered immune response resulting in increases of critical innate cytokines.


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)

siRNA knockdown of Kdm5b leads to increased cytokine and chemokine gene expression.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control siRNA (1 μM). Total RNA was extracted from the cells and gene expression levels measured by qPCR. n = 6 from two combined experiments. (B) Transcript levels of Ifnb, Tnf and Il6 were measured from Kdm5b-siRNA transfected BMDCs and were compared to scrambled siRNA control following infection with RSV. n = 3 samples/group and is representative of two independent experiments. (C) BMDCs were treated with 1μM 2,4-PDCA or 0.1% DMSO for 24 hours and subsequently infected with RSV for 24 hours. Transcript levels were measured by qPCR. n = 4 samples/group and is representative of two independent experiments. (D) 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. Data are representative of three different experiments, with each sample run in duplicate. n = 4 samples/group. *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.g002: siRNA knockdown of Kdm5b leads to increased cytokine and chemokine gene expression.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control siRNA (1 μM). Total RNA was extracted from the cells and gene expression levels measured by qPCR. n = 6 from two combined experiments. (B) Transcript levels of Ifnb, Tnf and Il6 were measured from Kdm5b-siRNA transfected BMDCs and were compared to scrambled siRNA control following infection with RSV. n = 3 samples/group and is representative of two independent experiments. (C) BMDCs were treated with 1μM 2,4-PDCA or 0.1% DMSO for 24 hours and subsequently infected with RSV for 24 hours. Transcript levels were measured by qPCR. n = 4 samples/group and is representative of two independent experiments. (D) 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. Data are representative of three different experiments, with each sample run in duplicate. n = 4 samples/group. *p<0.05, **p<0.01, ***p<0.001.
Mentions: To determine whether KDM5B affects DC function, specific siRNA was used to knock down Kdm5b resulting in >70% reduction in expression levels (Fig 2A). Previous reports have indicated that RSV, unlike many viruses, is a poor inducer of type I IFN, including IFN-β [9,10]. BMDCs infected with RSV produced low levels of IFN-β at both 4 and 24 hours, whereas H1N1 virus produced very high levels (S2 Fig). We therefore hypothesized that the increase in KDM5B in BMDCs contributed to the suppression of type I IFN production and that knocking down Kdm5b expression would result in increased IFN-β. Following in vitro treatment of BMDCs with Kdm5b-specific siRNA or with a scrambled siRNA control, significantly increased expression levels of Ifnb, as well as the pro-inflammatory cytokines Tnfa and Il6 were observed in in vitro RSV-infected cells compared to sham-infected BMDCs (Fig 2B). To determine whether APC function was affected by Kdm5b siRNA or inhibitor treatment, MHC-II expression on the cell surface of BMDCs was measured, as well as expression of the co-stimulatory molecules CD80 and CD86. No differences in any maturation markers were noticed in treated cells compared to controls (S3 Fig). Furthermore, when a chemical inhibitor, 2,4-pyridinedicarboxylic acid (2,4-PDCA), was used to block the function of KDM5B [24,25] prior to RSV infection, significantly higher levels of Ifnb, Tnfa and Il6 transcripts compared to controls were observed (Fig 2C). While this inhibitor also interacts with other KDM family members, it has the highest specificity for KDM5B. Thus, two independent approaches to block KDM5B function demonstrated an altered immune response resulting in increases of critical innate cytokines.

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