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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

Transfer of RSV-infected Kdm5b-deficient DCs leads to a decreased pathogenic pulmonary RSV challenge.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control, then infected with RSV overnight. These BMDCs were transferred intratracheally to the lungs of naïve mice 7 days prior to RSV infection. (B) At 8 days post infection, protein levels were measured from MLN cells restimulated with RSV in vitro. (C) Inflammation around the airways was measured by H&E staining in mice primed with BMDCs, including airway eosinophils (inset). (D) Eosinophils were counted in 100μm sections around the airways. (E) Lungs were dispersed into a single cell suspension using collagenase A and DNase. Esoinophils were measured using flow cytometry. (F) Total lung cells were counted following digestion of lung tissue. CD11c+CD11b+ and CD11c+CD103+ DC populations were measured by flow cytometry (G), as well as CD4+ T cells and activated CD4+CD69+ cells (H). (I) RNA was extracted from lung tissue using Trizol reagent, and transcripts of Muc5ac, Gob5 and Il13 were measured by qPCR. (J) Mucus was visualized in the lungs using PAS staining. Arrows indicate mucus production. (K) Identifying numbers on histology slides were blinded and slides were scored on a scale of 1–4 for mucus production. For all data n = 4-5samples/group and data are representative of two independent experiments. *p<0.05, **p<0.01.
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ppat.1004978.g004: Transfer of RSV-infected Kdm5b-deficient DCs leads to a decreased pathogenic pulmonary RSV challenge.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control, then infected with RSV overnight. These BMDCs were transferred intratracheally to the lungs of naïve mice 7 days prior to RSV infection. (B) At 8 days post infection, protein levels were measured from MLN cells restimulated with RSV in vitro. (C) Inflammation around the airways was measured by H&E staining in mice primed with BMDCs, including airway eosinophils (inset). (D) Eosinophils were counted in 100μm sections around the airways. (E) Lungs were dispersed into a single cell suspension using collagenase A and DNase. Esoinophils were measured using flow cytometry. (F) Total lung cells were counted following digestion of lung tissue. CD11c+CD11b+ and CD11c+CD103+ DC populations were measured by flow cytometry (G), as well as CD4+ T cells and activated CD4+CD69+ cells (H). (I) RNA was extracted from lung tissue using Trizol reagent, and transcripts of Muc5ac, Gob5 and Il13 were measured by qPCR. (J) Mucus was visualized in the lungs using PAS staining. Arrows indicate mucus production. (K) Identifying numbers on histology slides were blinded and slides were scored on a scale of 1–4 for mucus production. For all data n = 4-5samples/group and data are representative of two independent experiments. *p<0.05, **p<0.01.

Mentions: Pro-inflammatory cytokines, including IFN-β, are important factors in driving Th1 responses following viral infection in vivo [26]. The above data indicated siRNA knockdown of Kdm5b led to increased pro-inflammatory cytokines in vitro, suggesting that priming the immune system in vivo with these DCs would drive a stronger Th1 response to RSV. To test this hypothesis, BMDCs were treated with Kdm5b-specific siRNA or scrambled control siRNA for 48 hours and subsequently infected overnight with RSV. These cells were then transferred intratracheally into naïve C57Bl/6 mice to prime the immune system in the context of Kdm5b-deficient DCs. One week after transfer, mice were challenged with RSV (Fig 4A). Our lab has previously demonstrated that this sensitization protocol with myeloid DC elicits a pathogenic immune environment upon RSV reinfection, and that the RSV-infected DCs that are transferred begin migrating to the lymph nodes within 24 hours [27,28]. Eight days following challenge, the mediastinal lymph nodes (MLN) were removed and restimulated ex vivo to determine the effect that sensitizing mice with Kdm5b-knockdown DCs would have on the T cell response. The data indicated an increase in IFN-γ production from mice that had been sensitized with Kdm5b-specific siRNA treated DCs, accompanied by a significant decrease in IL-5 and a trend toward less IL-4 production (Fig 4B). Furthermore, it was found that the lungs of mice primed with DCs that had been transfected with Kdm5b-specific siRNA had decreased inflammatory infiltrates compared to mice primed with control siRNA-treated DCs, as observed by H&E stained sections (Fig 4C). High-power images revealed that the inflammation surrounding the airways of control RSV-infected DC-sensitized mice contained numerous eosinophils (Fig 4C, insets). The number of eosinophils surrounding the airways was enumerated by microscopy in 100μm sections demonstrating decreased numbers in the Kdm5b siRNA group (Fig 4D) and confirmed with flow cytometric analysis of Gr-1+ and Siglec-F+ cells (Fig 4E). Additionally, it was noted that the total number of cells in the lungs was increased in mice sensitized with control siRNA, but the lungs had fewer cells when the mice received Kdm5b siRNA (Fig 4F). Flow cytometric analysis showed that these differences in total lung cells were due to fewer numbers of both conventional CD11c+CD11b+ DCs, as well as CD11c+CD103+ DCs (Fig 4G) [29,30]. Furthermore, there was a decrease in total CD4+ T cells as well as activated CD4+CD69+ cells (Fig 4H). Increased numbers of total cells, as well as DCs, T cells and eosinophils were also observed in mice that where sensitized with uninfected DCs, but to a lesser degree. (S5 Fig). In these mice, the numbers of inflammatory cells were similar to the numbers of cells measured in mice that received Kdm5b-deficient DCs, but was lower than the number of cells noted with control-treated, RSV-infected DCs were used to prime mice prior to challenge. Thus, the inhibition of Kdm5b in RSV-infected DCs led to significant protection from immunopathology that often has been associated with immunization to RSV [8].


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)

Transfer of RSV-infected Kdm5b-deficient DCs leads to a decreased pathogenic pulmonary RSV challenge.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control, then infected with RSV overnight. These BMDCs were transferred intratracheally to the lungs of naïve mice 7 days prior to RSV infection. (B) At 8 days post infection, protein levels were measured from MLN cells restimulated with RSV in vitro. (C) Inflammation around the airways was measured by H&E staining in mice primed with BMDCs, including airway eosinophils (inset). (D) Eosinophils were counted in 100μm sections around the airways. (E) Lungs were dispersed into a single cell suspension using collagenase A and DNase. Esoinophils were measured using flow cytometry. (F) Total lung cells were counted following digestion of lung tissue. CD11c+CD11b+ and CD11c+CD103+ DC populations were measured by flow cytometry (G), as well as CD4+ T cells and activated CD4+CD69+ cells (H). (I) RNA was extracted from lung tissue using Trizol reagent, and transcripts of Muc5ac, Gob5 and Il13 were measured by qPCR. (J) Mucus was visualized in the lungs using PAS staining. Arrows indicate mucus production. (K) Identifying numbers on histology slides were blinded and slides were scored on a scale of 1–4 for mucus production. For all data n = 4-5samples/group and data are representative of two independent experiments. *p<0.05, **p<0.01.
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ppat.1004978.g004: Transfer of RSV-infected Kdm5b-deficient DCs leads to a decreased pathogenic pulmonary RSV challenge.(A) BMDCs were transfected with Kdm5b-specific siRNA or non-targeting control, then infected with RSV overnight. These BMDCs were transferred intratracheally to the lungs of naïve mice 7 days prior to RSV infection. (B) At 8 days post infection, protein levels were measured from MLN cells restimulated with RSV in vitro. (C) Inflammation around the airways was measured by H&E staining in mice primed with BMDCs, including airway eosinophils (inset). (D) Eosinophils were counted in 100μm sections around the airways. (E) Lungs were dispersed into a single cell suspension using collagenase A and DNase. Esoinophils were measured using flow cytometry. (F) Total lung cells were counted following digestion of lung tissue. CD11c+CD11b+ and CD11c+CD103+ DC populations were measured by flow cytometry (G), as well as CD4+ T cells and activated CD4+CD69+ cells (H). (I) RNA was extracted from lung tissue using Trizol reagent, and transcripts of Muc5ac, Gob5 and Il13 were measured by qPCR. (J) Mucus was visualized in the lungs using PAS staining. Arrows indicate mucus production. (K) Identifying numbers on histology slides were blinded and slides were scored on a scale of 1–4 for mucus production. For all data n = 4-5samples/group and data are representative of two independent experiments. *p<0.05, **p<0.01.
Mentions: Pro-inflammatory cytokines, including IFN-β, are important factors in driving Th1 responses following viral infection in vivo [26]. The above data indicated siRNA knockdown of Kdm5b led to increased pro-inflammatory cytokines in vitro, suggesting that priming the immune system in vivo with these DCs would drive a stronger Th1 response to RSV. To test this hypothesis, BMDCs were treated with Kdm5b-specific siRNA or scrambled control siRNA for 48 hours and subsequently infected overnight with RSV. These cells were then transferred intratracheally into naïve C57Bl/6 mice to prime the immune system in the context of Kdm5b-deficient DCs. One week after transfer, mice were challenged with RSV (Fig 4A). Our lab has previously demonstrated that this sensitization protocol with myeloid DC elicits a pathogenic immune environment upon RSV reinfection, and that the RSV-infected DCs that are transferred begin migrating to the lymph nodes within 24 hours [27,28]. Eight days following challenge, the mediastinal lymph nodes (MLN) were removed and restimulated ex vivo to determine the effect that sensitizing mice with Kdm5b-knockdown DCs would have on the T cell response. The data indicated an increase in IFN-γ production from mice that had been sensitized with Kdm5b-specific siRNA treated DCs, accompanied by a significant decrease in IL-5 and a trend toward less IL-4 production (Fig 4B). Furthermore, it was found that the lungs of mice primed with DCs that had been transfected with Kdm5b-specific siRNA had decreased inflammatory infiltrates compared to mice primed with control siRNA-treated DCs, as observed by H&E stained sections (Fig 4C). High-power images revealed that the inflammation surrounding the airways of control RSV-infected DC-sensitized mice contained numerous eosinophils (Fig 4C, insets). The number of eosinophils surrounding the airways was enumerated by microscopy in 100μm sections demonstrating decreased numbers in the Kdm5b siRNA group (Fig 4D) and confirmed with flow cytometric analysis of Gr-1+ and Siglec-F+ cells (Fig 4E). Additionally, it was noted that the total number of cells in the lungs was increased in mice sensitized with control siRNA, but the lungs had fewer cells when the mice received Kdm5b siRNA (Fig 4F). Flow cytometric analysis showed that these differences in total lung cells were due to fewer numbers of both conventional CD11c+CD11b+ DCs, as well as CD11c+CD103+ DCs (Fig 4G) [29,30]. Furthermore, there was a decrease in total CD4+ T cells as well as activated CD4+CD69+ cells (Fig 4H). Increased numbers of total cells, as well as DCs, T cells and eosinophils were also observed in mice that where sensitized with uninfected DCs, but to a lesser degree. (S5 Fig). In these mice, the numbers of inflammatory cells were similar to the numbers of cells measured in mice that received Kdm5b-deficient DCs, but was lower than the number of cells noted with control-treated, RSV-infected DCs were used to prime mice prior to challenge. Thus, the inhibition of Kdm5b in RSV-infected DCs led to significant protection from immunopathology that often has been associated with immunization to RSV [8].

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