Limits...
Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase.

Mishra A, Brown AL, Yao X, Yang S, Park SJ, Liu C, Dagur PK, McCoy JP, Keeran KJ, Nugent GZ, Jeffries KR, Qu X, Yu ZX, Levine SJ, Chung JH - Nat Commun (2015)

Bottom Line: Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species.We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens.Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Asthma and Lung Inflammation, Division of Intramural Research, NHLBI, NIH, Bethesda, Maryland 20892, USA.

ABSTRACT
DNA-dependent protein kinase (DNA-PK) mediates double-stranded DNA break repair, V(D)J recombination and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

No MeSH data available.


Related in: MedlinePlus

Characterization of HDM-challenged CD11c-specific DNA-PKcs KnockoutMiceA & B. The percentage of CD3+ T cells (A) andCD19+ B cells (B) in lungs and spleens of naïve, wild type (WT)C57BL/6 mice, which is the parental strain of bothDNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 6, *P < 0.05, one-way ANOVA with Sidak’smultiple comparison test). Pooled data from two independent experiments. C &D. Mean fluorescence intensity (MFI) of OX-40L, CD40, CD80 and CD86 expression byCD11c+/MHCIIhi/SSClo DCs in lungs (C) and mediastinallymph nodes (MLN) (D) of HDM-challenged DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice (n = 5 – 6 mice, * P< 0.05, Mann Whitney test). E & F. The percentage ofCD11c+/MHCIIhi/SSClo DCs that express CD11b in thelungs (E) and MLNs (F) of HDM-challenged DNA-PKcsfl/fl miceand DNA-PKcsfl/fl; CD11c-Cre mice (n = 13 mice, * P <0.0025, Mann Whitney test). G & H. Uptake of HDM byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungs(G) and MLNs (H) of DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice 72 hours afteradministration of HDM extract (50 µg) labeled with Alexa Fluor® 647 (n =21 mice, P = NS, unpaired t test). I & J. Thepercentage of CD11c+/MHCIIhi/SSClo/CD11b+ DCsthat express TLR4 or TLR5 (I) and the MFI of TLR4 and TLR5 expression (J) byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungsof DNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 8 mice, P = NS, Mann-Whitney test). K. BALFIL-10 (n = 9, * P < 0.01, DNA-PKcsfl/fl; CD11c-Cre +HDM vs. DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). L. IL-10 secretion by bone marrow-deriveddendritic cells (BMDCs) (n = 7 – 9, * P < 0.01,DNA-PKcsfl/fl; CD11c-Cre + HDM vs.DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). M. IL-10 secretion by BMDCs stimulated with HDM(100 ug/ml) with or without Akt inhibitors, GDC0068 (GDC) and MK2206 (MK), both at 1 uMfor 24 hrs (n = 8, * P < 0.05, HDM vs. HDM + Akt inhibitor, one way ANOVA withBonferroni multiple comparison test). Pooled data from two independent experiments.N. The percentage ofCD3+/CD4+/CD25+/Foxp3+ regulatory T cell(Tregs) in MLNs from saline- and HDM-challenged DNA-PKcsfl/fl;CD11c-Cre mice were compared to DNA-PKcsfl/fl mice,which served as a control (n = 12 mice, P = NS, Mann Whitney test).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4333735&req=5

Figure 6: Characterization of HDM-challenged CD11c-specific DNA-PKcs KnockoutMiceA & B. The percentage of CD3+ T cells (A) andCD19+ B cells (B) in lungs and spleens of naïve, wild type (WT)C57BL/6 mice, which is the parental strain of bothDNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 6, *P < 0.05, one-way ANOVA with Sidak’smultiple comparison test). Pooled data from two independent experiments. C &D. Mean fluorescence intensity (MFI) of OX-40L, CD40, CD80 and CD86 expression byCD11c+/MHCIIhi/SSClo DCs in lungs (C) and mediastinallymph nodes (MLN) (D) of HDM-challenged DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice (n = 5 – 6 mice, * P< 0.05, Mann Whitney test). E & F. The percentage ofCD11c+/MHCIIhi/SSClo DCs that express CD11b in thelungs (E) and MLNs (F) of HDM-challenged DNA-PKcsfl/fl miceand DNA-PKcsfl/fl; CD11c-Cre mice (n = 13 mice, * P <0.0025, Mann Whitney test). G & H. Uptake of HDM byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungs(G) and MLNs (H) of DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice 72 hours afteradministration of HDM extract (50 µg) labeled with Alexa Fluor® 647 (n =21 mice, P = NS, unpaired t test). I & J. Thepercentage of CD11c+/MHCIIhi/SSClo/CD11b+ DCsthat express TLR4 or TLR5 (I) and the MFI of TLR4 and TLR5 expression (J) byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungsof DNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 8 mice, P = NS, Mann-Whitney test). K. BALFIL-10 (n = 9, * P < 0.01, DNA-PKcsfl/fl; CD11c-Cre +HDM vs. DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). L. IL-10 secretion by bone marrow-deriveddendritic cells (BMDCs) (n = 7 – 9, * P < 0.01,DNA-PKcsfl/fl; CD11c-Cre + HDM vs.DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). M. IL-10 secretion by BMDCs stimulated with HDM(100 ug/ml) with or without Akt inhibitors, GDC0068 (GDC) and MK2206 (MK), both at 1 uMfor 24 hrs (n = 8, * P < 0.05, HDM vs. HDM + Akt inhibitor, one way ANOVA withBonferroni multiple comparison test). Pooled data from two independent experiments.N. The percentage ofCD3+/CD4+/CD25+/Foxp3+ regulatory T cell(Tregs) in MLNs from saline- and HDM-challenged DNA-PKcsfl/fl;CD11c-Cre mice were compared to DNA-PKcsfl/fl mice,which served as a control (n = 12 mice, P = NS, Mann Whitney test).

Mentions: Experiments were next conducted to assess the mechanisms by whichDNA-PK-deficient CD11c+ DCs have a reduced capability to induce allergicsensitization and Th2 immunity to HDM. First, we assessed whether the percentage of T andB cells in naïve DNA-PKcsfl/fl andDNA-PKcsfl/fl; Cd11c-Cre mice were reduced as compared tothe wild type (WT) parental strain of C57BL/6 mice, which could be indicative of a defectin V(D)J recombination with resultant altered lymphocyte development. As shown in Figure 6A and 6B, the percentage of CD3+ Tcells and CD19+ B cells were not significantly different in the lungs andspleens of naïve WT, DNA-PKcsfl/fl, andDNA-PKcsfl/fl; Cd11c-Cre mice, except for an increase inthe percentage of splenic CD19+ B cells in DNA-PKcsfl/fl;Cd11c-Cre mice as compared to WT mice. This finding suggests that lymphocytedevelopment was not significantly impaired in DNA-PKcsfl/fland DNA-PKcsfl/fl; Cd11c-Cre mice. Second, we found that cellsurface expression of the co-stimulatory molecule, CD80, was significantly reduced onCD11c+ DCs in the lungs (Figure 6C) andMLNs (Figure 6D) of HDM-challengedDNA-PKcsfl/fl; Cd11c-Cre mice as compared toHDM-challenged DNA-PKcsfl/fl mice. Although there werestatistically significant differences in the expression of OX-40L, CD40 and CD86 byCD11c+ DCs in the lung and MLNs, these differences appeared modest ascompared to the reduction in cell surface expression of CD80. In addition, as compared tothe murine BMDCs and human moDCs that were utilized for the experiments in Figure 1H, CD11c+ DCs from the lungs and MLNsof DNA-PKcsfl/fl and DNA-PKcsfl/fl;Cd11c-Cre mice had a different pattern of expression of CD40, CD80 and CD86, aswell as lower cell surface levels of these co-stimulatory molecules.


Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase.

Mishra A, Brown AL, Yao X, Yang S, Park SJ, Liu C, Dagur PK, McCoy JP, Keeran KJ, Nugent GZ, Jeffries KR, Qu X, Yu ZX, Levine SJ, Chung JH - Nat Commun (2015)

Characterization of HDM-challenged CD11c-specific DNA-PKcs KnockoutMiceA & B. The percentage of CD3+ T cells (A) andCD19+ B cells (B) in lungs and spleens of naïve, wild type (WT)C57BL/6 mice, which is the parental strain of bothDNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 6, *P < 0.05, one-way ANOVA with Sidak’smultiple comparison test). Pooled data from two independent experiments. C &D. Mean fluorescence intensity (MFI) of OX-40L, CD40, CD80 and CD86 expression byCD11c+/MHCIIhi/SSClo DCs in lungs (C) and mediastinallymph nodes (MLN) (D) of HDM-challenged DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice (n = 5 – 6 mice, * P< 0.05, Mann Whitney test). E & F. The percentage ofCD11c+/MHCIIhi/SSClo DCs that express CD11b in thelungs (E) and MLNs (F) of HDM-challenged DNA-PKcsfl/fl miceand DNA-PKcsfl/fl; CD11c-Cre mice (n = 13 mice, * P <0.0025, Mann Whitney test). G & H. Uptake of HDM byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungs(G) and MLNs (H) of DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice 72 hours afteradministration of HDM extract (50 µg) labeled with Alexa Fluor® 647 (n =21 mice, P = NS, unpaired t test). I & J. Thepercentage of CD11c+/MHCIIhi/SSClo/CD11b+ DCsthat express TLR4 or TLR5 (I) and the MFI of TLR4 and TLR5 expression (J) byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungsof DNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 8 mice, P = NS, Mann-Whitney test). K. BALFIL-10 (n = 9, * P < 0.01, DNA-PKcsfl/fl; CD11c-Cre +HDM vs. DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). L. IL-10 secretion by bone marrow-deriveddendritic cells (BMDCs) (n = 7 – 9, * P < 0.01,DNA-PKcsfl/fl; CD11c-Cre + HDM vs.DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). M. IL-10 secretion by BMDCs stimulated with HDM(100 ug/ml) with or without Akt inhibitors, GDC0068 (GDC) and MK2206 (MK), both at 1 uMfor 24 hrs (n = 8, * P < 0.05, HDM vs. HDM + Akt inhibitor, one way ANOVA withBonferroni multiple comparison test). Pooled data from two independent experiments.N. The percentage ofCD3+/CD4+/CD25+/Foxp3+ regulatory T cell(Tregs) in MLNs from saline- and HDM-challenged DNA-PKcsfl/fl;CD11c-Cre mice were compared to DNA-PKcsfl/fl mice,which served as a control (n = 12 mice, P = NS, Mann Whitney test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4333735&req=5

Figure 6: Characterization of HDM-challenged CD11c-specific DNA-PKcs KnockoutMiceA & B. The percentage of CD3+ T cells (A) andCD19+ B cells (B) in lungs and spleens of naïve, wild type (WT)C57BL/6 mice, which is the parental strain of bothDNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 6, *P < 0.05, one-way ANOVA with Sidak’smultiple comparison test). Pooled data from two independent experiments. C &D. Mean fluorescence intensity (MFI) of OX-40L, CD40, CD80 and CD86 expression byCD11c+/MHCIIhi/SSClo DCs in lungs (C) and mediastinallymph nodes (MLN) (D) of HDM-challenged DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice (n = 5 – 6 mice, * P< 0.05, Mann Whitney test). E & F. The percentage ofCD11c+/MHCIIhi/SSClo DCs that express CD11b in thelungs (E) and MLNs (F) of HDM-challenged DNA-PKcsfl/fl miceand DNA-PKcsfl/fl; CD11c-Cre mice (n = 13 mice, * P <0.0025, Mann Whitney test). G & H. Uptake of HDM byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungs(G) and MLNs (H) of DNA-PKcsfl/fl mice andDNA-PKcsfl/fl; CD11c-Cre mice 72 hours afteradministration of HDM extract (50 µg) labeled with Alexa Fluor® 647 (n =21 mice, P = NS, unpaired t test). I & J. Thepercentage of CD11c+/MHCIIhi/SSClo/CD11b+ DCsthat express TLR4 or TLR5 (I) and the MFI of TLR4 and TLR5 expression (J) byCD11c+/MHCIIhi/SSClo/CD11b+ DCs in the lungsof DNA-PKcsfl/fl mice and DNA-PKcsfl/fl;CD11c-Cre mice (n = 8 mice, P = NS, Mann-Whitney test). K. BALFIL-10 (n = 9, * P < 0.01, DNA-PKcsfl/fl; CD11c-Cre +HDM vs. DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). L. IL-10 secretion by bone marrow-deriveddendritic cells (BMDCs) (n = 7 – 9, * P < 0.01,DNA-PKcsfl/fl; CD11c-Cre + HDM vs.DNA-PKcsfl/fl + HDM, one way ANOVA with Bonferronimultiple comparison test). M. IL-10 secretion by BMDCs stimulated with HDM(100 ug/ml) with or without Akt inhibitors, GDC0068 (GDC) and MK2206 (MK), both at 1 uMfor 24 hrs (n = 8, * P < 0.05, HDM vs. HDM + Akt inhibitor, one way ANOVA withBonferroni multiple comparison test). Pooled data from two independent experiments.N. The percentage ofCD3+/CD4+/CD25+/Foxp3+ regulatory T cell(Tregs) in MLNs from saline- and HDM-challenged DNA-PKcsfl/fl;CD11c-Cre mice were compared to DNA-PKcsfl/fl mice,which served as a control (n = 12 mice, P = NS, Mann Whitney test).
Mentions: Experiments were next conducted to assess the mechanisms by whichDNA-PK-deficient CD11c+ DCs have a reduced capability to induce allergicsensitization and Th2 immunity to HDM. First, we assessed whether the percentage of T andB cells in naïve DNA-PKcsfl/fl andDNA-PKcsfl/fl; Cd11c-Cre mice were reduced as compared tothe wild type (WT) parental strain of C57BL/6 mice, which could be indicative of a defectin V(D)J recombination with resultant altered lymphocyte development. As shown in Figure 6A and 6B, the percentage of CD3+ Tcells and CD19+ B cells were not significantly different in the lungs andspleens of naïve WT, DNA-PKcsfl/fl, andDNA-PKcsfl/fl; Cd11c-Cre mice, except for an increase inthe percentage of splenic CD19+ B cells in DNA-PKcsfl/fl;Cd11c-Cre mice as compared to WT mice. This finding suggests that lymphocytedevelopment was not significantly impaired in DNA-PKcsfl/fland DNA-PKcsfl/fl; Cd11c-Cre mice. Second, we found that cellsurface expression of the co-stimulatory molecule, CD80, was significantly reduced onCD11c+ DCs in the lungs (Figure 6C) andMLNs (Figure 6D) of HDM-challengedDNA-PKcsfl/fl; Cd11c-Cre mice as compared toHDM-challenged DNA-PKcsfl/fl mice. Although there werestatistically significant differences in the expression of OX-40L, CD40 and CD86 byCD11c+ DCs in the lung and MLNs, these differences appeared modest ascompared to the reduction in cell surface expression of CD80. In addition, as compared tothe murine BMDCs and human moDCs that were utilized for the experiments in Figure 1H, CD11c+ DCs from the lungs and MLNsof DNA-PKcsfl/fl and DNA-PKcsfl/fl;Cd11c-Cre mice had a different pattern of expression of CD40, CD80 and CD86, aswell as lower cell surface levels of these co-stimulatory molecules.

Bottom Line: Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species.We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens.Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Asthma and Lung Inflammation, Division of Intramural Research, NHLBI, NIH, Bethesda, Maryland 20892, USA.

ABSTRACT
DNA-dependent protein kinase (DNA-PK) mediates double-stranded DNA break repair, V(D)J recombination and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

No MeSH data available.


Related in: MedlinePlus