17 research outputs found
IRF4-Dependent and IRF4-Independent Pathways Contribute to DC Dysfunction in Lupus
<div><p>Interferon Regulatory Factors (IRFs) play fundamental roles in dendritic cell (DC) differentiation and function. In particular, IRFs are critical transducers of TLR signaling and dysregulation in this family of factors is associated with the development of autoimmune disorders such as Systemic Lupus Erythematosus (SLE). While several IRFs are expressed in DCs their relative contribution to the aberrant phenotypic and functional characteristics that DCs acquire in autoimmune disease has not been fully delineated. Mice deficient in both DEF6 and SWAP-70 (= Double-knock-out or DKO mice), two members of a unique family of molecules that restrain IRF4 function, spontaneously develop a lupus-like disease. Although autoimmunity in DKO mice is accompanied by dysregulated IRF4 activity in both T and B cells, SWAP-70 is also known to regulate multiple aspects of DC biology leading us to directly evaluate DC development and function in these mice. By monitoring Blimp1 expression and IL-10 competency in DKO mice we demonstrate that DCs in these mice exhibit dysregulated IL-10 production, which is accompanied by aberrant Blimp1 expression in the spleen but not in the peripheral lymph nodes. We furthermore show that DCs from these mice are hyper-responsive to multiple TLR ligands and that IRF4 plays a differential role in in these responses by being required for the TLR4-mediated but not the TLR9-mediated upregulation of IL-10 expression. Thus, DC dysfunction in lupus-prone mice relies on both IRF4-dependent and IRF4-independent pathways.</p></div
Selective requirement for IRF4 in TLR stimulated DKO BMDCs.
<p>(<b>A</b>) WT, DKO and CD11c-Cre IRF4<sup>fl/fl</sup> DKO BMDCs were generated <i>in vitro</i> in presence of GM-CSF for 7–9 days prior to FACS analysis of MHCII, CD86 and PDL2. Histograms show relative expression of the indicated marker on WT (red), DKO (blue) and CD11c-Cre IRF4<sup>fl/fl</sup> DKO (green) total CD11c<sup>+</sup>DCs or GFP<sup>+</sup>CD11c<sup>+</sup>DCs. One representative experiment out of 3 independent experiments is shown. <b>(B-C)</b> BMDCs were generated <i>in vitro</i> for 7 days. CD11c<sup>+</sup>DCs were purified by magnetic sorting followed by in vitro stimulation with 0.1<b>μ</b>g/ml LPS or 3<b>μ</b>M CpG, for 24h. IL-10 and IFN<b>β</b> gene expression in LPS treated (<b>□</b>) or CpG treated (<b>C</b>) WT, DKO and CD11c-Cre IRF4<sup>fl/fl</sup> DKO BMDCs were assayed by qPCR. One representative experiment out of 4 independent experiments is shown. *: p<0.05; **: p<0.01, ***: p<0.001.</p
Increased IL-10 production by CD11b<sup>+</sup> DKO DCs.
<p>(<b>A</b>) Spleens (SPL) from 14–16 weeks old WT and DKO IL-10 and Blimp1 dual reporter mice were examined by flow cytometry. Splenocytes were gated on MHCII<sup>+</sup>CD11c<sup>+</sup>B220<sup>-</sup> conventional DCs followed by analysis of YFP (Blimp1) and Thy1.1 (IL-10) expression on CD11b<sup>+</sup>DCs and CD8<sup>+</sup>DCs. Percentages and numbers of Thy1.1<sup>+</sup>YFP<sup>-</sup>, Thy1.1<sup>+</sup>YFP<sup>+</sup> and Thy1.1<sup>-</sup>YFP<sup>+</sup> cells are shown. Scatter plots show data of individual mice and mean value of 4 independent experiments. *: p<0.05; **: p<0.01, ***: p<0.001. (<b>B</b>) IL-10 production by splenic CD11c<sup>+</sup> cells stimulated <i>in vitro</i> with 0.1 <b>μ</b>g/ml LPS for 24 hours was analyzed by qPCR and ELISA. Representative data of 3 independent experiments are shown **: p<0.01. (<b>C</b>) Skin draining lymph nodes (SDLN) from 14–16 weeks old WT and DKO IL-10 and Blimp1 dual reporter mice were examined by flow cytometry. Cells were gated on MHCII<sup>+</sup>CD11c<sup>+</sup>B220<sup>-</sup> conventional DCs followed by analysis of YFP (Blimp1) and Thy1.1 (IL-10) expression on CD11b<sup>+</sup>DCs and CD8<sup>+</sup>DCs. Percentages and numbers of Thy1.1<sup>+</sup>YFP<sup>-</sup>, Thy1.1<sup>+</sup>YFP<sup>+</sup>, and Thy1.1<sup>-</sup>YFP<sup>+</sup> cells are shown. Scatter plots show data of individual mice and mean value of 4 independent experiments. *: p<0.05; **: p<0.01, ***: p<0.001. (<b>D</b>) PDL2 cell surface expression on DCs from WT and DKO IL-10 and Blimp1 dual reporter mice was analyzed by flow cytometry. Splenic and skin draining lymph node (SDLN) cells were gated on MHCII<sup>+</sup>CD11c<sup>+</sup>CD11b<sup>+</sup> DCs and PDL2 expression was analyzed on Thy1.1<sup>-</sup>YFP<sup>-</sup>, Thy1.1<sup>+</sup>YFP<sup>-</sup>, Thy1.1<sup>+</sup>YFP<sup>+</sup> and Thy1.1<sup>-</sup>YFP<sup>+</sup> subsets. Histograms show representative expression of PDL2. Scatter plots show mean florescence intensity (MFI) data of individual mice and mean value of 3 independent experiments: ****: p<0.0001.</p
Effects of IRF4 deletion in CD11c<sup>+</sup> cells on DC, T, and B cell populations in DKO mice.
<p>(<b>A)</b> Spleens of 14–20 weeks old WT, DKO and CD11c-Cre IRF4<sup>fl/fl</sup> DKO mice were assayed for DC populations by flow cytometry. Splenocytes were gated on MHCII<sup>+</sup>CD11c<sup>+</sup>B220<sup>-</sup> conventional DCs and analyzed for the proportion and numbers of CD11b<sup>+</sup>DCs and CD8<sup>+</sup>DCs. (<b>B</b>) CD86 and PDL2 cell surface expression on CD11b<sup>+</sup>DCs. Histograms show relative expression of the indicated marker on WT (red), DKO (blue) and CD11c-Cre IRF4<sup>fl/fl</sup> DKO (green) mice. Representative data of at least 2 independent experiments with a total of 3–5 mice per group is shown. (<b>C-F</b>) WT, DKO and CD11c-Cre IRF4<sup>fl/fl</sup> DKO total splenocytes were analyzed for their CD4<sup>+</sup>T cell (<b>C-D</b>) and B cell (<b>E-F</b>) populations. Percentages and numbers of activated Tregs (CD4<sup>+</sup>Foxp3<sup>+</sup>CD44<sup>+</sup>), activated T cells (CD4<sup>+</sup>Foxp3<sup>-</sup>CD44<sup>+</sup>), Tfh (CD4<sup>+</sup>Foxp3<sup>-</sup>PD1<sup>+</sup>CXCR5<sup>+</sup>), germinal center B cells (GC; B220<sup>+</sup>GL7<sup>+</sup>Fas<sup>+</sup>) and plasma cells (PC; B220<sup>+</sup>CD138<sup>+</sup>) are shown. Scatter plots show data of individual mice and mean value of 4 independent experiments. *: p<0.05; **: p<0.01, ***: p<0.001, ****: p≤0.0001.</p
Relative expansion of CD11b<sup>+</sup> DCs in DKO mice.
<p>Spleen (SPL) (<b>A</b>) and skin draining lymph nodes (SDLN) (<b>B</b>) of 8 weeks old WT and DKO female mice were assayed for DC populations by flow cytometry. Splenocytes were gated on MHCII<sup>+</sup>CD11c<sup>+</sup> conventional DCs and analyzed for the proportion of CD8<sup>+</sup>DCs and CD11b<sup>+</sup>DCs. In SDLNs, MHCII<sup>Hi</sup>CD11c<sup>+</sup> migratory DCs were also examined. Spleen (<b>C</b>) and skin draining lymph nodes (<b>D</b>) of >24 weeks old mice were also assayed for DC populations by flow cytometry. Cells were gated on MHCII<sup>+</sup>CD11c<sup>+</sup>B220<sup>-</sup> conventional DCs and analyzed for the proportion of CD8+DCs and CD11b<sup>+</sup>DCs. In lymph nodes, MHCII<sup>Hi</sup>CD11c<sup>+</sup> migratory DC frequencies were also examined. Scatter plots show data of individual mice and mean value of at least 3 independent experiments. *: p<0.05; **: p<0.01, ***: p≤0.0001. <b>(E)</b> CD86, PDL2 and PDL1 cell surface expression was analyzed by flow cytometry on conventional CD11b<sup>+</sup>DCs and CD8<sup>+</sup> DCs in the spleen of >24 weeks old WT (red) and DKO (blue) mice. Histograms show relative expression of the indicated marker. Representative data of 2–4 independent experiments with a total of 5–9 mice per group are shown.</p
Increased IL-10 and IFNβ expression by TLR-stimulated DKO BMDCs.
<p>(<b>A</b>) WT and DKO BMDCs were generated <i>in vitro</i> in presence of GM-CSF for 7–9 days prior to FACS analysis of MHCII, CD86, CD80, PDL2 and PDL1. Histograms show relative expression of the indicated marker on WT (red) or DKO (blue) CD11c<sup>+</sup>DCs. One representative experiment out of 1 (CD80) or at least 2 independent experiments (MHCII, CD86, PDL2 and PDL1) is shown. <b>(B-E)</b> BMDCs were generated <i>in vitro</i> for 7 days. CD11c<sup>+</sup>DCs were purified by magnetic sorting followed by <i>in vitro</i> stimulation with 0.1<b>μ</b>g/ml LPS or 3<b>μ</b>M CpG for 24h. (<b>B</b>) Cells were harvested and MHCII, CD86 and PDL2 expression on WT and DKO LPS treated CD11c<sup>+</sup> DCs analyzed by FACS. One representative experiment out of 2 independent experiments is shown. IL-10 and IFN<b>β</b> production and gene expression in LPS treated (<b>C</b>) or CpG treated (<b>D</b>) WT and DKO BMDCs were assayed by ELISA and qPCR. One representative experiment out of at least 3 independent experiments is shown. *: p<0.05; **: p<0.01, ***: p<0.001 (<b>E</b>) Thy1.1 expression in BMDCs from IL-10 and Blimp1 dual reporter WT or DKO mice was analyzed by FACS. Histograms show relative expression of Thy1.1 on WT (red) or DKO (blue) CD11c+MHCII<sup>hi</sup> or CD11c+MHCII<sup>lo</sup> DCs. One representative experiment out of at least 2 independent experiments is shown.</p
Deletion of a Conserved <i>cis</i>-Element in the <i>Ifng</i> Locus Highlights the Role of Acute Histone Acetylation in Modulating Inducible Gene Transcription
<div><p>Differentiation-dependent regulation of the <i>Ifng</i> cytokine gene locus in T helper (Th) cells has emerged as an excellent model for functional study of distal elements that control lineage-specific gene expression. We previously identified a <i>cis</i>-regulatory element located 22 kb upstream of the <i>Ifng</i> gene (<u>C</u>onserved <u>N</u>on-coding <u>S</u>equence -22, or CNS-22) that is a site for recruitment of the transcription factors T-bet, Runx3, NF-κB and STAT4, which act to regulate transcription of the <i>Ifng</i> gene in Th1 cells. Here, we report the generation of mice with a conditional deletion of CNS-22 that has enabled us to define the epigenetic and functional consequences of its absence. Deletion of CNS-22 led to a defect in induction of <i>Ifng</i> by the cytokines IL-12 and IL-18, with a more modest effect on induction via T-cell receptor activation. To better understand how CNS-22 and other <i>Ifng</i> CNSs regulated <i>Ifng</i> transcription in response to these distinct stimuli, we examined activation-dependent changes in epigenetic modifications across the extended <i>Ifng</i> locus in CNS-22-deficient T cells. We demonstrate that in response to both cytokine and TCR driven activation signals, CNS-22 and other <i>Ifng</i> CNSs recruit increased activity of histone acetyl transferases (HATs) that transiently enhance levels of histones H3 and H4 acetylation across the extended <i>Ifng</i> locus. We also demonstrate that activation-responsive increases in histone acetylation levels are directly linked to the ability of <i>Ifng</i> CNSs to acutely enhance Pol II recruitment to the <i>Ifng</i> promoter. Finally, we show that impairment in IL-12+IL-18 dependent induction of <i>Ifng</i> stems from the importance of CNS-22 in coordinating locus-wide levels of histone acetylation in response to these cytokines. These findings identify a role for acute histone acetylation in the enhancer function of distal conserved <i>cis</i>-elements that regulate of <i>Ifng</i> gene expression.</p></div
Deletion of CNS-22 leads to prominent defects in IL-12+IL-18 dependent modulation of histone hyperacetylation.
<p>(<b>A</b>) Th1 cells generated from either WT or CNS-22<sup>−/−</sup> mice were either left unstimulated or activated with IL-12+IL-18 for 1.5 h or anti-CD3 and anti-CD28 for 3 h and then subject to ChIP-chip with antibodies against H4K12ac. Levels of H4K12ac were analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003969#pgen-1003969-g003" target="_blank">Fig. 3</a> and visualized using the IGB browser. (<b>B</b>) H4K12ac levels across the <i>Ifng</i> locus documented in WT Th1 cells were normalized against H4K12ac levels documented in CNS-22<sup>−/−</sup> Th1 cells to visualize in a semi-quantitative manner the magnitude to which locus-wide acquisition of H4K12ac marks was impaired in the absence of CNS-22. (<b>C</b>) Th1 cells generated from WT or CNS-22<sup>−/−</sup> mice were restimulated with anti-CD3+anti-CD28 for 3 h or IL-12+IL-18 for 1.5 h and recruitment of RNA-Pol II to the <i>Ifng</i> gene was assessed by ChIP-qPCR. RNA-Pol II recruitment is shown as a percentage of input DNA. Data represent means from at least three independent experiments. Statistical analyses were carried out on means and standard errors from three independent experiments * p<0.01, # p<0.05, stimulated WT versus CNS-22<sup>−/−</sup>.</p
Identification of putative regulatory elements greater than 100<i>Ifng</i> gene.
<p>(<b>A–B</b>) Naïve CD4<sup>+</sup> T cells from OT-II transgenic WT mice were differentiated under Th1, Th2 or Th17 polarizing conditions. Cells were left unstimulated or activated with IL-12+IL-18 for 1.5 h and subject to ChIP-chip. To identify STAT4, Smc3 and CTCF binding sites and to define H4K12ac-enriched regions across the <i>Ifng</i> locus, peak calling was carried out using a previously described algorithm for capturing microarray enrichment (ACME) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003969#pgen.1003969-Crawford2" target="_blank">[49]</a>. Peak-calling thresholds were set to a confidence limit of 95% and visualized with the IGB browser <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003969#pgen.1003969-Nicol1" target="_blank">[48]</a>. Data are representative of at least two independent experiments.</p
IL-12+IL-18 driven <i>Ifng</i> transcription is compromised in CNS-22<sup>−/−</sup> T cells and NK cells.
<p>(<b>A</b>) CD4<sup>+</sup> T cells derived from OT-II transgenic WT and CNS-22<sup>−/−</sup> mice were differentiated with 2 ng/ml IL-12, 5 µg/ml ova-peptide (ISQAVHAAHAEINEAGR) and cultured with CD4-depleted irradiated feeder cells derived from <i>Il12a</i><sup>−/−</sup> mice. Cells were reactivated for 4 h as described in methods and expression of IFN-γ was assessed by flow cytometric analysis following intracellular staining. Percentages of viable, IFN-γ<sup>+</sup> T cells are indicated by black numbers and mean fluorescence intensities (MFI) of IFN-γ<sup>+</sup> cells are indicated in grey. Data are representative of at least three independent experiments. (<b>B</b>) CD8<sup>+</sup> T cells isolated from WT and CNS-22<sup>−/−</sup> mice were differentiated with 2.5 µg/ml of anti-CD3 antibody, 2 ng/ml IL-12 for 3 days and reactivated for 4 h with IL-12+IL-18 and subject to intracellular staining. For evaluating IFN-γ expression in NK cells, following depletion of both CD4<sup>+</sup> and CD8<sup>+</sup> T cells bulk splenocytes were activated with IL-12+IL-18 for 4 h and subject to intracellular staining. Percentages of viable, IFN-γ<sup>+</sup> T/NK cells are indicated in black and MFI of IFN-γ<sup>+</sup> cells are indicated in grey. Data are representative of at least three independent experiments. (<b>C</b>) Total RNA from anti-CD3+anti-CD28 or IL-12+IL-18 stimulated WT and CNS-22<sup>−/−</sup> Th1 cells was isolated at indicated time points and reverse-transcribed to generate cDNA. Transcript levels were measured by RT-PCR and relative levels of spliced transcripts were calculated by normalization against levels of spliced transcripts in resting Th1 cells. Data represent means from at least three independent experiments. Statistical analyses were carried out on means and standard errors from three independent experiments * p<0.01, # p<0.05, stimulated WT versus CNS-22<sup>−/−</sup>.</p