8 research outputs found
Dynamic Expression of BCL6 in Murine Conventional Dendritic Cells during <i>In Vivo</i> Development and Activation
<div><p>The transcriptional repressor BCL6 plays an essential role in the development of germinal center B cells and follicular helper T cells. However, much less is known about the expression and function of BCL6 in other cell types. Here we report that during murine dendritic cell (DC) ontogeny in vivo, BCL6 is not expressed in bone marrow hematopoietic stem cells, common DC precursors and committed precursors of conventional DCs (pre-cDCs), but is elevated in peripheral pre-cDCs. BCL6 protein levels rise as pre-cDCs differentiate into cDCs in secondary lymphoid organs. Elevated protein levels of Bcl6 are observed in all cDC subsets, with CD8α<sup>+</sup> cDCs displaying the greatest levels. Co-staining of Ki-67 revealed BCL6<sup>hi</sup> cDCs to be more proliferative than BCL6<sup>lo</sup> cDCs. After adjuvant inoculation, BCL6 levels are significantly reduced in the CD11c<sup>int</sup> MHC class II<sup>hi</sup> CD86<sup>hi</sup> cDCs. Activation-induced BCL6 reduction correlated with reduced proliferation. A LPS injection study further confirmed that, in response to microbial stimuli, BCL6 levels are dynamically regulated during the maturation of CD11c<sup>int</sup> MHC class II<sup>hi</sup> splenic cDCs. This reduction of BCL6 levels in cDCs does not occur after LPS injection in MyD88<sup>−/−</sup> TRIF<sup>−/−</sup> mice. Thus, regulation of Bcl6 protein levels is dynamic in murine cDCs during development, maturation and activation <i>in vivo</i>.</p></div
BCL6 protein is weakly expressed in peripheral pre-cDCs during in vivo development.
<p>BM, peripheral blood, spleens and LNs were collected from naïve C57BL/6 mice. <i>(A)</i> BM HSCs and CDPs were gated as indicated. The Lin markers include CD3, CD4, CD8, CD19, B220 and CD11b. Representative FACS data were shown (n = 8). <i>(B)</i> Pre-cDCs were gated as indicated usingthe Lin markers including CD3, CD19 and B220. Representative FACS plots were shown (n = 6). Mouse IgG1 isotype control (solid grey), BCL6 expression in indicated DC population (open black). BCL6 expression gates were drawn based on the negative expression of isotype controls. Numbers in the FACS plots indicated the percent of population. <i>(C)</i> Overlay of BCL6 expression levels in different cell population. BCL6 expression in GC B cells obtained 7 days p.i. (gated as B220<sup>+</sup> Fas<sup>+</sup> CD38<sup>lo</sup> cells) served as positive controls (open grey line) and naïve B cells (gated as B220<sup>+</sup> Fas<sup>−</sup> CD38<sup>hi</sup> cells) as negative controls (open grey dashed line).</p
BCL6 is elevated in steady-state cDCs.
<p><i>(A)</i> Splenocytes from naïve C57BL/6 mice were stained for cDCs subsets or pDCs, and for intracellular BCL6 protein expression. Representative FACS plots were shown (n = 8): Isotype controls (solid grey), BCL6 expression of GC B cells gated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101208#pone-0101208-g001" target="_blank">Figure 1</a> as positive controls (dashed line), BCL6 expression of the indicated DC population (open black). BCL6 expression gates were drawn based on the negative expression of isotype controls, and the percentage of BCL6<sup>+</sup> cells of the gated cDC population indicated. <i>(B)</i> Representative immunofluorescence picture shows the staining pattern of CD11c (red), BCL6 (green), B220 (blue), and MOMA-1 (grey). Solid white arrows identify CD11c<sup>+</sup> cells expressing BCL6 with higher intensity, and open white arrows indentify CD11c<sup>+</sup> cells expressing BCL6 with lower intensity. <i>(C)</i> Mean fluorescence intensity (MFI)±SEM (n = 4) of CD11c (left panel) and I-A/E (right panel) in either BCL6<sup>hi</sup> (solid bar) or BCL6<sup>lo</sup> (open bar) cDCs. <i>(D)</i> BM and splenic pre-cDCs, spleen cDCs, and naïve B cells were sorted from naïve C57BL/6 mice; control GC B cells and T follicular helper (T<sub>FH</sub>) cells were sorted from immunized mice as described in the methods section. Bcl6 transcript levels were determined by quantitative RT-PCR. Normalized expression is relative to the corresponding β-actin control. Bar Graph shows the relative expression ± SEM (n = 3) of Bcl6 in different DC populations and control cells. *<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.001 (students T-test) as compared to Bcl6 transcripts in Naïve B cells.</p
LPS injection dynamically affects BCL6 expression, preferentially in splenic CD8α<sup>+</sup> cDCs.
<p>C57BL/6 mice were injected with TLR4 agonist LPS by <i>i.p.</i> route. <i>(A)</i> After LPS injection, representative FACS data (n = 4) showing transient segregation of two subpopulations of total cDCs. <i>(B)</i> The average percent ±SEM (n = 4) BCL6<sup>hi</sup> in splenic CD11c<sup>hi</sup> I-A<sup>int</sup> (balck solid bar) or CD11c<sup>int</sup> I-A<sup>hi</sup> (black open bar) cDCs before and after LPS injection. <i>(C)</i> The average BCL6 MFI ±SEM (n = 4) in splenic CD11c<sup>hi</sup> I-A<sup>int</sup> (balck solid bar) or CD11c<sup>int</sup> I-A<sup>hi</sup> (black open bar) cDCs before and after LPS injection. <i>(D)</i> Bar graph showing the average MFI ±SEM (n = 4) of BCL6 in cDC subsets (CD4<sup>+</sup>, CD8α<sup>+</sup> and CD4<sup>−</sup>CD8α<sup>−</sup>) of total splenic cDCs at 0 h, 2 h, 12 h, 24 h and 72 h after LPS injection. <i>(E)</i> and <i>(F)</i> The average MFI ±SEM (n = 4) of BCL6 in CD4<sup>+</sup>, CD8α<sup>+</sup> and CD4<sup>−</sup>CD8α<sup>−</sup> subsets within either CD11c<sup>hi</sup> I-A<sup>int </sup><i>(C)</i> or CD11c<sup>int</sup> I-A<sup>hi </sup><i>(D)</i> cells at indicated hours after LPS injection. *<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.001 (two way ANOVA) with multiple comparisons was made between groups <i>(B and C)</i> or within each group compared to values at 0 h <i>(B, C, D, E and F)</i>.</p
BCL6 protein levels correlates with the proliferation marker Ki-67.
<p>Splenic cDCs were further analyzed for intracellular BCL6 and Ki-67 expression. <i>(A)</i> Co-expression of BCL6 and Ki-67 in splenic total cDCs (right plot). Expression was gated based on staining of mouse IgG1 and Rabbit IgG isotype controls (left plot). <i>(B)</i> Bar Graph shows the MFI of Ki-67± SEM (n = 4) in BCL6<sup>hi</sup> (solid bar) or BCL6<sup>lo</sup> (open bar) cDCs. **<i>p</i><0.005 (Students T-test).</p
BCL6 downregulation in CD11c<sup>int</sup> I-A<sup>hi</sup> cells is correlated to reduced proliferation.
<p>Splenocytes or LN cells obtained before and after Alum/CFA inoculation were stained with BCL6, Ki-67 and cDC markers. <i>(A)</i> Representative FACS plots (n = 4) showing Ki-67 and BCL6 expression on gated either CD11c<sup>hi</sup> I-A<sup>int</sup> or CD11c<sup>int</sup> I-A<sup>hi</sup> splenic cDCs at day 0, 2 and 7 after Alum i.p. <i>(B)</i> The average MFI of Ki-67±SEM (n = 4) in either CD11c<sup>hi</sup> I-A<sup>int</sup> (solid black bar) or CD11c<sup>int</sup> I-A<sup>hi</sup> (open bar) splenic cDCs. <i>(C)</i> and <i>(D)</i> Representative flow cytometry data (n = 4) showing Ki-67 and BCL6 expression in CD11c<sup>hi</sup> I-A<sup>int</sup> or CD11c<sup>int</sup> I-A<sup>hi</sup> cells of mesenteric LNs under steady state <i>(C)</i> or of draining popliteal LNs two days post CFA injection <i>(D)</i>. *<i>p</i><0.05, ***<i>p</i><0.001 (Two way ANOVA) was compared either as indicated or with its counterpart.</p
LPS-induced rapid reduction of BCL6 levels does not occur in MyD88<sup>−/−</sup> TRIF<sup>−/−</sup> mice.
<p>WT or MyD88<sup>−/−</sup> TRIF<sup>−/−</sup> mice were <i>i.p.</i> injected with LPS. <i>(A)</i> Representative FACS plots (n = 4) showing CD11c and I-A/E expression on splenic cells in 0 h or 24 h post-LPS injected mice. <i>(B)</i> and <i>(C)</i> The MFI±SEM (n = 4) of CD11c <i>(B)</i> and CD86 <i>(C)</i> in gated splenic CD11c<sup>int</sup> I-A<sup>hi</sup> cells obtained from either WT or MyD88<sup>−/−</sup> TRIF<sup>−/−</sup> mice 0 h (black bar) or 24 h after (grey bar) LPS injection. <i>(D)</i> and <i>(E)</i> The average MFI ±SEM (n = 4) of BCL6 in CD11c<sup>hi</sup> I-A<sup>int </sup><i>(D)</i> and CD11c<sup>int</sup> I-A<sup>hi </sup><i>(E)</i> cells 0 h (black bar) and 24 h after (grey bar) LPS injection. ***<i>p</i><0.001 (two way ANOVA) was compared within each group.</p
BCL6 is transiently downregulated in CD11c<sup>int</sup> I-A<sup>hi</sup> subpopulation of cDCs in secondary lymphoid organs after Alum or CFA injection.
<p>C57BL/6 mice were immunized with Alum by intraperitoneal injection or with CFA by footpad injection. <i>(A)</i> The average percent ±SEM (n = 4) BCL6<sup>hi</sup> in splenic total cDCs (CD11c<sup>+</sup> I-A<sup>+</sup>) before and after Alum injection. <i>(B)</i> By D2 after immunization, representative FACS data shows splenic CD11c<sup>hi</sup> I-A<sup>int</sup> and CD11c<sup>int</sup>I-A<sup>hi</sup> subpopulations (gated on single live CD19<sup>−</sup> TCRβ<sup>−</sup> cells), which are absent in unimmunized mice, and their CD86 expression (n = 4). CD11c<sup>hi</sup> I-A<sup>int</sup> (grey line) and CD11c<sup>int</sup> I-A<sup>hi</sup> (black line). <i>(C)</i> The average percent ±SEM (n = 4) BCL6<sup>hi</sup> in splenic cDC subpopulations before and after alum injection. <i>(D)</i> The average BCL6 MFI ±SEM (n = 4) in splenic cDC subpopulations before and after alum injection. <i>(E)</i> The average BCL6 MFI ±SEM (n = 4) in LN cDC subpopulations before and after CFA footpad injection. <i>(F)</i> The average BCL6 MFI ±SEM (n = 4) in cDC subpopulations of mesenteric LNs under steady state. CD11c<sup>hi</sup>I-A<sup>int</sup> (solid bar) and CD11c<sup>int</sup>I-A<sup>hi</sup> (open bar); One way and two way ANOVA multiple comparasions were used (within and between cDC subpopulation), *<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.001 (ANOVA) was compared either as indicated or with their counterparts.</p