Effects of PU.1 knockdown on the histone acetylation and on HDAC recruitment at the <i>Gata3-1b</i> promoter.

<p>Histone acetylation status of the <i>Gata3-1b</i> promoter in BMDCs transfected with either PU.1 siRNA (siPU.1) or its control siRNA (siCTRL) (A), and the acetylation status in BMDCs cultured without (CTRL) or with 10 nM the histone deacetylation inhibitor trichostatin A (TSA) for 3 h (B). Quantification of acetyl-histone H3 at the <i>Gata3-1b</i> promoter was performed by ChIP assay. (C, D) GATA3 mRNA levels in BMDCs cultured with 10 nM trichostatin A for the indicated times (C) or with the indicated trichostatin A concentration for 3 h (D). Effects of HDAC inhibitors (E) and knockdown of HDACs (F) on GATA3 mRNA levels. BMDCs were treated with MS-275 (1 μM, 10 μM), Droxinostat (20 μM, 50 μM), or MC1568 (5 μM, 20 μM) for 6 h (E). Relative mRNA levels (GATA3-1b/GAPDH) were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. (G) The binding degree of HDAC3 on the <i>Gata3-1b</i> promoter was analyzed by a ChIP assay. Open circles, control rabbit IgG binding in control siRNA-transfected cells; closed circles, anti-HDAC3 antibody binding in control cells; open squares, control IgG binding in PU.1 siRNA-transfected cells; closed squares, anti-HDAC3 antibody binding in PU.1 siRNA-transfected cells. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

Effects of PU.1 knockdown on expression of GATAs, 1, 2, and 3.

<p>(A) BMDCs were transfected with either control (siCTRL; open bars) or PU.1 (siPU.1; closed bars) siRNA. After 48 h incubation, relative mRNA levels were determined by quantitative RT-PCR after normalization to GAPDH mRNA. Data are expressed as the ratio of the expression level of respective control siRNA-introduced cells. Results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in two-tailed paired Student’s <i>t</i> test. (B) Aliquots of total proteins (15 μg/lane) of the indicated cells were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. Similar results were obtained in three separate experiments.</p

PU.1 binds to the <i>Gata3-1b</i> promoter.

<p>(A) Upper carton: Schematic drawing of the mouse <i>Gata3</i> gene. Ex, exon; the arrows over exons 1a/1b and that under exon 2, indicate forward and reverse PCR primers, respectively. Lower graph: BMDCs were transfected with either control siRNA (siCTRL) or PU.1 siRNA (siPU.1). Relative mRNA levels (1a/GAPDH or 1b/GAPDH) were determined by quantitative RT-PCR after normalization to GAPDH mRNA and are expressed as the ratio of the expression level of GATA3-1b in control siRNA-introduced cells. (B, C) Quantification of PU.1 or control goat IgG binding to the <i>Gata3-1b</i> promoter in BMDCs (B) or in splenic DCs (C) was performed using a ChIP assay with the series of primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137699#pone.0137699.s001" target="_blank">S1 Table</a>. Binding is expressed as a percentage of the input for each ChIP assay. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

PU.1 knockdown enables GATA3 to transactivate the <i>Il13</i> promoter via affecting histone H3 modification of CGRE.

<p>(A) BMDCs from wild type (WT) or CGRE deletion (ΔCGRE) mice were transfected with either control siRNA (siCTRL) or PU.1 siRNA (siPU.1). After 48 h incubation, the cells were stimulated or not with 1 μg/ml LPS for 6 h. Relative mRNA levels (IL-13/GAPDH) were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test. (B) Quantification of the H3K4me3 degree around CGRE on the <i>Il13</i> promoter was performed by a ChIP assay with anti-H3K4me3 antibody or its control antibody, and a primer set described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137699#pone.0137699.s001" target="_blank">S1 Table</a>. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in another experiment. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

Increased expression of GATA3 is involved in the LPS-inducible IL-13 upregulation in PU.1 knockdown cells.

<p>BMDCs were transfected with the indicated siRNAs. After 48 h incubation, the cells were stimulated with (black bars) or without (white bars) of 1 μg/ml LPS for 6 h. (A, B) Relative mRNA levels of the indicated cytokines were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. Data are expressed as the ratio of the expression level of respective control siRNA-introduced cells without LPS stimulation. (C) IL-13 protein concentrations in the supernatant were measured using ELISA. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

Effects of PU.1 knockdown on Th2 cytokine expression.

<p>BMDCs were transfected with either control (siCTRL) or PU.1 (siPU.1) siRNA and were then incubated for 48 h prior to stimulation with (closed bars) or without (open bars) 1 μg/ml LPS for 6 h. (A) Relative mRNA levels were determined by quantitative RT-PCR after normalization to GAPDH mRNA. Data are expressed as the ratio of the expression level of respective control siRNA-introduced cells without LPS stimulation. (B) IL-13 protein concentrations in the supernatant were measured using ELISA. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

<i>Schistosoma mansoni</i> infection suppresses the growth of <i>Plasmodium yoelii</i> parasites in the liver and reduces gametocyte infectivity to mosquitoes

<div><p>Malaria and schistosomiasis are major parasitic diseases causing morbidity and mortality in the tropics. Epidemiological surveys have revealed coinfection rates of up to 30% among children in Sub-Saharan Africa. To investigate the impact of coinfection of these two parasites on disease epidemiology and pathology, we carried out coinfection studies using <i>Plasmodium yoelii</i> and <i>Schistosoma mansoni</i> in mice. Malaria parasite growth in the liver following sporozoite inoculation is significantly inhibited in mice infected with <i>S</i>. <i>mansoni</i>, so that when low numbers of sporozoites are inoculated, there is a large reduction in the percentage of mice that go on to develop blood stage malaria. Furthermore, gametocyte infectivity is much reduced in mice with <i>S</i>. <i>mansoni</i> infections. These results have profound implications for understanding the interactions between <i>Plasmodium</i> and <i>Schistosoma</i> species, and have implications for the control of malaria in schistosome endemic areas.</p></div

Gametocyte infectivity.

<p>(A) Parasitemia. Female BALB/c mice were each inoculated with 1 x 10⁶ <i>Plasmodium yoelii</i> parasitized erythrocytes intravenously with (n = 4 mice) or without (n = 5) pre-existing <i>Schistosoma mansoni</i> infection. Parasitaemia was determined by microscopic examination on days 3, 4, and 5 post-inoculation; day 3: Student’s two-tailed t-test; **P<0.01, t = -4.906, df = 7; Day5: *P<0.05, t = -2.922, df = 5 (<b>B</b>) Gametocyte density. Gametocyte density was determined on days 3 and 4 post-inoculation of 1 x 10⁶ <i>P</i>. <i>yoelii</i>-parasitized erythrocytes intravenously. Day 3: Student’s two-tailed t-test; **P<0.01, t = 3.813, df = 5; Day 4: **P<0.01, t = 3.608, df = 5. Error bars show the geometric mean with 95% confidence intervals. (C) Percentage of mosquitoes with one or more oocysts present on the midgut eight days post-feeding on infected mice. A minimum of eight mosquitoes were allowed to feed on each individual mouse in the group per day **P = 0.0003, (2-way ANOVA, F = 22.23, DFn = 1, DFd = 14). Error bars mar the standard error of the mean per mouse group. (D) Oocyst numbers per mosquito. The numbers of oocysts present on mosquito midguts were determined eight days post-mosquito feeding; day 3: Student’s two-tailed t-test, **P<0.01, t = 3.077, df = 25. Error bars show the geometric mean with 95% confidence intervals. Data is representative of three independent experiments.</p

Growth of malaria parasites in the liver and blood of mice following SPZ inoculation of <i>Plasmodium yoelii</i> with and without <i>Schistosoma mansoni</i> infection.

<p>(A) Copy number of <i>P</i>. <i>yoelii</i> 18s RNA gene per 1×10⁶mouse G3PDH gene measured at 42 h post SPZ inoculation. Female BALB/c mice (N = 6) infected with 50 <i>S</i>. <i>mansoni</i>-cercariae 10 weeks previously were challenged with 1,500 <i>P</i>. <i>yoelii</i> SPZ along with <i>S</i>. <i>mansoni</i>-non-infected controls. **P<0.01, Student’s two-tailed t-test, t = 4.362, df = 10. (B) Parasitaemia. Blood stage malaria parasites were monitored daily from day 2 to 8 post i.v. inoculation of 500 <i>P</i>. <i>yoelii</i> SPZ. (C) Percentage survival. Data from one representative experiment of three independent repeats are shown.</p

<i>Plasmodium yoelii</i> parasite density in the blood and liver.

<p>Copy number of <i>P</i>. <i>yoelii</i> 18s RNA gene per 1×10⁶ mouse G3PDH gene measured at 42 h post sporozoite inoculation. Female BALB/c mice (N = 5) infected with 50 <i>Schistosoma mansoni</i>-cercaria 10 weeks previously were challenged with 1,500 SPZ of <i>P</i>. <i>yoelii</i> along with <i>S</i>. <i>mansoni</i>-non-infected controls. (A) <i>P</i>. <i>yoelii</i> parasitaemia in the blood. (B) <i>P</i>. <i>yoelii</i> parasite density and proliferation in the liver. ***P<0.001, Student’s two-tailed t-test, t = -6.316, df = 8.</p