Regulation of bovine IL-12R beta 2 subunit mRNA expression in bovine lymph node cells

The beta 2 subunit of the interleukin (IL)-12 receptor (IL-12R beta 2) has been shown to play an essential role in differentiation of T helper 1 (Th1) cells in the murine and human system, and antibodies raised against IL-12R beta 2 recognized this molecule on human Th1 but not Th2 cells. However, while the cytokines secreted by clones of murine cells allowed the definition of distinct T helper cell subsets, bovine clones with polarized Th1 and Th2 cytokine profiles were rarely found. This raised important questions about the regulation of immune responses in cattle. We therefore cloned bovine IL-12R beta2 (boIL-12R beta 2) DNA complementary to RNA (cDNA) from the start codon to the 3' end of the mRNA. Comparison of boIL-12R beta 2 cDNA with human and murine IL-12R beta 2 cDNA sequences revealed homologies of 85 and 78%, respectively. The deduced protein sequence showed the hallmark motifs of the cytokine receptor superfamily including the four conserved cysteine residues, the WSXWS motif and fibronectin domains in the extracellular part as well as a STAT4 binding site in the intracellular part of the molecule. Using real-time reverse transcription-polymerase chain reaction, upregulation of mRNA expression of this molecule could be demonstrated in cultured bovine lymph node cells stimulated with phytohemagglutinin. Furthermore, cells with upregulated boIL-12R beta 2 mRNA responded with enhanced expression of interferon gamma to treatment with interleukin 12

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident

Inverted recruitment of autophagy proteins to the <i>Plasmodium berghei</i> parasitophorous vacuole membrane

<div><p>Selective autophagy and related mechanisms can act as variable defense mechanisms against pathogens and can therefore be considered as intracellular immune responses. When in hepatocytes, <i>Plasmodium</i> parasites reside in a parasitophorous vacuole (PV) and the PV membrane (PVM) is the main contact site between host cell and parasite. Early in infection, the PVM is directly labeled with host cell autophagy proteins LC3B and p62 (nucleoporin 62). We investigated the recruitment of different selective autophagy receptors and could show that mainly p62 and NBR1 (neighbour of BRCA1 gene 1) and to a lesser extent NDP52 (nuclear dot protein 52) associate with the PVM. To investigate the recruitment of these receptors to the PVM in <i>Plasmodium</i>-infected cells, we generated LC3B knock out HeLa cells. In these cell lines, autophagosome formation and autophagic flux are not different to those in WT cells. Unexpectedly, p62 and NBR1 recruitment to the PVM was strongly impaired in LC3B-negative host cells, suggesting that LC3B recruits both receptors to the PVM of <i>Plasmodium</i> parasites. We also noticed that LC3B recruited ubiquitin to the PVM. This indicates that, in comparison to classical selective autophagy, in <i>P</i>. <i>berghei</i>-infected cells the order of membrane labeling with autophagy proteins appears to be inverted from canonical ubiquitin-receptor-LC3B recruitment to LC3B-receptor and possibly ubiquitin.</p></div

LC3B recruits p62 to the PVM.

<p><b>(A)</b> HeLa WT, HeLa LC3B- and ATG5-knockout cells were infected with <i>Pb</i>mCherry (red). 6 hours post-infection, cells were fixed and stained with α-p62 antibodies (green). All cell lines were transfected with RFP-LC3B (two lower panels) and infected 17 hours after transfection with <i>P</i>. <i>berghei</i> sporozoites expressing mCherry (red). p62 (green) was visualised using a specific monoclonal α-p62 antibody. DNA was labeled with DAPI (blue). Cells were analyzed by confocal microscopy. Scale bar 10 μm. <b>(B)</b> Numbers of p62-labeled <i>P</i>. <i>berghei</i> parasites in non-transfected and in RFP-LC3B-transfected cells were determined by fluorescence microscopy. 100–130 parasites were analyzed in non-transfected HeLa cells and 60–120 parasites were analyzed for RFP-LC3B-transfected HeLa cells. Two individual experiments were carried out. Labeled parasites are expressed as percentages. Standard deviations are depicted. <b>(C)</b> Pearson’s correlation coefficient of p62 and RFP-LC3B were calculated for six individual <i>P</i>. <i>berghei</i> parasites in HeLa WT and LC3B^-/- cells transfected with RFP-LC3B. The mean values of 0.762 (HeLa WT) and 0.715 (LC3B^-/-) indicate a strong co-localization of p62 and RFP-LC3B. Depicted are standard deviations.</p

Generation of LC3B knockout HeLa cell lines.

<p><b>(A)</b> Genomic region of the <i>LC3B</i> gene. Exon 1 is shown in lowercase blue, the 5’-upstream region is printed in black capitals. Binding regions of the two gRNAs are highlighted in green and PAM sequences are shown in red. <b>(B)</b> Western blot of HeLa WT and three LC3B knock out cell lines to confirm the lack of LC3B protein in three clonal cell lines. GAPDH was used as a loading control. <b>(C)</b> IFA analysis of HeLa WT and three LC3B knock out cell lines. Cells were starved for 2 hours in EBSS, fixed, stained with anti-LC3B antibodies (green) and analysed by fluorescence microscopy. DNA was visualised with DAPI (blue). Scale bar 20 μm.</p

LC3B recruits ubiquitin to the PVM.

<p><b>(A)</b> HeLa WT, HeLa LC3B- and ATG5-knockout cells were infected with <i>Pb</i>mCherry (red). 6 hours post-infection, cells were fixed and stained with anti-ubiquitin antibodies (green) as a control. Control and knockout cell lines were transfected with RFP-LC3B (two lower panels) and infected 17 hours after transfection with <i>P</i>. <i>berghei</i> sporozoites expressing mCherry (red). Ubiquitin (green) was visualised using anti-ubiquitin antibodies. DNA was labeled with DAPI (blue). Cells were analyzed by confocal microscopy. Scale bar 10 μm. <b>(B)</b> Numbers of ubiquitin-labeled <i>P</i>. <i>berghei</i> parasites in non-transfected and in RFP-LC3B-transfected cells were determined by fluorescence microscopy. 77–103 parasites were analysed in the non-transfected HeLa cells and 53–83 parasites were analysed for the RFP-LC3B-transfected HeLa cells. Two individual experiments were carried out. Labeled parasites are expressed as percentages. Standard deviations are depicted. <b>(C)</b> Pearson’s correlation coefficients of ubiquitin and RFP-LC3B were calculated from seven individual <i>P</i>. <i>berghei</i> parasites in HeLa WT and LC3B^-/- cells transfected with RFP-LC3B. Mean values are 0.4829 (HeLa WT) and 0.6871 (LC3B^-/-). Depicted are standard deviations.</p

Autophagy receptors p62 and NBR1 localise to the parasitophorous vacuole membrane of <i>Plasmodium berghei</i>.

<p><b>(A)</b> HeLa cells were infected with <i>Plasmodium berghei</i>. The parasitophorous vacuole membrane (PVM) of the parasite was stained with antibodies (UIS4, red). To visualise the autophagy receptors, cells were stained with antibodies (p62, green) or transfected 24 hours before infection with plasmids expressing GFP fusion proteins (GFP-NBR1, GFP-NDP52, OPTN-GFP, GFP, all shown in green). Infected cells were fixed 6 hours post-infection, stained with antibodies and analysed by confocal microscopy. DNA was stained with DAPI. Scale bar 10 μm <b>(B)</b> Numbers of receptor-labeled <i>P</i>. <i>berghei</i> parasites were determined by fluorescence microscopy. 60–100 parasites were counted in two separate experiments. Numbers of labeled parasites are expressed as percentages, error bars show standard deviations, p values were calculated using a t-test. <b>(C)</b> Pearson's correlation coefficients were calculated from at least 5 images, except for cells expressing GFP only. Standard deviations are depicted, p values were calculated using a t-test.</p

LC3B knockout cells are able to undergo canonical autophagy.

<p><b>(A)</b> Representative western blot of non-infected HeLa WT, ATG5-knockout cells and one clonal LC3B-knockout cell line left untreated or treated simultaneously with 10 μM chloroquine and 250 ng/ml rapamycin for 4 hours. <b>(B)</b> HeLa WT and HeLa LC3B knockout cells ectopically expressing GFP-Gate16 were left untreated or treated with 10 μM chloroquine and 250 ng/ml rapamycin for 4 hours. Fixed cells were stained with anti-GFP antibodies to visualise Gate16 (green) or anti-LC3B antibodies (red). DNA was stained with DAPI (blue). White arrows in the LC3B panel indicate Gate16-transfected cells. Yellow arrowheads in the enlarged pictures indicate autophagic structures where Gate16 and LC3B colocalise. Scale bar 20 μm. Experiments using other clonal LC3B-knockout cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183797#pone.0183797.s001" target="_blank">S1B Fig</a>.</p