Changes in transcription patterns in BoMac cells in response to PDD spirochetes
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ABSTRACT:
ORGANISM(S): Bos taurus
PROVIDER: GSE6702 | GEO | 2007/06/22
SECONDARY ACCESSION(S): PRJNA99015
REPOSITORIES: GEO
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Project description:Papillomatous digital dermatitis (PDD) is a polymicrobial infection in soft tissue adjacent to the hoof and is the leading cause of lameness in dairy cattle. Treponema phagedenis-like (TPL) spirochetes are a constant feature of PDD lesions and are localized deep in infected tissue. Host-cell response mechanisms to TPL bacteria are poorly understood. To assess how bovine macrophages respond to cellular constituents of TPL spirochetes, changes in transcription were analyzed using serial analysis of gene expression (SAGE) and real time RT-PCR. This analysis revealed that bovine macrophages responded to stimulation with TPL-lysates by induction of several pro-apoptotic genes. Although several proinflammatory cytokines (eg. IL-6 and IL-8) are induced in treated macrophages, receptors and their accessory proteins for IL-1, IL-6, and IL-11 are either down regulated or unchanged. Surprisingly, IL-1beta is not induced and IL-18 is suppressed in treated macrophages. Two genes encoding proteins having negative effects on NF?B, I?B and Siva-1, are significantly induced in stimulated cells. Combined, these data suggest that the innate immune response of bovine macrophages exposed to TPL cellular constituents are impaired thereby enabling bacteria to resist clearance and induce lesion formation. Keywords: Bovine macrophage, transcription response, spirochete bacteria An immortalized bovine macrophage cell line (BoMac) was used throughout this study (supplied by Dr. Judith Stabel, USDA, ARS, NADC, Ames, IA). Cells were propagated in RPMI 1640 medium (GIBCO, Gaithersburg, MD) containing 10% fetal calf serum (Colorado Serum Company), 100µg/ml streptomycin, and 100U/ml penicillin (Sigma, St. Louis, MO) at 37C with 5% CO2. PDD spirochetes (Treponema phagedenis-like strain 1A) in the exponential phase of growth were harvested by centrifugation (10,000g for 15 min.) and washed twice with PBS. Cell lysates were prepared by sonication using short-wave pulses of 30s each (Vibra Cell, Sonics and Materials Inc, Danbury, Connecticut) until no intact cells were observed by dark field microscopy. Protein concentrations of whole cell sonicates (WCS) were determined using the Bradford assay (Bio-Rad, Hercules, CA) following the manufacturers instructions. Bovine macrophages grown to about 90% confluence were stimulated with WCS lysate of TPL strain 1A (1 µg/ml) suspended in complete RPMI 1040. Cells were incubated at 37oC for 4, 8, and 18 hrs. Cell monolayers were disrupted with RLT+ lysis solution and total cellular RNA isolated with RNeasy kits (Qiagen) according to the manufacturer’s instructions. Where appropriate cell suspensions were further disrupted by homogenization using Qiashredder columns (Qiagen). Residual DNA was removed by DNaseI treatment, followed by RNA purification (Qiagen). Cellular mRNA was enriched from total RNA using PolyATract mRNA isolation system II (Promega, Madison, WI) and used as starting material for preparation of SAGE libraries. Two SAGE libraries were prepared from 5 ug ds-cDNA, one each from non-stimulated and WCS-stimulated macrophages (4 hr treatment) following established protocols using NlaIII as the anchoring enzyme. Concatermerized ditags were size selected by electrophoresis through polyacrylamide gels, then ligated with SphI-digested pZERO-1 (Invitrogen Corp.) and used to transform E. coli to zeocin resistance. Transformed colonies were picked at random to 96-well plates and plasmid DNA prepared by alkaline lysis (Qiagen). Plasmid DNA was sequenced unidirectionally from the T7 primer region using fluorescent dideoxy chain-termination reactions separated in a 3700-sequence analyzer (Applied Biosystems, Inc.). Sequence data interpreted using Phred and Sequencher (Gene Codes Corporation, Ann Arbor, MI) were combined and edited manually. Inserts from a total of 2155 and 2131 plasmids were sequenced with average error rates of 0.00435 and 0.00977 from control and stimulated cell libraries, respectively. Data files for each library were processed with eSAGE software to extract ditags, remove duplicate sequences, and compare the two libraries to determine statistically significant differences in tag frequency. SAGE data were validated by quantifying transcription levels of selected genes using RNA isolated from three distinct biological replicates each of treated and control samples. Semiquantitative RT-PCR was done in a DNA Engine Opticon 2 detection system (MJ Research, Inc) using QuantiTect™ SYBR Green PCR Kit (Qiagen), gene specific primers (Table 1), with 200 ng/µl cDNA template. PCR primers were designed using Primer Design 5 software (Sci Ed Central) from sequences obtained from GenBank. All reactions were performed in triplicate and qRT-PCR data were analyzed by using the 2-(??Ct) method. The ?-actin gene was used as the control gene (?Ct) and non-stimulated samples were used as the calibrator (??Ct).
2007-06-22 | E-GEOD-6702 | biostudies-arrayexpress
Project description:Tumor Progression Locus 2 (TPL-2) kinase mediates Toll-like Receptor (TLR) activation of ERK1/2 and p38-alpha MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed bacteria. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Mass spectrometry analysis revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 was shown to stimulate the phosphorylation of DMXL1, a critical regulator of V-ATPases, to induce phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification, activation of phagosome acid-sensitive cathepsins and the efficient killing of Staphylococcus aureus following phagocytic uptake by macrophages. These results indicate that TPL-2 controls the innate immune response of macrophages to bacteria via MAP kinase regulation of gene expression and V-ATPase induction of phagosome maturation.
2021-02-11 | PXD020401 | Pride
Project description:Tumor Progression Locus 2 (TPL-2) kinase mediates Toll-like Receptor (TLR) activation of ERK1/2 and p38 MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a critical regulator of V-ATPases, to induce phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome maturation and the efficient killing of Staphylococcus aureus following phagocytic uptake by macrophages. These results indicate that TPL-2 controls the innate immune response of macrophages to bacteria via V-ATPase induction of phagosome maturation.
2021-03-24 | PXD020247 | Pride
Project description:IL-4 drives expansion of Th2 cells that cause generation of alternatively activated macrophages (AAMs). Filarial infections are established early in life, induce increased IL-4 production are co-endemic with tuberculosis (TB). We sought to understand, therefore, how mycobacteria are handled in the context of IL-4-induced AAM. Comparing IL-4 generated in vitro monocyte derived human AAMs to LPS and IFN-γ generated classically macrophages (CAMs), both infected with mycobacteria (BCG), we demonstrated increased early BCG uptake and increased IL-10 production in AAMs compared to CAMs. We further demonstrated that increased IL-10 production is mediated by upregulation of tumor progression locus 2 (TPL-2), an upstream activator of extracellular signal related kinases (ERKs) in AAMs but not in CAMs, both at the transcript as well as the protein level. Pharmacologic inhibition of TPL-2 significantly diminished IL-10 production only in BCG-infected AAMs. Finally, we validated our findings in an in vivo C57Bl/6 model of filarial infection, where an exaggerated Th2 induced lung-specific alternative activation led to TPL-2 and IL-10 upregulation on subsequent TB infection. These data show that in response to mycobacterial infection, IL-4 generated AAMs in chronic filarial infections have impaired immune responses to TB infection by increasing IL-10 production in a TPL-2 mediated manner. Experimental rationale (microarray): To understand the effect of a pre-existing filiarial infection on the immune response of mice to Mycobacterium tuberculosis
2017-06-17 | GSE100105 | GEO
Project description:Analysis of Mtb infected murine macrophages derived from C57Bl/6 WT, TPL2KO, IFNARKO & TPL2IFNAR DKO mice [Set 1] Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality and morbidity worldwide, causing approximately 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL?12, IL?1 and TNF??, as well as IFN?? and CD4+ Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I interferon have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL?10 is known to inhibit the immune response to Mtb in murine models through the negative regulation of key pro-inflammatory cytokines and the subsequent Th1 response. We show here, using a combination of transcriptomic analysis, genetics and pharmacological inhibitors that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I interferon production. The TPL-2-ERK1/2 signalling pathway regulated production by macrophages of several cytokines important in the immune response to Mtb as well as regulating induction of a large number of additional genes, many in a type I IFN dependent manner. In the absence of TPL-2 in vivo, excess type I interferon promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I interferon may promote susceptibility to this important disease. Macrophages were derived from C57Bl/6 bone marrow, plated and infected with Mtb H37Rv (or not) in duplicate wells. Samples were then harvested for RNA at time 0 (uninfected only), 15m, 30m, 1hr, 3hr, 6hr and 24hr.
2013-09-05 | E-GEOD-47672 | biostudies-arrayexpress
Project description:Purpose: To examine differential expression of genes in bovine macrophages under the influence of Toll-like receptor agonists. Goal: to study and understand the early phase of transcriptomic changes induced by Toll-like receptor in bovine macrophage cell line.
2018-12-22 | GSE106843 | GEO
Project description:Purpose: To examine differential expression of miRNA in bovine macrophages under the influence of Toll-like receptor agonists. Goal: to study and understand the miRNA regulation of early phase of transcriptomic changes induced by Toll-like receptor in bovine macrophage cell line.
2020-11-30 | GSE111162 | GEO
Project description:Analysis of Mtb infected murine macrophages derived from C57Bl/6 WT, TPL2KO, IFNARKO & TPL2IFNAR DKO mice [Set 2] Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality and morbidity worldwide, causing approximately 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL‐12, IL‐1 and TNF‐α, as well as IFN‐γ and CD4+ Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I interferon have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL‐10 is known to inhibit the immune response to Mtb in murine models through the negative regulation of key pro-inflammatory cytokines and the subsequent Th1 response. We show here, using a combination of transcriptomic analysis, genetics and pharmacological inhibitors that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I interferon production. The TPL-2-ERK1/2 signalling pathway regulated production by macrophages of several cytokines important in the immune response to Mtb as well as regulating induction of a large number of additional genes, many in a type I IFN dependent manner. In the absence of TPL-2 in vivo, excess type I interferon promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I interferon may promote susceptibility to this important disease. Macrophages were derived from C57Bl/6 WT, TPL2KO, IFNARKO & TPL2IFNARKO bone marrow, plated and infected with Mtb H37Rv (or not) in triplicate wells. The indicated samples were also treated with 5ng/ml IFNgamma at the time of infection. Samples were then harvested for RNA at time 0, 1hr and 6hr. Please note that *Sample and *Medium control samples (e.g. '0hr TPL2-/-IFNabR-/- Sample' vs '0hr TPL2-/-IFNabR-/- Medium Control Sample') are the same sample group (i.e. uninfected) with one group being a repeat re-run of the other (indicated in the correponding SAMPLE description field).
2013-09-05 | E-GEOD-47673 | biostudies-arrayexpress
Project description:Changes in transcription patterns in BoMac cells in response to PDD spirochetes
| PRJNA99015 | ENA
Project description:Comparison of Tpl-2 small molecule inhibitor (SMI) versus MEK SMI activities in LPS-treated human monocytes will reveal a subset of genes that require Tpl-2 but are independent of MEK. Tpl-2 is a highly conserved (94% human versus mouse) serine-threonine kinase expressed in cells important to the inflammatory response, including monocytes, macrophages, dendritic cells, and B/T cells. The role of Tpl-2 in monocytes and macrophages has been especially well-studied. It has been shown in these cells that Tpl-2 is required for the expression of cytokines in response to Toll-like receptor ligands, including LPS. In resting cells, Tpl-2 forms a complex with p105 and ABIN-2. Upon stimulation, this complex dissociates. Dissociated p105 is processed to p50, which impacts transcription. Dissociated Tpl-2 phosphorylates MEK, which, in turn, phosphorylates ERK. ERK activates the transcription factor AP-1 and its downstream gene targets. An open question in the field is whether Tpl-2 acts solely through MEK to drive gene expression, or does Tpl-2 have any MEK-independent targets. Answering this question is important both for understanding basic Tpl-2 biology as well as its role in disease. Based on published data, Tpl-2 is important for inflammatory cytokine production, and in animal models where these cytokines contribute to disease (septic shock, IBD), blocking Tpl-2 ameliorates disease symptoms. We have a highly selective SMI of Tpl-2 that effectively blocks cytokine production in purified human monocytes. This SMI can block production of the same cytokines that a MEK inhibitor blocks, which is expected given that Tpl-2 lies upstream of MEK. However, the MEK SMI only partially inhibits certain cytokines, while the Tpl-2 SMI fully blocks them, suggesting there are additional factors downstream of Tpl-2 that are not MEK-dependent.
2019-07-01 | GSE84161 | GEO