19 research outputs found
TLR2 ligands can selectively potentiate IFN-β production induced via TLR4.
<p>(A–C) Primary peritoneal macrophages were treated overnight with media or the indicated TLR ligands, and then re-stimulated with <i>E. coli</i> LPS (100 ng/ml) for 4 hrs (A and B) or 2 hrs (C) and gene expression for IL-6 (A), IL-12 p40 (B), or IFN-β (C) was analyzed by qRT-PCR. (D) Primary peritoneal macrophages were treated overnight with media or Pam3Cys or Pam2Cys (250 ng/ml) and re-stimulated after overnight incubation with <i>E. coli</i> LPS (100 ng/ml) for 2 hrs before extraction of RNA and analysis of gene expression. (E) Cell supernatants from macrophages treated as in (A–C) were analyzed for IL-12 p40 and IFN-β protein by ELISA. (F) Peritoneal macrophages were treated overnight with media or Pam3Cys (250 ng/ml) and re-stimulated either immediately with LPS for 2 hrs or following an additional 24, 48, or 72 hr resting period. Experiments were performed at least three times. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003479#s2" target="_blank">Results</a> from a single representative experiment are shown.</p
Gram positive bacteria and select viruses, but not Gram negative bacteria, can prime for TLR- and RLR-driven IFN-β production.
<p>(A–C) Primary peritoneal macrophages were incubated overnight with media alone or with the indicated heat killed bacteria (moi = 1). Following treatment, macrophages were washed 3X with PBS and re-stimulated either with <i>E coli</i> LPS (100 ng/ml) for 2 hours, or transfected with poly I:C (1 µg/ml) for 6 hours. (D) Macrophages were incubated with media or UV inactivated Vaccinia Virus (moi = 0.5) overnight and re-stimulated with E. coli LPS (100 ng/ml) for 2 hours or transfected with poly I:C for 6 hours. (E) BALB/cJ mice were injected i.p. with saline or heat-killed <i>S. aureus</i> 24 hours prior to i.n. infection with x10<sup>7</sup> pfu of VSV. N = 12 mice per group. Experiments were performed three times with a representative experiment shown for each.</p
TLR2 enhances RLR expression and signaling.
<p>(A) Primary peritoneal macrophages were treated overnight with media alone or 250 ng/ml Pam3Cys and were analyzed by Western blot for expression of RLR family members. (B–D) Macrophages treated with media or Pam3Cys overnight were transfected with HMW poly I:C, (B) LMW poly I:C, (C) or non-transfected extracellular poly I:C (D) and analyzed for IRF3 activation by Western blot. (E) Cells treated as in (B) were used to examine gene expression by qRT-PCR. (F) Cells treated as in (C) were used to examine gene expression by qRT-PCR. (G) Cells treated as in (D) were used to examine gene expression by qRT-PCR. Experiments were performed three times with a representative experiment shown for each.</p
Reprogramming of Murine Macrophages through TLR2 Confers Viral Resistance via TRAF3-Mediated, Enhanced Interferon Production
<div><p>The cell surface/endosomal Toll-like Receptors (TLRs) are instrumental in initiating immune responses to both bacteria and viruses. With the exception of TLR2, all TLRs and cytosolic RIG-I-like receptors (RLRs) with known virus-derived ligands induce type I interferons (IFNs) in macrophages or dendritic cells. Herein, we report that prior ligation of TLR2, an event previously shown to induce “homo” or “hetero” tolerance, strongly “primes” macrophages for increased Type I IFN production in response to subsequent TLR/RLR signaling. This occurs by increasing activation of the transcription factor, IFN Regulatory Factor-3 (IRF-3) that, in turn, leads to enhanced induction of IFN-β, while expression of other pro-inflammatory genes are suppressed (tolerized). <i>In vitro</i> or <i>in vivo</i> “priming” of murine macrophages with TLR2 ligands increase virus-mediated IFN induction and resistance to infection. This priming effect of TLR2 is mediated by the selective upregulation of the K63 ubiquitin ligase, TRAF3. Thus, we provide a mechanistic explanation for the observed antiviral actions of MyD88-dependent TLR2 and further define the role of TRAF3 in viral innate immunity.</p></div
TLR2 selectively potentiates TLR4 driven transcription factor activation.
<p>(A) and (B) Primary peritoneal macrophages were treated overnight with media alone or with Pam3Cys (250 ng/ml) and then re-stimulated with <i>E. coli</i> LPS (100 ng/ml) for 0, 20, 40, or 60 min before harvesting cell lysates for Western analysis with indicated antibodies. (C–E) These experiments were carried out as in (A) with the indicated time-course of LPS re-stimulation. (F) Cells were treated overnight as in (A) and then re-stimulated for 60 min with LPS. Nuclear lysates were harvested and used for Western analysis with indicated antibodies. (G) Nuclear lysates from cells treated overnight as indicated and then re-stimulated with LPS were used in EMSA. Where indicated, antibodies were added to reaction to super-shift complexes. Experiments were performed at least three times and the results of a representative experiment are shown.</p
TLR2 ligation enhances <i>in vitro</i> and <i>in vivo</i> viral resistance and IFN-β production.
<p>(A) WT C57BL/6 or IFN-β<sup>−/−</sup> primary peritoneal macrophages were treated overnight with media alone or with 250 ng/ml Pam3Cys and subsequently infected with the indicated MOI of Vesicular Stomatitis Virus. After 24 hrs of infection, adherent monolayers were stained with crystal violet. (B) (left panel) Supernatant from triplicate wells of macrophages infected with VSV (MOI = of 10) for 24 hours were harvested and viral titer was determined by plaque titration on BHK cells. (right panel) Trypan Blue was used to stain macrophages infected with VSV (MOI = 50) for 24 hrs. A minimum of 10 fields per well were counted. (C) Macrophages were pretreated with media alone or Pam3Cys for 24 hrs and subsequently infected with indicated MOI of VSV and harvested after six hrs for gene expression analysis. (D) Macrophages were pretreated with media alone or Pam3Cys for 24 hrs and subsequently infected with influenza strain PR8 at an MOI = 1 for 6 hrs and harvested for gene expression analysis. (E) Macrophages were pretreated with media alone or with Pam3Cys for 24 hrs and subsequently infected with Vaccinia Virus (WR strain) at an MOI of 5 for 8 hours prior to harvesting for gene expression analysis (F) WT 6–8 week female BALBc/J mice were injected i.p. with 0.5 ml saline or 100 µg Pam3Cys in saline 24 hrs prior to i.n. infection with 1×10<sup>6</sup> pfu VSV. N = 12 mice per treatment group (G) WT or TLR2<sup>−/−</sup> mice were infected i.n with 1×10<sup>7</sup> pfu of VSV in saline. N = 12 mice per group. All experiments were performed at least three times and data from a representative experiment is shown for each.</p
TRAF3 expression is elevated by TLR2 priming and over-expression is sufficient to potentiate TLR- and RLR-mediated IRF3 activation.
<p>(A) Primary peritoneal macrophages treated overnight with media alone or 250 ng/ml Pam3Cys were washed 3X with PBS and were harvested at the indicated times and analyzed by qRT-PCR for TRAF3 expression. (B) Macrophages were treated overnight with Pam3Cys (250 ng/ml) and analyzed by Western blot for TRAF3 expression. (C) Peritoneal macrophages were treated overnight with media or Pam3Cys and re-stimulated for 45 min with LPS (300 ng/ml). Cell lysates were immunoprecipitated with antibodies against IRF3 and blotted with antibody against TRAF3. (D and E, left panels) MAT4 HeLa cells transiently transfected with human TRAF3 were stimulated with <i>E. coli</i> LPS (300 ng/ml) for the times indicated and used in Western blot (D) or qRT-PCR (E). (D and E, right panels) MAT4 HeLa cells transiently transfected with human TRAF3 were transfected with Poly I:C and used in Western blotting (D) or qRT-PCR (E). (F) Peritoneal macrophages carrying a TRAF3 floxed gene and expressing CRE recombinase, or TRAF3-sufficient littermate control macrophages, were primed as in (A) and assayed for IFN-β induction or IL-12 p40 induction. All experiments were performed at least three times and data from a representative experiment is shown for each.</p
Enhanced transcription factor occupancy on the IFN-β promoter.
<p>(A) and (B). Primary peritoneal macrophages were treated for 8 hrs with media alone, or with 250 ng/ml Pam3Cys, or Pam3Cys plus 50 nM Trichostatin A (TSA). Following stimulation, cells were washed and allowed to rest overnight, at which time the cells were re-stimulated with 100 ng/ml <i>E. coli</i> LPS for 4 (A) or 2 (B) hrs. Gene expression was analyzed by qRT-PCR. (C) and (D) Macrophages treated overnight with media alone or 250 ng/ml Pam3Cys were re-stimulated with <i>E. coli</i> LPS (250 ng/ml) for 60 min and used in Chromatin Immuno-precipitation (ChIP) assays with antibodies against the indicated transcription factors. Precipitated DNA was amplified using primers directed against a region of the IFN-β promoter. Experiments were performed at least three times and the results were obtained from a single representative experiment.</p
Serotype-independent binding to KP strains by the new antibodies.
<p>Flow cytometry experiment was used to gauge the binding of the four new antibodies against three WT KP strains of different serotype as indicated. KP3 serves as the positive control and R347 is a human IgG<sub>1</sub> isotype control.</p
K<sub>D</sub> measurement in Fab format against monomeric MrkA.
<p>ND, not detectable; N/A, not applicable.</p