Neither Lys- and DAP-type peptidoglycans stimulate mouse or human innate immune cells via Toll-like receptor 2.

Peptidoglycan (PGN), a major component of bacterial cell walls, is a pathogen-associated molecular pattern (PAMP) that causes innate immune cells to produce inflammatory cytokines that escalate the host response during infection. In order to better understand the role of PGN in infection, we wanted to gain insight into the cellular receptor for PGN. Although the receptor was initially identified as Toll-like receptor 2 (TLR2), this receptor has remained controversial and other PGN receptors have been reported. We produced PGN from live cultures of Bacillus anthracis and Staphylococcus aureus and tested samples of PGN isolated during the purification process to determine at what point TLR2 activity was removed, if at all. Our results indicate that although live B. anthracis and S. aureus express abundant TLR2 ligands, highly-purified PGN from either bacterial source is not recognized by TLR2

Lipid modification of contaminating peptides in purified <i>S</i>. <i>aureus</i> PGN is important for TLR2 signaling.

<p>PGN from Newman <i>S</i>. <i>aureus</i> (A) and Newman <i>S</i>. <i>aureus Δlgt</i> (B) was digested with mutanolysin. After digestion samples were run in triplicate for amino acid analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193207#pone.0193207.g002" target="_blank">Fig 2</a>. (C-D) Differentiated BMDMs were stimulated with LPS (1 μg/ml), <i>B</i>. <i>anthracis</i> PGN (10 μg/ml), Newman <i>S</i>. <i>aureus</i> PGN (10 μg/ml), or Newman <i>S</i>. <i>aureus Δlgt</i> PGN (10 μg/ml) in the presence of Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of three independent experiments (solid grey peak is unstimulated sample). Results are mean ± SEM from three independent experiments. The <i>p</i> values were calculated using a one-way ANOVA using Bonferroni post hoc test for multiple comparisons. ^# <i>p</i> < .05 compared to unstimulated control, ** <i>p</i> = .001, **** <i>p</i> < .0001.</p

Amino acid analysis of <i>S</i>. <i>aureus and B</i>. <i>anthracis</i> and PGN preparations before and after proteinase K digestion.

<p><i>S</i>. <i>aureus</i> amino acids were analyzed before (A) and after (B) Proteinase K digestion. The final <i>S</i>. <i>aureus</i> PGN product was additionally treated with HF, extracted with SDS, and analyzed (C). <i>B</i>. <i>anthracis</i> amino acids were analyzed before (D) and after (E) proteinase K digestion. Two separate PGN preparations for each species were analyzed, with three replicates of each sample. Data are expressed as the mean ± SEM of amino acid content, normalized to lysine for <i>S</i>. <i>aureus</i> PGN and to DAP for <i>B</i>. <i>anthracis</i> PGN.</p

Phosphate content of <i>B</i>. <i>anthracis</i> and <i>S</i>. <i>aureus</i> PGN.

<p><i>B</i>. <i>anthracis</i> (A) and <i>S</i>. <i>aureus</i> (B) PGN samples were taken after the major steps of the purification process, washed three times with endotoxin free water, dried, weighed, resuspended tested for phosphate content. Two separate PGN preparations for each species were tested, with two replicates for each species. Data are expressed as the mean ± SEM of the nmoles of phosphate per μg/PGN. Statistical significance was determined by ANOVA with Bonferroni post test. *p<0.05, **p<0.01, ***p<0.001 <i>versus</i> heat killed bacteria.</p

Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax

TLR2 activity of <i>S</i>. <i>aureus</i> and <i>B</i>. <i>anthracis</i> PGNs.

<p><i>B</i>. <i>anthracis</i> (A) and <i>S</i>. <i>aureus</i> (B) PGN samples were taken after the indicated steps of the purification process. The samples were washed three times with endotoxin free (LAL) water, dried, weighed and tested for TLR2 activity at a concentration of 10 μg/ml, using the HEK-TLR2 cell assay. The data are expressed as the mean ± SEM fold difference TLR2 activity over the negative control, endotoxin free water. Statistical significance was determined by ANOVA with Bonferroni post test. *, **p<0.01, ***p<0.001 <i>versus</i> endotoxin free water. C & D: Freshly prepared peripheral blood mononuclear cells were stimulated for 12 hours with 10 μg/mL <i>B</i>. <i>anthracis</i> (Panel C) or <i>S</i>. <i>aureus</i> (Panel D) PGN samples taken at defined steps during the purification procedure, as described in Panels A & B. The cells were stained for intracellular TNFα and then analyzed by flow cytometry. The internalization requirement was assessed in the presence of 15 μM cytochalasin D (CytoD). Data are expressed as the mean ± SEM of TNFα+CD14+ cells. Statistical significance was determined by paired two-way ANOVA with Bonferroni post hoc test for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ns p>0.05.</p

Mouse macrophage response to <i>B</i>. <i>anthracis</i>- and <i>S</i>. <i>aureus</i>-derived PGN.

<p>(A-B) Differentiated BMDMs from WT or TLR2^-/- mice were plated into wells of a 96-well plate (1.5 x 10⁵ cells per well) and stimulated with LPS (1 μg/ml), <i>B</i>. <i>anthracis</i> PGN (10 μg/ml), or <i>S</i>. <i>aureus</i> PGN (10 μg/ml) in the presence of Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of 3 independent experiments (solid grey peak is unstimulated sample). Results are mean ± SEM from three independent experiments. The <i>p</i> values were calculated using a two-way ANOVA using Bonferroni post hoc test for multiple comparisons. ^# <i>p</i> < .0001 compared to unstimulated control. **** <i>p</i> < .0001 between WT and TLR2^-/- BMDMs.</p

TLR4 activity of <i>B</i>. <i>anthracis</i> and <i>S</i>. <i>aureus</i> PGN.

<p><i>B</i>. <i>anthracis</i> (open bars) and <i>S</i>. <i>aureus</i> (closed bars) PGN samples were taken after the major steps of the purification process, washed three times with endotoxin free water, dried, weighed, resuspended in water and tested for TLR4 activity at a concentration of 10 μg/ml, using the HEK-TLR4 cell assay. Two separate PGN preparations for each species were tested. The data are expressed as the mean ± SEM fold difference TLR4 activity over the negative control, endotoxin free water. Statistical significance was determined by ANOVA with Bonferroni post test. All PGN samples were not significantly different in TLR4 activity than 10 pg/ml LPS.</p

Human monocyte responses to PGN from lipoprotein deficient <i>S</i>. <i>aureus</i> require serum opsonins.

<p>(A) PBMCs were plated into wells of a 96-well plated (4–8 x 10⁵ cells per well) and stimulated with <i>B</i>. <i>anthracis</i> PGN (10 μg/ml) in presence of FCS (1% v/v) with or without normal human serum (1% v/v) and with Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. (B) PBMCs were plated into wells of a 96-well plated (4–8 x 10⁵ cells per well) and stimulated with Newman <i>S</i>. <i>aureus</i> PGN (10 μg/ml), or Newman <i>S</i>. <i>aureus Δlgt</i> PGN (10 μg/ml) in presence of FCS (1% v/v) with or without normal human serum (1% v/v) and with Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of experiments from three donors (solid grey peak is unstimulated sample). Results are mean ± SEM from three donors. The <i>p</i> values were calculated using either a two-way (B) or one-way (D) ANOVA using Bonferroni post hoc test for multiple comparisons. ^# <i>p</i> ≤ .0001 compared to unstimulated control, ** <i>p</i> = .001, ***<i>p</i> = 0001, **** <i>p</i> < .0001.</p