Lipoteichoic Acid Induces Unique Inflammatory Responses when Compared to Other Toll-Like Receptor 2 Ligands

Departments of Oncology, Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Canada.
PLoS ONE (Impact Factor: 3.23). 02/2009; 4(5):e5601. DOI: 10.1371/journal.pone.0005601
Source: PubMed


Toll-like receptors (TLRs) recognize evolutionarily-conserved molecular patterns originating from invading microbes. In this study, we were interested in determining if microbial ligands, which use distinct TLR2-containing receptor complexes, represent unique signals to the cell and can thereby stimulate unique cellular responses. Using the TLR2 ligands, R-FSL1, S-FSL1, Pam2CSK4, Pam3CSK4, and lipoteichoic acid (LTA), we demonstrate that these ligands activate NF-kappaB and MAP Kinase pathways with ligand-specific differential kinetics in murine macrophages. Most strikingly, LTA stimulation of these pathways was substantially delayed when compared with the other TLR2 ligands. These kinetics differences were associated with a delay in the LTA-induced expression of a subset of genes as compared with another TLR2 ligand, R-FSL1. However, this did not translate to overall differences in gene expression patterns four hours following stimulation with different TLR2 ligands. We extended this study to evaluate the in vivo responses to distinct TLR2 ligands using a murine model of acute inflammation, which employs intravital microscopy to monitor leukocyte recruitment into the cremaster muscle. We found that, although R-FSL1, S-FSL1, Pam2CSK4, and Pam3CSK4 were all able to stimulate robust leukocyte recruitment in vivo, LTA remained functionally inert in this in vivo model. Therefore distinct TLR2 ligands elicit unique cellular responses, as evidenced by differences in the kinetic profiles of signaling and gene expression responses in vitro, as well as the physiologically relevant differences in the in vivo responses to these ligands.

9 Reads
  • Source
    • "For example both lipopolysaccharide and Pam2CSK4 at 100 ng/ml (as used in the present study) induced activation of MAP kinase and NF-B signalling pathways and inflammatory cytokine expression in the murine macrophage/ monocyte cell line Raw 264.7, and bone marrow-derived macrophages from C57BL/6 mice (Long et al., 2009). Importantly, the effects of Pam2CSK4 were TLR2 specific as Pam2CSK4 was unable to induce activation of the above signalling pathways in macrophages from Tlr2 À / À mice (Long et al., 2009). However, assessment of a more extensive dose range of the agonists used in the study should be the focus of future experiments to compliment these studies. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Improving neurological outcome after spinal cord injury is a major clinical challenge because axons, once severed, do not regenerate but 'dieback' from the lesion site. Although microglia, the immunocompetent cells of the brain and spinal cord respond rapidly to spinal cord injury, their role in subsequent injury or repair remains unclear. To assess the role of microglia in spinal cord white matter injury we used time-lapse two-photon and spectral confocal imaging of green fluorescent protein-labelled microglia, yellow fluorescent protein-labelled axons, and Nile Red-labelled myelin of living murine spinal cord and revealed dynamic changes in white matter elements after laser-induced spinal cord injury in real time. Importantly, our model of acute axonal injury closely mimics the axonopathy described in well-characterized clinically relevant models of spinal cord injury including contusive-, compressive- and transection-based models. Time-lapse recordings revealed that microglia were associated with some acute pathophysiological changes in axons and myelin acutely after laser-induced spinal cord injury. These pathophysiological changes included myelin and axonal spheroid formation, spectral shifts in Nile Red emission spectra in axonal endbulbs detected with spectral microscopy, and 'bystander' degeneration of axons that survived the initial injury, but then succumbed to secondary degeneration. Surprisingly, modulation of microglial-mediated release of neurotoxic molecules failed to protect axons and myelin. In contrast, sterile stimulation of microglia with the specific toll-like receptor 2 agonist Pam2CSK4 robustly increased the microglial response to ablation, reduced secondary degeneration of central myelinated fibres, and induced an alternative (mixed M1:M2) microglial activation profile. Conversely, Tlr2 knock out: Thy1 yellow fluorescent protein double transgenic mice experienced greater axonal dieback than littermate controls. Thus, promoting an alternative microglial response through Pam2CSK4 treatment is neuroprotective acutely following laser-induced spinal cord injury. Therefore, anti-inflammatory treatments that target microglial activation may be counterintuitive after spinal cord injury.
    Brain 12/2013; 137(3). DOI:10.1093/brain/awt341 · 9.20 Impact Factor
  • Source
    • "PCSK binds to TLR-2/TLR-1 heterodimers and does not interact with TLR-6, unlike other TLR-2 agonists [30]. Long et al [31] compared effects of four different TLR-2 agonists, including PCSK. Though all tested TLR-2 ligands were shown to activate NFκB and MAPK signalling pathways in murine macrophages, the degree of activation differed among the tested compounds. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation). In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life.
    PLoS ONE 06/2013; 8(6):e66897. DOI:10.1371/journal.pone.0066897 · 3.23 Impact Factor
  • Source
    • "IκBα was partially degraded by 15 minutes and remained at low levels throughout the 8-hour time period, except for a brief return to pre-stimulation levels at the 1 hour time point. To our knowledge, such a prolonged absence of cytoplasmic IκBα has not been observed after stimulation with any other TLR2 ligand, including the triacylated lipopeptide, Pam3CSK4 (TLR1/TLR2) or the diacylated lipopeptides, Pam2CSK4 (TLR2/TLR6), S-FSL-1 (TLR2/TLR6) and R-FSL1 (TLR2/TLR6/CD-36) [44]. Hb activation also resulted in IκBα levels that were lower than pre-stimulatory levels at some time points. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-β mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.
    PLoS ONE 10/2012; 7(10):e47333. DOI:10.1371/journal.pone.0047333 · 3.23 Impact Factor
Show more

Preview (3 Sources)

9 Reads
Available from