Early p38 Activation Regulated by MKP-1 Is Determinant for High Levels of IL-10 Expression Through TLR2 Activation

Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. It has been suggested that depending on the nature of the pathogens, TLRs activation induce a distinct cytokine profile that may contribute to the polarization of the acquired immune response. Here, we investigated the early MAPK signaling activation via TLR4 and TLR2 receptors and its impact in differential cytokine profile by macrophages. We found that TLR2 ligands activated MAPKs p38 and ERK earlier compared to the TLR4 ligand LPS in macrophages. Higher IL-10/IL-12 and IL-10/TNF-α ratios were also observed at later time points in response to TLR2 ligands compared to LPS. The results also indicate an earlier activation of the phosphatase MKP-1 and that MKP-1 KO macrophages show a prolongation in p38 phosphorylation in response to TLR2 stimulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production, likely as a virulence strategy to suppress host Th1 response against certain types of pathogens.

TLR2 on CD4+ and CD8+ T cells promotes late control of Mycobacterium tuberculosis infection

Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout (ko) mice is due to its role in macrophages, on T cells or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-g, TNF-a) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells did not affect early control but did result in decreased late control of Mtb in the lungs of infected mice. This suggests that T cell co-stimulation by mycobacterial TLR2 ligands in vivo is important for control of infection during the chronic phase of Mtb infection in the lung.

Time-lapse monitoring of TLR2 ligand internalization with newly developed fluorescent probes

Bacterial lipopeptide fluorescent probes were developed as TLR2 ligands, and their time-lapse monitoring of cellular internalization was performed.

Heterodimer-Specific Stimulation Of Toll-Like Receptor 2 Induces Divergent Downstream Effects In Primary Samples Of Precursor B Cell Acute Lymphoblastic Leukemia

Introduction Reports of spontaneous acute lymphoblastic leukemia (ALL) remissions following severe bacterial infections or administration of Coley's bacteria extract intriguingly suggest that bacterial lipopeptides may trigger effective immune-mediated eradication of ALL. Coley's extract is now known to have delivered a “perfect storm of Toll-like receptor (TLR) agonists” providing potent immune-stimulation. Considerable preclinical and clinical data verified strong anticancer effects for purified TLR ligands. To date, however, TLR2 ligands remain the least studied despite several unique characteristics of TLR2: i) recognition of most bacterial and other pathogens through hetero-dimerization with TLR1 or 6 (all surface-localized, easily ligand-accessible, consistently expressed on most immune cells); ii) mediation of direct cytotoxicity against malignant cells; and iii) distinction of healthy from damaged self by recognizing endogenous ligands from damaged cells. Building on work previously presented at ASH, the aim of this study was to investigate the effects of TLR2 stimulation on primary ALL and to delineate whether heterodimer-specific TLR2 stimulation resulted in divergent functional downstream effects that could contribute to the generation of anti-leukemia immune activity. Methods Pam3CSK4 (Pam3) and Pam2CSK4 (Pam2) are two well-characterized heterodimer-specific synthetic TLR2 ligands not known to cause overwhelming sepsis in vivo. Recently, their exact binding properties were identified by crystallography, offering a reliable in-vitro model to study the potentially divergent functional downstream effects of TLR2/1 (Pam3) vs TLR2/6 (Pam2) stimulation. We investigated whether both TLR2 ligands a) modulate CD40 expression and b) induce death in 4 primary ALL samples and 4 ALL cell-lines. Furthermore, using the best responding cell-line, we investigated which downstream signaling pathways were activated by either ligand. Results TLR1, 2 and 6 expression was confirmed on all primary ALL and ALL cell lines. As shown in Table 1, all 4 primary ALL samples were highly responsive to stimulation with TLR2 ligands as both Pam2 and Pam3 strongly upregulated CD40 expression. However, only Pam3 induced death of leukemic blasts. A similar response pattern was observed with cell lines, albeit stronger and more consistent in primary ALL. Antibody blockade of TLR2 abrogated Pam2-mediated CD40 expression. Blocking of both TLR2 and TLR1 was required to reduce Pam3-mediated CD40 upregulation. Pam3-induced cell death was identified as caspase-mediated apoptosis without ROS activation. Both Pam2 and Pam3 strongly induced NFkB and PI3K pathways, though with distinct signaling kinetics that may underlie their divergent effects on ALL blasts. Conclusions Our results reveal that synthetic TLR2 ligands are potent stimulators of primary ALL blasts and we demonstrate that heterodimer-specific stimulation resulted in distinct functional downstream responses not previously reported. Close association of danger signals with dying cells critically shapes the immune response: although both TLR2 ligands stimulate ALL cells, the ability of Pam3 to augment CD40 expression while simultaneously inducing cell death achieves the conditions recognized as necessary for generating potent immunological responses. Our study, therefore, indicates that the TLR2/1 ligand Pam3 possesses significant potential for generating anti-ALL immune activity through its direct effects on leukemic blasts. Disclosures: No relevant conflicts of interest to declare.