Induction of Inflammatory Chemokines: RANTES/CCL5, MCP-1/CCL2 and MIP-1β/CCL4 in Response to Toll-Like Receptor 2 Agonists
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Toll-Like Receptors (TLRs) belong to signaling class of pattern recognition receptors (PRRs) and are involved in the recognition of invariant pathogen associated molecular patterns (PAMPs), which are not found in vertebrates. Upon recognition of PAMPs, TLRs initiate downstream signaling cascades to induce inflammatory cytokines like TNF-α, IL-1β etc., chemokines and type I interferons through activation of transcription factors. TLR signaling is mediated by Toll/interleukin-1 receptor domain (TIR) containing adaptor proteins. All TLRs except TLR3 use Myeloid differentiation factor 88 (MyD88) for downstream signaling. TIR-domain-containing adaptor-inducing interferon-β (TRIF) is another adaptor molecule used by TLR3 and TLR4 for signaling. TLR4 is a unique TLR as it uses both MyD88 and TRIF for downstream signaling. Activation of MyD88 pathway induces inflammatory cytokines like TNF-α and IL-1β, while TRIF typically activates the transcription factor interferon regulatory factor 3 (IRF-3) and subsequent interferon β (IFN-β) induction. TLR2 recognizes bacterial lipopeptides and their synthetic analogues (Pam3CSK4 and FSL-1) mycoplasmal polypeptides, mycobacterial proteins and fungal zymosan. Additionally it is known that Lactobacillus.Acidophilus (L.Acid) is able to use TLR2 as a receptor for certain responses. TLR2 mediates its downstream signaling pathway in MyD88 dependent manner. Induction of inflammatory chemokines like regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) is one of the many TLR2 mediated responses. TLR4 mediated RANTES induction is TRIF dependent and previously, TLR2 mediated RANTES induction was found to be partially affected by TRIF absence. Therefore, this study was carried out to determine if TRIF is also involved in other similar chemokines like MCP-1 and MIP-1β induction by TLR2 agonists. Intriguingly, MIP-1β was also found to be TRIF dependent when mouse macrophages were stimulated with TLR2 agonists. Since, TRIF activates IRF-3, therefore TLR2 agonists, Mycobacterium.Avium (M.Avium) and L.Acid were utilized to show the ability to activate IRF-3 and interferon-β (IFN-β) in a TLR2 dependent manner in transfected human kidney epithelial (HEK293) cells. Indeed, M.Avium activated both IRF-3 and IFN-β in TLR2 dependent manner in HEK293 cells. In contrast, L.Acid only activated IFN-β in a TLR2 dependent manner. In contrast to HEK293 cells, in mouse macrophages (MACs), M.Avium and L.Acid induced MIP-1β was not found to be mediated by TLR2. On the other hand, MCP-1 induction was found to be regulated differently from MIP-1β. In mouse MACs TLR2 mediated MCP-1 induction was TRIF independent. Also, M.Avium and L.Acid induced MCP-1 in MACs in TLR2 dependent manner. During this study, bone marrow derived immortalized macrophages (B6IMOs) were also compared with RAW264.7 and peritoneal MACs from C57BL/6 mice in context of TLR2 mediated chemokine induction. B6IMOs generally exhibited heightened responses on TLR stimulation compared to peritoneal MACs. Also, in B6IMOs MIP-1β was found to be TRIF independent compared to peritoneal MACs. TLR2 agonists, M.Avium and L.Acid induced RANTES, MCP-1 and MIP-1β were also measured in human peripheral blood mononuclear cells (hPBMC). Interestingly, L.Acid potently induced TNF, MIP-1b and RANTES in hPBMC, but failed to induce MCP-1. Taken together, these results unravel a new aspect of TLR2 signaling; implying a role of TRIF in certain TLR2 mediated responses and suggests that complex control mechanisms are involved in TLR2 dependent chemokine induction.