TLR4 and S1P receptors cooperate to enhance inflammatory cytokine production in human gingival epithelial cells.

Toll-like receptors (TLR) are pattern recognition receptors for highly conserved microbial molecular patterns. Activation of TLR is a pivotal step in the initiation of innate, inflammatory, and immune defense mechanisms. Recent findings indicate that G protein-coupled receptors (GPCR) may modulate TLR signaling, but it is unclear which GPCR are involved in this process. One such cooperation between GPCR and TLR can be attributed to the sphingosine 1-phosphate (S1P) receptor family. The S1P receptors (S1P1-5) are a family of GPCR with a high affinity for S1P, a serum-borne bioactive lipid associated with diverse biological activities such as inflammation and healing. In this study, we show that pro-inflammatory cytokine production, including IL-6 and IL-8, was increased with LPS and concomitant S1P stimulation. Furthermore, elevated cytokine production following LPS and S1P challenge in human gingival epithelial cells (HGEC) was significantly reduced when TLR4, S1P1 or S1P3 signaling was blocked. Our study also shows that S1P1 and S1P3 expression was induced by LPS in HGEC, and this elevated expression enhanced the influence of S1P in its cooperation with TLR4 to increase cytokine production. This cooperation between TLR4 and S1P1 or S1P3 demonstrates that TLR4 and GPCR can interact to enhance cytokine production in epithelial cells.

TLR2 transmodulates monocyte adhesion and transmigration via Rac1- and PI3K-mediated inside-out signaling in response to Porphyromonas gingivalis fimbriae.

We present evidence for a novel TLR2 function in transmodulating the adhesive activities of human monocytes in response to the fimbriae of Porphyromonas gingivalis, a pathogen implicated in chronic periodontitis and atherosclerosis. Monocyte recruitment into the subendothelium is a crucial step in atherosclerosis, and we investigated the role of P. gingivalis fimbriae in stimulating monocyte adhesion to endothelial cells and transendothelial migration. Fimbriae induced CD11b/CD18-dependent adhesion of human monocytes or mouse macrophages to endothelial receptor ICAM-1; these activities were inhibited by TLR2 blockade or deficiency or by pharmacological inhibitors of PI3K. Moreover, this inducible adhesive activity was sensitive to the action of Clostridium difficile toxin B, but was not affected by Clostridium botulinum C3 exoenzyme, pertussis toxin, or cholera toxin. Accordingly, we subsequently showed through the use of dominant negative signaling mutants of small GTPases, that Rac1 mediates the ability of fimbria-stimulated monocytes to bind ICAM-1. A dominant negative mutant of Rac1 also inhibited the lipid kinase activity of PI3K suggesting that Rac1 acts upstream of PI3K in this proadhesive pathway. Furthermore, fimbriae stimulated monocyte adhesion to HUVEC and transmigration across HUVEC monolayers; both activities required TLR2 and Rac1 signaling and were dependent upon ICAM-1 and the high-affinity state of CD11b/CD18. P. gingivalis-stimulated monocytes displayed enhanced transendothelial migration compared with monocytes stimulated with nonfimbriated isogenic mutants. Thus, P. gingivalis fimbriae activate a novel proadhesive pathway in human monocytes, involving TLR2, Rac1, PI3K, and CD11b/CD18, which may constitute a mechanistic basis linking P. gingivalis to inflammatory atherosclerotic processes.

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2.

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.

Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins.

The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1beta (IL-1beta), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-kappaB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities.