Toll-like receptor (TLR) activation is dictated by a number of factors including the ligand itself and the localization of the receptor, in terms of expression profile and subcellular localization and the signal transduction pathway that has been activated. Recent work into TLR signal transduction has revealed complex regulation at a number of different levels including regulation by phosphorylation, targeted degradation, and sequestration of signaling molecules. Here, we describe recent advances that have been made in our understanding of how TLR signaling is regulated at the biochemical level.

The human thromboxane A2 receptor (TP), a G protein-coupled receptor, exists as two isoforms, TPalpha and TPbeta, which arise by alternative mRNA splicing and differ exclusively in their carboxyl terminal cytoplasmic regions. In this study, a reverse transcriptase-polymerase chain reaction (RT-PCR)-based strategy was developed to examine the expression of the TPs in tissues of physiologic relevance to TXA2. Although most of the 17 different cell/tissue types examined expressed both TP isoforms, the liver hepatoblastoma HepG2 cell line was found to exclusively express TPalpha mRNA. In most cell types, TPalpha mRNA predominated over TPbeta mRNA. Moreover, although the levels of TPalpha mRNA expression were similar in most of the cell/tissue types examined, extensive differences in the levels of TPbeta mRNA were observed. Consequently, the relative expression of TPalpha: TPbeta mRNA varied considerably due to extensive differences in TPbeta mRNA expression. Most strikingly, primary HUVECs were found to express: (i) low levels of TPbeta and (ii) approximately 6-fold greater levels of TPalpha than TPbeta. These data were confirmed in the spontaneously transformed HUVEC derived ECV304 cell line. Expression of TP mRNAs in the various tissue/cells correlated with protein expression, as assessed by radioligand binding using the selective TP antagonist [3H]SQ29,548.

The authors note that Fig. 3 appeared incorrectly. Panel B (Top) depicting the time course of NF-κB activation in wild type and caspase-1 deficient peritoneal macrophages is not the same version as the authors submitted originally to PNAS. A correct version including vertical rules to indicate splicing is shown below. Regarding panel C, during the preparation of the manuscript, we inadvertently repeated a set of p38 blots corresponding to p-p38 Western blots. The correct p38 Western blots are shown below. The findings of the paper have not been affected by the error and the authors apologize to the editors and readers. The corrected figure and its legend appear below. . Caspase-1 is required for Mal to signal. (A) (Upper) U373 cells were transfected with a 5x NF-κB reporter gene plasmid. Cells were left untreated or pretreated with YVAD-Cmk (100 μM) or IL-1 receptor antagonist (1 μg/ml) for 1 h. Thereafter, cells were untreated or incubated with LPS (1 μg/ml) or IL-1 (100 ng/ml) for 6 h. Shown is the mean relative stimulation of luciferase activity ± SD for a representative experiment from three separate experiments. (Lower) THP-1 cells were left untreated or pretreated with YVAD-Cmk (100 μM) or IETD-Fmk (50 μM) for 1 h followed by treatment with Pam 3 Cys (1 μg/ml) or IL-1 (1 μg/ml) for 0-120 min. Activation of p38 was analyzed by using an anti-phospho-p38-specific antibody. (B) (Top) Time course of NF-κB activation in wild-type and caspase-1-deficient peritoneal macrophages stimulated with LPS (10 ng/ml), Malp-2 (10 nM), and R848 (10 μM) as detected by EMSA. (Middle) Supershift assay was performed by using an anti-p65 antibody for 1 h before analysis by EMSA. Protein:DNA complexes are shown. (Bottom) Wild-type and caspase-1-deficient murine embryonic fibroblasts were treated with LPS (100 ng/ml), lipid A (100 ng/ml), or Malp-2 (10 nM) as indicated, followed by immunoblot analysis of the cell lysates with antibodies directed against IκBα or β-actin. (C) Time course of p38 activation in wild-type and caspase-1-deficient peritoneal macrophages stimulated with LPS (100 ng/ml), Malp-2 (10 nM), and IL-1 (1 μg/ml) analyzed by immunoblotting with phospho-p38-specific antibodies. Total p38 levels are also shown. (D) Time course of p38 activation in wild-type and caspase-1-deficient peritoneal murine embryonic fibroblasts stimulated with LPS (100 ng/ml) or Malp-2 analyzed by immunoblotting with phospho-p38-specific antibodies. Total p38 levels are also shown.

The pathogenesis and complications of type 2 diabetes (T2DM) are closely linked with defective glucose metabolism, obesity, cardiovascular disease and an inability to mount an effective immune response to certain pathogenic organisms. Perturbations in key innate immune receptors known as Toll-like receptors (TLRs) and inflammatory mediators such as IL-6, TNFα and IL-1β have been linked with T2DM. Herein, we sought to establish whether patients with T2DM and underlying complications exhibit perturbations in cytokine and TLR expression. Serum cytokine and mRNA levels of cytokines/TLRs in monocytes (M) and neutrophils (N) were measured in a cohort of 112 diabetic patients: good glycaemic control without complications (GC), good glycaemic control with complications (GCC), poor glycaemic control without complications (PC) and poor glycaemic control with complications (PCC) and compared them with 34 non-diabetic volunteers (NGT). Serum cytokine levels were normal in all study participants. In the GC group, cytokine and TLR gene expression were enhanced compared to NGT. In contrast, suppressed cytokine and TLR gene expression were evident in PC, GCC & PCC groups when compared to the GC. In conclusion, whereas serum pro-inflammatory cytokine levels are unaltered in T2DM patients, differences in inflammatory gene profiles exist among the T2DM patient groups.

Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in “danger” recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN-β. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN-β induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF-α induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN-β and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKKε-, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN-β induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN-β. Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKKε-mediated induction of IRF3 and concomitant IFN-β and RANTES production.