Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness.

Toll-like receptors (TLRs) are innate immune receptors for sensing microbial molecules and damage-associated molecular patterns released from host cells. Double-stranded RNA and the synthetic analog polyinosinic:polycytidylic acid (poly(I:C)) bind and activate TLR3. This stimulation leads to recruitment of the adaptor molecule TRIF (Toll/IL-1 resistance (TIR) domain-containing adapter-inducing interferon ß) and activation of the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3), classically inducing IFNß production. Here we report that, unlike non-metastatic intestinal epithelial cells (IECs), metastatic IECs express TLR3 and that TLR3 promotes invasiveness of these cells. In response to poly(I:C) addition, the metastatic IECs also induced the chemokine CXCL10 in a TLR3-, TRIF-, and IRF3-dependent manner but failed to produce IFNß. This was in contrast to healthy and non-metastatic IECs, which did not respond to poly(I:C) stimulation. Endolysosomal acidification and the endosomal transporter protein UNC93B1 was required for poly(I:C)-induced CXCL10 production. However, TLR3-induced CXCL10 was triggered by immobilized poly(I:C), was only modestly affected by inhibition of endocytosis, and could be blocked with an anti-TLR3 antibody, indicating that TLR3 can still signal from the cell surface of these cells. Furthermore, plasma membrane fractions from metastatic IECs contained both full-length and cleaved TLR3, demonstrating surface expression of both forms of TLR3. Our results imply that metastatic IECs express surface TLR3, allowing it to sense extracellular stimuli that trigger chemokine responses and promote invasiveness in these cells. We conclude that altered TLR3 expression and localization may have implications for cancer progression.

TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-ß Production via a TAK1-IKKß-IRF5 Signaling Pathway.

Staphylococcus aureus may cause serious infections and is one of the most lethal and common causes of sepsis. TLR2 has been described as the main pattern recognition receptor that senses S. aureus and elicits production of proinflammatory cytokines via MyD88 -: NF-κB signaling. S. aureus can also induce the production of IFN-ß, a cytokine that requires IFN regulatory factors (IRFs) for its transcription, but the signaling mechanism for IFN-ß induction by S. aureus are unclear. Surprisingly, we demonstrate that activation of TLR2 by lipoproteins does not contribute to IFN-ß production but instead can suppress the induction of IFN-ß in human primary monocytes and monocyte-derived macrophages. The production of IFN-ß was induced by TLR8-mediated sensing of S. aureus RNA, which triggered IRF5 nuclear accumulation, and this could be antagonized by concomitant TLR2 signaling. The TLR8-mediated activation of IRF5 was dependent on TAK1 and IκB kinase (IKK)ß, which thus reveals a physiological role of the recently described IRF5-activating function of IKKß. TLR8 -: IRF5 signaling was necessary for induction of IFN-ß and IL-12 by S. aureus, and it also contributed to the induction of TNF. In conclusion, our study demonstrates a physiological role of TLR8 in the sensing of entire S. aureus in human primary phagocytes, including the induction of IFN-ß and IL-12 production via a TAK1 -: IKKß -: IRF5 pathway that can be inhibited by TLR2 signaling.

Toll-like receptor 3-elicited MAPK activation induces stabilization of interferon-ß mRNA.

Prolonged release of cytokines after activation of the innate immune system may lead to systemic infection and inflammatory diseases. Many cytokines with short half-lives contain adenine- and uridine-rich elements (AREs) in their 3'-untranslated region (UTR), which mediate mRNA destabilization. The Toll-like receptors (TLRs) TLR3 and TLR4 induce immune responses via the adaptor proteins TRIF or TRIF and MyD88, respectively, leading to IFN-ß production. The 3'-UTR of IFN-ß mRNA contains an ARE sequence. We demonstrate that the TLR3 ligand dsRNA and the TLR4 ligand LPS induce stabilization of IFN-ß mRNA transcripts in monocyte-derived dendritic cells. In cells from TRIF(-/-) and MyD88(-/-) mice we found that dsRNA-induced stabilization of IFN-ß mRNA is TRIF-dependent. MAPK-activated protein 2 (MK2) has previously been found to regulate mRNA stabilization. We show that dsRNA elicits increased MK2 activation, mediated by TRIF and p38 MAPK. Chemical inhibition of p38 and MK2, and siRNA knockdown of MK2 relieved dsRNA-triggered prolongation of IFN-ß mRNA half-life. Taken together, these results suggest that TLR3 induces signaling mechanisms involving TRIF, p38 MAPK and MK2 to enhance stabilization of IFN-ß mRNA contributing to enhanced IFN-ß levels during pathogen infections.

Coactivation of TLR2 and TLR8 in Primary Human Monocytes Triggers a Distinct Inflammatory Signaling Response.

Innate immune signaling is essential to mount a fast and specific immune response to pathogens. Monocytes and macrophages are essential cells in the early response in their capacity as ubiquitous phagocytic cells. They phagocytose microorganisms or damaged cells and sense pathogen/damage-associated molecular patterns (PAMPs/DAMPs) through innate receptors such as Toll-like receptors (TLRs). We investigated a phenomenon where co-signaling from TLR2 and TLR8 in human primary monocytes provides a distinct immune activation profile compared to signaling from either TLR alone. We compare gene signatures induced by either stimulus alone or together and show that co-signaling results in downstream differences in regulation of signaling and gene transcription. We demonstrate that these differences result in altered cytokine profiles between single and multi-receptor signaling, and show how it can influence both T-cell and neutrophil responses. The end response is tailored to combat extracellular pathogens, possibly by modifying the regulation of IFNß and IL12-family cytokines.