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.

Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

INTRODUCTION: Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll-like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase-8 is involved in activation of NF-kB downstream of TLRs in immune cells. Here we investigated the role of caspase-8 in regulating TLR-induced cytokine production from human bone marrow-derived mesenchymal stromal cells (hBMSCs). METHODS: Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase-8 were silenced using siRNA. Caspase-8 was also inhibited using a caspase-8 inhibitor, z-IEDT. RESULTS: We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro-inflammatory cytokines in a TLR-dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti-inflammatory cytokines HGF and TGFß whereas LPS reduced HGF expression only. Notably, caspase-8 was involved in the induction of IL- IL-1ß, IL-6, CXCL10, and in the inhibition of HGF and TGFß. CONCLUSION: Caspase-8 appears to modulate hBMSCs into gaining a pro-inflammatory phenotype. Therefore, inhibiting caspase-8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders.

A role for the adaptor proteins TRAM and TRIF in toll-like receptor 2 signaling.

Toll-like receptors (TLRs) are involved in sensing invading microbes by host innate immunity. TLR2 recognizes bacterial lipoproteins/lipopeptides, and lipopolysaccharide activates TLR4. TLR2 and TLR4 signal via the Toll/interleukin-1 receptor adaptors MyD88 and MAL, leading to NF-κB activation. TLR4 also utilizes the adaptors TRAM and TRIF, resulting in activation of interferon regulatory factor (IRF) 3. Here, we report a new role for TRAM and TRIF in TLR2 regulation and signaling. Interestingly, we observed that TLR2-mediated induction of the chemokine Ccl5 was impaired in TRAM or TRIF deficient macrophages. Inhibition of endocytosis reduced Ccl5 release, and the data also suggested that TRAM and TLR2 co-localize in early endosomes, supporting the hypothesis that signaling may occur from an intracellular compartment. Ccl5 release following lipoprotein challenge additionally involved the kinase Tbk-1 and Irf3, as well as MyD88 and Irf1. Induction of Interferon-ß and Ccl4 by lipoproteins was also partially impaired in cells lacking TRIF cells. Our results show a novel function of TRAM and TRIF in TLR2-mediated signal transduction, and the findings broaden our understanding of how Toll/interleukin-1 receptor adaptor proteins may participate in signaling downstream from TLR2.

Enhanced expression of CXCL10 in inflammatory bowel disease: potential role of mucosal Toll-like receptor 3 stimulation.

BACKGROUND: We explored the gene expression in colonic biopsies of active and inactive inflammatory bowel disease (IBD) in an extensive material of ulcerative colitis (UC) and Crohn's disease (CD). The chemokine CXCL10 and its receptor CXCR3 were among the upregulated genes. This study examined the expression of CXCL10 and the mechanisms for its release in patients with UC or CD and in intestinal epithelial cell (IEC) lines. METHODS: A microarray gene expression analysis was done on colonic biopsies (n = 133) from patients with IBD. Biopsies were studied with immunohistochemistry for CXCL10 and CXCR3 expression. Mechanisms for CXCL10 release in peripheral blood mononuclear cells (PBMCs) and in the colonic epithelial cell lines HT-29 and SW620 were studied upon pattern recognition receptor (PRR) stimulation. RESULTS: CXCL10 and CXCR3 mRNA abundances were increased in biopsies from active UC and CD compared to inactive disease and controls. CXCL10 was mainly localized to mucosal epithelial cells, with increased immunostaining in active IBD. CXCR3-positive cells were scattered in the lamina propria. CXCL10 was secreted from the colonic epithelial cell lines in response to the Toll-like receptor 3 (TLR3) ligand polyinosinic: polycytidylic acid (poly(I:C)). This ligand also induced a marked release of CXCL10 in PBMCs from IBD patients and controls. CONCLUSIONS: We identified CXCL10 and CXCR3 as upregulated genes in colonic mucosa in active IBD. The TLR3-ligand poly(I:C) markedly increased release of CXCL10 in colonic epithelial cell lines, suggesting a TLR3-mediated CXCL10 release from mucosal epithelial cells in IBD patients.

The Rab11a GTPase controls Toll-like receptor 4-induced activation of interferon regulatory factor-3 on phagosomes.

Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and interferon regulatory factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust interferon-ß induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IRF3 activation from this compartment.

Cellular trafficking of lipoteichoic acid and Toll-like receptor 2 in relation to signaling: role of CD14 and CD36.

Lipoteichoic acid (LTA) is a central inducer of inflammatory responses caused by Gram-positive bacteria, such as Staphylococcus aureus, via activation of TLR2. Localization of TLR2 in relation to its coreceptors may be important for function. This study explores the signaling, uptake, and trafficking pattern of LTA in relation to expression of TLR2 and its coreceptors CD36 and CD14 in human monocytes. We found TLR2 expressed in early endosomes, late endosomes/lysosomes, and in Rab-11-positive compartments but not in the Golgi apparatus or endoplasmic reticulum (ER). Rapid internalization of fluorescently labeled LTA was observed in human monocytes, colocalizing with markers for early and late endosomes, lysosomes, ER, and Golgi network. Blocking CD14 and CD36 with antibodies inhibited LTA binding and LTA-induced TNF release from monocytes, emphasizing an important role for both molecules as coreceptors for TLR2. Importantly, blocking CD36 did not affect TNF release induced by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine or LPS. Expression of CD14 markedly enhanced LTA binding to the plasma membrane and also enhanced NF-kappaB activation. LTA internalization, but not NF-kappaB activation, was inhibited in Dynamin-I K44A dominant-negative transfectants, suggesting that LTA is internalized by receptor-mediated endocytosis but that internalization is not required for signaling. In fact, immobilizing LTA and thereby inhibiting internalization resulted in enhanced TNF release from monocytes. Our results suggest that LTA signaling preferentially occurs at the plasma membrane, is independent of internalization, and is facilitated by CD36 and CD14 as coreceptors for TLR2.

TLR9 signals after translocating from the ER to CpG DNA in the lysosome.

Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA-containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.