Neisserial porin-induced dendritic cell activation is MyD88 and TLR2 dependent

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin's ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin's immune stimulatory activity.     

TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.     

TLR2, TLR4 and the MyD88 Signaling Are Crucial For the In Vivo Generation and the Longevity of Long-Lived Antibody-Secreting Cells

This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220^pos. TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220^pos (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220^neg in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220^pos (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220^pos (MyD88-independent) and ASC B220^neg into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220^neg in peritoneum and BM. Terminally differentiated ASC B220^neg required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220^pos rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220^pos and ASC B220^neg.     

MyD88 plays a unique role in host defense but not arthritis development in Lyme disease

To assess the contribution of TLR signaling in the host response to Borrelia burgdorferi, mice deficient in the common TLR adaptor protein, myeloid differentiation factor 88 (MyD88), were infected with B. burgdorferi. MyD88-deficient mice harbored extremely high levels of B. burgdorferi in tissues when compared with wild-type littermates and greater amounts of spirochetes in tissues than TLR2-deficient mice. These findings suggest that, in addition to TLR2, other MyD88-dependent pathways play a significant role in the host defense to B. burgdorferi. MyD88(-/-) mice maintained the ability to produce Abs directed against B. burgdorferi. Partial clearance of spirochetes was evident in long term infection studies and immune sera from MyD88-deficient mice were able to protect naive mice from infection with B. burgdorferi. Thus, the acquired immune response appeared to be functional in MyD88(-/-) mice, and the inability to control spirochete numbers was due to a failure of cells involved in innate defenses. Although macrophages from MyD88(-/-) mice responded poorly to Borrelia sonicate in vitro, MyD88(-/-) mice still developed an inflammatory arthritis after infection with B. burgdorferi characterized by an influx of neutrophils and mononuclear cells. The findings presented here point to a dichotomy between the recruitment of inflammatory cells to tissue and an inability of these cells to kill localized spirochetes.     

The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease

Abnormal T cell responses to commensal bacteria are involved in the pathogenesis of inflammatory bowel disease. MyD88 is an essential signal transducer for TLRs in response to the microflora. We hypothesized that TLR signaling via MyD88 was important for effector T cell responses in the intestine. TLR expression on murine T cells was examined by flow cytometry. CD4(+)CD45Rb(high) T cells and/or CD4(+)CD45Rb(low)CD25(+) regulatory T cells were isolated and adoptively transferred to RAG1(-/-) mice. Colitis was assessed by changes in body weight and histology score. Cytokine production was assessed by ELISA. In vitro proliferation of T cells was assessed by [(3)H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling. CD4(+)CD45Rb(high) T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules. MyD88(-/-) CD4(+) T cells showed decreased proliferation compared with WT CD4(+) T cells both in vivo and in vitro. CD4(+)CD45Rb(high) T cells from MyD88(-/-) mice did not induce wasting disease when transferred into RAG1(-/-) recipients. Lamina propria CD4(+) T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice receiving MyD88(-/-) cells than mice receiving WT cells. In vitro, MyD88(-/-) T cells were blunted in their ability to secrete IL-17 but not IFN-gamma. Absence of MyD88 in CD4(+)CD45Rb(high) cells results in defective T cell function, especially Th17 differentiation. These results suggest a role for TLR signaling by T cells in the development of inflammatory bowel disease.     

Candesartan attenuates Angiotensin II-induced mesangial cell apoptosis via TLR4/MyD88 pathway

Angiotensin II (Ang II) can stimulate Toll-like receptor 4 (TLR4) expression in mesangial cells (MCs), but the role of TLR4 in the Ang II-induced apoptosis and the effect of candesartan on TLR4 expression remain unclear. Here, we report that Ang II-induced MC apoptosis in a time-dependent manner and up-regulated TLR4/MyD88 expression, and that the intracellular ROS was subsequently increased. We also show that candesartan attenuated the Ang II-induced MC apoptosis, and that this protective effect was dependent on decreased TLR4/MyD88 expression as well as reduced intracellular ROS formation. Furthermore, Ang II increased the apoptosis inducing factor protein level, while candesartan markedly reduced this increase. These results demonstrate that TLR4/MyD88 pathway was involved in the Ang II promoted MC apoptosis, which was related to TLR4/MyD88 mediated oxidative stress. These data also suggest that candesartan exerted anti-apoptotic effect as an antioxidant by modulating this pathway.     

Long noncoding RNA ANRIL contributes to the development of ulcerative colitis by miR-323b-5p/TLR4/MyD88/NF-κB pathway

The aim of this study was to investigate the role and possible mechanism of long noncoding RNA ANRIL in the development of ulcerative colitis (UC). The expression of ANRIL in colonic mucosa tissues collected from the sigmoid colon of UC patients and healthy control was determined. Subsequently, fetal human cells (FHCs) were treated with lipopolysaccharide (LPS) to stimulate UC-caused inflammatory injury, followed by detection of the effects of suppression of ANRIL on cell viability, apoptosis and cytokines production in LPS-stimulated FHCs. Moreover, the regulatory relationship between ANRIL and miR-323b-5p as well as the target relationship between miR-323b-5p and TLR4 were investigated. Furthermore, the effects of ANRIL/miR-323b-5p axis on the activation of TLR4/MyD88/NF-κB pathway in LPS-stimulated FHCs were investigated. LncRNA ANRIL was highly expressed in colonic mucosa tissues of UC patients. In addition, LPS markedly induced cell injury in FHC cells (inhibited cell viability and promoted cell apoptosis and cytokine production). Suppression of ANRIL alleviated LPS-induced injury in FHC cells, which was achieved by negatively regulating miR-323b-5p. Moreover, miR-323b-5p negatively regulated TLR4 expression and TLR4 was a target of miR-323b-5p. Knockdown of TLR4 reversed the effects of miR-323b-5p suppression on LPS-induced injury in LPS-stimulated FHCs. Furthermore, the effects of ANRIL on LPS-induced cell injury were achieved by TLR4/MyD88/NF-κB pathway. Our data indicate that suppression of ANRIL may inhibit the development of UC by regulating miR-323b-5p/TLR4/MyD88/NF-κB pathway. ANRIL/miR-323b-5p/TLR4/MyD88/NF-κB pathway may provide a new strategy for UC therapy.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

R753Q Polymorphism Inhibits Toll-like Receptor (TLR) 2 Tyrosine Phosphorylation,Dimerization with TLR6, and Recruitment of Myeloid Differentiation Primary Response Protein 88

The R753Q polymorphism in the Toll-IL-1 receptor domain of Toll-like receptor 2 (TLR2) has been linked to increased incidence of tuberculosis and other infectious diseases, but the mechanisms by which it affects TLR2 functions are unclear. Here, we studied the impact of the R753Q polymorphism on TLR2 expression, hetero-dimerization with TLR6, tyrosine phosphorylation, and recruitment of myeloid differentiation primary response protein (MyD) 88 and MyD88 adapter-like (Mal). Complementation of HEK293 cells with transfected WT or R753Q TLR2 revealed their comparable total levels and only minimal changes in cell surface expression of the mutant species. Notably, even a 100-fold increase in amounts of transfected R753Q TLR2 versus WT variant did not overcome the compromised ability of the mutant TLR2 to activate nuclear factor κB (NF-κB), indicating that a minimal decrease in cell surface levels of the R753Q TLR2 cannot account for the signaling deficiency. Molecular modeling studies suggested that the R753Q mutation changes the electrostatic potential of the DD loop and results in a discrete movement of the residues critical for protein-protein interactions. Confirming these predictions, biochemical assays demonstrated that R753Q TLR2 exhibits deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6, and recruitment of Mal and MyD88. These proximal signaling deficiencies correlated with impaired capacities of the R753Q TLR2 to mediate p38 phosphorylation, NF-κB activation, and induction of IL-8 mRNA in transfected HEK293 cells challenged with inactivated Mycobacterium tuberculosis or mycobacterial components. Thus, the R753Q polymorphism renders TLR2 signaling-incompetent by impairing its tyrosine phosphorylation, dimerization with TLR6, and recruitment of Mal and MyD88.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway

We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-gamma by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses.     

Membrane-tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.     

Role for MyD88 signaling in murine gammaherpesvirus 68 latency

Toll-like receptors (TLRs) are known predominantly for their role in activating the innate immune response. Recently, TLR signaling via MyD88 has been reported to play an important function in development of a B-cell response. Since B cells are a major latency reservoir for murine gammaherpesvirus 68 (MHV68), we investigated the role of TLR signaling in the establishment and maintenance of MHV68 latency in vivo. Mice deficient in MyD88 (MyD88(-/-)) or TLR3 (TLR3(-/-)) were infected with MHV68. Analysis of splenocytes recovered at day 16 postinfection from MyD88(-/-) mice compared to those from wild-type control mice revealed a lower frequency of (i) activated B cells, (ii) germinal-center B cells, and (iii) class-switched B cells. Accompanying this substantial defect in the B-cell response was an approximately 10-fold decrease in the establishment of splenic latency. In contrast, no defect in viral latency was observed in TLR3(-/-) mice. Analysis of MHV68-specific antibody responses also demonstrated a substantial decrease in the kinetics of the response in MyD88(-/-) mice. Analysis of wild-type x MyD88(-/-) mixed-bone-marrow chimeric mice demonstrated that there is a selective failure of MyD88(-/-) B cells to participate in germinal-center reactions as well as to become activated and undergo class switching. In addition, while MHV68 established latency efficiently in the MyD88-sufficient B cells, there was again a ca. 10-fold reduction in the frequency of MyD88(-/-) B cells harboring latent MHV68. This phenotype indicates that MyD88 is important for the establishment of MHV68 latency and is directly related to the role of MyD88 in the generation of a B-cell response. Furthermore, the generation of a B-cell response to MHV68 was intrinsic to B cells and was independent of the interleukin-1 receptor, a cytokine receptor that also signals through MyD88. These data provide evidence for a unique role for MyD88 in the establishment of MHV68 latency.     

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion, our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4 signaling, indicating that hypoxia might impair the host cell's ability to eliminate the Acanthamoeba invasion and that hypoxia could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.     

Flavopiridol inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.     

Toll-Like Receptor 4 Prompts Human Breast Cancer Cells Invasiveness via Lipopolysaccharide Stimulation and Is Overexpressed in Patients with Lymph Node Metastasis

Toll-like receptor (TLR)4-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study investigated the expression and biological role of TLR4 in human breast cancer metastasis. MCF-7 and MDA-MB-231 are human breast cancer cell lines with low and high metastatic potential, respectively. Using lipopolysaccharide (LPS) to stimulate MCF-7 and MDA-MB-231 cells, expression of TLR4 mRNA and protein increased compared with that in control cells. TLR4 activation notably up-regulated expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor(VEGF) mRNA and their secretion in the supernatants of both cell lines. LPS enhanced invasion of MDA-MB-231 cells by transwell assay and MCF-7 cells by wound healing assay. LPS triggered increased expression of TLR4 downstream signaling pathway protein myeloid differentiation factor 88(MyD88) and resulted in interleukin (IL)-6 and IL-10 higher production by human breast cancer cells. Stimulation of TLR4 with LPS promoted tumorigenesis and formed metastatic lesions in liver of nude mice. Moreover, expression of TLR4 and MyD88 as well as invasiveness and migration of the cells could be blocked by TLR4 antagonist. Combined with clinicopathological parameters, TLR4 was overexpressed in human breast cancer tissue and correlated with lymph node metastasis. These findings indicated that TLR4 may participate in the progression and metastasis of human breast cancer and provide a new therapeutic target.