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PubMed2003

Salmonella septicemia in rheumatoid arthritis patients receiving anti-tumor necrosis factor therapy: association with decreased interferon-gamma production and Toll-like receptor 4 expression.

OBJECTIVE: Patients treated with antibodies to tumor necrosis factor alpha (TNFalpha) have an increased susceptibility to intracellular infections. We describe 2 patients with rheumatoid arthritis (RA) who developed Salmonella septicemia during anti-TNF treatment. The aim of this study was to identify the mechanisms involved in the increased susceptibility of anti-TNF-treated patients to intracellular microorganisms. METHODS: We evaluated an additional 6 RA patients receiving anti-TNF antibodies, 5 RA patients not receiving anti-TNF therapy, and 6 age- and sex-matched healthy volunteers. The in vitro production of cytokines (interleukin-1beta [IL-1beta], IL-6, interferon-gamma [IFNgamma], and IL-10) upon bacterial stimulation of whole blood and the expression of Toll-like receptor 4 (TLR-4) on dendritic cells from RA patients treated with infliximab, RA patients not treated with infliximab, and healthy controls were compared. RESULTS: Stimulation with heat-killed Salmonella typhimurium or Candida albicans led to a significantly decreased production of IFNgamma, but not to a decreased production of IL-10, IL-beta, or IL-6, in anti-TNF-treated RA patients compared with RA patients who were not receiving anti-TNF antibodies and compared with healthy controls. TNF-blocking treatment ex vivo significantly inhibited TLR-4 expression on dendritic cells from RA patients and healthy controls. CONCLUSION: Since recognition of microorganisms by TLR-4 and activation of phagocytes by IFNgamma are essential mechanisms for the defense against intracellular and fungal pathogens, we propose that this pathway is crucial for the increased susceptibility to these microorganisms in patients receiving anti-TNF therapy.

Adult↗
PubMed2000

The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes.

The central regulatory role of the adipocyte in whole body energy homeostasis is well established. However, recent findings suggest that preadipocytes and adipocytes may play an important physiological role in the regulation of both the innate and adaptive immune response. To systematically characterize the molecular machinery of the adipocyte that mediates the recognition of pathogens, we have focused our analysis on the recently identified Toll-like receptors (TLRs). These receptors have been implicated as mediators of the cellular response to bacterial lipopolysacharides (LPSs). Here, we report the cloning and functional characterization of mouse TLR-2 from 3T3-L1 adipocytes. TLR-2 synthesis is strongly induced in the adipocyte by LPS, TNFalpha, and the yeast cell wall extract zymosan. TLR-2 undergoes a lengthy intracellular maturation process with a half-life of exit from the ER of approximately 3 h. Furthermore, LPS treatment of adipocytes results in dramatic changes at the level of gene expression, including the synthesis of a distinct set of secretory proteins such as interleukin-6. Our studies demonstrate the presence of a fully intact pathway of innate immunity in the adipocyte that can be activated by LPS binding to the cell surface and results in the secretion of immunomodulatory molecules.

3T3 Cells↗
PubMed2003

Two distinct mechanisms for induction of dendritic cell apoptosis in response to intact Streptococcus pneumoniae.

Apoptotic dendritic cells (DCs) are ineffective at inducing immunity. Thus, parameters that regulate DC viability during a primary infection will help to determine the outcome of the subsequent immune response. In this regard, pathogens have developed strategies to promote DC apoptosis to counterbalance the nascent primary immune response. We demonstrate, using cultured bone marrow-derived DCs, that Streptococcus pneumoniae can induce DC apoptosis through two distinct mechanisms: 1) a rapid, caspase-independent mechanism of apoptosis induction, critically dependent on bacterial expression of pneumolysin, and 2) a delayed-onset, caspase-dependent mechanism of apoptosis induction associated with terminal DC maturation. Delayed-onset apoptosis does not require bacterial internalization, but rather is triggered by the interaction of bacterial subcapsular components and bone marrow-derived DC (likely Toll-like) receptors acting in a myeloid differentiation factor 88-dependent manner. In this regard, heavy polysaccharide encapsulation interferes with both DC maturation and apoptosis induction. In contrast, neither CD95/CD95 ligand interactions nor TNF-alpha appear to play a role in the delayed onset of apoptosis. These data are the first to define two mechanistically distinct pathways of DC apoptosis induction in response to an extracellular bacterium that likely have important consequences for the establishment of antibacterial immunity.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Regulation and signal transduction of toll-like receptors in human chorioncarcinoma cell lines.

PROBLEM: Toll-like receptors (TLRs) are expressed on placental cells. The aim of this study is to analyze signaling components activated in placenta cells after TLR ligand engagement. METHODS OF STUDY: In chorioncarcimoma cell lines the regulation of TLRs was determined by real time polymerase chain reaction as well as by fluorescence-activated cell sorter analysis. Activation of NF-kappaB was determined in a reporter assay system and the activation of the mitogen-activated protein kinase signaling pathways by immunoblot analysis. RESULTS: Both lipopolysaccharide (LPS) and DNA oligonucleotides containing unmethylated CpG motifs (CpG) induced the enhanced expression of TLR2 mRNA as well as a TLR2 surface protein expression. Functionally, incubation of JAR cells with microbial stimuli such as LPS activated NF-kappaB, as well as the phosphorylation of ERK1/2 and p38 MAP kinases and secretion of interleukin-8. CONCLUSION: The functional expression of TLRs on placental cells may play an important role in the initiation of an immune response in the developing fetus.

Cell Line, Tumor↗
PubMed2004

Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C.

BACKGROUND & AIMS: Protein kinase C (PKC) has been implicated in regulation of intestinal epithelial integrity in response to lumenal bacteria. Intestinal epithelial cells (IECs) constitutively express Toll-like receptor (TLR)2, which contains multiple potential PKC binding sites. The aim of this study was to determine whether TLR2 may activate PKC in response to specific ligands, thus potentially modulating barrier function in IECs. METHODS: TLR2 agonist (synthetic bacterial lipopeptide Pam(3)CysSK4, peptidoglycan)-induced activation of PKC-related signaling cascades were assessed by immunoprecipitation, Western blotting, immunofluorescence, and kinase assays-combined with functional transfection studies in the human model IEC lines HT-29 and Caco-2. Transepithelial electrical resistance characterized intestinal epithelial barrier function. RESULTS: Stimulation with TLR2 ligands led to activation (phosphorylation, enzymatic activity, translocation) of specific PKC isoforms (PKCalpha and PKCdelta). Phosphorylation of PKC by TLR2 ligands was blocked specifically by transfection with a TLR2 deletion mutant. Ligand-induced activation of TLR2 greatly enhanced transepithelial resistance in IECs, which was prevented by pretreatment with PKC-selective antagonists. This effect correlated with apical tightening and sealing of tight junction (TJ)-associated ZO-1, which was mediated via PKC in response to TLR2 ligands, whereas morphologic changes of occludin, claudin-1, or actin cytoskeleton were not evident. Downstream the endogenous PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS), but not transcriptional factor activator protein-1 (AP-1), was activated significantly on stimulation. CONCLUSIONS: The present study provides evidence that PKC is an essential component of the TLR2 signaling pathway with the physiologic consequence of directly enhancing intestinal epithelial integrity through translocation of ZO-1 on activation.

Caco-2 Cells↗
PubMed2006

New players in TLR-mediated innate immunity: PI3K and small Rho GTPases.

Toll-like receptors (TLRs) play a crucial role in the innate immune system as a first line of defense against pathogens. TLR activation in phagocytes produces pro-inflammatory cytokines and chemokines that contribute directly to elimination of infectious agents and activation of adaptive immune responses. However, a sustained inflammatory response can result in tissue damage and generalized sepsis. This review summarizes the complex and sometimes conflicting links of TLR signaling with two important regulators of immune cells functions: phosphoinositide 3-kinases (PI3Ks) and small GTPases of the Rho family. A unified model of hierarchical organization of these signaling participants is still premature, given that the tools for delineating how control of TLRmediated pathways is achieved are just emerging. Critical progress in our understanding of spatial-temporal propagation of TLR signaling will certainly be provided in the near future by pharmacological targeting of PI3Ks using recently characterized, second-generation PI3K inhibitors in combination with gene-targeting strategies for PI3K subunits and Rho GTPases targeted to the murine myeloid compartment.

Animals↗
PubMed2003

p38 activation through Toll-like receptors modulates IFN-gamma-induced expression of the Tap-1 gene only in macrophages.

Although interferon-gamma (IFN-gamma) induces the transporter associated with antigen processing (Tap)-1 expression in macrophages, cooperation with lipopolysaccharide signaling through Toll-like receptor 4 (TLR4) accelerates the kinetics and increases the overall levels of this gene. In this report, we show that peptidoglycan signaling through TLR2 and bacterial CpG DNA signaling through TLR9 are functionally equivalent at synergizing with IFN-gamma in regulating Tap-1 expression in macrophages. Activation of the p38 mitogen-activated protein kinase is necessary for this response, which correlates with increased phosphorylation of signal transducer and activator of transcription-1 on serine 727. Activation of p38, however, is not sufficient, as this signaling event does not affect the response to IFN-gamma in HeLa cells. The cooperation between these different signaling pathways also requires membrane fluidity. These data suggest that macrophages possess an ability to coordinate the signaling between the IFN-gamma and TLR receptors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Immunomodulatory effects of Toll-like receptor-7 activation on chronic lymphocytic leukemia cells.

Weak immunogenicity of chronic lymphocytic leukemia (CLL) cells may contribute to disease progression and inhibit effective immunotherapy. Accordingly, agents that enhance the immunogenicity of CLL cells may be useful in immunotherapeutic approaches to this disease. Since Toll-like receptors (TLRs) are major regulators of innate immunity and initiation of adaptive immunity, we studied the effects of viral pathogen associated molecular pattern agonists (that are recognized by TLRs) on the costimulatory phenotype and function of CLL cells. CLL cells (especially those with high endogenous expression of CD38) responded to TLR7-activating imidazoquinolines and guanosine analogs by increasing costimulatory molecule expression, producing inflammatory cytokines, and becoming more sensitive to killing by cytotoxic effectors. Additional activation of protein kinase C pathways increased the ability to stimulate T-cell proliferation, blocked phosphorylation of the transcription factor, signal transducer and activator of transcription (STAT)3, and resulted in the acquisition of a dendritic cell surface phenotype by TLR7-activated CLL cells. Normal B cells also responded to TLR7 activation by increasing costimulatory molecule expression and cytokine production. These findings suggest a potential role for TLR7 agonists in CLL immunotherapy.

Adult↗
PubMed2006

Myeloid differentiation primary response gene (88)- and toll-like receptor 2-deficient mice are susceptible to infection with aerosolized Legionella pneumophila.

BACKGROUND: Toll-like receptors (TLRs) are a family of proteins that orchestrate innate immune responses to microbes. Although pathogens are typically recognized by multiple TLRs, the specific roles of individual TLRs in mediating host protection during in vivo infection are not well understood. METHODS: We compared the roles of myeloid differentiation primary response gene (88) (MyD88), which regulates signaling through multiple TLRs, and TLR2 in mediating resistance to aerosolized Legionella pneumophila infection in vivo. RESULTS: In comparison with wild-type mice, MyD88-deficient (MyD88(-/-)) mice had dramatically higher bacterial counts in the lungs, with decreased neutrophil counts in the bronchoalveolar lavage fluid as well as absent cytokine and chemokine production at early time points. By day 6 of infection, the MyD88(-/-) mice developed organizing pneumonia with dissemination of L. pneumophila to the lymph nodes and spleen. TLR2(-/-) mice were also more susceptible to L. pneumophila, with higher bacterial counts in the lung. However, TLR2(-/-) mice produced proinflammatory cytokines, recruited neutrophils to the lung alveoli, and cleared the infection without progression to organizing pneumonia and disseminated disease. CONCLUSIONS: These results suggest that a MyD88-dependent pathway is required for eradication of L. pneumophila and prevention of organizing pneumonia. TLR2 mediates partial resistance to L. pneumophila, which indicates that additional MyD88-dependent, TLR2-independent pathways are essential for full protection.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The Flagellin-TLR5 Axis: Therapeutic Opportunities.

Motile bacteria synthesize large-sized surface structures known as flagella through the ordered polymerization of protein subunits. Flagellin, a protein of 40-60 kDa, is the principal constituent of the flagellum; each flagellum consists of approximately 20,000 flagellin molecules. An alignment of the amino acid sequences from different Gram-negative species shows a high degree of similarity in the amino and carboxy terminal domains. In contrast, the central hypervariable regions of these proteins are quite divergent. Recent work reveals that--in addition to playing a role in bacterial adhesion--monomeric flagellin, a protein component of flagellated bacteria, can also act as a soluble immunostimulatory and proinflammatory factor, activating the immune/inflammatory axis via the Toll-like receptor 5-nuclear factor-kappaB axis. Monocytes and macrophages, as well as intestinal and pulmonary epithelial cells, respond to monomeric flagellin at low concentrations. Administration of flagellin at doses comparable to or lower than that of bacterial lipopolysaccharide (endotoxin) can induce prominent local and systemic immune/inflammatory responses in vivo. Recognition of the flagellin-TLR5 pathway offers novel opportunities for the experimental therapy of various forms of shock, sepsis, acute respiratory distress syndrome, bacterial inflammation and infection. (c) Prous Science 2002. All rights reserved.

Journal Article↗
PubMed2002

An integrated view of the roles and mechanisms of heat shock protein gp96-peptide complex in eliciting immune response.

Heat shock protein (HSP) gp96, or grp94 is an endoplasmic reticular (ER) paralog of the cytosolic HSP90. Being abundant and non-polymorphic, gp96 plays significant roles in maintaining protein homeostasis in the secretory pathway. This "house-keeping" role of gp96 has now been overshadowed by the intriguing findings that gp96 modulates both the innate and adaptive components of the immune system. It has been found that, (i) gp96 is one of the major peptide binding proteins in the ER, (ii) gp96 interacts specifically with receptors including CD91 and possibly toll-like receptors (TLRs), on the surface of professional antigen presenting cells (APCs), (iii) interaction with APCs leads to re-presentation of gp96-chaperoned peptides to the major histocompatibility complex (MHC) molecules of APCs, (iv) direct access of gp96 to APCs triggers functional activation of APCs. In this review, we will examine each of these immunological attributes of gp96 critically. As experimentalists, we will also propose specific experiments to examine the argument that gp96, perhaps along with other members of HSP family, is the antigenic carrier for mediating cross priming of antigen-specific T lymphocytes in vertebrates.

Amino Acid Sequence↗
PubMed2004

TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function.

Natural interferon-producing cells (IPC) respond to viruses by secreting type I interferon (IFN) and interleukin-12 (IL-12). Toll-like receptor (TLR) 9 mediates IPC recognition of some of these viruses in vitro. However, whether TLR9-induced activation of IPC is necessary for an effective antiviral response in vivo is not clear. Here, we demonstrate that IPC and dendritic cells (DC) recognize murine cytomegalovirus (MCMV) through TLR9. TLR9-mediated cytokine secretion promotes viral clearance by NK cells that express the MCMV-specific receptor Ly49H. Although depletion of IPC leads to a drastic reduction of the IFN-alpha response, this allows other cell types to secrete IL-12, ensuring normal IFN-gamma and NK cell responses to MCMV. We conclude that the TLR9/MyD88 pathway mediates antiviral cytokine responses by IPC, DC, and possibly other cell types, which are coordinated to promote effective NK cell function and MCMV clearance.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Toll-like receptor 9-independent aggravation of glomerulonephritis in a novel model of SLE.

The generation of anti-DNA auto-antibodies is characteristic for the human autoimmune condition systemic lupus erythematosus (SLE) and its animal models. However, the contribution of the toll-like receptor (TLR) system of innate immunity receptors and, in particular, TLR9 to this B cell-mediated autoimmune process remains controversial. Here we report that in a novel murine model of SLE, based on hyper-reactive B cell activation mediated by mutant phospholipase Cg2, the genetic deficiency of TLR9 does not protect from spontaneous anti-DNA auto-antibody formation and glomerulonephritis. On the contrary, disease induction is aggravated and additional nucleolar antibody specificity develops in autoimmune TLR9-deficient mice. In vitro studies demonstrate that, in autoimmune-prone mice, dual signaling via the B cell receptor and non-CpG DNA results in synergistic B cell activation in a TLR9-independent manner. These results suggest that engagement of a TLR9-independent DNA activation pathway may promote autoimmunity, while TLR9 signaling can ameliorate SLE-like immune pathology in vivo.

Animals↗
PubMed2004

Dorsoventral patterning: a serpin pinned down at last.

Dorsoventral patterning in Drosophila has long been known to involve a cascade of proteases, held in the inactive zymogen state prior to signaling. At long last, the prediction that a protease inhibitor is involved in this pathway has been shown to be true, with the identification of a serpin that plays a key part in Drosophila embryonic patterning.

Animals↗
PubMed2002

Association of changes in D-dimer and other coagulation markers with changes in Marder score after treatment of acute venous thrombosis.

AIM: Coagulation markers are sensitive tools to assess ongoing thrombus formation. An association between changes in these markers and changes in venographic Marder scores in patients with acute deep vein-thrombosis treated with low-molecular-weight (LMWH) or unfractionated heparin (UFH) has not been reported. METHODS: We investigated differences in coagulation parameters before and at the end of a twelve days the treatment of patients with an improvement versus no improvement of the venographic findings at the end of the treatment with LMWH (n = 48) and UFH (n = 41). RESULTS: Patients with lower values in the Marder score had lower D-dimer levels at day 12 compared to entry treated with UFH and LMWH (p < 0.001). Not improved Marder scores paralleled unchanged D-dimer levels at end of both treatments. Higher values of factor-Xa inhibition and Heptest assay (p < 0.001) were measured at the end of treatment in LMWH- in contrast to UFH-patients. Thrombin inhibition was lower and unchanged at day 12 in patients treated with LMWH and UFH, respectively. Thrombin generation inhibition and release of tissue-factor pathway inhibitor remained unchanged in both groups. CONCLUSION: An improved Marder score is associated with a decrease of D-dimer during UFH and LMWH treatment of deep vein-thrombosis.

Acute Disease↗
PubMed2006

TLR2, TLR4, CD14, CD11B, and CD11C expressions on monocytes surface and cytokine production in patients with sepsis, severe sepsis, and septic shock.

Bacterial recognition and induced cellular activation are fundamental for the host control of infection, yet the limit between protective and harmful response is still inexact. Forty-one patients were enrolled in this study: 14 with sepsis, 12 with severe sepsis, and 15 with septic shock. Seventeen healthy volunteers (HV) were included as control. The expression of TLR2, TLR4, CD14, CD11b, and CD11c was analyzed on monocytes surface in whole blood. sCD14 was measured in serum, and TNF-alpha, IL-6, and IL-10 cytokine levels were measured in PBMC supernatants after LPS, IL-1beta, and TNF-alpha stimuli by ELISA. An increase in sCD14 and a decreased mCD14 were found in patients as compared with HV (P < 0.001). However, no differences in the expression of TLR2, TLR4, and CD11c were found among the groups. A trend toward differential expression of CD11b was observed, with higher values found in patients with sepsis as compared with HV. A negative regulation of the inflammatory cytokine production was observed in patients with severe sepsis and shock septic in relation to sepsis and HV, regardless of the stimulus. No significant difference in IL-10 production was found among the groups. In this study, we show that the inflammatory response is associated with the continuum of clinical manifestations of sepsis, with a strong inflammatory response in the early phase (sepsis) and a refractory picture in the late phases (severe sepsis and septic shock). Correlation between cell surface receptors and cytokine production after IL-1beta and TNF-alpha stimuli and the observation of a single and same standard response with the different stimulus suggest a pattern of immunology response that is not dependent only on the expression of the evaluated receptors and that is likely to have a regulation in the intracellular signaling pathways.

Antigens, CD↗
PubMed2004

Preservation of eumelanin hair pigmentation in proopiomelanocortin-deficient mice on a nonagouti (a/a) genetic background.

The original strain of proopiomelanocortin (POMC)-deficient mice (Pomc-/-) was generated by homologous recombination in 129X1/SvJ (A(w)/A(w))-derived embryonic stem cells using a targeting construct that deleted exon 3, encoding all the known functional POMC-derived peptides including alpha MSH, from the Pomc gene. Although these Pomc-/- mice exhibited adrenal hypoplasia and obesity similar to the syndrome of POMC deficiency in children, their agouti coat color was only subtly altered. To further investigate the mechanism of hair pigmentation in the absence of POMC peptides, we studied wild-type (Pomc+/+), heterozygous (Pomc+/-), and homozygous (Pomc-/-) mice on a nonagouti (a/a) 129;B6 hybrid genetic background. All three genotypes had similar black fur pigmentation with yellow hairs behind the ears, around the nipples, and in the perianal area characteristic of inbred C57BL/6 mice. Histologic and electron paramagnetic resonance spectrometry examination demonstrated that hair follicles in back skin of Pomc-/- mice developed with normal structure and eumelanin pigmentation; corresponding molecular analyses, however, excluded local production of alpha MSH and ACTH because neither Pomc nor putative Pomc pseudogene mRNAs were detected in the skin. Thus, 129;B6 Pomc null mutant mice produce abundant eumelanin hair pigmentation despite their congenital absence of melanocortin ligands. These results suggest that either the mouse melanocortin receptor 1 has sufficient basal activity to trigger and sustain eumelanogenesis in vivo or that redundant nonmelanocortin pathway(s) compensate for the melanocortin deficiency. Whereas the latter implies feedback control of melanogenesis, it is also possible that the two mechanisms operate jointly in hair follicles.

Adrenal Glands↗
PubMed2006

Intracellular TLR signaling: a structural perspective on human disease.

TLRs are crucial sensors of microbial infection. Maintaining structural integrity of TLR signaling components is essential for subsequent immunological protection. Alterations to the structure of these signaling molecules are often associated with profound clinical outcomes and susceptibility to various infectious diseases. These changes in structure are sometimes the result of a single nucleotide polymorphism (SNP). Numerous SNPs have been found in components of the TLR signaling pathway. Recently, the medical consequences and effects on TLR signaling of several of these SNPs have been elucidated. In addition, there have been numerous structures solved that are important to our understanding of the TLR signaling pathway at the molecular level. The scope of this review is to tie together current structural, biochemical, and genetic information of TLR signaling.

Animals↗