CD14 impairs host defense against gram-negative sepsis caused by Burkholderia pseudomallei in mice

BACKGROUND: CD14 is a pattern-recognition receptor that can facilitate the presentation of bacterial components to either Toll-like receptor 2 (TLR2) or TLR4. We have recently shown that during melioidosis, a severe infection caused by the gram-negative bacterium Burkholderia pseudomallei, TLR2 but not TLR4 impacts the immune response of the intact host in vivo. METHODS: The function of CD14 in melioidosis was analyzed by means of in vitro and in vivo approaches, using wild-type (WT) and CD14 knockout (KO) mice. RESULTS: CD14-deficient macrophages and whole blood leukocytes released less tumor necrosis factor (TNF)-alpha on stimulation with B. pseudomallei or B. pseudomallei lipopolysaccharide in vitro, compared with WT cells. Strikingly, CD14 KO mice intranasally inoculated with B. pseudomallei demonstrated reduced lethality and significantly decreased bacterial outgrowth, compared with WT mice. Administration of recombinant soluble CD14 to CD14 KO mice partially reversed their phenotype to that of WT mice. Lastly, CD14 deficiency did not alter the capacity of macrophages or neutrophils to phagocytose or kill B. pseudomallei. CONCLUSION: CD14 is crucially involved in the recognition of B. pseudomallei by innate immune cells but plays a remarkable detrimental role in the host response against B. pseudomallei. Inhibition of CD14 may be a novel treatment strategy in melioidosis.     

Differential expression and action of Toll-like receptors in human adrenocortical cells

During sepsis, an intact adrenal gland glucocorticoid stress response is critical for survival. Recently, we have shown that Toll-like receptors, particularly TLR2 and TLR4, are crucial in HPA axis regulation following inflammation, establishing a direct link between bacterial and viral ligands and the endocrine stress response. However, the exact role which TLRs play in adrenal homeostasis and malfunction is not yet sufficiently known. Using quantitative real-time PCR, confocal microscopy and the NF-κB reporter gene assay, we aimed to analyse both, expression and function of all relevant TLRs in the human adrenocortical cell line—NCI-H295R and adrenal cells in primary culture. Our results demonstrate a differential expression pattern of TLR1–9 in human adrenocortical cells as compared to immune cells and adrenocortical cancer cells. Consequently, activation of these cells by bacterial ligands leads to differential induction of cytokines including IL6, IL8 and TNF-α. Therefore, Toll-like receptors expression and function is a novel feature of the adrenal stress system contributing to adrenal tissue homeostasis, regeneration and tumorigenesis.     

Defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists

Neonates exhibit an increased risk of sepsis mortality compared with adults. We show that in contrast to adults, survival from polymicrobial sepsis in murine neonates does not depend on an intact adaptive immune system and is not improved by T cell-directed adaptive immunotherapy. Furthermore, neonates manifest an attenuated inflammatory and innate response to sepsis, and have functional defects in their peritoneal CD11b(+) cells. Activation of innate immunity with either a Toll-like receptor 4 (TLR4) or TLR7/8 agonist, but not a TLR3 agonist, increased the magnitude, but abbreviated the early systemic inflammatory response, reduced bacteremia, and improved survival to polymicrobial sepsis. TLR4 agonist pretreatment enhanced peritoneal neutrophil recruitment with increased oxidative burst production, whereas the TLR7/8 agonist also enhanced peritoneal neutrophil recruitment with increased phagocytic ability. These benefits were independent of the adaptive immune system and type I interferon signaling. Improving innate immune function with select TLR agonists may be a useful strategy to prevent neonatal sepsis mortality.     

Failure of TLR4-driven NF-kappa B activation to stimulate virus replication in models of HIV type 1 activation

The interaction of HIV-1 with Toll-like receptors (TLR) on host target cells is incompletely understood. Data from several in vivo and in vitro model systems suggest that TLR2, TLR4, and TLR9 remain functional and if stimulated, cause an upregulation of viral replication. In the present studies employing two different chronically HIV-1-infected cell lines and highly purified TLR agonists, we found ligation of TLR2 and TLR9, but not TLR4, resulted in significant upregulation of HIV-1 production. This result was not due to a lack of TLR4 expression or impaired NF-kappa B activation. Using HEK293 cells transfected with individual TLRs and an HIV-1 LTR reporter confirmed that TLR4 signaling does not directly activate the HIV-1 LTR. Finally, ultrapurified LPS did not enhance production of IL-1 beta or IL-6 in chronically infected U1 cells, whereas significant cytokine production was observed in uninfected U937 cells. These results confirm the biological activity of ultrapurified LPS and raise the possibility that TLR4 signaling pathways may be altered during chronic HIV-1 infection. Collectively, these studies suggest that although several TLR can upregulate NF-kappaB in HIV-1-infected cells, upregulation of NF-kappaB alone is insufficient to activate the viral LTR. Further dissection of the TLR signaling pathways is necessary to determine how TLR stimulation leads to LTR activation and whether HIV-1 infection can alter signaling through TLR4.     

Hypothalamo-pituitary and immune-dependent adrenal regulation during systemic inflammation

Inflammation-related dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immune–adrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune–adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPA-axis activation mediated by LPS were found in Mx1^Cre+-MyD88^fl/fl mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.Sepsis and septic shock are major causes of death in intensive-care units worldwide and show an increasing incidence (1). In sepsis, excessive, uncontrolled activation of the immune system is harmful to the host and leads to multiorgan failure and death. Adrenal glucocorticoid production plays a beneficial role in response to systemic inflammation by counteracting hyperactivation of the immune system. However, in many critically ill patients, this homeostatic activation of adrenocortical hormone secretion is impaired (2). It has been estimated that 60% of critically ill patients show an abnormal adrenal glucocorticoid response to administration of exogenous adrenocorticotropic hormone (ACTH) (3).Adrenal hormone production in sepsis is thought to be regulated by cytokines that elevate hypothalamic corticotropin releasing hormone (CRH) levels. CRH, in turn, produces the release of pituitary ACTH—the main regulator of synthesis of adrenal glucocorticoid hormones (4). It is generally accepted that pattern-recognition receptors such as Toll-like receptors (TLRs) play a substantial role in hypothalamic–pituitary–adrenal (HPA) axis activation induced by pathogens. This activation, in turn, may be attributable to functions of these receptors on both myeloid and nonmyeloid cells (e.g., endothelial cells of the blood–brain barrier) (5). Furthermore, besides activation of immune cells, bacterially derived TLR ligands, such as lipopolysaccharides (LPSs), can directly affect both neuronal cells (6) and steroid-producing cells in the adrenal gland (7).Although most LPS actions on adrenal-gland glucocorticoid production have been attributed to activation of HPA axis by cytokines at the level of the hypothalamus and pituitary gland, a critical involvement of an intrinsically regulated immune–adrenal cross talk has also been implicated. In fact, we and others have demonstrated that adrenocortical cells express several TLRs and secrete multiple proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)6 in response to bacterial endotoxin stimulation (7). Using mice deficient in either TLR2 or TLR4, we demonstrated a critical function of these receptors as regulators of the immune–adrenal cross talk during LPS-mediated systemic inflammation. The absence of these receptors correlates with increased basal levels of either ACTH (for TLR2) or corticosterone (for TLR4) and impaired adrenal glucocorticoid responses to injection of LPS (8, 9). However, the exact contributions of the TLRs on different cell types to inflammation-mediated adrenal dysfunction as well as to activation of HPA axis are not entirely clear. Furthermore, because both TLR2 and TLR4 were shown to be involved in early neurogenesis (10), the deficiency of these receptors from the very early developmental stage might potentially influence the basal function of the HPA axis. Myeloid differentiation factor 88 (MyD88) is a central component of the signaling pathway of TLRs, promoting nuclear factor kappa B (NF-κB) activation in response to LPS or IL1 β. Thus, blocking of MyD88-dependent signaling in a cell-specific manner represents an attractive experimental tool to address the aforementioned issue.These data and observations prompted us to investigate the role of systemic and local adrenal TLR signaling in the activation of the adrenal glucocorticoid response to stress and regulation of the immune–adrenal cross talk during the systemic inflammatory response syndrome (SIRS) phenomenon. Our results suggest that immune cells, rather than adrenal cells, are the major regulators of the systemic and intraadrenal inflammatory response to LPS. However, the full HPA axis activation in SIRS is not entirely dependent on systemic TLR signaling.     

Obstructive jaundice increases sensitivity to lipopolysaccharide via TLR4 upregulation: possible involvement in gut-derived hepatocyte growth factor-protection of hepatocytes

BACKGROUND: Patients with obstructive jaundice are prone to sepsis after biliary tract surgery. The present study was designed to determine the effect of biliary obstruction on cytokine responses to lipopolysaccharide (LPS). METHODS: Wister rats were allocated into two groups; the BDL group underwent bile duct ligation, followed 2 weeks later by administration of LPS into the duodenum. The control group underwent sham operation, and similarly received enteral LPS. Specimens were collected serially, and applied for the assays. RESULTS: Serum aspartate aminotransferase and alanine aminotransferase levels were significantly increased in BDL rats. High tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 levels in peripheral blood were observed 2 h after LPS administration in BDL rats. In contrast, no increases in both cytokines were noted in peripheral and portal blood in control rats. Baseline HGF levels in portal and peripheral blood in BDL rats were significantly higher than in control rats. LPS significantly increased hepatocyte growth factor (HGF) levels in portal blood and decreased in peripheral blood in BDL rats, but not in control rats. Immunohistochemical analysis revealed that BDL increased expressions of Toll-like receptor (TLR)4, CD14 and CD68 both in the small intestine and liver. Both TLR4 and CD14 mRNAs were upregulated in the small intestine and liver after LPS administration in BDL rats. CONCLUSION: Obstructive jaundice and LPS stimulation induced TLR4 upregulation both in the liver and small intestine, which led to increased TNF-alpha and IL-6 production in liver and HGF production in the small intestine. The upregulation of TLR4 may lead to pathological and host defense reactions in obstructive jaundice complicated with Gram-negative bacterial infection.     

慢性乙型肝炎患者外周血单核细胞Toll样受体2和4的变化

目的探讨慢性乙型肝炎和慢性重型乙型肝炎患者外周血单核细胞Toll样受体2 （TLR2）和Toll样受体4（TLR4）的变化情况及其意义。方法用流式细胞仪检测正常对照、慢性乙型肝炎患者和慢性重型乙型肝炎患者外周血单核细胞表面TLR2和TLR4的表达,ELISA法检测上述患者血清TNFα的水平。Student-t检验比较3组间TLR2、TLR4和TNFα表达的差异;3组病例TLR2、TLR4的表达水平间及它们与血清TNFα水平间的相关性分析采用线性相关分析。结果正常对照组（30例）、慢性乙型肝炎组（31例）和慢性重型乙型肝炎组（30例）外周血单核细胞TLR2和TLR4的平均免疫荧光强度（MFI）分别为21．5±2．7、39．0±4．1．47．7±21．4和2．3±1．1、3．7±2．3、6．9±4．1;外周血清TNFα（ng／L）水平分别为53．8±38．1、164．3±89．9、359．8±1 40．0,自正常对照组到慢性乙型肝炎组及慢性重型乙型肝炎组外周血单核细胞TLR2、TLR4表达强度和外周血清TNFα水平均依次显著升高;经线性相关分析研究发现,外周血单核细胞TLR2、TLR4表达水平和血清TNFα表达水平间均呈现显著的正相关性。结论TLR2和TLR4可能与慢性乙型肝炎及慢性重型乙型肝炎的发病有关。     

Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance

TLR4 and MD-2 are necessary for conferring cellular responsiveness to LPS. Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS re-stimulation, known as 'endotoxin tolerance'. While induction of LPS tolerance has been reported to correlate with down-regulation of cell surface expression of TLR4/MD-2, other mechanisms of LPS tolerance have been revealed that target intracellular intermediates downstream of the TLR4/MD-2 complex. In this study, we sought to examine whether endotoxin tolerance could be induced under conditions where expression of TLR4 and MD-2 proteins is not affected by LPS. Human HEK 293T cells are completely unresponsive to LPS, but acquire high LPS sensitivity following transient transfection with CD14, TLR4, and MD-2 (293T/CD14/TLR4/MD-2 cells), as judged by NF-kappaB activation, ERK 1/2 phosphorylation, and TNF-alpha gene expression. Prior exposure of 293T/CD14/TLR4/MD-2 cells to LPS resulted in a significant decrease of LPS-mediated responses, yet failed to affect expression levels of TLR4 and MD-2. Thus, altered expression and/or function of intracellular mediators downstream of the TLR4/MD-2 complex play an important role in mediating LPS tolerance.     

The functional variant (Asp299gly) of toll-like receptor 4 (TLR4) influences TLR4-mediated cytokine production in rheumatoid arthritis

OBJECTIVE: To investigate functional consequences of the Toll-like receptor 4 (TLR4) variant (Asp299Gly) in rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells from 28 patients with RA carrying or not carrying the TLR4 variant were incubated with lipopolysaccharide (LPS) and heat shock protein B8 (HSPB8). Concentrations of interleukin 6 (IL-6), tumor necrosis factor-alpha(TNF-alpha), and IL-10 were determined along with TLR4 and CD14 expression. RESULTS: TLR4 expression was similar in patients carrying or not carrying the variant. In contrast, both LPS and HSPB8 resulted in significantly lower secretion of IL-6, TNF-alpha, and IL-10 in those who carried the variant, whereas the frequency of CD14+ cells was higher in these individuals. CONCLUSION: TLR4 variant clearly reduces its potency to mediate signaling. Correction for CD14+ cells is necessary in comparable experiments.     

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain–containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868–880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 “pocket,” completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.Toll-like receptor 4 (TLR4) signaling plays a crucial role in host defense against Gram-negative bacteria by recognizing the outer membrane component, lipopolysaccharide (LPS) (1–3). TLR4 signaling is initiated by transfer of an LPS monomer from LPS binding protein (LBP) to cluster of differentiation 14 (CD14) (GPI-linked or soluble). In turn, CD14 transfers monomeric LPS to myeloid differentiation factor 2 (MD-2), a protein that associates noncovalently with TLR4 (4). Appropriate ligand binding to MD2 results in dimerization of two TLR4/MD2 complexes (4). TLR4 is unique in that it is the only TLR that activates both myeloid differentiation primary response 88 (MyD88) and TIR-domain–containing adapter-inducing IFN-β (TRIF)-dependent signaling pathways (5, 6). MyD88-mediated, TLR4 signaling occurs mainly at plasma membranes and involves IL-1R–associated kinases phosphorylation, association of TNF-receptor–associated factor 6, and downstream signaling that results in NF-κB activation and induction of proinflammatory mediators such as TNF-α and IL-6 (7). In contrast, TRIF-mediated signaling in response to LPS occurs at the endosomal membrane after internalization of the TLR4 that, in turn, activates IFN regulatory factor 3 (IRF3), resulting in production of IFN-β, IP-10, and other IRF-3–dependent genes, as well as delayed NF-κB activation (8). Recent studies have shown that the endocytosis of TLR4 is tightly controlled by several molecules. Rab11a, ARF6, and p120-catenin have been implicated in Escherichia coli/LPS-induced TLR4 endocytosis and IRF3 activation (9–11). Zanoni et al. showed that CD14 plays critical roles in translocation of TLR4 into endosomes and in activation of IRF3 that are dependent upon the enzymatic activities of PLCγ2 and Syk (12). However, CD14-independent TLR4 endocytosis and TRIF signaling have not been reported.UT12 is a monoclonal antibody (MAb) with specificity for the mouse TLR4/MD2 complex and mediates LPS-like signaling (13). It has been shown that UT12 induces endotoxic shock-like symptoms in mice including augmentation of TNF-α and IL-6. Furthermore, UT12 induced long-term tolerance and protection against LPS-induced lethal shock in mice (14). However, the ability of UT12 to induce translocation of TLR4/MD2 into endosomes, as well as its potential for mediating TRIF-dependent signaling, has not been reported. Recently, a group of substituted pyrimido[5,4-b]indoles, synthetic ligands for TLR4 that activate NF-κB that act in a CD14-independent manner, were discovered by high-throughput screening (15). These synthetic ligands induced IL-6 and IP-10 in a TLR4/MD2-dependent, but CD14-independent manner (16). They, too, have not been tested for TLR4 endocytosis and TRIF-dependent intermediates.In this study, we report, for the first time to our knowledge, CD14-independent translocation of TLR4 to endosomes and TRIF signaling by UT12 and small synthetic TLR4 ligands (1Z105). A TLR4 antagonist, Eritoran, that binds to a deep hydrophobic pocket in MD2 and blocks signaling induced by LPS, UT12, and 1Z105, blocked only TLR4 internalization and dimerization induced by LPS and 1Z105. Despite TLR4/MD2 internalization, endotoxin-tolerized macrophages fail to activate TRIF-mediated signaling. These findings reveal previously unidentified insights into the possible role of CD14 in LPS-mediated TLR4 endocytosis and signaling and demonstrate that TLR4 endocytosis and signaling are dissociable processes.     

HIV Type 1 Infection Up-Regulates TLR2 and TLR4 Expression and Function in Vivo and in Vitro

Abstract Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens. Their stimulation induces the activation of NF-κB, an important inducer of HIV-1 replication. In recent years, an increasing number of studies using several cells types from HIV-infected patients indicate that TLRs play a key role in regulating the expression of proinflammatory cytokines and viral pathogenesis. In the present study, the effect of HIV-1 stimulation of monocyte-derived macrophage (MDM) and peripheral blood mononuclear cell (PBMC) subpopulations from healthy donors on the expression and functions of TLR2 and TLR4 was examined. In addition, and to complete the in vitro study, the expression pattern of TLR2 and TLR4 in 49 HIV-1-infected patients, classified according to viral load and the use of HAART, was determined and compared with 25 healthy subjects. An increase of TLR expression and production of proinflammatory cytokines were observed in MDMs and PBMCs infected with HIV-1 in vitro and in response to TLR stimulation, compared to the mock. In addition, an association between TLR expression and up-regulation of CD80 in plasmacytoid dendritic cells (pDCs) was observed. The ex vivo analysis indicated increased expression of TLR2 and TLR4 in myeloid dendritic cells (mDCs), but only of TLR2 in monocytes obtained from HIV-1-infected patients, compared to healthy subjects. Remarkably, the expression was higher in cells from patients who do not use HAART. In monocytes, there was a positive correlation between both TLRs and viral load, but not CD4(+) T cell numbers. Together, our in vitro and ex vivo results suggest that TLR expression and function can be up-regulated in response to HIV-1 infection and could affect the inflammatory response. We propose that modulation of TLRs represents a mechanism to promote HIV-1 replication or AIDS progression in HIV-1-infected patients.     

The equine TLR4/MD-2 complex mediates recognition of lipopolysaccharide from Rhodobacter sphaeroides as an agonist

Lipopolysaccharide (LPS) antagonists inhibit the response of inflammatory cells to LPS, presumably by competitive inhibition, and may be of therapeutic value in the treatment of endotoxemia and sepsis. The inhibitory effects of some LPS antagonists are restricted to certain host species, however, as the same molecules can have significant endotoxic activity in other species. This species-specific recognition appears to be mediated by Toll-like receptor 4 (TLR4) and/or MD-2. We have shown previously that LPS from Rhodobacter sphaeroides ( RsLPS) is an LPS antagonist in human cells but an agonist (or LPS mimetic) in equine cells. In the present study, HEK293 cells were transfected with combinations of human and equine CD14, TLR4 and MD-2, and incubated with either RsLPS or with LPS from Escherichia coli as an endotoxin control. NF-kappaB activation was measured in a dual luciferase assay as an indicator of cellular activation. Our results indicate that E. colic LPS activated NF-kappaB in cells transfected with all combinations of the three receptor proteins, whereas RsLPS activated NF-kappaB only in cells expressing the single combination of equine TLR4 and equine MD-2. We conclude that the TLR4/MD-2 complex is responsible for recognition of RsLPS as an agonist in equine cells.     

Myeloid related proteins activate Toll-like receptor 4 in human acute coronary syndromes

Introduction

We previously reported increased expression of TLR4 on monocytes in thrombi from patients with acute coronary syndromes (ACS). In mice, myeloid related protein (MRP) 8 and MRP14, cytoplasmic proteins of neutrophils and monocytes, activate Toll-like receptor (TLR) 4 during sepsis. In human ACS, we investigated now whether the pro-inflammatory action of MRPs occurs through TLR4 in monocytes derived from thrombi.

Methods

Coronary thrombi and peripheral blood of 27 ACS patients were analyzed. CD14⁺ monocytes were isolated and incubated with TLR2 ligand PM3SKA, TLR4 ligand lipopolysaccharide (LPS), MRP8, MRP14, or MRP8/14 heterocomplex. Anti-TLR4 antibodies (HTA125) were used to block TLR4 and polymyxin B (PMB) was employed to inhibit endotoxins. Before and after stimulation, the release of TNFα was measured by ELISA and the expression of TLR4 on CD14⁺ monocytes was determined by flow cytometry. Further, selected pathways of downstream signaling were analyzed.

Results

MRP8 and MRP8/14 increased release of TNFα in cultures of CD14⁺ monocytes, more in cells derived from thrombi compared with matched peripheral blood cells (p < 0.001). LPS, MRP8, and MRP8/14, but much less PM3SKA and MRP14 alone, stimulated TNFα release, which can be inhibited by HTA125. MRP8/14 enhanced TLR4 expression on monocytes from thrombi (p < 0.001), but not on monocytes from peripheral blood of the same patients.

Conclusion

In ACS, MRP8 and MRP8/14 complex are specific ligands of TLR4, which induce the release of TNFα and probably other pro-inflammatory agents from monocytes. This specific MRP8/14-dependent pathway with striking similarities to sepsis increasing expression of TLR4 in thrombi appears to be involved in the pathogenesis of coronary occlusion and may represent a novel therapeutic target in ACS.     

Ligand-dependent Toll-like receptor 4 (TLR4)-oligomerization is directly linked with TLR4-signaling

Toll-like receptor 4 (TLR4) and MD-2 recognize lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4/MD-2. We here showed, by using in vitro transfectants, ligand-induced TLR4-oligomerization, which required both membrane CD14 and MD-2. We previously reported that lipid IVa, a lipid A precursor, is agonistic on mouse TLR4/MD-2 but antagonistic on human TLR4/MD-2 and chimeric mouse TLR4/human MD-2. Lipid IVa triggered oligomerization of mouse TLR4/MD-2 but not human TLR4/MD-2 or chimeric mouse TLR4/human MD-2. Further, lipid IVa inhibited lipid A-dependent oligomerization of chimeric mouse TLR4/human MD-2. These results demonstrate that ligand-induced TLR4-oligomerization is directly linked with TLR4-signaling and suggest that MD-2 has an important role in regulating TLR4-oligomerization.     

CD14和Toll样受体4的表达在内毒素致肝炎重型化中的意义

目的 探讨CD14和Toll样受体4(TLR4)在内毒素(LPS)诱导机体炎性因子分泌过程中的作用,阐明乙型肝炎重型化的机制. 方法 采用流式细胞学方法和逆转录聚合酶链反应等技术,检测30例慢性重型乙型肝炎患者,30例慢性乙型肝炎患者和20名健康者(对照组)的外周血单核细胞的mCD14水平及CD14 mRNA,TLR4 mRNA表达水平,同时应用动态浊度法测定患者血浆LPS水平,应用酶联免疫吸附法检测患者血清肿瘤坏死因子(TNF)α、白细胞介素(IL)-1 β,IL-6的含量.数据的统计分析采用SPSS11.5统计软件完成,分别采用单因素方差分析、非参数检验、Nemenyi法和直线相关分析.结果 外周血单个核细胞mCD14表达水平:慢性重型乙型肝炎组为74.2%±12.3%,慢性乙型肝炎组为63.6%±11.8%,对照组为60.3%±7.20/;CD14mRNA和TLR4 mRNA的相对表达:慢性重型乙型肝炎组分别为2.92±0.67和1.86±0.45,慢性乙型肝炎组分别为1.34±0.51和0.93±0.18,对照组分别为0.92±0.58和0.73±0.16,慢性重型乙型肝炎组均明显高于慢性乙型肝炎组和对照组,F值分别为11.473、85.037和102.328,P值均<0.01,差异有统计学意义.血浆LPS、IL-l,IL-6水平:慢性重型乙型肝炎组分别为(1.87±1.61)Eu/ml,(0.96±0.16)pg/ml和(68.34±48.30)pg/ml,慢性乙型肝炎组分别为(0.11±0.11)Eu/ml、(0.19±0.02)pg/ml,(19.28±4.65)pg/ml,对照组分别为(0.03±0.03)Eu/ml、(0.15±0.01)pg/ml、(12.01±3.88)pg/ml,慢性重型乙型肝炎组均明显高于慢性乙型肝炎组和对照组,χ2值分别为32.065、83.472、36.236,P值均<0.01.外周血TNFα表达水平:慢性重型乙型肝炎组为(19.78±9.25)pg/ml、慢性乙型肝炎组分别为(7.26±6.52)pg/ml、对照组分别为(4.15±4.06)pg/ml、F=35.092,P<0.01.相关分析显示,在慢性重型乙型肝炎组LPS水平与mCD14、CD14:mRNA,TLR4 mRNA表达水平呈明显的相关性,n=0.865、r2=0.415、r3=0.524,P值均<0.05.结论 LPS可能通过以CD14和TLR4为主的LPS受体激活及其信号传导,刺激炎性因子分泌的途径,在肝炎重型化过程中发挥重要作用.     

A designed TLR4/MD-2 complex to capture LPS

The family of Toll-like receptors (TLRs) is involved in the defense of an organism to microbial attack. TLR4-induced signaling is involved in infectious diseases, chronic inflammatory diseases and sepsis; therefore, we aimed at modulating TLR4-signaling via ligand-binding soluble receptors. Because recognition of microbial structures shows some species-specific traits, we specifically selected the mouse model for later in vivo studies. We first prepared the N-terminally Flag-tagged mouse (m) recombinant (r) soluble (s) fusion proteins mrsTLR4-IgGFc (T4Fc) and mrsMD-2 in Drosophila melanogaster Schneider 2 (S2) cells. The function of these molecules was tested by inhibition of synthesis of pro-inflammatory cytokines after stimulation of mouse macrophage RAW 264.7 cells with purified lipopolysaccharide (LPS). T4Fc alone had no inhibitory activity; however, a T4Fc/MD-2 complex blocked LPS activity. By analogy with 'cytokine traps', we then prepared a designer molecule (LPS-Trap) by fusing MD-2 to the C-terminus of soluble TLR4 via a flexible linker. LPS-Trap significantly inhibited TNF production by LPS-stimulated RAW 264.7 cells. Thus, the T4Fc/MD-2 complex as well as the LPS-Trap blocked LPS activity in vitro and might thus represent a new therapeutic option in sepsis by neutralization of TLR4-activating ligands.     

Wnt-4 deficiency alters mouse adrenal cortex function,reducing aldosterone production

Wnt-4 is a signaling factor with multiple roles in organogenesis, a deficiency that leads to abnormal development of the kidney, pituitary gland, female reproductive system, and mammary gland. Wnt-4 is expressed in the cortical region of the developing adrenal gland from embryonic d 11.5 onward, especially in the outermost part. Expression of Cyp11B2 and preadipocyte factor 1 is lowered in the glands of Wnt-4 mutant animals, resulting in significantly reduced aldosterone production in the newborn mutants, suggesting that Wnt-4 may be needed for proper formation of the zona glomerulosa. On the other hand, both proopiomelanocortin-derived peptide beta-endorphin and corticosterone concentration levels are elevated in Wnt-deficient mice, and the expression of Cyp17 is altered in Wnt-4 mutant females, so that it mimics the pattern specific for males. Finally, some cells that are positive for Cyp21, which is normally expressed only in the adrenal gland, are found in the gonads of Wnt-4-deficient embryos, indicating that Wnt-4 may play a role in cell migration or in the sorting of adrenal and gonadal cells during early development. In summary, these results point to a role for Wnt-4 in adrenal gland development and function.     

Immune responses of TLR5+ lamina propria dendritic cells in enterobacterial infection

Toll-like receptors (TLRs) recognize distinct microbial components and induce innate immune responses. TLR5 has been shown to recognize bacterial flagellin. Unlike other TLRs, TLR5 is not expressed on conventional dendritic cells or macrophages. By contrast, TLR5 is mainly expressed on intestinal CD11c⁺ lamina propria cells (LPCs), which do not express TLR4. These cells detect pathogenic bacteria and secreted proinflammatory cytokines, mainly in a TLR5-dependent manner. Notably, transport of pathogenic Salmonella typhimurium from the intestinal tract to mesenteric lymph nodes was impaired in Tlr5 ^−/− mice, suggesting that CD11c⁺ LPCs expressing TLR5 are used by S. typhimurium for systemic infection. CD11c⁺ LPCs consist of four subsets distinguished by differential expression patterns of CD11c and CD11b. CD11c^hiCD11b^hi LPDCs have been identified as TLR5-expressing cells. In response to flagellin, these LPDCs induce the differentiation of naive B cells into IgA⁺ plasma cells via a mechanism independent of gut-associated lymphoid tissue (GALT), and trigger the differentiation of antigen-specific Th17 and Th1 cells. These LPDCs have unique properties in that they can induce acquired immunity as well as innate immunity via TLR5 in the intestine.