Ergosta-7, 9 (11), 22-trien-3ß-ol Interferes with LPS Docking to LBP, CD14, and TLR4/MD-2 Co-Receptors to Attenuate the NF-κB Inflammatory Pathway In Vitro and Drosophila.

Ergosta-7, 9 (11), 22-trien-3ß-ol (EK100) was isolated from Cordyceps militaris, which has been used as a traditional anti-inflammatory medicine. EK100 has been reported to attenuate inflammatory diseases, but its anti-inflammatory mechanism is still unclear. We were the first to investigate the effect of EK100 on the Toll-like receptor 4 (TLR4)/nuclear factor of the κ light chain enhancer of B cells (NF-κB) signaling in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells and the green fluorescent protein (GFP)-labeled NF-κB reporter gene of Drosophila. EK100 suppressed the release of the cytokine and attenuated the mRNA and protein expression of pro-inflammatory mediators. EK100 inhibited the inhibitor kappa B (IκB)/NF-κB signaling pathway. EK100 also inhibited phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) signal transduction. Moreover, EK100 interfered with LPS docking to the LPS-binding protein (LBP), transferred to the cluster of differentiation 14 (CD14), and bonded to TLR4/myeloid differentiation-2 (MD-2) co-receptors. Compared with the TLR4 antagonist, resatorvid (CLI-095), and dexamethasone (Dexa), EK100 suppressed the TLR4/AKT signaling pathway. In addition, we also confirmed that EK100 attenuated the GFP-labeled NF-κB reporter gene expression in Drosophila. In summary, EK100 might alter LPS docking to LBP, CD14, and TLR4/MD-2 co-receptors, and then it suppresses the TLR4/NF-κB inflammatory pathway in LPS-stimulated RAW264.7 cells and Drosophila.

CD14 is a key mediator of both lysophosphatidic acid and lipopolysaccharide induction of foam cell formation.

Macrophage uptake of oxidized low-density lipoprotein (oxLDL) plays an important role in foam cell formation and the pathogenesis of atherosclerosis. We report here that lysophosphatidic acid (LPA) enhances lipopolysaccharide (LPS)-induced oxLDL uptake in macrophages. Our data revealed that both LPA and LPS highly induce the CD14 expression at messenger RNA and protein levels in macrophages. The role of CD14, one component of the LPS receptor cluster, in LPA-induced biological functions has been unknown. We took several steps to examine the role of CD14 in LPA signaling pathways. Knockdown of CD14 expression nearly completely blocked LPA/LPS-induced oxLDL uptake in macrophages, demonstrating for the first time that CD14 is a key mediator responsible for both LPA- and LPS-induced oxLDL uptake/foam cell formation. To determine the molecular mechanism mediating CD14 function, we demonstrated that both LPA and LPS significantly induce the expression of scavenger receptor class A type I (SR-AI), which has been implicated in lipid uptake process, and depletion of CD14 levels blocked LPA/LPS-induced SR-AI expression. We further showed that the SR-AI-specific antibody, which quenches SR-AI function, blocked LPA- and LPS-induced foam cell formation. Thus, SR-AI is the downstream mediator of CD14 in regulating LPA-, LPS-, and LPA/LPS-induced foam cell formation. Taken together, our results provide the first experimental evidence that CD14 is a novel connecting molecule linking both LPA and LPS pathways and is a key mediator responsible for LPA/LPS-induced foam cell formation. The LPA/LPS-CD14-SR-AI nexus might be the new convergent pathway, contributing to the worsening of atherosclerosis.

Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes.

We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand-receptor (antigen-antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct ( k+= (5.6 ± 0.2) × 107 M-1 min-1 ) and reverse ( k-= (1.3 ± 0.2) × 10-2 min-1 ) rate constants of ligand-receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

Interaction of CD14 haplotypes and soluble CD14 on pulmonary function in agricultural workers.

BACKGROUND: Agricultural environments are contaminated with organic dusts containing bacterial components. Chronic inhalation of organic dusts is implicated in respiratory diseases. CD14 is a critical receptor for gram-negative lipopolysaccharide; however, its association with respiratory disease among agricultural workers is unknown. The objective of this study was to determine if serum soluble CD14 (sCD14) levels are associated with lung function among agricultural workers and if this association is modified by genetic variants in CD14. METHODS: This cross-sectional study included 584 veterans with >2 years of farming experience and that were between the ages of 40 and 80 years. Participants underwent spirometry and were genotyped for four tagging CD14 polymorphisms (CD14/-2838, rs2569193; CD14/-1720, rs2915863; CD14/-651, rs5744455; and CD14/-260, rs2569190). Serum sCD14 was assayed by ELISA. RESULTS: Subjects were 98% white males with a mean age 64.5 years. High soluble CD14 levels (> median sCD14) were associated decreased lung function (FEV1/FVC, p = 0.011; % predicted FEV1, p = 0.03). When stratified by COPD (yes/no) and smoking status (ever/never), high sCD14 levels (> median sCD14) were associated with low lung function among ever smokers with COPD (% predicted FEV1, padj = 0.0008; FEV1/FVC, padj = 0.0002). A similar trend was observed for never smokers with COPD; however, results did not reach statistical significance due to small sample size. There was a significant sCD14 x COPD/smoking interaction with lung function (% predicted FEV1, pinter = 0.0498; FEV1/FVC, pinter = 0.011). Regression models were adjusted for age, body mass index, education, sex, race and years worked on a farm. No association was found between CD14 polymorphisms/haplotypes (CD14/-2838; CD14/-1720; CD14/-651; CD14/-260) and sCD14 levels. The final model included the variables sCD14 and haplotypes and a haplotype x sCD14 interaction term. Individuals with the GTTG haplotype (CD14/-2838 → CD14/-260) and high sCD14 levels (> median sCD14) had on average 6.94 lower % predicted FEV1 than individuals with the GCCA haplotype and low sCD14 levels (≤ median sCD14, padj = 0.03). CONCLUSION: CD14 haplotypes and sCD14 are important mediators of lung function among those with COPD in this occupationally-exposed population.

The crystal structure of human soluble CD14 reveals a bent solenoid with a hydrophobic amino-terminal pocket.

Human monocyte differentiation Ag CD14 is a pattern recognition receptor that enhances innate immune responses to infection by sensitizing host cells to bacterial LPS (endotoxin), lipoproteins, lipoteichoic acid, and other acylated microbial products. CD14 physically delivers these lipidated microbial products to various TLR signaling complexes that subsequently induce intracellular proinflammatory signaling cascades upon ligand binding. The ensuing cellular responses are usually protective to the host but can also result in host fatality through sepsis. In this work, we have determined the x-ray crystal structure of human CD14. The structure reveals a bent solenoid typical of leucine-rich repeat proteins with an amino-terminal pocket that presumably binds acylated ligands including LPS. Comparison of human and mouse CD14 structures shows great similarity in overall protein fold. However, compared with mouse CD14, human CD14 contains an expanded pocket and alternative rim residues that are likely to be important for LPS binding and cell activation. The x-ray crystal structure of human CD14 presented in this article may foster additional ligand-bound structural studies, virtual docking studies, and drug design efforts to mitigate LPS-induced sepsis and other inflammatory diseases.

Ingested soluble CD14 from milk is transferred intact into the blood of newborn rats.

BACKGROUND: Milk acts as an edible immune system that is transferred from mother to newborn. Soluble Cluster of Differentiation 14 (sCD14) is a protein found in significant quantities in human milk (~8-29 µg/ml). At a 10-fold lower concentration in the blood (~3 µg/ml), the most notable role of sCD14 is to sequester lipopolysaccharides of Gram-negative bacteria from immune cells. METHODS: To explore the pharmacodynamics of this milk protein and its biological fate, the biodistribution of radiolabeled sCD14 ((14)C, (125)I) was monitored in 10-d-old rat pups. RESULTS: Up to 3.4 ± 2.2% of the radiolabeled sCD14 administered was observed, intact, in the pup blood for up to 8 h post-ingestion. Additionally, 30.3 ± 13.0% of the radiolabeled sCD14 administered was observed degraded in the stomach at 8 h post-ingestion. A reservoir of intact, administered sCD14 (3.2 ± 0.3%), however, remained in the stomach at 8 h post-ingestion. Intact sCD14 was observed in the small intestine at 5.5 ± 1.6% of the dose fed at 8 h post-ingestion. CONCLUSION: The presence of intact sCD14 in the blood and the gastrointestinal tract of newborns post-ingestion has implications in the development of allergies, obesity, and other inflammation-related pathogeneses later in life.

Probing the cell membrane by magnetic particle actuation and Euler angle tracking.

The mechanical properties of the cell membrane and the subjacent actin cortex are determinants of a variety of processes in immunity and cell division. The lipid bilayer itself and its connection to the actin cortex are anisotropic. An accurate description of the mechanical structure of the cell membrane and the involved dynamics therefore necessitates a measurement technique that can capture the inherent anisotropy of the system. Here, we combine magnetic particle actuation with rotational and translational particle tracking to simultaneously measure the mechanical stiffness of monocytic cells in three rotational and two translational directions. When using particles that bind via integrins to the cell membrane and the subjacent cortex, we measured an isotropic stiffness and a characteristic power-law dependence of the shear modulus on the applied frequency. When using particles functionalized with immunoglobulin G, we measured an anisotropic stiffness with a 10-fold-reduced value in one dimension. We suggest that the observed reduced stiffness in the plane of the cell membrane is caused by a local detachment of the lipid bilayer from the subjacent cytoskeletal cortex. We expect that our technique will enable new insights into the mechanical properties of the cell membrane that will help us to better understand membrane processes such as phagocytosis and blebbing.

Usefulness of presepsin (sCD14-ST) measurements as a marker for the diagnosis and severity of sepsis that satisfied diagnostic criteria of systemic inflammatory response syndrome.

CD14 is present in macrophage, monocyte, and granulocyte cells and their cell membranes, and it is said to be responsible for intracellular transduction of endotoxin signals. Its soluble fraction is present in blood and is thought to be produced in association with infections. It is called the soluble CD14-subtype (sCD14-ST), and in the following text it is referred to by its generic name, presepsin. We have previously reported that presepsin is produced in association with infection and that it is specifically expressed in sepsis. In the present study we developed a new rapid diagnostic method by using a chemiluminescent enzyme immunoassay that allowed making automated measurements in a shorter time. The results of using this method to measure presepsin values in different pathological conditions were normal, 294.2 ± 121.4 pg/ml; local infection, 721.0 ± 611.3 pg/ml; systemic inflammatory response syndrome, 333.5 ± 130.6 pg/ml; sepsis, 817.9 ± 572.7 pg/ml; and severe sepsis, 1,992.9 ± 1509.2 pg/ml; the presepsin values were significantly higher in patients with local infection, sepsis, and severe sepsis than in patients who did not have infection as a complication. In a comparative study with other diagnostic markers of sepsis based on ROC curves, the area under the curve (AUC) of presepsin was 0.845, and greater than the AUC of procalcitonin (PCT, 0.652), C-reactive protein (CRP, 0.815), or interleukin 6 (IL-6, 0.672). In addition, a significant correlation was found between the APACHE II scores, an index of disease severity, and the presepsin values, suggesting that presepsin values can serve as a parameter that closely reflects the pathology.

Mass spectrometry enables the discovery of inhibitors of an LPS transport assembly via disruption of protein-protein interactions.

We developed a native mass spectrometry-based approach to quantify the monomer-dimer equilibrium of the LPS transport protein LptH. We use this method to assess the potency and efficacy of an antimicrobial peptide and small molecule disruptors, obtaining new information on their structure-activity relationships. This approach led to the identification of quinoline-based hit compounds representing the basis for the development of novel LPS transport inhibitors.

The antifungal agent itraconazole induces the accumulation of high mannose glycoproteins in macrophages.

Bacterial lipopolysaccharide (LPS) is a key mediator in the development of Gram-negative septic shock, which is a major health problem. The effect of LPS on myeloid cells is mediated by a multicomplex receptor system in which CD14, a glycosylphosphatidylinositol-anchored glycoprotein, and Toll-like receptor 4 are the major players. We have found that incubation of macrophages with itraconazole (ICZ), an azole antifungal commonly used in humans, altered both the expression and glycosylation of CD14. This glycoprotein, which is endo H-resistant in untreated cells, becomes endo H-sensitive following ICZ treatment. The effect of ICZ on glycan processing was observed in all newly synthesized glycoproteins as indicated by incorporation of [2-(3)H]mannose. In addition, cells treated with ICZ increased surface concanavalin A (ConA) binding, corroborating an increase in high mannose surface glycoproteins. Although the glycosylation pattern of CD14 was altered, this glycoprotein was delivered to the cell surface or was secreted. Moreover, it appeared functional as demonstrated by the release of LPS-induced tumor necrosis factor-alpha under conditions specific for a CD14-mediated activation process. The effect of ICZ on glycosylation was not dependent on inhibition of the cholesterol biosynthetic pathway and was specific for this drug because other azole antifungals, such as ketoconazole and econazole, did not alter glycan processing. These results suggest a possible secondary effect of ICZ that impacts the processing of glyconjugates and may alter cellular function and homeostasis.

Improved characterization of nod factors and genetically based variation in LysM Receptor domains identify amino acids expendable for nod factor recognition in Lotus spp.

Formation of functional nodules is a complex process depending on host-microsymbiont compatibility in all developmental stages. This report uses the contrasting symbiotic phenotypes of Lotus japonicus and L. pedunculatus, inoculated with Mesorhizobium loti or the Bradyrhizobium sp. (Lotus), to investigate the role of Nod factor structure and Nod factor receptors (NFR) for rhizobial recognition, infection thread progression, and bacterial persistence within nodule cells. A key contribution was the use of 800 MHz nuclear magnetic resonance spectroscopy and ultrahigh-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry for Nod factor analysis. The Nod factor decorations at the nonreducing end differ between Bradyrhizobium sp. (Lotus) and M. loti, and the NFR1/NFR5 extracellular regions of L. pedunculatus and L. japonicus were found to vary in amino acid composition. Genetic transformation experiments using chimeric and wild-type receptors showed that both receptor variants recognize the structurally different Nod factors but the later symbiotic phenotype remained unchanged. These results highlight the importance of additional checkpoints during nitrogen-fixing symbiosis and define several amino acids in the LysM domains as expendable for perception of the two differentially carbamoylated Nod factors.

Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses.

Mammals mount a rapid inflammatory response to gram-negative bacteria by recognizing lipopolysaccharide (LPS, endotoxin). LPS binds to CD14, and the resulting LPS-CD14 complex induces synthesis of cytokines and up-regulation of adhesion molecules in a variety of cell types. Gram-positive bacteria provoke a very similar inflammatory response, but the molecules that provoke innate responses to these bacteria have not been defined. Here we show that protein-free, phenol extracts of Staphylococcus aureus contain a minor component that stimulates adhesion of neutrophils and cytokine production in monocytes and in the astrocytoma cell line, U373. Responses to this component do not absolutely require CD14, but addition of soluble CD14 enhances sensitivity of U373 cells by up to 100-fold, and blocking CD14 on monocytes decreases sensitivity nearly 1,000-fold. Deletion of residues 57-64 of CD14, which are required for responses to LPS, also eliminates CD14-dependent responses to S. aureus molecules. The stimulatory component of S. aureus binds CD14 and blocks binding of radioactive LPS. Unlike LPS, the activity of S. aureus molecules was neither enhanced by LPS binding protein nor inhibited by bactericidal/permeability increasing protein. The active factor in extracts of S. aureus is also structurally and functionally distinct from the abundant species known as lipoteichoic acid (LTA). Cell-stimulating activity fractionates differently from LTA on a reverse-phase column, pure LTA fails to stimulate cells, and LTA antagonizes the action of LPS in assays of IL-6 production. These studies suggest that mammals may use CD14 in innate responses to both gram-negative and gram-positive bacteria, and that gram-positive bacteria may contain an apparently unique, CD14-binding species that initiates cellular responses.

Alpha-lactalbumin in human milk alters the proteolytic degradation of soluble CD14 by forming a complex.

Mother's milk represents a foundational step in the proper development of newborn immunity. This is achieved, in part, through the action of numerous regulatory proteins such as soluble cluster of differentiation 14 (sCD14) found in significant quantities in human milk (~25-50 µg/mL). In adults, CD14 stimulates cytokine production in response to lipopolysaccharide (LPS), the major lipid component found in the outer membrane of Gram-negative bacteria. However, the fate and function of sCD14 in the neonatal gastrointestinal (GI) tract are unknown and may function differently from adults. Therefore, we administered human sCD14 to experimental animals and observed that it persisted in the upper GI tract after feeding. In our search for potential proteolytic protectants, immunoprecipitation of sCD14 from human milk revealed a 15-kD novel protein that copurified with sCD14. Mass spectrometry analysis of the protein identified alpha-lactalbumin. CD14 was also identified by immunoblot after immunoprecipitation of alpha-lactalbumin from milk. In vitro digestion assays revealed that purified alpha-lactalbumin decreases the proteolytic degradation of human milk derived sCD14 in vitro, suggesting a mechanism by which this key LPS receptor may remain functional in the neonate gut.

Evidence of a specific interaction between new synthetic antisepsis agents and CD14.

Synthetic molecules derived from natural sugars with a positively charged amino group or ammonium salt and two lipophilic chains have been shown to inhibit TLR4 activation in vitro and in vivo. To characterize the mechanism of action of this class of molecules, we investigated possible interactions with the extracellular components that bind and shuttle endotoxin [lipopolysaccharide (LPS)] to TLR4, namely, LBP, CD14, and MD-2. Molecules that inhibited TLR4 activation inhibited LBP.CD14-dependent transfer of endotoxin monomers derived from aggregates of tritiated lipooligosaccharide ([(3)H]LOS) from Neisseria meninigitidis to MD-2.TLR4, resulting in a reduced level of formation of a ([(3)H]LOS.MD-2.TLR4(ECD))(2) (M(r) approximately 190000) complex. This effect was due to inhibition of the transfer of [(3)H]LOS from aggregates in solution to sCD14 with little or no effect on [(3)H]LOS shuttling from [(3)H]LOS.sCD14 to MD-2. These compounds also inhibited transfer of the [(3)H]LOS monomer from full-length CD14 to a truncated, polyhistidine-tagged CD14. Dose-dependent inhibition of the transfer of [(3)H]LOS between the two forms of CD14 was observed with each of three different synthetic compounds that inhibited TLR4 activation but not by another structurally related analogue that lacked TLR4 antagonistic activity. Saturation transfer difference (STD) NMR data showed direct binding to CD14 by the synthetic TLR4 antagonist mediated principally through the lipid chains of the synthetic compound. Taken together, our findings strongly suggest that these compounds inhibit TLR4 activation by endotoxin by competitively occupying CD14 and thereby reducing the level of delivery of activating endotoxin to MD-2.TLR4.

NMR spectral mapping of Lipid A molecular patterns affected by interaction with the innate immune receptor CD14.

Soluble CD14 (sCD14) is a serum glycoprotein that binds to the Lipid A moiety of lipopolysaccharides (LPS) with high affinity as part of the innate immune response to bacterial endotoxins. In order to investigate structural interactions of Lipid A with sCD14, we have prepared an isotopically labeled form of a fully active and chemically defined endotoxin, Kdo(2)-Lipid A, which allowed us to carry out detailed NMR spectral mapping of this agonist ligand bound to sCD14 and identify for the first time structural regions that are strongly affected during complex formation with sCD14. These map to two adjacent areas comprising the lower portions of the sugar headgroup and upper half of the acyl chains I, III, and V, which are spatially proximal to the 1- and 4'-phosphate ends. Additionally, we have detected for the first time, presence of differential dynamic behavior for the affected resonances, suggesting a likely role for dynamics in the mechanism of Lipid A pattern recognition by sCD14.

Chicken CD14, unlike mammalian CD14, is trans-membrane rather than GPI-anchored.

A cDNA encoding the chicken homologue of the human myelomonocytic differentiation antigen, CD14, was cloned by RT-PCR from chicken bone marrow cell RNA, using oligonucleotide primers based on the predicted cDNA sequence. The cloned chicken CD14 (chCD14) cDNA encodes an open reading frame of 465 amino acids (aa), with 31-34% aa identity to mouse, bovine and human (hu) CD14. As in mouse and man, chCD14 is a leucine-rich protein. In mammals, CD14 is a GPI-anchored protein. Protein structure analysis suggested that chCD14, by contrast, was potentially a trans-membrane protein. The predicted aa sequence comprises an extracellular domain of 417 aa, followed by a 23-aa trans-membrane segment, and a 25-aa intracytoplasmic region, the latter containing no obvious signalling motifs. COS-7 cells were transfected with p3XFLAG-CMV-8::chCD14 or pCDM8::huCD14, incubated with or without PI-PLC and stained with anti-FLAG or anti-huCD14 antibody respectively. PI-PLC cleaved huCD14 but not chCD14, suggesting that chCD14 is not GPI-anchored. Real-time quantitative RT-PCR analysis revealed that chCD14 mRNA was expressed in most lymphoid and non-lymphoid tissues, except muscle. ChCD14 mRNA was also expressed in most cells examined but strongly expressed in chicken peripheral blood monocyte/macrophages and KUL01+ splenocytes.

CD14: Biology and role in the pathogenesis of disease.

Human monocyte differentiation antigen CD14 is a pattern recognition receptor (PRR) that enhances innate immune responses. CD14 was first identified as a marker of monocytes to signal intracellular responses upon bacterial encounters. Given the absence of an intracellular tail, CD14 was doubted to have the signaling capacities. Later CD14 was confirmed as the TLR co-receptor for the detection of pathogen-associated molecular patterns. However, CD14 has been revealed as a multi-talented receptor. In last decade, CD14 was identified to activate NFAT to regulate the life cycle of myeloid cells in a TLR4-independent manner and to transport inflammatory lipids to induce phagocyte hyperactivation. And its influences on multiple related diseases have been further considered. In this review, we summarize advancements in the basic biology of the CD14 including its structure, binding ligands, signaling pathways, and its roles in the pathogenesis of inflammation, atherosclerosis, tumor and metabolic diseases. We also discuss the therapeutic potential of targeting the CD14 in related diseases.

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14.

CD14 functions as a key pattern recognition receptor for a diverse array of Gram-negative and Gram-positive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy, the binding of three different endotoxin ligands, lipopolysaccharide, lipoteichoic acid, and a PGN-derived compound, muramyl dipeptide to a 15N isotopically labeled 152-residue N-terminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site.

Pattern recognition receptors and their role in innate immunity: focus on microbial protein ligands.

Antigen-presenting cells, such as macrophages and dendritic cells, represent a central and important part of the immune defence against invading microorganisms, as they participate in initial capture and processing of microbial antigens (innate immunity) and then activation of specific T and B cell effector mechanisms (acquired immunity). Recognition of microbial molecules by antigen-presenting cells occurs through so called pattern recognition receptors (PRRs), which recognize conserved structures, or pathogen-associated molecular patterns, in pathogenic microbes. The Toll-like receptors are the most extensively studied of these receptors, but accumulating evidence shows that other PRRs, such as scavenger receptors, C-type lectin receptors and NOD-like receptors, also play important roles in the innate immune defence. Here, we summarize current knowledge of the role of various PRRs in the defence against pathogenic microorganisms and we report recent advances in studies of different receptor-ligand interactions. In particular, we focus on the importance of microbial proteins as ligands for PRRs.

CD14: a soluble pattern recognition receptor in milk.

An innate immune system capable of distinguishing among self, non-self, and danger is a prerequisite for health. Upon antigenic challenge, pattern recognition receptors (PRRs), such as the Toll-like receptor (TLR) family of proteins, enable this system to recognize and interact with a number of microbial components and endogenous host proteins. In the healthy host, such interactions culminate in tolerance to self-antigen, dietary antigen, and commensal microorganisms but in protection against pathogenic attack. This duality implies tightly regulated control mechanisms that are not expected of the inexperienced neonatal immune system. Indeed, the increased susceptibility of newborn infants to infection and to certain allergens suggests that the capacity to handle certain antigenic challenges is not inherent. The observation that breast-fed infants experience a lower incidence of infections, inflammation, and allergies than formula-fed infants suggests that exogenous factors in milk may play a regulatory role. There is increasing evidence to suggest that upon exposure to antigen, breast milk educates the neonatal immune system in the decision-making processes underlying the immune response to microbes. Breast milk contains a multitude of factors such as immunoglobulins, glycoproteins, glycolipids, and antimicrobial peptides that, qualitatively or quantitatively, may modulate how neonatal cells perceive and respond to microbial components. The specific role of several of these factors is highlighted in other chapters in this book. However, an emerging concept is that breast milk influences the neonatal immune system's perception of "danger." Here we discuss how CD14, a soluble PRR in milk, may contribute to this education.