Endotoxin Recognition Research Articles

Human inflammatory response of endotoxin affected by particulate matter-bound transition metals

Bacterial endotoxins are a component of particulate matter (PM) with anticipated health implications, yet we know little about how host reception of endotoxin through toll-like receptor 4 (TLR4) is affected by its association with other PM components. Subsequently, we investigated the relationship between endotoxin concentration (recombinant Factor C (rFC) assay) and host recognition (HEK Blue-TLR4 NF-kB reporter cell line based assay) in various compositions of urban PM, including road traffic, industrial and urban green land use classes. While the assays did not correlate strongly between each other, the TLR4 reporter cell line was found to be better correlated to the IL-8 response of PM. Furthermore, the ability of the quantified endotoxin (rFC assay) to stimulate the TLR4/MD-2 complex was significantly affected by the urban land use class, where traffic locations were found to be significantly higher in bioactive endotoxin than the industrial and green locations. We subsequently turned our attention to PM composition and characterized the samples based on transition metal content (through ICP-MS). The effect of nickel and cobalt – previously reported to activate the hTLR4/MD-2 complex – was found to be negligible in comparison to that of iron. Here, the addition of iron as a factor significantly improved the regression model between the two endotoxin assays, explaining 77% of the variation of the TLR4 stimulation and excluding the significant effect of land use class. Moreover, the effect of iron proved to be more than a correlation, since dosing LPS with Fe2+ led to an increase up to 64% in TLR4 stimulation, while Fe2+ without LPS was unable to stimulate a response. This study shows that endotoxin quantification assays (such as the rFC assay) may not always correspond to human biological recognition of endotoxin in urban PM, while its toxicity can be synergistically influenced by the associated PM composition.

Endotoxin-Free E. coli-Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production.

Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated.

A Thermodynamic Funnel Drives Bacterial Lipopolysaccharide Transfer in the TLR4 Pathway

The Gram-negative bacterial outer membrane contains lipopolysaccharide, which potently stimulates the mammalian innate immune response. This involves a relay of specialized complexes culminating in transfer of lipopolysaccharide from CD14 to Toll-like receptor 4 (TLR4) and its co-receptor MD-2 on the cell surface, leading to activation of downstream inflammatory responses. In this study we develop computational models to trace the TLR4 cascade innear-atomic detail. We demonstrate through rigorous thermodynamic calculations that lipopolysaccharide molecules traversing the receptor cascade fall into a thermodynamic funnel. An affinity gradient for lipopolysaccharide is revealed upon extraction from aggregates or realistic bacterial outer membrane models and transfer through CD14 to the terminal TLR4/MD-2 receptor-co-receptor complex. We subsequently assemble viable CD14/TLR4/MD-2 oligomers at the plasma membrane surface, and observe lipopolysaccharide exchange between CD14 and TLR4/MD-2. Collectively, this work helps to unravel the key structural determinants governing endotoxin recognition in the TLR4 innate immune pathway.

Diverse pro-inflammatory endotoxin recognition systems of mammalian innate immunity.

In humans and other mammals, recognition of endotoxins—abundant surface lipopolysaccharides (LPS) of Gram-negative bacteria—provides a potent stimulus for induction of inflammation and mobilization of host defenses. The structurally unique lipid A region of LPS is the principal determinant of this pro-inflammatory activity. This region of LPS is normally buried within the bacterial outer membrane and aggregates of purified LPS, making even more remarkable its picomolar potency and the ability of discrete variations in lipid A structure to markedly alter the pro-inflammatory activity of LPS. Two recognition systems—MD-2/TLR4 and “LPS-sensing” cytosolic caspases—together confer LPS responsiveness at the host cell surface, within endosomes, and at sites physically accessible to the cytosol. Understanding how the lipid A of LPS is delivered and recognized at these diverse sites is crucial to understanding how the magnitude and character of the inflammatory responses are regulated.

High-affinity caspase-4 binding to LPS presented as high molecular mass aggregates or in outer membrane vesicles.

Caspases of the non-canonical inflammasome (caspases -4, -5, and -11) directly bind endotoxin (LOS/LPS) and can be activated in the absence of any co-factors. Models of LPS-induced caspase activation have postulated that 1:1 binding of endotoxin monomers to caspase trigger caspase oligomerization and activation, analogous to that established for endotoxin-induced activation of MD-2/TLR4. However, using metabolically radiolabeled LOS and LPS, we now show high affinity and selective binding of caspase-4 to high molecular mass aggregates of purified endotoxin and to endotoxin-rich outer membrane vesicles without formation of 1:1 endotoxin:caspase complexes. Thus, our findings demonstrate markedly different endotoxin recognition properties of caspase-4 from that of MD-2/TLR4 and strongly suggest that activation of caspase-4 (and presumably caspase-5 and caspase-11) are mediated by interactions with activating endotoxin-rich membrane interfaces rather than by endotoxin monomers.

The Effect of Elevated Intra-Abdominal Pressure on TLR4 Signaling in Intestinal Mucosa and on Intestinal Bacterial Translocation in a Rat.

Recent evidence suggests that elevated intra-abdominal pressure (IAP) may adversely affect the intestinal barrier function. Toll-like receptor 4 (TLR-4) is responsible for the recognition of bacterial endotoxin or lipopolysaccharide and for initiation of the Gram-negative septic shock syndrome. The objective of the current study was to determine the effects of elevated IAP on intestinal bacterial translocation (BT) and TLR-4 signaling in intestinal mucosa in a rat model. Male Sprague-Dawley rats were randomly assigned to one of two experimental groups: sham animals (Sham) and IAP animals who were subjected to a 15 mmHg pressure pneumoperitoneum for 30 minutes. Rats were sacrificed 24 hours later. BT to mesenteric lymph nodes, liver, portal vein blood, and peripheral blood was determined at sacrifice. TLR4-related gene and protein expression (TLR-4; myeloid differentiation factor 88 [Myd88] and TNF-α receptor-associated factor 6 [TRAF6]) expression were determined using real-time PCR, western blotting, and immunohistochemistry. Thirty percent of sham rats developed BT in the mesenteric lymph nodes (level I) and 20% of control rats developed BT in the liver and portal vein (level II). abdominal compartment syndrome (ACS) rats demonstrated an 80% BT in the lymph nodes (Level I) and 40% BT in the liver and portal vein (Level II). Elevated BT was accompanied by a significant increase in TLR-4 immunostaining in jejunum (51%) and ileum (35.9%), and in a number of TRAF6-positive cells in jejunum (2.1%) and ileum (24.01%) compared to control animals. ACS rats demonstrated a significant increase in TLR4 and MYD88 protein levels compared to control animals. Twenty-four hours after the induction of elevated IAP in a rat model, increased BT rates were associated with increased TLR4 signaling in intestinal mucosa.

Treatment of endotoxaemia and septicaemia in the equine patient.

Endotoxins, constituents of the cell wall of gram-positive and gram-negative bacteria, regularly result in severe illness and death in horses. In endotoxaemia, these constituents are present in the systemic circulation; in septicaemia, whole microbes invade normally sterile parts of the body. Interaction of these endotoxins with pathogen recognition receptors leads to an inflammatory response that cannot always be sufficiently contained and hence needs direct treatment. Over the last decennia, our understanding of the pathophysiology of endotoxaemia and septicaemia has significantly increased. Based on improved understanding of the interaction between receptors and endotoxins as well as the subsequent downstream signalling pathways, new therapeutic targets have been identified in laboratory animal species and humans. Important species differences in the recognition of endotoxins and pathogens by their receptors as well as the inflammatory response to receptor activation hamper extrapolation of this information to the horse (and other species). Historically, horses with endotoxaemia and septicaemia have been treated mainly symptomatically and supportively. Based on the identified therapeutic targets, this review describes the current knowledge of the treatment for endotoxaemia and septicaemia in the horse with reference to the findings in other animal species and humans.

Ultrasensitive detection of endotoxins using computationally designed nanoMIPs

Novel molecularly imprinted polymer nanoparticles (nanoMIPs) were designed for endotoxin from Escherichia coli 0111:B4, using computational modeling. The screening process based on binding energy between endotoxin and each monomer was performed with 21 commonly used monomers, resulting in the selection of itaconic acid, methacrylic acid and acrylamide as functional monomers due to their strong binding interaction with the endotoxin template. The nanoMIPs were successfully synthesized with functional groups on the outer surface to aid in the immobilization onto sensor surface. The solid phase photopolymerization approach used for the synthesis of nanoMIPs ranging from 200 to 235 nm in diameter. The limit of detection and KD were significantly improved when endotoxin samples were prepared using a novel triethylamine method. This improved the efficiency of gold nanoparticle functionalization by targeting the subunits of the endotoxin. Compared to the vancomycin MIP control, the endotoxin MIPs displayed outstanding affinity and selectivity towards the endotoxin with KD values in the range of 4.4–5.3 × 10−10 M, with limits of detection of 0.44 ± 0.02 ng mL−1 as determined by surface plasmon resonance (SPR) sensor when itaconic acid was used as the functional monomer. The MIP surface can be regenerated >30 times without significant loss of binding activity making this approach highly cost effective for expensive analyte templates. The combination of molecular modeling and solid phase synthesis enabled the successful synthesis of nanoMIPs capable of recognition and ultrasensitive detection of endotoxins using the highly sensitive SPR biosensor with triethylamine method.

Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response.

Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the “membrane-like” nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics.

Vasopressin inhibits endotoxin binding in activated macrophages

BackgroundVasopressin possesses potent anti-inflammatory effects. Endotoxin recognition (mediated by cluster of differentiation 14 [CD14]), endotoxin binding, and subsequent nuclear factor-κB (NF-κB) activation are essential mechanisms for initiation of the inflammatory response. We elucidated the effects of vasopressin on these essential mechanisms of inflammation with the hypothesis that vasopressin could inhibit CD14 expression, endotoxin binding, and NF-κB activation in activated macrophages. MethodsMurine macrophage-like cell line RAW264.7 cells were stimulated with endotoxin (lipopolysaccharide [LPS]; 100 ng/mL) or LPS plus vasopressin (1000 pg/mL; designated as the LPS and the LPS + V groups, respectively). After reaction, between-group differences in inflammatory molecule concentrations and levels of NF-κB activation, endotoxin-macrophages binding, and CD14 expression were compared. Analysis of variance was performed for statistical analysis. ResultsThe concentrations of chemokine macrophage inflammatory protein 2 and cytokine interleukin 6 of the LPS + V group were significantly lower than those of the LPS group (P = 0.004 and P < 0.001). The nuclear concentration of phosphorylated NF-κB p65 and cytosolic concentration of phosphorylated inhibitor-κBα of the LPS + V group were significantly lower than those of the LPS group (all P < 0.05). In addition, the level of endotoxin-macrophages binding of the LPS + V group was significantly lower than that of the LPS group (P < 0.001). The level of surface CD14 expression of the LPS + V group was also significantly lower than that of the LPS group (P = 0.019). ConclusionsThis study confirmed the potent anti-inflammatory effects of vasopressin. The mechanisms underlying the anti-inflammatory effects of vasopressin may involve its effects on inhibiting CD14 expression, endotoxin binding, and subsequent NF-κB activation.

Neutrophil and monocyte toll-like receptor 4, CD11b and reactive oxygen intermediates, and neuroimaging outcomes in preterm infants.

Activated leukocytes and infection are implicated in neonatal brain injury. Leukocyte surface receptors are increased in stroke models and may be targets for future adjunctive therapies. Serial blood samples were analyzed from preterm infants (n = 51; <32 wk gestation) on days 0, 1, 2, and 7 of life. Monocyte and neutrophil activation were evaluated via flow cytometry at baseline and following endotoxin stimulation ex vivo by measuring CD11b (activation), toll-like receptor 4 (TLR-4; endotoxin recognition) expression, and intracellular reactive oxygen intermediate (ROI) production (function). Control preterm infants with normal neuroimaging had elevated baseline CD11b and TLR-4 expression and ROI production compared with adults as well as a robust immune response following endotoxin stimulation. Preterm infants with abnormal neuroimaging had increased neutrophil TLR-4 and ROI compared with all controls. Preterm infants have a robust immune response compared with adults. Increased TLR-4 expression in preterm infants with abnormal neuroimaging is similar to findings in adult stroke. In addition, ROI production may cause tissue injury. The modulation of these responses may be beneficial in preterm inflammatory disorders.

Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy

Aim: Circulating immune cell activation is associated with worse outcome in adult and animal models of brain injury. Our aim was to profile the systemic inflammatory response over the first week of life in infants at risk of neonatal encephalopathy and correlate early neutrophil and monocyte endotoxin and activation responses with outcome. Methods: Prospective observational study in a tertiary referral university hospital including 22 infants requiring resuscitation at birth who had serial (five time points) neutrophil and monocyte CD11b (marker of cell adhesion) (intracellular Reactive oxygen intermediates) ROI (cell activation), and Toll-like receptor (endotoxin recognition) before and after endotoxin stimulation ex vivo compared to neonatal controls. Results: All neonates requiring resuscitation at delivery (n = 122 samples) had higher neutrophil and monocyte CD11b and TLR-4 expressions compared with adults and neonatal controls. Neonates with abnormal neuroimaging and/or severe neonatal encephalopathy had increased CD11b, ROI and TLR-4. Increased PMN TLR-4 expression was associated with increased mortality in infants with neonatal encephalopathy (NE). Conclusion: Innate immune dysregulation in the first week of life is associated with severity of outcome in neonatal brain injury in this cohort and may be amenable to immunomodulation.

Species-specific activation of TLR4 by hypoacylated endotoxins governed by residues 82 and 122 of MD-2.

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.

Role of endotoxin in the etiology of periapical lesions: molecular mechanisms involved in endotoxin's recognition and cell activation

Periapical lesions are diseases resulting from contamination and necrosis of dental pulp and the progression of this infection toward the periapical tissues. Technological advances in microbiological culture and identification have shown that anaerobic microorganisms, especially Gram-negative, are predominate in root canals of teeth with pulp necrosis and chronic periapical lesions. Gram-negative bacteria not only have different factors of virulence and generate products and sub-products that are toxic to apical and periapical tissues, but also contain endotoxin in the outer membrane of their cell wall. This information is important because endotoxin is released during multiplication or bacterial death, causing a series of biological effects that lead to an inflammatory reaction and resorption of mineralized tissues. Thus, the aim of this work is to carry out a review of the literature on the role of bacterial endotoxin in the etiology of periapical lesions, its mechanism of action, and to elucidate molecular mechanisms involved in endotoxin's recognition by the immune system and cell activation.

Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins.

A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

Microcantilever array biosensors for detection and recognition of Gram-negative bacterial endotoxins

Gram-negative sepsis is an increasingly common medical problem, in particular in developed countries. Toxic effects associated with Gram-negative sepsis are driven mainly by lipopolysaccharides (endotoxin), a species specific component of the outer membrane of bacterial cell wall. Quick and accurate detection of the endotoxin and determination of the pathogen species responsible for the infection are crucial for prompt and effective treatment. Micromechanical biosensor arrays can be an effective approach for construction of endotoxin (lipopolysaccharides) biosensors. In this work, we present the preparation and application of a microcantilever array for the detection and recognition of Gram-negative bacterial endotoxins. Self-assembling monolayers on sensing elements were functionalized with monoclonal antibodies specific to endotoxins of choice. Purity and cross-reactivity of antibodies were verified using SDS-PAGE and ELISA analyses. A home-made micromechanical sensors analysis platform was used to test the performance of prepared cantilever biosensor arrays. The sensor array was shown to recognize endotoxins from strains of Hafnia alvei PCM 1185 and PCM 1186. The proposed functionalization procedure and the applied measurement system form a promising platform for further investigations, including that of different antigens.

The molecular mechanism of cell activation by cobalt ions. Comment on Ninomiya et al.: Metal ions activate vascular endothelial cells and increase lymphocyte chemotaxis and binding

Dear Editors: We read the recent paper by Ninomiya et al.1 with considerable interest and share the authors conclusion that cobalt ions released by orthopaedic metals can directly activate cells leading to the release of pro-inflammatory chemokines. Although Ninomiya et al. argue against a “need for immunological sensitization by metal ions,” thereby excluding the adaptive immune response, the biological basis for the innate immune response to cobalt ions was not identified. Our group has recently demonstrated that clinically relevant concentrations of cobalt ions can activate human toll-like receptor 4 (TLR4) in a manner entirely analogous to innate immune recognition of the bacterial endotoxin, lipopolysaccharide.2 Downstream consequences of the activation of this receptor include the release of multiple pro-inflammatory cytokines (including chemokines) and upregulation of the expression of immunologically relevant adhesion molecules. We feel that the work of our group and others 3, 4 in this important area provide a clear basis to explain the observations reported in this journal by Ninomiya et al. Further studies will certainly lead to the development of strategies to mitigate the innate immune response to cobalt ions, allowing safer use of metal-on-metal bearings in arthroplasty. Alison J. Tyson-Capper1 Helen Lawrence1 James P. Holland2 David J. Deehan2 John A. Kirby1 1 Institute of Cellular Medicine Faculty of Medical Sciences Newcastle University, Newcastle upon Tyne NE2 4HH, UK 2 Department of Orthopaedics Freeman Hospital, Newcastle upon Tyne NE7 7DN UK

Analysis of the Enzymatic Properties of a Broad Family of Alanine Aminotransferases

Alanine aminotransferase (AlaAT) has been studied in a variety of organisms due to the involvement of this enzyme in mammalian processes such as non-alcoholic hepatocellular damage, and in plant processes such as C4 photosynthesis, post-hypoxic stress response and nitrogen use efficiency. To date, very few studies have made direct comparisons of AlaAT enzymes and fewer still have made direct comparisons of this enzyme across a broad spectrum of organisms. In this study we present a direct kinetic comparison of glutamate:pyruvate aminotransferase (GPAT) activity for seven AlaATs and two glutamate:glyoxylate aminotransferases (GGAT), measuring the KM values for the enzymes analyzed. We also demonstrate that recombinant expression of AlaAT enzymes in Eschericia coli results in differences in bacterial growth inhibition, supporting previous reports of AlaAT possessing bactericidal properties, attributed to lipopolysaccharide endotoxin recognition and binding. A probable lipopolysaccharide binding region within the AlaAT enzymes, homologous to a region of a lipopolysaccharide binding protein (LBP) in humans, was also identified in this study. The AlaAT enzyme differences identified here indicate that AlaAT homologues have differentiated significantly and the roles these homologues play in vivo may also have diverged significantly. Specifically, the differing kinetics of AlaAT enzymes and how this may alter the nitrogen use efficiency in plants is discussed.

Structural and Thermodynamic Insights into Bacterial Outer Membrane Lipid Signaling by the Innate Immune System

Lipopolysaccharide (LPS) from bacterial outer membranes is a potent early indicator of microbial infection and the primary inducer of fatal septic shock syndrome. Its recognition by the immune system is carried out by Toll-like receptor 4 (TLR4) when associated with its co-receptor MD-2, an immunoglobulin-like protein. MD-2 adopts a characteristic “beta-cup” fold with a large hydrophobic cavity, and is able to bind a variety of lipid species. Subtle alterations in the structure of LPS derivatives can profoundly alter the resultant immunological response, hampering the rational design of TLR4 immunomodulators. To unravel the associated structure-activity relationships, we have performed long-timescale, all-atom molecular dynamics simulations and free-energy calculations of the isolated MD-2 co-receptor and the entire signaling-active receptor complex in the presence of a variety of LPS species, as well as an LPS membrane. Unbiased simulations revealed that the MD-2 cavity is highly conformationally flexible, identifying spontaneous switching between active signalling-competent and inactivated states dependent upon the presence of different ligands, leading us to propose a conserved receptor activation mechanism. To gain insights into the thermodynamic determinants of endotoxin recognition, extensive umbrella sampling has been applied to estimate the potential of mean force (PMF) for the binding of LPS molecules to MD-2 co-receptor. Strikingly, stronger binding to signalling-inactivate MD-2 were observed for antagonists, and conversely, stronger binding to active MD-2 for agonists. Comparison of this data to the first ever PMF calculated for extraction of LPS from a model of the bacterial outer membrane has revealed how MD-2 creates a “membrane-like” environment within its protein cavity, providing a mechanism for sensitive LPS recognition by the innate immune system.

Immunity and Inflammation in the Uterus

Microbes often infect the uterus and particularly the endometrium of animals. Infections are most commonly associated with natural service, pregnancy and the post-partum period, leading to inflammation with the elaboration of cytokines, chemokines and prostaglandins. Clinical diseases such as metritis, endometritis and abortion are important causes of infertility. The adaptive immune response to infection has been characterized previously, so the present review aims to highlight the emerging role for innate immunity in the endometrium. The detection of microbes and the innate immune response depends on the detection of pathogen-associated molecular patterns by pattern recognition receptors. The main families of pattern recognition receptors are Toll-like receptors (TLRs), nucleotide oligomerization domain-like receptors, retinoic acid-inducible gene I-like receptors and C-type lectin receptors. These receptors are most often expressed by hematopoietic cells, but the epithelial and stromal cells of the endometrium also possess functional receptors. For example, endometrial cells express TLR4 for recognition of the lipopolysaccharide endotoxin of Gram-negative bacteria, leading to secretion of IL-6, IL-8 and prostaglandin E(2) . It is likely that the epithelial and stromal cells provide a first line of defence in the endometrium to alert hematopoietic cells to the presence of microbes within the uterus.