Endotoxin recognition: in fish or not in fish?
The interaction between pathogens and their multicellular hosts is initiated by activation of pathogen recognition receptors (PRRs). These receptors, that include most notably members of the toll-like receptor (TLR) family, recognize specific pathogen-associated molecular patterns (PAMPs). TLR4 is a central part of the receptor complex that is involved in the activation of the immune system by lipopolysaccharide (LPS) through the specific recognition of its endotoxic moiety (Lipid A). This is a critical event that is essential for the immune response to Gram-negative bacteria as well as the etiology of endotoxic shock. Interestingly, compared to mammals, fish are resistant to endotoxic shock. This in vivo resistance concurs with in vitro studies demonstrating significantly lowered sensitivity of fish leukocytes to LPS activation. Further, our in vitro analyses demonstrate that in trout mononuclear phagocytes, LPS fails to induce antiviral genes, an event that occurs downstream of TLR4 and is required for the development of endotoxic shock. Finally, an in silico approach that includes mining of different piscine genomic and EST databases, reveals the presence in fish of all of the major TLR signaling elements except for the molecules specifically involved in TLR4-mediated endotoxin recognition and signaling in mammals. Collectively, our analysis questions the existence of TLR4-mediated cellular responses to LPS in fish. We further speculate that other receptors, in particular beta-2 integrins, may play a primary role in the activation of piscine leukocytes by LPS. (+info
Microbial pattern recognition receptors mediate M-cell uptake of a gram-negative bacterium.
The receptors involved in the sampling of particulate microbial antigens by the gut are largely unknown. Here we demonstrate for the first time in an in vitro M-cell model and in situ in isolated murine intestinal segments that the receptors TLR-4, PAF-R, and alpha5beta1 integrin are all involved in mediating bacterial uptake associated with transcytosis. The pattern of expression of TLR-4 and alpha5beta1 integrin differed between M cells and enterocytes. There was increased apical expression of TLR-4 in M-cell cultures, and it was present on the apical surface of murine M cells but not enterocytes in situ. In contrast, PAF-R was expressed equally by both cell types in vitro and was abundantly expressed throughout the intestinal epithelium. Inhibition of TLR-4 and PAF-R, but not TLR-2, reduced gram-negative bacterial uptake by both cell types, whereas inhibition of the apically expressed alpha5beta1 integrin significantly reduced the ability of M cells to translocate bacteria. Hence, the involvement of each receptor was dependent not only on differences in the level of receptor expression but the cellular localization. Using bacteria that had mutations that affected the bacterial lipooligosaccharide structure indicated that the oligosaccharide moiety was important in bacterial uptake. Taken together, the data suggest that pathogen-associated molecular pattern interactions with pattern recognition receptors are key factors in M-cell recognition of intestinal antigens for mucosal immune priming. (+info
Functional genomics of innate host defense molecules in normal human monocytes in response to Aspergillus fumigatus.
Aspergillus fumigatus induces the release of innate immune-related molecules from phagocytic cells early in the course of infection. Little is known, however, about the complex expression profiles of the multiple genes involved in this response. We therefore investigated the kinetics of early gene expression in human monocytes (HMCs) infected with conidia of A. fumigatus using DNA microarray analysis. Total RNA from HMCs at 0, 2, 4, and 6 h was extracted, linearly amplified, hybridized onto Affymetrix HG133 Plus 2.0 gene chips, and analyzed with an Affymetrix scanner. Changes in gene expression were calculated as a ratio of those expressed by infected versus control HMCs. Aspergillus fumigatus induced differential regulation of expression in 1,827 genes (P < 0.05). Genes encoding cytokines and chemokines involved in host defense against A. fumigatus, including interleukin-1beta (IL-1beta), IL-8, CXCL2, CCL4, CCL3, and CCL20, as well as the opsonin long pentraxin 3, were up-regulated during the first 2 to 6 h, coinciding with an increase in phagocytosis. Simultaneously, genes encoding CD14, ficolin1, and MARCO were down-regulated, and genes encoding IL-10 and matrix metalloproteinase 1 were up-regulated. Up-regulation of the genes encoding heat shock proteins 40 and 110 and connexins 26 and 30 may point to novel molecules whose role in the pathogenesis of aspergillosis has not been previously reported. Verification of the transcriptional profiling was obtained for selected genes by reverse transcription-PCR and enzyme immunoassay. Thus, A. fumigatus conidia induced a coordinated expression of genes important in host defense and immunomodulation. (+info
IFN-gamma enhances production of nitric oxide from macrophages via a mechanism that depends on nucleotide oligomerization domain-2.
Pattern recognition receptors are central to the responsiveness of various eukaryotic cell types when they encounter pathogen-associated molecular patterns. IFN-gamma is a cytokine that is elevated in humans and other animals with bacterial infection and enhances the LPS-induced production of antibacterial mediators by macrophages. Mice lacking the pattern recognition receptor, TLR4, respond very poorly to stimulation by LPS, but administration of IFN-gamma has been described as restoring apparent sensitivity to this stimulatory ligand. In this study, we show that IFN-gamma primes murine macrophages stimulated by crude LPS preparations to produce the antibacterial mediator NO, a proportion of which is independent of TLRs 2 and 4. This response is lost in tlr4-/- IFN-gamma-primed murine macrophages when the LPS preparation is highly purified. NO is also induced if chemically synthesized muramyl dipeptide, an intermediate in the biosynthesis of peptidoglycan, is used to stimulate macrophages primed with IFN-gamma. This is absolutely dependent on the presence of a functional nucleotide oligomerization domain-2 (NOD-2) protein. IFN-gamma increases NOD-2 expression and dissociates this protein from the actin cytoskeleton within the cell. IFN-gamma priming of macrophages therefore reveals a key proinflammatory role for NOD-2. This study also shows that the effect of IFN-gamma in restoring inflammatory responses to gram-negative bacteria or bacterial products in mice with defective TLR4 signaling is likely to be due to a response to peptidoglycan, not LPS. (+info
Central role for MyD88 in the responses of microglia to pathogen-associated molecular patterns.
Microglia, the innate immune effector cells of the CNS parenchyma, express TLR that recognize conserved motifs of microorganisms referred to as pathogen-associated molecular patterns (PAMP). All TLRs identified to date, with the exception of TLR3, use a common adaptor protein, MyD88, to transduce activation signals. Recently, we reported that microglial activation in response to the Gram-positive bacterium Staphylococcus aureus was not completely attenuated following TLR2 ablation, suggesting the involvement of additional receptors. To assess the functional role of alternative TLRs in microglial responses to S. aureus and its cell wall product peptidoglycan as well as the Gram-negative PAMP LPS, we evaluated primary microglia from MyD88 knockout (KO) and wild-type mice. The induction of TNF-alpha, IL-12 p40, and MIP-2 (CXCL2) expression by S. aureus- and peptidoglycan-stimulated microglia was MyD88 dependent, as revealed by the complete inhibition of cytokine production in MyD88 KO cells. In addition, the expression of additional pattern recognition receptors, including TLR9, pentraxin-3, and lectin-like oxidized LDL receptor-1, was regulated, in part, via a MyD88-dependent manner as demonstrated by the attenuated expression of these receptors in MyD88 KO microglia. Microglial activation was only partially inhibited in LPS-stimulated MyD88 KO cells, suggesting the involvement of MyD88-independent pathways. Collectively, these findings reveal the complex mechanisms for microglia to respond to diverse bacterial pathogens, which occur via both MyD88-dependent and -independent pathways. (+info
AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system.
Activation of the insect innate immune system is dependent on a limited number of pattern recognition receptors (PRRs) capable of interacting with pathogen-associated molecular pattern. Here we report a novel role of an alternatively spliced hypervariable immunoglobulin domain-encoding gene, Dscam, in generating a broad range of PRRs implicated in immune defense in the malaria vector Anopheles gambiae. The mosquito Down syndrome cell adhesion molecule gene, AgDscam, has a complex genome organization with 101 exons that can produce over 31,000 potential alternative splice forms with different combinations of adhesive domains and interaction specificities. AgDscam responds to infection by producing pathogen challenge-specific splice form repertoires. Transient silencing of AgDscam compromises the mosquito's resistance to infections with bacteria and the malaria parasite Plasmodium. AgDscam is mediating phagocytosis of bacteria with which it can associate and defend against in a splice form-specific manner. AgDscam is a hypervariable PRR of the A. gambiae innate immune system. (+info
Lung epithelium as a sentinel and effector system in pneumonia--molecular mechanisms of pathogen recognition and signal transduction.
Pneumonia, a common disease caused by a great diversity of infectious agents is responsible for enormous morbidity and mortality worldwide. The bronchial and lung epithelium comprises a large surface between host and environment and is attacked as a primary target during lung infection. Besides acting as a mechanical barrier, recent evidence suggests that the lung epithelium functions as an important sentinel system against pathogens. Equipped with transmembranous and cytosolic pathogen-sensing pattern recognition receptors the epithelium detects invading pathogens. A complex signalling results in epithelial cell activation, which essentially participates in initiation and orchestration of the subsequent innate and adaptive immune response. In this review we summarize recent progress in research focussing on molecular mechanisms of pathogen detection, host cell signal transduction, and subsequent activation of lung epithelial cells by pathogens and their virulence factors and point to open questions. The analysis of lung epithelial function in the host response in pneumonia may pave the way to the development of innovative highly needed therapeutics in pneumonia in addition to antibiotics. (+info
Fine discrimination in the recognition of individual species of phosphatidyl-myo-inositol mannosides from Mycobacterium tuberculosis by C-type lectin pattern recognition receptors.
The Mycobacterium tuberculosis (M.tb) envelope is highly mannosylated with phosphatidyl-myo-inositol mannosides (PIMs), lipomannan, and mannose-capped lipoarabinomannan (ManLAM). Little is known regarding the interaction between specific PIM types and host cell C-type lectin pattern recognition receptors. The macrophage mannose receptor (MR) and dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells engage ManLAM mannose caps and regulate several host responses. In this study, we analyzed the association of purified PIM families (f, separated by carbohydrate number) and individual PIM species (further separated by fatty acid number) from M.tb H(37)R(v) with human monocyte-derived macrophages (MDMs) and lectin-expressing cell lines using an established bead model. Higher-order PIMs preferentially associated with the MR as demonstrated by their reduced association with MDMs upon MR blockade and increased binding to COS-1-MR. In contrast, the lower-order PIM(2)f associated poorly with MDMs and did not bind to COS-1-MR. Triacylated PIM species were recognized by MDM lectins better than tetra-acylated species and the degree of acylation influenced higher-order PIM association with the MR. Moreover, only higher-order PIMs that bind the MR showed a significant increase in phagosome-lysosome fusion upon MR blockade. In contrast with the MR, the PIM(2)f and lipomannan were recognized by DC-SIGN comparable to higher-order PIMs and ManLAM, and the association was independent of their degree of acylation. Thus, recognition of M.tb PIMs by host cell C-type lectins is dependent on both the nature of the terminal carbohydrates and degree of acylation. Subtle structural differences among the PIMs impact host cell recognition and response and are predicted to influence the intracellular fate of M.tb. (+info