Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.

Microglia subpopulations were studied in mouse experimental autoimmune encephalomyelitis and toxoplasmic encephalitis. CNS inflammation was associated with the proliferation of CD11b+ brain cells that exhibited the dendritic cell (DC) marker CD11c. These cells constituted up to 30% of the total CD11b+ brain cell population. In both diseases CD11c+ brain cells displayed the surface phenotype of myeloid DC and resided at perivascular and intraparenchymatic inflammatory sites. By lacking prominent phagocytic organelles, CD11c+ cells from inflamed brain proved distinct from other microglia, but strikingly resembled bone marrow-derived DC and thus were identified as DC. This brain DC population comprised cells strongly secreting IL-12p70, whereas coisolated CD11c− microglia/brain macrophages predominantly produced TNF-α, GM-CSF, and NO. In comparison, the DC were more potent stimulators of naive or allogeneic T cell proliferation. Both DC and CD11c− microglia/macrophages from inflamed brain primed naive T cells from DO11.10 TCR transgenic mice for production of Th1 cytokines IFN-γ and IL-2. Resting microglia that had been purified from normal adult brain generated immature DC upon exposure to GM-CSF, while CD40 ligation triggered terminal maturation. Consistently, a functional maturation of brain DC was observed to occur following the onset of encephalitis. In conclusion, these findings indicate that in addition to inflammatory macrophage-like brain cells, intraparenchymatical DC exist in autoimmune and infectious encephalitis. These DC functionally mature upon disease onset and can differentiate from resident microglia. Their emergence, maturation, and prolonged activity within the brain might contribute to the chronicity of intracerebral Th1 responses.

The mucosal host defence discriminates pathogens from commensals, and prevents infection while allowing the normal flora to persist. Paradoxically, Toll-like receptors (TLR) control the mucosal defence against pathogens, even though the TLR recognise conserved molecules like LPS, which are shared between pathogens and commensals. This study proposes a mechanism of pathogen-specific mucosal TLR4 activation, involving adhesive ligands and their host cell receptors. TLR4 signalling was activated in CD14-negative, LPS-unresponsive epithelial cells by P fimbriated, uropathogenic Escherichia coli but not by a mutant lacking fimbriae. Epithelial TLR4 signalling in vivo involved the glycosphingolipid receptors for P fimbriae and the adaptor proteins Toll/IL-1R (TIR) domain-containing adaptor inducing IFN-b (TRIF)/TRIF-related adaptor molecule (TRAM), but myeloid differentiation protein 88 (MyD88)/TIR domain-containing adaptor protein were not required for the epithelial response. Substituting the P fimbriae with type 1 fimbriae changed TLR4 signalling from the TRIF to the MyD88 adaptor pathway. In addition, the adaptor proteins and the fimbrial type were found to influence bacterial clearance. Trif -/-and Tram -/-mice remained infected with P fimbriated E. coli but cleared thetype 1 fimbriated strain, while Myd88 -/-mice became carriers of both the P and the type 1 fimbriated bacteria. Thus, TLR4 may be engaged specifically by pathogens, when the proper cell surface receptors are engaged by virulence ligands. IntroductionToll-like receptors (TLR) control the innate host defence at mucosal surfaces and yet commensals do not induce an inflammatory response at those sites. The relative inertia to the commensals is paradoxical, as the TLR recognise conserved pathogen-associated molecular patterns (PAMP), which are present on both pathogenic and commensal bacteria. Lipolysaccharide (LPS), flagellin, peptidoglycan or DNA may bind directly to the TLR but in addition, co-receptors are needed to enhance the TLR response to these conserved ligands [1][2][3]. Myeloid cells use CD14 and MD-2 as co-receptors for LPS in TLR4 signalling, and minute amounts of LPS may elicit a strong systemic inflammatory response [4,5]. However, the TLR response to pathogen attack at mucosal surfaces is controlled by different interactions. Epithelial cells must be protected from constant TLR activation by commensals and their PAMP, in order for mucosal integrity to be maintained. The epithelial cells thus lack co-receptors like CD14 [6][7][8][9][10][11], and in addition, commensals have been suggested to actively suppress epithelia responses through NF-jB [12,13]. Yet, mucosal pathogens evoke rapid TLR4-dependent responses at mucosal sites, suggesting that alternative ligands and receptors might be involved in mucosal TLR activation. Pathogens use adhesive surface ligands to break the inertia of the mucosal barrier [14][15][16]. The innate defence is activated as a direct result of these interactions and epithelial cells produce the first wave of me...

In literature and alchemy the salamander is a fabled creature. Salamanders have long been known to be toxic animals, the poisons they produce usually being associated with secretions of specialized glands or of the skin itself. An extraordinarily powerful neurotoxin, called tarichatoxin, has recently been isolated in crystalline form from the eggs of various species of western American newts of the genus Taricha. This toxin, present in adult newts as well as in newt eggs and embryos, is very different chemically and pharmacologically from other known salamander toxins. This makes all the more remarkable the finding, in recent work, that tarichatoxin is identical to a toxin known as tetrodotoxin which occurs in the Japanese Fugu or puffer fish. The substance appears to occur only in one family of Amphibia (the Salamandridae) and one suborder of fishes (the Tetraodontoidae). This extremely limited distribution is a remarkable biogenetic finding.Here we discuss the course of the investigations which led to the isolation of the toxin from the California newt, the history of tetrodotoxin, the evidence, both physical and physiological, which points to the conclusion that tarichatoxin and tetrodotoxin are one and the same substance, and the deductions which can be made concerning the chemical structure of the toxin. Tarichatoxin Discovery and history. In the early 1930's Victor C. Twitty, an experimental embryologist, came to Stanford University from Yale. At New Haven he had worked with the eastern salamander, Ambystoma punctatum, and at 1100 Dr. Mosher is professor of chemistry, Dr. Fuhrman is professor of experimental medicine, and Drs. Buchwald and Fischer are research associates in chemistry at

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3−/− mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3−/− mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.

During chronic infection of mice with Toxoplasma gondii, gene message for IL-12p40, CD86, and the potassium channel Kv1.3 was detected in brain mononuclear cells, suggesting the presence of dendritic cells (DC) in the CNS. Consistently, cells bearing the DC markers CD11c and 33D1 were localized at inflammatory sites in the infected brain. The number of isolated CD11c+ brain cells increased until peak inflammation. The cells exhibited the surface phenotype of myeloid DC by coexpressing 33D1 and F4/80, little DEC-205, and no CD8α. These brain DC were mature, as indicated by high-level expression of MHC class II, CD40, CD54, CD80, and CD86. They triggered Ag-specific and primary allogeneic T cell responses at very low APC/T cell ratios. Among mononuclear cells from encephalitic brain, DC were the main producers of IL-12. Evidence for a parasite-dependent development of DC from CNS progenitors was obtained in vitro: after inoculation of primary brain cell culture with T. gondii, IL-12-secreting dendriform cells emerged, and DC marker genes were expressed. Different stimuli elicited the generation and maturation of brain DC: neutralization of parasite-induced GM-CSF prevented outgrowth of dendriform cells and concomitant release of IL-12. IL-12 production was up-regulated by external IFN-γ but was stopped by inhibiting parasite replication. Consistently, DC isolated from GM-CSF-treated brain cell culture were activated to secrete IL-12 by exposure to parasite lysate. In sum, these results demonstrate T. gondii-induced expansion and functional maturation of DC in the CNS and, thus, highlight a mechanism that may contribute to the chronicity of the host response.

SummaryFimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae-dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll-like receptor 4 (TLR4)-dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS-CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co-staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co-localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4-dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co-receptors.

SummaryFimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae-dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll-like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS-and TLR4-dependent signal to the CD14-negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS-dependent response in those cells. A similar LPS-and fimbriaedependent response was observed in the urinary tract of TLR4-proficient mice, but not in TLR4-defective mice. The moderate inflammatory response in the TLR4-defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14-negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host`sees' LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.