MD-2 Residues Tyrosine 42, Arginine 69, Aspartic Acid 122, and Leucine 125 Provide Species Specificity for Lipid IVA

Lipopolysaccharide (LPS) activates the innate immune response through the Toll-like receptor 4 (TLR4)·MD-2 complex. A synthetic lipid A precursor, lipid IV_A, induces an innate immune response in mice but not in humans. Both TLR4 and MD-2 are required for the agonist activity of lipid IV_A in mice, with TLR4 interacting through specific surface charges at the dimerization interface. In this study, we used site-directed mutagenesis to identify the MD-2 residues that determine lipid IV_A species specificity. A single mutation of murine MD-2 at the hydrophobic pocket entrance, E122K, substantially reduced the response to lipid IV_A. Combining the murine MD-2 E122K with the murine TLR4 K367E/S386K/R434Q mutations completely abolished the response to lipid IV_A, effectively converting the murine cellular response to a human-like response. In human cells, however, simultaneous mutations of K122E, K125L, Y41F, and R69G on human MD-2 were required to promote a response to lipid IV_A. Combining the human MD-2 quadruple mutations with the human TLR4 E369K/Q436R mutations completely converted the human MD-2/human TLR4 receptor to a murine-like receptor. Because MD-2 residues 122 and 125 reside at the dimerization interface near the pocket entrance, surface charge differences here directly affect receptor dimerization. In comparison, residues 42 and 69 reside at the MD-2/TLR4 interaction surface opposite the dimerization interface. Surface charge differences there likely affect the binding angle and/or rigidity between MD-2 and TLR4, exerting an indirect influence on receptor dimerization and activation. Thus, surface charge differences at the two MD-2/TLR4 interfaces determine the species-specific activation of lipid IV_A.     

Macrophages recognize streptococci through bacterial single-stranded RNA

Group B streptococcus (GBS) is a leading cause of both neonatal sepsis and meningitis, two diseases that are characterized by inflammation. However, the manner in which GBS organisms are recognized by monocytes and macrophages is poorly understood. In this study, we report that the recognition of GBS and other Gram-positive bacteria by macrophages and monocytes relies on bacterial single-stranded RNA (ssRNA). ssRNA interacts with a signalling complex, which comprises the Toll-like receptor adaptors MyD88 and UNC-93B, but not the established MyD88-dependent ssRNA sensors. The role of ssRNA in the recognition of Gram-positive bacteria--leading to the induction of inflammatory cytokines--has potential implications for sepsis pathogenesis, diagnosis and treatment.     

Defective pro-IL-1β responses in macrophages from aged mice

Background

Cytokines regulated by the inflammasome pathway have been extensively implicated in various age-related immune pathologies. We set out to elucidate the contribution of the nod-like receptor protein 3 (NLRP3) inflammasome pathway to the previously described deficiencies in IL-1β production by macrophages from aged mice. We examined the production of pro-IL-1β and its conversion into IL-1β as two separate steps and compared these cytokine responses in bone marrow derived macrophages from young (6–8 weeks) and aged (18–24 months) C57BL/6 mice.

Findings

Relative to macrophages from young mice, macrophages from aged mice produced less pro-IL-1β after TLR4 stimulation with LPS. However upon activation of the NLRP3 inflammasome with ATP, macrophages from young and aged mice were able to efficiently convert and secrete intracellular pro-cytokines as functional cytokines.

Conclusions

Lower levels of IL-1β production are a result of slower and lower overall production of pro-IL-1β in macrophages from aged mice.

     

Cutting edge: cells that carry A null allele for toll-like receptor 2 are capable of responding to endotoxin.

Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.     

Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.     

Lipoproteins are critical TLR2 activating toxins in group B streptococcal sepsis

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.     

Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium

Background  

Many bacteria swim by rotating helical flagellar filaments [1]. Waterbury et al. [15] discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces [2,7]. The hypothesis that Synechococcus might swim using traveling surface waves [6,13] prompted this investigation.     

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.     

Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers

The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.     

Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF,unlike IL-12p40 secretion,is Toll-like receptor 2 dependent

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.