A potential pathogenic factor from Mycoplasma hominis is a TLR2-dependent, macrophage-activating, P50-related adhesin.

PROBLEM: Mycoplasma hominis has been implicated in many inflammatory conditions of the human urogenital tract in particular amniotic infections that lead to fetal and neonatal disease and pre-term labor. The mechanisms responsible are poorly defined. METHOD OF STUDY: Biochemical and immunological methods were used to extract, purify, and characterize an inflammatory component present in M. hominis. RESULTS: We isolated and purified to homogeneity a 40-kDa bioactive lipoprotein from M. hominis that was a potent TLR2-dependent, CD14-independent activator of the human THP-1 macrophage cell line. Homology searches of the N-terminal sequence revealed that 22 of the first 23 residues were identical to those seen for the phase-variable M. hominis p50 adhesin. The truncated P50t lipoprotein importantly retained its adhesive properties for human macrophages. CONCLUSION: The unique adhesin/macrophage activator may play a key role in M. hominis infections by triggering an inflammatory cytokine cascade.

Mycoplasma superantigen initiates a TLR4-dependent Th17 cascade that enhances arthritis after blocking B7-1 in Mycoplasma arthritidis-infected mice.

Mycoplasma arthritidis is a natural pathogen of rodents causing arthritis, toxic shock and necrotizing fasciitis. It secretes a potent superantigen (SAg), MAM, that differentially affects the immune system depending upon presence or absence of TLR4, thus potentially influencing disease outcomes. Here, we establish that antibody to co-stimulatory molecule B7-1(CD80) enhances arthritis in wild-type C3H/HeSnJ (TLR2+4+) mice but suppresses arthritis in TLR4-defect C3H/HeJ (TLR2+4-) mice. Also, blockade of the B7-1/CD28 co-stimulatory pathway in C3H/HeSnJ mice resulted in a marked increase in an alternative co-stimulatory pathway ICOS/ICOSL that was associated with elevation of the IL-17/Th17cascade with enhanced IL-23, IL-6, and the RORγt and STAT3 transcriptional factors on CD4+ T cells. Anti- B7-1 also increased inflammatory chemokines and the stress protein HMGB1 that promotes cellular infiltration to joints. Using a MAM-deficient strain of M. arthritidis, a monoclonal antibody to TLR4 and a TLR4-defective mouse strain, we established that both MAM and TLR4 are required for the systemic and local joint triggering of the Th17/IL-17 cascade in mice treated with anti-B7-1 antibody. Importantly, blocking of IL-17 with anti-IL-17 antibody suppressed the elevated arthritis in M. arthritidis-infected mice treated with anti-B7-1 antibody. Thus, this unique model of arthritis illustrates how microbial agonists can bridgeinnate and adaptive immune responses to redirect signalling pathways, thus promoting chronic inflammatory and autoimmune disease.

Novel interactions of a microbial superantigen with TLR2 and TLR4 differentially regulate IL-17 and Th17-associated cytokines.

Mycoplasma arthritidis, an inflammatory murine pathogen, secretes a potent superantigen, Mycoplasma arthritidis mitogen (MAM) that contributes to toxic shock, arthritis and skin necrosis. Previously we showed that MAM induced type 2 T-cell cytokines in mice that express functional TLR2 and TLR4, but type 1 cytokines in mice that lack TLR4 function. We show here that IL-17, pSTAT3 and retinoid-related orphan nuclear receptorγt are rapidly expressed in wild-type C3H/HeSnJ (TLR2+/4+) mice but are significantly delayed in mutant C3H/HeJ (TLR2+/4-) mice. This marked kinetic difference was associated with a high level of IL-6 in TLR2+/4+ mice versus high levels of IL-1ß and TNFα in TLR2+/4- mice. Also antibodies to IL-6 and IL-23, suppressed IL-17 responses to MAM in TLR2+/4+ mice whereas anti-IL-1ß, but not anti-TNFα, enhanced IL-17 in TLR2+/4- mice. Antibody blocking of TLR4 in TLR2+/4+ mice decreased IL-17 and IL-6 but not IL-23. In addition both IL-17 and IL-6 but not IL-23 were elevated in TLR2 KO mice versus wild-type TLR2+/4+ mice given MAM. We conclude that the MAM interaction with TLR2 versus TLR4 leads to distinct cytokine pathways mediated primarily by IL-1ß or IL-6/IL-17 signalling respectively. Our findings suggest that the differential interaction of MAM with different TLRs might play an important role in disease outcomes by M. arthritidis.

Characterization of the macrophage-stimulating activity from Ureaplasma urealyticum.

PROBLEM: Intra-amniotic infection is the most common cause of preterm labor. Infections are thought to cause preterm labor by increasing the production of proinflammatory cytokines at the maternal-fetal interface. Experiments with cell culture and animal models have indicated that bacterial lipopolysaccharide (LPS) increases the production of proinflammatory cytokines in reproductive tissues. The majority of intrauterine infections, however, are associated with Ureaplasma urealyticum, which does not contain LPS. Therefore, we performed a series of experiments to understand better the bacterial factor(s) that are responsible for the proinflammatory effects of U. urealyticum. METHOD OF STUDY: U. urealyticum was cultivated in 3-4 L 10B broth, harvested by centrifugation, washed with saline and frozen at -85 degrees C until use. Cells were then extracted with Triton X-114 and the macrophage-stimulating activity (MSA) of the preparations was studied by evaluating their ability to stimulate tumor necrosis factor-alpha production by a monocytic cell line (THP-1 cells). Additional studies involved testing the sensitivity of the detergent extracts to heating, alkaline hydrolysis and proteinase K digestion. Interaction of Triton X-114 extracts with Toll-like receptor (TLR)-2 and TLR-4 was evaluated using cell lines transfected with one of these receptors, CD14 and a reporter gene. RESULTS: Extraction of U. urealyticum with Triton X-114 demonstrated that the MSA preferentially partitioned to the detergent phase. The MSA of the detergent extracts was abrogated by proteinase K digestion or alkaline hydrolysis but only partially inhibited by heating. Further studies suggested that the detergent extracts could activate both TLR-2 and TLR-4. CONCLUSION: These experiments suggest that the MSA of U. urealyticum is lipophilic, sensitive to alkaline hydrolysis and proteinase K digestion, partially sensitive to heating. These properties are consistent with the activity being due to a lipoprotein. Unlike other Mycoplasma species, the MSA of U. urealyticum appears to interact with both TLR-2 and TLR-4. Purification of the molecule(s) that regulate this activity may provide good therapeutic targets for anti-inflammatory strategies to prevent preterm labor caused by intrauterine infection with U. urealyticum.

Inflammatory lipoproteins purified from a toxigenic and arthritogenic strain of Mycoplasma arthritidis are dependent on Toll-like receptor 2 and CD14.

Mycoplasma arthritidis is a naturally occurring murine pathogen, and the disease model has been used extensively to understand inflammatory mechanisms. Recently, Triton X-114 extracts of a virulent strain of M. arthritidis were found to be more potent in activating macrophages than were those from an avirulent strain, suggesting a role in disease. Here, octyl glucoside extraction of cells was used to identify four distinct bioactive moieties, with molecular masses of approximately 41, 37, 34, and 17 kDa. Their bioactivities were resistant to proteinase K but were destroyed by alkaline hydrolysis and oxidation. As for MALP-2, all were dependent upon Toll-like receptor 2, but unlike MALP-2, they were also dependent upon CD14. The M. arthritidis lipoproteins exhibited infrared absorbances at 2,900 cm(-1) and 1,662 cm(-1), similar to those seen in Pam(3)-Cys-Ser-(Lys)(4). Edman degradation failed to reveal N-terminal sequences, suggesting that they were blocked and therefore might be triacylated. However, mass spectrometry of fragments revealed that the 41-kDa moiety, which binds to serum apolipoprotein A-1, had similarity with the recently described MlpD lipoprotein of M. arthritidis.

A microbial TLR2 agonist imparts macrophage-activating ability to apolipoprotein A-1.

There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H(2)O(2) oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-alpha and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.

TLR2 and TLR4 differentially regulate B7-1 resulting in distinct cytokine responses to the mycoplasma superantigen MAM as well as to disease induced by Mycoplasma arthritidis.

Mycoplasma arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis, an agent of murine arthritis and toxicity. We previously demonstrated that C3H mouse sub-strains differing in expression of Toll-like receptor 4 (TLR4), differed in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Here we examine the role of B7 co-stimulatory molecules in immune outcome and disease manifestations resulting from these different MAM/TLR2 and MAM/TLR4 interactions. Injections of MAM into C3H/HeJ mice upregulated expression of B7-1 but not B7-2 on peritoneal adherent cells, whereas B7-1 expression was lower on cells from C3H/HeSnJ mice. Anti-B7-1 antibody but not anti-B7-2, injected in vivo, changed the type 1 cytokines in MAM-injected C3H/HeJ mice to a type 2 cytokines and, conversely, the type 2 response in C3H/HeSnJ mice injected with anti-B7-1 shifted to a type 1 pattern. Whereas anti-B7-2 exerted no effect on disease in either mouse strain, anti-B7-1 significantly delayed the lethal toxicity of M. arthritidis in C3H/HeJ mice but enhanced arthritis in C3H/HeSnJ mice. Thus, TLR-mediated regulation of B7-1 results in diverse cytokine profiles in C3H sub-strains, and that the interaction of MAM with different TLR(s) may differentially affect cytokine responses and ultimately, M. arthritidis disease.

Characterization and partial purification of a macrophage-stimulating factor from Mycoplasma hominis.

PROBLEM: Mycoplasma hominis is one of the most common pathogens of the genital tract and is associated with increased production of proinflammatory cytokines in reproductive tissues during preterm labor. The mechanism by which M. hominis, an organism lacking cell walls, increases the production of proinflammatory cytokines is unknown. METHOD OF STUDY: We characterized and purified a macrophage-activating factor from this organism. RESULTS: Extraction of whole organisms with Triton-X-114 demonstrated that the activity was primarily associated with the detergent phase. Macrophage-stimulating activity (MSA) of detergent extracts of M. hominis was not inhibited by polymyxin B or heating but was completely abrogated by alkaline hydrolysis and partially reduced by proteinase K digestion. Further experiments that utilized Toll-like receptor (TLR)-2- and TLR-4-transfected cells, revealed that the detergent extracts activate TLR-2 but not TLR-4 signal transduction. Purification of the activity using preparative SDS-PAGE and reverse phase chromatography experiments led to the isolation of a 29-kDa protein. CONCLUSIONS: These experiments suggest that the MSA of M. hominis is due to a lipophillic factor that interacts with TLR-2 rather than TLR-4 (as does lipopolysaccharide), to increase tumor necrosis factor (TNF)-alpha by macrophages. It is known that TNF-alpha can cause preterm labor and intrauterine fetal death and that it is upregulated in amniotic fluid samples infected with M. hominis.

Isolation and partial purification of macrophage- and dendritic cell-activating components from Mycoplasma arthritidis: association with organism virulence and involvement with Toll-like receptor 2.

Mycoplasma arthritidis induces toxicity, arthritis, and dermal necrosis in mice. Virulence factors include a superantigen and membrane adhesins and possibly also a bacteriophage component. Here we compare the biological properties of Triton X-114 extracts derived from avirulent and virulent M. arthritidis strains. Macrophage cell lines and resident peritoneal macrophages were used to assess inflammatory potential as indicated by production of tumor necrosis factor alpha, interleukin-6, and/or nitric oxide. The activity resided exclusively within the hydrophobic detergent phase, was unaffected by heat treatment at 100 degrees C for 30 min, and was resistant to proteinase K digestion, suggesting involvement of a lipopeptide. Contamination of extracts with endotoxin or superantigen was excluded. Extracts of the more virulent strain had higher activity than did those of the avirulent strain. Using CHO cells expressing Toll-like receptor 2 (TLR2) or TLR4, both with transfected CD14, we showed that extracts activated these cells via TLR2 but not by TLR4. Also, macrophages from C57BL/6 TLR2(-/-) mice failed to respond to the extracts, whereas those from TLR2(+/+) cells did respond. The preparations from the virulent strain of M. arthritidis were also more potent in activating dendritic cells, as evidenced by up-regulation of major histocompatibility complex class II, CD40, B7-1, and B7-2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent elution of gel slices revealed the presence of three active moieties which corresponded to molecular masses of approximately 24, 28, and 40 kDa. Three active components were also found by reverse-phase chromatography. We suggest that macrophage activation by M. arthritidis could play a significant role in the inflammatory response induced in the host by this organism.