Simultaneous Detection of Mycoplasma pneumoniae, Mycoplasma hominis and Mycoplasma arthritidis in Synovial Fluid of Patients with Rheumatoid Arthritis by Multiplex PCR.

BACKGROUND: It has been recognized that infectious agents, such as different bacteria and viruses, may play a role in the developing of rheumatoid arthritis (RA). Recently, the mycoplasma species has been implicated in the pathogenesis of RA. AIM: The aim of this study was to design a multiplex PCR for rapid and simultaneous detection of Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma arthritidis in the synovial fluid of patients with rheumatoid arthritis (RA). METHODS: A total of 131 synovial fluid (SF) samples from patients with RA were assayed. Mycoplasma pneumoniae (ATCC: 29342), M. hominis (native strain), and the synthetic complete genome of M. arthritidis mitogen (MAM) superantigen were used as controls. All SF samples were subjected to DNA extraction separately and multiplex PCR was performed. The PCR products were confirmed by sequencing. RESULTS: The designed multiplex PCR was able to detect M. pneumoniae, M. hominis, and M. arthritidis in the SF of patients with RA with a frequency of 30 (22.9%), 23 (17.5%) and 13 (9.9%), respectively. CONCLUSION: In this study, the overall detection of the Mycoplasma species in RA patients was 53.4%; thus, we recommend the application of multiplex PCR assays when searching for a specific anti mycoplasma treatment for RA patients.

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.

O-linked protein glycosylation in Mycoplasma.

Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high-resolution mass spectrometry. O-linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O-linked modification, and glucose was inferred by (13) C-labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.

Rhamnose biosynthesis in mycoplasmas requires precursor glycans larger than monosaccharide.

Despite the apparent absence of genes coding for the known pathways for biosynthesis, the monosaccharide rhamnose was detected in the d configuration in Mycoplasma pneumoniae and Mycoplasma pulmonis, and in both the d and l configurations in Mycoplasma arthritidis. Surprisingly, the monosaccharide glucose was not a precursor for rhamnose biosynthesis and was not incorporated at detectable levels in glucose-containing polysaccharides or glycoconjugates. In contrast, carbon atoms from starch, a polymer of glucose, were incorporated into rhamnose in each of the three species examined. When grown in a serum-free medium supplemented with starch, M. arthritidis synthesized higher levels of rhamnose, with a shift in the relative amounts of the d and l configurations. Our findings suggest the presence of a novel pathway for rhamnose synthesis that is widespread in the genus Mycoplasma.

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.

Crystal structure of the Mycoplasma arthritidis-derived mitogen in apo form reveals a 3D domain-swapped dimer.

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vbeta elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-A resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the "reconstituted" MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM(wt) molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.

Identification of an isoschizomer of the HhaI DNA methyltransferase in Mycoplasma arthritidis.

The genome of Mycoplasma arthritidis strain 158 has modified cytosine residues at AGCT sequences that render the DNA resistant to digestion with the AluI restriction endonuclease. The DNA methyltransferase responsible for the base modification has previously been designated MarI. From the complete genome sequence of M. arthritidis, we identify Marth_orf138 as a candidate marI gene. Marth_orf138 was cloned in Escherichia coli and its TGA codons converted to TGG. DNA isolated from E. coli cells expressing the modified Marth_orf138 gene was degraded by the AluI nuclease, indicating that Marth_orf138 does not code for MarI. However, the DNA from E. coli was found to have acquired resistance to the restriction endonuclease HhaI. Genomic DNA from M. arthritidis was also found to be resistant to HhaI (recognizes GCGC). The M. arthritidis isoschizomer of the HhaI DNA methyltransferase, coded by Marth_orf138, is designated MarII. Transformation of M. arthritidis was not significantly affected by modification of plasmid at HhaI sites, indicating that the mycoplasma lacks a restriction endonuclease that recognizes GCGC sites.

Crucial cytokine interactions in nitric oxide production induced by Mycoplasma arthritidis superantigen.

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57Bl/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies.

Association of Mycoplasma arthritidis mitogen with lethal toxicity but not with arthritis in mice.

Mycoplasma arthritidis induces an acute to chronic arthritis in rodents. Arthritis induced in mice histologically resembles human rheumatoid arthritis and can be associated with lethal toxicity following systemic injection. The M. arthritidis mitogen (MAM) superantigen has long been implicated as having a role in pathogenesis, but its significance with respect to toxicity and arthritogenicity in mycoplasma-induced disease is unclear. To study the pathogenic significance of MAM, M. arthritidis mutants that overproduced or failed to produce MAM were developed. MAM overproduction and knockout mutants were more and less mitogenic, respectively, than the wild-type strain. The degree of mitogenic activity correlated with lethal toxicity in DBA/2J mice. In contrast, histopathological studies detected no correlation between MAM production and the severity of arthritis induced in DBA/2J and CBA/J mice.

Mutation of two Mycoplasma arthritidis surface lipoproteins with divergent functions in cytadherence.

Mycoplasma arthritidis is a natural pathogen of rats, causing an acute polyarthritis. Previous studies identified two membrane-bound lipoproteins, Maa1 and Maa2, thought to be associated with cytadherence of M. arthritidis strain 158p10p9. We have since confirmed that Maa1 is a major adhesin, although the role of Maa2 has proven more elusive. Both proteins were capable of eliciting protective immunity in rats against challenge with the virulent strain 158p10p9, suggesting that they may be important in pathogenesis. The purpose of this study was to better understand the roles of Maa1 and Maa2 in cytadherence in vitro. Insertion mutants were created for both genes by transposon mutagenesis. In vitro adherence of the Maa1 mutant KOMaa1 to rat L2 lung cells was reduced to the level previously reported for a spontaneous low-adherence mutant of 158p10p9 in which Maa1 is truncated and nonfunctional. Surprisingly, adherence of the Maa2 mutant KOMaa2 was approximately fivefold greater than that of the wild type. Complementation of KOMaa1 and KOMaa2 with wild-type alleles of maa1 and maa2, respectively, returned adherence to wild-type levels. This work confirms our earlier observation that Maa1 is a major adhesin for M. arthritidis strain 158p10p9. Maa2, on the other hand, may play a suppressive or modulatory role, possibly serving to release organisms from microcolonies at certain stages of infection.

Genome of Mycoplasma arthritidis.

The genomes of several species of mycoplasma have been sequenced. Most of these species rely on the glycolytic pathway for energy production, with the one exception of Ureaplasma, a species that breaks down urea as its principle source of acquiring energy. Several species, including as Mycoplasma arthritidis, are nonglycolytic and can use arginine as their source of energy. Described here are the genome sequence and a transposon library of M. arthritidis. The genome of 820,453 bp is typical in size for a mycoplasma and contains two large families of genes that are predicted to code for phase-variable proteins. The transposon library was constructed using a minitransposon that inserts stably into the mycoplasma genome. Of the 635 predicted coding regions, 218 were disrupted in a library of 1,100 members. Dispensable genes included the gene coding for the MAM superantigen and genes coding for ribosomal proteins S15, S18, and L15.

Superantigen-like effects of a Candida albicans polypeptide.

The amino terminal sequence of the Candida albicans cell wall protein Int1 exhibited partial identity with the major histocompatibility complex (MHC) class II binding site of the Mycoplasma arthritidis superantigen MAM. Int1-positive C. albicans blastospores activated human T lymphocytes and expanded Vbeta subsets 2, 3, and/or 14; Int1-negative strains were inactive. Release of interferon-gamma (IFN-gamma) but not of tumor necrosis factor-alpha or interleukin-6 was Int1 dependent; interleukin-4 and interleukin-10 were not detected. T lymphocyte activation, Vbeta expansion, and IFN-gamma release were associated with a soluble polypeptide that encompassed the first 263 amino acids of Int1 (Pep(263)). Monoclonal antibody 163.5, which recognizes an Int1 epitope that overlaps the region of identity with MAM, significantly inhibited these activities when triggered by Int1-positive blastospores or Pep(263) but not by staphylococcal enterotoxin B. Histidine(263) was required. Pep(263) bound to T lymphocytes and MHC class II and was detected in the urine of a patient with C. albicans fungemia. These studies identify a candidal protein that displays superantigen-like activities.

Suppurative polyarthritis in striped skunks (Mephitis mephitis) from Cape Cod, Massachusetts: detection of mycoplasma DNA.

Striped skunks (Mephitis mephitis) from Cape Cod, Massachusetts, U.S.A. were necropsied (n=34; 1995-1997) or clinically evaluated (n=25, 2002-2003) to characterize a lameness and polyarthritis, reported by wildlife veterinarians and rehabilitators, and unsuccessfully treated with antibiotics. Overall, 22 affected skunks had one or multiple swollen joints, swollen paws, and subcutaneous abscesses. Purulent exudate was located in joint spaces, in periarticular connective tissue between muscle fascicles and tendons, and between and along flexor and extensor tendons of the paws. Histologic examination revealed suppurative arthritis, with necrosis and erosion of articular cartilage, and suppurative osteomyelitis. Special stains failed to reveal a causative microorganism within affected joints, and routine bacteriologic cultures failed to isolate a pathogen with any significant frequency or consistency. Polymerase chain reaction (PCR) experiments were performed using DNA extracted from archived, formalin-fixed joint samples of 11 affected skunks, and DNA from joints of 7 of 11 affected skunks yielded amplicons with sequences highly similar to sequences of Mycoplasma fermentans within the Mycoplasma bovis cluster, whereas DNA samples from joints of four unaffected skunks were negative by PCR. Skunks from Connecticut, U.S.A. (n=21; 1995-2003) were similarly examined and were found not to have suppurative polyarthritis, suggesting a unique geographic distribution of this condition. Concurrent pathologic conditions in adult skunks from both Cape Cod and Connecticut included verminous pneumonia, gastric nematodiasis, arthropod ectoparasitism, and canine distemper. Amyloidosis was present in skunks with and without suppurative polyarthritis, and the amyloid was immunohistochemically identified as AA-amyloid. This is the first report of suppurative polyarthritis in wild skunks with evidence of a mycoplasmal etiology.

Mycoplasma arthritidis-derived superantigen (MAM) displays DNase activity.

Bacterial superantigens are potent stimulators of the immune system. In this study, we expressed recombinant superantigens, which were then affinity purified and used for growth curves and DNase activity assays. Overexpression of Mycoplasma arthritidis-derived superantigen in Escherichia coli reduced bacterial growth. This is unique, as staphylococcal enterotoxin A and toxic shock syndrome toxin-1, expressed in the same vector system, showed no growth impairment. The observed growth inhibition was caused by the DNase activity of recombinant M. arthritidis-derived superantigen, thus describing the first superantigen showing enzymatic activity, which may be a result of the separate evolution of this toxin.

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.

Crystal structure of a complete ternary complex of TCR, superantigen and peptide-MHC.

'Superantigens' (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-A resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR Vbeta but with TCR Valpha.

Zinc induces dimerization of the class II major histocompatibility complex molecule that leads to cooperative binding to a superantigen.

Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vbeta elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306-318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn(2+) is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn(2+). However, in the presence of Zn(2+), a dimerized MAM/HLA-DR1/HA complex can arise through the Zn(2+)-induced DR1 dimer. In the presence of Zn(2+), cooperative binding of MAM to the DR1 dimer was also observed.

Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.

Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics. Specifically, ADI with either 12 kDa or 20 kDa PEG attachments on 33% of the primary amines retained about 60% or 48% of enzyme activity, respectively; the Km and pH profiles were nearly unchanged; IC50 values were diminished by less than 30%; while stability studies demonstrated full retention of activity at 4 degrees C for 5 months. A comparison of the enzymatic properties of a second ADI from Pseudomonas putida illustrated the superior characteristics of the M. arthritidis ADI enzyme.

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.