Ureaplasma Urealyticum Infection In The Pathogenesis Of Rheumatoid Arthritis

Background and objectiveRheumatoid arthritis is a chronic inflammatory disease characterized by chronic inflammation in the polyarthritis. There will be joint stiffness varying degrees of deformity and with bone destruction, muscle atrophy and joint dysfunction, with a very high morbidity. Rheumatoid arthritis is characterized by the synovial proliferation and inflammatory cytokines. Because of the unclear disease causes and unknown mechanism, it's difficult to do the secondary prevention better, even the first primary prevention is impossible. So we need to clear the predisposing factors, then better to guide doctor-patient involved in the disease prevention and treatment.The common genetic factor was put forward as an important pathogenic mechanism by researchers. P Stastny found that 60% to 80% of white RA patients carrying HLA-DR4 haplotype, while 20% to 25% in control group. But so far we haven't found the specific antigen yet. Furthermore, some normal expressing homozygous HLA-DR4 alleles do not get RA, which illustrates genetic factors account for about 35% of causes of pathogenic factors, not single pathogenic factor but other factors play an effective influence.The researchers fixed attention on inflammatory cells like macrophages, neutrophils, T cells, B cells and cytokines like TNF-aand IL-1. Many kinds of monoclonal antibody and soluble receptor antagonist aimed at these cells and cytokines have been applied to clinical practice,such as Adalimumab,Infiiximab and Eternacept and achieved the good effect in the early stage. However, after 10 years of the above-mentioned biologics using, more and more clinical doctors found the rate of patients reaching American Rheumatism Association(ACR)20 was less than 60 percent. People have to rethink whether there is a factor which acts the initiation factor except inflammatory cells and cytokines. Then synovial gradually comes into our sight.Ureaplasma urealyticum (Uu) is a species which belong to mycoplasmatales, Mycoplasmataceae, Ureaplasma. In 1954, Uu was isolated from the patients with non-gonococcal urethritis (NGU) by Shepard for the first time and was called "tiny strain"(T strain). Uu is a range between viruses and bacteria, the smallest self-propagation prokaryote in the artificial medium, without cell wall. According to the characteristics of molecular biology, Uu can be divided into two major biovars and 14 serotypes. Biovar 1, also known as the parvo biovar, contains serotypes 1,3,6, and 14, while biovar 2, also known as the T960 biovar, contains serotypes 2,4,5,7,8,9, 10,11,12, and 13. The two biovars were designated as distinct species, because whose DNA homology is less than 60%. Biovar 1 became U. parvum and biovar 2 became U. urealyticum, and they account for 90% to 92% and 8% to 10% respectively. And the serotype 4 is dominant in the patients. UuLAMPs is identified as the key player which can activate the immune response and cause immune disorders such as prostatitis, epididymitis, infertility, preterm births, spontaneous abortions and pelvic infection from birth. With further researching, it has been found that UuLAMPs has influence on some autoimmune disease, including RA.Additional several studies showed there was a connection existed between mycoplasma infection and RA. Stuckey et al. found a 10 year-old male patient with congenital agammaglobulinemia, while suffering from polyarthritis. And synovium puncture cultured was identified with positive Uu, from which the hypothesis that Uu may induce arthritis was proposed. Mycoplasmas were detected in 19 out of 24 RA patients by nested-PCR, which was higher than OA, post-traumatic arthritis and normal controls. Significant differences were found in symptom morbidity between the control group and contrast group. The present study collected case-control studies from domestic and overseas to compare the positive rates of mycoplasma infection between RA and other groups. This work would confirm the relationship between the mycoplasma infection and RA. We gathered and compared the data about general conditions, clinical and imaging indexes of RA patients with Uu infection and RA patients without Uu infection. Some characteristics of serum antibodies, non-special and imaging indexes were found. In them, the anti-Cyclic citrullinated peptide(CCP) antibody was recognized as key factor in the RA pathogenesis, then we speculated Uu may play a role in RA pathogenesis.The synovium pathology sections of rheumatoid arthritis showed the high proliferation of synovial fibroblasts. However, synovial fibroblastsis was always considered a passive role in rheumatoid arthritis, which is always shown as a "victim". Owing to unsatisfactory effect of biologics, synovial fibroblasts as an initiative factor in RA is gaining increasing interest. Another key part the infiltration and cytokines secretion of synovial macrophages was another pathological features of RA synovium. These two cells were supposed as the target cells of RA pathology. Because the synovial macrophages are less viable in vitro, THP-1 macrophages induced with PMA was used to be the cell model of synovial macrophages to explore the mechanism of the effects of UuLAMPs on RA.UuLAMPs is actually a group of proteins, and it has been studied always as a whole. Only few groups studied some special protein factions of the UuLAMPs. Liu W and Gong M found that a membrane ipoprotein p37 can induce ipoprotein of mammalian cell and inhance the abilities of metastasis and invasion of tumor cells. To explore which protein fraction participate in the RA pathogenesis, UuLAMPs will be divided into several fractions by SDS-PAGE. The anti UuLAMPs fractions IgM and IgG antibodies will be detected with the divided protein fractions by Western blot in RA and controls. Some strong immunogenic proteins will be found and some proteins which may paticipate in the RA pathogenesis will be screened out from the UuLAMPs.Objects and methodsThe first part of the reasearch studied the relationship between mycoplasma infection and RA with meta-analyses. Mycoplasma or ureaplasma and arthritis, rheumatoid, with limited terms of human and English are used as the search terms to serch in the English and Chinese databases. The articles on case-control studies about the relationship between mycoplasmas infection and RA were collected according to the search criteria. Data on methodological quality and trial information was extracted by two researchers separately. Meta-analyses were performed with RevMan 4.3.2 software. The odds ratio and its corresponding 95% confidence interval were calculated by Meta-analysis. Fixed-effect approach was took unless there's significant heterogeneity, while random-effects statistical model was used if there's no significant heterogeneity. Sensitivity analysis was performed to look at the possible contribution of differences in the methodological quality. Funnel plot was used for the analysis of publication bias.The second part of the research studied the relationship between Uu infection and RA through clinical case material. Reviewed 74 cases of RA. Uu detection was performed by liquid culture. According to the Uu infection, two groups were divided, one was RA with Uu infection, while the other was RA without Uu infection. Then compared the general conditions, clinical indexes including autoantibodies and imaging study between the two groups.The third part of the research investigated the effects of UuLAMPs on RASFs isolated from RA patients and THP-1 mcrophages induced by PMA. UuLAMPs were prepared from Uu serovar4. Stimulated RASFs with different concentrations of UuLAMPs at different time points. RASFs proliferation was measured by MTT. TNF-a and IL-1βin were detected with RT-PCR and ELISA.studied the immunogenic protein fragments of UuLAMPs. Collected 17 Uu positive patients,18 ReA with Uu infection,16 RA with Uu infection,17 RA without Uu infection and 22 healthy control with Uu negative. In the analyse of UuLAMPs by SDS-PAGE, two gels were prepared, one was used for coomassie brilliant blue R250 staining, the other was used for Western blot, UuLAMPs fractions were used as antigens, while dilute serum of patients and control groups was used as the primary antibody, and rabbit anti-human IgG and goat anti-human IgM antibodies was used as the secondary antibodies. Screen out the immunogenic protein fragments of UuLAMPs, and some fractions may play a role in RA.Results1. The results of meta-analyses showed that there was a significant difference in the infection ratio of mycoplasma between the patients with RA and non-RA (OR=4.40,95%CI 2.26 to 8.56, P<0.0001), and sensitivity analysis according to different publish date and detection methods showed no difference in the results. There were also significant differences between RA patients and OA patients (OR=11.29,95%CI 5.55 to 22.97, P<0.00001) and healthy controls (OR=6.80,95%CI 3.96 to 11.67,P<0.00001).2. The clinical data showed of 74 RA patients with initial diagnosis and treatment, Uu positive RA patients were 41(55.4%). Uu infection had high-positive correlation with the anti-CCP antibody, RA33, ESR, CRP, and the positive rater of declining bone density or osteoporosis, Median Disease Activity Score in 28 joints (DAS28) and imaging changes.3. UuLAMPs improved RASFs proliferation. Over 0.2μg/mL, RASFs proliferation presented down trend.0.05μg/mL UuLAMPs could promote RASFs proliferate to some extent. UuLAMPs could induce the upstream of TNF-αand IL-1βlevels in THP-1 mcrophages. Using UuLAMPs as antigens to detect the special antibodies by westernblot method. Some immunogenic protein fragments were screened out. After compared to the gel with coomassie brilliant blue R250 staining, 25kD protein fragment was proposed to play role in RA.Conclusions1. Mycoplasma infection is associated with RA. More evidences are needed to prove whether mycoplasma plays an equal role in RA as that in ReA, and the significant difference of mycoplasma infection between RA and GA groups.2. Analysis of clinical cases confirms that Uu infection is related to RA, which reveals Uu infection may be involved in RA pathogenesis.3. The present study reveals that UuLAMPs can improve the proliferation of RASFs. And UuLAMPs upregulate the mRNA and protein expression of TNF-αand IL-1βlevels in THP-1 mcrophages. There are immunogenic protein fragments in UuLAMPs, some of which may play roles in RA pathogenesis.