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R forms in correlation with TLR4 (Thr399Ile) gene polymorphism in rheumatoid arthritis
Clinical Biochemistry 45 (2012) 1374–1382
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The presence of anti-LPS antibodies and human serum activity against Proteus mirabilis S/R forms in correlation with TLR4 (Thr399Ile) gene polymorphism in rheumatoid arthritis Michal Arabski a,⁎, Rafal Fudala a, b, Anna Koza a, Slawomir Wasik c, Bozena Futoma-Koloch d, Gabriela Bugla-Ploskonska d, Wieslaw Kaca a a
Institute of Biology, Department of Microbiology, Jan Kochanowski University, 25‐406, Kielce, Poland Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76197, USA c Institute of Physics, Jan Kochanowski University, Swietokrzyska 15, 25‐406, Kielce, Poland d Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63‐77, 51‐148 Wroclaw, Poland b
a r t i c l e
i n f o
Article history: Received 13 December 2011 Received in revised form 14 June 2012 Accepted 16 June 2012 Available online 27 June 2012 Keywords: Proteus mirabilis, lipopolysaccharides Rheumatoid arthritis Complement Laser interferometry TLR4 gene polymorphism LAL
a b s t r a c t Objectives: Proteus mirabilis strains are human pathogens responsible for urinary tract infections, which may also be involved in rheumatoid arthritis (RA). Design and methods: We determined whether the binding site of anti-LPS antibodies on the O-polysaccharide part of P. mirabilis LPS correlates with the level of TLR4 (Thr399Ile) gene polymorphism in the sera of RA patients. We investigated the deposition of C3d and C5b complement components on the P. mirabilis LPS. The ELISA method used in this study was optimized with LAL test and laser interferometry. Results: Depending on LPS P. mirabilis used in these studies, the amount of antibodies in RA patients sera varied. We did not observe a correlation between anti-LPS antibodies binding and the level of TLR4 (Thr399Ile) gene polymorphism. We found that the lower complement components deposition by O49 in contrast to O9 LPS correlates with its reduced sensitivities to human complement-mediated killing. Conclusion: The immunological response against P. mirabilis LPS might play a role in rheumatoid arthritis. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory arthritis which primarily causes a symmetric polyarthritis, clinically manifesting in joint pain, stiffness and swelling. The prevalence of RA reaches 0.5%–1.1% (20 to 50 cases per 100 000 population) in North America and Northern Europe and is higher than in Southern Europe (0.3%–0.7%; 9 to 24 cases per 100 000 population). The total cost of RA in 2006 was estimated at €45 billion in Europe and €42 billion in the US, representing around 1.4% of total health care expenditures [1]. RA is a crippling joint disease with environmental and genetic components. The cause of RA is most probably linked to several microbial triggers (Borrelia burgdorferi, Chlamydia pneumoniae, Klebsiella pneumoniae, Mycoplasma, Mycobacterium tuberculosis, Escherichia coli, Porphyromonas gingivalis, Proteus mirabilis), viral triggers (human parvovirus B1, Rubella virus, human retrovirus 5, alphaviruses, hepatitis B virus, Epstein-Barr virus), genetic predisposition (tumor necrosis factor-receptor associated factor 1 and complement component 5 ⁎ Corresponding author. Fax: +48 42 349 63 07. E-mail address: [email protected] (M. Arabski).
(TRAF1-C5), tumor necrosis factor, alpha-induced protein 3 (TNFAIP3), signal transduction and activation of transcription 4 (STAT4), cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene polymorphism) and autoimmunity (protein tyrosine phosphatase 22 (PTPN22) gene polymorphism) [1–3]. All of these factors play important roles in the etiopathogenesis of RA, thusly described as a multifactorial disease. The role of P. mirabilis rods in RA is still unclear. Some evidences of P. mirabilis in pathogenesis of rheumatoid arthritis are associated with the presence of anti-Proteus antibodies in urine and sera of RA patients in contrast to control sera [4]. Molecular mimicry has been observed between P. mirabilis enzymes (haemolysin and urease) and selfantigens (human leukocyte antigen DR4 (HLA-DR1/4) and collagen type XI), while cross-reactivity between HLA-DR4 and P. mirabilis has also been shown [3]. P. mirabilis produces several virulence factors to be able to invade and successfully establish on the uroepithelial cells of the host, e.g. hemolysins, leukocidin, IgA and/or IgG proteases, urease, siderophores. It has also been suggested that Proteus cell wall surface antigens play a role in the pathogenesis of RA [5]. Various components of the bacterial cells of P. mirabilis interplay with the tissue of the host to determine the bacterial virulence, such as polysaccharide capsules (CPS), peritrichous flagella, different types of pili/fimbriae as well as lipopolysaccharide (LPS, endotoxin) [6–8].
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.06.021
M. Arabski et al. / Clinical Biochemistry 45 (2012) 1374–1382
LPS is considered as an important virulence factor of Gram-negative bacteria, such as P. mirabilis rods. The presence of Gram-negative bacteria in the blood stream results in releasing of LPS from the bacterial cell wall, the activation of the immunological system of the host, blood coagulation (thrombin, plasmin), complement cascade (anaphylatoxins C3a, C5a) and kininogenesis compounds (bradykinin, kininogen, kallikrein). Also, neutrophils, macrophages and monocytes are activated. The activated cells release mediators such as cytokines (interleukins (IL) 1, IL 6, IL 8, tumor necrosis factor (TNF), interferon), proteases (elastase, cathepsins), phospholipids (platelet activating factor (PAF), thromboxanes, leukotrienes) and free radicals [9,10]. The presence of enterobacterial LPS might lead to the high and non-specific activation of the human immunological systems in patients with RA. The final outcome of inflammatory responses is a release of cytokine and antibodies against LPS production which is associated with pathogen-recognition mechanisms, including toll-like receptor 4 (TLR4). LPS is composed of O-specific polysaccharide (O-PS; O-antigen), core oligosaccharide and lipid A [11]. The O-antigen and chain length are diversified in LPS and these variances may contribute to their resistance of bacteria against serum. It is believed that bacteria, which produce long and branched O-PS may be protected against serum complement activity by avoiding the development of a membrane attack complex (MAC). The long O-PS chains of LPS may activate the complement system. However, the MAC components are deposited far away from cell membrane. A weak hydrophilic interaction with the bacterial cell is created, but it leads to the removal of complex from the bacterial cell surface. It is also known that a long O-specific chain of LPS inhibits the interaction of C1q with the bacterial cell wall and protects the Proteus against activation of a classical pathway of complement system. Rough strains (R) that posses LPS lacking in
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O-PS chain are generally susceptible to the bactericidal effects of serum in contrast to smooth (S) strains, which are on the whole more resistant [12,13]. In our previous studies we demonstrated that the level of antibodies recognizing the P. mirabilis O36 LPS in healthy donors correlates with Tlr399Ile TLR4 gene polymorphism types [14]. Hence, in this work we report a series of experiments investigating interactions of a new panel of anti-LPS P. mirabilis O3, O23, O9, O40, O49, O10, R110 and R45 antibodies with Tlr399Ile TLR4 gene polymorphism in RA patients cells. Specifically, we have examined (i) whether the presence of the specific structure of the O-polysaccharide part of P. mirabilis LPS (Table 1) or lack of this structure (R mutants) might be correlated with the presence of anti-LPS antibodies in the sera of RA patients, (ii) if the Limulus Amebocyte Lysate assay (LAL) test and the novel laser interferometry method can be used to verify the immunoassay determining antibodies level in human; (iii) whether human complement deposition correlates with the presence of the specific O-polysaccharide part of LPS. 2. Material and methods 2.1. Human sera of control and rheumatoid arthritis donors Blood samples were taken from 50 healthy donors and 50 patients with rheumatoid arthritis (hospitalized in the Department of Rheumatology, SPZZOZ in Lipsko, Poland) by vein puncture and collected into dry vacutainer tubes. The samples were then allowed to clot for about 1 h at room temperature and centrifuged at 4 °C. The serum was frozen and stored in 0.5 mL aliquots at − 20 °C until tested. The patients (37 females and 13 males) with a positive C-reactive protein,
Table 1 Structures of the O-specific polysaccharide of P. mirabilis using in this study: with non-sugar constituent (O3), with peculiar structures (O10), linear (O9, O40) and branched (O23, O49) [29]. Non-sugar constituent are presented in bold (L-Lys) and peculiar structures for Proteus in frame (L-Alt).
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rhematoid factor (RF) and Waaler–Rose reaction tests ranged in the age from 33 to 86 years (median age 59 years), while healthy donors (28 females and 22 males) ranged from 43 to 67 with a median age of 52 years (Table 2). Experiments were carried out in accordance with the Ethical Committees for Human Subjects guidance. 2.2. Bacteria growth and isolation of the lipopolysaccharides P. mirabilis O9 (PrK 18/57), O40 (PrK 66/57), O49 (PrK 75/57), O23 (PrK 41/57), and O10 (PrK 19/57) were obtained from the Czech National Collection of Type Cultures (Institute of Epidemiology and Microbiology, Prague). Strains S1959 (OXK, O3), R110 and R45 were from the Institute of Microbiology and Immunology University of Lodz, Poland. P. mirabilis strains were cultivated under aerobic conditions in a fermenter (Chemap AG, Switzerland) in nutrient broth (BTL, Poland) under controlled conditions (37 °C, pH 7.4–7.6, pO2 75–85%). The LPS was isolated by the phenol–water procedure and purified by treatment with DNAse and RNAse (Boehringer Mannheim, Germany) as described [13,15,16]. The Proteus LPS of R form was extracted through the phenol–chloroform–petroleum ether (PCP) method [17]. The LPS preparations thus obtained were free of nucleic acid and contained less than 2.5% proteins. 2.3. Verification of ELISA for the determination of antibody levels in human sera The level of anti-LPS antibody detected by enzyme-linked immunosorbent assay (ELISA) depends on well surface covering by LPS from methodological point of view. Degree of well surface covering is associated with the hydrophobic properties of LPS (estimated with laser interferometry method), which determine its solubility, and might be estimated with LAL assay. The comparison of LPS binding intensity (ELISA) and percentage LPS fixed to microplates detected with LAL were presented as coefficient A (arbitrary units) calculated according to formula:
A¼
E L
Table 2 The individual values of C-reactive protein (CRP), RF factor and Waaler–Rose reaction (WR) of RF patients. M—male, F—female. Serum number
Sex
Age
CRP
RF
WR
Serum number
Sex
Age
CRP
RF
WR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
F F F F M F M F F F F F F F F F F F F M F F F F M
37 41 54 27 70 67 64 86 69 56 69 67 25 78 56 84 51 42 82 56 47 68 70 56 81
2 23.6 31.1 4.5 32 40.8 187.7 7.9 35.2 8.3 12.5 25.2 97 6.7 32.6 29.7 16.4 3.6 15.9 75.6 12.5 16.7 8.2 10.6 43.3
56 44 402 43 71 53 42 52 1050 42 41 56 37 36 48 210 55 21 57 1176 40 51 45 49 29
24 – 64 – 32 8 – – – 16 8 – – 64 32 64 64 – – 128 16 32 32 8 16
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
M F M M F M M F F F F F F M F F F F F M M F F M F
45 45 51 74 54 61 61 73 69 44 38 59 74 40 33 36 70 69 81 56 49 69 77 56 63
52.4 3.3 15 43.4 3.2 198 8.7 101 9.7 30.4 5.3 47.6 53.1 90.8 70.7 – 30.7 121 86 45 289 9.8 31.9 98.5 30.8
23 49 77 127 50 39 36 29 33 43 47 309 429 27 455 59 28 87 98 64 114 33 72 28 –
8 8 64 64 8 – – – 32 – – 64 192 16 64 8 16 32 64 24 16 – 64 – 32
where E is the absorbance measured at 490 nm and responds to anti-LPS P. mirabilis antibodies level in RA sera detected by ELISA, L is % of LPS binds to microplates and measured by LAL test. 2.3.1. Limulus amebocyte lysate assay The level of well surface covering (NUNC MaxiSorp microtitre F-bottom plates (black)) by O9, O49, O3, R110 or R45 P. mirabilis endotoxins was determined by the fluorogenic endotoxin detection assay PyroGene rFC purchased from Lonza (Walkersville, USA). The reaction mixtures contained 5 ng LPS per well in 100 μL PBS and left at 4 °C overnight. The plates were washed with phosphate buffered saline (PBS) and all procedures of PyroGene rFC assay were done in accordance with the test instruction. Percent of well surface covering was calculated in comparison to solution of LPS at concentration 5 ng/100 μL (100%). The fluorescence was measured with Microplate Reader TECAN Infinite 200 PRO (Tecan Group Ltd., Switzerland). 2.3.2. Laser interferometry The hydrophobic properties of LPS isolates (Section 2.2) are unfavorably correlated with their solubility and consequently with the degree of well surface covering by tested endotoxins. The hydrophobic properties of LPS were analyzed by laser interferometry method. The measurement set-up for the interferometric investigations was presented previously [18,19]. The interferograms are determined by the refraction coefficient of the solute, which in turn depends on the concentration of the substance. When the solute is uniform, the interference fringes are straight (water compartment) and they bend when a concentration gradient appears (aqueous LPS solution/water interface). The membrane system in this study consists of two glass cuvettes separated by the horizontally located membrane (polymeric nuclear track membrane (nucleopore) with different pore diameters 0.9 μm purchased from Joint Institute for Nuclear Research in Dubna, Russia). In our experiments the upper cuvette held pure water while the lower was filled with aqueous LPS solution. In this system, water migrating through the membrane, diffusing in aqueous LPS solution and lead to the formation of concentration boundary layers (CBLs). The applied computer program to analyse these images allows finding the concentration profiles and the CBL thicknesses (δ). The CBL thickness δ is defined as the distance from the LPS/water interface to the point at which the deviation of the interference fringe from its straight line run is 10% of the fringe thickness (Figs. 1AB). The interferograms were recorded from 2 to 40 min. with a time interval of Δt = 2 min. and the profiles for a given initial LPS concentrations 0.5 mg/mL were reconstructed. Results are presented in CBL thicknesses (δ) in function of time (min). This method permits to compare the hydrophobicity of O3, O49, O9, R110 and R45 LPS isolates. The hydrophobicity of LPS solutions at the same initial concentration is negatively correlated with the δ in function of time. All experiments were performed at a temperature of 37 °C. 2.4. Analysis of anti-LPS P.mirabilis antibodies in RA sera by ELISA The level of anti-LPS P. mirabilis antibodies was determined in healthy donors and RA sera (Table 2) by ELISA. NUNC MaxiSorp microtitre U-bottom plates were coated with 5 ng LPS per well in 100 μL PBS (15 mM Na2HPO4, 150 mM NaCl, pH 7.2) and left at 4 °C overnight. The plates were washed with PBS, blocked with 2% BSA (Sigma, USA) in PBS at 37 °C for 2 h in damp baths and then washed three times with PBS. The plates were incubated with human sera (diluted in PBS 1:1000) for 1 h at 37 °C and washed three times with PBS. Diluted (1:1000) rabbit anti-human IgG conjugated with peroxidase (Sigma, USA) in 1% BSA in PBS was used as the second antibody. Next the plates were washed three times with PBS. A solution of 0.4 mg o-phenylenediamine dihydrochloride (Sigma, USA) in 1 mL of substrate buffer (0.05 M phosphate-citrate buffer pH 5.0) and 40 μL of 0.1% H2O2 (POCH, Poland) was freshly prepared and added. Adding
M. Arabski et al. / Clinical Biochemistry 45 (2012) 1374–1382
0.5 M H2SO4, (after 25 min. of incubation at ambient temperature) stopped the reaction. Then the absorbance was measured at 490 nm with an EL312e Microplate Reader (BIO-TEK Instruments, USA). Each analysis was made in three independent experiments. 2.5. Correlation of complement deposition with O-polysaccharide part of LPS with ELISA We have chosen these two serotypes of LPS (O9 and O49) to correlate the complement deposition with O-polysaccharide part of LPS by ELISA. In this part of study we used three kinds of human sera. The normal human serum (NHS), obtained from healthy donors, was frozen in liquid nitrogen and stored in 0.2 mL aliquots at −70 °C until tested [20]. In some assays, the human sera were chelated with 10 mM ethylene glycol tetraacetic acid (EGTA) containing 10 mM MgCl2 to block activation of the classical complement pathway (bC-NHS). To block the alternative pathway of complement activation, the serum was incubated for 20 min at 50 °C (bA-NHS). Heatinactivated serum (30 min. at 56 °C) was used as a control serum with inactive complement system (T-NHS). We used above two types of sera to detect deposition of C3 or C5 complement molecules on immobilized O9 or O49 LPS. 2.5.1. Detection of deposition C3 complement molecules on immobilized LPS NUNC MaxiSorp microtitre flat-bottom plates were coated with LPS obtained after preparative electrophoresis diluted to 0.5 μg/50 μL in saline phosphate buffer (PBS), pH 7.4 for 18 h at 4 °C. The plates were washed with PBS containing 0.05% Tween 80 (PBS/Tween) (Sigma, USA), blocked with 0.5% gelatin in PBS at 37 °C for 1 h in damp baths and then washed four times with PBS/Tween. Human serum diluted from 100% to 12.5% in PBS was added and incubated with immobilized LPS at 37 °C for 30 min. After washing with PBS/Tween (4×), the deposition of C3 protein was detected with HRP-conjugated rabbit antihuman C3d (DAKO, Denmark), diluted 1/5000 in PBS containing 0.1% gelatin. Human inactivated serum was used as negative control. After washing, the enzymatic activity was estimated by adding 0.1 mL OPD solution (Sigma, USA) and stopping the reaction after 25 min. with 0.5 M H2SO4 (POCH, Poland). The absorbance at 490 nm was measured using a multichannel photometer (BIO-TEK Instruments, USA). Each sample was analyzed in triplicate. The experiment was performed three times, and representative data are presented. In some experiments, before human serum was added, antigens were coated for 45 min. with polyclonal anti-P. mirabilis O9 and O49 rabbit serum (diluted with PBS to give the absorbance A492 = 1.0), respectively and washed 4 times with PBS. Polyclonal anti-P. mirabilis O9 and O49 were obtained by immunization of rabbits with heat-inactivated bacteria (100 °C, 2.5 h). To prepare the immunogen, lyophilized bacteria were suspended in sodium chlorine (0.85% NaCl) at a concentration of 1 mg/mL. Animals were injected intravenously with 50, 100, 100, 200 and 500 μg of immunogen in 500 μL of saline solution on days 1, 4, 7, 11 and 16, respectively. Seven days after the last immunization, the blood was sampled by the cardiac puncture, and then serum was separated and stored in 0.5 mL aliquots at − 20 °C. Experiments were carried out in accordance with the Animal Ethical Committees guidance. 2.5.2. Detection of deposition C5b complement molecules on immobilized LPS The beginning steps were performed as described above. After incubation in human serum and washing with PBS/Tween, the deposition of C5b was detected with mouse monoclonal antibody (clone aE11) (DAKO, Denmark), diluted 1:100 in PBS containing 0.1% gelatin. After incubation (60 min. 37 °C) and washing with PBS/Tween 80 buffer (4×) secondary antibodies anti-mouse goat IgG with HRP-conjugated (DAKO, Denmark), diluted 1:2000 in PBS containing 0.1% gelatin
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was used. As negative control, human inactivated serum was used. The enzymatic activity was estimated using the above methods. In some experiments, before human serum was added, antigens were coated for 45 min with polyclonal anti-P. mirabilis O9 and O49 rabbit serum (diluted with PBS to give the absorbance A492 =1.0), respectively and washed 4 times with PBS. Polyclonal anti- P. mirabilis O9 and O49 rabbit sera were obtained according to description in Section 2.5.1. 2.6. Correlation of anti-LPS antibodies levels with tlr4 gene polymorphism with RFLP-PCR Genomic DNA was prepared by Miniprep/Qiagen kit (QIAGEN, Germany). The tlr4 (Thr399Ile) gene polymorphism was determined by the Restriction Fragment Length Polymorphism-Polymerase Chain Reaction (RFLP-PCR) with the following primers: sense 5′-GCT GTT TTC AAA GTG ATT TTG GGA GAA-3′, antisense 5′‐CAC TCA TTT GTT TCA AAT TGG AAT G-3′. This single nucleotide polymorphism (SNP) is a C/T transition causing a threonine/isoleucine switch at amino acid in 1196 position of tlr4 gene. The 146-bp PCR product was digested 18 h with 2 U of the restriction enzyme HinfI (Promega, Madison, Wisconsin, USA). The Ile allele was digested into 127-bp fragments, whereas the Thr variant remained intact. The PCR products were run on a 12% of polyacrylamide gel [21]. 2.7. Data analysis The data were analyzed using the Statistica (StatSoft, Tulsa, OK, USA) software package. Values of anti-LPS P. mirabilis antibodies in control and RA sera are expressed as mean± standard device. The individual level of anti-LPS P. mirabilis antibodies of control donors and RA patients detected by ELISA was performed in triplicate. The summary of the results of anti-LPS antibodies in control and RA sera is expressed as a mean of fifty or three independent experiments, respectively. The evaluation of the amount of LPS bind to microtiter plates with LAL was performed in triplicate. The differences between anti-LPS level in control and RA groups were compared with One-way ANOVA test. One-way ANOVA is a useful test in comparing two or more means because we can omit the type I error associated with false selection of test, like t-test. We used this analysis previously and might compare this study with our earlier conclusions [14]. Genotype frequencies were tested for Hardy–Weinberg equilibrium. Distribution of genotypes was analyzed using the χ2-test. The odds ratio (OR) and 95% confidence interval (CI) were estimated by logistic regression analysis. To evaluate the goodness of fit of the logistic regression model, Nagelkerke's R-square was calculated. P values less than 0.05 were considered statistically significant. For the statistical analysis, differences between controls and RA patients were calculated with the two-tailed Fisher exact probability test. 3. Results 3.1. Serological reaction of human sera with LPS and genotype analysis In order to investigate serological reaction of human sera with LPS, levels of antibodies in 50 sera samples from patients with RA and 50 healthy blood donors were measured by ELISA, as shown in Fig. 2 and Fig. 3. We observed that levels of anti-O9, O40, O49, O23 and O3 LPS P. mirabilis antibodies are statistically, significantly higher in RA patients than in control, which is in contrast to anti-O10 and R forms (Fig. 3). Analysis of individual levels of anti-LPS P. mirabilis antibodies of S forms shows that the levels of anti-O9 antibodies in sera are higher than in control for 28% of RA patients, 78% for anti-O40, 74% for anti-O49, 54% for anti-O23, and 68% for anti-O3 (Fig. 2). Tables 3 and 4 show genotype analysis of the tlr4 gene polymorphism (Thr399Ile) in 50 RA patients versus 50 controls and selected 34 candidates from 50 RA patients versus 50 controls, respectively.
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Fig. 1. Laser interferometry analysis of CBL thicknesses (δ) for S and R forms of aqueous LPS solutions (C). A—scheme of membrane system, denotes δ, B—examples of interferograms for O3 and R45 LPS.
We have selected 34 candidates from 50 RA patients on the basic of two criteria. Firstly, we have chosen only these RA patients for whom the level of anti-LPS antibodies in sera was statistically higher than in control healthy donors (Fig. 2A). Secondly, we have chosen only these RA patients in whose sera the individual levels of anti-LPS P. mirabilis antibodies was statistically higher than in control (at least three of them) (Fig. 2B denoted by *). The logistic regression model including all tested anti-LPS antibodies level in control and RA sera as well as the tlr4 gene variants had a Nagelkerke's R-square of 0.903 (indicating that it explained approximately 90.3% of the variation in the data). We did not find any correlation between anti-LPS P. mirabilis antibodies in RA patient's blood and TLR4 gene polymorphism. Taking into consideration odds ratio (OR) and probability P (calculated by two-tailed Fisher test) values, we did not find any correlation between anti-LPS P. mirabilis antibodies in RA sera (Table 3) or selected RA patient's blood and tlr gene polymorphism Thr399Ile (Table 4). The distribution of genotypes in control and RA groups was consistent with the Hardy–Weinberg equilibrium in both studies (Tables 3 and 4). 3.2. Verification of ELISA with LAL test and laser interferometry method In order to confirm that the level of anti-LPS antibodies in RA patients sera does not depend on the amount of antigen bound to microplate, the LAL test was done. Assessed by the LAL test, the amount of LPS fixed were 1.27% for R110, 4.26% for R45, 18.39% for O9, 15.65% for O49 and 17.11% for O3 (Table 5). The differences in binding strengths could be due to different solubility properties of smooth
(O9, O49, and O3) and rough (R45 and R110) LPS (Fig. 1C). Laser interferometry analysis of CBL thicknesses (δ) for S and R forms of aqueous LPS solutions shows that δ= 1.653 mm for O9 after 40 min., 0.975 for O49, 0.880 for O3, 0.133 for R110 and 0.399 for O45, respectively (Table 5). The higher value of δ is positively correlated with better solubility of LPS aggregates, its binding to microtitre plates, and finally the level of anti-LPS P. mirabilis antibodies in sera measured with ELISA. We have observed that the summary reactions of sera from RA patients with R forms of P. mirabilis (R45 and R110) LPS are higher than with S forms (O3 and O9, in contrast to O49) (Table 5). 3.3. Deposition of C3d and C5b-9 on native P. mirabilis O9 or O49 LPS The pre-incubation of LPS with rabbit antisera caused the increase in C3d deposition onto both LPS (Fig. 4). LPS pre-incubated with homologous rabbit antisera bound much more amounts of C3d component of human complement in comparison to not pre-incubated LPS (data not shown). It is probably caused by the reaction of the C1q component with a heavy chain of specific anti-O-chain rabbit antibodies binding to LPS during pre-incubation. The deposition of C3d depends on the concentration of serum used as a source of the complement. The beginning of the reaction above control (T-NHS) was visible when the concentration of serum reached 12.5%. Native P. mirabilis O9 LPS strongly activated complement, which was estimated with C3d complement component deposition assay. The structural investigation showed that the difference in the O-antigen structure of both LPS. LPS from P. mirabilis O49 is branched in contrast to LPS from P. mirabilis O9
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Fig. 2. The individual level of anti-LPS P. mirabilis antibodies of RA patients compared to 50 healthy donors measured by ELISA (panel A). Error bars denote standard device. Panel B presents only these RA patients (denote by grey frame) in whose sera the individual levels of anti-LPS P. mirabilis antibodies was statistically higher than in control (at least three of them; denote by *).
(Table 1). The inhibition of the alternative pathway of the complement activation via inactivation of factor B slightly influenced the amount of C3d component bounded with P. mirabilis O9 LPS when compared to
native complement activation results. However, inhibition of alternative pathway in the case of P. mirabilis O49 LPS resulted in a significant decrease of C3d binding.
Fig. 3. The summary level of anti-LPS P. mirabilis antibodies of RA patients compared to 50 healthy donors measured by ELISA. Error bars denote standard device.
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Table 3 The allele and genotype frequencies and odds ratio (OR) of the Thr399Ile polymorphism of the TLR4 gene in RA patients in comparison to control healthy donors. Genotype or allele
RA patients (n = 50)
C/C C/G G/G C G a
Controls (n = 50)
OR (95% CI)
P valuea
Number
Frequency
Number
Frequency
42 8 0 χ2 = 0.378; p = 0.539 92 8
0.840 0.160 –
44 6 0 χ2 = 0.204; p = 0.651 94 6
0.880 0.120 –
0.716 (0.229–2.238) 1.397 (0.447–4.367) –
0.774 0.774 –
0.940 0.060
0.734 (0.245–2.198) 1.362 (0.455–4.080)
0.782 0.783
0.920 0.080
Two-tailed Fisher exact probability test.
Table 4 The allele and genotype frequencies and odds ratio (OR) of the Thr399Ile polymorphism of the TLR4 gene in RA patients with highest individual level of anti-LPSs P. mirabilis antibodies (at least three of them) in comparison to control healthy donors. Genotype or allele
RA patients (n = 34)
C/C C/G G/G C G a
Controls (n = 50)
OR (95% CI)
P valuea
Number
Frequency
Number
Frequency
30 4 0 χ2 = 0.133; p = 0.716 64 4
0.882 0.118 –
44 6 0 χ2 = 0.204; p = 0.651 94 6
0.880 0.120 –
1.022 (0.266–3.936) 0.978 (0.254–3.762) –
1.000 1.000
0.940 0.060
1.021 (0.277–3.764) 0.979 (0.266–3.609)
1.000 1.000
0.941 0.059
Two-tailed Fisher exact probability test.
The bactericidal activity of serum is related to the complement cascade activation, leading to the lytic complex C5b-9 creation. In order to confirm that the native LPS P. mirabilis O9, O49 indeed activated human complement, the depositions of C5b with monoclonal antibodies (clone aE11) were done. As a result of complement activation with P. mirabilis O9 LPS, higher deposition of C5b was observed. However deposition of C5b on P. mirabilis O49 LPS was significantly lower (Fig. 5). As in the case of C3d deposition, the amounts of binding C5b by native LPS were strongly enhanced in the presence of anti-O9 and O49 antibodies (data not shown). The complement resistance of P. mirabilis O9 and O49 rods was tested in 50% PBSdiluted normal human serum. After 3 h incubation at 37 °C only 45% bacterial cell of P. mirabilis O9 survived. In contrary, in the same conditions intensive growth of P. mirabilis O49 (above 100% used, 10 7 CFU/mL) was observed. Similarly, in NHS with inhibited alternative complement activation pathway, P. mirabilis O9 were sensitive to bactericidal effect of serum in contrast to O49 strain. 4. Discussion 4.1. Correlation of anti-LPS antibodies with TLR4 (Thr399Ile) gene polymorphism The TLR4 receptor is specific for lipopolysaccharide and is important for pathogen-recognition mechanisms. Host–pathogen interactions are involved in RA pathophysiology and TLR genes influence both
proinflammatory cytokine production and autoimmune responses. TLRs are expressed by B lymphocytes and T lymphocytes, antigenpresenting cells, regulatory T cells and are found in the rheumatoid synovium. It is suggested that innate immunity may be involved in initiating the inflammatory process or in inhibiting regulation mechanisms that normally prevent chronic inflammation [22]. We did not observe a correlation between the levels of all tested anti-LPS with different structures of O-polysaccharide region P. mirabilis antibodies and TLR4 (Thr399Ile) gene polymorphism. This result is in accordance with some studies, in which more specific markers for RA diagnosis were used as anti-cyclic citrullinated peptide and erosions autoantibodies [23,24]. 4.2. The level of anti-LPS P. mirabilis antibodies in RA sera The individual and summary level of anti-LPS P. mirabilis S forms antibodies is higher in sera of RA patients than healthy blood donors. This result is in accordance with some studies [4] and indicates that P. mirabilis infection might be correlated with human susceptibility to RA. Additionally, LPS from Proteus strains have uncommon constituents: 4-amino-4-deoxy-L-arabinosyl residues (AraN) in the 3-deoxyoctulosonic acid (KDO) and lipid A regions, D-galacturonic acid in the core region, and amino and uronic acids and unusual sugars in the O-polysaccharide domain. These constituents are not present in other members of the Enterobacteriaceae [25]. In this study, the serological reaction of human sera with LPS was measured with ELISA and we
Table 5 Correlation of summary anti-LPSs P. mirabilis antibodies level measured by ELISA with amount of LPSs bind to micotitre plates (%) evaluated by LAL test and solubility of LPSs isolates by laser interferometry (δ in mm). δ is defined as the distance from the LPS/water interface to the point at which the deviation of the interference fringe from its straight line measured by laser interferometry; δ value is positvely correlated with hydrophobic properties of LPS isolates Coefficient A is the relation of absorbance (A490 nm) and proper % of LPS binds to micotitre plates. LPS
O9 O49 O3 R110 R45
ELISA (A490 nm) [mean ± standard device] control
RA
0.195 ± 0.115 0.237 ± 0.059 0.129 ± 0.075 0.132 ± 0.083 0.156 ± 0.091
0.301 ± 0.289 0.990 ± 0.635 0.415 ± 0.293 0.161 ± 0.098 0.155 ± 0.082
LAL (%) [mean ± standard device]
Laser interferometry (δ)
Coefficient A (arbitrary units)
18.39 ± 4.29 15.65 ± 5.43 17.11 ± 4.65 1.27 ± 0.97 4.26 ± 2.84
1.653 0.975 0.880 0.133 0.399
0.016 0.063 0.024 0.127 0.036
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4.4. Correlation between complement deposition and O-polysaccharide structure of LPS
Fig. 4. Deposition of C3d on native P. mirabilis O9 (A) or P. mirabilis O49 (B) LPS pre‐ incubated with rabbit antisera and after incubation with: normal human serum NHS (●), human serum with blocked alternative complement pathway bA-NHS (▼), human serum with blocked classic complement pathway bC-NHS (■) or inactivated human serum without complement activity T-NHS (♦, control).
observed the level of antibodies against antigen O, core, and lipid A regions of LPS. These regions contain unique components for Proteus and serological reactions confirm the role of Proteus infection in rheumatoid arthritis. The level of anti-LPS O10 P. mirabilis antibodies stayed at the same level in RA and control sera. It might be associated with peculiar (L-alturonic acid; L-Alt) structure in antigen O. The immune system might be highly activated by the O-polysaccharide domain with very unique L-alturonic acid in both investigated groups.
4.3. Verification of ELISA with LAL assay and laser interferometry method It is important that the ELISA method used in this study was optimized with LAL test and laser interferometry. The CBL thicknesses (δ) measured by interferometry method was positively correlated with better solubility of LPS aggregates, its binding to microtitre plates evaluated with LAL test and finally level of anti-LPS P. mirabilis antibodies in sera measured by ELISA (coefficient A). This complex correlation confirmed the role of anti-LPS P. mirabilis antibody in RA. The high value of coefficient A for R forms of P. mirabilis LPS indicated that we had detected antibodies against the most conservative structure of LPS–core-lipid A parts of LPS. The detection of anti-LPS R forms antibodies in RA sera suggests that the LPS of Enterobacteriaceae may play a significant role in RA. Unstable immune system of RA patients could unexpectedly produce antibodies against weak immunogenic antigens, like lipid A in RA patients in contrast to healthy blood donors.
Fig. 5. Deposition of C5b-9 on native P. mirabilis O9 (▼) or P. mirabilis O49 (●) LPS after incubation with normal human serum NHS. ■—inactivated human serum without complement activity T-NHS (control).
The level of anti-LPS O9 P. mirabilis antibodies was on the same level in RA and control sera. It might be associated with the linear structure in antigen O9. We have chosen these two serotypes of LPS (O9 and O49) to the next step of broad investigations on the deposition of C3d and C5b complement components on P. mirabilis LPS. Immunochemical analysis of the outer membrane of the different Gram-negative bacteria showed that the differences in the structure of LPS can play an important role in determining the susceptibility of Gram-negative strains to the action of serum [12,26]. Our previous observations that P. mirabilis Ra mutant (R110) and P. mirabilis Re mutant (R45) demonstrated sensitivity to the bactericidal activity of serum than the P. mirabilis S form (S1959), resistant to NHS [27]. The LPS O9 deposited C3d and C5b molecules and made the P. mirabilis O9 rods sensitive to bactericidal effect of complement, in contrast to P. mirabilis O49. The LPS O49 deposited much less C3d and C5b complement components. Both investigated strains differs on their O-antigen structures and our result indicated that both: the length as well as O-specific chain of LPS structures may play the crucial role in the complement binding and bactericidal affect of human sera [28]. In conclusion, we suggest that the chemical structure, length of the O49-polysaccharide chains of LPS protects P. mirabilis O49 strain against the bactericidal effect of human serum. It is contrary to P. mirabilis O9 cells lysed through by deposited MAC complexes. The prolonged presence of P. mirabilis O49 cells in human intestine or in the lower part of the renal tract resulted in the high level of anti-O49 antibodies presence. It was observed that only 28% of RA patients sera reacted with O9 LPS, whereas 74% with O49 LPS. One may postulate that prolonged complement activation by complement-resistant enterobacterial cells i.e. P. mirabilis O49 resulted in increased C3ca and other complement mediators released. The constant proinflammatory activities of LPS and its part may cause native immune system to be overactivated with the final outcome of non-specific autoantibodies generation in RA diseases. The verification of methods sensitive and valuable in detection of antibodies needs to be stressed. In the presented results, the binding to microtitre plates abilities of LPS varied significantly (tested by LAL method). Also hydrophobicity of Proteus LPS varied (determined by laser interferometry). This may indicate that human cells stimulation via TLR 4 receptors depends on unique structures of the polysaccharide part of the LPS, that modulates lipid A part binding to TLR 4, MD2 receptor complexes drafts. Abbreviations AraN 4-amino-4-deoxy-L-arabinosyl residue bA-NHS serum with blocked activation of the alternative complement pathway bC-NHS serum with blocked activation of the classical complement pathway BSA bovine serum albumin C1q subcomponent of the C1 complex of the classical pathway of complement activation C3d complement component 3d C5b complement component 5b CBL concentration boundary layer CPS polysaccharide capsules CTLA-4 cytotoxic T-lymphocyte antigen 4 EGTA ethylene glycol tetraacetic acid ELISA enzyme-linked immunosorbent assay HLA-DR4 human leukocyte antigen DR4 HRP horseradish peroxidase IL interleukin KDO 3-deoxyoctulosonic acid LAL limulus amebocyte lysate assay LPS lipopolysaccharide
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MAC MD2
membrane attack complex glycoprotein associating with the extracellular domain of TLR4 NHS normal human serum O-PS O specific polysaccharide PAF platelet activating factor PBS phosphate buffered saline PTPN22 protein tyrosine phosphatase 22 R rough strains without antigen O in LPS structure RA rheumatoid arthritis RF rhematoid factor RFLP-PCR restriction fragment length polymorphism-polymerase chain reaction S smooth strains with antigen O in LPS structure SNP single nucleotide polymorphism TLR4 toll-like receptor 4 TNF tumor necrosis factor TNFAIP3 tumor necrosis factor; alpha-induced protein 3 STAT4 signal transduction and activation of transcription 4 protein T-NHS serum with inactive complement system TRAF1-C5tumor necrosis factor-receptor associated factor 1 and complement component 5
Acknowledgments The work was supported by grant BS/2012 from the Jan Kochanowski University, Poland. We would like to thank Krystyna Surma, Iwona Koprucha, Halina Bakalarska, Wojciech Garbacz, Łukasz Madej, Ewelina Nowak and Agnieszka Matusiak for sample collection and technical support. References [1] Tobón GJ, Youinou P, Saraux A. The environment, geo-epidemiology, and autoimmune disease: rheumatoid arthritis. J Autoimmun 2010;35:10–4. [2] Newkirk MM. Rheumatoid factors: host resistance or autoimmunity? Clin Immunol 2002;104:1–13. [3] Rashid T, Ebringer A. Rheumatoid arthritis is linked to Proteus—the evidence. Clin Rheumatol 2007;26:1036–43. [4] Senior EBW, Anderson GA, Morley KD, Kerr MA. Evidence that patients with rheumatoid arthritis have asymptomatic 'non-significant' Proteus mirabilis bacteriuria more frequently than healthy controls. J Infect 1999;38:99–106. [5] Tiwana H, Wilson C, Pirt J, Cartmell W, Ebringer A. Autoantibodies to brain components and antibodies to Acinetobacter calcoaceticus are present in bovine spongiform encephalopathy. Infect Immun 1999;67:6591–5. [6] Rozalski A, Sidorczyk Z, Kotelko K. Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 1997;61:65–89. [7] Mishra M, Thakar YS, Pathak AA. Haemagglutination, haemolysin production and serum resistance of Proteus and related species isolated from clinical sources. Indian J Med Microbiol 2001;19:5–11. [8] Rocha SP, Elias WP, Cianciarullo AM, Menezes MA, Nara JM, Piazza RM, et al. Aggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cells. FEMS Immunol Med Microbiol 2007;51:319–26.
[9] Gutierrez-Ramos JC, Bluethmann H. Molecular and mechanisms operating in septic shock: lessons from knockout mice. Immunol Today 1997;18:329–34. [10] Sehic S, Li AL, Ungar CM. Blatteis, Complement reduction impairs the febrile response of guinea pigs to endotoxin. Am J Physiol 1998;274:1594–603. [11] Knirel YA, Kochetkov NK. The structure of the lipopolysaccharides of Gram‐ negative bacteria. III. The structure of O‐antigens. Biochemistry (Moscow) 1994;59: 1325. [12] Mielnik G, Gamian A, Doroszkiewicz W. Bactericidal activity of normal cord serum (NCS) against Gram-negative rods with sialic acid-containing lipopolysaccharides (LPS). FEMS Immunol Med Microbiol 2001;31:169–73. [13] Vinogradov EV, Radziejewska-Lebrecht J, Kaca W. The structure of the carbohydrate backbone of core-lipid A region of the lipopolysaccharide from Proteus mirabilis wildtype strain S1959 (serotype O3) and its Ra mutant R110/1959. Eur J Biochem 2000;267:262–8. [14] Arabski M, Grabowski S, Konieczna I, Kaca W, Kondakova AN, Perepelov A, et al. Serotyping of clinical isolates belonging to Proteus mirabilis serogroup O36 and structure elucidation of the O36-antigen polysaccharide. FEMS Immunol Med Microbiol 2008;5:395–403. [15] Westphal O, Jann K, Himmelspach K. Chemistry and immunochemistry of bacterial lipopolysaccharides as cell wall antigens and endotoxins. Prog Allergy 1983;33:9–39. [16] Kondakova AN, Fudala R, Senchenkova SN, Shashkov AS, Knirel YA, Kaca W. Structural and serological studies of the O-antigen of Proteus mirabilis O-9. Carbohydr Res 2003;338:1191–6. [17] Galanos C, Luderitz O, Westphal O. A new method for the extraction of Rlipopolysaccharides. Eur J Biochem 1969;9:245–9. [18] Arabski M, Wasik S, Dworecki K, Kaca W. Laser interferometric determination of ampicillin and colistin transfer through cellulose biomembrane in the presence of Proteus vulgaris O25 lipopolysaccharide. J Membr Sci 2007;299: 268–75. [19] Wasik S, Arabski M, Dworecki K, Slezak A, Kaca W. Influence of gravitational field on substance transport in gels. J Membr Sci 2010;365:341–6. [20] Andersson J, Ekdahl KN, Lambris JD, Nilsson B. Binding of C3 fragments on top of adsorbed plasma proteins during complement activation on a model biomaterial surface. Biomaterial 2005;26:1477–85. [21] Schippers EF, van't Veer C, van Voorden S, Martina CA, le Cessie S, van Dissel JT. TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin. Cytokine 2004;26:16–24. [22] Jaen O, Petit-Teixeira E, Kirsten H, Ahnert P, Semerano L, Pierlot C, et al. European Consortium on Rheumatoid Arthritis Families. No evidence of major effects in several Toll-like receptor gene polymorphisms in rheumatoid arthritis. Arthritis Res Ther 2009;11:1–10. [23] Zheng B, Li Q, Wei C, Qin J, Shou T, Zhou R, et al. Lack of association of TLR4 gene Asp299Gly and Thr399Ile polymorphisms with rheumatoid arthritis in Chinese Han population of Yunnan Province. Rheumatol Int 2010;30:1249–52. [24] Sánchez E, Orozco G, López-Nevot MA, Jiménez-Alonso J, Martín J. Polymorphisms of toll-like receptor 2 and 4 genes in rheumatoid arthritis and systemic lupus erythematosus. Tissue Antigens 2004;63:54–7. [25] Swierzko A, Kirikae T, Kirikae F, Hirata M, Cedzynski M, Ziolkowski A, et al. Biological activities of lipopolysaccharides of Proteus spp. and their interactions with polymyxin B and an 18-kDa cationic antimicrobial protein (CAP18)-derived peptide. J Med Microbiol 2000;49:127–38. [26] Simmons DAR. The structural basis of serological specificity in Shigella flexneri O‐ antigens. Biochem Soc Trans 1990;18:1271–2. [27] Kaca W, Arabski M, Fudała R, Holmström E, Sjöholm A, Weintraub A, et al. Human complement activation by smooth and rough Proteus mirabilis strains: the role of lipopolysaccharides and outer membrane proteins. Arch Immunol Ther Exp (Warsz) 2009;57:383–91. [28] Futoma-Koloch B, Bugla-Ploskonska G, Doroszkiewicz W, Kaca W. Survival of Proteus mirabilis O3 (S1959), O9 and O18 strains in normal human serum (NHS) correlates with the diversity of their outer membrane proteins (OMPs). Pol J Microbiol 2006;55:153–6. [29] Knirel YA, Perepelov AV, Kondakova AN, Senchenkova SN, Sidorczyk Z, Rozalski A, et al. Structure and serology of O-antigens as the basis for classification of Proteus strains. Innate Immun 2011;17:70–96.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
The presence of anti-LPS antibodies and human serum activity against Proteus mirabilis S/R forms in correlation with TLR4 (Thr399Ile) gene polymorphism in rheumatoid arthritis Michal Arabski a,⁎, Rafal Fudala a, b, Anna Koza a, Slawomir Wasik c, Bozena Futoma-Koloch d, Gabriela Bugla-Ploskonska d, Wieslaw Kaca a a
Institute of Biology, Department of Microbiology, Jan Kochanowski University, 25‐406, Kielce, Poland Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76197, USA c Institute of Physics, Jan Kochanowski University, Swietokrzyska 15, 25‐406, Kielce, Poland d Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63‐77, 51‐148 Wroclaw, Poland b
a r t i c l e
i n f o
Article history: Received 13 December 2011 Received in revised form 14 June 2012 Accepted 16 June 2012 Available online 27 June 2012 Keywords: Proteus mirabilis, lipopolysaccharides Rheumatoid arthritis Complement Laser interferometry TLR4 gene polymorphism LAL
a b s t r a c t Objectives: Proteus mirabilis strains are human pathogens responsible for urinary tract infections, which may also be involved in rheumatoid arthritis (RA). Design and methods: We determined whether the binding site of anti-LPS antibodies on the O-polysaccharide part of P. mirabilis LPS correlates with the level of TLR4 (Thr399Ile) gene polymorphism in the sera of RA patients. We investigated the deposition of C3d and C5b complement components on the P. mirabilis LPS. The ELISA method used in this study was optimized with LAL test and laser interferometry. Results: Depending on LPS P. mirabilis used in these studies, the amount of antibodies in RA patients sera varied. We did not observe a correlation between anti-LPS antibodies binding and the level of TLR4 (Thr399Ile) gene polymorphism. We found that the lower complement components deposition by O49 in contrast to O9 LPS correlates with its reduced sensitivities to human complement-mediated killing. Conclusion: The immunological response against P. mirabilis LPS might play a role in rheumatoid arthritis. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory arthritis which primarily causes a symmetric polyarthritis, clinically manifesting in joint pain, stiffness and swelling. The prevalence of RA reaches 0.5%–1.1% (20 to 50 cases per 100 000 population) in North America and Northern Europe and is higher than in Southern Europe (0.3%–0.7%; 9 to 24 cases per 100 000 population). The total cost of RA in 2006 was estimated at €45 billion in Europe and €42 billion in the US, representing around 1.4% of total health care expenditures [1]. RA is a crippling joint disease with environmental and genetic components. The cause of RA is most probably linked to several microbial triggers (Borrelia burgdorferi, Chlamydia pneumoniae, Klebsiella pneumoniae, Mycoplasma, Mycobacterium tuberculosis, Escherichia coli, Porphyromonas gingivalis, Proteus mirabilis), viral triggers (human parvovirus B1, Rubella virus, human retrovirus 5, alphaviruses, hepatitis B virus, Epstein-Barr virus), genetic predisposition (tumor necrosis factor-receptor associated factor 1 and complement component 5 ⁎ Corresponding author. Fax: +48 42 349 63 07. E-mail address: [email protected] (M. Arabski).
(TRAF1-C5), tumor necrosis factor, alpha-induced protein 3 (TNFAIP3), signal transduction and activation of transcription 4 (STAT4), cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene polymorphism) and autoimmunity (protein tyrosine phosphatase 22 (PTPN22) gene polymorphism) [1–3]. All of these factors play important roles in the etiopathogenesis of RA, thusly described as a multifactorial disease. The role of P. mirabilis rods in RA is still unclear. Some evidences of P. mirabilis in pathogenesis of rheumatoid arthritis are associated with the presence of anti-Proteus antibodies in urine and sera of RA patients in contrast to control sera [4]. Molecular mimicry has been observed between P. mirabilis enzymes (haemolysin and urease) and selfantigens (human leukocyte antigen DR4 (HLA-DR1/4) and collagen type XI), while cross-reactivity between HLA-DR4 and P. mirabilis has also been shown [3]. P. mirabilis produces several virulence factors to be able to invade and successfully establish on the uroepithelial cells of the host, e.g. hemolysins, leukocidin, IgA and/or IgG proteases, urease, siderophores. It has also been suggested that Proteus cell wall surface antigens play a role in the pathogenesis of RA [5]. Various components of the bacterial cells of P. mirabilis interplay with the tissue of the host to determine the bacterial virulence, such as polysaccharide capsules (CPS), peritrichous flagella, different types of pili/fimbriae as well as lipopolysaccharide (LPS, endotoxin) [6–8].
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.06.021
M. Arabski et al. / Clinical Biochemistry 45 (2012) 1374–1382
LPS is considered as an important virulence factor of Gram-negative bacteria, such as P. mirabilis rods. The presence of Gram-negative bacteria in the blood stream results in releasing of LPS from the bacterial cell wall, the activation of the immunological system of the host, blood coagulation (thrombin, plasmin), complement cascade (anaphylatoxins C3a, C5a) and kininogenesis compounds (bradykinin, kininogen, kallikrein). Also, neutrophils, macrophages and monocytes are activated. The activated cells release mediators such as cytokines (interleukins (IL) 1, IL 6, IL 8, tumor necrosis factor (TNF), interferon), proteases (elastase, cathepsins), phospholipids (platelet activating factor (PAF), thromboxanes, leukotrienes) and free radicals [9,10]. The presence of enterobacterial LPS might lead to the high and non-specific activation of the human immunological systems in patients with RA. The final outcome of inflammatory responses is a release of cytokine and antibodies against LPS production which is associated with pathogen-recognition mechanisms, including toll-like receptor 4 (TLR4). LPS is composed of O-specific polysaccharide (O-PS; O-antigen), core oligosaccharide and lipid A [11]. The O-antigen and chain length are diversified in LPS and these variances may contribute to their resistance of bacteria against serum. It is believed that bacteria, which produce long and branched O-PS may be protected against serum complement activity by avoiding the development of a membrane attack complex (MAC). The long O-PS chains of LPS may activate the complement system. However, the MAC components are deposited far away from cell membrane. A weak hydrophilic interaction with the bacterial cell is created, but it leads to the removal of complex from the bacterial cell surface. It is also known that a long O-specific chain of LPS inhibits the interaction of C1q with the bacterial cell wall and protects the Proteus against activation of a classical pathway of complement system. Rough strains (R) that posses LPS lacking in
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O-PS chain are generally susceptible to the bactericidal effects of serum in contrast to smooth (S) strains, which are on the whole more resistant [12,13]. In our previous studies we demonstrated that the level of antibodies recognizing the P. mirabilis O36 LPS in healthy donors correlates with Tlr399Ile TLR4 gene polymorphism types [14]. Hence, in this work we report a series of experiments investigating interactions of a new panel of anti-LPS P. mirabilis O3, O23, O9, O40, O49, O10, R110 and R45 antibodies with Tlr399Ile TLR4 gene polymorphism in RA patients cells. Specifically, we have examined (i) whether the presence of the specific structure of the O-polysaccharide part of P. mirabilis LPS (Table 1) or lack of this structure (R mutants) might be correlated with the presence of anti-LPS antibodies in the sera of RA patients, (ii) if the Limulus Amebocyte Lysate assay (LAL) test and the novel laser interferometry method can be used to verify the immunoassay determining antibodies level in human; (iii) whether human complement deposition correlates with the presence of the specific O-polysaccharide part of LPS. 2. Material and methods 2.1. Human sera of control and rheumatoid arthritis donors Blood samples were taken from 50 healthy donors and 50 patients with rheumatoid arthritis (hospitalized in the Department of Rheumatology, SPZZOZ in Lipsko, Poland) by vein puncture and collected into dry vacutainer tubes. The samples were then allowed to clot for about 1 h at room temperature and centrifuged at 4 °C. The serum was frozen and stored in 0.5 mL aliquots at − 20 °C until tested. The patients (37 females and 13 males) with a positive C-reactive protein,
Table 1 Structures of the O-specific polysaccharide of P. mirabilis using in this study: with non-sugar constituent (O3), with peculiar structures (O10), linear (O9, O40) and branched (O23, O49) [29]. Non-sugar constituent are presented in bold (L-Lys) and peculiar structures for Proteus in frame (L-Alt).
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rhematoid factor (RF) and Waaler–Rose reaction tests ranged in the age from 33 to 86 years (median age 59 years), while healthy donors (28 females and 22 males) ranged from 43 to 67 with a median age of 52 years (Table 2). Experiments were carried out in accordance with the Ethical Committees for Human Subjects guidance. 2.2. Bacteria growth and isolation of the lipopolysaccharides P. mirabilis O9 (PrK 18/57), O40 (PrK 66/57), O49 (PrK 75/57), O23 (PrK 41/57), and O10 (PrK 19/57) were obtained from the Czech National Collection of Type Cultures (Institute of Epidemiology and Microbiology, Prague). Strains S1959 (OXK, O3), R110 and R45 were from the Institute of Microbiology and Immunology University of Lodz, Poland. P. mirabilis strains were cultivated under aerobic conditions in a fermenter (Chemap AG, Switzerland) in nutrient broth (BTL, Poland) under controlled conditions (37 °C, pH 7.4–7.6, pO2 75–85%). The LPS was isolated by the phenol–water procedure and purified by treatment with DNAse and RNAse (Boehringer Mannheim, Germany) as described [13,15,16]. The Proteus LPS of R form was extracted through the phenol–chloroform–petroleum ether (PCP) method [17]. The LPS preparations thus obtained were free of nucleic acid and contained less than 2.5% proteins. 2.3. Verification of ELISA for the determination of antibody levels in human sera The level of anti-LPS antibody detected by enzyme-linked immunosorbent assay (ELISA) depends on well surface covering by LPS from methodological point of view. Degree of well surface covering is associated with the hydrophobic properties of LPS (estimated with laser interferometry method), which determine its solubility, and might be estimated with LAL assay. The comparison of LPS binding intensity (ELISA) and percentage LPS fixed to microplates detected with LAL were presented as coefficient A (arbitrary units) calculated according to formula:
A¼
E L
Table 2 The individual values of C-reactive protein (CRP), RF factor and Waaler–Rose reaction (WR) of RF patients. M—male, F—female. Serum number
Sex
Age
CRP
RF
WR
Serum number
Sex
Age
CRP
RF
WR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
F F F F M F M F F F F F F F F F F F F M F F F F M
37 41 54 27 70 67 64 86 69 56 69 67 25 78 56 84 51 42 82 56 47 68 70 56 81
2 23.6 31.1 4.5 32 40.8 187.7 7.9 35.2 8.3 12.5 25.2 97 6.7 32.6 29.7 16.4 3.6 15.9 75.6 12.5 16.7 8.2 10.6 43.3
56 44 402 43 71 53 42 52 1050 42 41 56 37 36 48 210 55 21 57 1176 40 51 45 49 29
24 – 64 – 32 8 – – – 16 8 – – 64 32 64 64 – – 128 16 32 32 8 16
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
M F M M F M M F F F F F F M F F F F F M M F F M F
45 45 51 74 54 61 61 73 69 44 38 59 74 40 33 36 70 69 81 56 49 69 77 56 63
52.4 3.3 15 43.4 3.2 198 8.7 101 9.7 30.4 5.3 47.6 53.1 90.8 70.7 – 30.7 121 86 45 289 9.8 31.9 98.5 30.8
23 49 77 127 50 39 36 29 33 43 47 309 429 27 455 59 28 87 98 64 114 33 72 28 –
8 8 64 64 8 – – – 32 – – 64 192 16 64 8 16 32 64 24 16 – 64 – 32
where E is the absorbance measured at 490 nm and responds to anti-LPS P. mirabilis antibodies level in RA sera detected by ELISA, L is % of LPS binds to microplates and measured by LAL test. 2.3.1. Limulus amebocyte lysate assay The level of well surface covering (NUNC MaxiSorp microtitre F-bottom plates (black)) by O9, O49, O3, R110 or R45 P. mirabilis endotoxins was determined by the fluorogenic endotoxin detection assay PyroGene rFC purchased from Lonza (Walkersville, USA). The reaction mixtures contained 5 ng LPS per well in 100 μL PBS and left at 4 °C overnight. The plates were washed with phosphate buffered saline (PBS) and all procedures of PyroGene rFC assay were done in accordance with the test instruction. Percent of well surface covering was calculated in comparison to solution of LPS at concentration 5 ng/100 μL (100%). The fluorescence was measured with Microplate Reader TECAN Infinite 200 PRO (Tecan Group Ltd., Switzerland). 2.3.2. Laser interferometry The hydrophobic properties of LPS isolates (Section 2.2) are unfavorably correlated with their solubility and consequently with the degree of well surface covering by tested endotoxins. The hydrophobic properties of LPS were analyzed by laser interferometry method. The measurement set-up for the interferometric investigations was presented previously [18,19]. The interferograms are determined by the refraction coefficient of the solute, which in turn depends on the concentration of the substance. When the solute is uniform, the interference fringes are straight (water compartment) and they bend when a concentration gradient appears (aqueous LPS solution/water interface). The membrane system in this study consists of two glass cuvettes separated by the horizontally located membrane (polymeric nuclear track membrane (nucleopore) with different pore diameters 0.9 μm purchased from Joint Institute for Nuclear Research in Dubna, Russia). In our experiments the upper cuvette held pure water while the lower was filled with aqueous LPS solution. In this system, water migrating through the membrane, diffusing in aqueous LPS solution and lead to the formation of concentration boundary layers (CBLs). The applied computer program to analyse these images allows finding the concentration profiles and the CBL thicknesses (δ). The CBL thickness δ is defined as the distance from the LPS/water interface to the point at which the deviation of the interference fringe from its straight line run is 10% of the fringe thickness (Figs. 1AB). The interferograms were recorded from 2 to 40 min. with a time interval of Δt = 2 min. and the profiles for a given initial LPS concentrations 0.5 mg/mL were reconstructed. Results are presented in CBL thicknesses (δ) in function of time (min). This method permits to compare the hydrophobicity of O3, O49, O9, R110 and R45 LPS isolates. The hydrophobicity of LPS solutions at the same initial concentration is negatively correlated with the δ in function of time. All experiments were performed at a temperature of 37 °C. 2.4. Analysis of anti-LPS P.mirabilis antibodies in RA sera by ELISA The level of anti-LPS P. mirabilis antibodies was determined in healthy donors and RA sera (Table 2) by ELISA. NUNC MaxiSorp microtitre U-bottom plates were coated with 5 ng LPS per well in 100 μL PBS (15 mM Na2HPO4, 150 mM NaCl, pH 7.2) and left at 4 °C overnight. The plates were washed with PBS, blocked with 2% BSA (Sigma, USA) in PBS at 37 °C for 2 h in damp baths and then washed three times with PBS. The plates were incubated with human sera (diluted in PBS 1:1000) for 1 h at 37 °C and washed three times with PBS. Diluted (1:1000) rabbit anti-human IgG conjugated with peroxidase (Sigma, USA) in 1% BSA in PBS was used as the second antibody. Next the plates were washed three times with PBS. A solution of 0.4 mg o-phenylenediamine dihydrochloride (Sigma, USA) in 1 mL of substrate buffer (0.05 M phosphate-citrate buffer pH 5.0) and 40 μL of 0.1% H2O2 (POCH, Poland) was freshly prepared and added. Adding
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0.5 M H2SO4, (after 25 min. of incubation at ambient temperature) stopped the reaction. Then the absorbance was measured at 490 nm with an EL312e Microplate Reader (BIO-TEK Instruments, USA). Each analysis was made in three independent experiments. 2.5. Correlation of complement deposition with O-polysaccharide part of LPS with ELISA We have chosen these two serotypes of LPS (O9 and O49) to correlate the complement deposition with O-polysaccharide part of LPS by ELISA. In this part of study we used three kinds of human sera. The normal human serum (NHS), obtained from healthy donors, was frozen in liquid nitrogen and stored in 0.2 mL aliquots at −70 °C until tested [20]. In some assays, the human sera were chelated with 10 mM ethylene glycol tetraacetic acid (EGTA) containing 10 mM MgCl2 to block activation of the classical complement pathway (bC-NHS). To block the alternative pathway of complement activation, the serum was incubated for 20 min at 50 °C (bA-NHS). Heatinactivated serum (30 min. at 56 °C) was used as a control serum with inactive complement system (T-NHS). We used above two types of sera to detect deposition of C3 or C5 complement molecules on immobilized O9 or O49 LPS. 2.5.1. Detection of deposition C3 complement molecules on immobilized LPS NUNC MaxiSorp microtitre flat-bottom plates were coated with LPS obtained after preparative electrophoresis diluted to 0.5 μg/50 μL in saline phosphate buffer (PBS), pH 7.4 for 18 h at 4 °C. The plates were washed with PBS containing 0.05% Tween 80 (PBS/Tween) (Sigma, USA), blocked with 0.5% gelatin in PBS at 37 °C for 1 h in damp baths and then washed four times with PBS/Tween. Human serum diluted from 100% to 12.5% in PBS was added and incubated with immobilized LPS at 37 °C for 30 min. After washing with PBS/Tween (4×), the deposition of C3 protein was detected with HRP-conjugated rabbit antihuman C3d (DAKO, Denmark), diluted 1/5000 in PBS containing 0.1% gelatin. Human inactivated serum was used as negative control. After washing, the enzymatic activity was estimated by adding 0.1 mL OPD solution (Sigma, USA) and stopping the reaction after 25 min. with 0.5 M H2SO4 (POCH, Poland). The absorbance at 490 nm was measured using a multichannel photometer (BIO-TEK Instruments, USA). Each sample was analyzed in triplicate. The experiment was performed three times, and representative data are presented. In some experiments, before human serum was added, antigens were coated for 45 min. with polyclonal anti-P. mirabilis O9 and O49 rabbit serum (diluted with PBS to give the absorbance A492 = 1.0), respectively and washed 4 times with PBS. Polyclonal anti-P. mirabilis O9 and O49 were obtained by immunization of rabbits with heat-inactivated bacteria (100 °C, 2.5 h). To prepare the immunogen, lyophilized bacteria were suspended in sodium chlorine (0.85% NaCl) at a concentration of 1 mg/mL. Animals were injected intravenously with 50, 100, 100, 200 and 500 μg of immunogen in 500 μL of saline solution on days 1, 4, 7, 11 and 16, respectively. Seven days after the last immunization, the blood was sampled by the cardiac puncture, and then serum was separated and stored in 0.5 mL aliquots at − 20 °C. Experiments were carried out in accordance with the Animal Ethical Committees guidance. 2.5.2. Detection of deposition C5b complement molecules on immobilized LPS The beginning steps were performed as described above. After incubation in human serum and washing with PBS/Tween, the deposition of C5b was detected with mouse monoclonal antibody (clone aE11) (DAKO, Denmark), diluted 1:100 in PBS containing 0.1% gelatin. After incubation (60 min. 37 °C) and washing with PBS/Tween 80 buffer (4×) secondary antibodies anti-mouse goat IgG with HRP-conjugated (DAKO, Denmark), diluted 1:2000 in PBS containing 0.1% gelatin
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was used. As negative control, human inactivated serum was used. The enzymatic activity was estimated using the above methods. In some experiments, before human serum was added, antigens were coated for 45 min with polyclonal anti-P. mirabilis O9 and O49 rabbit serum (diluted with PBS to give the absorbance A492 =1.0), respectively and washed 4 times with PBS. Polyclonal anti- P. mirabilis O9 and O49 rabbit sera were obtained according to description in Section 2.5.1. 2.6. Correlation of anti-LPS antibodies levels with tlr4 gene polymorphism with RFLP-PCR Genomic DNA was prepared by Miniprep/Qiagen kit (QIAGEN, Germany). The tlr4 (Thr399Ile) gene polymorphism was determined by the Restriction Fragment Length Polymorphism-Polymerase Chain Reaction (RFLP-PCR) with the following primers: sense 5′-GCT GTT TTC AAA GTG ATT TTG GGA GAA-3′, antisense 5′‐CAC TCA TTT GTT TCA AAT TGG AAT G-3′. This single nucleotide polymorphism (SNP) is a C/T transition causing a threonine/isoleucine switch at amino acid in 1196 position of tlr4 gene. The 146-bp PCR product was digested 18 h with 2 U of the restriction enzyme HinfI (Promega, Madison, Wisconsin, USA). The Ile allele was digested into 127-bp fragments, whereas the Thr variant remained intact. The PCR products were run on a 12% of polyacrylamide gel [21]. 2.7. Data analysis The data were analyzed using the Statistica (StatSoft, Tulsa, OK, USA) software package. Values of anti-LPS P. mirabilis antibodies in control and RA sera are expressed as mean± standard device. The individual level of anti-LPS P. mirabilis antibodies of control donors and RA patients detected by ELISA was performed in triplicate. The summary of the results of anti-LPS antibodies in control and RA sera is expressed as a mean of fifty or three independent experiments, respectively. The evaluation of the amount of LPS bind to microtiter plates with LAL was performed in triplicate. The differences between anti-LPS level in control and RA groups were compared with One-way ANOVA test. One-way ANOVA is a useful test in comparing two or more means because we can omit the type I error associated with false selection of test, like t-test. We used this analysis previously and might compare this study with our earlier conclusions [14]. Genotype frequencies were tested for Hardy–Weinberg equilibrium. Distribution of genotypes was analyzed using the χ2-test. The odds ratio (OR) and 95% confidence interval (CI) were estimated by logistic regression analysis. To evaluate the goodness of fit of the logistic regression model, Nagelkerke's R-square was calculated. P values less than 0.05 were considered statistically significant. For the statistical analysis, differences between controls and RA patients were calculated with the two-tailed Fisher exact probability test. 3. Results 3.1. Serological reaction of human sera with LPS and genotype analysis In order to investigate serological reaction of human sera with LPS, levels of antibodies in 50 sera samples from patients with RA and 50 healthy blood donors were measured by ELISA, as shown in Fig. 2 and Fig. 3. We observed that levels of anti-O9, O40, O49, O23 and O3 LPS P. mirabilis antibodies are statistically, significantly higher in RA patients than in control, which is in contrast to anti-O10 and R forms (Fig. 3). Analysis of individual levels of anti-LPS P. mirabilis antibodies of S forms shows that the levels of anti-O9 antibodies in sera are higher than in control for 28% of RA patients, 78% for anti-O40, 74% for anti-O49, 54% for anti-O23, and 68% for anti-O3 (Fig. 2). Tables 3 and 4 show genotype analysis of the tlr4 gene polymorphism (Thr399Ile) in 50 RA patients versus 50 controls and selected 34 candidates from 50 RA patients versus 50 controls, respectively.
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Fig. 1. Laser interferometry analysis of CBL thicknesses (δ) for S and R forms of aqueous LPS solutions (C). A—scheme of membrane system, denotes δ, B—examples of interferograms for O3 and R45 LPS.
We have selected 34 candidates from 50 RA patients on the basic of two criteria. Firstly, we have chosen only these RA patients for whom the level of anti-LPS antibodies in sera was statistically higher than in control healthy donors (Fig. 2A). Secondly, we have chosen only these RA patients in whose sera the individual levels of anti-LPS P. mirabilis antibodies was statistically higher than in control (at least three of them) (Fig. 2B denoted by *). The logistic regression model including all tested anti-LPS antibodies level in control and RA sera as well as the tlr4 gene variants had a Nagelkerke's R-square of 0.903 (indicating that it explained approximately 90.3% of the variation in the data). We did not find any correlation between anti-LPS P. mirabilis antibodies in RA patient's blood and TLR4 gene polymorphism. Taking into consideration odds ratio (OR) and probability P (calculated by two-tailed Fisher test) values, we did not find any correlation between anti-LPS P. mirabilis antibodies in RA sera (Table 3) or selected RA patient's blood and tlr gene polymorphism Thr399Ile (Table 4). The distribution of genotypes in control and RA groups was consistent with the Hardy–Weinberg equilibrium in both studies (Tables 3 and 4). 3.2. Verification of ELISA with LAL test and laser interferometry method In order to confirm that the level of anti-LPS antibodies in RA patients sera does not depend on the amount of antigen bound to microplate, the LAL test was done. Assessed by the LAL test, the amount of LPS fixed were 1.27% for R110, 4.26% for R45, 18.39% for O9, 15.65% for O49 and 17.11% for O3 (Table 5). The differences in binding strengths could be due to different solubility properties of smooth
(O9, O49, and O3) and rough (R45 and R110) LPS (Fig. 1C). Laser interferometry analysis of CBL thicknesses (δ) for S and R forms of aqueous LPS solutions shows that δ= 1.653 mm for O9 after 40 min., 0.975 for O49, 0.880 for O3, 0.133 for R110 and 0.399 for O45, respectively (Table 5). The higher value of δ is positively correlated with better solubility of LPS aggregates, its binding to microtitre plates, and finally the level of anti-LPS P. mirabilis antibodies in sera measured with ELISA. We have observed that the summary reactions of sera from RA patients with R forms of P. mirabilis (R45 and R110) LPS are higher than with S forms (O3 and O9, in contrast to O49) (Table 5). 3.3. Deposition of C3d and C5b-9 on native P. mirabilis O9 or O49 LPS The pre-incubation of LPS with rabbit antisera caused the increase in C3d deposition onto both LPS (Fig. 4). LPS pre-incubated with homologous rabbit antisera bound much more amounts of C3d component of human complement in comparison to not pre-incubated LPS (data not shown). It is probably caused by the reaction of the C1q component with a heavy chain of specific anti-O-chain rabbit antibodies binding to LPS during pre-incubation. The deposition of C3d depends on the concentration of serum used as a source of the complement. The beginning of the reaction above control (T-NHS) was visible when the concentration of serum reached 12.5%. Native P. mirabilis O9 LPS strongly activated complement, which was estimated with C3d complement component deposition assay. The structural investigation showed that the difference in the O-antigen structure of both LPS. LPS from P. mirabilis O49 is branched in contrast to LPS from P. mirabilis O9
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Fig. 2. The individual level of anti-LPS P. mirabilis antibodies of RA patients compared to 50 healthy donors measured by ELISA (panel A). Error bars denote standard device. Panel B presents only these RA patients (denote by grey frame) in whose sera the individual levels of anti-LPS P. mirabilis antibodies was statistically higher than in control (at least three of them; denote by *).
(Table 1). The inhibition of the alternative pathway of the complement activation via inactivation of factor B slightly influenced the amount of C3d component bounded with P. mirabilis O9 LPS when compared to
native complement activation results. However, inhibition of alternative pathway in the case of P. mirabilis O49 LPS resulted in a significant decrease of C3d binding.
Fig. 3. The summary level of anti-LPS P. mirabilis antibodies of RA patients compared to 50 healthy donors measured by ELISA. Error bars denote standard device.
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Table 3 The allele and genotype frequencies and odds ratio (OR) of the Thr399Ile polymorphism of the TLR4 gene in RA patients in comparison to control healthy donors. Genotype or allele
RA patients (n = 50)
C/C C/G G/G C G a
Controls (n = 50)
OR (95% CI)
P valuea
Number
Frequency
Number
Frequency
42 8 0 χ2 = 0.378; p = 0.539 92 8
0.840 0.160 –
44 6 0 χ2 = 0.204; p = 0.651 94 6
0.880 0.120 –
0.716 (0.229–2.238) 1.397 (0.447–4.367) –
0.774 0.774 –
0.940 0.060
0.734 (0.245–2.198) 1.362 (0.455–4.080)
0.782 0.783
0.920 0.080
Two-tailed Fisher exact probability test.
Table 4 The allele and genotype frequencies and odds ratio (OR) of the Thr399Ile polymorphism of the TLR4 gene in RA patients with highest individual level of anti-LPSs P. mirabilis antibodies (at least three of them) in comparison to control healthy donors. Genotype or allele
RA patients (n = 34)
C/C C/G G/G C G a
Controls (n = 50)
OR (95% CI)
P valuea
Number
Frequency
Number
Frequency
30 4 0 χ2 = 0.133; p = 0.716 64 4
0.882 0.118 –
44 6 0 χ2 = 0.204; p = 0.651 94 6
0.880 0.120 –
1.022 (0.266–3.936) 0.978 (0.254–3.762) –
1.000 1.000
0.940 0.060
1.021 (0.277–3.764) 0.979 (0.266–3.609)
1.000 1.000
0.941 0.059
Two-tailed Fisher exact probability test.
The bactericidal activity of serum is related to the complement cascade activation, leading to the lytic complex C5b-9 creation. In order to confirm that the native LPS P. mirabilis O9, O49 indeed activated human complement, the depositions of C5b with monoclonal antibodies (clone aE11) were done. As a result of complement activation with P. mirabilis O9 LPS, higher deposition of C5b was observed. However deposition of C5b on P. mirabilis O49 LPS was significantly lower (Fig. 5). As in the case of C3d deposition, the amounts of binding C5b by native LPS were strongly enhanced in the presence of anti-O9 and O49 antibodies (data not shown). The complement resistance of P. mirabilis O9 and O49 rods was tested in 50% PBSdiluted normal human serum. After 3 h incubation at 37 °C only 45% bacterial cell of P. mirabilis O9 survived. In contrary, in the same conditions intensive growth of P. mirabilis O49 (above 100% used, 10 7 CFU/mL) was observed. Similarly, in NHS with inhibited alternative complement activation pathway, P. mirabilis O9 were sensitive to bactericidal effect of serum in contrast to O49 strain. 4. Discussion 4.1. Correlation of anti-LPS antibodies with TLR4 (Thr399Ile) gene polymorphism The TLR4 receptor is specific for lipopolysaccharide and is important for pathogen-recognition mechanisms. Host–pathogen interactions are involved in RA pathophysiology and TLR genes influence both
proinflammatory cytokine production and autoimmune responses. TLRs are expressed by B lymphocytes and T lymphocytes, antigenpresenting cells, regulatory T cells and are found in the rheumatoid synovium. It is suggested that innate immunity may be involved in initiating the inflammatory process or in inhibiting regulation mechanisms that normally prevent chronic inflammation [22]. We did not observe a correlation between the levels of all tested anti-LPS with different structures of O-polysaccharide region P. mirabilis antibodies and TLR4 (Thr399Ile) gene polymorphism. This result is in accordance with some studies, in which more specific markers for RA diagnosis were used as anti-cyclic citrullinated peptide and erosions autoantibodies [23,24]. 4.2. The level of anti-LPS P. mirabilis antibodies in RA sera The individual and summary level of anti-LPS P. mirabilis S forms antibodies is higher in sera of RA patients than healthy blood donors. This result is in accordance with some studies [4] and indicates that P. mirabilis infection might be correlated with human susceptibility to RA. Additionally, LPS from Proteus strains have uncommon constituents: 4-amino-4-deoxy-L-arabinosyl residues (AraN) in the 3-deoxyoctulosonic acid (KDO) and lipid A regions, D-galacturonic acid in the core region, and amino and uronic acids and unusual sugars in the O-polysaccharide domain. These constituents are not present in other members of the Enterobacteriaceae [25]. In this study, the serological reaction of human sera with LPS was measured with ELISA and we
Table 5 Correlation of summary anti-LPSs P. mirabilis antibodies level measured by ELISA with amount of LPSs bind to micotitre plates (%) evaluated by LAL test and solubility of LPSs isolates by laser interferometry (δ in mm). δ is defined as the distance from the LPS/water interface to the point at which the deviation of the interference fringe from its straight line measured by laser interferometry; δ value is positvely correlated with hydrophobic properties of LPS isolates Coefficient A is the relation of absorbance (A490 nm) and proper % of LPS binds to micotitre plates. LPS
O9 O49 O3 R110 R45
ELISA (A490 nm) [mean ± standard device] control
RA
0.195 ± 0.115 0.237 ± 0.059 0.129 ± 0.075 0.132 ± 0.083 0.156 ± 0.091
0.301 ± 0.289 0.990 ± 0.635 0.415 ± 0.293 0.161 ± 0.098 0.155 ± 0.082
LAL (%) [mean ± standard device]
Laser interferometry (δ)
Coefficient A (arbitrary units)
18.39 ± 4.29 15.65 ± 5.43 17.11 ± 4.65 1.27 ± 0.97 4.26 ± 2.84
1.653 0.975 0.880 0.133 0.399
0.016 0.063 0.024 0.127 0.036
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4.4. Correlation between complement deposition and O-polysaccharide structure of LPS
Fig. 4. Deposition of C3d on native P. mirabilis O9 (A) or P. mirabilis O49 (B) LPS pre‐ incubated with rabbit antisera and after incubation with: normal human serum NHS (●), human serum with blocked alternative complement pathway bA-NHS (▼), human serum with blocked classic complement pathway bC-NHS (■) or inactivated human serum without complement activity T-NHS (♦, control).
observed the level of antibodies against antigen O, core, and lipid A regions of LPS. These regions contain unique components for Proteus and serological reactions confirm the role of Proteus infection in rheumatoid arthritis. The level of anti-LPS O10 P. mirabilis antibodies stayed at the same level in RA and control sera. It might be associated with peculiar (L-alturonic acid; L-Alt) structure in antigen O. The immune system might be highly activated by the O-polysaccharide domain with very unique L-alturonic acid in both investigated groups.
4.3. Verification of ELISA with LAL assay and laser interferometry method It is important that the ELISA method used in this study was optimized with LAL test and laser interferometry. The CBL thicknesses (δ) measured by interferometry method was positively correlated with better solubility of LPS aggregates, its binding to microtitre plates evaluated with LAL test and finally level of anti-LPS P. mirabilis antibodies in sera measured by ELISA (coefficient A). This complex correlation confirmed the role of anti-LPS P. mirabilis antibody in RA. The high value of coefficient A for R forms of P. mirabilis LPS indicated that we had detected antibodies against the most conservative structure of LPS–core-lipid A parts of LPS. The detection of anti-LPS R forms antibodies in RA sera suggests that the LPS of Enterobacteriaceae may play a significant role in RA. Unstable immune system of RA patients could unexpectedly produce antibodies against weak immunogenic antigens, like lipid A in RA patients in contrast to healthy blood donors.
Fig. 5. Deposition of C5b-9 on native P. mirabilis O9 (▼) or P. mirabilis O49 (●) LPS after incubation with normal human serum NHS. ■—inactivated human serum without complement activity T-NHS (control).
The level of anti-LPS O9 P. mirabilis antibodies was on the same level in RA and control sera. It might be associated with the linear structure in antigen O9. We have chosen these two serotypes of LPS (O9 and O49) to the next step of broad investigations on the deposition of C3d and C5b complement components on P. mirabilis LPS. Immunochemical analysis of the outer membrane of the different Gram-negative bacteria showed that the differences in the structure of LPS can play an important role in determining the susceptibility of Gram-negative strains to the action of serum [12,26]. Our previous observations that P. mirabilis Ra mutant (R110) and P. mirabilis Re mutant (R45) demonstrated sensitivity to the bactericidal activity of serum than the P. mirabilis S form (S1959), resistant to NHS [27]. The LPS O9 deposited C3d and C5b molecules and made the P. mirabilis O9 rods sensitive to bactericidal effect of complement, in contrast to P. mirabilis O49. The LPS O49 deposited much less C3d and C5b complement components. Both investigated strains differs on their O-antigen structures and our result indicated that both: the length as well as O-specific chain of LPS structures may play the crucial role in the complement binding and bactericidal affect of human sera [28]. In conclusion, we suggest that the chemical structure, length of the O49-polysaccharide chains of LPS protects P. mirabilis O49 strain against the bactericidal effect of human serum. It is contrary to P. mirabilis O9 cells lysed through by deposited MAC complexes. The prolonged presence of P. mirabilis O49 cells in human intestine or in the lower part of the renal tract resulted in the high level of anti-O49 antibodies presence. It was observed that only 28% of RA patients sera reacted with O9 LPS, whereas 74% with O49 LPS. One may postulate that prolonged complement activation by complement-resistant enterobacterial cells i.e. P. mirabilis O49 resulted in increased C3ca and other complement mediators released. The constant proinflammatory activities of LPS and its part may cause native immune system to be overactivated with the final outcome of non-specific autoantibodies generation in RA diseases. The verification of methods sensitive and valuable in detection of antibodies needs to be stressed. In the presented results, the binding to microtitre plates abilities of LPS varied significantly (tested by LAL method). Also hydrophobicity of Proteus LPS varied (determined by laser interferometry). This may indicate that human cells stimulation via TLR 4 receptors depends on unique structures of the polysaccharide part of the LPS, that modulates lipid A part binding to TLR 4, MD2 receptor complexes drafts. Abbreviations AraN 4-amino-4-deoxy-L-arabinosyl residue bA-NHS serum with blocked activation of the alternative complement pathway bC-NHS serum with blocked activation of the classical complement pathway BSA bovine serum albumin C1q subcomponent of the C1 complex of the classical pathway of complement activation C3d complement component 3d C5b complement component 5b CBL concentration boundary layer CPS polysaccharide capsules CTLA-4 cytotoxic T-lymphocyte antigen 4 EGTA ethylene glycol tetraacetic acid ELISA enzyme-linked immunosorbent assay HLA-DR4 human leukocyte antigen DR4 HRP horseradish peroxidase IL interleukin KDO 3-deoxyoctulosonic acid LAL limulus amebocyte lysate assay LPS lipopolysaccharide
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MAC MD2
membrane attack complex glycoprotein associating with the extracellular domain of TLR4 NHS normal human serum O-PS O specific polysaccharide PAF platelet activating factor PBS phosphate buffered saline PTPN22 protein tyrosine phosphatase 22 R rough strains without antigen O in LPS structure RA rheumatoid arthritis RF rhematoid factor RFLP-PCR restriction fragment length polymorphism-polymerase chain reaction S smooth strains with antigen O in LPS structure SNP single nucleotide polymorphism TLR4 toll-like receptor 4 TNF tumor necrosis factor TNFAIP3 tumor necrosis factor; alpha-induced protein 3 STAT4 signal transduction and activation of transcription 4 protein T-NHS serum with inactive complement system TRAF1-C5tumor necrosis factor-receptor associated factor 1 and complement component 5
Acknowledgments The work was supported by grant BS/2012 from the Jan Kochanowski University, Poland. We would like to thank Krystyna Surma, Iwona Koprucha, Halina Bakalarska, Wojciech Garbacz, Łukasz Madej, Ewelina Nowak and Agnieszka Matusiak for sample collection and technical support. References [1] Tobón GJ, Youinou P, Saraux A. The environment, geo-epidemiology, and autoimmune disease: rheumatoid arthritis. J Autoimmun 2010;35:10–4. [2] Newkirk MM. Rheumatoid factors: host resistance or autoimmunity? Clin Immunol 2002;104:1–13. [3] Rashid T, Ebringer A. Rheumatoid arthritis is linked to Proteus—the evidence. Clin Rheumatol 2007;26:1036–43. [4] Senior EBW, Anderson GA, Morley KD, Kerr MA. Evidence that patients with rheumatoid arthritis have asymptomatic 'non-significant' Proteus mirabilis bacteriuria more frequently than healthy controls. J Infect 1999;38:99–106. [5] Tiwana H, Wilson C, Pirt J, Cartmell W, Ebringer A. Autoantibodies to brain components and antibodies to Acinetobacter calcoaceticus are present in bovine spongiform encephalopathy. Infect Immun 1999;67:6591–5. [6] Rozalski A, Sidorczyk Z, Kotelko K. Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 1997;61:65–89. [7] Mishra M, Thakar YS, Pathak AA. Haemagglutination, haemolysin production and serum resistance of Proteus and related species isolated from clinical sources. Indian J Med Microbiol 2001;19:5–11. [8] Rocha SP, Elias WP, Cianciarullo AM, Menezes MA, Nara JM, Piazza RM, et al. Aggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cells. FEMS Immunol Med Microbiol 2007;51:319–26.
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