Fecal IgA coating bacteria are increased in antiphospholipid syndrome.

Abstract

Background: The antiphospholipid syndrome (APS) is an autoimmune thrombophilic condition that is manifested by vascular thrombosis and pregnancy complications . It is marked by the presence in blood of antibodies that recognize phospholipid-binding proteins. While the nature of the autoantibodies and their significance has been better understood through decades of research, the disease trigger and pathogenesis still remain elusive. The autoimmune diseases share epidemiological, genetical, immunological features and recent evidence suggests that several inflammatory disorders may be caused by specific bacterial species, so called ‘pathobiont’ found in most healthy hosts. The latest studies showed that the patients with inflammatory bowel disease (IBD) had an increased percentage of immunoglobulin A(IgA)-coated fecal bacteria in active disease, compared with healthy controls and intestinal bacteria of high coating with IgA conferred dramatic susceptibility to colitis in germ free mice. We hypothesized that members of gut microbiota could represent a chronic trigger in APS patients and that they exhibit heightened adaptive immune responses to the microbiota. The objective of this study was to analyze levels of IgA-coated fecal bacteria and testify the hypothesis in APS patients. Method: Altogether 32 subjects were included in this study: 15 patients with APS (M:F=5:10, mean age 48.8 years, range 27 - 70 years), 5 patients with non-autoimmune thrombotic states (M:F=1:4, mean age 45.4 years, range 28 - 68 years), and 12 healthy normal donors (M:F=4:8, mean age 37 years, range 23-60 years) (total of 17 non-APS controls). Stool was collected at each of three monthly visits at the maximum. Fresh fecal samples were collected at home, stored at freezer pack in an insulated foam shipper, mailed and stored at -80°C for further analysis. Approximately 100mg of frozen human fecal material were placed in 2mL microtube (Sarstedt, Numbrecht, Germany) containing ceramic beads (Big D Lysing Matrix, MP Biomedicals, Santa Ana, CA, USA) and incubated in 1 mL phosphate buffered saline (PBS) per 100mg fecal material on ice for 30 minutes. Fecal bacteria in the supernatants were removed (100uL/sample), washed and then stained with 100 uL staining buffer containing PE-conjugated Anti-Human IgA (1:10; Miltenyl Biotec clone IS11-8E10, San Diego, CA, USA) for 30 min on ice. Samples were then washed two times with 1 mL staining buffer before flow cytometric analysis (FACSAria, BD Biosciences, San Jose, CA, USA). Unstained sample was used as a negative control and IgA-positive fraction among total bacteria was measured as %IgA. Result: A total of seventy samples were measured with 38 for patients with APS and 32 for non-APS control subjects. One control subject showed zero percent in all three fecal samples at different time points, which was excluded from the analysis. The analysis showed a median percentage of 16.1% (range, 0.2-57.1%) for APS and 10.4% (range, 2.2-34.9%) for control. The average percentage was 19.0 ± 2.4% (mean ± standard deviation) for APS and 11.3 ± 1.3% (mean ± standard deviation) for control. The Mann-Whitney U analysis showed significant difference between APS and control groups (p=0.02). Conclusion: According to our knowledge, this is the first study of fecal IgA coating in antiphospholipid syndrome. Our study showed that the mean percentage and range of IgA coat in controls was 11.3% and 2.2-34.9%, which are similar to those reported by Waiij et al (13.4% and 4-30%). The mean difference between APS patients and controls was about 8% which is significant but lower than that between IBD patients and controls reported by Waaij et al (29%). Based on previous findings from IBD research, the higher percentage of fecal IgA in APS patients than controls supports that there may be a breakdown of mucosal tolerance to the commensal gut flora. The exact background behind the lower mean difference of fecal IgA coating percentage in this APS study than in IBD study remained a question for future reasearch, it may be supposedly from different pathophysiological mechanisms between the two disease entities. The anti-β2-glycoprotein I-mediated thrombogenesis is main effector mechanism in APS in contrast to that TNF-mediated procolitogenecity is main effector mechanism in IBD.