Role of PR3-homologous bacterial proteases in the formation of C-ANCA as a diagnostic marker for granulomatosis with polyangiitis

Abstract

Objectives Molecular mimicry between microbial antigens and autoantigens has a role in many autoimmune diseases. Granulomatosis with polyangiitis (GPA) is a complex autoimmune syndrome that affects small- and medium-sized blood vessels in many tissues such as those in the upper respiratory tract, lung, and kidney. Anti-neutrophil cytoplasmic autoantibody (ANCA) antigens, especially those directed against proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA), are the hallmark of the GPA and are involved in the pathogenesis of vasculitis. In this study, the potential role of bacterial proteases in the production of PR3-ANCA was investigated. Following a homology search of a bacterial protein database, two bacterial proteases homologous to PR3 were selected: the S1A family peptidase of Myxococcus xanthus (Mx) and the trypsin-like serine protease of Saccharomonospora viridis (Sv), which had 34%/44% and 30%/45% identities/similarities, respectively, with human PR3 and 33%/41% and 32%/43% identities/similarities, respectively, with mouse PR3. To elucidate the pathogenesis of GPA, two questions were addressed. First, although ANCA-mediated pathogenesis of vasculitis is well established, how is ANCA produced? Second, what is the pathogenesis of granuloma formation?

Materials and Methods For this study, recombinant types of human PR3 (rhPR3), mouse pr3 (rmPR3), Sv protease (rSvPr), and Mx protease (rMxPr) were expressed in Escherichia coli and purified. NZBWF1 and C57BL/6 mice were intranasally mono-infected with either Sv or Mx bacteria or co-infected together with Staphylococcus aureus (Sa). In addition, mice were subcutaneously immunized with either rSvPr or Sv bacteria. In other experiments, mice were challenged once with Sa (nasal), Sv (nasal), or LPS (intraperitoneal) two weeks after immunization with rSvPr. Levels of IgG antibodies to purified human PR3 (phPR3), rhPR3, rmPR3, rMxPr, and rSvPr in sera were measured by using enzyme-linked immunosorbent assay (ELISA). For histological examination, sections of paraffin-embedded kidney and lung tissues were stained by using H&E or Verhoeff-Van Gieson (VVG) stains. Levels of antibodies to pHPR3, rHPR3, rmPR3, rMxPr, and rSvPr in GPA patient sera were measured by ELISA.

Results Although all infected mice produced antibodies to bacterial lysates, only a small percentage of mice developed antibodies to bacterial proteases. Autoantibodies to rmPR3 were detected in 22.7% of NZBWF1 and 9% of C57BL/6 mice infected with Mx and in 33.3% of NZBWF1 and 13% of C57BL/6 mice infected with Sv. Differences in the percentage of autoantibody production associated with mouse strain or bacteria were not statistically significant. Mice infected with bacteria exhibited perivascular inflammation in the lung, but no granulomas or vasculitis were observed, even in mice that developed a high level of anti-mPR3 autoantibodies. Co-infection of Sa with Mx or Sv did not result in a significant change in the percentage of autoantibody production. Therefore, co-infection with Sa did not enhance the production of ANCA in mice. Interestingly, one C57BL6 mouse co-infected with Sv and Sa developed a large necrotic granulomatous lung lesion. In contrast to the results of bacterial infection, immunization with rSvPr resulted in the production of autoantibodies to rmPR3 in 75% of NZBWF1 mice and 100% of C57BL/6 mice. Mice immunized with rSvPr developed multiple inflammatory infiltrating sites in lung parenchyma and exhibited perivascular inflammation and vasculitis. The mice challenged once with Sa, Sv, or LPS following immunization with rSvPr developed vasculitis and small granulomatous lesions in lung parenchyma. Interestingly, antibodies to rSvPr were detected in 23% of PR3-ANCA GPA patients, but none were observed in control subjects. However, the percentage (63%) of control subjects with antibodies to Sv lysates in sera was higher than the percentage (38%) in PR3-ANCA GPA patients.

Conclusion In mice, infection with bacteria containing PR3-homologous proteases or immunization with bacterial PR3 homologous proteases can induce PR3-ANCA production via molecular mimicry. Although elevated ANCA production did not induce granuloma development, ANCA alone could produce inflammation in the lung, which presented as inflammatory cell infiltration in the perivascular areas and vasculitis involving small- to medium-sized blood vessels.