The Role And Mechanism Of Bacterial Protein FimH In ANCA-associated Vasculitis

BackgroudAnti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) is a localmanifestation of systemic vessel vasculitis (SVV), with the presence of myeloperoxidaseANCA antibodies (MPO) and proteinase3antibodies (PR3). The renal involvement iscommonly characterized by focal necrotizing glomerulonephritis (FNGN) with crescent. Itoften leads to rapid progression of renal failure and poor prognosis[1]. As a new subtype ofANCA, the lysosomal membrane protein-2(LAMP-2), highly correlated with kidneydamage[2-3]. It provided new field to explore pathogenesis of AAV. Kain reported that theanti-LAMP-2antibody can induce FNGN in rats through molecular mimicry. The LAMP-2epitope (P41-49) has100%homology with amino acids72–80of mature FimH, an adhesinfrom Gram-negative bacteria. Rats developed cross-reactive antibodies to LAMP-2whenimmunized with FimH[2].Th17cells are novel CD4+helper T cells (T helper, Th) subpopulations, which differsfrom Th1cells and Th2cells. They are widely involved in physiological and pathologicalprocesses through the secretion of interleukin-17(IL-17), tumor necrosis factor-α (TNF-α)and some other inflammatory cytokines. IL-17plays an important role in the AAV.Nogueira[4]found that the IL-17A were significantly elevated in serum and overexpressedin renal tissue of patients with acute phase AAV, what’s more, the serum IL-23, MPO andPR3-specific Th17cells were also increased visiblely. There was specific MPO-ANCA orPR3-ANCA cell clones responses to Th17in ANCA-positive vasculitis patients or mousemodel. As a new subtype of ANCA, however, whether LAMP-2antibodies can be as aserological markers of clinical diagnosis of Chinese AAV is remain elusive. There is noreport that whether IL-17is elevated or associated with kidney damage after FimH proteininfection.Objective1. The LAMP-2antibodies, as an new subtype of ANCA, are presented inANCA-associated vasculitis（AAV）, but the prevalence has considerable controversy.This study aims to determine the diagnostic value of anti-LAMP-2antibodies in apopulation of Chinese patients with AAV.2. To construct and express a prokaryotic expression vector carrying the gene of FimH1-156that comprises human lysosome membrane protein2P41-49gene, and to express andpurify the fusion protein. And to investigate the mechanism of FNGN induced by FimHfusion protein.Method1. ANCA-associated vasculitis (AAV) patients, diagnosed according to inclusion criteria,involved in this study during September2010to June2013. ANCA negative subjectswith various chronic glomerulonephritis and renal diseases served as disease controls.Sera from healthy individuals were used as healthy controls. Recombinant humanLAMP-2/CD107b（R&D Systems） as coating antigen, anti-hLAMP-2antibodies weredetected by indirect enzyme-linked immune sorbent assay (ELISA) for all serumsamples. Optical density （OD）values more than mean+2SD （cut off value）greaterthan the healthy controls were regarded as positive. Referring to the kit instructions(Aumont Medical Diagnosis,Germany), the antibodies to MPO, PR3and GBM weredetected by EUROLine and indirect immunofluorescence method. Finally, we list thefourfold table, and calculate diagnostic evaluation, including sensitivity (Sen),specificity (Spe), positive/negative predictive value (PPV/NPV), positive/negativelikelihood ratio (PLR/NLR), and Youden index (YI), etc. 2. FimH1-156gene was cloned from plasmid pPKL241by PCR, and inserted into vectorpET-28a (+) to obtain prokaryotic expression plasmid pET-28a-FimH. Aftertransforming Escherichia coli BL21(DE3) with pET-28a-FimH, fusion proteinFimH1-156was expressed under IPTG (1mmol/L) induction. After cracking bacterium byultrasound,the target fusion protein was purified with Ni-NTA, and its antigenicitywas detected through SDS-PAGE and Western blotting.3. Wistar-Kyoto (WKY) rats immunized with purified FimH fusion protein （150μg）emulsified in Titermax Gold（150μl）. Controls received PBS（150μl） in Titermaxalone. Glomerular injuries were assessed by24-hour urinary protein, urea nitrogen(BUN), serum creatinine (Scr), serum uric acid (UA) and histomorphology. The levelsof interleukin-17a (IL-17A) were detected by ELISA assay. Additionally, we alsoimmunized WKY rats with complete Freund’s adjuvant combined LAMP-2peptide P41-49as control.Results1. According to inclusion criteria, thirty-two ANCA-associated vasculitis (AAV) patients,forty-two ANCA negative subjects with various chronic glomerulonephritis, and fiftyhealthy individuals involved in this study. The anti-hLAMP-2antibodies were detectedin30out of32(93.8%) patients with AAV and7out of42(16.67%) in disease controlsgroup. The anti-hLAMP-2antibodies had sensitivity, specificity, PPV, NPV, PLR, NLRand YI for a diagnosis of AARC of93.8%,83.3%,81.1%,94.6%,5.625,0.075and0.771, respectively.2. We successfully constructed expression vector pET28a-FimH1-156, and inducedthe highest expression of the target protein when IPTG concentration1mmol/L after4hours. The expressed recombinant protein was purified，it wasexpressed as Inclusion-Body，and the expressed protein was identified to be His-probeby Western-blotting．3. The levels of24-hour urinary protein began to rise at3rdday after immunization with FimH fusion protein, and significantly higher than control group on7th,35thand50thday (P<0.05). The serum levels of BUN、Scr、UA in model rats were increasedsignificantly at50thday (P<0.05) compared to control group. WKY rats immunizedwith FimH fusion protein showed segmental necrosis of glomerular capillaries, alveolarwall thickening, and significant inflammatory cells infiltration at35thday, andglomerular crescent formation after50days. The serum levels of IL-17A wereincreased significantly compared to control group on35thand50thday (P <0.05). TheIL-17A levels were positively correlated with24-hour urinary protein in model group(R=0.5566, P=0.0210). In addition, we used complete Freund’s adjuvantcombined LAMP-2peptide P41-49immunized WKY rats. Similar results, thelevels of24-hour urinary protein at13thday and IL-17A were alsosignificantly higher than controls (p<0.05), but there is no significant crescentformation in rat models.Conclusion1. We suggest that anti-LAMP-2antibodies can be detected in most Chinese patientswith AAV, even in ANCA-negative FNGN, however, large-scale studies are needed forfurther research.2We cloned FimH1-156fusion protein expressed genes successfully, constructed toprokaryotic expression vector, and won the inclusion body purification of FimH1-156fusionprotein.3. Bacteria FimH protein can induce glomerular focal necrotic lesion and lung injuryin WKY rats, and IL-17A may involve in the damage process.