Experimental Antiglomerular Basement Membrane GN Induced by a Peptide from Actinomyces

Abstract

Background: Antiglomerular basement membrane (anti-GBM) disease is associated with HLA-DRB1*1501 (the major predisposing genetic factor in the disease), with α3_127-148 as a nephritogenic T and B cell epitope. Although the cause of disease remains unclear, the association of infections with anti-GBM disease has been long suspected.

Methods: To investigate whether microbes might activate autoreactive T and B lymphocytes via molecular mimicry in anti-GBM disease, we used bioinformatic tools, including BLAST, SYFPEITHI, and ABCpred, for peptide searching and epitope prediction. We used sera from patients with anti-GBM disease to assess peptides recognized by antibodies, and immunized WKY rats and a humanized mouse model (HLA-DR15 transgenic mice) with each of the peptide candidates to assess pathogenicity.

Results: On the basis of the critical motif, the bioinformatic approach identified 36 microbial peptides that mimic human α3_127-148. Circulating antibodies in sera from patients with anti-GBM recognized nine of them. One peptide, B7, derived from Actinomyces species, induced proteinuria, linear IgG deposition on the GBM, and crescent formation when injected into WKY rats. The antibodies to B7 also targeted human and rat α3_127-148. B7 induced T cell activation from human α3_127-148-immunized rats. T cell responses to B7 were detected in rats immunized by Actinomyces lysate proteins or recombinant proteins. We confirmed B7's pathogenicity in HLA-DR15 transgenic mice that developed kidney injury similar to that observed in α3_135-145-immunized mice.

Conclusions: Sera from patients with anti-GBM disease recognized microbial peptides identified through a bioinformatic approach, and a peptide from Actinomyces induced experimental anti-GBM GN by T and B cell crossreactivity. These studies demonstrate that anti-GBM disease may be initiated by immunization with a microbial peptide.

Keywords: glomerular disease; pathology; renal injury.

Graphical abstract

Figure 1.

Schematic diagram for the screening of candidate microbial peptides mimicking the critical motif of the anti-GBM epitope. The pathogenic motif of α3_127–148 (WIxLW) was applied as the query sequence to search microbial peptides using BLASTp suite. Candidate peptides were selected when they fulfilled the following three criteria. Firstly, the peptide contained at least one residue from GFxF, which is crucial for antibody binding. Secondly, the peptides were derived from probiotics or microbes associated with human infections. Thirdly, the peptides had a relatively high probability of being both T cell and B cell epitopes.

Figure 2.

The microbial peptide B7 induced anti-GBM GN in WKY rats. WKY rats were immunized with B7 (n=11), α3_127–148 (n=12), or CFA (control, n=12) on day 0, and experiments ended on day 49. Disease was assessed by proteinuria (A), serum creatinine (B), serum urea (C), crescent formation (D), and infiltrating cells (E–G). Illustrative photomicrographs of glomeruli showing crescent formation by periodic acid–Schiff stain, immunofluorescent staining of linear IgG deposits, and cellular infiltrates of CD4+ T cells, CD8+ T cells, and macrophages (H). Infiltrating cells are expressed as cells per gcs for T cells and IOD per gcs for macrophages. No pulmonary lesion was observed (H). *P<0.05, **P<0.01, and *** P<0.001.

Figure 3.

Immune response induced by B7 in WKY rats. Sera (A) and kidney eluates (D) of B7-immunized rats crossreacted with human α3_127–148 and rat α3_127–148, but not human α3(IV)NC1. Sera (B) and kidney eluates (D) of α3_127–148-immunized rats crossreacted with rat α3_127–148 and human α3(IV)NC1. No antibody was found in the negative control group (C). Splenocytes from α3_127–148- and B7-immunized rats were cultured and restimulated with α3_127–148 and B7 for 20 hours; supernatant was collected and measured for IFN-γ by ELISpot. Splenocytes from α3_127–148-immunized rats could respond to α3_127–148 and B7 (E). Splenocytes from B7-immunized rats could respond to α3_127–148 and B7 (F). Antibodies toward B7 were detected in the Actinomyces lysate protein-immunized group (G). Splenocytes from both the Actinomyces lysate protein- and recombinant protein-immunized rats could respond to B7 (H). SFC, spot-forming cells. *P<0.05, **P<0.01, and ***P<0.001.

Figure 4.

HLA-DR15 (Dr15⁺) transgenic mice immunized with B7 developed anti-GBM GN. DR15⁺.Fcgr2b^−/− mice were immunized with OVA_323–339 (n=8), α3135–145 (n=8), or B7 (n=7) on days 0, 7, and 14, and experiments ended on day 42. Disease was assessed by albuminuria (A), BUN (B), glomerular IgG deposition (C), segmental glomerular necrosis (D), crescent formation (E), glomerular fibrin (F), and infiltrating cells (G–I). Illustrative photomicrographs of glomeruli showing necrosis and early crescent formation by periodic acid–Schiff stain, immunofluorescent staining of linear IgG deposits, and immunoperoxidase staining of fibrin deposition and cellular infiltrates with macrophages as an example (J). IgG deposition is expressed semiquantitatively on the basis of intensity; infiltrating cells are expressed as cells per gcs. *P<0.05, **P <0.01, and *** P<0.001.

Figure 5.

Immune response induced by B7 in HLA-DR15 transgenic mice. B7-specific T cell immune responses do not crossreact with α3_135–145. Splenocytes from immunized DR15⁺Fcgr2b^−/− mice were cultured and restimulated with α3_135–145, recombinant α3(IV)NC1, and B7 for 72 hours; supernatant was collected and measured for IFN-γ and IL-17A by ELISA (A). Early antigen-specific T cell responses 10 days postimmunization were assessed in DR15⁺.Fcgr2b^+/+ mice by IFN-γ and IL-17A production measured by ELISpot (B). *P<0.05, **P<0.01, and ***P<0.001.

Figure 6.

Sequence alignment of B7, mouse α3_135–145, and human α3_127–148. Two of the four critical amino acids (V₁₃₇, W₁₄₀, G₁₄₂, and F₁₄₃) of mouse α3_135–145 were identical to those of B7. Six of the seven critical residues of α3_127–148 (W₁₃₆, I₁₃₇, L₁₃₉, W₁₄₀, G₁₄₂, F₁₄₃, and F₁₄₅) were identical to those of B7.