A Highly Expressed Human Protein, Apolipoprotein B-100, Serves as an Autoantigen in a Subgroup of Patients With Lyme Disease

Abstract

To discover novel autoantigens associated with Lyme arthritis (LA), we identified T-cell epitopes presented in vivo by human leukocyte antigen (HLA)-DR molecules in patients' inflamed synovial tissue or joint fluid and tested each epitope for autoreactivity. Using this approach, we identified the highly expressed human protein, apolipoprotein B-100 (apoB-100), as a target of T- and B-cell responses in a subgroup of LA patients. Additionally, the joint fluid of these patients had markedly elevated levels of apoB-100 protein, which may contribute to its autoantigenicity. In patients with antibiotic-refractory LA, the magnitude of apoB-100 antibody responses correlated with increased numbers of plasma cells in synovial tissue, greater numbers and activation of endothelial cells, and more synovial fibroblast proliferation. Thus, a subset of LA patients have high levels of apoB-100 in their joints and autoreactive T- and B-cell responses to the protein, which likely contributes to pathogenic autoimmunity in patients with antibiotic-refractory LA.

Keywords: Borrelia burgdorferi; Lyme arthritis; Lyme disease; apolipoprotein B; autoantibodies; autoimmunity; erythema migrans.

Figure 1.

Initial screening process for identification of candidate autoantigens. Abbreviations: apoB-100, apolipoprotein B-100; HLA, human leukocyte antigen; LA, Lyme arthritis; LC-MS/MS, liquid chromatography–tandem mass spectrometry; PBMC, peripheral blood mononuclear cell.

Figure 2.

Testing of PMBCs for T-cell autoreactivity against full-length apoB-100. PBMCs were stimulated with the full -length apoB-100 and T-cell reactivity was measured using the human IFN-γ ELISpot assay. A, A positive response was defined as a mean SFU/10⁶ cells that was 3 standard deviations above the mean of healthy controls (area above the gray shaded region). P values were calculated using an unpaired t test. B, The number of patients with positive responses and percentage of positive responses for each patient group. Groups are compared using Fisher exact test. Abbreviations: apoB-100, apolipoprotein B-100; ELISpot, enzyme-linked immunospot; EM, erythema migrans; HC, healthy control; IFN-γ, interferon-γ; PBMCs, peripheral blood mononuclear cells; SFU, spot forming units.

Figure 3.

IgG anti-apoB-100 autoantibody responses. A, The levels of apoB-100 autoantibodies in serum samples were measured by ELISA. The shaded gray area corresponds to 3 standard deviations above the mean of healthy control subjects. P values for antibody amounts were calculated using an unpaired t test. B, The number of positive responses and percentages for each patient group. Groups are compared using Fisher exact test. C and D, Anti-apoB-100 antibody measurements for patient's paired serum and JF samples. Analysis for antibiotic-responsive LA patients (C); analysis for antibiotic-refractory LA patients (D). The P value was calculated using a paired t test. Abbreviations: apoB-100, apolipoprotein B-100; ELISA, enzyme-linked immunosorbent assay; EM, erythema migrans; HC, healthy control; IgG, immunoglobulin G; JF, joint fluid; LA, Lyme arthritis; OD, optical density; RA, rheumatoid arthritis.

Figure 4.

ApoB-100 protein levels in patients with Lyme disease or rheumatoid arthritis and in healthy control subjects. A, The concentration of apoB-100 in serum and JF samples were measured by sandwich ELISA. The shaded gray area corresponds to 3 standard deviations above the mean value in healthy control subjects. P values were calculated using unpaired t test. B, The number of patients >3 SD healthy controls and percentages for each patient group examined. Abbreviations: apoB-100, apolipoprotein B-100; ELISA, enzyme-linked immunosorbent assay; EM, erythema migrans; HC, healthy control; JF, joint fluid; RA, rheumatoid arthritis.

Figure 5.

Correlation between histologic rankings (CXCL13 staining, plasma cells, vascularity in the tissue, and ECGF) and anti-apoB-100 antibody rankings in patients with antibiotic-refractory Lyme arthritis who underwent synovectomies. The correlations were determined by Pearson's correlation test. Abbreviations: apoB-100, apolipoprotein B-100; ECGF, endothelial cell growth factor; IgG, immunoglobulin G.

Figure 6.

Correlation between histologic rankings (endothelial cells, expression of markers for endothelial cell activation, ICAM and VCAM, and fibroblasts) and anti-apoB-100 antibody rankings in patients with antibiotic-refractory Lyme arthritis who underwent synovectomies. The correlations were determined by Pearson's correlation test. Abbreviations: apoB-100, apolipoprotein B-100; ICAM, intercellular adhesion molecule; IgG, immunoglobulin G; VCAM, vascular cell adhesion molecule.

Figure 7.

Immunohistochemical staining of synovial tissue from representative patients with antibiotic-refractory LA who had the highest or lowest rankings for serum apoB-100 IgG antibodies. The patient with the highest ranking for apoB-100 antibodies had marked staining for CXCL13, plasma cells, endothelial cells, and synovial fibroblasts, whereas the patient with the lowest ranking had minimal staining for these histologic findings. Brown indicates specific staining of the immune cells, and purple is the counter-stain (hematoxylin). A, Staining for CXCL13 and plasma cells. B, Staining for endothelial cells and synovial fibroblasts. Bars = 100 µm. Abbreviations: apoB-100, apolipoprotein B-100; IgG, immunoglobulin G; LA, Lyme arthritis.