Parvovirus B19V Nonstructural Protein NS1 Induces Double-Stranded Deoxyribonucleic Acid Autoantibodies and End-Organ Damage in Nonautoimmune Mice

Abstract

Background: Viral infection is implicated in development of autoimmunity. Parvovirus B19 (B19V) nonstructural protein, NS1, a helicase, covalently modifies self double-stranded deoxyribonucleic acid (dsDNA) and induces apoptosis. This study tested whether resulting apoptotic bodies (ApoBods) containing virally modified dsDNA could induce autoimmunity in an animal model.

Methods: BALB/c mice were inoculated with (1) pristane-induced, (2) B19V NS1-induced, or (3) staurosporine-induced ApoBods. Serum was tested for dsDNA autoantibodies by Crithidia luciliae staining and enzyme-linked immunosorbent assay. Brain, heart, liver, and kidney pathology was examined. Deposition of self-antigens in glomeruli was examined by staining with antibodies to dsDNA, histones H1 and H4, and TATA-binding protein.

Results: The B19V NS1-induced ApoBod inoculation induced dsDNA autoantibodies in a dose-dependent fashion. Histopathological features of immune-mediated organ damage were evident in pristane-induced and NS1-induced ApoBod groups; severity scores were higher in these groups than in staurosporine-treated groups. Tissue damage was dependent on NS1-induced ApoBod dose. Nucleosomal antigens were deposited in target tissue from pristane-induced and NS1-induced ApoBod inoculated groups, but not in the staurosporine-induced ApoBod inoculated group.

Conclusions: This study demonstrated proof of principle in an animal model that virally modified dsDNA in apoptotic bodies could break tolerance to self dsDNA and induce dsDNA autoantibodies and end-organ damage.

Keywords: B19V; SLE; anti-dsDNA antibody; apoptotic bodies; glomerulonephritis.

Figure 1.

Parvovirus B19 (B19V) apoptotic bodies (ApoBods) are responsible for production of double-stranded deoxyribonucleic acid (dsDNA) autoantibody. (A) Fluorescence images of Crithidia lucilae immunofluorescent test (CLIFT) illustrate anti-dsDNA antibody levels in sera from differently treated mice at week 8 postimmunization. The presence of anti-dsDNA antibodies is determined by the positive green fluorescence of the kinetoplast (yellow arrowheads); an individual C lucilae cell representative of the positive population in each group is enlarged and displayed at the left lower corner of each panel. Kinetoplast is considered positive when its fluorescence intensity is the same or greater than the basal body (B), nucleus (N), or flagellum (F). Without fluorescence of kinetoplast, the cells are defined as negative. Bars, 10 μm. (B) Positive kinetoplasts stained with each mouse serum are counted out of 300 C lucilae cells. Percentages of positive kinetoplasts in each group (n = 6 mice/group) are calculated and presented as mean ± standard error of the mean (SEM). (C) Relative absorbance value of anti-dsDNA antibodies in each group assessed by the enzyme-linked immunosorbent assay and determined as mean ± SEM. Seropositive autoantibodies for each group are examined at week 1, 4, and 8 postimmunization, respectively. Results are compared with negative controls, untreated and phosphate-buffered saline (PBS)-treated groups, and all staurosporine (ST)-induced ApoBods groups, respectively. Significant difference considered as ^#P < .05 and *P < .01, whereas – indicates nonsignificance.

Figure 2.

Apoptotic bodies stimulates inflammation and damage in major organs. Inflammation and cellular degeneration in mice organs are illustrated by the representative bright-field images of hematoxylin and eosin-stained sections of each treatment group. (A) Suspected demyelination (arrowheads), degenerating neurons with more than 6 cells surrounding the neuropil (asterisks), and neuropil vacuolation are indicated in the brain. (B and C) Infiltrated immune cells (arrowheads) and cellular vacuolation (arrows) are revealed in the heart and liver, respectively. (D) Infiltrated immune cells (arrowheads) and proliferated glomerular tissues (arrows) are observed in the kidney. Bars, 100 μm in A–C and 50 μm in D. B19V, parvovirus B19V; NS1, nonstructural protein 1; PBS, phosphate-buffered saline.

Figure 3.

Significant histopathological destruction in major organs triggered by parvovirus B19 (B19V) apoptotic bodies (ApoBods). Histopathological severity of organs, including (A) brain, (B) heart, (C) liver, and (D) kidney were examined in every mouse. The severity scores are graded according to the histological features of inflammation and cellular degeneration. A severity score for each group (n = 6 mice/group) is determined as mean ± standard error of the mean. Results are compared with the negative controls, untreated and phosphate-buffered saline (PBS)-treated groups, and all staurosporine (ST)-induced ApoBods groups, respectively. Significant difference determined as ^#P < .05 and *P < .01, whereas, – refers to nonsignificance.

Figure 4.

Parvovirus B19 (B19V) apoptotic bodies (ApoBods) induced glomerulonephritis (GN). (A) Bright-field microscopy images of hematoxylin and eosin-stained sections demonstrate histological alterations in a representative glomerulus of each group of controls and B19V nonstructural protein 1 (NS1)-induced ApoBods-treated groups. Histological features of GN, including infiltrated inflammatory cells (yellow arrowheads), glomerular cell proliferation (long blue arrows), mesangial proliferation (blue arrowheads), and glomerular capillary thickening (short blue arrows), were observed. Bars, 20 μm. Fluorescence images illustrate immunolabeled (B) cellular deoxyribonucleic acid (DNA) (4’,6-diamidino-2-phenylindole, blue) and (C) double-stranded (ds)DNA deposition in glomerular tissues (green). (D) Merge of B and C with differential interference contrast images demonstrate the morphology and location of deposited dsDNA self-antigen in the glomerulus. (E) Higher magnification of the area in white boxes in D indicate green signal of deposited dsDNA in glomerular basement membrane surrounding the glomerular capillary (asterisks). Turquoise represents the red blood cells. Bars, 20 μm. PBS, phosphate-buffered saline.

Figure 5.

Deposition of self-antigens is detected in viral-induced glomerulonephritis. Nucleosomes deposition in glomeruli of each group was quantified from confocal microscopy images. Deposition of (A) double-stranded deoxyribonucleic acid (dsDNA) and (B) histone 1 (H1), H4, and TATA-binding protein (TBP) in the glomerular membrane were indicated by green and red fluorescence, respectively. A total of 30 glomeruli per group were analyzed. A triangle on a column scatter represents the fluorescence intensity of each glomerulus. Intensity from each group is determined as mean ± standard error of the mean (SEM). The mean intensity of each group is indicated in the scattered column. Results are compared with untreated and phosphate-buffered saline (PBS)-treated groups (left hand/ * or #), and then all staurosporine (ST)-induced apoptotic bodies (ApoBods) groups (/right hand * or #), respectively. Significant difference was defined as ^#P < .05 and *P < .01.