Thymic alterations in GM2 gangliosidoses model mice

Abstract

Background: Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb(-/-) mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies.

Methodology/principal findings: In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb(-/-) mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4(+)/CD8(+) T cells and a significantly increased number of CD4(+)/CD8(-) T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb(-/-) mice.

Conclusions/significance: These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.

Figure 1. Hexb ^−/− FcRγ ^+/+ mice show progressive, age-related thymus atrophy.

(A) The thymus was removed from 15 week old Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice. Scale bar, 1 cm. (B) Thymic tissues from Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcR^+/+ and Hexb ^−/− FcR ^−/− mice at 11, 13 and 15 weeks of age were removed and weighed. The data shown are expressed as the mean (S.D. of 3–14 mice for each group per time point. *P<0.01. (C) Thymic cells from Hexb ^+/− FcR ^+/+, Hexb ^−/− FcR ^+/+ and Hexb ^−/− FcR ^−/− mice were stained with CD4 and CD8 antibodies. The percentages of T cell subsets were then determined by flow cytometry. The profiles for each genotype are indicated in the top panels. The absolute cell numbers for each subpopulation of the thymus were calculated from the total number of thymic cells (mean (S.D.; n = 3–4). *P<0.05.

Figure 2. Histological examination of the thymus from Hexb ^+/− FcRγ ^+/+, Hexb ^−/− FcRγ ^+/+ and Hexb ^−/− FcRγ ^−/− mice.

H&E staining of paraffin-embedded thymic sections from 13 week old Hexb ^+/− FcRγ^+/+, 15 week old Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice. The bottom panels show a higher magnification image of the framed area in the top panels. Cor, cortex; Med, medulla. Arrows indicate vacuolated cells. Scale bars, top panel, 100 µm; bottom panel, 50 µm.

Figure 3. Histological examination of a thymus from an SD patient.

H&E staining of a paraffin-embedded thymic section from an 11 month-old SD patient. The right panel shows a higher magnification of the framed area in the left panel. Scale bars, left panel, 100 µm; right panel, 50 µm.

Figure 4. Increased cell death in the thymus of the 15 week old Hexb ^−/− FcRγ ^+/+ mouse.

(A) Thymic sections from 15 week old Hexb ^−/− FcRγ^+/+ and Hexb ^+/− FcRγ^+/+ mice were analyzed by TEM. Left panel, at lower magnification many electron dense materials and vacuoles are evident. Middle panel, nucleus-like electron dense particles and vacuoles are visible in the cytoplasm of the macrophages at higher magnification. Right panel, nuclear particles were not confirmed in the thymus of the Hexb ^+/− FcRγ^+/+ mouse.N, nucleus particle-like structure; MN, macrophage nucleus. (B) Paraffin-embedded thymic sections from Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice were stained using the TUNEL method. Scale bar, 50((m. A higher magnification image of the arrow position in the middle panel is shown in the right shoulder frame. (C) Thymic cells from Hexb ^+/− FcR^+/+, Hexb ^−/− FcR^+/+ and Hexb ^−/− FcR ^−/− mice were stained with FITC conjugated anti-Annexin V antibody and PI, and the percentages of dying cells were determined by flow cytometry (n = 4–8). *P<0.01. (D) IgG deposition in TCR(-positive T cells analyzed by flow cytometry (n = 3–5). *P≤0.01.

Figure 5. Macrophage activation and expansion.

(A) Thymic tissues from 13 week old Hexb ^+/− FcR^+/+, 15 week old Hexb ^+/− FcR^+/+, Hexb ^−/− FcR^+/+ and Hexb ^+/− FcR^+/+ mice were stained with antibodies against the macrophage marker Iba1 and processed using the avidin-biotin-peroxidase complex (ABC) method. The bottom panels show higher magnifications of the framed areas in the top panels. Scale bars, top panel, 100(µm; bottom panel, 50 µm. (B) Thymic cells from15 week old Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice were stained with anti-Iba1 followed by Alexa Fluor 488® conjugated anti-rabbit IgG (H+L), and the percentages of macrophages were determined by flow cytometry (n = 4). *p<0.01. (C) Mip-1α mRNA levels in the thymus from 15week old Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice were assayed by Real-time RT-PCR and these values were compared with those of the Hexb ^+/− FcRγ^+/+ mouse (mean ± S.D.; n = 5–9). *P<0.05.

Figure 6. CXCL13 expression is markedly increased in the thymus of the 15 week old Hexb ^−/− FcRγ ^+/+ mouse.

(A) The CXCL13 mRNA levels in the thymus from each of the Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice at 11, 13 and 15 weeks of age were determined by real-time RT-PCR and compared with the levels in the Hexb ^+/− FcRγ^+/+ mouse (mean ± S.D.;n = 3–8). *P<0.05. (B) The CXCL13 mRNA levels in the thymus, spleen and lymph nodes from 15 weeks old Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice was also measured using real-time RT-PCR and compared with the levels in Hexb ^+/− FcRγ^+/+ mice (mean ± S.D.; n = 4–8). *P<0.05. (C) Thymus cryosections from 13 week old Hexb ^+/− FcRγ^+/+, 15 week old Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice were stained with CXCL13 antibodies and detected using DAB. Arrows indicate CXCL13-positive cells. Scale bar, 50 µm. (D) Thymic cells from 15 week old Hexb ^−/− FcRγ^+/+ mice were cell sorted using MACS. The CXCL13 mRNA levels in the CD11b⁺ and CD11b⁻ cell populations are expressed relative to the normalized CXCL13 levels in whole thymic cells (mean ± S.D.; n = 4). *P<0.05.

Figure 7. Significant increase in the B1 and B2 cell number in the thymus of 15 week old Hexb ^−/− FcRγ ^+/+ mouse.

(A) Thymus cryosections from 13 week old Hexb ^+/− FcRγ^+/+, 15 week old Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice were stained with CD19 monoclonal antibodies and detected using the ABC method. The bottom panels show higher magnification images of the framed areas in the top panels. Scale bars, 50 µm. (B) Thymic cells from 15 week old Hexb ^+/− FcRγ^+/+, Hexb ^−/− FcRγ^+/+ and Hexb ^−/− FcRγ ^−/− mice were stained with FITC-CD5, PE-B220 and PE-Cy5-CD19 antibodies and the percentages of the B cell subsets were determined by flow cytometry (mean ± S.D.; n = 6–9). *P<0.01.