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. 2015;46(2):365-74.
doi: 10.3233/JAD-142184.

APOE Genotype Alters Immunoglobulin Subtypes in Knock-In Mice

Ye Zhou  1 , Wenjuan Zhao  2 , Nour Al-Muhtasib  3 , G William Rebeck  4
Affiliations

APOE Genotype Alters Immunoglobulin Subtypes in Knock-In Mice

Ye Zhou et al. J Alzheimers Dis. 2015.

Abstract

Apolipoprotein E (APOE) alleles are strongly related to the risk of Alzheimer's disease (AD). APOE genotype also affects inflammatory processes in response to damage. We tested whether APOE genotype affected the levels of specific immunoglobulins in healthy, uninfected APOE knock-in mice. We measured specific immunoglobulins in brain, spleen, and plasma. Levels of total IgG in brain and spleen were highest in APOE-ɛ3 mice, significantly higher than in APOE-ɛ2 and APOE-ɛ4 mice; no differences were observed for levels of total IgG in plasma. We also measured specific subtypes of IgG. IgG1 was only detectable in plasma and did not differ by APOE genotype. IgG3 was detectable in plasma and spleen, and also did not differ by APOE genotype. IgG2b showed the same pattern as levels of total IgG by APOE genotype, with the highest levels of IgG2b in brain, spleen, and plasma of APOE-ɛ3 mice. IgG2a showed an entirely different pattern, with significantly higher levels in spleen and plasma of APOE-ɛ4 mice compared to APOE-ɛ2 and APOE-ɛ3 mice. We also measured IgM and IgA in spleens and plasma of these mice. In spleen, APOE-ɛ4 mice had the lowest IgA levels and the highest levels of IgM; both being significantly different from APOE-ɛ2 mice. In total, murine IgG2a and IgM were highest in APOE-ɛ4 mice, while total IgG and Ig2b were highest in APOE-ɛ3 mice. These dramatically different distributions of immunoglobulins could allow for human AD risk biomarkers based on specific immunoglobulin subtypes.

Keywords: Apolipoprotein E; brain; immunoglobulin; inflammation; plasma; spleen.

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Figures

Figure 1
Figure 1. APOE3 mice have higher levels of IgG in spleen and brain
Nine-month old female APOE2 (n=4), APOE3 (n=6) and APOE4 (n=6) mice were euthanized and IgG levels measured by Western blot. Brain (A) and spleen (B) tissues were homogenized sequentially in TBS (for soluble proteins) and TBS-X (for membrane-associated proteins); IgG was detected primarily in the soluble tissue fractions as a 50 kDa protein. (C, D) APOE3 mice demonstrated the highest level of IgG in soluble brain fractions (3.0±0.9 in APOE2 mice, 42.0±17.9 in APOE3 mice, 6.0±0.9 in APOE4 mice; E2 vs E3, *p<0.05; E3 vs E4, p=0.056). (E, F) APOE3 mice also showed a significantly higher level of IgG in soluble spleen fractions (4.4±1.3 in APOE2 mice, 9.0±0.9 in APOE3 mice, 5.8±0.9 in APOE4 mice; E2 vs E3, *p<0.05; E3 vs E4, *p<0.05). (G, H) No significant differences of IgG levels were detected in plasma among the APOE groups (1.4±0.1 in APOE2 mice, 1.6±0.2 in APOE3 mice, 1.5±0.1 in APOE4 mice). Data are presented as mean ± SEM.
Figure 2
Figure 2. IgG1 was detected only in plasma
Plasma from APOE2 (n=4), APOE3 (n=4) and APOE4 (n=4) were analyzed for IgG1 by immunoblot. Quantification of films demonstrated that no significant differences in levels of IgG1 were detected across APOE genotypes. Data are presented as mean ± SEM.
Figure 3
Figure 3. APOE4 mice have significantly higher levels of IgG2a in plasma and spleen
(A) Spleen samples were analyzed for IgG2a. APOE4 mice showed a significantly higher level of Ig2a in the soluble spleen fractions (1.03±0.39 in APOE2 mice, 1.87±0.93 in APOE3 mice, 3.56±0.77 in APOE4 mice; E2 vs E4, ##p<0.01; E3 vs E4, #p<0.05). (B) In plasma samples, IgG2a was significantly higher in APOE4 mice (0.98±0.50 in APOE2 mice, 1.78±1.49 in APOE3 mice, 5.75±1.15 in APOE4 mice; E2 vs E4, ###p<0.001; E3 vs E4, ###p<0.001). Data are presented as mean ± SEM.
Figure 4
Figure 4. IgG2b levels are highest in APOE4 tissues
IgG2b was measured in brain (A), spleen (B), and plasma (C) by western blotting (n=3–4). (A) APOE3 mice showed significantly higher levels of brain IgG2b compared to APOE4 mice (1.1±0.4 in APOE2 mice, 1.6±0.3 in APOE3 mice, 0.69±0.12 in APOE4 mice; E3 vs E4, *p<0.05). (B) APOE3 mice also showed highest levels of spleen IgG2b, although not significantly different from APOE2 or APOE4 mice (1.5±0.9 in APOE2 mice, 2.3±1.1 in APOE3 mice, 0.72±0.2 in APOE4 mice; ns). (C) APOE3 mice showed significantly higher levels of plasma IgG2b compared to APOE2 or APOE4 mice (1.6±0.5 in APOE2 mice, 3.4±0.9 in APOE3 mice, 0.87±0.42 in APOE4 mice; E3 vs E2, *p<0.05; E3 vs E4, **p<0.01). Data are presented as mean ± SEM.
Figure 5
Figure 5. APOE genotype had no effects on IgG3 levels
IgG3 was measured in TBS-soluble spleen samples (A), and plasma (B) by western blotting. There were no significant differences in IgG3 levels by APOE genotype.
Figure 6
Figure 6. APOE4 mice have lower levels of IgA in spleen
IgA levels were examined by Western blot. (A) IgA bands were detected in the TBS fraction of immunoblots of spleen samples (inset). APOE4 mice showed a significantly lower level of IgA in the soluble spleen fractions compared to APOE2 mice (94.8±17.7 in APOE2 mice, 53.3±21.0 in APOE3 mice, 15.8±12.5 in APOE4 mice; E2 vs E4, #p<0.05). (B) No significant differences of IgA in plasma were observed among groups (8.4±1.9 in APOE2 mice, 6.8±2.6 in APOE3 mice, 8.9±2.0 in APOE4 mice). Error bars expressed as SEM.
Figure 7
Figure 7. APOE4 mice have higher levels of IgM in spleen and plasma
Western blots of IgM in soluble fractions of spleen revealed proteins of 50 and 75 kDa (A). (B) Quantification of soluble brain fractions revealed that APOE4 mice had the highest levels of IgM, but differences with APOE2 and APOE3 mice were statistically not significant. (C) APOE4 mice showed a significantly higher level of IgM in spleen (44±33 in APOE2 mice, 136±38 in APOE3 mice, 275±33 in APOE4 mice; E2vsE3, p<0.05, E3vsE4, #p<0.05, E2vsE4, ##p<0.01). (D) APOE2 mice showed a significantly lower level of IgM in plasma (1.4±0.3 in APOE2 mice, 2.2±0.2 in APOE3 mice, 2.5±0.1 in APOE4 mice; E2vsE3, *p<0.05, E2vsE4, ##p<0.01). Error bars expressed as SEM.
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