Immune and Inflammatory Determinants Underlying Alzheimer's Disease Pathology

Abstract

This study examines the link between peripheral immune changes in perpetuation of the Alzheimer's disease (AD) neuropathology and cognitive deficits. Our research design using human AD patients and rodent model is supported by past evidence from genomic studies. We observed an active immune response against Aβ as indicated by the increased Aβ specific IgG antibody in the serum of AD and patients with mild cognitive impairments as compared to healthy controls. A similar increase in IgG and decrease in IgM antibody against Aβ was also confirmed in the 5xFAD mouse model of AD. More importantly, we observed a negative correlation between reduced IgM levels and cognitive dysfunction that manifested as impaired memory consolidation. Strong peripheral immune activation was supported by increased activation of microglia in the brain and macrophages in the spleen of AD mice compared to wild type control littermates. Furthermore, inflammatory cytokine IL-21 that is involved in antibody class switching was elevated in the plasma of AD patients and correlated positively with the IgG antibody levels. Concurrently, an increase in IL-21 and IL-17 was observed in spleen cells from AD mice. Further investigation revealed that proportions of T follicular helper (Tfh) cells that secrete IL-21 are increased in the spleen of AD mice. In contrast to Tfh, the frequency of B1 cells that produce IgM antibodies was reduced in AD mice. Altogether, these data indicate that in AD the immune tolerance to Aβ is compromised leading to chronic immune/inflammatory responses against Aβ that are detrimental and cause neuropathology. Graphical Abstract Healthy subjects are tolerant to Aβ and usually react weakly to it resulting the in the production of IgM class of antibodies that are efficient at clearing up self-antigens such as Aβ without causing inflammation. In contrast, Alzheimer's disease patients mount a strong immune response against Aβ probably in an effort to clear up excessive Aβ. There is enhanced production of inflammatory cytokines such as IL-21 as well as an increase in Tfh cells that cause antibody class switching form IgM to IgG. The strong immune response is inefficient at clearing up Aβ and instead exacerbates inflammation that causes AD neuropathology and cognitive dysfunction.

Keywords: Alzheimer’s disease; Aβ; Cognition; IL-21; Inflammation; Tfh.

Figure 1:. Aβ (1–42) peptide specific antibodies of IgM isotype are decreased in AD patients and in 5xFAD mice compared to littermate controls.

The levels of Aβ peptide (1–42) specific antibodies, as well as Aβ scrambled peptide, were measured in the plasma samples of AD, MCI and age matched healthy controls (HC). (A) Dot plots depict the Aβ specific IgM to IgG antibodies. Mean ± S.D. of 26 AD patients, 26 MCI patients and 26 HC. Equal number of males and females in each group. (B) The levels of Aβ peptide (1–42) specific antibodies as well as Aβ scrambled peptide were also measured in the plasma samples of 5xFAD mice compared to WT littermate controls at 4 and 8 months of age. Dot plots depict the ratio of Aβ specific IgG and IgM antibodies to scrambled peptide antibodies. Mean ± S.D. For 4 mo: N=7M, 2F mice per group; 8 mo: N=7M, 7F mice per group. P values are derived from one way ANOVA in A and Student’s t test in B.

Figure 2:. Correlation of IgM and IgG response with cognitive function.

(A) The cognitive testing of 6–8 month old WT and AD mice using the Fear Extinction (FE) memory test, showed significant cognitive impairments as indicated by elevated freezing during the extinction test. (B) The AD-related memory deficits correlated with the reduced IgM levels. AD mice showing higher freezing display higher IgM levels. Mean ± SE (n=14 mice per group, A; n=8, mice per group B). P values are derived from Student’s t test (A) and Pearson’s test (B).

Figure 3:. Macrophages/microglia display increased activation in 5xFAD mice compared to littermate controls.

(A-C) Representative high-resolution confocal micrographs from the basal lateral amygdala (BLA) of WT and AD mouse brains (red, CD68; blue, DAPI nuclear counterstain; Scale 40 μm). (D) Quantification of CD68 immunohistochemical staining demonstrated that compared to controls, AD mice had increased microglial activation at both 4 and 8 months of age as compared to WT controls where data are Mean ± SE (n=4 mice per group); *p<0.01. (E) Dot plot depict the MFI (Mean fluorescence intensity) of MHC-II on macrophages in the spleen of AD and WT mice at 4 and 8 months of age. Histogram is representative of macrophage-MHC staining. For 4 mo: n=7M, 2F mice per group; 8 mo: n=7M, 7F mice per group. P values are derived from Student’s t test.

Figure 4:. Inflammatory cytokines are increased in AD patients and 5xFAD mice.

(A) Dot plots depict the levels of cytokines in the plasma of human AD, MCI and HC samples. Mean ± S.D. of 26 AD patients, 26 MCI patients and 26 HC. (B) Dot plots display the levels of cytokines secreted by PMA, ionomycin stimulated spleen cells from 5xFAD mice and WT littermate controls at 4 and 8 months of age. For 4 mo: 7M, 2F mice per group; 8 mo: 7M, 7F mice per group. P values are derived from Student’s t test.

Figure 5:. Tfh cells are increased and B1 cells are decreased in 5xFAD mice compared to littermate controls.

(A) Spleen cells were stained for Tfh cells (CD4⁺, CXCR5⁺, PD-1⁺) and analyzed by flow cytometry. Contour plots depict the percentage of Tfh cells in AD and WT mice. Dot plots depict the % of CD4⁺, CXCR5⁺, PD-1⁺ Tfh cells at 4 and 8 months of age. (B) Spleen cells were stained for B1 cells (CD19+CD5+CD43+) and analyzed by flow cytometry. Contour plot depicts the percentage of B1 cells in AD and WT mice. Dot plot depicts the percentage of B1 cells at 4 and 8 months of age. Mean +/−S.E. For 4 mo: 7M, 2F mice per group; 8 mo: 7M, 7F mice per group. P values are derived from Student’s t test.