Agostini, Alessandra
(2020)
Investigating genotype and sex susceptibility to a systemic infection in APPswe/PS1ΔE9 mouse model of Alzheimer’s disease, at an early stage of amyloid plaque deposition.
PhD thesis, University of Nottingham.
Abstract
Systemic bacterial infection can worsen Alzheimer’s disease (AD) progression by compromising the central microglia and astrocytes immune response implicated in the clearance of amyloid-β (Aβ) plaques, one of the AD hallmarks. In the present thesis, I investigated whether systemic infection with lipopolysaccharide (LPS) enhanced glial activation, increased amyloid plaque load and infiltration of peripheral macrophages into the brain and whether tolerance occurred following repeated LPS injections. The incidence of AD is higher in women than in men, thus sex-differences in the immune response were also investigated.
Method. 4.5 months old females and males, APPswe/PS1ΔE9 and wild-type littermates were used. LPS (100 μg/kg, i.v) or vehicle (PBS) were systemically administered. Behavioural changes were investigated both at baseline and post-injection assessing spontaneous alternation, food burrowing and open field, to measure spatial working memory daily living and locomotor activity respectively.
In the first experiment (n=5-6/ group) (chapter 3) brain tissue and blood were collected from the mice 4 hours after the challenge to study the acute effect of LPS. In the second experiment (n=8/group) (chapter 4) brain tissue, blood and peritoneal macrophages were collected 7 days after the treatment to examine the long effect of an acute immune challenge on amyloid plaque load and glial response. In the third study (chapter 5), mice (n=8/group) received an LPS or PBS injection at 4.5months of age, and a second LPS or PBS challenge 2 weeks later, yielding to 3 experimental groups: PBS-PBS- treated (control), PBS-LPS- treated and LPS-LPS-treated mice. The long effect of an acute challenge was analysed on slightly older animals (5 months old) than the previous study (PBS-LPS-treated group). The effect of repeated exposure to LPS (LPS-LPS-treated group) was also determined. Quantification of amyloid plaque load, microglia and astrocytes density in cortex and hippocampus was assessed by immunostaining of Aβ, Iba-1 and GFAP, respectively. Microglia and astrocytes clusters were also measured to analyse the glial response to the presence of plaques. Identification of the possible infiltration of peripheral macrophages into the brain was performed by flow cytometry staining for the surface receptors CD11b and CD45, to distinguish between infiltrated macrophages (CD11b+ CD45+Hi) and resident microglia (CD11b+ CD45+Low). Characterization of the immune phenotype was assessed by flow cytometry: the expression of the chemotactic receptor CCR2, the pro-inflammatory marker CD80, and anti-inflammatory marker CD206, as well as the presence of intracellular Aβ in resident microglia and macrophages were analysed.
Results. LPS induced an increase in the pro-inflammatory cytokines IL-6 and TNF-α, and an increase in the anti-inflammatory cytokine IL-10, after 4 hours. A single systemic LPS challenge induced an increase in the number of microglial cells in APPswe/PS1ΔE9 males and females challenged at 4.5 months of age, after 7 days (LPS-treated group). A single systemic LPS challenge, given at 5 months of age (PBS-LPS-treated group), induced an increase in the number of microglial cells, a reduction in the size of small amyloid plaques and a reduction in the levels of IFN-γ in the plasma, in APPswe/PS1ΔE9 female mice after 7 days.
Two challenges of LPS within 3 weeks did not affect amyloid plaque load but induced a reduction in IFN-γ plasma levels in APPswe/PS1ΔE9 females, 7 days after the last injection. This reduction was associated with increased microglia density in the brain. LPS-LPS-treated females developed tolerance to LPS after the second injection, compared to the first challenge. Improvement in spatial working memory was seen in LPS-LPS-treated APPswe/PS1ΔE9 females, 7 days after the second challenge. Unexpectedly, LPS did not affect the migration of peripheral macrophages into the brain and the expression of the immune phenotypic markers in any of the studies.
Further investigation is needed to evaluate genotype susceptibility to the immune challenge and the impact of the systemic LPS infection on the age-dependent amyloid plaque accumulation, in order to clarify the effect of the central inflammatory response on disease progression.
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