Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD

Abstract

Background: Microglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an "alternative" activation state.

Methods: To study alternative activation of microglia, we used quantitative RT-PCR to identify genes associated with alternative activation in microglia, including arginase I (AGI), mannose receptor (MRC1), found in inflammatory zone 1 (FIZZ1), and chitinase 3-like 3 (YM1).

Results: Our findings confirmed that treatment of microglia with anti-inflammatory cytokines such as IL-4 and IL-13 induces a gene profile typical of alternative activation similar to that previously observed in peripheral macrophages. We then used this gene expression profile to examine two mouse models of AD, the APPsw (Tg-2576) and Tg-SwDI, models for amyloid deposition and for cerebral amyloid angiopathy (CAA) respectively. AGI, MRC1 and YM1 mRNA levels were significantly increased in the Tg-2576 mouse brains compared to age-matched controls while TNFalpha and NOS2 mRNA levels, genes commonly associated with classical activation, increased or did not change, respectively. Only TNFalpha mRNA increased in the Tg-SwDI mouse brain. Alternative activation genes were also identified in brain samples from individuals with AD and were compared to age-matched control individuals. In AD brain, mRNAs for TNFalpha, AGI, MRC1 and the chitinase-3 like 1 and 2 genes (CHI3L1; CHI3L2) were significantly increased while NOS2 and IL-1beta mRNAs were unchanged.

Conclusion: Immune cells within the brain display gene profiles that suggest heterogeneous, functional phenotypes that range from a pro-inflammatory, classical activation state to an alternative activation state involved in repair and extracellular matrix remodeling. Our data suggest that innate immune cells in AD may exhibit a hybrid activation state that includes characteristics of classical and alternative activation.

Figure 1

Alternative activation genes are induced by treatment of microglia with IL4 or IL-13. BV2 cells were treated with IL-4 or IL-13 for 24 hrs and the mRNA expression levels for AG1 (Panel A); MRC1 (Panel B); FIZZ and YM1 (Panel C) were determined using quantitative RT-PCR. mRNA levels for each of these genes significantly increased compared to untreated alone. IL-4 or IL-13 treatment failed to induce TNFα (Panel D) or NOS2 (Panel E) mRNA expression. To determine if the alternative activation genes were induced by classical activation agents, cells were treated with IFNγ (panels A-C). In this case, no induction was observed with IFNγ treatment and, with the exception of AG1 (A), treatment of BV2 cells with the combination of IFNγ and IL-4 reduced mRNA expression of each gene studied; Panel F- Primary microglia obtained from neonatal mouse cortex also demonstrated increased mRNA expression for alternative activation genes (AG1; MRC1) on stimulation with IL-4. NOS2 was not increased by IL-4 treatment but was increased with IFNγ treatment. * = p < 0.001 compared to IL-4 treated alone; ** = p < 0.001 compared to IFNγ treated alone; *** = p < 0.001 compared to untreated alone.

Figure 2

Alternative activation genes in mouse models of amyloid pathology. The transcripts of activation-related genes were measured in cortical extracts from Tg-2576 (AD model) and Tg-SwDI mice (CAA model) using quantitative RT-PCR. The data are presented as the average (± SEM) fraction of control levels where the appropriate aged-matched wild type littermate mice served as the comparator control. A.Tg-2576 mice- mRNA for TNFα (p < 0.01), AGI (p = 0.05), MRC1 (p < 0.01), and YM1 (p < 0.02) were significantly increased in the Tg-2576 mice brain B. Tg-SwDI mice- Only TNFα mRNA levels were significantly increased (p < 0.05) in Tg-SwDI mouse brain.

Figure 3

Alternative activation genes in Alzheimer's disease. mRNA expression was determined for NOS2, TNFα, IL-1β, AGI, MRC1, CHI3L1, CHI3L2, AG2, CD45, CAT 2 and CAT3 in frontal lobe cortical extracts from AD patients and age-matched, cognitively normal controls. The real-time PCR results are expressed as the average (± SEM) fraction of control where age-matched, cognitively normal brain served as the comparator control. The data show significant elevations in mRNA expression levels for AG1 (* = p < 0.04), CHI3L1 (** = p < 0.006), and CHI3L2 (* = p < 0.04). NOS2 and IL-1β mRNA did not change but TNFα mRNA increased significantly (* = p < 0.04) in AD. CAT2 mRNA, which encodes the inducible arginine transporter, also significantly increased (# = p < 0.02) in AD compared to control. AG2, MRC1, CD45 and CAT3 mRNA expression levels were equivalent between the AD and control brains.