Sustained hippocampal IL-1β overexpression mediates chronic neuroinflammation and ameliorates Alzheimer plaque pathology (original) (raw)
Engineering of the IL-1βXAT mouse model. In order to study the role of sustained cytokine expression within the brain, we employed an excisional activation transgene (XAT) (9) cassette to drive eventual transcription of IL-1β in the CNS (10). We subcloned the hybrid cDNA ssIL-1β — which incorporates the signal sequence (ss) from human IL-1 receptor antagonist (hIL-1ra) fused in frame to the coding sequence for mature hIL-1β, thus directing extracellular release and eliminating the need for caspase-1 cleavage of IL-1β (11) — into a universal XAT vector along with a LacZ reporter gene (9). We chose a human transgene because of its ability to signal through IL-1 receptor 1 (IL-1R1) in the mouse and because of the capability of distinguishing the transgene from its murine counterpart. Functionality of the transgene construct was established in the 293GLVP/CrePr stable cell line, which is capable of producing Cre when treated with RU486 (Figure 1A) (12). Cre expression led to DNA excisional recombination and dropout of the approximately 2.5-kb loxP-flanked transcriptional stop sequence as well as induction of hIL-1β relative to GAPDH. Additionally, increased LacZ activity was observed using X-gal histochemistry (Figure 1A).
Engineering and testing of the IL-1βXAT mouse. (A) The 293GLVP/CrePr stable cell line was transiently transfected with CMV/IL-1βXAT and cultured with or without RU486. RU486 caused DNA excision and expression of hIL-1β and β-galactosidase by RT-PCR and X-gal histochemistry, respectively. (B) The linear IL-1βXAT construct (~10 kb) consisted of a murine GFAP promoter; a transcriptional stop element flanked by loxP sequences; downstream ssIL-1β cDNA (11); and an internal ribosome entry site (IRES) followed by the β-gal coding sequence (LacZ) and bovine growth hormone polyadenylation signal (pA). Exposure to Cre recombinase caused excision of the transcriptional stop and subsequent transcriptional activation of ssIL-1β. (C) PCR screening of the 11 live-born IL-1βXAT pups using ssIL-1β–specific primers. Transgenic founder lines A/a and B/b are shown. (D) ELISA quantification (mean ± SEM) of hIL-1β protein supernatant concentration in individual primary astrocyte (n = 4) cultures from B/b and WT astrocytes transduced with FIV-Cre or FIV-GFP. ND, not detected (i.e., below detection limits). (E) The epicenter of viral transduction, the dentate gyrus, was bounded by NeuN-positive neurons (red stain). Colocalization of the epitope tag V5 (green stain) expressed by FIV-Cre demonstrated transduction of both neuronal (arrows) and non-neuronal cells (arrowheads). (F) Demonstration of hIL-1β expression (green stain) by astrocytes (red stain) in the dentate gyrus. Hoechst (blue stain) labeled cell nuclei. (G) MHC class II–stained coronal section from a B/b animal 1 week after intrahippocampal FIV-Cre injection (right hemisphere). Scale bars: 25 μm (E); 10 μm (F); 1 μm (G).
We substituted a GFAP promoter to direct CNS-specific expression of the transgene and used the resulting IL-1βXAT construct to create parallel lines of transgenic mice (Figure 1B). Following microinjections, we identified 2 heterozygous transgenic founders designated A/a and B/b (Figure 1C). We used an infection-competent, replication-incompetent feline immunodeficiency virus (FIV) to deliver Cre to the brain, which allowed for both temporal and spatial control of the initiation of IL-1β production. Production of hIL-1β protein in FIV-Cre–infected IL-1βXAT primary astrocyte cultures confirmed the function of the transgene construct (Figure 1D). Stereotactic injection of FIV-Cre into the dentate gyrus of IL-1βXAT mice led to viral transduction of both neuronal and non-neuronal cells (Figure 1E). FIV-Cre–mediated hIL-1β protein expression was detected in astrocytes (Figure 1F) but not in microglia or neurons (data not shown). LacZ activity was not detected in transgenic animals (data not shown). Finally, following stereotactic injections of FIV-Cre into the dentate gyrus of the mouse brain, we demonstrated spatially restricted MHC class II expression in the injected ipsilateral hippocampus (Figure 1G).
Neuroinflammatory phenotype of IL-1βXAT mice. After successful creation of the IL-1βXAT model, our initial efforts were directed at defining the downstream effects of sustained IL-1β expression in the adult murine CNS (Figure 2). Two weeks following intrahippocampal FIV-Cre injections, we sought to characterize phenotypic and transcriptional changes in glia residing in the hippocampus of IL-1βXAT A/a and B/b animals compared with WT animals. FIV-Cre injections in WT mice controlled for inflammation resulting from the stereotactic surgeries and host response to the viral vector. Most of the phenotypic changes were seen in the region of the dentate gyrus, where the stereotaxic injections were directed (Figure 2, A and B). Microglial activation was most prominent in B/b animals, as demonstrated by dramatic increases in ionized calcium-binding adaptor molecule 1 (Iba-1) staining through much of the hippocampus (Figure 2A). Many microglia assumed a highly activated amoeboid state, with fewer cellular processes than in WT animals (Figure 2A, insets). The B/b animals also exhibited robust MHC class II expression — in both perivascular and parenchymal sites — that colabeled with Iba-1. In the A/a animals there was very mild activation of microglia and scant MHC class II expression limited to perivascular Iba-1–positive cells (Figure 2A). Astrocyte activation and increased GFAP expression was evident only in B/b mice and was confined to the dentate gyrus (Figure 2B). Despite robust glial activation, we detected no evidence of neuronal death in the dentate gyrus of A/a or B/b animals using either Fluoro-Jade or TUNEL staining 2 weeks after FIV-Cre injections (data not shown).
Induction of hIL-1β mediates a robust neuroinflammatory response in the mouse hippocampus. IL-1βXAT A/a and B/b mice and WT controls were injected unilaterally in the dentate gyrus with approximately 1.5 × 104 infectious units of FIV-Cre. Inflammatory indices were assayed 2 weeks later. (A) Microglial activation was demonstrated by increased staining intensity of Iba-1 (green) and MHC class II (red) in B/b and A/a relative to WT mice, with colocalization appearing yellow. Insets show higher-magnification morphologic changes among Iba-1–positive cells residing in the dentate gyrus. (B) Astrocyte activation, as evidenced by increased GFAP expression, was demonstrated in the dentate gyrus of B/b animals only. Scale bars: 50 μm (A and B); 10 μm (insets in A). (C and D) qRT-PCR analysis compared relative abundance of gene transcripts in ipsilateral (I) and contralateral (C) hippocampi. Analysis revealed significant upregulation of MHC class II in A/a and B/b mice (C), but significant upregulation of GFAP in B/b mice only (D), compared with WT controls. n = 3–4 per group. Data are mean ± SEM. *P < 0.05 versus respective WT.
Quantitative real-time PCR (qRT-PCR) was used to determine relative changes in copies of gene transcripts in B/b and A/a mice relative to WT animals (Figure 2, C and D). MHC class II expression was significantly increased in the FIV-Cre–injected ipsilateral hippocampi of both A/a and B/b animals (6.8- and 34.1-fold, respectively; Figure 2C). GFAP expression was significantly increased only in B/b mice (2.9-fold; Figure 2D). Intrahippocampal injections using GFP-expressing FIV in A/a and B/b animals failed to precipitate a neuroinflammatory response, establishing its dependence upon exposure to Cre (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI31450DS1). Based on this demonstration of a more potent neuroinflammatory phenotype in B/b versus A/a IL-1βXAT mice, we used only B/b animals in our subsequent experiments.
In order to prove that IL-1β was in fact mediating the inflammatory response seen, we crossed B/b mice with il1r1–/– animals (Figure 3). These animals lack IL-1R1, the sole biologically active receptor for IL-1β (13). As expected, il1r1–/– animals failed to demonstrate increases in neuroinflammatory indices 2 weeks following transgene activation. Relative MHC class II expression was unchanged in _Il1r1_–/– B/b compared with WT animals (0.84-fold that of WT), whereas Il1r1+/– B/b animals demonstrated an intermediate phenotype compared with Il1r1+/+ B/b mice (20.4- and 46.3-fold increases, respectively, relative to WT; Figure 3, A and B). GFAP mRNA expression in _Il1r1_–/– B/b mice was 1.03-fold that of WT controls, but this was significantly upregulated in both Il1r1+/– B/b and Il1r1+/+ B/b mice (2.7- and 2.5-fold, respectively, that of WT; Figure 3C).
The neuroinflammatory response requires IL-1R1. IL-1βXAT B/b animals that were lacking (_Il1r1_–/–), heterozygous for (Il1r1+/–), or with 2 copies (Il1r1+/+) of the gene encoding IL-1R1 were examined 2 weeks following FIV-Cre hippocampal injections. qRT-PCR analysis of ipsilateral hippocampi was performed relative to WT controls. (A) MHC class II induction was absent in Il1r1–/– B/b animals, whereas Il1r1+/– B/b mice exhibited a significant, intermediate phenotype compared with WT mice. (B) Histochemical analysis of MHC class II expression within the dentate gyrus of B/b mice revealed a pattern of expression mirroring the results in A. Scale bar: 25 μm. (C) GFAP induction was absent in Il1r1–/– B/b mice, whereas Il1r1+/– and Il1r1+/+ B/b mice displayed significant GFAP upregulation. n = 3–5 animals per group. Data are mean ± SEM. *P < 0.05 versus WT.
IL-1βXAT model of chronic neuroinflammation. To establish the IL-1βXAT mouse as a useful model of chronic CNS inflammation, we followed cohorts of adult B/b and WT mice for an extended time course after transgene activation (Figure 4). Expression of MHC class II gene transcripts, representative of sustained microglial activation, remained significantly elevated compared with WT controls between 1 week and 10 months after FIV-Cre injection (Figure 4A). An analogous pattern of expression was determined for GFAP expression, attributable to chronic astrocyte activation (Figure 4C). Elevated MHC class II and GFAP expression were detected histochemically 1 year after FIV-Cre injection, the latest time point at which the mice were examined (Figure 4, B and D). The chronology of glial activation was mirrored by expression of the activated hIL-1β transgene, which peaked early in the time course and was last detected 4 months after transgene activation (Figure 4E). In addition to glial activation markers, we sought to determine whether transgene activation could drive induction of inflammatory cytokines classically associated with acute hIL-1β activity. Indeed, transgene activation resulted in chronic, increased expression of all members of the murine IL-1 (mIL-1) family: mIL-1α, mIL-1β, and mIL-1ra (Figure 4F). In addition, hIL-1β activity drove chronic expression of the proinflammatory cytokines TNF-α and IL-6 (Figure 4G).
Transgene activation in the IL-1βXAT mouse elicits a chronic neuroinflammatory response. IL-1βXAT B/b and WT control mice received intrahippocampal injections of FIV-Cre and were analyzed over a prolonged time course for neuroinflammatory indices. (A, C, F, and G) qRT-PCR generated a ratio of gene expression in the ipsilateral hippocampi of B/b compared with WT mice at the same time point, except for MHC class II analysis, in which the 4-week time point was used for all comparisons. (A) MHC class II expression was significantly upregulated at all time points assayed. (B) MHC class II staining in the dentate gyrus of a B/b mouse 1 year after FIV-Cre injection. (C) GFAP expression was also significantly upregulated at all time points examined. (D) GFAP expression in the dentate gyrus at 1 year. Scale bar: 20 μm. (E) GFAP and MHC class II upregulation coincided with prolonged expression of ssIL-1β. (F and G) In addition to glial activation markers, hIL-1β expression caused significant increases in qRT-PCR gene transcripts coding for all members of the mIL-1 family (F) and for proinflammatory cytokines IL-6 and TNF-α (G). n = 4–5 animals per group. Data are mean ± SEM. *P < 0.05 versus WT as described.
IL-1β overexpression reduces amyloid pathology. Our primary impetus for creating the IL-1βXAT mouse model was a desire to understand the functional role of sustained IL-1β upregulation in AD. Having confirmed their ability to mount an IL-1β–driven chronic neuroinflammatory response, we next crossed the IL-1βXAT B/b animals with the APP/PS1 mouse model of AD (14). This model coexpresses a chimeric mouse/human amyloid precursor protein (APP) along with human mutant presenilin 1 (PS1) and features accelerated plaque deposition beginning at 4 months of age (15, 16). We hypothesized that sustained IL-1β overexpression would exacerbate the plaque pathology seen in these animals based on studies linking IL-1β expression to disease pathogenesis (17). Pathologic lesions were characterized in 7-month-old animals 4 weeks after FIV-Cre injections (Figure 5). We were surprised to discover substantially reduced pathologic indices in animals exposed to sustained levels of IL-1β expression. Using Aβ antibody and Congo red staining, we observed dramatically reduced plaque pathology in the injected ipsilateral hippocampi of mice expressing the APP/PS1 and IL-1βXAT transgenes (APP/PS1+IL-1β mice; Figure 5, A and B).
IL-1β overexpression ameliorates plaque pathology in a mouse model of AD. IL-1βXAT B/b animals were crossed with APP/PS1 mice, generating APP/PS1+IL-1β animals heterozygous for all 3 transgenes. Intrahippocampal FIV-Cre injections were performed at 6 months of age in APP/PS1 and APP/PS1+IL-1β animals to control both for the injection and for viral transduction. After 4 weeks, (7 months of age) the ratio of pathologic indices between the ipsilateral (FIV-Cre injected) and contralateral (uninjected) hemispheres within individual animals was determined. (A and B) Histochemical analysis using the 6E10 antibody (A) and Congo red (B; shown inverted) revealed a reduction in amyloid deposition in the injected ipsilateral hippocampi of APP/PS1+IL-1β mice compared with that of the uninjected contralateral hemispheres. Scale bars: 100 μm (A); 200 μm (B). (C) hIL-1β induction caused significant reductions in Congo red plaque area fraction and frequency. (D and E) Furthermore, hIL-1β overexpression mediated significant reductions in both insoluble Aβ40 and Aβ42 peptides (D), but did not significantly alter levels of their soluble forms (E), as assessed by ELISA. For additional data from C–E, see Supplemental Table 1. n = 6–7 per group. Graphs represent mean ± SEM. *P < 0.05.
To account for wide variability in pathologic indices in this mouse model at the time point assayed, we determined the ratio of pathologic indices between the FIV-Cre–injected ipsilateral and uninjected contralateral hemispheres within individual animals. Detailed histological analysis of fibrillar plaques stained with Congo red revealed 59% and 46% reductions in plaque area fraction and frequency, respectively, in APP/PS1+IL-1β compared with APP/PS1 mice (Figure 5C). Analogous observations were made using Thioflavine-S staining (data not shown). Amelioration of plaque pathology was further substantiated by determining the concentration of hippocampal-insoluble amyloid β (Aβ) peptide, the major component of plaques. Aβ40 and Aβ42 peptides were reduced by 46% and 36%, respectively, in APP/PS1+IL-1β compared with APP/PS1 mice (Figure 5D). No significant differences were observed for their soluble counterparts (Figure 5E and Supplemental Table 1). This finding, together with the lack of evidence that IL-1β modulates APP expression or BACE-1 expression and activity (Supplemental Figure 2), suggests that the observed reduction in plaque pathology in this model is likely not mediated through regulation of Aβ synthesis.
Enhanced microglial activation may underlie reductions in plaque pathology. Based in part on their physical association with plaques in AD (4), ability to efficiently phagocytose amyloid (18, 19), and heightened activation states in the IL-1βXAT mouse model as demonstrated in the present study, microglia represent an attractive candidate cell type for mediating the observed reductions in plaque pathology in APP/PS1+IL-1β mice. Using confocal microscopy and To-Pro-3 nuclear stain, we demonstrated that direct interaction readily occurred between microglial processes and amyloid plaques in APP/PS1 mice (Figure 6A). IL-1β overexpression caused both a dramatic spatial shift in the relationship between microglial cell nuclei and amyloid plaque and an increased Iba-1 staining intensity within these cells (Figure 6A). Quantitative analysis revealed a 4.4-fold increase in the number of microglial nuclei overlapping plaques in APP/PS1+IL-1β compared with APP/PS1 mice (4.27 and 0.96 nuclei per plaque, respectively; Figure 6B). MHC class II, a classic marker of activated scavenger cells (20), was highly expressed among amoeboid Iba-1–expressing microglia directly contacting amyloid plaques (Figure 6, C and D). Moreover, monocyte chemotactic protein–1 (MCP-1) expression was significantly increased within the hippocampi of APP/PS1+IL-1β mice (Figure 6C).
IL-1β driven microglial activation likely underlies reductions in plaque pathology. Histological analysis was performed in 7-month-old APP/PS1 mice and APP/PS1+IL-1β mice 4 weeks following viral injections. (A) Confocal microscopy revealed an increase in Iba-1–positive (green) microglial cells (nuclei stained blue) directly in contact with Congophilic plaques (red) in APP/PS1+IL-1β mice compared with their APP/PS1 counterparts. (B) Quantitative analysis of mice in A revealed a greater than 4-fold increase in the number of Iba-1–positive cell nuclei overlapping Congophilic plaques. (C) qRT-PCR analysis showed robust upregulation of MHC class II and MCP-1 in APP/PS1+IL-1β mice compared with APP/PS1 mice. (D) 6E10 amyloid staining (blue) in conjunction with Iba-1 (green) and MHC class II (red) staining revealed activation of microglia in contact with amyloid plaque in an APP/PS1+IL-1β mouse. Scale bars: 5 μm. n = 6–7 per group. Data are mean ± SEM. *P < 0.05.