Acute systemic inflammation exacerbates neuroinflammation in Alzheimer's disease: IL‐1β drives amplified responses in primed astrocytes and neuronal network dysfunction

Neuroinflammation contributes to Alzheimer's disease (AD) progression. Secondary inflammatory insults trigger delirium and can accelerate cognitive decline. Individual cellular contributors to this vulnerability require elucidation. Using APP/PS1 mice and AD brain, we studied secondary inflammatory insults to investigate hypersensitive responses in microglia, astrocytes, neurons, and human brain tissue. The NLRP3 inflammasome was assembled surrounding amyloid beta, and microglia were primed, facilitating exaggerated interleukin‐1β (IL‐1β) responses to subsequent LPS stimulation. Astrocytes were primed to produce exaggerated chemokine responses to intrahippocampal IL‐1β. Systemic LPS triggered microglial IL‐1β, astrocytic chemokines, IL‐6, and acute cognitive dysfunction, whereas IL‐1β disrupted hippocampal gamma rhythm, all selectively in APP/PS1 mice. Brains from AD patients with infection showed elevated IL‐1β and IL‐6 levels. Therefore, amyloid leaves the brain vulnerable to secondary inflammation at microglial, astrocytic, neuronal, and cognitive levels, and infection amplifies neuroinflammatory cytokine synthesis in humans. Exacerbation of neuroinflammation to produce deleterious outcomes like delirium and accelerated disease progression merits careful investigation in humans.

inflammation and elevated serum tumor necrosis factor α (TNF-α) are associated with an increased rate of cognitive decline in patients with AD. 10 Therefore, systemic inflammation can trigger delirium and significantly affect the dementia trajectory, but the mechanisms by which it drives deleterious outcomes in patients with underlying dementia remain poorly understood. Addressing the cellular levels at which the amyloid-laden brain shows disproportionate responses to secondary inflammatory insults is the central scientific question in the current study.
The existing neuroinflammatory state of the brain appears to influence subsequent responses to secondary inflammation. 11 Microglia surrounding amyloid plaques in AD become activated, and these cells can produce interleukin 1β (IL-1β), which may contribute to neuronal degeneration. 12 More recently, the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which cleaves immature pro-IL-1 to allow the release of mature IL-1β, has been shown to mediate key aspects of neuronal and cognitive dysfunction in the APP/PS1 model of AD. 13 Notwithstanding these descriptions, IL-1β production is relatively muted in AD and in associated animal models. However, IL-1β can be induced acutely in the brain following peripheral bacterial or viral infections 14 and microglia have been shown to be "primed" by evolving brain pathology, 15 facilitating exaggerated IL-1β production upon exposure to acute systemic inflammation induced by bacterial lipopolysaccharide (LPS). 16 This is a potentially important mechanism for clinically relevant brain sequelae of acute systemic inflammation, since it has been shown that microglia and IL-1β contribute to new Tau pathology, acute cognitive deficits, and new brain injury after acute systemic inflammation in models of neurodegeneration, delirium, and post-operative cognitive dysfunction. [17][18][19][20][21] These brain responses to a single episode of acute systemic inflammation are distinct from studies of repeated dosing with LPS, [22][23][24][25] which, cumulatively, affect amyloidosis and neuroinflammation (see review 26 ). Although those studies sought to influence neuroinflammation from outside the brain, with largely detrimental consequences, chronic dosing regimens address questions that are fundamentally different from those posed by the interrogation of deleterious effects of a single acute systemic inflammatory episode on the vulnerable brain, leading to delirium and acute brain injury. The current study also does not seek to address the hypothesis that amyloidosis is insufficient for disease and that a hypothetical "first hit" or "second hit" infectious insult [27][28][29] is necessary for disease expression. The key motivation in the current study is that, There is, as discussed above, evidence to implicate microglia and IL- proportionate responses to IL-1β in the amyloid-laden brain is largely unstudied.
Astrocytes are known to be activated in proximity to amyloid plaques, showing hypertrophy and upregulation of glial fibrillary acidic protein (GFAP). 30 Astrocytes become "primed" by neurodegeneration in prion disease, such that they produce exaggerated chemokine responses to subsequent acute IL-1β stimulation, 31 and astrocyte chemokine transcription was heightened in the aged brain upon LPS-induced sepsis. 32 AD-like pathology may leave astrocytes hypersensitive to secondary inflammatory challenge, and this represents a significant gap in our knowledge, with potential implications for delirium and exacerbation of dementia upon acute systemic inflammation.
In hippocampal neuronal networks of rodents and non-human primates, gamma oscillations support dynamic cognitive function, decision-making, and coding of novel stimuli. [33][34][35] Prior studies in APP/PS1 slices revealed a mixture of persistent gamma and intermittent burst discharges, indicating a state of network hyperexcitability 36 and suggesting mechanisms compensating for amyloid-related changes in function. IL-1β has been implicated in reduced power of hippocampal gamma oscillations in a multiple sclerosis model 37 and pyramidal neurons are hypersensitive to IL-1β in a chronic hippocampal neurodegeneration model. 19 Therefore, the possibility that acutely elevated IL-1β might disrupt gamma rhythm selectively in the APP/PS1 brain could illustrate network vulnerability and might represent an important link between acute inflammatory challenge and acute alterations in cognitive function.
Although episodes of systemic inflammation induce delirium and exacerbate underlying dementia, we understand neither the cellular and molecular changes that underpin delirium nor how prior amyloidosis influences the wave of neuroinflammatory consequences that follows acute systemic inflammation. This represents a significant unmet clinical need because there are limited evidence-based strategies for neurologists or geriatric psychiatrists to actually manage patients with dementia when they become acutely ill and/or delirious. This is largely because of the significant gaps in our knowledge about what happens to microglia, astrocytes, and neurons during the period of acute inflammatory activation occurring during secondary inflammation.
Here, we hypothesized that microglia in the APP/PS1 brain would produce exaggerated levels of IL-1β upon secondary inflammatory challenge and, in turn, that both astrocytes and neuronal networks would respond in an exaggerated manner to locally applied, or locally produced, IL-1β. These questions were approached through intracerebral and intra-peritoneal challenges of APP/PS1 and wild-type (WT) animals with bacterial LPS or IL-1β, and examination of appropriate cellular readouts in microglia, astrocytes, and neuronal networks, as well as examination of acute cognitive dysfunction under these challenges. AD patients who died with infection versus those who died without infection were also examined to assess whether central cytokine tenets of that neuroinflammatory exacerbation hypothesis were preserved in humans.

Study conclusions and implications
The data herein indicate that both microglia and astrocytes are primed in the APP/PS1 model. Microglia show exaggerated IL-1β production upon LPS challenge and astrocytes show an exaggerated chemokine and IL-6 response to acutely elevated IL-1β, suggesting an amplification loop that drives exaggerated inflammatory responses to acute stimulation in the amyloid-laden brain ( Figure 1). In addition, IL-1β was sufficient to disrupt gamma rhythm selectively in brain slices from APP/PS1 mice and these mice were also more vulnerable to acute cognitive dysfunction triggered by bacterial LPS. Analysis of human AD brains showed that both IL-1β and IL-6 were elevated in AD patients who died with infection with respect to those without infection, and that these two cytokines were correlated, supporting their sequential induction, as shown in the mouse experiments. Thus vulnerability of the diseased brain to exacerbation of neuroinflammation upon acute systemic inflammation occurs in mice and in humans and this is mediated by multiple cellular populations.
The study results emphasize that, in brains with substantial amyloid deposition, microglia, astrocytes, and neuronal networks adopt new properties that leave them vulnerable to producing hypersensitive responses to subsequent secondary inflammation events. Upon sec-ondary inflammation, microglia produce exaggerated IL-1β, and both astrocytes and neuronal networks show hypersensitive responses to acutely elevated IL-1β.
The microglia of APP/PS1 mice ( Figure 2) increased in number, were altered in morphology, showed an elevation of microglial priming and recently described disease-associated microglia (DAM) phenotype transcripts (Clec7a, Itgax, Tyrobp, Trem2) 15,38 and suppression of the homeostatic gene Sall1. 39 Functionally, microglia proximal to amyloid beta (Aβ) produced exaggerated IL-1β responses to acute stimulation with either LPS or IL-1β ( Figure 3). Elegant single cell RNAseq studies show microglial heterogeneity within the AD transgenic brain, 38,40 but the current studies using classical immunohistochemistry are better able to show the precise spatial resolution of acute change in microglial phenotype. These microglia were "primed" before the secondary challenge was applied and already showed evidence of inflammasome assembly, as has been described previously in APP transgenics and in patients with AD. 13 However, in the absence of secondary inflammation, Il1b mRNA was limited and there was no visible nuclear localization of NFκB p65 in plaque-associated microglia. There are prior reports of IL-1β expression in AD tissue and in animal models, 12,37 and we do not dispute those findings. However, our data support the idea that the inflammasome is already assembled in APP/PS1 brain (classically "signal II"), but Il1b transcription is restrained, so there is a weak supply of pro-IL-1β to cleave. However, later "pulses" of pro-IL-1β arising from secondary inflammation (induced by LPS or IL-1β; Figure 3) provide much higher levels of IL-1β for processing. This inflammasome assembly, but with limited pro-IL-1β throughput, may represent a significant dimension of the primed microglial phenotype.
We also demonstrate, for the first time, in any AD transgenic mouse model, that astrocytes become primed to produce exaggerated chemokine synthesis upon secondary inflammatory stimulation.
Although both microglia and astrocytes can synthesize chemokines, astrocytes were the stronger producer of CC chemokine ligand 2 (CCL2), CXCL1, and CXCL10 in response to IL-1β both in vitro (supplemental data) and in vivo ( Figure 5). These chemokines were not detectable in reactive astrocytes proximal to the plaques at base- Cxcl10, S1pr3) phenotypes. This phenotypic switching replicates the recent description of primed astrocytes in the ME7 prion-diseased F I G U R E 1 Acute inflammatory events occurring in humans and mice with evolving amyloid pathology have disproportionate effects on neuroinflammation and cognitive and neurophysiological function compared to those in normal individuals. When APP/PS1 double transgenic mice and age-matched controls are exposed to equivalent acute LPS challenge, microglia (μ) surrounding amyloid plaques (Aβ) in APP/PS1 mice show exaggerated IL-1β responses, whether the LPS challenges were intracerebral (i.c.) or intraperitoneal (i.p.). In turn, astrocytes (*) from the APP/PS1 brain show exaggerated chemokine and IL-6 responses when exposed to acute IL-1β or LPS challenge. Acute peripheral LPS challenge was sufficient to produce acute cognitive impairment in a Y-maze task of cognitive flexibility, and directly applied IL-1β was sufficient to disrupt gamma rhythm in ex vivo cortical-hippocampal networks. Both of these functional impairments occur selectively in acutely challenged APP/PS1 mice. Systemic infection also exacerbated brain inflammation in human Alzheimer's disease (AD) cases: in patients who died with acute systemic infection, brain levels of IL-1β and IL-6 were higher than in those who did not experience infection, and the levels of these two cytokines were directly correlated. Therefore the amyloid-laden brain is "primed" at multiple cellular levels, causing heightened vulnerability to acute inflammatory events. Placing this within the context of the slowly evolving progression of AD, one can propose that these cellular and molecular events, occurring within the "black box" of proximal factors, are contributing to episodes of delirium and to the accelerated cognitive trajectory that has been observed in patients who experience delirium before or during their dementia 7,8 brain, 31 perhaps indicating that such priming of astrocytes may be a generic feature of astrocytes exposed to prior neurodegenerative stimuli. In our studies in prion disease 31 in APP/PS1 mice (this study), in aged mice, 32 and in earlier in vitro studies, 46 increased chemokine output was a key measure of exaggerated astrocyte responses. Elevated chemokine synthesis had consequences for neutrophil, monocyte, and T-cell infiltration in the ME7 model, 31 and such leukocyte infiltration to the AD brain could have significant consequences for disease given reports that T cells, 47 50 and we reasoned that using older animals may therefore maximize their relevance to human disease and would be more appropriate to our aim: not to determine "first events" in development of disease, but to elucidate the development of vulnerability to secondary stressors as disease progresses. Priming of both astrocytes and microglia by their proximity to human amyloid (formed from mutated human forms of APP and PSEN1), even in the absence of Tau, supports the idea that these may occur in the human AD brain, even though Tau and other neurodegenerative pathology may lead to additional glial phenotypes. There are also many differences between immune responses in humans and mice 51 and significant differences between microglial profiles in AD 52,53 and mouse models thereof. 38 However, Habib et al. 54 describe activated astrocytes in the 5xFAD model of AD that overlap with aged astrocyte phenotypes in both mice and humans, and Grubman et al. 55 show enrichment in human AD of several transcripts involved in the regulation of cytokine production and in the response to cytokine stimulation. Those findings support the idea that astrocytes in human AD might indeed show hyper-responsiveness to cytokine stimulation. Moreover, despite their differences, human and mouse immune responses still show more convergence than divergence 56 : Expression patterns of most orthologous inflammatory genes are conserved among humans and mice 57 and many are shared between APP/PS1 mice and humans with AD. 58 In particular, pathways of IL-1 expression, maturation, and action are well conserved and are demonstrated to be up-regulated in both AD patients and mouse models. 13 It is obviously essential to now address the extent to which these glial priming and phenotype switching phenomena, replicated across multiple mouse models, 15   far from clear at this point, there is evidence that IL-6, IL-8, CCL2, [64][65][66] and soluble TREM2 67 are increased in the CSF of hip-fracture patients, suggesting acute changes in cytokine, chemokine, and microglial markers in the brain upon acute systemic inflammatory insults.
Although many of our findings use intracerebral challenges (in order to address specific hypotheses about glial hypersensitivity), LPS gains access to the brain parenchyma to a very limited extent [68][69][70] so it is important that we have also shown that both primed astrocytes and microglia can switch phenotype upon systemic inflammation ( Figure 6). Brain effects of systemic infection and sepsis often clinically manifest as septic encephalopathy or delirium 71,72 but pathophysiological understanding of inflammation-induced delirium remains limited. The demonstration here that moderate dose LPS produces acute cognitive deficits selectively in APP/PS1 animals ( Figure 6E) resonates with recent mouse data revealing acute attentional deficits following surgery (tibial fracture). 73  Pursuing ways in which these acute elevations of cytokines might contribute to neuropsychiatric changes, we show that acute application of IL-1β to hippocampal brain slices also revealed a selective vulnerability, triggering a disruption of gamma network activity in CA3 of slices from APP/PS1 mice that does not occur in WT animals ( Figure 6). Although one cannot conclude that this neurophysiological disruption is that which underpins the acute cognitive deficits, disruption of gamma rhythm does interfere with dynamic cognitive functions like working memory and executive function. 33 injury markers, such as neurofilament light and synaptic proteins, 87,88 are clearly necessary.
Although falling short of defining the signaling processes that cause the delirium-like deficits observed here, the hypersensitivity of the three cell populations examined here and the central involvement of IL-1β at all three levels, as well as increased vulnerability at the brain endothelium, 73 identifies these hypersensitivities as likely contributors to neuroinflammatory exacerbation. This is also consistent with dual roles for IL-1β in acute cognitive dysfunction and in acute brain injury already shown in an alternative model for delirium superimposed on dementia. 19 The cellular and molecular vulnerabilities demonstrated in the current study are summarized in Figure 1 and seated within our overarching hypothesis for how systemic inflammation triggers delirium and contributes to long-term cognitive trajectories in AD. Targeting this interaction between underlying disease and secondary inflammation to limit the impacts of secondary inflammation on exacerbation of disease should, therefore, become an area of significant therapeutic interest 89 and may also synergize with the current focus on disease modification using new anti-inflammatory medications.

Design
The overall aim of the study was to elucidate the multiple cellular levels at which the amyloid-laden brain becomes hypersensitive to secondary acute inflammation. APP/PS1 double transgenic mice (mixed sex, 16 ± 1 months and 19 ± 3 months, in separate experiments) were exposed to secondary inflammation in order to directly measure the in vivo responses of microglia, astrocytes and neurons to acute proinflammatory stimulation. We used immunohistochemistry and quantitative PCR to measure key descriptors of the responses of these cells and then sought to verify the effects of acute systemic inflammation on cognitive function in mice. We also validated, in brain tissue from human AD cases, key brain inflammatory outcomes of acute systemic inflammation.

Human validation
Key outcomes of acute systemic infection in human AD cases were then examined: brain tissue expression of IL-1β and IL-6.

Findings
Microglial activation was verified around plaques in the APP/PS1 brain at 19 ± 3 months of age, with increased numbers (by Pu.1 labeling), condensed morphology (by ionized calcium-binding adapter molecule 1 [Iba1] labeling) and altered transcriptional phenotype (elevated levels of Tyrobp, Trem2, Itgax, and Clec7a) compared to age-matched controls ( Figure 2). This previously described set of core genes 15,38 suggested that these microglia were "primed" to show exaggerated responses to subsequent stimulation, and therefore, intracerebral LPS was administered to examine this hypothesis. LPS induced limited IL-1β in microglia in WT brains but exaggerated levels in the APP/PS1 brain. The IL-1β was clearly expressed in microglia, and not in astrocytes, specifically around amyloid plaques ( Figure 3A-C). Activation of the IL-1maturing enzyme complex, the NLRP3 inflammasome, was demonstrated in plaque-associated microglia in APP/PS1 mice in the absence of secondary inflammation, 13 as described previously, but p65 labeling showed that the NFκB transcription factor was not activated (ie, was not localized to the nucleus) until secondary inflammation was induced.
Consistent with this muted NFκB activation, several NFκB-dependent genes were minimally expressed in APP/PS1 mice per se, but Il1b, Il1a, Cd14, and Nlrp3 all showed exaggerated induction 2 hours after acute LPS or IL-1β challenge in APP/PS1 mice ( Figure 3E,F). Therefore, secondary inflammation switches the microglial phenotype toward prominent IL-1β expression and processing selectively in APP/PS1 mice.
Astrocytes also showed morphological evidence of activation around plaques ( Figure 4A) and qPCR on hippocampal homogenates revealed elevated expression of astrocyte markers Gfap, Serping1, and Ctss ( Figure 4B). We used astrocytes and microglia, freshly isolated from 16-month-old APP/PS1 and WT mice, to validate a panel of specific astrocyte transcripts: Ptx3, Gbp2, Tgm1, and Gfap ( Figure 4C) and others identified in transcriptomic studies of isolated astrocytes. 30,44 Thereafter, these were pursued in transcriptional analysis of astrocyte phenotype after acute challenge with IL-1β (astrocytes respond directly to microglial IL-1β in vivo, but not to LPS itself). Two hours after acute challenge with IL-1β, several genes were induced in both WT and APP/PS1 mice but a substantial panel showed exaggerated induction in APP/PS1 mice, including A1 and A2 astrocyte transcripts and the chemokine Cxcl10 ( Figure 5A,B). Based on our prior demonstration of astrocyte "priming" in a prion model of neurodegeneration, 31 we used immunohistochemistry to show exaggerated production of the chemokines CCL2, CXCL1, and CXCL10 and double-labeling with confocal microscopy to show that CCL2 and CXCL10 were expressed in astrocytes, specifically proximal to amyloid plaques ( Figure 5C).
This showed that astrocytes were "primed" to produce exaggerated chemokine responses with respect to astrocytes from non-diseased animals.
To verify that these glial populations could be triggered to switch phenotype even during systemic inflammation, we challenged animals with LPS (100 μg/kg, i.p.) and showed that microglia made exaggerated levels of IL-1β ( Figure 6A) and isolated astrocytes made exaggerated levels of all of the chemokines above and also the cytokine Il6 Caveats with transgenic models prompted us to validate some key findings in human AD cases. In grey matter tissue from AD patients F I G U R E 5 Astrocytes are primed to show exaggerated chemokine production in APP/PS1. A, Transcriptional changes induced by intracerebral (i.c.) IL-1β challenge (10 ng) in hippocampal mRNA levels of selected astrocyte-associated genes, 2 hours after challenge, expressed as fold change with respect to WT + Sal. Aged 19 ± 3 months. Mean ± SEM (n = 8 to 10). B, Exaggerated response to IL-1β challenge (10 ng, i.c.) in hippocampal mRNA levels of selected astrocyte-associated genes, 2 hours after challenge, expressed as fold change with respect to WT + Sal. Aged 19 ± 3 months. Mean ± SEM (n = 8 to 10). C, Chemokine response to IL-1β challenge ( which is expressed in astrocytes in mice, also showed elevated expression in patients with infection and was strongly correlated with IL-1β expression ( Figure 7).
These data confirm that acute systemic inflammation in mice and in AD patients switches the brain inflammatory phenotype to one producing elevated IL-1β and IL-6, and these hypersensitive inflammatory responses, at least in APP/PS1 mice, are sufficient to drive neuronal and cognitive dysfunction.

Microglia
Microglia are known to increase in number and reactivity around Aβ plaques. 96 Figure 2B). RT-PCR was used to assess a set of microglial-specific genes that are part of the core gene signature for microglial priming reported to change in AD models. 15 We observed a significant decrease in Sall1 (P = .048) but no significant change in Sparc, P2ry12, and Tmem119, all of which have a role in the homeostatic maintenance of the resident microglia phenotype. 38,39 Because the numbers of microglia are significantly increased, the unchanged tissue levels on P2ry12 and Tmem119 actually indicate decreased expression of these markers on a "per cell" basis, as described elsewhere. 38 However, there was a marked increase in Tyrobp (P < .001), which reliably correlates with increased microglial numbers 15,99,100 and significant increases in Trem2, Itgax, and Clec7a (P < .001; Figure 5C). These data suggest that these microglia have lost regulatory control and have become primed.

Microglial priming
Therefore we examined whether superimposed acute challenges (LPS or IL-1β, i.c.) would produce exaggerated IL-1β responses in these Because administering LPS directly to the brain is a relatively unphysiological experiment, which we used to address a specific hypothesis predicting exaggerated microglial response to LPS when the stimulus was applied proximally, we also assessed whether microglia would show exaggerated responses to IL-1β itself, since this cytokine frequently arises in the brain following traumatic or infectious episodes. We directly injected IL-1β, i.c., and showed that only microglia in the Tg brain presented detectable de novo IL-1β protein expression at 2 hours ( Figure 3B). Examining transcriptional changes 2 hours after IL-1β challenge ( Figure 3F), we showed that IL-1β elevated Il1b, Il1a, Tnf, CD14, and Nlrp3 mRNA levels in both genotypes but

Astrocytes
Using immunohistochemical techniques, we show that astrocytes show a more activated phenotype and strong GFAP staining in Tg astro-cytes encircling 6E10-positive Aβ plaques ( Figure 4A). We analyzed a small panel of astrocyte genes in brain homogenates ( Figure 4B) and showed that Tg mice have significantly higher levels of Gfap, Ctss, Ser-ping1, and Il1r1 than WT animals (all P values < .036). To validate further astrocyte transcripts identified by previous investigators as being elevated in models of stroke, sepsis, and AD 30,43,104 while also attending to recently described A1, A2, and pan-reactive signatures, 44 we isolated astrocytes and microglia from WT and Tg animals (14 ± 2 months old) ( Figure 4C). Sorted microglia and astrocytes showed high

Astrocyte priming
Having demonstrated exaggerated microglial production of IL-1β we then asked whether astrocytes would, downstream, show heightened responses to IL-1β. Using the genes validated above, we investigated the hypothesis that astrocytic "priming" also occurs in the APP/PS1  Figure 5B). Most data were non-parametric and using Kruskal-Wallis test followed by Mann-Whitney analyses, we found an effect of genotype and treatment exclusively in Tg + IL-1β group (interaction between treatment and genotype for all transcripts in Figure 5B; P < .035). Several of those, including Ptx3, Tgm1, and Gbp2, were shown to be astrocytic using isolated cells ( Figure 4C). These data support the idea that the astrocyte population is primed to show phenotypic switching in response to acute IL-1 stimulation. To confirm this hypothesis, we focused on chemokine expression (CXCL1, CCL2, and CXCL10) in light of their implication in our prior demonstration of astrocyte priming in chronic neurodegeneration. 31 This was assessed by light microscopy ( Figure 5C, upper panel) and confocal imaging ( Figure 5C, bottom) in animals (19 ± 3 months) challenged with IL-1β (10 ng, i.c.).
These chemokines are induced at the brain endothelium by IL-1β, serving as a positive control for the immunolabeling (insets in Figure 5C). was evident in astrocytes of WT animals, even when challenged with IL-1β. Highly pure primary astrocytes were also shown to be more robust than primary microglia in their synthesis of these chemokines upon acute IL-1β treatment in vitro (2.5 ng/mL for 6 hours; Figure S1).
Thus astrocytes proximal to Aβ plaques in Tg mice are primed to show exaggerated chemokine responses to acute IL-1β stimulation.

Systemic inflammation
Because these acute challenges were intracerebral, it was important to show that key tenets of glial priming were demonstrable even when acute inflammatory challenges were made systemically, using LPS (250 μg/kg, i.p.) in a different cohort of animals (14 ± 2 months).
We focused on the transcription of these three chemokines together with Il6 and Stat3 (a transcript directly downstream of IL-6 signaling), as measured by qPCR on fluorescence-activated cell sorting astrocytes ( Figure 5D). Data were not normally distributed and were analyzed by Exaggerated microglial responses could also be triggered by systemic LPS, using CD68 and C1qa mRNA levels ( Figure 6A) as reliable measures of plaque-associated microglia activation 105  was significantly reduced relative to that in the absence of IL-1β (baseline 4437 ± 1854 μV 2 vs IL-1β 1782 ± 818.5 μV 2 , six slices from five animals, P < .03). This represents an IL-1-induced, 44% reduction, in gamma oscillation power. This reduction in power was not observed in brain slices from WT mice, similarly challenged with IL-1β (baseline 1768 ± 1043 μV 2 vs IL-1β 1920 ± 929.5 μV 2 , P < .25, eight slices from three animals). Acute challenge with IL-1β slightly reduced the mean peak frequency of the oscillations in the gamma band (4.3 ± 3.29 Hz in WT vs 3.2 ± 3.93 in Tg), but this effect does not represent a band shift in the type of oscillatory activity present and was not strain-dependent (two-way analysis of variance [ANOVA] main effect of IL-1β: P < .0071, no effect of genotype P < .34; WT: eight slices from three animals, Tg: six slices from five animals). The data suggest that hippocampal gamma frequency oscillations in APP/PS1 brain are more susceptible to disruption by IL-1β than is the case in age-matched control brain.

Human validation
The data from our mouse experiments (Figures 3 and 6) led to the a priori hypothesis that IL-1β would be elevated in AD patients with infection. Fresh frozen tissue from AD patients with and without infection at the time of death was used for IL-1β and IL-6 analysis using Mesoscale assays. IL-1β in brain homogenates from AD patients with infection (n = 39) was significantly higher than in AD patients without infection (n = 28, P = .0484; Figure 7A). IL-6 was also elevated in patients with infection ( Figure 7B; P = .0236). Consistent with the hypothesis of IL-1β-dependent IL-6 synthesis, the levels of these two cytokines were significantly positively correlated only in AD patients with infection (r 2 = 0.5239, P < .0001), whereas no correlation was found in AD patients without infection (r 2 = 0.020, P = .4656) ( Figure 7C).
In summary, we present evidence that upon secondary inflammatory challenge, microglia in APP/PS1 mice, produce acutely elevated IL-1β. In turn, IL-1β is sufficient to trigger exaggerated levels of chemokines and IL-6 in astrocytes. These acutely elevated IL-1β and IL-6 changes are replicated in AD patients who died with systemic infection. LPS, in mice, acutely disrupted cognitive flexibility, and IL-1β was sufficient to disrupt hippocampal gamma rhythm, selectively in the APP/PS1 mouse brain.

Animals and pro-inflammatory stimuli
APPSwe/PS1dE9 mice (Jax strain #005864, +/0, hereafter referred to as Tg) of 19 ± 3 months were housed at 21 • C with a 12hour light/dark cycle. We used relatively old animals, since amyloid transgenic mice are thought to better represent the mild cognitive impairment stage of disease, 50 and we reasoned that using older animals may therefore maximize their relevance to human disease.
Although some animals in the colony did die at earlier ages, likely due to reported seizure activity, 92 none died as result of the secondary inflammatory challenges made here. Food and water access was ad libitum.

Reference memory and cognitive flexibility
To investigate reference memory and cognitive flexibility, the "paddling" Y-maze visuospatial task was used as described in 84   A power spectrum (FFT size 8192, Hanning window) was generated for the final 60 seconds before the IL-1β was added to the slice as well as for the final 60 seconds of the IL-1β application. These power spectra were analyzed by measuring the area under the curve in the gamma frequency band (20 to 80 Hz). The peak frequency was also recorded in this band. The change induced by IL-1β was normalized relative to the baseline values recorded in the absence of IL-1β. The number and average amplitude of the interictal events in these periods were also quantified.

3.2.9
Human tissue collection Autopsy-acquired brain tissue from donors was sourced from the South West Dementia Brain Bank (University of Bristol) and BRAIN UK

3.2.11
Statistical analyses For multiple comparisons two-way analysis of variance (ANOVA) was performed, with factors being genotype (WT or transgenic) and treatment (LPS, saline, or IL-1β). Data were not always normally distributed, and, in these cases, non-parametric tests were used (Kruskal-Wallis and post hoc pair-wise comparisons with Mann-Whitney U test). Post hoc comparisons were performed with a level of significance set at P ≤ .05. For data that were normally distributed and homoscedastic, we used a standard parametric post hoc test (Bonferroni test), and for those that were normally distributed, but non-homoscedastic, we performed non-parametric post hoc comparisons (Games-Howell test).
For two-group comparisons, data were analyzed using Student t test when they were normally distributed, and the Mann-Whitney U test was run if data did not pass the assumptions for parametric analyses.
Data are presented as mean ± standard error of the mean (SEM

CONFLICTS OF INTEREST
The authors have no competing interests.