Systemic inflammation impairs microglial Aβ clearance through NLRP 3 inflammasome
2019; Springer Nature; Volume: 38; Issue: 17 Linguagem: Inglês
10.15252/embj.2018101064
ISSN1460-2075
AutoresDarío Tejera, Dilek Mercan, J. M. Sanchez-Caro, Mor Hanan, David Greenberg, Hermona Soreq, Eicke Latz, Douglas T. Golenbock, Michael T. Heneka,
Tópico(s)Adenosine and Purinergic Signaling
ResumoArticle30 July 2019Open Access Transparent process Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome Dario Tejera Dario Tejera Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Dilek Mercan Dilek Mercan Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany Search for more papers by this author Juan M Sanchez-Caro Juan M Sanchez-Caro Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany Search for more papers by this author Mor Hanan Mor Hanan Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author David Greenberg David Greenberg Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Hermona Soreq Hermona Soreq orcid.org/0000-0002-0955-526X Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Eicke Latz Eicke Latz German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA Institute of Innate Immunity, University Hospitals Bonn, Bonn, Germany Search for more papers by this author Douglas Golenbock Douglas Golenbock Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Michael T Heneka Corresponding Author Michael T Heneka [email protected] orcid.org/0000-0003-4996-1630 Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Dario Tejera Dario Tejera Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Dilek Mercan Dilek Mercan Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany Search for more papers by this author Juan M Sanchez-Caro Juan M Sanchez-Caro Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany Search for more papers by this author Mor Hanan Mor Hanan Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author David Greenberg David Greenberg Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Hermona Soreq Hermona Soreq orcid.org/0000-0002-0955-526X Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Eicke Latz Eicke Latz German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA Institute of Innate Immunity, University Hospitals Bonn, Bonn, Germany Search for more papers by this author Douglas Golenbock Douglas Golenbock Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Michael T Heneka Corresponding Author Michael T Heneka [email protected] orcid.org/0000-0003-4996-1630 Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Author Information Dario Tejera1,2,‡, Dilek Mercan1,‡, Juan M Sanchez-Caro1, Mor Hanan3, David Greenberg3, Hermona Soreq3, Eicke Latz2,4,5, Douglas Golenbock4 and Michael T Heneka *,1,2,4 1Department of Neurodegenerative Disease and Geriatric Psychiatry, University Hospitals Bonn, Bonn, Germany 2German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 3Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 4Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA 5Institute of Innate Immunity, University Hospitals Bonn, Bonn, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 228 28713091; Fax: +49 228 28713166; E-mail: [email protected] The EMBO Journal (2019)38:e101064https://doi.org/10.15252/embj.2018101064 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Alzheimer's disease is the most prevalent type of dementia and is caused by the deposition of extracellular amyloid-beta and abnormal tau phosphorylation. Neuroinflammation has emerged as an additional pathological component. Microglia, representing the brain's major innate immune cells, play an important role during Alzheimer's. Once activated, microglia show changes in their morphology, characterized by a retraction of cell processes. Systemic inflammation is known to increase the risk for cognitive decline in human neurogenerative diseases including Alzheimer's. Here, we assess for the first time microglial changes upon a peripheral immune challenge in the context of aging and Alzheimer's in vivo, using 2-photon laser scanning microscopy. Microglia were monitored at 2 and 10 days post-challenge by lipopolysaccharide. Microglia exhibited a reduction in the number of branches and the area covered at 2 days, a phenomenon that resolved at 10 days. Systemic inflammation reduced microglial clearance of amyloid-beta in APP/PS1 mice. NLRP3 inflammasome knockout blocked many of the observed microglial changes upon lipopolysaccharide, including alterations in microglial morphology and amyloid pathology. NLRP3 inhibition may thus represent a novel therapeutic target that may protect the brain from toxic peripheral inflammation during systemic infection. Synopsis Microglia morphology and Aβ clearance is negatively regulated by systemic inflammation in a NLRP3-dependent fashion. Collectively, these results shed light on the mechanism underlying Alzheimer disease progress and aggravation upon peripheral inflammation. Systemic inflammation activates microglia in a transient and NLRP3-dependent manner. Systemic inflammation impairs amyloid-beta microglial clearance. APP/PS1 but not APP/PS1xNLRP3ko mice show accelerated amyloid-beta deposition upon systemic immune challenge. Collectively these data suggest that NLRP3 inhibition may protect from peripheral inflammation driven aggravation of cerebral amyloidosis. Introduction Traditionally, the brain has been conceived as an immune-privileged organ. However, it is now widely accepted that several factors including obesity, acute injuries, aging, and neurodegenerative disease can trigger a sustained immune response in the central nervous system (CNS) leading to neuronal dysfunction and demise by microglia activation and release of neuroinflammatory mediators (Lucin & Wyss-Coray, 2009; Villeda et al, 2014; Heneka et al, 2015). Alzheimer's disease (AD) is the most prevalent type of dementia affecting approximately 45 million people worldwide. Pathologically, AD is characterized by the deposition of amyloid-β (Aβ), the formation of neurofibrillary tangles and neuroinflammation (Heneka et al, 2014). The hypothesis that innate immune activation contributes to AD pathogenesis has recently been supported by genome-wide association studies which have identified several immune-related gene variants, including Trem2 (Guerreiro et al, 2013) and Cd33 (Bradshaw et al, 2013), that modify the risk of developing AD. Under physiological conditions, microglia, the immune resident cells of the CNS, exhibit highly ramified and motile cell processes that allow continued surveillance of their environment for tissue damage, cell debris, or pathogens. Once microglia sense signals indicating such challenges, they react in order to maintain cerebral homeostasis (Davalos et al, 2005; Tremblay et al, 2010). During aging and neurodegeneration, microglia acquire an activated phenotype, morphologically characterized by a reduction in branch number accompanied by an increase in cell soma volume. Functionally, microglial activation is defined by the release of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 (Biber et al, 2007; Wyss-Coray & Rogers, 2012; Tejera & Heneka, 2016). The initiation of the inflammatory response by microglia involves the multiprotein complexes termed "inflammasomes". Comprising a cytosolic multiprotein platform, the inflammasome enables the activation of pro-inflammatory caspases, mainly caspase-1. NACHT-, LRR-, and pyrin (PYD)-domain-containing protein 3 (NLRP3) inflammasome is the best-characterized and most widely implicated regulator of IL-1β and IL-18 (Lu et al, 2014; Walsh et al, 2014). In AD, microglia are activated upon deposition of fibrillar Aβ presumably as an attempt to remove Aβ aggregates. It has been shown that this process is highly dependent on the NLRP3 inflammasome (Halle et al, 2008; Heneka et al, 2013). Because Aβ deposition is an early event, preceding the development of mnestic and cognitive deficits by years if not decades (Jack et al, 2013), it is likely that microglial activation influences the pathogenesis of AD during this clinically silent period. It is therefore important to identify exogenous and endogenous factors that influence these microglial responses and thereby the pathogenesis of AD. There is considerable evidence suggesting that systemic inflammation triggers a neuroinflammatory response, characterized by sustained microglial activation with deleterious consequences for learning and memory in rodent models (Semmler et al, 2005, 2007; Weberpals et al, 2009) and in human patients (Qin et al, 2007; Semmler et al, 2008, 2013; Iwashyna et al, 2010; Gyoneva et al, 2014; Widmann & Heneka, 2014). Additionally, it has been proposed that systemic inflammation could influence the pathogenesis of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, and multiple sclerosis (Cunningham et al, 2005; Qin et al, 2007; Cardoso et al, 2015). While microglia-driven neuroinflammation has been identified as a key process during systemic inflammation, aging, and neurodegenerative diseases, its dynamics in vivo and mechanism remain poorly understood. Here, using in vivo two-photon laser scanning microscopy (2PLSM), we describe the effects of systemic inflammation and aging on microglia activation. Moreover, we determine how systemic inflammation alters Aβ pathology by negatively regulating microglial clearance capacity. On a mechanistic level, we identify the NLRP3 inflammasome-signaling pathway as a key mediator of detrimental microglial effects during aging and systemic inflammation. Results Systemic inflammation affects microglia in an age-dependent manner Previous reports demonstrated that the peripheral administration of a single dose of lipopolysaccharide (LPS) ranging from systemic inflammation (0.5–1 mg/kg) to septic shock dosages (5–10 mg/kg) causes an immune response in the CNS, characterized by neuroinflammatory changes (Semmler et al, 2005, 2007; Qin et al, 2007; Gyoneva et al, 2014), identifying that microglia are affected by systemic immune processes. Using in vivo 2PLSM, we sought to determine the microglial dynamics behind these changes. Hence, we performed cranial window surgery on 15-month-old (mo) Cx3cr1-eGFP−/+ mice and 3 weeks later injected them with a single dose of the bacterial cell wall component LPS (1 mg/kg i.p). Following this peripheral challenge, we assessed effects on microglial morphology within the first 48 h post-LPS. 2PLSM revealed that 24 h after LPS injection, microglia cells showed morphological signs of activation, characterized by a significant reduction in the number, length, and maximum order of the branches when compared to control mice (Fig 1A and B). Moreover, we found that changes in microglial morphology peaked at 48 h, with a 50% reduction in all parameters measured (Fig 1A and B). To determine whether these changes were of transient or a more permanent nature and also to assess whether aging, a major priming factor for microglial activation (Cunningham, 2013; Raj et al, 2014; Fonken et al, 2016), influences these results, 5-month- and 15-month-old Cx3cr1-eGFP−/+ mice were analyzed longitudinally at 2 and 10 days post-LPS (Fig 1C). Comparison between 5 and 15mo showed that 15mo animals already presented signs of microglia activation (Fig 1D and E) prior to any LPS administration, which was defined by a significant reduction in the number of processes as well as a reduction in the length of processes and maximum branch order (Fig 1D and E). In addition, we found that as a consequence of the above-described changes in the branches, the brain volume covered (total processes length/cell volume) was significantly reduced at 15mo compared to 5mo already in the absence of any LPS challenge, suggesting that age compromised microglial surveying functions already. As shown for 15mo mice, 5mo animals also had a significant reduction in the morphological parameters analyzed 2 days upon LPS challenge. Interestingly, we found that there was a significant increase for all the morphological parameters 10 days after immune challenge compared to 2 days after LPS injection for 5mo mice (Fig 1D and E). In the case of the 15mo mice, there were no morphological changes between 2 and 10 days post-LPS in the morphological parameters, but also the parameters were indistinguishable from baseline conditions (Fig 1D and E). In order to corroborate the changes observed on the morphological level, microglia activation marker CD68 (Hickman et al, 2013) was evaluated by immunohistochemistry. A transient increase in CD68 immunoreactivity was observed 2 days after immune challenge in 15mo mice (Fig EV1). Importantly, 10 days after LPS injection, immunoreactivity levels were the same as PBS-treated mice (Fig EV1). Figure 1. Systemic inflammation transiently affects microglia in an age-dependent manner Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 h. Scale bar: 10 μm. Morphological parameters quantification within 48 h after LPS injection (mean of 5–6 ± SEM; one-way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). Two-photon microscopy experimental design. Representative 3D microglia reconstructions from 5- and 15-month-old mice showing microglia changes 2 and 10 days post-LPS injection. Scale bar: 20 μm. Morphological parameters quantification for 5- and 15-month-old mice after LPS injection (mean of 5–6 ± SEM; two-way ANOVA followed by Tukey's post hoc test, #*P < 0.05, **P < 0.01, ***P < 0.001). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Transient increase in CD68 immunoreactivity upon LPS injection CD68 staining in cortex of 5 and 15 months old of wild-type and Nlrp3−/− mice. Scale bar: 20 μm. CD68 integrated density in wild-type and Nlrp3−/− mice (mean of 5 ± SEM; two-way ANOVA followed by Tukey's post hoc test, ***P < 0.001). Download figure Download PowerPoint NLRP3 ko mice are refractory to peripheral immune challenge and age-associated changes Inflammasomes form in response to microbial or danger signals, which leads to the cleavage of pro-caspase-1 into the active caspase-1 enzyme. Active caspase-1 then cleaves the pro-forms of the inflammatory cytokines, IL-1β and IL-18, into their active forms (Vanaja et al, 2015; Man et al, 2016). To assess if the NLRP3 inflammasome is involved in the observed changes during aging and LPS challenge, Nlrp3−/− mice were crossed with Cx3cr1-eGFP−/+ mice to assess microglial dynamics using 2PLSM (Fig 2A). No morphological differences between 5 and 15mo animals were observed at baseline for the assessed parameters, suggesting that Nlrp3 deficiency protects against age-induced microglial alterations (Fig 2B and C). Moreover, microglia from Nlrp3−/− mice were refractory to LPS injection (Fig 2B and C), since no morphological changes were observed in any of the tested time points after LPS challenge. In line with these findings, no changes were observed in the levels of CD68 immunoreactivity after peripheral immune challenge (Fig EV1). Figure 2. Nlpr3 knockout mice are refractory to peripheral immune challenge or age-associated changes Schematic representation of two-photon microscopy experimental design. Two-photon representative images of wild-type and Nlrp3−/− mice (5 and 15 months old). Scale bar: 20 μm. Quantification of morphological parameters for wild-type and Nlpr3−/− mice after LPS injection (mean of 5-6 ± SEM; two-way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01). Download figure Download PowerPoint The activation of the inflammasome and subsequent release of IL-1β requires the adaptor protein ASC, which in turn leads to the recruitment of the effector caspase-1 (Baroja-Mazo et al, 2014). Interaction of ASC with caspase-1 could lead to the formation of an ASC speck (Venegas et al, 2017). We found that 2 days post-LPS challenge leads to an increase in the formation of ASC specks in 15mo mice concordant with inflammasome activation (Appendix Fig S1A). Notably, at 10 days after peripheral immune challenge, a reduction in ASC speck was observed (Appendix Fig S1A and C). It is important to mention that ASC speck formation was dependent on the activation of NLRP3 inflammasome, since no specks were detectable in Nlrp3−/− mice (Appendix Fig S1A and C). Accordingly, no changes in IL-1β levels were observed in Nlrp3−/− mice after LPS injection (Fig EV2A), whereas wild-type mice exhibited an increase 2 days after peripheral challenge, and then, consistent with morphological results, we observed a return to basal levels by 10 days post-LPS challenge. When TNF-α levels were measured, it was found to increase in all the experimental groups, 2 days after peripheral challenge (Fig EV2A). Importantly, this increase was transient; since 10 days after LPS injection, TNF-α levels were found to return to basal levels (Fig EV2A). Remarkably, the liver, as a measurement of the peripheral response, mirrored the immune response observed in the brain but with higher levels of both cytokines (Fig EV2B). Click here to expand this figure. Figure EV2. LPS injection triggers a transient increase in pro-inflammatory cytokines in both brain and periphery IL-1β and TNF-α ELISA measurement in brain lysates of wild-type and Nlrp3−/−. A significant NLRP3-dependent increase of IL-1β levels was observed 2 days after LPS injection and then a return to control levels. For TNF-α, all groups showed a transient increase in its levels (mean of 6 ± SEM; two-way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). IL-1β and TNF-α ELISA measurement in liver lysates of wild-type and Nlrp3−/− mice. A transient increase in NLRP3-dependent IL-1β levels is observed after immune challenge. LPS injection triggered a transient increase in TNF-α in all groups evaluated (mean of 6 ± SEM; two-way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). Download figure Download PowerPoint Astrocytes are transiently activated by LPS peripheral injection Astrocytes are CNS cells that participate in a myriad of processes, mainly providing trophic support to neurons and promoting synapse formation and elimination (Jäkel & Dimou, 2017). It has recently been shown that a subset of astrocytes become reactive by neuroinflammatory microglia (Liddelow et al, 2017). Considering the microglial response to peripheral immune challenge, it is plausible to imagine that astrocytes could undergo a process of reactive astrocytosis. Indeed, we found increased GFAP immunoreactivity 2 days after LPS challenge (Fig EV3A and B) in both, 5- and 15-month-old wild-type mice. However, 10 days after peripheral injection, immunoreactivity levels of GFAP were similar to PBS-treated mice (Fig EV3A and B). In line with these results described, no changes in GFAP immunoreactivity were observed in Nlrp3−/− mice after LPS injection (Fig EV3A and B), corroborating that neuroinflammatory microglia are required to promote reactive astrocytosis. Click here to expand this figure. Figure EV3. Astrocytes are transiently activated after peripheral immune challenge Representative cortical pictures of 5 and 15 months old wild-type and Nlrp3−/− stained with GFAP. A transient increase in GFAP immunoreactivity is observed 2 days after LPS injection in wild-type but not in Nlrp3−/− mice. Scale bar: 20 μm. GFAP integrated density in wild-type and Nlrp3−/− mice (mean of 5 ± SEM; two-way ANOVA followed by Tukey's post hoc test, ***P < 0.001). Download figure Download PowerPoint Peripheral immune challenge affects amyloid deposition in APP/PS1 mice Since systemic inflammation represents a risk for developing neurodegeneration particularly for AD (for review see: Heneka et al, 2015), we analyzed the effects of a peripheral immune challenge on pathological hallmarks of AD using APP/PS1 mice. Additionally, we tested whether these effects were mediated by the NLRP3 inflammasome. Therefore, APP/PS1 and APP/PS1/Nlrp3−/− mice underwent the same experimental protocols as described above for non-APP/PS1 mice (Fig 1C). While number and size of Aβ deposits were increased in APP/PS1 compared to APP/PS1/Nlrp3−/− mice (Fig 3A and B, and Appendix Fig S2A and B) at 15mo, APP/PS1 but not APP/PS1/Nlrp3/− revealed a significant increase in Aβ deposition upon LPS challenge at both time points investigated (Fig 3A and B). At 5mo, the time Aβ deposits begin to appear in this model, and no apparent differences were detectable (Maia et al, 2013). These results were confirmed by ELISA measurements of Aβ1–40 and Aβ1–42 (Fig 3B). Of note, this increase was not caused by any modification in the APP processing machinery (Appendix Fig S2C and D). Of note, we observed an increase in the number of ASC specks at both 5 and 15mo of age 2 days post-LPS challenge (Appendix Fig S1B and D). It is also important to mention that as previously described (Venegas et al, 2017), ASC specks were also observed in PBS-treated mice (Appendix Fig S1B and D). Moreover, these observations are appeared to be NLRP3-dependent, since no changes in ASC speck formation were observed in APP/PS1/Nlrp3/− (Appendix Fig S1B and D). Together, these results suggest that a LPS elicited peripheral immune challenge affects amyloid deposition in aged APP/PS1 mice in an NLRP3-dependent manner. Figure 3. Peripheral immune challenge affects amyloid deposition in APP/PS1 mice Representative cortical images of MXO4 staining for APP and APP/Nlrp3−/− 15-month-old mice. Scale bar: 50 μm. Cortical amyloid plaque number and size quantification, and Amyloid-beta1–40 and 1–42 ELISA quantification for APP and APP/Nlrp3−/− mice (5- and 15-month-old) (mean of 8 ± SEM; two-way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). Download figure Download PowerPoint Microglia dynamics depend on distance to Aβ deposition in APP/PS1 mice Microglia cluster around amyloid plaque deposits with their processes being retracted and less dynamic as compared to plaque-free or distant microglia (Condello et al, 2015). This suggests that on a functional level, at least two different populations of microglial cells have to be distinguished by location in murine AD models, those cells located near Aβ, and cells more distantly located. Analysis by 2PLSM revealed that LPS did not lead to further morphological changes in microglia located in the vicinity of Aβ deposits (Fig 4A and B), which is defined as the microglia cells in a 60 μm radius form the amyloid deposit core. This observation is most likely due to the already existing high-level of activation by Aβ itself (Appendix Fig S3). However, the peripheral LPS challenge impaired microglial uptake of Aβ in APP/PS1 but not APP/PS1/Nlrp3−/− mice (Fig 4C and D, and Appendix Fig S4). In this sense, the presence or absence of the NLRP3 inflammasome proofed to be a determinant factor, since we have found a significant interaction between time after LPS injection and strain of the mice (F = 6.44. DFn = 2 DFd = 13 and a P value of 0.014). Altogether, these results suggest that systemic inflammation affects the functional status particularly the Aβ clearance capacity of these cells. Figure 4. Microglia dynamics depend on distance to Aβ deposition in APP/PS1 mice Two-photon images of microglia (eGFP) cells clustering around amyloid plaque for APP and APP/Nlrp3−/− (5 and 15 months old). Scale bar: 20 μm. Quantification of morphological parameters in (A) (mean of 5–6 ± SEM). Flow cytometry plots from APP and APP/Nlrp3−/− mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation. Relative Aβ microglia uptake quantification (mean of 5 ± SEM; two-way ANOVA followed by Tukey's post hoc test, *P < 0.05). Download figure Download PowerPoint Since a functional role of beclin-1 for microglial Aβ clearance had been previously demonstrated, we analyzed whether systemic inflammation would affect beclin-1 expression (Lucin et al, 2013). In the present study, we found decreased expression of beclin-1 in APP/PS1 but not APP/PS1/Nlrp3−/− mice challenged with LPS (Fig EV4). Click here to expand this figure. Figure EV4. Systemic inflammation alters beclin-1 expression in an NLRP3-dependent manner Western blot analysis of whole brain lysate from 15-month-old APP and APP/Nlrp3−/− using beclin-1 antibody. Quantification of beclin-1 expression levels (mean of 2–5 ± SEM; two-way ANOVA followed by Tukey's post hoc test, *P < 0.05). Download figure Download PowerPoint In contrast to plaque-associated microglia, the morphological dynamics of plaque-distant microglia were more reminiscent to non-APP/PS1 microglia, with respect to their time-dependent morphological changes after peripheral immune challenge, age, and presence of the NLRP3 inflammasome. In 5mo APP/PS1 mice, the number of branches and the maximum branch order were reduced 2 days post-LPS, followed by a recovery in the number of branches by 10 days (Fig 5A and B). Of note, plaque-distant microglial dynamics in aged APP/PS1 (15mo) mice showed no changes upon peripheral immune challenge. These cells, despite not being in direct contact to Aβ deposits, already exhibited morphological signs of microglial activation (Fig 5A and B), likely to be caused by the presence of soluble Aβ species or inflammatory factors, which may render these cells less responsive to any further immune stimulation. In case of APP/PS1/Nlrp3−/− and in line with the results shown for Nlrp3−/−, we observed that these mice were largely refractory to peripheral immune challenge and aging, since no morphological changes were observed upon LPS challenge. Figure 5. Microglia dynamics depend on distance to Aβ deposition in APP/PS1 mice Two-photon microglia (eGFP) images from APP and APP/Nlrp3−/− in areas free of plaque (5 and 15 months old mice). Scale bar: 20 μm. Quantification of APP and APP/Nlrp3−/− morphological parameters for microglia in areas free of plaque (mean of 5 ± SEM; two-way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01). Download figure Download PowerPoint Peripheral myeloid cells infiltrate brains of APP/PS1 mice upon peripheral LPS challenge Myeloid cell infiltration into the brain occurs in several models of inflammation (Wohleb et al, 2014; Jay et al, 2015; Lévesque et al, 2016; Wattananit et al, 2016). To investigate this phenomenon, brain sections were immunostained for Iba-1 and the peripheral myeloid marker CD169 (Rice et al, 2015; Perez et al, 2017; Shinde et al, 2018). CD169 has been suggested to be a specific marker for bone marrow-derived monocytes and therefore may help to differentiate microglia from infiltrating myeloid cells (Butovsky et al, 2012). Analysis of young and aged WT and Nlrp3−/− mice did not reveal myeloid cell infiltration into the brain upon LPS (Appendix Fig S5A and B). Similarly, LPS did not induce myeloid cell infiltration in young or aged APP/PS1 or APP/PS1/Nlrp3−/− mice in brain areas free of Aβ deposits (Appendix Fig S5C and D). Likewise, young APP/PS1 and APP/PS1/Nlrp3−/− showed no CD169 immunopositive cells in response to LPS. In strong contrast, CD169-positive cells became detectable in aged, 15mo APP/PS1 mice at 2 days post-LPS and were mostly located in close vicinity to Aβ depo
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