Aged lipid‐laden microglia display impaired responses to stroke

Article21 December 2022Open Access Transparent process Aged lipid-laden microglia display impaired responses to stroke Maria Arbaizar-Rovirosa Maria Arbaizar-Rovirosa orcid.org/0000-0002-1647-8258 Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: ​Investigation, Methodology Search for more papers by this author Jordi Pedragosa Jordi Pedragosa orcid.org/0000-0001-8493-4545 Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Juan J Lozano Juan J Lozano orcid.org/0000-0001-7613-3908 Bioinformatics Platform, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain Contribution: Formal analysis Search for more papers by this author Carme Casal Carme Casal orcid.org/0000-0002-4898-7671 Microscopy Service, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Contribution: Methodology Search for more papers by this author Albert Pol Albert Pol orcid.org/0000-0002-1750-1085 Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Department of Biomedical Sciences, Faculty of Medicine, Universitat de Barcelona, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Mattia Gallizioli Corresponding Author Mattia Gallizioli [email protected] orcid.org/0000-0001-9245-914X Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: Conceptualization, Formal analysis, Supervision, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Anna M Planas Corresponding Author Anna M Planas [email protected] orcid.org/0000-0002-6147-1880 Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Maria Arbaizar-Rovirosa Maria Arbaizar-Rovirosa orcid.org/0000-0002-1647-8258 Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: ​Investigation, Methodology Search for more papers by this author Jordi Pedragosa Jordi Pedragosa orcid.org/0000-0001-8493-4545 Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Juan J Lozano Juan J Lozano orcid.org/0000-0001-7613-3908 Bioinformatics Platform, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain Contribution: Formal analysis Search for more papers by this author Carme Casal Carme Casal orcid.org/0000-0002-4898-7671 Microscopy Service, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Contribution: Methodology Search for more papers by this author Albert Pol Albert Pol orcid.org/0000-0002-1750-1085 Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Department of Biomedical Sciences, Faculty of Medicine, Universitat de Barcelona, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Mattia Gallizioli Corresponding Author Mattia Gallizioli [email protected] orcid.org/0000-0001-9245-914X Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: Conceptualization, Formal analysis, Supervision, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Anna M Planas Corresponding Author Anna M Planas [email protected] orcid.org/0000-0002-6147-1880 Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Maria Arbaizar-Rovirosa1,2, Jordi Pedragosa1,2, Juan J Lozano3, Carme Casal4, Albert Pol2,5,6, Mattia Gallizioli *,1,2 and Anna M Planas *,1,2 1Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain 2Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 3Bioinformatics Platform, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain 4Microscopy Service, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain 5Department of Biomedical Sciences, Faculty of Medicine, Universitat de Barcelona, Barcelona, Spain 6Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain *Corresponding author. Tel: +34 932275400 ext 4816; E-mail: [email protected] *Corresponding author. Tel: +34 933638327; E-mail: [email protected] EMBO Mol Med (2023)15:e17175https://doi.org/10.15252/emmm.202217175 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. 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 Microglial cells of the aged brain manifest signs of dysfunction that could contribute to the worse neurological outcome of stroke in the elderly. Treatment with colony-stimulating factor 1 receptor antagonists enables transient microglia depletion that is followed by microglia repopulation after treatment interruption, causing no known harm to mice. We tested whether this strategy restored microglia function and ameliorated stroke outcome in old mice. Cerebral ischemia/reperfusion induced innate immune responses in microglia highlighted by type I interferon and metabolic changes involving lipid droplet biogenesis. Old microglia accumulated lipids under steady state and displayed exacerbated innate immune responses to stroke. Microglia repopulation in old mice reduced lipid-laden microglia, and the cells exhibited reduced inflammatory responses to ischemia. Moreover, old mice with renewed microglia showed improved motor function 2 weeks after stroke. We conclude that lipid deposits in aged microglia impair the cellular responses to ischemia and worsen functional recovery in old mice. Synopsis Stroke outcome is impaired in old subjects. Here, microglia of aged mice are shown to accumulate lipids and display exacerbated inflammation after stroke. The microglia lipid droplet content was reduced by microglia depletion/repopulation in old mice, and motor function was improved after stroke. Ischemic stroke induced acute lipid droplet biogenesis in microglia. Aging caused lipid droplet accumulation in microglia and changes in lipid pathways under a steady state. Old mice showed worse outcomes after stroke, and their microglia displayed exaggerated innate immune reactions. Renewal of microglia in old mice by transient treatment with a CSF1R inhibitor reduced lipid droplets. Renewed microglia of old mice showed less inflammation after stroke, and motor function was improved. The paper explained Problem Over 12 million people suffer a new stroke every year in the world. Stroke is the second-leading cause of mortality and a prominent cause of permanent disability worldwide. Age is a nonmodifiable stroke risk factor, and stroke outcome is worse in aged individuals. Novel strategies for the restoration of cerebral blood flow by removing or dissolving the arterial blood clots have improved dramatically the treatment of ischemic stroke. However, these interventions are only useful in the hours that follow stroke onset and within the first day at the most. Beyond this, no drugs have shown efficacy to improve stroke disability so far. Innovative therapeutic strategies are needed to reduce the disability burden of stroke sufferers. Results Our study focused on microglia, which is a population of immune cells resident in the brain. After stroke, these cells carry out important functions like removing the damaged tissue. To this end, these cells suffer metabolic and immune changes, and we noticed that they accumulate little organelles of fat, called lipid droplets. Moreover, we found that microglia of old but healthy mice do also have lipid droplets. After stroke, old mice show larger brain lesions and worse neurological outcomes, and their microglia show a more inflammatory profile. We argued that microglia of old mice may be dysfunctional and hypothesized that renewing this cell population may be beneficial. We used a pharmacological strategy to deplete and then repopulate the brain microglia of old mice. Notably, the renewed microglia showed less lipid droplets, and after stroke, the old mice showed a better recovery of motor function. Impact Our experimental findings show the involvement of lipid droplets in the response of microglia to stroke and suggest that lipid disposal may be impaired in microglia of aged individuals. Altered lipid metabolism in the elderly may deteriorate the cell functions and may contribute to worsen stroke outcome. Strategies preventing cellular lipid accumulations in aging may preserve microglia function and increase the resilience to stroke. Introduction Age is the main nonmodifiable stroke risk factor, and the stroke outcome is worse in the elderly. The higher vulnerability of the aged brain to ischemia has been attributed to differences in the mechanisms of ischemic brain injury between aged and young individuals (Chen et al, 2010). Moreover, elderly patients with stroke show worse responses to reperfusion therapies (Mishra et al, 2010). Several lines of evidence support that exacerbated brain inflammation and altered immunological response underlie the worse neurological impairment in aged subjects after cerebral ischemia/reperfusion (Ritzel et al, 2018). The brain of aged mice shows a differential transcriptional profile to that of the young (Androvic et al, 2020). The expression of genes encoding pro-inflammatory and innate immune response molecules is exaggerated in the elderly, whereas genes involved in axonal and synaptic structure and activity are downregulated (Hickman et al, 2013; Hammond et al, 2019; Androvic et al, 2020; Tabula Muris Consortium, 2020). Aging impairs microglia functions (Sierra et al, 2007) particularly affecting white matter-associated microglia (Cantuti-Castelvetri et al, 2018; Safaiyan et al, 2021). Recent studies identified subsets of dysfunctional microglia in the aging brain characterized by increased oxidative stress, inflammatory profile, abnormal lipid accumulation (Marschallinger et al, 2020), and increased lysosomal storage (Burns et al, 2020). Microglial cells play critical roles in the central nervous system during brain development (Wu et al, 2015) and in adulthood to maintain brain homeostasis via microglia–neuron interactions (Cserép et al, 2020, 2021). Microglia prevent excessive neuronal depolarization following excitotoxic insults (Kato et al, 2016) and exert some protective effects in cerebral ischemia (Szalay et al, 2016; Otxoa-de-Amezaga et al, 2019; Marino Lee et al, 2021). Microglia survival is dependent on colony-stimulating factor 1 receptor (CSF1R). In rodents, genetic strategies to prevent microglial Csf1r expression or treatment with pharmacological inhibitors of CSF1R cause microglia depletion (Waisman et al, 2015). Upon removal of the CSF1R inhibitor, microglia repopulate the brain and several lines of evidence support that repopulated microglia exert beneficial effects (Elmore et al, 2018; Han et al, 2019). Proliferating and nonproliferating microglia with a distinct transcriptomic profile are detected in the adult mouse brain during repopulation following microglia depletion (Belhocine et al, 2021). Notably, microglia repopulation in the aged brain reversed some age-induced changes in microglia gene expression, and reduced lysosome enlargement and lipofuscin accumulation (O'Neil et al, 2018). In an experimental model of traumatic brain injury, repopulating microglia stimulated neurogenesis and promoted recovery mediated by IL-6-dependent neuroprotection (Willis et al, 2020). We hypothesized that restoring the microglia phenotype in the elderly may improve the functional outcome of stroke. We investigated the microglia response to stroke in young (3–4 months) and aged (21–22 months) mice and studied whether microglia renewal improved stroke outcome in the elderly. Our work shows that drug-induced microglia repopulation in the elderly modifies some phenotypic traits of old microglia and increases the resilience of old individuals to the motor impairment caused by stroke. Results Cerebral ischemia/reperfusion changes the transcriptomic profile and function of microglia Microglia undergo strong phenotypic alterations due to brain injury following stroke. In line with the need to remove dead cells and cell debris, microglia morphology became more reactive and displayed engulfing phagosomes (Fig 1A). In the periphery of infarction, electron microscopy showed microglia surrounding swollen synaptic vesicles (Fig 1B). We studied the transcriptomic profile of microglia obtained via fluorescence-activated cell sorting (FACS) from the brain of mice after an episode of transient ischemia induced by 45-min intraluminal middle cerebral artery occlusion (MCAo) followed by reperfusion (Fig 1C), as we previously reported (Gallizioli et al, 2020). RNAseq analysis showed marked changes in the microglia transcriptomic profile 4 days postischemia versus controls (Fig 1D). According to gene ontology (GO) pathways and gene set enrichment analysis (GSEA), ischemia upregulated innate immune pathways in microglia with prominent enrichment of genes regulating lipid storage and defense response to virus, particularly interferon (IFN)-α and IFN-β (Fig 1E and F). Other biological processes highly enriched in ischemic microglia included terms related to phagocytosis, lysosomes, and cholesterol storage (Fig 1G). Notably, some of the abovementioned ischemia-induced differentially expressed genes (DEGs) in microglia are typically reported in disease-associated microglia (DAM) under diverse neurodegenerative conditions (Keren-Shaul et al, 2017; Deczkowska et al, 2018). Expression of the DAM genes was similarly upregulated or downregulated in ischemic microglia. Therefore, microglia acquire features of a DAM-like profile within hours/days of an acute ischemic insult (Fig 1H). Figure 1. Transcriptomic and phenotypic changes in microglia after brain ischemia Representative P2YR12 immunostaining (green) of microglia of wild-type male mice showing morphological differences between control microglia (a) and ischemic microglia (b–d). Ischemic microglial cells show typical phagocytic pouches (arrows). Nuclei are labeled with TO-PRO-3 (blue). Image (d) is a magnification of the area marked with a square in (c). Scale bar (a, b): 20 μm; (c): 10 μm; (d): 4 μm. Transmission electron microscopy showing a microglial cell at the periphery of infarction 1 day postischemia surrounding remarkably swollen postsynaptic vesicles. Scale bar: 2 μm. Microglia were obtained using fluorescence-activated cell sorting (FACS) from the brain of control and ischemic young (3–4 months) male CX3CR1creERT2:Rosa26-tdT mice 4 days postischemia (n = 4 mice per group). Microglia RNA was extracted for RNAseq analysis (GSE136856), as reported (Gallizioli et al, 2020). Principal components analysis (PCA) shows sample distribution clearly separating microglia from control and ischemic mice. Gene Ontology analysis illustrating pathways enriched in microglia after brain ischemia. GSEA highlights the IFN-α response pathway as highly upregulated after ischemia. Genes of the Kegg pathway: Phagosomes are upregulated in microglia after ischemia. Color scale as in (F). Most genes described as upregulated or downregulated in disease-associated microglia (DAM) are accordingly regulated in microglia 4 days postischemia. Download figure Download PowerPoint Ischemia induces the formation of lipid droplets in microglia Conditions involving infection or inflammation in certain cell types cause the accumulation of neutral lipids in the cytoplasm forming lipid droplets. Interestingly, lipid droplets are surrounded by a monolayer of phospholipids decorated with innate immune molecules, particularly proteins regulated by type I IFN that play a critical role in immune defense (Bosch et al, 2020; Monson et al, 2021). Given the conspicuous type I IFN response induced by cerebral ischemia in microglial cells, as well as the accompanying inflammatory response and enrichment in genes regulating lipid storage, we hypothesized that ischemia could induce lipid droplet biogenesis in microglia. We checked for ischemia-induced DEGs encoding typical lipid droplet membrane protein components, such as PLIN1-5 family, and the reported IFN-induced lipid droplet-associated proteins (Bosch et al, 2020). Brain ischemia in young mice increased at day 4 the microglial mRNA expression of Adipophilin (Plin2), Perilipin (Plin3), Hypoxia-Inducible Lipid Droplet-Associated (Hilpda), granulin precursor (Grn), Abhydrolase Domain Containing 5, Lysophosphatidic Acid Acyltransferase (Abhd5), Synaptosome Associated Protein 23 (Snap23), and lipoprotein lipase (Lpl; Fig 2A). Moreover, we detected ischemia-induced microglial mRNA upregulation of IFN-induced lipid droplet-associated molecules such as ISG15 Ubiquitin-Like Modifier (Isg15), Ubiquitin-Conjugating Enzyme E2 L6 (Ube2L6), Ubiquitin-Specific Peptidase 18 (Usp18), Viperin (Rsad2), Ring Finger Protein 213 (Rnf213), Immunity-Related GTPase M (Irgm1), and Interferon-Inducible GTPase 1 (Iigp1; Fig 2A). We investigated whether this response was also upregulated after human stroke using postmortem brain tissue. Characteristics of the stroke patients are shown in Table EV1. Stroke increased the mRNA expression of PLIN2 and ISG15, one of the typical type I IFN responsive genes, suggesting that the molecular machinery related to lipid droplet biogenesis was activated after stroke in the human brain too (Fig 2B). We then validated the ischemia-induced expression of PLIN2 at the protein level by Western blotting (Fig 2C) and immunofluorescence (Fig 2D) in mouse tissue. Figure 2. Microglia accumulate lipid droplets after brain ischemia Expression of a selection of genes encoding lipid droplet-associated proteins in microglia sorted from control (n = 4) and ischemic (n = 4) male mice at day four postischemia (obtained from RNAseq data shown in Fig 1). Heatmap illustrates ischemia-induced upregulation of genes marked with * indicating adjusted P-value < 0.001. mRNA expression of PLIN2 and ISG15 in postmortem human brain tissue of nine stroke patients (points are values of individual patients); eight women (red points) and one man (black point). Samples were obtained from the ischemic tissue (Stroke) and nonaffected (NA) tissue. The graph shows boxes from the 25th to 75th percentiles, the median line, and whiskers down to the minimum and up to the maximum value, showing all points. Values are expressed as fold versus mean control (i.e., nonaffected tissue) Wilcoxon matched-pairs signed-rank test, **P = 0.0039; *P = 0.0195. PLIN2 protein expression in mouse brain tissue, as assessed by Western blotting. Values were obtained from the ipsilateral hemisphere 1 h (n = 3), 4 h (n = 3), 15 h (n = 4), 24 h (n = 5) and 96 h (n = 5) postischemia, and 15 h (n = 4), 24 h (n = 2), and 96 h (n = 2) after sham operation. Values of sham mice were pooled together since they did not differ between time points. Samples of the contralateral hemisphere (Contra) of ischemic mice (1 h, n = 2; 24 h, n = 3; and 96 h, n = 3) were also evaluated. Ischemia increased PLIN2 expression at day 4 versus the sham group (**P = 0.0032, Kruskal–Wallis test and Dunn's multiple comparisons test). β-tubulin is the protein loading control. The “Std” lane indicates the molecular weight standard. Quantification of band intensity where values are expressed as fold versus control (nonischemic). Points correspond to independent male mice and group values are expressed as a violin plot. Values were obtained 1 h (n = 3), 4 h (n = 3), 15 h (n = 4), 24 h (n = 5), and 96 h (n = 5) postischemia, and 15 h (n = 4), 24 h (n = 2), and 96 h (n = 2) after sham operation. Values of sham mice were pooled together since they did not differ between time points. Samples of the contralateral hemisphere (Contra) of ischemic mice (1 h, n = 2; 24 h, n = 3; and 96 h, n = 3) were also evaluated. Ischemia increased PLIN2 expression at day 4 (**P = 0.0032, Kruskal–Wallis test and Dunn's multiple comparisons test versus the sham group). Data are presented as violin plots with lines at the median and quartiles (dashed lines). Immunofluorescence with antibodies against PLIN2 (green) and Iba-1 (red) in brain tissue 1 day after induction of ischemia in female mice. Nuclei are stained with DAPI (blue). Control is the contralateral hemisphere. Scale bar: 10 μm. Transmission electron microscopy showing microglial cells in control or ischemic tissue 1 day postischemia. Ischemia induces the formation of lipid droplets (LD) in microglia. LD are often seen near lysosomes (Lys). Insets in the lower panels are magnifications of the regions marked with a square. Scale bar 2 μm. Flow cytometry gates to identify Bodipy+ microglia for control and ischemic brain tissue. Gates were set based on fluorescence minus one (FMO) intensity values. Quantification of the percentage of CD11b+CD45low microglia containing lipid droplets as Bodipy+ microglia using flow cytometry in male mice deficient in Stat1 (Stat1−/−; n = 4) and corresponding Stat1+/+ mice (n = 7) shows ischemia-induced increases in Stat1+/+ mice (**P = 0.0086) but not in Stat1−/− mice (P = 0.927; Two-way ANOVA and Šídák's multiple comparisons test). The graph shows values of the nonischemic (−) and ischemic (+) brain hemispheres of individual mice and the mean ± SD. Download figure Download PowerPoint We identified the presence of lipid droplets in microglia by transmission electron microscopy of mouse brain tissue 24 h after ischemia (Fig 2E). Furthermore, we observed lipid droplets in contact with lysosomes, supporting the occurrence of macrolipophagy as a mechanism that could supply energy to the cells. The presence of lipid droplets in ischemic microglia was quantified by flow cytometry after staining the cells with fluorescent Bodipy (Fig 2F). The percentage of Bodipy+ CD45lowCD11b+ microglia increased 4 days postischemia in Stat1+/+ mice, but not in Stat1−/− mice (Fig 2G). Stat1 is a critical factor mediating the transduction of cellular responses to several types of IFNs (Van Boxel-Dezaire et al, 2006). Therefore, these results show that the IFN response is involved in ischemia-induced lipid droplet biogenesis in microglia. Old mice show worse neurological outcomes than young mice, and microglia of old mice show exacerbated innate immune responses to brain ischemia Stroke caused more severe neurological deficits and larger infarct volumes in old (21–22 months) than in young (3–4 months) female mice (Fig 3A). Given the protective effect of estrogens (Koellhoffer & McCullough, 2013), it is possible that differences in plasma estrogen levels in individual young female mice (Appendix Fig S1), attributable to the various stages of the estrus cycle, contributed to infarct volume variability. Comparison of the transcriptomic profile of microglia FACS-sorted from the brain 4 days postischemia in young and old female mice showed 1,980 DEGs. Of these, 1,153 genes (58%) were upregulated in old ischemic mice, and so were most GSEA pathways indicating a gain of function associated with age (Dataset EV1). Compared with microglia of young ischemic mice, microglia of old ischemic mice showed enrichment of GO pathways related to the innate immune system and antigen presentation, amongst others (Fig 3B). Enrichment of innate immune system and inflammatory pathways was highlighted by the GO terms “Response to interferon-beta,” “Response to interferon-gamma,” and “Response to bacterium” (Figs 3C and EV1). Many of these responses were already induced by ischemia in microglia of young mice (Fig 1E and F), but they were upregulated further in microglia of old mice. Inflammatory responses were accompanied by upregulation of genes involved in the “Regulation of necroptotic cell death,” and microglia of ischemic old mice also showed a prominent enrichment of antigen-presenting machinery (Fig EV1). Figure 3. Worse stroke outcome in old mice and corresponding transcriptional profile of microglia A. Ischemia was induced in young and old female C57BL/6 mice (n = 17 per group) and infarct volume, as assessed with T2w MRI, and the neurological score was evaluated at day 4. The neurological score was higher (worse) in old mice (Mann–Whitney test, ****P < 0.0001), which also showed larger infarct volume (Mann–Whitney test, *P = 0.041) compared with young mice. The graph shows boxes from the 25th to 75th percentiles, the median line, and whiskers down to the minimum and up to the maximum value, showing all points. B. Microglia mRNA was obtained from young (3–4 months; n = 7) and old (21–22 months; n = 5) female mice 4 days postischemia and studied by RNAseq (GSE196737). Cnetplot illustrates the network of genes enriched after ischemia in old versus young mice linked to GO terms mainly related to innate immunity, inflammatory responses, and antigen presentation. C. Heatmaps of genes of representative innate immunity pathways enriched in microglia of old versus young ischemic mice. D, E. Upregulation of genes related to long-chain fatty acid binding and downregulation of genes related to peroxisomal long-chain fatty acid metabolism in microglia of old mice. F. Microglia obtained from young and old male (gray) and female (pink) mice 4 days postischemia (n = 16 mice; 4 mice per age group and sex) and sham operation (n = 12 mice; 3 mice per age group and sex) was studied by flow cytometry. After sham operation old mice showed a higher proportion of microglia containing lipid droplets after sham operation (***P < 0.001) and ischemia (**P = 0.0022; t-test). Values show the mean ± SD. G. Microglia from adult young and old male mice using CD11b+ beads were kept in culture for 7 days and then exposed to red fluorescent pHrodo Escherichia coli bioparticles and stained with Bodipy. Bodipy+ lipid droplets (white in the upper panels -raw intensity- and green in the corresponding lower panels) are clearly seen in microglia from old mice, but not young mice. The images at the bottom also show phagocytosed red bioparticles, which are hardly seen in lipid droplets containing cells. The square in each image in the center is magnified in the images on the left and right sides for young and old microglia, respectively. Scale bar = 20 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Enrichment of genes in GO pathways in microglia of old versus young ischemic female mice. Related to Fig 3RNAseq analysis of microglia obtained by FACS from the brain of old (n = 5) versus young (n = 7) female mice 4 days after ischemia (GSE196737). A–C. Heatmaps illustrate genes upregulated in microglia of old ischemic mice versus young ischemic female mice for the following GO terms: “Response to IFN-γ” (A), “Response to bacterium” (B), and “Antigen binding