SFRP1 modulates astrocyte‐to‐microglia crosstalk in acute and chronic neuroinflammation

Article27 September 2021Open Access Transparent process SFRP1 modulates astrocyte-to-microglia crosstalk in acute and chronic neuroinflammation Javier Rueda-Carrasco Javier Rueda-Carrasco orcid.org/0000-0002-9280-6612 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Search for more papers by this author María Jesús Martin-Bermejo María Jesús Martin-Bermejo Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain These authors contributed equally to this work Search for more papers by this author Guadalupe Pereyra Guadalupe Pereyra Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain These authors contributed equally to this work Search for more papers by this author María Inés Mateo María Inés Mateo Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain These authors contributed equally to this work Search for more papers by this author Aldo Borroto Aldo Borroto Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Frederic Brosseron Frederic Brosseron orcid.org/0000-0003-3137-7516 Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Markus P Kummer Markus P Kummer Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Stephanie Schwartz Stephanie Schwartz Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author José P López-Atalaya José P López-Atalaya orcid.org/0000-0001-6064-7584 Instituto de Neurociencias, CSIC-UMH, Sant Joan d'Alacant, Spain Search for more papers by this author Balbino Alarcon Balbino Alarcon orcid.org/0000-0001-7820-1070 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Pilar Esteve Pilar Esteve Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Search for more papers by this author Michael T Heneka Michael T Heneka orcid.org/0000-0003-4996-1630 Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Paola Bovolenta Corresponding Author Paola Bovolenta [email protected] orcid.org/0000-0002-1870-751X Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Search for more papers by this author Javier Rueda-Carrasco Javier Rueda-Carrasco orcid.org/0000-0002-9280-6612 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Search for more papers by this author María Jesús Martin-Bermejo María Jesús Martin-Bermejo Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain These authors contributed equally to this work Search for more papers by this author Guadalupe Pereyra Guadalupe Pereyra Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain These authors contributed equally to this work Search for more papers by this author María Inés Mateo María Inés Mateo Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain These authors contributed equally to this work Search for more papers by this author Aldo Borroto Aldo Borroto Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Frederic Brosseron Frederic Brosseron orcid.org/0000-0003-3137-7516 Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Markus P Kummer Markus P Kummer Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Stephanie Schwartz Stephanie Schwartz Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author José P López-Atalaya José P López-Atalaya orcid.org/0000-0001-6064-7584 Instituto de Neurociencias, CSIC-UMH, Sant Joan d'Alacant, Spain Search for more papers by this author Balbino Alarcon Balbino Alarcon orcid.org/0000-0001-7820-1070 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Pilar Esteve Pilar Esteve Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Search for more papers by this author Michael T Heneka Michael T Heneka orcid.org/0000-0003-4996-1630 Neurology, Universitätsklinikum Bonn, Bonn, Germany German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Paola Bovolenta Corresponding Author Paola Bovolenta [email protected] orcid.org/0000-0002-1870-751X Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Search for more papers by this author Author Information Javier Rueda-Carrasco1,2, María Jesús Martin-Bermejo1,2, Guadalupe Pereyra1,2, María Inés Mateo1,2, Aldo Borroto1, Frederic Brosseron3,4, Markus P Kummer3,4, Stephanie Schwartz3,4, José P López-Atalaya5, Balbino Alarcon1, Pilar Esteve1,2, Michael T Heneka3,4 and Paola Bovolenta *,1,2 1Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain 2CIBER de Enfermedades Raras (CIBERER), Madrid, Spain 3Neurology, Universitätsklinikum Bonn, Bonn, Germany 4German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 5Instituto de Neurociencias, CSIC-UMH, Sant Joan d'Alacant, Spain *Corresponding author. Tel: +34 91 1964718; E-mail: [email protected] EMBO Reports (2021)22:e51696https://doi.org/10.15252/embr.202051696 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 Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron–microglia–astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases. Synopsis In response to neuroinflammation, astrocytes secrete more SFRP1 protein. SFRP1 enhances glial activation and microglia responses by sustaining robust expression of the down-stream targets of the HIF pathway. Astrocytes produce SFRP1 in response to neuroinflammation. Increased SFRP1 levels are sufficient to trigger and sustain glial cell activation. Astrocyte-derived SFRP1 enhances the response of microglial cells to damage. SFRP1 is required for robust microglial expression of down-stream targets of HIF transcription factors. Introduction Degeneration of neurons occurs in a variety of rare and common pathological conditions of the central nervous system (CNS) including Alzheimer’s disease (AD) or multiple sclerosis (MS). CNS function is supported by a robust neuron–glia crosstalk (Jha et al, 2019; Marinelli et al, 2019) so that neuronal damage is almost invariably associated with the activation of two types of glial cells: microglia and astrocytes. The prompt glial response to insults brings about an inflammatory reaction that favours tissue healing and helps restoring CNS homeostasis (Ransohoff, 2016; Escartin et al, 2019). However, excessive glial activation, as often occurs in neurodegeneration, is itself a cause of neuronal damage, which, in turn, establishes a state of pernicious chronic neuroinflammation (Frank-Cannon et al, 2009; Glass et al, 2010; Perry & Holmes, 2014; Escartin et al, 2019). Consistent with an important cellular crosstalk, glial cell dysfunction, either as a consequence of hyper- or hypofunctionality, can also be the cause of neuronal damage, rather than its consequence, strongly contributing to the progression of neurodegenerative diseases (Ransohoff, 2016; Sarlus & Heneka, 2017; Hickman et al, 2018; Escartin et al, 2019). Although this is nowadays a widely accepted idea, there is still only partial information on the molecular components that sustain a dys- or hyperfunctional state of glial cells and an abnormal glia–neuron crosstalk. Here, we have investigated whether SFRP1 may represent one of such components. SFRP1 is a small, secreted and dispersible protein with functions that have been related to Wnt signalling (Esteve & Bovolenta, 2010) and ADAM10 activity (Esteve et al, 2011a), although its interaction with other factors such as RANKL (receptor activator of nuclear factor-kappa B ligand), integrins (Bovolenta et al, 2008) and thrombospondin-1 (Martin-Manso et al, 2011) has also been reported. In particular, Wnt signalling not only has been mostly implicated in moulding neurodegenerative synaptic changes (Palomer et al, 2019) but also contributes to neuroinflammation, although this role is still ill-defined (Aghaizu et al, 2020). ADAM10, a member of the A Disintegrin and Metalloprotease family of plasma membrane proteins (Saftig & Lichtenthaler, 2015), acts as an α-secretase (or sheddase; Lichtenthaler et al, 2018) for many neuronal or glial expressed substrates, which participate in the control of microglial activation (Marinelli et al, 2019). These include TREM2 (triggering receptor expressed on myeloid) (Kleinberger et al, 2014) and CD200 and CX3CL1 (Hundhausen et al, 2007; Wong et al, 2016). ADAM10 also sheds proteins involved in synaptic plasticity, such as N-cadherin or the amyloid precursor protein (APP) (Musardo et al, 2014; Saftig & Lichtenthaler, 2015). Consistently, genetic inactivation of Adam10 in mice causes neuroinflammation and loss of synaptic plasticity (Prox et al, 2013), whereas genetic studies in humans have demonstrated a link between impaired ADAM10 activity and AD (Suh et al, 2013; Kunkle et al, 2019), in part linked to its non-amyloidogenic processing of APP (Kuhn et al, 2010). Supporting this possibility, we have recently shown that elevated levels of SFRP1 contribute to AD pathogenesis, acting as an endogenous negative modulator of ADAM10 (Esteve et al, 2019b). SFRP1 upregulation correlates with poor ADAM10-mediated processing of APP and N-cadherin, whereas neutralization of its activity prevents the appearance of AD pathological traits, including glial cell activation (Esteve et al, 2019b). Sfrp1 is abundantly expressed in mammalian radial glial progenitors of the developing CNS (Campanelli et al, 2008; Esteve et al, 2011a, 2019a) but is largely downregulated in the adult brain with the exception of restricted neurogenic areas (Augustine et al, 2001; Zhang et al, 2016; Esteve et al, 2019a). Besides in human neurodegenerative diseases (Blalock et al, 2004; Esteve et al, 2019b; Folke et al, 2019; Bai et al, 2020; preprint: Johnson et al, 2021), SFRP1-upregulated expression has been reported in several other inflammatory conditions such as periodontitis, rheumatoid arthritis, uropathies or pulmonary emphysema (Esteve & Bovolenta, 2010; Claudel et al, 2019). An upregulation has been also observed in aged brains (Folke et al, 2019), in which a low-grade chronic inflammation is present (Youm et al, 2013). Notwithstanding, whether SFRP1 is directly involved in the modulation of neuroinflammation remains unexplored. By addressing this issue, here we show that SFRP1 is a novel mediator of the astrocyte-to-microglia crosstalk that underlies mammalian CNS inflammation. In mice, SFRP1, largely astrocyte-derived, is sufficient to activate microglial cells and to amplify their response to distinct acute and chronic neuroinflammatory challenges, sustaining their chronic activation. From a molecular point of view, SFRP1 allows for the full expression of downstream targets of the transcription factors hypoxia-induced factors (HIFs) and, to a lesser extent, nuclear factor-kappa B (NF-κB), which are mediators of neuroinflammatory responses (Helton et al, 2005; Kaltschmidt & Kaltschmidt, 2009). Thus, neutralizing SFRP1 function may represent a strategy to counteract pernicious chronic neuroinflammation that contributes to many human neurodegenerative conditions. Results Acute brain neuroinflammation elevates astrocyte-specific levels of SFRP1 expression To evaluate a possible link between SFRP1 and neuroinflammation, we induced acute brain inflammation by injecting bacterial lipopolysaccharides (LPS; Wright, 1999) or control saline into the somatosensory cortex of 3-month-old Sfrp1+/βgal;CX3CR1+/GFP mice (n = 4), which allow the simultaneous identification of microglial (GFP; Jung et al, 2000) and Sfrp1-producing (nuclear βgal-positive; Satoh et al, 2006) cells. In a separate set of experiments, we performed similar injections in wt and Sfrp1+/βgal mice (n = 5). Immunofluorescence of the brains three days after injection, when inflammation is at its peak (Rivest, 2009), revealed a broader βgal immunoreactivity (reporter of Sfrp1 expression) in LPS vs saline-treated animals (Fig 1A), largely localized in GFAP+ astrocytes (Fig 1A), as also reported in demyelinating or kainic acid-induced CNS lesions (Huang et al, 2020; García-Velázquez & Arias, 2021). Using RNAscope, we had previously detected Sfrp1 mRNA in some Iba+ microglial cells surrounding amyloid plaques present in an AD-like mouse model (Esteve et al, 2019b). However, we failed to detect βgal+ signal in these cells after LPS injection in neither Sfrp1+/βgal;CX3CR1+/GFP (Fig 1A) or Sfrp1+/βgal (Fig 1C). This observation was confirmed by RNA-seq analysis of isolated microglial cells (see the last section of the results) and likely reflects the reported heterogeneous and disease-dependent activation of microglial cell (Bachiller et al, 2018). Figure 1. Astrocytes upregulate SFRP1 expression upon LPS stimulation A–D. Confocal image analysis of cryostat sections from adult CX3CR1+/GFP; Sfrp1+/βgal (A, B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1−/− (D) and mouse brains three days after intracortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A, B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar: 25 μm. E. ELISA determination of Sfrp1 levels in brain extracts from 3-month-old wt and Sfrp1−/− mice. WT mice were injected either with saline or with LPS. Three days after injection, the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1−/− mice used as negative control (n = 5 mice for each group). Error bars represent standard error. Statistical significance: ns P > 0.5 ****P < 0.0001; one-way ANOVA followed by the Bonferroni multiple comparisons test. Download figure Download PowerPoint Immunodetection of SFRP1 with specific antibodies (Esteve et al, 2019b) confirmed an increased SFRP1 production after LPS compared with saline injections (Fig 1B). Notably, SFRP1 protein distribution in saline-injected animals was comparable to that of non-injected mouse brains (Esteve et al, 2019b). Consistent with the secreted and dispersible nature of SFRP1 (Mii & Taira, 2009; Esteve et al, 2011b), immunosignal was widely distributed in the brain parenchyma (arrows, Fig 1B) and localized to the choroid plexus (not shown) but completely absent in Sfrp1−/− brains (Fig 1D), as previously reported (Esteve et al, 2019b). The use of a highly specific ELISA (Esteve et al, 2019b) further confirmed a significant increase of SFRP1 levels in extracts of small cortical tissue samples—dissected in the proximity of the injection site—from LPS-treated animals (Fig 1E) as compared to that present in equivalent brain regions from non-injected or saline-injected animals (Fig 1E). These data indicate that the level of brain SFRP1 increases in response to a bacterial lipopolysaccharide and that astrocytes are likely the most abundant source of SFRP1. In vivo Sfrp1 gene addition is sufficient to trigger and sustain glial cell activation We next reasoned that if SFRP1 is indeed associated with neuroinflammation, its forced expression should be sufficient to activate glial cells. To test this possibility, we next infused lentiviral particles (LV) containing Sfrp1-IRES-Gfp or control IRES-Gfp into the lateral ventricle of 8- to 10-week-old wt mice (n = 13 per group; Figs 2 and EV1A). As expected by the injection site, GFP+ (used to determine infection efficiency) LV-transduced cells included cells lining along the wall of the lateral ventricle, the choroid plexus, GFAP+ astrocytes and, to a lesser extent, cells of the rostral migratory stream (Fig EV1A and B). Immunohistochemistry of cortical sections 1 month after injection showed a significantly higher presence of SFRP1 protein at the infected site (Fig 2A) associated with Sox9+, GFAP+ and S100 β+ reactive astrocytes (Escartin et al, 2021) and CD45+, Clec7a+ and Iba1+ microglial cells as compared to LV-IRES-Gfp control animals (Figs 2A and B, and EV1C). Iba1 immunoreactivity was accumulated around the injection site (Fig 2A and B), and many cells had a round amoeboid morphology and a significantly higher CD45 expression (Figs 2B and EV1D), characteristics of activated microglia (Heneka et al, 2014). CD45hi round cells could also represent blood-borne infiltrating macrophages (Fig EV1D), although the molecular and functional difference between the two cell types is currently a matter of debate (Grassivaro et al, 2020; Honarpisheh et al, 2020). A wider distribution of hyperphosphorylated tau, which often appears as a brain response to inflammation and degeneration (Ising et al, 2019), was also detected in LV-Sfrp1-IRES-Gfp-transduced vs control brains with a significantly wider distribution (Fig 2A and B). Analysis of a different set of LV-transduced animals (n = 4) 5 months after LV delivery showed a persistent microglial activation in SFRP1- vs GFP-treated animals, with more CD45+ cells than those observed at 1-month post-injection (Fig EV1D). Furthermore, several CD45hi round-shaped microglia/macrophages were detected in the parenchyma, especially in the proximity of transduced cells (white arrowheads in Fig EV1D). Figure 2. SFRP1 is sufficient and required to enhance glial cell activation upon LPS treatment A. Coronal cryostat sections of LV-IRES-Gfp- or LV-Sfrp1-IRES-Gfp-infected brains 1 month post-infusion immunostained for SFRP1 (magenta), Iba1 (green), GFAP (cyan) or TauP (red). Lv, lateral ventricle. Scale bar: 100 μm. B. The graph shows the normalized level of GFAP, Iba1 and CD45 immunoreactivity (IR) and the area occupied by TauP signal (n = 24 acquisitions, white dots; N = 3 mice, black dots, for each group), normalized to LV-IRES-Gfp-infected brains. Error bars represent standard error. Statistical significance: *P < 0.05; **P < 0.01; ****P < 0.0001 by two-sided Student’s t-test. C, D. Coronal sections from wt and Sfrp1−/− mouse brains three days after infusion of saline or LPS immunostained for GFAP (cyan, C) or CD45 (magenta, D). The images are high-power views of the somatosensory cortex (lower power view in Fig EV1D). Scale bar: 60 μm. E, F. The graphs show the normalized levels of immunoreactivity (IR) for GFAP (E) and CD45 (F, P = 0.006) present in cortical sections (n = 24 acquisitions white dots; from N = 3 animals, black dots, per group) from wt and Sfrp1−/− animals infused with saline or LPS. Bars represent standard error. Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black. ** or ##P < 0.01; *** or ###P < 0.001; and **** or ####P < 0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. * and # indicate significance between genotypes and treatments, respectively. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. SFRP1 enhances glial cell activation On the left, diagram of the observed GFP distribution (green dots) after lentiviral (LV) particles' delivery into the lateral ventricle (Lv). The dashed boxes represent areas shown in (B) and (D), 1 points to the wall of the lateral ventricle, and 2 to the Rostral Migratory Stream. Images on the right show examples of immunostaining against GFP protein (GFP signal was no longer visible, unless detected with immunohistochemistry) in the Lv of brains transduced with LV-IRES-Gfp or LV-Sfrp1-IRES-Gfp 5 months (m) post-infusion. Scale bar: 100 μm. Double immunostaining against GFP (green) and GFAP (magenta) of brains transduced with LV-Sfrp1-IRES-Gfp, indicating that transduced cells include astrocytes. Arrowheads indicate GFP+ astrocytes. Scale bar: 50 μm. The graphs show the number of Sox9 immunopositive cells, S100β and Cleac7a immune intensity level and CD45 immunopositive area in LV-Sfrp1-IRES-Gfp-infected brains normalized against the mean value obtained in LV-IRES-Gfp-infected control brains. Dots represent the mean value obtained by the analysis of 6 sections for each biological replica (n = 4–6, mice per group). Error bars represent standard error. Statistical significance: *P < 0.05; **P < 0.01 by two-sided Student’s t-test. Coronal sections from LV-IRES-Gfp- or LV-Sfrp1-IRES-Gfp-infected brains 1 or 5 months (m) post-infusion (PI). Sections were immunostained for CD45. Arrowheads indicate CD45hi-positive macrophages or lymphocytes infiltrated in the parenchyma after prolonged LV-Sfrp1-IRES-Gfp infection. High-power images 1 and 2 were taken from the regions indicated with grey dotted lines in A. Scale bar: 50 μm. Coronal sections from wt and Sfrp1−/− mouse brains three days after infusion of saline or LPS, immunostained for GFAP. The white arrows indicate the injection site; the area boxed with white lines is represented at high power in Fig 2. Scale bar: 100 μm. Download figure Download PowerPoint Together, these results indicate that SFRP1 is sufficient to trigger an inflammatory response in glial cells, which persists for prolonged periods of time. Sfrp1 is required for amplifying CNS inflammatory response Given that SFRP1 was sufficient to induce glial cell activation, we next asked whether it was also a necessary component of the CNS inflammatory response. To this end, we took advantage of Sfrp1−/− mice, in which SFRP1 protein is completely undetectable (Esteve et al, 2019b) (Fig 1B and C). These mice have a slightly shorter and thicker cortex that however does not affect their life span, reproduction rate or cognitive and motor behaviour and present no evident neuronal defects (Esteve et al, 2019a, 2019b). Furthermore, their content of astrocytes and microglial cells was undistinguishable from that of wt mice (Fig 2C–F). We thus compared the effect of intracortical LPS infusion into the brains of 3-month-old wt and Sfrp1−/− mice (Fig 2C and D). In the somatosensory cortex of wt brains (Fig EV1E), LPS but not saline treatment caused the appearance of GFAP+ reactive astrocytes (Fig 2C) and CD45+ reactive microglia (Fig 2D). In contrast, Sfrp1−/− littermates presented fewer and less immunopositive astrocytes and a significant reduction in CD45+ reactive microglia/macrophages (Fig 2C and D), further supporting that SFRP1 is relevant for the activation of both astrocytes and microglial cells. Quantitation of immunoreactivity among different genotypes and treatments confirmed no significant differences between the two saline-treated genotypes but showed a significantly lower response of Sfrp1−/− mice to LPS as compared to wt (Fig 2E and F). Nevertheless, Sfrp1−/− mice do not completely lose their response to LPS, given that this is significantly different from that observed in saline-injected mice (Fig 2E and F). This suggests that SFRP1 is involved in boosting inflammation. To evaluate a possible specificity of this response, we induced experimental autoimmune encephalomyelitis (EAE) in wt and Sfrp1−/− mice. This is a widely used experimental model for MS, a human inflammatory-demyelinating disease. In EAE, CNS inflammation and gliosis occur as a consequence of a strong autoimmune response against the peripheral exposure to myelin components (Constantinescu et al, 2011), thus representing a neuroinflammatory paradigm quite different from the direct LPS intracerebral infusion. Female littermates of the two genotypes were immunized following a standard protocol, and animals were scored for the development/remission of their clinical symptoms over the course of a month (Borroto et al, 2016). Animals were classified with a standard 0 to 5 rank based on their paralysis degree, with 0 corresponding to the absence of symptoms and 5 to a moribund condition (Borroto et al, 2016). In wt mice (n = 19), tail limping—the first symptom of the disease—became apparent around 8 days after immunization with a subsequent rapid progression so that, by 16 days, most of the wt animals presented hindlimb paralysis followed by a slow recovery (Fig 3A). Notably, 47% of the immunized wt mice developed extreme and protracted symptoms (Fig 3A and B). The response of Sfrp1−/− mice (n = 19) to immunization was instead slower and milder: only 16% of them developed extreme symptoms, and their recovery was significantly faster (Fig 3A and B). Immunostaining of spinal cord sections before immunization showed no difference between wt and Sfrp1−/− in astrocytes, microglia or myelin distribution (not shown). In contrast, in animals (n = 4) sacrificed 16 days after immunization, there was a significant reduction in pathological signs in Sfrp1−/− vs wt mice, including infiltration of CD4+/Iba- lymphocytes (no CD4+/Iba+ monocytes were instead detected), presence of Iba1+ macrophages/activated microglial cells and loss of MBP+ myelin (Fig 3C and D). There was no significant difference in the amount of GFAP labelling between wt and Sfrp1−/−; however, in all null mice analysed there was basically no disruption of the astrocytic pial surface otherwise observed in all wt mice (Fig 3C). Figure 3. Sfrp1−/− mice develop a milder form of EAE A, B. Time course analysis and severity of the symptoms in wt and Sfrp1−/− mice after EAE induction. In A, means are depicted with black (wt, n = 19) and cyan (Sfrp1−/−, n = 19) lines. In B, data are expressed as % of the total number of analysed animals (n = 19 per genotype). In (A), error bars represent standard error. C. Wt and Sfrp1−/− mice immunized with MOG and sacrificed 16 days post-immunization. Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4, Iba1, GFAP and MBP. Images show the region dorsal fasciculus. White arrowheads point to the disruption of the pia surface in wt. Scale bar: 100μm. D. Quantification of CD4+ infiltrated lymphocytes, Iba1-normalized immunoreactivity, MBP-normalized immunoreactivity and MBP immune-reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1−/− mice 16 days after immunization (n = 16 acquisitions, white dots; from N = 4 animals, black dots, per genotype). Error bars represent standard error. Statistic refers to number of acquisitions. Data information: *P < 0.05, **P < 0.01 and ***P < 0.001 by the Kolmogorov–Smirnov test followed by the Mann–Whitney U nonparametric test comparing mice of the same day after immunization (A) or two-sided Student’s t-test (D). Download figure Download PowerPoint All in all, these data indicate that SFRP1 is commonly required for a robust neuroinflammatory response, likely contributing to astrocyte-to-microglia crosstalk. Astrocyte-derived Sfrp1 is required for a robust response of microglial cells to damage The latter possibility found support in the observation that astrocytes but not microglial cells are the main SFRP1 source in the brain, although the protein has a widespread distribution including around microglial cells (Fig 1), as well as in the remarkably poor microglial activation (Figs 2 and 3) and myeloid cell recruitment (Fig 3C) observed in the absence of Sfrp1. To further explore this possibility, we used flow cytometric analysis to determine the proportion of the diff