In vivo characterization of functional states of cortical microglia during peripheral inflammation

Journal Pre-proofs In vivo characterization of functional states of cortical microglia during peripheral inflammation Karin Riester, Bianca Brawek, Daria Savitska, Nicole Fröhlich, Elizabeta Zirdum, Nima Mojtahedi, Michael T. Heneka, Olga Garaschuk PII: DOI: Reference:

S0889-1591(19)31294-2 https://doi.org/10.1016/j.bbi.2019.12.007 YBRBI 3924

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Brain, Behavior, and Immunity

Received Date: Revised Date: Accepted Date:

8 October 2019 5 December 2019 9 December 2019

Please cite this article as: Riester, K., Brawek, B., Savitska, D., Fröhlich, N., Zirdum, E., Mojtahedi, N., Heneka, M.T., Garaschuk, O., In vivo characterization of functional states of cortical microglia during peripheral inflammation, Brain, Behavior, and Immunity (2019), doi: https://doi.org/10.1016/j.bbi.2019.12.007

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In vivo characterization of functional states of cortical microglia during peripheral inflammation

Karin Riestera,#, Bianca Braweka,#, Daria Savitskaa, Nicole Fröhlicha, Elizabeta Zirduma, Nima Mojtahedia, Michael T. Henekab,c, Olga Garaschuka,* a

Institute of Physiology, Department of Neurophysiology, Eberhard Karls University

Tübingen, Tübingen, Germany b

Department of Neurodegenerative Disease and Geriatric Psychiatry, University of

Bonn, Bonn, Germany. c

German Center for Neurodegenerative Diseases, Bonn, Germany.

#

These authors contributed equally to this work.

* Corresponding author

Keywords: Ca2+ signaling / in vivo / microglia / peripheral inflammation Word count: 11,999 Corresponding author: Prof. Dr. Olga Garaschuk Institute of Physiology, Department of Neurophysiology Eberhard Karls University Tübingen Keplerstr. 15, 72074 Tübingen, Germany Tel: +49-07071 29 73640 Fax: +49-07071 29 5395 E-mail: [email protected]

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Abstract Peripheral inflammation is known to trigger a mirror inflammatory response in the brain, involving brain’s innate immune cells - microglia. However, the functional phenotypes, which these cells adopt in the course of peripheral inflammation, remain obscure. In vivo two-photon imaging of microglial Ca2+ signaling as well as process motility reveals two distinct functional states of cortical microglia during a lipopolysaccharide-induced peripheral inflammation: an early “sensor” state” characterized by dramatically increased intracellular Ca2+ signaling but ramified morphology and a later “effector state” characterized by slow normalization of intracellular Ca2+ signaling but hypertrophic morphology, substantial IL-1β production in a subset of cells as well as increased velocity of directed process extension and loss of coordination between individual processes. Thus, lipopolysaccharide-induced microglial Ca2+ signaling might represent the central element connecting receptive and executive functions of microglia.

1. Introduction During the lifetime, any organism is frequently subjected to infections or other harmful stimuli, causing a stereotyped protective response called “inflammation”. The term inflammation describes the body’s defense reaction against foreign or altered endogenous substances for the sake of elimination of the initial cause of injury, as well as regeneration and tissue repair (Netea et al., 2017; Yong and Rivest, 2009). It has long been thought that the brain is not involved in this inflammatory reaction. Nowadays, however, it is clear that the peripheral and the central immune systems closely communicate with each other and a systemic challenge of the peripheral 2

immune system produces a mirror inflammatory response in the brain, which eventually leads to a physiological response called “sickness behavior”, characterized by reduced appetite, social isolation, lethargy and fever. Sickness behavior is triggered by cytokines (predominantly Interleukin (IL)-1β and Tumor necrosis factor-α (TNF-α)), which are initially produced by immune cells in the site of infection (Dantzer et al., 2008; Yong and Rivest, 2009). The brain senses peripheral elevation of cytokines, which can either cross the blood brain barrier (BBB) by specific transport mechanisms, or enter the brain in regions where the BBB is or becomes permeable (Dantzer et al., 2008; Lucas et al., 2006). Further neuronal and cellular routes (e.g. activation of vagal and trigeminal nerves or cytokine receptors on endothelial cells leading to local prostaglandin E2 production) also help to transmit the inflammation signals from the periphery to the brain (Dantzer et al., 2008). The central inflammatory reaction is mostly driven by microglia, the resident immune cells of the brain. As shown by single-cell RNA sequencing and RT-PCR data, during a systemic immune challenge microglia down-regulate homeostatic genes and upregulate inflammatory genes, such as Il1β, Tnf, Il6 and Ccl2, (Chemokine ligand 2, also known as Monocyte chemoattractant protein-1 (MCP-1)) (Norden et al., 2016; Sousa et al., 2018). Indeed, already several hours after induction of a peripheral inflammation cytokine (e.g. TNF-α, IL-6, MCP-1) levels in the brain are up-regulated (Biesmans et al., 2013; Norden et al., 2016; Qin et al., 2008). In addition, microglial cells undergo prominent morphological alterations characterized by deramification and enlargement of their cell bodies (Gyoneva et al., 2014; Kozlowski and Weimer, 2012). The functional significance of these morphological changes is not completely understood, although it is thought that morphological alterations are associated with an activation of microglia and the enhanced ability to phagocytose 3

pathogens or cell debris (Kettenmann et al., 2011). Whereas basal process motility of in vivo microglia increased after induction of a peripheral inflammation (Gyoneva et al., 2014), the speed of directed process movement towards the injured tissue was lower, indicating a dysfunctional phenotype (Gyoneva et al., 2014; Pozner et al., 2015). The latter studies, however, used laser-induced damage of the tissue, which is much more destructive than the death of individual neurons or other small insults (e.g. rupture of tiny blood vessels) regularly occurring within the brain. Therefore, how systemic inflammation modifies the reaction of microglia to such minor insults remains obscure. Several lines of evidence suggest that an increase of the intracellular free Ca2+ concentration ([Ca2+]i) in microglia represents a central signaling pathway for the regulation of the immune response. First, in vitro activation of receptors for several inflammatory mediators such as IL-1β, MCP-1, TNF-α is associated with an increase in [Ca2+]i (Boddeke et al., 1999; Goghari et al., 2000; Pollock et al., 2002). Second, an increase in microglial [Ca2+]i in vitro can initiate gene transcription and release of cytokines, like TNF-α and IL-1β, or nitric oxide (Hoffmann et al., 2003). Third, microglia increase their basal [Ca2+]i in the presence of lipopolysaccharide (LPS) in vitro (Hoffmann et al., 2003) and in conditions of tissue damage/dissociation, like tissue slicing and cell culturing (Brawek et al., 2017), identifying an increase in [Ca2+]i as an integral component of their activation. During central or peripheral (neuro)inflammation in addition to sustained rises in [Ca2+]i, microglia also increase the incidence of “spontaneous” Ca2+ transients (Brawek et al., 2014; Olmedillas Del Moral et al., 2019; Pozner et al., 2015). Indeed, being rather silent in terms of spontaneous Ca2+ signaling under homeostatic conditions, microglia vividly react with Ca2+ transients to (i) inflammaging (Franceschi et al., 2007; Olmedillas Del Moral et al., 2019), (ii) damage

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of individual cells in their microenvironment (Eichhoff et al., 2011), (iii) accumulation of misfolded or aggregated proteins (Brawek et al., 2014), etc. Peripheral injections of LPS have been routinely used to induce a systemic immune response and to study the reaction of microglia in the brain. Despite being extremely heterogeneous in terms of serotype, origin and concentration of LPS used, injection protocols (single vs. repeated, intraperitoneal vs. intravenous vs. subcutaneous), and post-injection time analyzed (for review see Hoogland et al., 2015), these studies suggested that in the course of peripheral inflammation microglia sequentially undergo different biochemical and morphological alterations, likely defining distinct functional states of these cells (Norden et al., 2016). The current study aimed to characterize in vivo the functional states, which microglia adopt in the course of systemic inflammation, mainly focusing on microglial morphology, Ca2+ signaling, and microglial process motility.

2. Methods 2.1 Mice All experiments were performed in the frontal/motor cortex of 4-6 months old C57BL/6, CX3CR1GFP/+ (Jung et al., 2000), Iba-1GFP/+ (Hirasawa et al., 2005), TNF-α-/(Pasparakis et al., 1996), and NLRP3-/- (Kanneganti et al., 2006) mice of either sex. Animals were housed under standard conditions with a 12 h light-dark cycle and free access to water and food. Before and between the imaging sessions, the mice were housed in the animal facility in standard conditions, i.e. temperature of 22°C and humidity of 55%. During the imaging sessions, mice were head-fixed in the experimental setup in the imaging room, in which the temperature and humidity are 5

kept constant at 20°C and 25%, respectively. All experimental procedures were carried out in accordance with institutional animal welfare guidelines and approved by the government of Baden-Württemberg, Germany. To stay comparable with previous studies (Gyoneva et al., 2014; Kozlowski and Weimer, 2012), CX3CR1GFP/+ mice were used to study LPS-induced alterations in morphology and cell density. To ensure that in vivo functional properties of microglia are not distorted by diminished interaction between the OFF signal fractalkine and its CX3CR1 receptor, Iba-1GFP/+ mice were used for functional studies (i.e. in vivo microglial Ca2+ signaling and process motility). To study the role of cytokine production for microglial Ca2+ signaling, we used NLRP3-/- and TNF-α-/- mice, in which the production of IL-1β or TNF-α, respectively, is compromised. In NLRP3-/- mice, the NLRP3 (NOD-, LRR- and pyrin domain- containing 3) inflammasome, a key innate immune sensor for danger signals causing the caspase-1-dependent maturation of IL1β, is deleted (Kanneganti et al., 2006). In TNF-α-/- mice, the production of TNF-α is abolished (Pasparakis et al., 1996). For all other experiments, C57BL/6 mice were used.

2.2 Induction of inflammation To induce a peripheral inflammatory response, mice received an intraperitoneal (i.p.) injection of the bacterial endotoxin lipopolysaccharide (LPS) derived from Escherichia Coli, Serotype O111:B4 (Sigma-Aldrich; USA). LPS was diluted in sterile PBS and injected once at a dose of 1.5 mg/kg body weight (BW). Control animals received an equivalent amount of sterile PBS. This dose induced a moderate sickness behavior reflected by slightly reduced weight (95.62 ± 3.30% 5 h after LPS and 87.88 ± 10.74% 30 h after LPS), lethargy, and reduced grooming behavior. 6

2.3 Cytokine enzyme-linked immunosorbent assay (ELISA) 5 or 30 h after PBS/LPS injection, C57BL/6, NLRP3-/- or TNF-α-/- mice were deeply anesthetized with a combination of ketamine (200 mg/kg BW; Fagron, Netherlands) and xylaxine (20 mg/kg BW; Sigma-Aldrich, USA). Blood samples were obtained from the orbital sinus and stored on ice for 1 h before centrifugation for 15 min at 14,000 g. To obtain cortical homogenates, mice were perfused with cold sterile PBS and the brains were immediately dissected and snap frozen in 2-methylbutan on dry ice. A Dounce homogenizer was used to prepare brain homogenates in N-PER neuronal extraction reagent (1 g brain tissue per 5 ml N-PER reagent; Thermo Fisher Scientific, USA) containing 13.3% protease inhibitor cocktail (Sigma-Aldrich, USA). The brain homogenates were incubated on ice for 10 min, before centrifugation for 10 min at 10,000 g and 4°C. The supernatants were collected and the protein concentration determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA), according to the manufacturer’s guidelines. ELISA Kits (Quantikine® ELISA, R & D systems, USA) were used to measure the cytokine levels of IL-1β (sensitivity 4.8 pg/ml), TNF-α (sensitivity 7.21 pg/ml), MCP-1 (sensitivity 2 pg/ml), IL-6 (sensitivity 1.8 pg/ml) and IL-10 (sensitivity 5.22 pg/ml). A total volume of 100 µl of the serum samples or brain homogenates was used, according to the manufacturer’s guidelines. Each sample was tested in duplicate. The chemiluminescent protein signal was measured as optical density (OD) using a plate reader (PowerWave XS2, BioTek, USA). Serum levels of MCP-1, IL-6 and IL-10 were determined by microBIOMix GmbH (Germany) with the Luminex xMAP™-Technology (www.microbiomix.de). 7

2.4 Immunohistochemistry 5 or 30 h after PBS/LPS injections, C57BL/6 mice were deeply anesthetized with a combination of ketamine (200 mg/kg BW) and xylazine (20 mg/kg BW) and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA; Roth, Germany). Brains were dissected and post-fixed overnight in 4% PFA at 4°C. After washing the brains 3 x 10 min with PBS, they were dehydrated in 25% sucrose in PBS for 12 h at 4°C and stored at -80°C until further use. The staining procedure was performed on free-floating brain slices at room temperature. The brain tissue was cut into 50 µm sagittal sections using a cryostat (Leica, Germany) and washed 3 x 10 min with PBS. For co-labeling of Iba-1 and CD68, the slices were treated with a blocking solution (5% normal donkey serum and 1% Triton-X in PBS) for 1 h before incubation with the primary anti-Iba-1 (1:500; Wako, USA) and anti-CD68 (1:1,000; Abd Serotec, UK) antibodies overnight at room temperature. After rinsing the slices 3 x 10 min in PBS, they were incubated in a solution containing Alexa-488 and Alexa-494 conjugated secondary antibodies (1:1,000 for both Alexa-488 and Alexa-594) for 2 h at room temperature. The slices were mounted on fluorescence-free Superfrost microscope glass slides (Langenbrinck, Germany) in Vectashield mounting medium (Vector laboratories, USA). For co-labeling of Iba-1 and IL-1β, the slices were treated with a blocking solution (10% normal donkey serum and 1% Triton-X in PBS) for 2 h before incubation with the primary anti-Iba-1 (1:3,000; Wako, USA) and anti-IL-1β (1:200; R & D Systems, USA) antibodies for 60 h at room temperature. In this case, the antibodies were diluted in a solution containing 0.04% NaN3. After washing the slices 5 x 10 min in PBS, they were 8

incubated for 3 h in a solution containing Alexa-488 and Alexa-494 conjugated secondary antibodies (1:2,000 for Alexa-488, 1:1,000 for Alexa-594). Slices were mounted on glass slides in Prolong Gold Antifade mounting medium (Thermo Fisher Scientific, USA).

2.5 Implantation of a chronic cranial window For longitudinal in vivo experiments, a chronic cranial window was installed above the frontal/motor cortex as described previously (Kovalchuk et al., 2015). Briefly, CX3CR1GFP/+ mice were deeply anesthetized with a combination of fentanyl (0.05 mg/kg BW; Eurovet Animal Health, Netherlands), midazolam (5 mg/kg BW; Hameln Pharma Plus, Germany), and medetomidin (0.5 mg/kg BW; Alfavet, Germany). Additionally, the mice received a subcutaneous injection of carprofen (5 mg/kg; Pfizer, USA) to provide a persistent analgesia and an i.p. injection of dexamethasone (4 mg/kg BW; Sigma-Aldrich, USA) to prevent swelling of the brain. After removing the fur from the area of surgery, the animals were placed on a warming plate under a dissecting microscope and fixed in a stereotactic frame. During surgery, body temperature was kept at 36-37°C and eye ointment was applied to prevent the eyes from drying out. The skin above the area of surgery was disinfected with a povidone-iodine solution (Braunol, B. Braun, Germany), and a local anesthetic (2% Xylocaine®, AstraZeneca, UK) was injected subcutaneously before the skin above the skull was removed. An area with a diameter of ~ 3 mm was thinned using a dental drill. Then, the skull was removed with forceps without damaging the dura. Cold Ringer solution (B. Braun, Germany) was used to rinse the opening of the skull, and a glass coverslip (3 mm diameter; Warner Instruments, USA) was used to cover the exposed brain tissue, centered 1.5 mm anterior and 1.5 mm lateral from bregma. The gap between the 9

coverslip and the skull was filled with cyanoacrylate glue (UHU, Germany) and then strengthened with dental cement (Ivoclar Vivadent, Liechtenstein). A custom made titanium holder for head fixation during two-photon imaging was embedded into the dental cement between the ears. At the end of surgery, mice received a subcutaneous injection of antidote containing flumazenil (0.5 mg/kg BW; Fresenius, Germany) and atipamezol 2.5 mg/kg BW (Alfavet, Germany). Postoperative care included subcutaneous injections of carprofen (5 mg/kg BW, Pfizer, USA) once per day for the next three days and the antibiotic Baytril® (1:100 v/v; Bayer, Germany) in drinking water for 10 days. After the surgery, animals were allowed to recover for 3-4 weeks.

2.6 Preparation of an acute cranial window Surgery was performed in C57BL/6, Iba-1GFP/+, NLRP3-/-, and TNF-α-/- mice as described previously (Brawek and Garaschuk, 2019; Brawek et al., 2019; Brawek et al., 2014; Eichhoff et al., 2011; Schwendele et al., 2012). Briefly, mice were anesthetized using isoflurane (2-2.5% in O2 for induction, 0.8-1.25 in O2 for surgery; CP-Pharma, Germany) and placed on a warming plate. During the procedure, body temperature was monitored and kept at 36-37°C. After subcutaneous injection of a local anesthetic (2% Xylocaine®, AstraZeneca, UK), the skin above the skull was removed. A recording chamber with a central hole was glued to the exposed skull using cyanoacrylate glue (UHU, Germany). The skull was carefully thinned using a dental drill. Afterwards, the mouse was placed on a warming plate in the imaging setup and fixed. The recording chamber was continuously perfused with pre-warmed (36-37°C) extracellular solution containing 125 mM NaCl, 4.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2 and 20 mM glucose, pH 7.4 when bubbled with 10

95% O2 and 5% CO2. A small craniotomy with a size of ~ 1 mm2 was cut above the frontal/motor cortex using a 30 G syringe needle without damaging the dura.

2.7 In vivo visualization of microglia using Tomato lectin After an acute craniotomy (see above) microglia in TNF-α-/- and NLRP3-/- were visualized using the Tomato lectin conjugated to DyLight-594 as described previously (Brawek et al., 2019; Schwendele et al., 2012). Briefly, TomatoLectin-DyLight-594 (Vector labs, USA) was dissolved to a concentration of 25 µg/ml in a standard pipette solution containing 150 mM NaCl, 2.5 mM KCl and 10 mM HEPES (pH 7.4), and the solution was filtered using a centrifugal filter (Merck, USA). The Tomato lectin-DyLight594 solution was pressure injected (30 s, 10-55 kPa) into the brain parenchyma 100 µm below the dura from a glass micropipette (diameter 1 µm) using a pressure application system (PDES-02D, NPI Electronic, Germany). After dye injection, the pipette was removed and the experiment commenced after a 20 min-long washout period.

2.8 Single-cell electroporation of microglia using Oregon Green BAPTA-1 For measurements of in vivo Ca2+ signaling, microglial cells were loaded with a Ca2+ sensor Oregon Green BAPTA-1 (OGB-1) using single-cell electroporation as described previously (Brawek and Garaschuk, 2019; Eichhoff et al., 2011). Briefly, GFP- or TomatoLectin-DyLight-594 labeled microglia were approached using a glass 11

micropipette (diameter < 1µm) filled with 10 mM OGB-1 hexapotassium salt (Invitrogen, USA) in a solution containing 175 mM potassium gluconate, 17.5 mM KCl, 5 mM NaCl and 12.5 mM HEPES (pH 7.3). As soon as the pipette touched the membrane, a negative current pulse (600 nA, 10 ms) was applied using a MVCS-02C iontophoresis system (NPI Electronic, Germany). After electroporation, the pipette was carefully withdrawn and the experiment commenced 10 min later to allow distribution of the dye within the cell. To verify the viability of cells, they were tested for the ability to generate Ca2+ signals in response to brief (50-100 ms) pressure applications of ATP (50 µM) or UDP (100 µM) after imaging of spontaneous activity. Only responding cells (84% of all cells tested) were included in the analysis.

2.9 In vivo labeling of astrocytes with a Ca2+ indicator Astrocytes were labeled with OGB-1 AM by means of multi cell bolus loading, as described previously (Garaschuk et al., 2006; Stosiek et al., 2003). Briefly, the Ca2+ indicator was dissolved in DMSO containing 20% Pluronic F-127 and further diluted using standard pipette solution (composition see above) to obtain a final concentration of 0.5 mM. The dye solution was pressure-injected (60 kPa for 2 min) into the brain parenchyma approximately 200 µm below the surface. After a washout period of 1 h, the dye was completely taken up by neurons and astrocytes. Astrocytes were easily distinguished from neurons based on their bright appearance and cell type-specific morphology.

2.10 ATP-evoked microglial process dynamics

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After performing an acute craniotomy (see above) in PBS-/LPS-injected Iba-1GFP/+ mice, a glass micropipette (diameter 1 µm) containing 5 mM ATP dissolved in standard pipette solution (composition see above) was inserted into cortical layer 2/3 close to the microglial cells of interest. To improve visibility of the pipette, Alexa-594 (100 µM) was added to the pipette solution. At the beginning of the imaging session, a short pressure pulse (50 ms, 30-35 kPa) was applied to release ATP from the pipette.

2.11 Two-photon imaging For in vivo imaging, mice were fixed in the imaging setup by either attaching the titanium holder (chronic experiments) or the recoding chamber (acute experiments) to the stage of the microscope. All imaging procedures were performed under light isoflurane anesthesia (0.8-1.5% in pure O2) using an upright laser-scanning microscope (FV300; Olympus, Japan) coupled to a mode-locked Ti:sapphire laser (Mai Tai, Spectra Physics, USA; 710-990 nm wavelength) equipped with a water-immersion objective (40x, 0.80 NA; Nikon, Japan). OGB-1, DyLight-594 and Alexa-594 were excited at 800 nm. GFP was excited at 900 nm. However, if GFP was recorded in parallel with Sulforhodamine B, both dyes were excited at a wavelength of 870 nm. Emitted light was separated by a dichroic mirror at 580 nm. For recording of spontaneous microglial Ca2+ signaling 5 or 30 h after PBS/LPS injection, images were collected at a frame rate of 1 Hz with a zoom factor of 160 over a time period of 15 min. Astrocytic Ca2+ signals were recorded 5 h after PBS/LPS injection at a frame rate of 1 Hz with a zoom factor of 120 over a time period of 10 min. Microglial process dynamics was recorded in 4D (20 µm stack, 0.89 frames/s, step size 2 µm) with a zoom factor of 160 during a total time period of 13-16 min.

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For analysis of microglial morphology, CX3CR1GFP/+ mice with implanted cranial window were injected with Sulforhodamine B (10 µg/kg BW; Sigma-Aldrich, USA) to visualize the blood vessel pattern, used for the longitudinal alignment of data. Microglial morphology was assessed before as well as 5, 30, 55, and 72 h after PBS/LPS injection (see above) by acquiring z-stacks 50-200 µm below the dura with a zoom factor of 100. Unless otherwise indicated, all images are shown as maximum intensity projections (MIPs) of a 50-µm-thick image stack collected with a step size of 1 µm and averaged over 2 frames. Microglial cells were identified in vivo as recently proliferating using “doublet technique” (Askew et al., 2017). Two neighboring cells were identified as doublets if the three-dimensional soma-to-soma distance between two cells was ≤ 20 µm. Of note, similar proliferation rates were found by the authors using either doublet imaging in vivo or Iba1/BrdU staining in vitro (Askew et al., 2017). For imaging immunofluorescent brain sections all fluorophores used (e.g. Alexa-488, Alexa-594) were excited at a wavelength of 800 nm, and emitted light was separated with a dichroic mirror at 570 nm. Additionally, a 470/100 nm bandpass filter as well as a 586 longpass filter were used. All images were acquired as 25-µm-thick z-stack images with a step size of 1 µm and a zoom factor of 100.

2.12 Data analysis 2.12.1 ELISA Analysis

was

performed

using

the

ElisaAnalysis

software

(http://www.elisaanalysis.com/). The mean optical density measured from the two sample duplicates was used to estimate the cytokine concentration of the specific sample. Concentrations were calculated by generating a cytokine calibration curve with 14

a 4 parameter logistic curve fitting. For brain samples, obtained concentrations were normalized to the total protein concentrations in the sample.

2.12.2 Immunofluorescent staining The CD68-specific fluorescence in the somata of Iba-1 positive cells was evaluated using the “GECIquant” macro in ImageJ (http://imagej.nih.gov/ij/). Briefly, the channels were split and the somata of Iba-1-stained cells were detected as regions of interest (ROIs) after background subtraction with the “ROI detection” module in the Alexa-488 channel. Afterwards, the intensity of the CD68 staining was measured in these ROIs in the background subtracted z-stacks of the Alexa-594 channel. Here and below, cells located at the borders of the image were excluded from the analysis. The fraction of the IL-1β positive microglia (Iba-1-positive cells) was analyzed using the “Cell Counter” macro in ImageJ. Microglia were considered to be IL-1β positive if the background subtracted IL-1β signal reflected a clear morphology of the microglial cell.

2.12.3 Analysis of microglial morphology Microglial cell number was determined using the “Cell Counter” macro in ImageJ. Microglial soma volume and distance between individual cells were analyzed using the “3D Object Counter” macro in ImageJ. Briefly, a threshold value was determined based on the fluorescence intensity of the MIP image to enable the detection of microglial somata without contribution of processes. This threshold was determined as mean 15

grey value of the image plus 3-6 times the standard deviation and was kept constant for a series of z-stacks imaged at different time points after PBS/LPS injection in the same animal. Microglia soma volumes at the different time points after PBS/LPS injection were normalized to the soma volumes before PBS/LPS injection. Distances between the cells were measured between the centers of individual cells.

2.12.4 In vivo analysis of spontaneous Ca2+ signaling ROIs delineating microglial or astrocytic somata were manually drawn in ImageJ and the mean intensity values were measured over time. The relative change of fluorescence (ΔF/F) was calculated after background subtraction in Igor Pro (http://www.wavemetrics.com). A change in ΔF/F was considered a Ca2+ transient if its amplitude was higher than three (microglia) or five (astrocytes) times the standard deviation of background noise.

2.12.5 In vivo analysis of microglial process extension The velocity of microglial processes moving towards an ATP-pipette was quantified using the “MTrackJ” macro in ImageJ. GFP-labeled processes were tracked over time and the corresponding mean velocity was determined. When microglial processes converged around the tip of the pipette, the diameter of the containment remained stable over time, and the mean of these values was read out as the final diameter.

2.13 Statistical analysis

16

Statistical analyses were performed using GraphPad Prism 7 (GraphPad, USA), MATLAB (The Mathworks, USA), and JASP (https://jasp-stats.org/). If not otherwise indicated, data are shown as box-and-whisker plots with boxes from the 75th percentile to 25th percentile and whiskers representing 90th and 10th percentile. Data sets were tested for normality using the Shapiro-Wilk test. Multiple comparisons of repeated measurements were performed with repeated measures ANOVA with a GreenhouseGeisser correction in case of violation of the assumption of sphericity. A post-hoc Bonferroni correction was used for multiple comparisons. The Kruskal-Wallis test followed by a post-hoc Dunn’s correction for multiple comparisons was used for the comparison of data that was not normally distributed. The Wilcoxon Signed-rank test was used to compare pairs of repeated measurements. The Mann-Whitney test was used to compare two independent datasets. The Fisher’s exact test was used to compare categorical data. The two-sample Kolmogorov-Smirnov test or MANOVA (Zhang, 2011) was used to compare distributions. Differences were considered as significant if p-values were below 0.05.

3. Results 3.1 Time course of the mirror inflammatory response in the brain following peripheral LPS injection As already mentioned above, changes in microglial morphology are well known consequences of peripheral inflammation (Gyoneva et al., 2014; Kozlowski and Weimer, 2012) and were previously used as the main determinant of the microglial 17

functional state (Norden et al., 2016). Therefore, we established the temporal framework of our experiments based on changes in microglial morphology using CX3CR1GFP/+ mice, as this mouse strain was also used in the studies mentioned above (Gyoneva et al., 2014; Kozlowski and Weimer, 2012). To do so, in longitudinal in vivo experiments soma volume of individual microglial cells was analyzed in CX3CR1GFP/+ mice before, as well as 5, 30, 55, and 72 h after a single peripheral injection of 1.5 mg/kg LPS, inducing moderate systemic inflammation (see methods for details). Here and below, PBS (phosphate buffered saline)-treated mice were used as controls. In control mice, microglial cells exhibited a regular morphology, characterized by small somata and ramified processes, throughout the entire experiment. At all time points after PBS injection soma volume of individual cells was similar to that encountered before injection (Fig. 1A and B). In contrast, we detected a considerable variability in microglial morphology after LPS injection. The enlargement of cell somata was especially pronounced 30 and 55 h after LPS injection (Fig. 1A and B). It has been suggested that inflammatory processes in the brain can be accompanied by edema due to an increase in the permeability of the BBB (Banks, 2015; Jangula and Murphy, 2013). Although we never observed a leakage of Sulforhodamine B, which was used to label blood vessels (Fig. 1A), into the brain parenchyma, in some animals visibility of the GFP-labeled cells through the cranial window 5 h after LPS injection was compromised, as suggested by a slight non-significant decrease in soma size (Fig. 1B). To avoid such confounding factors we repeated the analysis of microglial soma volume in fixed brain slices of WT animals that were sacrificed 5 and 30 h after either PBS or LPS injection. As expected from our in vivo data, microglial cells showed no detectable difference between PBS- and LPS-injected mice 5 h after treatment (Fig.

18

1C and D). However, 30 h after induction of inflammation the soma volume increased significantly (Fig. 1C and E), confirming our in vivo results. Inflammatory processes might lead to microglial proliferation, which is an integral part of the activation process under various conditions (Fuger et al., 2017; Fukushima et al., 2015; Shankaran et al., 2007). To test if proliferation takes place under our experimental conditions, we first quantified in longitudinal experiments the number of microglial doublets (cell-to-cell distance ≤ 20 µm, Suppl. Fig. S1A), an indicator of recent cell division in vivo (Askew et al., 2017), before as well as 5, 30, 55, and 72 h after peripheral PBS/LPS injection. As shown in Suppl. Fig. S1B, there was no increase in cell doublets after PBS injection, ruling out any effects of repeated imaging or i.p. injections of Sulforhodamine B. In contrast, we observed an increase in the number of doublets after LPS injection, which was especially pronounced at later time points (Suppl. Fig. S1C). Due to a variable reaction of individual mice to LPS these changes, however, did not reach the level of statistical significance. Similar data were obtained when estimating microglial cell density, which was stable in control conditions and showed a slight non-significant increase after injection of LPS (Suppl. Fig. S1D and E). As expected, 5 h after LPS injection we encountered high levels of inflammationinduced cytokines IL-1β, TNF-α, IL-6, MCP-1, and IL-10 both in the serum (Suppl. Fig. S2A-E) and in the brain (Suppl. Fig. S2F-J). In contrast, 30 h after LPS injection the levels of many of these inflammatory mediators dropped to almost control levels (Suppl. Fig. S2A-J). This underscores the bell-shaped profile of the peripheral and central levels of (proinflammatory) cytokines in the course of peripheral inflammation. In summary, the above described data suggest that under our experimental conditions 5 h after LPS injection correspond to an early initiation phase of the inflammation (high levels of inflammatory cytokines in the serum and brain, no alteration of microglial 19

morphology) whereas 30 h after LPS injection correspond to an advanced resolution phase (low levels of inflammatory cytokines in the serum and brain, altered microglial morphology and mild proliferation of microglia) (Kozlowski and Weimer, 2012; Norden et al., 2016).

3.2 Potentiation of microglial Ca2+ signaling during the early initiation phase Next, we analyzed in Iba-1GFP/+ mice in vivo spontaneous microglial Ca2+ signaling (Fig. 2), a known marker of the activated state (see above). 5 h after LPS injection, with microglia still retaining their ramified morphology with small somata and highly branched processes (Fig. 2A), we detected a profound and significant increase in the fraction of spontaneously active microglial cells (Fig. 2B and C). Whereas under control conditions (5 h after PBS injection) only 14% of microglia showed spontaneous Ca2+ transients during the 15-min-long recording period (see also (Brawek et al., 2014; Eichhoff et al., 2011)), the picture looked almost inverted 5 h after LPS injection (Fig. 2C). Here, 79% of microglia were spontaneously active. Surprisingly, 30 h after LPS injection, i.e. at the time when microglia started to alter their morphology (Fig. 1), the microglial Ca2+ signaling was already decreasing (43% of spontaneously active cells, Fig. 2C). Astrocytes are also endowed with the ability to communicate with the immune system (Jensen et al., 2013) and were previously shown to react with Ca2+ transients to cell damage in their microenvironment (Eichhoff et al., 2011). Therefore, we recorded spontaneous Ca2+ transients in cortical astrocytes 5 h after either PBS or LPS injection. In contrast to data obtained in microglia, we did not observe any change in the fraction of astrocytes showing spontaneous Ca2+ transients during the 10-min-long recording 20

period in LPS-treated mice (Fig. 2D, E and F). We concluded, therefore, that the ability to respond with Ca2+ transients to the initiation of peripheral inflammation is a characteristic feature of microglia, and they don't share this property with other glial cells. Because an increase in microglial Ca2+ signaling coincided in time with a massive presence of inflammatory mediators, in the next series of experiments we examined the causal relationship between the two events. To this end, we used two different mouse strains in which the production of either IL-1β or TNF-α is compromised. First, we used NLRP3-/- mice. In microglia the NLRP3 (NOD-, LRR- and pyrin domaincontaining 3) inflammasome is the key innate immune sensor for danger signals causing the caspase-1-dependent maturation of IL-1β (Heneka et al., 2018), but this inflammasome is also expressed in peripheral cells like tissue macrophages and dendritic cells (Elliott and Sutterwala, 2015; Strowig et al., 2012). Second, we used TNF-α-/- mice, in which the production of TNF-α is completely abolished (Pasparakis et al., 1996). To start with, we characterized the cytokine profiles induced by peripheral LPS injection in these two knockout strains and compared them to the situation in WT mice. Serum levels of IL-1β and IL-6 as well as brain levels of IL-1β, TNF-α, and IL-6 in LPS-injected NLRP3-/- mice were reduced when compared to WT mice, albeit to variable extent (Suppl. Fig. S3). Surprisingly, serum levels of TNF-α in NLRP3-/- mice were significantly higher compared to LPS-injected WT mice (Suppl. Fig. S3B). In LPSinjected TNF-α-/- mice, peripheral and central levels of all measured cytokines were lower than in WT mice (Suppl. Fig. S4A, B). Of note, in NLRP3-/- mice the fraction of in vivo microglia showing spontaneous Ca2+ transients 5 h after LPS injection was significantly reduced compared to LPS-injected control (Iba-1GFP/+) mice and did not differ significantly from the fraction observed in 21

PBS-injected control mice (Fig. 2G). In contrast, the fraction of spontaneously active microglia in the brains of TNF-α-/- mice was higher, still significantly enhanced in comparison to that encountered in PBS-injected control mice and not significantly different from the one measured in LPS-injected control mice (Fig. 2G). In summary, our data identify increased microglial Ca2+ signaling as a reliable biomarker of the early initiation phase of peripheral inflammation and point to activation of the NLRP3-/- inflammasome as the main underlying mechanism.

3.3 Consequences of increased Ca2+ signaling in microglia We next analyzed possible functional consequences of the observed increase in microglial Ca2+ signaling, i.e. production and release of cytokines, e.g. IL-1β (Brough et al., 2003; Lee et al., 2012; Murakami et al., 2012), phagocytosis (Koizumi et al., 2007), and cytoskeletal rearrangement (Madry and Attwell, 2015). Using immunocytochemistry, IL-1β producing microglia were selectively detected in the cortex of LPS- but not of PBS-injected mice (Fig. 3A). In LPS-treated mice the fraction of IL-1β positive cells started to increase already 5 h after LPS injection but reached the level of statistical significance 30 h after injection (Fig. 3B). Still, even at this time point IL-1β positive cells represented a minority of cortical microglia. Moreover, IL-1β positive cells were unevenly distributed throughout the brain parenchyma, suggesting that microglial cells are functionally heterogeneous. Interestingly, all IL-1β positive cells were also Iba-1 positive, we never observed any IL-1β positivity in other cells (e.g. astrocytes). Next, we analyzed somatic expression of the lysosomal protein CD68, to test for any signs of increased phagocytosis. However, microglial CD68 expression was similar in PBS- and LPS-injected mice 22

during the early initiation as well as the advanced resolution phase of the inflammatory reaction (Suppl. Fig. S5). Finally, we studied microglial process extension, a well-known functional response of these cells to the presence of danger signals in their vicinity (Fig. 4A). As it has recently been shown that the process velocity towards the tip of a pipette filled with ATP in young CX3CR1GFP/+ mice depends on the initial distance of the respective process to the pipette tip, reflecting a high coordination between the individual processes (Olmedillas Del Moral et al., 2019), we analyzed whether this dependence is altered in our Iba-1GFP/+ LPS-injected mice. To do so, we determined the averaged velocity of single microglial processes (7-47 processes per area) during ATP-induced process movement and plotted these values against the initial distance of the respective process to the tip of the pipette (Fig. 4B). Then, we created contour plots reflecting the bivariate normal density distribution of the data points (ellipsoid contour lines in Fig. 4B), and estimated the mean values of both parameters (initial distance and process velocity) in all data sets (red dashed lines in Fig. 4B). As shown in Fig. 4B, in PBSinjected mice there seemed to be a linear relationship between process velocity and the distance to the pipette tip, although, likely due to the lower and more heterogeneous brightness of individual processes and/or slightly older age of experimental animals, the linearity was not so tight as seen previously in CX3CR1GFP/+ mice (Olmedillas Del Moral et al., 2019). Interestingly, we did observe small but significant differences when comparing 2-dimensional distributions obtained 5 and 30 h after PBS injection, with 30 h PBS data showing on average a somewhat increased process velocity and somewhat decreased initial distance to the pipette tip. Similarly, small but significant changes were also observed between PBS and LPS injection groups 5 h after the induction of inflammation. Here, the mean initial distance to the 23

pipette tip was largely unchanged, but processes seemed to move on average somewhat faster. However, the changes were much more pronounced in the data recorded 30 h after LPS injection. Here, both the mean initial distance to the pipette tip and the mean process velocity were clearly increased (Fig. 4B, lower right panel). Furthermore, the process movement appeared much less coordinated, with individual processes moving at any speed independent of their initial location. To control for interindividual variability we next analyzed the data on a per mouse basis (Fig. 4C-E). Consistent with data shown in Fig. 4B, microglial process velocity showed a tendency to increase already during the initiation phase but became significantly different between PBS- and LPS-injected mice during the advanced resolution phase of inflammation (Fig. 4C). Similarly, the initial distance between the microglial processes and the pipette tip became significantly larger 30 h after LPS injection (Fig. 4D), likely reflecting the activation-mediated reduction in the length of microglial processes and, as a consequence, the reduction in the degree of coverage of the brain parenchyma. The final diameter of the containment formed by the microglial processes around the pipette tip did not differ significantly between PBS- and LPS-treated mice (Fig. 4E). In summary, our data show that specific effector functions of microglia, including IL-1β production and the targeted process extension, are induced or accelerated in the course of a moderate inflammatory reaction, whereas others, like increased expression of the lysosomal protein CD68, indicative of increased phagocytosis, are not affected. Interestingly, these effector functions are selectively altered in the advanced resolution phase of inflammation.

4. Discussion

24

This in vivo study characterizes functional states of microglia during a moderate systemic inflammation. Based on their morphological and functional properties, we distinguish “sensor microglia”, found during the initiation phase of inflammation and characterized by profoundly increased spontaneous Ca2+ signaling but ramified morphology, as well as “effector microglia”, present during the resolution phase of systemic inflammation and characterized by hypertrophic morphology, IL-1β production, and increased mobility of their processes (Fig. 5). Hereby, microglial [Ca2+]i is identified as the earliest and a highly reliable biomarker of systemic inflammation. Indeed, as previously shown in vivo by us and others, under physiological conditions [Ca2+]i in microglia is rather stable and low (Brawek et al., 2017; Brawek et al., 2014; Eichhoff et al., 2011; Olmedillas Del Moral et al., 2019; Pozner et al., 2015), in line with our present data obtained in PBS-injected mice (Fig. 2C). If the damage(danger)associated molecular pattern (DAMP) signal is brief and local, as in case of a damage of a single cell, the change in [Ca2+]i is also brief and local, restricted to the microglia residing in the immediate vicinity of a damaged cell (Eichhoff et al., 2011). If the DAMP signal is local but persisting, as in the case of parenchymal accumulation of amyloid β, the nature of the stimulus is also reflected in microglial Ca2+ signaling, which now becomes long-lasting and fluctuating but still localized, restricted to the vicinity of amyloid plaques (Brawek et al., 2014). Finally, if the DAMP signal is prolonged and widespread, as in the case of aging (Olmedillas Del Moral et al., 2019) or systemic inflammation (this study), the increase in microglial Ca2+ signaling is also prolonged and widespread. The observed reduction in the intensity of the population Ca2+ signal (i.e. the number of cells involved) 30 h after LPS injection suggests that by this time the underlying DAMP signal gets weaker and the organism moves over to the recovery phase. Taken together, the data show that somatic microglial Ca2+ signals are not 25

spontaneous but DAMP-triggered and firmly identify the “sensor” function of this signal reflecting the strength, the location and the duration of the damage/danger-associated event. The molecular mechanisms underlying the various DAMP-induced Ca2+ transients described above are likely to differ. In the case of peripheral inflammation, based on our data obtained in TNF-α-/- and NLRP3-/- mice we assume that microglia are detecting soluble mediators like IL-1β, IL-6, TNF-α, IL-10, and MCP-1, known to possess receptors on microglia (Kettenmann et al., 2011). According to in vitro data, activation of receptors for IL-1β and MCP-1 induces an increase in microglial [Ca2+]i (Boddeke et al., 1999; Goghari et al., 2000; Kettenmann et al., 2011), thus providing a direct mechanistic link to the “sensor” signal. Because LPS-mediated increases in the concentration of peripheral as well as brain-derived cytokines were observed as early as 1-2 hours after LPS injection (Biesmans et al., 2013; Qin et al., 2008), we hypothesize that by this time DAMP-triggered somatic microglial Ca2+ signals also become visible. In contrast, astrocytes, which are also immune-competent and in principle able to sense DAMP signals within the brain by means of Ca2+ signaling (Colombo and Farina, 2016; Eichhoff et al., 2011), did not show any alterations in [Ca2+]i in our study. However, as astrocytes were only studied 5 h after LPS injection, we cannot exclude the possibility that changes in astrocytic Ca2+ signaling occur later, during the resolution of inflammation. Indeed, whereas the LPS-induced changes in mRNA levels of astrocytic IL-6 and IL-1β were much smaller than the ones observed in microglia, the changes in mRNA levels of TNF-α and MCP-1 were large but delayed, peaking 1224 h after LPS injection (Norden et al., 2016). In any case, these data identify microglia

26

as the first line immune-competent brain cell sensing the initiation of peripheral inflammation. Interestingly, the LPS-induced increase in microglial Ca2+ signaling occurred without a significant change in morphology, as active cells had small cell somata with long elaborate processes resembling “resting” microglia. These results provide additional evidence that morphology per se is an unreliable readout of the functional state of microglia (Cunningham et al., 2005; Norden et al., 2016). Moreover, as remodeling of the actin cytoskeleton is a Ca2+-dependent process (Bader et al., 1994; Madry and Attwell, 2015), morphological alterations of microglia are likely to be a consequence of alterations in [Ca2+]i. In this way, changes in microglial [Ca2+]i likely act as a bridge connecting receptive and executive functions of these cells. Here, we investigated several microglial functions known to be linked to alterations in [Ca2+]i: proliferation (Hooper et al., 2005; Moller et al., 2000), phagocytosis (Koizumi et al., 2007), IL-1β production, strongly associated with the activation of the NLRP3 inflammasome (Lee et al., 2012; Murakami et al., 2012), and cytoskeletal rearrangements necessary for process extension (Bader et al., 1994; Madry and Attwell, 2015). During the resolution phase of the peripheral inflammation, we detected a tendency towards higher numbers of microglial doublets, indicative of some proliferative activity. These data aligns well with previous finding showing that during moderate peripheral inflammation considerable proliferation of microglia is rather restricted to deeper brain regions,

especially

those

located

in

the

immediate

neighborhood

of

the

circumventricular organs (Dantzer et al., 2008; Furube et al., 2018), the substantia nigra (Yang et al., 2013) and the hippocampus (Fukushima et al., 2015), and is negligible in the cortex (Chen et al., 2012; Furube et al., 2018). Similarly, together with other studies (Cazareth et al., 2014; Kozlowski and Weimer, 2012; Tejera et al., 2019), 27

our results indicate that moderate systemic inflammation is not accompanied by microglial phagocytosis, as suggested by constant levels of the lysosomal protein CD68. We therefore conclude that under our experimental conditions the increased Ca2+ signaling in cortical microglia is driving neither a profound proliferation nor an increase in phagocytic activity of these cells. In the healthy brain, endogenous levels of inflammatory cytokines are generally low (Allan et al., 2005). Consistently, we did not find any IL-1β positive cells in PBS-injected mice. In LPS-injected mice, however, IL-1β-positive microglia were seen already during the early initiation phase (Fig. 3) and became even more numerous during the resolution phase of the peripheral inflammation. Microglial IL-1β reactivity in the course of a peripheral inflammation has been previously described in areas close to the circumventricular organs, where it was implicated in the fever response (Konsman et al., 1999). In rat cortical microglia, IL-1β was detected 8 h after intraperitoneal LPS injection on the mRNA (Eriksson et al., 2000) and protein level (Van Dam et al., 1995). In the latter study, IL-1β was predominantly found in microglial cell clusters around blood vessels. This is in contrast to our results as IL-1β-positive microglia in this study still occupied their own territory, indicating a weaker activation state. In fact, the inflammatory stimulus used in our experiments did not induce any clustering of cortical microglia. Microglial IL-1β is first produced in an inactive pro-form and the formation of the NLRP3 inflammasome is necessary for maturation of this protein (Heneka et al., 2018). Both the formation of NLRP3 inflammasome (Lee et al., 2012; Murakami et al., 2012) as well as the release of IL-1β (Brough et al., 2003) are known to be Ca2+dependent. Hence, it is tempting to speculate that rises in [Ca2+]i observed in this study promote IL-1β maturation and/or release. Indeed, components of the NLRP3 inflammasome are up-regulated in microglia after intraperitoneal LPS injection (Zhang 28

et al., 2014) and a mixture of cytokines (IL-1β, TNF-α, Interferon-γ) is able to induce NLRP3 expression in microglia in vitro (Gustin et al., 2015). Interestingly, morphological activation of microglia, induced by systemic inflammation, is abolished in NLRP3-/- mice (Tejera et al., 2019). This suggests that NLRP3 formation is critical not only for IL-1β production but also for typical inflammation-induced change in microglial morphology. Together with our data showing that the early increase in microglial [Ca2+]i is inhibited in NLRP3-/- mice, these data strongly suggest a causal relationship between microglial Ca2+ signaling, NLRP3-/- formation and morphological remodeling of activated microglia. Noteworthy, all IL-1β-positive cells identified in our study were also Iba-1 positive. Together with studies showing that in vitro stimulation with TNF-α or IL-1β triggers IL1β production in purified microglia but not astrocytes (Lee et al., 1993), and the lack of a functional NLRP3 inflammasome in astrocytes (Gustin et al., 2015), this suggests that microglia are the predominant IL-1β producing cell type in the murine brain. However, not all microglia in our study were IL-1β-positive, suggesting that during the inflammatory challenge the microglial population does not behave uniformly. This observation is consistent with a transcriptome analysis confirming the existence of specific microglial subpopulations (Deczkowska et al., 2018; Stratoulias et al., 2019) and a profound heterogeneity of microglia isolated from the brains of LPS-injected mice (Sousa et al., 2018). Directed movement of microglial processes towards the source of the DAMP signal with its subsequent insulation from the brain parenchyma represents an important neuroprotective function of microglia (Davalos et al., 2005; Hanisch and Kettenmann, 2007). In our study, microglia, affected by systemic inflammation, exhibited a pronounced increase in the velocity of directed process extension (Fig. 4). This 29

increase was already apparent during the initiation phase of inflammation and became even more profound during the resolution phase, indicating an increased microglial alertness triggered by the inflammatory insult. These results are in contrast with previous studies reporting a decrease in velocity of directed process extension in LPSinjected mice (Gyoneva et al., 2014; Pozner et al., 2015). However, the authors of both studies used a laser-induced lesion of the tissue to provoke process extension in microglia, which is generally more destructive than minute insults occurring under physiological conditions. Furthermore, in contrast to our experiments, in which a parenchymal purine microgradient was used to mimic DAMPs (Deczkowska et al., 2018), different factors released from the tissue lesion site likely activate multiple signaling pathways. Indeed, the microglial response towards a laser-induced tissue lesion is much faster and less coordinated than the reaction to a point source of an agonist for P2Y12 receptor, the receptor that is predominantly involved in this process (Avignone et al., 2015). In a model of status epilepticus, directed microglial process movement towards a laser lesion was not affected, whereas the velocity of the process movement towards a point source of ATP analog was markedly increased (Avignone et al., 2015). Together, these data suggest that microglia in the same activation state react differently to different DAMP signals. Interestingly, faster microglial process extension towards ATP or its analogs was observed in brains suffering from aging (Olmedillas Del Moral et al., 2019), epilepsy (Avignone et al., 2015; Avignone et al., 2008) or amyloid-induced inflammation (Brawek et al., 2014), indicating that microglia in the inflamed brain react faster to minor tissue injury. A property, which is likely to be beneficial for the restoration of tissue homeostasis. At the same time, the LPS-induced retraction of microglial processes, leaving tissue gaps without microglial coverage, as well as the loss of coordination 30

between the individual processes (Fig. 4B), are less beneficial and point towards the more dysfunctional microglia. In summary, microglia have a dual dynamic role in the course of systemic inflammation (Fig. 5). During the initiation phase of the peripheral inflammation they adopt a “sensor” state, characterized by preserved morphology but significantly increased Ca2+ signaling, whereas during the resolution phase they switch to an “effector” state, characterized by reactive morphology, IL-1β production, and the enhanced velocity of directed process movement. Hereby, altered microglial Ca2+ signaling might represent the central element connecting receptive and executive functions of microglia.

Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgements We thank A. Weible, G. Heck, and K. Schöntag for technical assistance. This work was partially supported by VolkswagenStiftung Grant 90233 to O.G.

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Figure legends Fig. 1. Morphological activation of microglia during peripheral inflammation. A

Maximum intensity projection (MIP) images showing GFP-labeled microglial

cells (green) in vivo, in the cortex of PBS-treated (upper panels) and LPS-treated (lower panels) CX3CR1GFP/+ mice before as well as 5, 30, 55, and 72 h post-injection. Sulforhodamine B labeling of blood vessels (red) was used as a landmark for the identification of the same microglial cells over time. Scale bars: 10 µm. B

Box-and-whisker plots displaying median (per mouse) soma volume of microglia

after treatment with PBS (upper panel) and LPS (lower panel; n = 5 mice per group). Soma volume of a given cell at different time points was normalized to the soma volume of the respective cell before treatment. Whereas there was no change in soma volume after PBS injection (F1.481 = 2.190, p = 0.194, ANOVA with a GreenhouseGeisser correction), it was significantly increased 30 h after LPS injection (F4 = 5.374, p = 0.006, ANOVA; p = 0.03, post-hoc Bonferroni correction for multiple comparisons). C

MIP images showing cortical microglia in fixed brain slices, obtained from WT

mice 5 h (left panels) and 30 h (right panels) after PBS or LPS injection, labeled with an antibody against Iba-1 (green). Scale bar: 10 µm. D, E

Cumulative probability histograms illustrating distributions of microglial soma

volume in fixed brain slices of PBS- (blue) and LPS-treated (red) mice 5 h (D) and 30 h (E) post-injection. 30 h after LPS injection, soma volume of LPS-treated mice was significantly shifted to higher values (p < 0.001; Kolmogorov-Smirnov test; PBS: 121 cells, LPS: n = 109 cells), whereas there was no difference between PBS- and LPSinjected animals 5 h after treatment (p = 0.198; Kolmogorov-Smirnov test; PBS: 120 cells, LPS: n = 116 cells). Insets: Box-and-whisker plots depicting median (per mouse) 36

soma volume of cortical microglia 5 h (D) and 30 h (E) after PBS or LPS injection (n = 6-10 areas per mouse in 5 mice per group). Soma volume was significantly larger in microglia 30 h after LPS injection when compared to microglia after PBS injection (p = 0.012; Mann-Whitney test), whereas there was no significant difference in soma volume 5 h after LPS and 5h after PBS injection (p = 0.92; Mann-Whitney test).

Fig. 2. Cytokine-dependent transient up-regulation of in vivo Ca2+ signaling in microglia but not in astrocytes. A

MIP image of a microglial cell loaded with OGB-1 by means of single cell

electroporation in the cortex of an Iba-1GFP/+ mouse 5 h after peripheral LPS injection. Scale bar: 10 µm. B

Representative spontaneous Ca2+ transients recorded in vivo from the microglial

cell shown in (A). C

Pie charts showing the fractions of spontaneously active (red) and inactive

(green) microglial cells in Iba-1GFP/+ mice 5 h after PBS (left; n = 22 cells in 5 mice) or LPS (middle; n = 14 cells in 5 mice) injection as well as 30 h after LPS injection (right; n = 14 cells in 5 mice). The fractions of active and inactive cells were significantly different 5 h after LPS injection compared to those 5 h after PBS injection (p = 0.0002, Fisher’s exact test). 30 h after LPS injection, the fractions of active and inactive cells were not significantly different from those 5 h after PBS or LPS injection (30 h LPS vs. 5 h PBS: p = 0.111; 30 h LPS vs. 5 h LPS: p = 0.12, Fisher’s exact test). D

MIP image of cortical astrocytes and neurons labeled in vivo with OGB-1 by

means of multi cell bolus loading 5 h after peripheral LPS injection in a WT mouse. Scale bar: 10 µm. 37

E

Representative somatic Ca2+ recordings obtained in vivo from astrocytes

marked with respective numbers in (D) 5 h after peripheral LPS injection. F

Box-and-whisker plot showing the fractions of active astrocytes (per mouse) 5

h after PBS (n = 69 cells in 4 mice) or LPS (n = 57 cells in 4 mice) injection. The fraction of active cells was not significantly different between the groups (p > 0.05; MannWhitney test). G

Summary bar graph comparing the fraction of spontaneously active microglial

cells 5 h after PBS or LPS injection in Iba-1GFP/+, NLRP3-/-, and TNF-α-/- mice. For Iba1GFP/+ mice the same data set as the one shown in (C) is used. Fractions of spontaneously active cells were significantly different in LPS-treated NLRP3-/- mice (n = 20 cells in 5 mice) compared to respective Iba-1GFP/+ mice (p = 0.002; Fisher’s exact test) and resembled those measured in PBS-injected Iba-1GFP/+ mice (p = 0.445; Fisher’s exact test). In TNF-α-/- mice (n = 18 cells in 6 mice) fractions of active cells were significantly different when compared to PBS-treated Iba-1GFP/+ mice (p = 0.018; Fisher’s exact test) but similar to those measured in LPS-treated Iba-1GFP/+ mice (0.147; Fisher’s exact test).

Fig. 3. Microglial IL-1β production is up-regulated 30 h after induction of a peripheral inflammation. A

MIP images obtained in brain slices of WT mice fixed 5 or 30 h after either PBS

or LPS injection and stained with anti-IL-1β (red) and anti-Iba-1 (green) antibodies. Arrowheads point to IL-1β positive microglia, which could only be detected in LPSinjected mice. Scale bar: 10 µm.

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B

Box-and-whisker plot showing the median (per mouse) fraction of IL-1β positive

microglial cells 5 h and 30 h after PBS or LPS injection (n = 15 areas per mouse in 5 mice per group, except 30 h after LPS: n = 9 mice per group). Compared to PBSinjected mice, the fraction of IL-1β positive microglia started to increase already 5 h after LPS injection. However, this increase did not reach the level of statistical significance (p < 0.001, Kruskal Wallis test; p > 0.05, post-hoc Dunn’s test for multiple comparisons). However, the fraction of IL-1β positive microglia was significantly higher 30 h after LPS injection when compared to the corresponding PBS control (p ≤ 0.01; post-hoc Dunn’s test for multiple comparisons).

Fig. 4. Process velocity in microglia is enhanced 30 h after induction of peripheral inflammation. A

MIP images showing GFP-labeled microglia (green) in an Iba-1GFP/+ mouse

recorded 0 min (left), 7 min (middle) and 14 min (right) after a brief pressure application (50 ms, 30-35 kPa) of 5 mM ATP and 100 µM Alexa-594 (red) 30 h after LPS injection. Scale bar: 10 µm. B

Scatter plots showing the relationship between the initial distance of the

microglial process from the tip of the ATP-containing pipette and the average velocity of the respective process 5 h after PBS (left upper panel, 244 processes) or LPS (right upper panel, 304 processes) injection, as well as 30 h after PBS (left lower panel, 335 processes) or LPS (right lower panel, 439 processes) injection. Ellipses in the superimposed contour plots indicate levels of multivariate normal probability density function estimated from the original data. Dashed red lines indicate mean values of initial distance and process velocity, respectively. Statistical analysis revealed a 39

significant effect of time (5h/30h; F2, 1229.9 = 22.066, p < 0.001; two-way MANOVA) as well as treatment (PBS/LPS; F2, 1229.9 = 112.78, p < 0.001; two-way MANOVA) among the four groups. Pair-wise comparisons showed that there was a highly significant difference for all possible pairs (5h/PBS vs. 30h/PBS: F2, 485.4165 = 43.0488, p < 0.001; 5h/LPS vs. 30h/LPS: F2, 638.2587 = 29.1104, p < 0.001; 5h/PBS vs. 5h/LPS: F2, 474.3395 = 46.1718, p < 0.001; 30h/PBS vs. 30h/LPS: F2, 684.600 = 102.7704, p < 0.001; one-way Johansen’s MANOVA). C

Box-and-whisker plot comparing the mean (per mouse) process velocities 5 h

and 30 h after PBS or LPS injection (n = 5 mice per group). Compared to PBS-treated mice, 5 h after injection microglia in LPS-treated mice showed a tendency towards an increased process velocity (p = 0.002, Kruskal-Wallis test; p = 0.171, post-hoc Dunn’s test for multiple comparisons). This trend reached significance 30 h after injection (p = 0.039, post-hoc Dunn’s test for multiple comparisons). D

Box-and-whisker plot comparing the median (per mouse) initial distance

between the tip of the ATP-pipette and the microglial process 5 h and 30 h after PBS or LPS injection (n = 5 mice per group). Initial distance was significantly higher 30 h after LPS compared to 30 h after PBS injection (p = 0.014, Kruskal-Wallis test; p = 0.008, post-hoc Dunn’s test for multiple comparisons. E

Box-and-whisker plot comparing the median (per mouse) final diameter of the

spherical containment formed by microglial processes around the tip of the pipette 5 h and 30 h after either PBS or LPS injection (n = 5 mice per group). The final diameter was not significantly different between the groups (p = 0.095, Kruskal-Wallis test). Fig. 5. Microglial functional states at different phases of peripheral inflammation. A schematic drawing summarizing main findings of the present study. 40

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Highlights: - In vivo microglia adopt distinct functional states during systemic inflammation - Early sensor state is marked by increased Ca2+ signaling but ramified morphology - Late effector microglia produce IL-1β, change morphology and process motility - Cytosolic Ca2+ signaling likely links receptive and executive functions of microglia

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