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Nature Immunology doi:1.138/ni.2477 Supplementary Information Capillary and arteriolar pericytes attract innate leukocytes exiting through venules and instruct them with pattern recognition and motility programs Konstantin Stark, Annekathrin Eckart, Selgai Haidari, Anca Tirniceriu, Michael Lorenz, Marie-Luise von Brühl, Florian Gärtner, Alexander Georg Khandoga, Kyle R. Legate, Robert Pless, Ingrid Hepper, Kirsten Lauber, Barbara Walzog, and Steffen Massberg

Nature Immunology doi:1.138/ni.2477 Supplementary Video Legends Supplementary Video 1. NG2 + pericytes surrounding CD31 + endothelial cells. The video shows a whole mount staining of a microvascular vessel in the ear of an NG2DsRed mouse. NG2 + pericytes (red) in close contact to CD31 + endothelial cells (green), immunostained by a FITC labelled anti- CD31 antibody. Visualized by 2-photon microscopy. Still image is shown in Supplementary Figure 1d. Supplementary Video 2. Interaction of a CX3CR1 + macrophage with a microvascular pericyte during interstitial migration in vivo. The video shows a vessel in the ear skin of an NG2DsRed- CX3CR1eGFP chimera with an NG2 + pericyte (red) around the capillary, stained by i.a. injection of FITC-Dextran (green). The colocalization (yellow, arrow) is zoomed with decreased density in the green and red channel. Inflammation was induced by s.c. injection of TNF and intravital 2-photon microscopy was performed 4 hrs later. A CX3CR1 + macrophage (green, arrowhead) is interacting with a pericyte over a time period of 2 min. Images were acquired at 2 images per minute and the sequence shows a 3 min time period. Still images are shown in Figure 1e. Supplementary Video 3. Interaction of a LysM + neutrophil with a microvascular pericyte during interstitial migration in vivo. The video shows a polarized LysM + neutrophil (green) sequentially interacting with NG2 + cells (red) in the ear skin of an NG2DsRed-LysMeGFP chimera. The track of the cell is shown in blue, colocalization is indicated by yellow. Inflammation was induced by s.c. injection of fmlp 2 hrs before the 2-photon imaging was performed. Images were acquired every 45 sec and the sequence shows a 15 min time period. Still image is shown in Figure 1f. Supplementary Video 4. LysM + cells contacting NG2 + cells during undirected interstitial migration. The video shows LysM + neutrophils (green, cell 1-3) orientating towards NG2 + cells (red) during interstitial migration in the ear skin of an NG2DsRed-LysMeGFP chimera 2 hrs after injection of fmlp. After interaction cell 1 and cell 2 stay in close contact to NG2 + cells and sequentially interact (colocalization in yellow, arrowheads) with them (yellow/cyan track). Cell 3 interacts with a NG2 + cell (green track) and then establishes a long lasting contact (red track). Inflammation was induced by s.c. injection of fmlp 2 hrs before the 2-photon imaging was performed. Images were acquired every 3 sec and the sequence shows a time period of 1 hr. Still image is shown in Figure 2a. Supplementary Video 5. CX3CR1 + cells contacting NG2 + cells during random interstitial migration. The video shows the ear skin of an NG2DsRed-CX3CR1eGFP chimera 4 hrs after s.c. injection of TNF. During undirected migration CX3CR1 + macrophages (green) interact with NG2 + cells (red) and stay in close contact to them. Interaction is visualized by colocalization (yellow). The tracks of cells are shown in different colors. Images were acquired at a rate of 2 images per minute and the sequence shows a time period of 5 min. Still image is shown in Figure 2c. Supplementary Video 6. Heatmap visualization of LysM + cell density over time after fmlp or fmlp and ISO-1 injection. Heatmap visualizations are superimposed on the video, color coding the density of interstitial LysM + cells in the ear skin of NG2DsRed-LysMeGFP chimeras. Black/blue indicating low density, red/white marking areas of high density. Images were acquired at a rate of 2 images per minute by 2-PIVM and the sequence shows a time period of 55 min. Left: The video shows the ear skin 2 hrs after injection of fmlp. Initially, LysM + cells (green) are diffusely distributed in the interstitial space. Over time, there is a concentration around NG2 + (red) cells. Still images are shown in Figure 4c. Right: The video shows the ear skin 2 hrs after injection of fmlp and 3 min after injection of ISO-1. LysM + cells are diffusely distributed over the imaging area and there is no orientation towards NG2 + cells. Still images are shown in Figure 4f. Supplementary Video 7. Heatmap visualization of CX3CR1 + cell density over time after injection of TNF and ISO-1. The video shows the ear skin of an NG2DsRed- CX3CR1eGFP chimera 4 hrs after injection of TNF and 3 min after injection of ISO-1. A heatmap visualization is superimposed on the video, color coding the density of interstitial CX3CR1 + macrophages. Black/blue indicating low

Nature Immunology doi:1.138/ni.2477 density, red/white marking areas of high density. The distribution of CX3CR1 + cells over the whole imaging area remains stable over the observation period and there is no concentration around NG2 + cells. Images were acquired at a rate of 2 images per minute and the sequence shows a time period of 45 min. Still images are shown in Supplementary Figure 4f. Supplementary Video 8. LysM + cells contacting NG2 + cells during directed interstitial migration induced by laser injury. The video shows the ear skin of an NG2DsRed-LysMeGFP chimera 45 min after induction of a focal necrosis (yellow) by laser treatment. On their way to the laser injury LysM + neutrophils (green) are contacting NG2 + cells (red) and migrate along them. Interaction is visualized by colocalization (yellow). Images were acquired at a rate of 2 images per minute and the sequence shows a time period of 3 min. Still image is shown in Figure 6a. Supplementary Video 9. CX3CR1 + cells contacting NG2 + cells during interstitial migration induced by laser injury. The video shows the ear skin of an NG2DsRed-CX3CR1eGFP chimera 4 hrs after induction of localized necrosis by laser treatment. During directed migration CX3CR1 + macrophages (green) interact with NG2 + cells (red) and continue their path to the focus of sterile inflammation. Interaction is visualized by colocalization (yellow). The tracks of cells are shown in different colors. Images were acquired at a rate of 2 images per minute and the sequence shows a time period of 75 min. Still image is shown in Supplementary Figure 6g. Supplementary Video 1. LysM + cells contacting NG2 + cells during interstitial migration induced by laser injury after treatment with ISO-1. The video shows the ear skin of an NG2DsRed-LysMeGFP chimera 45 min after induction of a focal necrosis (yellow) by laser treatment. ISO-1 was injected s.c. 3 min before the laser treatment. On their way to the laser injury LysM + neutrophils (green) are contacting NG2 + cells (red), but do not follow them. The tracks of cells are shown in different colors. Images were acquired at a rate of 2 images per minute and the sequence shows a time period of 45 min. Supplementary Video 11. CX3CR1 + cells contacting NG2 + cells during interstitial migration induced by laser injury after treatment with ISO-1. The video shows the ear skin of an NG2DsRed-CX3CR1eGFP chimera 4 hrs after induction of localized necrosis by laser treatment. ISO- 1 was injected 3 min before the laser injury. CX3CR1 + macrophages (green) shortly interact with NG2 + cells (red) on their way to the focus of sterile inflammation. The tracks of cells are shown in different colors. Images were acquired at a rate of 2 images per minute and the sequence shows a time period of 5 min. Still image is shown in Supplementary Figure 7g.

Nature Immunology doi:1.138/ni.2477

Nature Immunology doi:1.138/ni.2477

Supplementary Figure 3 1 merge b 8 6 MIF ctrl TNF 6h LPS 6h NG2 4 2 CXCR4 CD74 TNF permeabilized TNF x-fold increase in mrna expression of pericytes 12 DAPI ctrl permeabilized a Nature Immunology doi:1.138/ni.2477 NG2 MIF d merge DAPI NG2 CXCL5 merge DAPI NG2 CCL2 merge 5 µm ctrl TNF c ctrl TNF e NG2+CCL2 NG2+CXCL5 NG2+ pericytes express and secrete chemoattractants after stimulation with DAMPs and PAMPs in vivo. (a) rt-pcr for the MIF-receptors CXCR4 and CD74, mrna was obtained from human placental pericytes stimulated with TNF!, LPS or control for 6 hrs. Relative increase in mrna compared to resting pericytes, set as 1. Data from n=3 experiments shown as mean ± s.e.m., P<.5. (b) Immunofluorescence staining of ear tissue sections from NG2DsRed mice after injection of TNF! or normal saline as control (ctrl) with or without permeabilization. Single images of Fig. 3f. Arrowheads indicating MIF expressing NG2+ cells. Bar 25µm. (c) MIF (green) shows a strong signal on NG2+ microvascular pericytes (red) in the ear skin 6 hrs after s.c. injection of TNF!. 3D rendering of images acquired by 2-photon microscopy. Image representative of n=3 experiments. Bar 5 µm. (d) Staining for the chemokines CXCL5 and CCL2 in sections from the ear skin of NG2DsRed mice 6 hrs after s.c. injection of TNF! or normal saline as control (ctrl). CXCL5 is only expressed in NG2+ cells after injection of TNF! (upper panel). CCL2 is localized intracellularly in the control group and exposed on the surface of NG2+ cells after injection of TNF! (lower panel). Bar 25 µm. Epidermal keratinocytes indicated by dashed line. Images representative of n=4 experiments. (e) Whole mount stainings of NG2DsRed ear tissue after stimulation with TNF! visualized by 2-photon microscopy. Stainings for the chemokines CCL2 and CXCL5. NG2+ cells (red) expressed CCL2 and CXCL5 (green) and were surrounded by a strong signal for the chemokines. Orthogonal slices and 3D reconstruction (top, left). Images representative of n=3 experiments. Bar 25 µm.

Nature Immunology doi:1.138/ni.2477

Nature Immunology doi:1.138/ni.2477

Supplementary Figure 6 25 1. a b c velocity (µm/min) interaction d e f mean displacement g 2 15 1 5 16 14 12 1 8 6 4 2 before during interacting tracks no interaction after none 1 2 3 4 5 6 time interval (square root of time) Laser injury 3 h meandering index motility coefficient (µm 2 /min).8.6.4.2 4 3 2 1 interacting CX3CR1 + cells 15µm injury displacement rate (µm/min) + - + - track interaction track interaction + - track interaction fast CX3CR1 + cells slow CX3CR1 + cells 2 15 1 Nature Immunology doi:1.138/ni.2477 5 non-interacting CX3CR1 + cells 15µm injury high density of cells low high density of cells low 2 1-15µm 15µm -15µm 15µm -15µm -15µm i 25 j 1. k 2 1.2 NS l velocity (µm/min) 2 15 1 5 before during interaction after none meandering index.8.6.4.2 displacement rate (µm/min) 15 1 + - + - track interaction track interaction 5 time to target (relative to non-interacting cells).8.4 + - track interaction NG2 + pericytes support the interstitial migration of CX3CR1 + macrophages. (a) Velocity of individual CX3CR1 + cells before, during, and after interaction with NG2 + pericytes or non-interacting cells (ANOVA/LSD). (b-e) Comparison of several cell motility parameters between interacting and non-interacting tracks of CX3CR1 + cells (Student s t-test): meandering index (b), displacement rate (c), mean displacement plot calculated from tracks <1 min duration (data are shown as mean ± s.e.m.) (d), and motility coefficient (e). (f) Distribution of fast and slow migrating CX3CR1 + cells in the interstitial space. NG2 + cells are outlined by dotted lines, hair follicles are marked by asterisks (). Bar 1 µm. (g) Imaging of macrophage migration 4 hrs after induction of a focal necrosis by laser treatment in an NG2DsRed-CX3CR1eGFP chimera. Tracks of individual macrophages (green) interacting with NG2 + pericytes (red) during interstitial migration on their way to the focus of sterile injury (yellow, arrowheads). Still image of Supplementary Video 1. Bar 6µm. (h) Track plots of CX3CR1 + cells interacting with NG2 + cells (left) and non-interacting cells (right). The relative localization of the injury is depicted in red. (i) Velocity of individual CX3CR1 + cells before, during, and after interaction with NG2 + cells or without interaction during the imaging period (ANOVA/LSD). Comparison of meandering index (j) and displacement rate (k) between interacting and non-interacting tracks of CX3CR1 + cells (Student s t-test) (l) Time to the focus of laser injury for CX3CR1 + cells (n=1) starting at the same distance from the laser injury. Relative migration time of interacting CX3CR1 + cells relative to non-interacting cells (data are shown as mean ± s.e.m.). (a-l) Data from n=4 independent experiments.

Nature Immunology doi:1.138/ni.2477

Schematic presentation of the role of NG2+ pericytes in sterile inflammation. (a) Sensing of sterile inflammation by pericytes. After cell necrosis, DAMPs are released from dead cells. Microvascular NG2+ pericytes express TLR4, TLR2, FPR2, TNFR1, and NLRP3, which allow them to sense mediators of sterile inflammation. (b) Reaction of pericytes to mediators of sterile inflammation. In response to inflammatory stimuli, pericytes acquire a proinflammatory phenotype, characterized by the up-regulation of chemokines, adhesion molecules, and NLRP3. In addition, pericytes release chemokines and present MIF as well as ICAM-1 on their surface. (c) Pericytes attract and interact with extravasated innate immune cells, thereby supporting their interstitial trafficking to foci of sterile inflammation. After crossing the basement membrane regulated by NG2 pericytes (yellow) and extravasation from postcapillary venules (top right), myeloid leukocytes face a complex field of DAMPs and other chemoattractants in the interstitial space. If they choose to migrate distant from NG2+ pericytes (blue), their path is undirected and slow. However, if innate immune cells interact with NG2+ pericytes (blue) along arterioles and capillaries (bottom), thereby entering the compartment highly enriched in pericyte-derived MIF, they become activated and sensitized to subsequent inflammatory stimuli. This results in a straighter and faster migration through the interstitial space, allowing interacting myeloid leukocytes to scan larger areas and to find a focus of sterile inflammation more efficiently. Pericyte Supplementary Figure 8 a c b DAMPs Directed and fast migration in the pericyte compartment Myeloid leukocyte Indirect and slow migration through the interstitial tissue ICAM-1 MIF Nature Immunology doi:1.138/ni.2477

Nature Immunology doi:1.138/ni.2477

Nature Immunology doi:1.138/ni.2477

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