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8 Supplemental Figures legends Supplementary Figure 1 (A) Additional dot plots from CyTOF analysis from untreated group. (B) Gating strategy for assessment of CD11c + NK cells frequency in liver non-parenchymal cells samples. CD11c + NK cells were defined as CD3 - CD11c + NKp46 + NK1.1 + events. (C) Additional dot plots from CyTOF analysis from mice 2 days after CLL treatment. Supplementary Figure 2 Identification of main hepatic vascular arrangements. Snapshots of chronological distribution of FITC-albumin within the liver (5mg/Kg). Portal spaces were identified as the first vascular region evidenced by fluorescence, and central vein were defined as the secondary draining vessels. Scale bars, 120 µm. Supplementary Figure 3 Expression of different cell surface markers in liver phagocytes. (A) Liver macrophage expression of lysozyme M (LysM-EGFP mice) and F4/80. (B) Same for A, but for CD11c (CD11c-EYFP mice). (C) Depletion of both intravascular CD11c + F4/80 + and extravascular CD11c + cells upon diphtheria toxin (DT) treatment in CD11c-DTR-EYFP mice. (D) Expression of EYFP and DTR under control of CX3CR1 in liver dendritic cells and selective depletion following diphtheria toxin administration. Note that only CX3CR1+ cells are depleted after DT treatment, with no effect on intravascular F4/80+ cells. Scale bars, 120 µm. Illustrative images from different experiments (N>5). Supplementary Figure 4 Liver dendritic cells are located in the subcapsular compartment and are CD11c + (A) Liver intravital microscopy of CX3CR1 gfp/wt mouse showing the distribution of CD11c + dendritic (anti-cd11) cells and
9 (B,C) confirmation of sub-mesothelial location of CX3CR1 + cells by intravital microscopy (B) immunohistochemistry (C). Illustrative images from different experiments (N>5). Supplementary Figure 5 Characterization of different cell surface markers and CCR2 role in phagocytes (A) Liver ex vivo transversal fragment showing that subcapsular neither CX3CR1 + nor CD11c + cells are positive for desmin (anti-desmin antibody), excluding this population as hepatic stellate cells. (B) Real-time PCR from isolated macrophages and dendritic cells showing that hepatic stellate cells (HSCs) did not contaminate samples. The following HSCs genes were measured: lecithin retinol acyltransferase (Lrat), desmin (Des) and alpha-actin-2 (Acta2). Data were normalized by a constitutive gene (Gapdh) and Itgax (CD11c) was used as positive control. (C) Liver intravital microscopy showing that neither CX3CR1 + cells nor F4/80 + have vitamin A granules (auto-fluorescence in 405nm laser). (D) Flow cytometry investigation of the absence of vitamin A in CX3CR1 + population (auto-fluorescence in 405nm laser). (E) CCR2 -/- mouse have normal KC density (anti-f4/80) and (F) location. Scale bars in A, 10 µm; B, 65µm and E, 120µm. Supplementary Figure 6 Additional dot plots from CyTOF analysis from mice 7 and 17 days after phagocyte depletion. Expression of cell surface markers in liver non-parenchymal cells clodronate depleted mice after 7 days (A) and after 17 days (B). CyTOF was performed and events were clustered as described in Methods and Results. Supplementary Figure 7 (A) Replenishment of liver phagocytes is driven by a bone marrow-derived precursor. Different focal planes imaged by intravital
10 confocal microscopy from GFP-expressing bone marrow chimeras showing that all Kupffer cells (F4/80 + GFP + cells in intravascular focus) and dendritic cells (GFP + cells in capsule focus) were derived from the bone marrow. (B) Acetaminophen (APAP) treatment caused a significant depletion of KCs (red; F4/80 + cells) and normal cell density and location were restored after days. In blue, DAPI stains for necrosis. Scale bars in KC focus, 35 µm, in DC focus, 20 µm and in APAP-treated group 120 µm. *P < 0.05 (unpaired t-test) in comparison to vehicle. Legends for Tables Table 1 Antibody panel for CyTOF and for genes for Real Time PCR. For PCR, cells were isolated and RNA was extracted with a mirneasy kit (Qiagen), then was reverse-transcribed with a high capacity cdna reverse transcription kit and analyzed by quantitative RT-PCR with a Vii 7 Real-time PCR system with Taqman primers. The following HSCs genes were measured: lecithin retinol acyltransferase (Lrat), desmin (Des) and alphaactin-2 (Acta2). Data were normalized by a constitutive gene (Gapdh) and Itgax (CD11c) was used as positive control. The comparative threshold cycle method and the internal control Gapdh was used for normalization of the target genes. Table 2 Absolute cell numbers (events) from time-of-flight flow cytometry (CyTOF) experiments. Liver samples from different groups were collected and processed for CyTOF. Events were normalized in all samples.
11 Table 3 Quantification of liver cytokine expression by multiplexed Luminex array. Liver samples from different groups were collected and processed for multiplexed cytokine array. Data are displayed as individual samples from different experiments. GEO accession number: GSE Gene expression assessed by Nanostring experiments. Intravascular CX3CR1 - F4/80 + and extravascular CX3CR1 + F4/80 - cells were isolated by sorting (FACS) and immune systemrelated genes expression was quantified using Nanostring. Statically relevant results consist in p-value < 0.05 and a fold change of at least 50% higher or lower. Pathways and functional classification were done by cross association using KEGG Pathways and KEGG Brite databases. Legends for Supplementary Movies Supplementary Movie 1 Identification of main hepatic vascular arrangements. Temporal distribution of FITC-albumin (green) within the liver (5mg/Kg). Portal spaces were identified as the first vascular region evidenced by fluorescence, and central vein were defined as the secondary draining vessels. Total experimenta time: 1 minute. Supplementary movie 2 Three-dimensional rendering of liver intravital imaging. Spatial distribution of Kupffer cells. Vessels are in blue (anti-pecam- 1 antibody) and KCs in red (anti-f4/80 antibody). Images were collect from a 3D section of µm of depth.
12 Supplementary movie 3 Three-dimensional rendering of liver intravital imaging. Liver dendritic cell (DCs) morphology and distribution in CD11c- EYFP mice. Vessels are in blue (anti-pecam-1 antibody) and DCs in yellow (EYFP). Images were collect from a 3D section of µm of depth. Supplementary movie 4 Three-dimensional rendering of liver intravital imaging. Liver dendritic cell morphology and distribution in CX3CR1-EGFP mice. Vessels are in blue (anti-pecam-1 antibody) and DCs in green (EGFP). Images were collect from a 3D section of µm of depth. Supplementary movie 5: KCs instantaneously trap circulating E. coli from the circulation. Merged video comparing E. coli arresting by a control KC (non-depleted mouse) versus an immature KC (7 days after CLL). Note that E. coli are arrested at the first passage through the liver, while immature KC are unable to proper catch and internalize bacteria. Kupffer cells (KCs) are in red and E. coli in green. Total video time: 10 minutes E.coli were injected in the beginning of the imaging procedure. Supplementary movie 6 Circulating E. coli capture by Kupffer cells. Merged video showing E. coli arresting by control group (non-depleted mouse) and after different timepoints of replenishment period (7, 17, 30 and 60 days). Kupffer cells (KCs) are in red and E. coli in green. Due to photobleaching issues, a still frame from KC channel was used as reference during the video. Total video time: 10 minutes each experimental group E.coli were injected in the beginning of the imaging procedure
13 Supplementary movie 7 E. coli are exclusively arrested by intravascular cells Three-dimensional rendering of liver confocal intravital microscopy showing GFP expressing E coli inside sinusoids. Sinusoids are in blue (anti-pecam-1 antibody) and E. coli gfp in green. Images were collect from a 3D section of µm of depth.
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