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HEP_25801_sm_SuppFig1.tif4434KSupporting Information Figure 1: Liver histopathology from APAP-treated mice. Mice were challenged with a toxic dose of APAP (acetaminophen, 500mg/Kg) and following 24 hours liver fragments were extracted, processed and stained by hematoxylin and eosin method. Histological score was estimated by the sum of the attributed standard values to morphological findings. 0: no lesion present. ½: individual necrotic cells seen at the first cell layer adjacent to the central vein, and hyaline degeneration present. 1: necrotic cells extending two or three cell layers from the central veins. 2: necrotic cells extending three to six cell layers from the central veins, but limited in peripheral distribution. 3: the same as 2, but with necrosis extending from one central vein to another. 4: more severe than 3, with extensive centrilobular necrosis throughout the section. Control mice received only saline by gavage, while WT/APAP mice received APAP. APAP+BOC1+DF group were previously treated with combined CXCR2-FPR1 antagonists (DF2156a, a CXCR2 antagonist, 30mg/Kg; and BOC1, a FPR1 antagonist, 2mg/Kg). Histological scores were plotted as mean± SEM. * - indicates statistical significance in comparison to control. ** - indicates statistical significance in comparison to APAP-wild type treated mice (WT). (ANOVA, Tukey post-test, P<0.05).
HEP_25801_sm_SuppFig2.tif3431KSupporting Information Figure 2: In vitro neutrophil and HepG2 incubation. (A) – Human neutrophils were purified by dextran sedimentation and further Ficoll gradient centrifugation. Cells were counted and incubated in different densities (105 and 106 cells/well) and viability was assessed over the time by Trypan Blue exclusion method. (B) Purified neutrophils were incubated with HepG2 cells in a 1:1 proportion (105 HepG2 and 105 neutrophils/well) in the presence of FPR1 antagonist (BOC1, 10-5M), CXCR2 antagonist (DF2156a, 10-6M) or with a combination of these two drugs (BOC1+DF). HepG2 viability was estimated by MTT assay. * - indicates statistical significance in comparison to vehicle (medium) treated cells. (C) Neutrophils were purified and incubated for 1 hour with supernatant from APAP (acetaminophen)-incubated HepG2 cells (5mM, 24 hours of incubation) or with supernatant from untreated HepG2 (medium alone, 24 hours of incubation). Neutrophil reactive oxygen species (ROS) production ex vivo was measured by the 2′,7′ dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence emission. In different groups, neutrophils were incubated throughout the experiment with FPR1 antagonist (BOC1, 10-5M), CXCR2 antagonist (DF2156a, 10-6M) or with a combination of these two drugs (BOC1+DF). * - indicates statistical significance in comparison to supernatant from untreated HepG2 cells. ** - indicates statistical significance in comparison to APAP-treated HepG2 supernatant. (ANOVA, Tukey post-test, P<0.05).
HEP_25801_sm_SuppFig3.tif3268KSupporting Information Figure 3: – In vitro model of APAP cytotoxicity on HepG2 cells. (A) HepG2 cells were incubated with different doses of APAP (acetaminophen; 1-25 mM) during 24 hours and cell viability was assessed by MTT assay. (B) Time course of APAP-induced HepG2 cytotoxicity (APAP 5mM; 105cells/well). * - indicates statistical significance in comparison to control or time zero (ANOVA, Tukey post-test, P<0.05).
HEP_25801_sm_SuppFig4.tif5830KSupporting Information Figure 4: Lung histology from APAP-treated mice. Mice were challenged with a toxic dose of APAP (acetaminophen; 500mg/Kg) and following 24 hours lung fragments were prepared for histology processing and stained by hematoxylin and eosin method. Control mice received only saline by gavage, while WT/APAP mice received APAP. APAP+BOC1+DF group were previously treated with combined CXCR2-FPR1 antagonists (DF2156a, a CXCR2 antagonist, 30mg/Kg; and BOC1, a FPR1 antagonist, 2mg/Kg). Note that APAP treatment led to an increased leukocyte infiltration in the perivascular regions (black arrow heads) and alveolar edema in comparison to controls. However, animals treated with BOC1+DF or TLR9-/ - showed reduced or absent pulmonary damage.
HEP_25801_sm_SuppFig5.tif3502KSupporting Information Figure 5: Lung levels of chemokines and pro-inflammatory cytokines during ALF in mice: effect of CXCR2 and FPR1 blockage. Mice were overdosed with a toxic dose of APAP (acetaminophen; 500mg/Kg) and lung fragments were collected after 24 hours and processed for quantification of chemokines and cytokines levels. In different groups, mice were previously treated with the CXCR2 antagonist DF2156a (DF; 30mg/Kg), FPR1 antagonist (BOC1, 2mg/Kg) or with a combined dose of these two antagonists (BOC1+DF). (A) Quantification by ELISA of lung levels of CXCL1, (B) CXCL2, (C) IL-1β and (D) TNF-α. * - indicates statistical significance in comparison to control (ANOVA, Tukey post-test, P<0.05).
HEP_25801_sm_SuppFig6.tif3514KSupporting Information Figure 6: TLR9-/- mice are completely protected from lung remote injury during APAP-induced acute liver failure. Lung samples were collected from TLR9-/- mice challenged with APAP (acetaminophen; 500mg/Kg). Wild-type or vehicle treated mice were studied as controls. Bronchial-alveolar lavage (BAL) was collected and lung parenchyma processed to further analyzes. (A) Total leukocyte number in BAL was counted in Neubauer chamber. (B) Neutrophils, (C) macrophages and (D) lymphocytes were counted under light microscopy using cytospin-prepared slides, stained with regular haematological dye kit. (E) Neutrophil accumulation into the lung parenchyma was estimated by myeloperoxidase activity assay (MPO). (F) Protection from APAP overdose was confirmed by assessing liver neutrophil accumulation by MPO assay. * - indicates statistical significance in comparison to control. ** - indicates statistical significance in comparison to wild type treated mice. (ANOVA, Tukey post-test, P<0.05).
HEP_25801_sm_SuppFig7.tif3468KSupporting Information Figure 7: TLR9-/- mice are resistant to systemic inflammatory response during APAP-induced acute liver failure: Serum levels of chemokines and pro-inflammatory cytokines. Mice were overdosed with a toxic dose of APAP (acetaminophen; 500mg/Kg) and blood samples were collected after 24 hours. Serum was separated for quantification of chemokines and cytokines levels. (A) Quantification by ELISA of CXCL1, (B) CXCL2, (C) IL-1β and (D) TNF-α. * - indicates statistical significance in comparison to vehicle treated mice. **- indicates statistical significance in comparison to wild type APAP-treated mice. (ANOVA, Tukey post-test, P<0.05).
HEP_25801_sm_SuppFig8.tif3620KSupporting Information Figure 8: Proposed mechanism for neutrophil-mediated liver injury amplification and lung remote inflammation. (1) Following administration of toxic doses of APAP, hepatocytes accumulate the reactive metabolite NAPQI (N-acetyl-p-benzoquinone-imine), (2) which leads to oxidative stress, DNA and mitochondria damage, and ultimately acute liver necrosis. (3) Massive cell death generates an abrupt local increase of necrotic products which attract circulating neutrophils to emigrate into the liver. (4) Chemokine releasing by resident cells may be amplified by recognition of necrotic products by innate immune receptors, but they can also be directly released from intracellular stocks in the course of hepatocyte lysis. In this context, mitochondrial products (formyl-peptides and mitDNA) and chemokines collaborate to liver neutrophil migration and activation, promoting additional damage. (5) Necrosis-derived products may be collected to systemic circulation and their recognition by lung resident cells or leukocytes retained into the pulmonary vasculature elicits a remote inflammatory response (6), which can be an adjuvant morbidity factor during acute liver failure.
HEP_25801_sm_SuppVideo1.mov173KSupporting Information Video 1: Liver confocal intravital microscopy – time zero. Lysm-eGFP mice were used to visualize neutrophil dynamics within liver microvasculature. Sinusoids wer stained by i.v. injection of PE-coupled anti-PECAM-1. Mice were imaged by sequential laser scans (2.71 seconds, with an interval of 8 seconds). Note the complete stained sinusoidal vasculature, which is an indicative of efficient liver perfusion. Few neutrophils adhered following imaging procedure. Mice were imaged for the period of 1 hour, and videos were accelerated to improve visualization of neutrophil chemotaxis. Representative movie of 3 similar experiments.
HEP_25801_sm_SuppVideo2.mov1408KSupporting Information Video 2: Liver confocal intravital microscopy – 6 hours following APAP overdose. Lysm-eGFP mice were used to visualize neutrophil dynamics within liver microvasculature. Sinusoids were stained by i.v. injection of PE-coupled anti-PECAM-1. Mice were imaged by sequential laser scans (2.71 seconds, with an interval of 8 seconds). After 6 hours of APAP administration, dark areas, which were weakly stained by anti-PECAM-1 were observed. Note the complete stained sinusoidal vasculature, which is an indicative if efficient liver perfusion. Neutrophils adhered and crawled within vasculature, and a significant accumulation in all field of view was observed. Mice were imaged for the period of 1 hour, and videos were accelerated to improve visualization of neutrophil chemotaxis. Representative movie of 3 similar experiments.
HEP_25801_sm_SuppVideo3.mov163KSupporting Information Video 3: Liver confocal intravital microscopy – 24 hours following APAP overdose. Lysm-eGFP mice were used to visualize neutrophil dynamics within liver microvasculature. Sinusoids were stained by i.v. injection of PE-coupled anti-PECAM-1. After 24 hours of APAP administration, are which were weakly stained by anti-PECAM-1 could still be noted. Neutrophil accumulation was markedly increased, with clusters of cells in the field of view. Mice were imaged by sequential laser scans (2.71 seconds, with an interval of 8 seconds). Mice were imaged for the period of 1 hour, and videos were accelerated to improve visualization of neutrophil chemotaxis. Representative movie of 3 similar experiments.
HEP_25801_sm_SuppVideo4.mov1419KSupporting Information Video 4: Three-dimensional Z-stack rendering from liver confocal intravital microscopy – Control in comparison to APAP-treated mice after 24 hours. Lysm-eGFP mice were used to visualize neutrophil dynamics within liver microvasculature. Sinusoids were stained by i.v. injection of PE-coupled anti-PECAM-1. Note the formation of neutrophil clusters within liver microvasculature, with were not observed in controls. Z-stacks were made (depth: 60?m) and TIFF-acquired images were mounted by using ImageJ software (NIH, USA). Mice were imaged by sequential laser scans (2.71 seconds).

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