quantitative imagining systems https: / / www. qi - tissue. com genexyz polylux resolve biosciences single - cell signature explorer algorithm ( pont et al., 2019 ) https: / / sites. google. com / site / fredsoftwares / products / single - cell - signature - explorer web - based gene set analysis toolkit ( liao et al., 2019 ) http: / / www. webgestalt. org / venn diag ram generator psb, vib - ghent university https: / / bioinformatics. psb. ugent. be / webtools / venn / zen black zeiss microscopy www. zeiss. com experimental models mouse: b6 ( c ) - ccr2 the jackson laboratory jax: 027619 mouse: c57bl / 6j spf janvier labs n / a mouse: b6 - clec4f ( scott et al., 2016 ) n / a mouse: b6 - clec4f ( scott et al., 2018 ) n / a mouse: alk1 - flox ( park et al., 2008 ) n / a source paper: pmc8809252 further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, martin guilliams ( martin. guilliams @ irc. vib - ugent. be ). this study did not generate new unique reagents. wt c57bl / 6j mice ( janvier ) were used for this study. male and female mice were used for all experiments, with the exception of nafld experiments were only male mice were used. for nafld experiments, mice were put on the sd or wd at 5 weeks of age and sacrificed after either 24 or 36 weeks on the diet as indicated. fcgr1 - cre mice ( scott et al., 2018 ) were obtained from prof. bernard malissen, ciml, marseille and clec4f - cre mice ( scott et al., 2018 ) were crossed with acvrl1 mice ( park et al., 2008 ) obtained from paul oh, barrow neurological institute, florida, usa. clec4f - dtr mice ( scott et al., 2016 ) were crossed to cd45. 1 mice ( janvier ) for kc depletion and development experiments all mice were used between 6 and 12 weeks of age unless otherwise stated. all mice maintained at the vib ( ghent university ) under specific pathogen free conditions. all animals were randomly allocated to experimental groups. all experiments were performed in accordance with the ethical committee of the faculty of science, ugent and vib. piglets ( female, 10 weeks old ) were purchased at a local farm and transported to the animal facilities of the faculty of veterinary medicine. the animals were housed in isolation units as blood donors and had access to feed and water ad libitum. at 30 weeks of age, the animals were euthanized by intravenous injection of sodium pentobarbital 20 % ( 60mg / 2. 5kg ) and livers were collected. the animal study was reviewed and approved by the ethical committee of the faculty of veterinary medicine ( ec2018 / 55 ). study animals were clinically healthy leghorn hens of approximately 58 weeks old collected from a commercial farm. the hens were housed at the faculty of veterinary medicine according to acceptable welfare standards and were observed at least twice daily for health problems. feed and water was offered ad libitum. the chickens were euthanized through intravenous injection ( in the wing ) with sodium pentobarbital. the ec approval number of this trial was ec2019 / 015. male cynomolgus macaques ( ≥2 years ) were sourced from china and supplied by guangzhou xiangguan biotech co., ltd and confirmed healthy before being assigned to the study. animal handling, husbandry and euthanasia was performed by wuxiapptec co., ltd., china according to local ethical guidelines ( aaalac accredited 2010 ). study animals consists of 4 groups, orally dosed once with a janssen proprietary immune modulator or vehicle control. vehicle control animals only were used in this study. liver tissue samples were snap - frozen immediately after euthanasia. samples were thawed once before shipping to ghent for snrna - seq analysis. female syrian hamsters ( janvier ) were housed per one or two in ventilated isolator cages at a temperature of 21°c, humidity of 55 % and 12: 12 dark / light cycles, with access to food and water ad libitum and cage enrichment. all hamsters had spf status at arrival and manipulations were performed in a laminar flow cabinet. housing conditions and experimental procedures were approved by the ethical committee of ku leuven ( license p015 - 2020 ). animals were euthanized at 6 - 8 weeks of age by intraperitoneal injection of 200 mg / ml sodium pentobarbital and livers were collected for analysis. zebrafish were maintained under standard conditions, according to felasa guidelines ( alestrom et al., 2019 ). all experimental procedures were approved by the ethical committee for animal welfare ( cebea ) from the ulb ( universite libre de bruxelles ) ( protocol # 594n ). the following transgenic lines at 6 months of age were used: tg ( mpeg1: egfp ) ( ellett et al., 2011 ); tg ( kdrl: cre ) ( bertrand et al., 2010 ); tg ( actb2: loxp - stop - loxp - dsred ) ( bertrand et al., 2010 ) enabling macs to be sorted for sequencing as dsred, gfp double positive cells. patient studies were run in collaboration with ghent university hospital. liver biopsies ( 1 – 2mm ) were isolated with informed consent from patients undergoing cholecystectomy or gastric bypass. in addition, liver biopsies were isolated from healthy adjacent tissue removed during liver resection due to colorectal cancer metastasis. in most cases, a second biopsy was also taken to evaluate liver histology. a full overview of all patient samples used in this study can be found in table s6. paraffin - embedded human liver samples were obtained through collaboration with dr. jan lerut ( universite catholique de louvain, ucl ). all studies were performed in accordance with the ethical committee of the ghent university hospital ( study numbers: 2015 / 1334 and 2017 / 0539 ). liver cells were isolated by either ex vivo digestion ( all species, except zebrafish ) or in vivo liver perfusion ( mice only ) and digestion as described previously ( bonnardel et al., 2019; scott et al., 2016 ). briefly, for ex vivo digestion, livers were isolated, cut into small pieces and incubated with 1mg / ml collagenase a and 10u / ml dnase at 37 c for 20 mins with shaking. for in vivo digestion, after retrograde cannulation, livers were perfused for 1 - 2mins with an egta - containing solution, followed by a 5min ( 6ml / min ) perfusion with 0. 2mg / ml collagenase a. livers were then removed, minced and incubated for 20mins with 0. 4mg / ml collagenase a and 10u / ml dnase at 37°c. all subsequent procedures were performed at 4°c. samples were filtered over a 100μm mesh filter and red blood cells were lysed. samples were again filtered over a 40μm mesh filter. at this point in vivo digestion samples only were subjected to two centrifugation steps of 1 min at 50g to isolate hepatocytes. remaining liver cells ( leukocytes, lsecs and hscs; in vivo protocol ) and total cells from the ex vivo digests were centrifuged at 400g for 5mins before proceeding to antibod y staining for flow cytometry. a combination of collagenase a and dnase were used to digest livers in both protocols to minimize cleavage of surface epitopes. dissected livers from 6 months old transgenic zebrafish were triturated and treated with liberase tm at 33°c for 20 min. cells were then filtered through 40μm nylon mesh and washed with 2 % fbs in pbs by centrifugation. sytox red was then added to the samples at a final concentration of 5nm to exclude nonviable cells before proceeding to flow cytometry. dsred gfp cells were then facs - purified. nuclei were isolated from snap frozen liver tissue with a sucrose gra dien t as previously described ( habib et al., 2016 ). briefly, frozen liver tissue is homogenized using kimble douncer grinder set in 1ml homogenization buffer with rnase inhibitors. homogenised tissue is then is then subjected to density gra dien t ( 29 % cushion – optiprep ) ultracentrifugation ( 7700rpm, 4 c, 30 mins ). after resuspension, nuclei are stained with dapi and intact nuclei were facs - purified from remaining debris.