as in the healthy mouse, visium identified human lams in portal zones of non - steatotic livers. however, in steatotic human livers, lams were primarily located peri - centrally, in zones with steatosis ( figure 5 e ), suggesting that monocytes are recruited to distinc t locations in the healthy and obese liver where they then differentiate into lams. this altered location of lams was further validated by confocal microscopy and molecular cartography ( figures 5 f – 5h ). however, this analysis did not identify any capsule macs, suggesting that these cells may be absent from our umap, likely as a result of the small amount of capsule tissue on a biopsy. the mac1 population expressing igsf21 was present in very low numbers throughput the tissue ( figures 5 g and 5h ). this coupled with their similar transcriptomic profile to mokcs could suggest that these are mokc precursors as observed in the mouse, but this requires further study. focusing on the lams, the change in their location in the steatotic human liver was also observed in the murine nafld model. here, lams were found across portal, periportal, and mid zones ( figures 5 i, s8 e, and s8f ), fitting with the presence of steatosis in these regions and consistent with our previous report ( remmerie et al., 2020 ). comparison of degs between lams in standard diet ( sd ) and wd - fed mice identified that lams had a more mature phenotype in wd - fed mice, downregulating their expression of some monocyte genes and increasing their expression of prototypical mac markers, consistent with the presence of both immature and mature lams in the human liver ( figure 5 j ). fitting with a more mature phenotype, wd - derived lams also expressed lower levels of il1b, tnf, and il10 compared with sd lams ( figure 5 k ). while further studies are required to assess the precise functions of these cells in nafld, this could further suggest a protective rather than a pathogenic role for lams ( daemen et al., 2021 ). given the altered localization of kcs in the healthy murine versus human liver and lams in the healthy versus steatotic liver of mice and humans, we next sought to investigate how the cells of the mac niches differed in these settings. analysis of cd45 cells in the human liver identified similar ( sub ) populations of ecs, scs, and hepatocytes as observed in the healthy mouse liver ( figures 6 a and 6b; table s3 ). however, while the 100 - plex mics analysis detected a second subset of portal fibroblasts expressing desmin ( figure s6 h ), we did not detect these cells in our umap, likely due to difficulties detecting desmin with snrna - seq. no significant differences were found in terms of localization of the identified cd45 cells that could explain the altered location of kcs compared with the mouse ( figures 6 c and 6d ). with this in mind, we next utilized nichenet to examine potential ligand - receptor pairs between kcs and cd45 cells present only in the human that could regulate kc location. this analysis identified ccl23 and ccl14 expression by lsecs and ccl16 by hepatocytes that binds to ccr1 expressed by human kcs but not murine kcs ( figure 6 e ). crucially, these ligands were preferentially located peri - centrally ( figure 6 f ) and thus represent interesting targets for further study regarding the potential regulation of kc location in the human liver. we next aimed to investigate the role of cd45 cells in the regulation of lam location in the healthy versus steatotic liver. given the low number of steatotic human samples in our sc / snrna - seq analysis and the variation between patients, we turned to the murine nash model to investigate this. zooming in on scs ( figure 6 g; table s10 ), we identified that there was an increase in fibroblasts in the wd - fed mice. there was also a considerable overlap between fibroblasts and hscs in terms of their gene expression profiles suggesting there may be a differentiation trajectory between the hscs and the fibroblasts, although this remains to be validated in vivo. we also noted a sub - population of fibroblasts expressing ccl2, a known ligand for ccr2 which is expressed on monocytes recruited to the liver during nafld ( remmerie et al., 2020 ), as well as cd44 and vcam1, two genes involved in monocyte recruitment and adhesion ( johnson and ruffell, 2009; meerschaert and furie, 1995 ). both fibroblast populations were enriched in the periportal steatotic regions ( figure 6 i ), in which we also observe enrichment of the lams ( figure 5 i ). we also found high ccl2 and cd44 expression in human fibroblasts ( table s3 ), potentially recruiting bile - duct lams. together, this suggests that fibroblasts may play an important role in recruiting lams. having identified a potential role for the mac niche cells in regulating mac subset location, we next sought to determine the involvement of these cells in regulating mac phenotypes. to assess the roles of conserved cell - cell interactions in driving mac heterogeneity across species, we performed a differential nichenet ( browaeys et al., 2019 ) analysis between the distinc t hepatic macs and the cd45 cells present in their respective niches focusing on ligands and receptors conserved in both human and mouse. this revealed very few specific ligand - receptor pairs for lams ( figure s8 g; data not shown ), hinting that local factors such as metabolites rather than unique cell - cell interactions may drive the lam phenotype. indeed, this would be consistent with the presence of lam - like cells in multiple tissues including the obese adipose tissue, the brain, the lung, and the heart ( jaitin et al., 2019; keren - shaul et al., 2017; liao et al., 2020; rizzo et al., 2020 ). in line with this, bm monocytes cultured with acetylated low - density lipoprotein expressed lam - associated genes ( figure 7 a ), demonstrating a dominant role for lipids in inducing the lam phenotype. conversely, for kcs, we found multiple ligand - receptor pairs to be conserved between human and mouse ( figures 7 b and s8 g ). one of these, an activin receptor - like kinase ( alk1 ) - bone morphogenic protein ( bmp ) 9 / 10 circuit between kcs ( alk1; encoded by acvrl1 ) and stellate cells ( bmp ) 9 / 10 encoded by gdf2 / bmp10 respectively ) was found to be conserved in all 7 species and was predicted to control the expression of a number of the conserved kc genes ( figures 7 b, 7c, and s8 h ). to validate a role for this axis in kcs, we generated fcgr1 - cre × acvrl1 mice, eliminating alk1 specifically from cd64 - expressing macs ( scott et al., 2018 ). this led to an almost complete loss of vsig4 kcs ( figures 8d – 8f ), demonstrating that evolutionarily conserved alk1 signaling is crucial for kcs. to determine if alk1 is required for kc maintenance we generated clec4f - cre × acvrl1 mice, eliminating alk1 only from differentiated kcs ( scott et al., 2018 ). this revealed a relatively similar phenotype than observed in the fcgr1 - cre mice, suggesting that alk1 is also required for kc maintenance ( figure s8 i ). to directly test the need for alk1 in kc development, we generated bm chimeras whereby cd45. 1 clec4f - dtr mice were irradiated with their livers shielded to avoid any radio damage. these mice were then reconstituted with either cd45. 2 acvrl1 or fcgr1 - crexacvrl1 bm. 4 weeks later, mice were given a single i. p. injection of dt to deplete the kcs and 7 or 13 days thereafter chimerism within the kc population was determined ( figure 7 g ). we observed that, already by day 7, ko bm - derived cells were almost completely outcompeted by wt bm - derived cells within the kc pool. this was not observed in monocytes but a similar pattern was observed in vsig4 macs demonstrating that alk1 is critically required for early kc development ( figure 7 h ).