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How to Find a Resident Kidney seeded macrophages, often affect macrophages and DCs, and utilize global rather than kidney-specific macrophage depletion. Macrophage: the Single-Cell However, even with these caveats, the overall body of evidence suggests that resident kidney macrophages are important players Sequencing Solution in injury responses, with both positive and negative effects.6 One of the problems of translating mouse studies of Menna R. Clatworthy1,2,3 kidney macrophages to humans is that some of the markers 1 Molecular Immunity Unit, Department of Medicine, University of used to delineate mouse macrophages are not expressed by Cambridge, Cambridge, UK; 2Cellular Genetics Programme, Wellcome Sanger Institute, Cambridge, UK; and 3Transplantation their human counterparts, making it difficult to identify and Regenerative Medicine Theme, Cambridge National Institute analogous populations across specifies. For example, the for Health Research Biomedical Research Centre, Cambridge, UK F4/80 antibody (binding to Adgre1) is mouse macrophage– J Am Soc Nephrol 30: ccc–ccc, 2019. specific. Further, it has emerged that markers that were doi: https://doi.org/10.1681/ASN.2019030245 historically considered to distinguish DCs from macro- phages such as CD11c and MHCII, are frequently expressed by tissue macrophages. Indeed, monocyte-derived macro- 1 Mononuclear phagocytes comprise monocytes, macro- phages in human skin and kidney are CD11c / 1 1 phages, and dendritic cells (DCs) with several tissue-resident MHCII /CD14 .7,8 In clinical histopathology, CD68 is of- subsets described according to surface markers, function, and ten used as a macrophage marker in human kidney, and 1 ontogeny. Macrophages alone are entirely tissue-resident, do CD206 expression has also been described on human kid- not circulate, and form a network of immune sentinels in all ney macrophages, but the specificity of these markers is organs, poised to detect a range of perturbations, from in- unclear. This highlights the problem that Zimmerman fection to tissue damage. Although initially thought to be et al.9 and colleagues attempt to address: is it possible to via derived from hematopoietic stem cells monocyte differenti- identify a cross-species kidney macrophage–specific “sig- ation, recent fate-mapping studies in mice have established that nature” or marker set that will facilitate the translation of mu- some tissue macrophages are seeded prenatally, and may origi- rine models to human biology and disease? nate from yolk-sac or fetal liver progenitors, with the proportion To tackle this problem, they turned to single-cell RNA of monocyte-derived or prenatally seeded macrophages varying sequencing (scRNAseq). To date, transcriptomic studies of 1 in different organs. Regardless of ontogeny, murine studies sug- immune cells have largely relied on the measurement of fl gest that macrophages are profoundly in uenced by their tissue mRNA in subsets isolated by flow sorting or magnetic bead fi environment and take on organ-speci c phenotypes and tran- selection, requiring prior knowledge of subset-specificmark- fl scriptional signatures in uenced by the local cell composition ers. However, advances in single-cell technologies10 now en- 2 and by sampling the milieu. able immune cells to be profiled in an unbiased manner, such Why should nephrologists care about macrophages? that those with a similar transcriptome can be clustered with- Several studies show that, at least in mice, tissue macro- out any preconceived idea about their canonical markers. phages can play an important role in organ homeostasis; Zimmerman et al.9 leveraged this strength in their 1 for example, in the heart, macrophages are intimately asso- study. They isolated kidney CD45 (immune) cells from ciated with the conducting system and are required for nor- onemouse,rat,pig,andhuman,andperformedscRNAseq 3 mal electrical conduction. In the gut, macrophages interact on a mononuclear phagocyte–enriched cell suspension. Us- 4 with enteric neurons to promote peristalsis. Murine data also ing the data generated, they clustered cells according to tran- suggest that kidney macrophages are likely to modulate suscep- scriptional similarity, allowing them to identify putative tibility to, and the resolution of, kidney infection, ischemia- “monocyte-derived” and “resident” kidney macrophage cell reperfusion injury, GN, diabetic nephropathy, and progression clusters, and confirmed overlap with the transcriptional sig- fi 5 to brosis. Notably, the methods used in these studies largely do natures obtained from flow-sorted F4/80lowCD11bhi and not distinguish between monocyte-derived or embryonically F4/80highCD11blow mouse kidney macrophages, respec- tively (Figure 1). They used the most differentially expressed in the mouse resident macrophage cluster, C1qc,to Published online ahead of print. Publication date available at www.jasn.org. identify analogous clusters in the human, pig, and rat kidney fi Correspondence: Dr. Menna R. Clatworthy, Department of Medicine, Uni- scRNAseq datasets, and con rmed that the C1q-expressing versity of Cambridge, Cambridge Biomedical Campus, Francis Crick Avenue, clusters across species expressed other that were highly Cambridge, CB2 0QH, UK. Email: [email protected] expressed in mouse macrophages: Cd81, Cd74,andApoe.To Copyright © 2019 by the American Society of Nephrology validate these markers, they identified commercially

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Figure 1. Summary of experimental setup used by Zimmerman et al.9 to identify a cross-species renal-resident macrophage signature. The resident macrophage cell cluster in mouse kidney (pink) was identified by comparing the within each cluster to that of sorted resident macrophages (top panel). Marker genes for the resident macrophage population were identified from this mouse cluster and applied to the data obtained from other species, allowing the resident macrophage population to be identified across species. available antibodies for the products of three of these DISCLOSURES genes: C1q, CD74, and CD81. The antibodies were applied None. to flow cytometric studies in mouse, rat, and human kid- neys, and although C1q was not highly expressed in human kidney macrophages, they were able to identify CD74 and REFERENCES CD81 expression on a population of kidney immune cells 1. Guilliams M, Ginhoux F, Jakubzick C, Naik SH, Onai N, Schraml BU, across species. Finally, they performed a parabiosis experi- et al.: Dendritic cells, monocytes and macrophages: A unified no- 1 ment in mouse, which suggested that the CD74/CD81 cells menclature based on ontogeny. Nat Rev Immunol 14: 571–578, 2014 in the kidney were not exchanged, confirming the utility of 2. A-Gonzalez N, Quintana JA, García-Silva S, Mazariegos M, González de these markers to identify a bona fide resident macrophage la Aleja A, Nicolás-Ávila JA, et al.: Phagocytosis imprints heterogeneity in tissue-resident macrophages. JExpMed214: 1281–1296, 2017 population. 3. Hulsmans M, Clauss S, Xiao L, Aguirre AD, King KR, Hanley A, et al.: Of note, this study included cells from only one kidney from Macrophages facilitate electrical conduction in the heart. Cell 169: each species in their scRNAseq experiment. Because humans are 510–522.e20, 2017 genetically heterogeneous and have variable environmental ex- 4. De Schepper S, Verheijden S, Aguilera-Lizarraga J, Viola MF, Boesmans W, Stakenborg N, et al.: Self-maintaining gut macrophages are es- posures, the extent to which the proportions of immune cell Cell – fi sential for intestinal homeostasis. 175: 400 415.e13, 2018 identi ed from this single human kidney, and the magnitude 5. Nelson PJ, Rees AJ, Griffin MD, Hughes J, Kurts C, Duffield J: The renal of expression of any given marker gene in each cell population, mononuclear phagocytic system. J Am Soc Nephrol 23: 194–203, 2012 may not be generally applicable. However, even with this ca- 6. Rogers NM, Ferenbach DA, Isenberg JS, Thomson AW, Hughes J: veat, the study demonstrates the power of scRNAseq to Dendritic cells and macrophages in the kidney: A spectrum of good and Nat Rev Nephrol – identify a core resident macrophage signature as well as novel evil. 10: 625 643, 2014 7. McGovern N, Schlitzer A, Gunawan M, Jardine L, Shin A, Poyner E, et al.: 1 cross-species kidney macrophage markers that can be vali- Human dermal CD14 cells are a transient population of monocyte- dated in future studies, and will facilitate clinically relevant derived macrophages. Immunity 41: 465–477, 2014 translational research. 8. Berry MR, Mathews RJ, Ferdinand JR, Jing C, Loudon KW, Wlodek E, et al.: Renal sodium gradient orchestrates a dynamic antibacterial de- fense zone. Cell 170: 860–874.e19, 2017 ACKNOWLEDGMENTS 9. Zimmerman KA, Bentley MR, Lever JM, Li Z, Crossman DK, Song CJ, et al.: Single-cell RNA sequencing identifies candidate renal resident macrophage gene expression signatures across species. JAmSoc Dr. Clatworthy is supported by the National Institutes of Health Re- Nephrol 30: xxx–xxx, 2019 search (NIHR) Cambridge Biomedical Research Centre, by a Chan 10. Potter SS: Single-cell RNA sequencing for the study of development, Zuckerberg Initiative Human Cell Atlas Technology Development physiology and disease. Nat Rev Nephrol 14: 479–492, 2018 grant, a Medical Research Council New Investigator research grant (MR/N024907/1), an Arthritis Research UK Cure Challenge re- search grant, 21777, and an NIHR Research Professorship (RP-2017- See related article, “Single-Cell RNA Sequencing Identifies Candidate Renal Resident 08-ST2-002). Macrophage Gene Expression Signatures across Species,” on pages XXX–XXX.

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