Single-Cell RNA Sequencing Identifies Candidate Renal Resident Macrophage Gene Expression Signatures across Species

Zimmerman, Kurt A.; Bentley, Melissa R.; Lever, Jeremie M.; Li, Zhang; Crossman, David K.; Song, Chun-Yan; Liu, Shanrun; Crowley, Michael R.; George, James F.; Mrug, Michal; Yoder, Bradley K.

doi:10.1681/asn.2018090931

Cited by 146 publications

(174 citation statements)

References 31 publications

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“…As expected, the patrolling monocyte cluster did not expand in the kidney following injury (Fig 3a, e). We annotated the second cluster as inflammatory Ly6C + monocytes as they expressed Ly6c2, Ccr2, F13a1, and Chil3 genes (23). Cells in this cluster increased at UUO-2 (Fig 3a, e) and did not express the macrophage marker F4/80 as expected (Fig.…”

Section: Early Accumulation Of Ly6c2 + and Arg1 + Monocytes Followingmentioning

confidence: 59%

“…Most studies have used panels of cell surface markers to characterise myeloid cell subsets on flow cytometry; however, this approach is inherently biased and is unlikely to capture the full phenotypic spectrum. Recent advances in transcriptomics including single-cell RNA sequencing (scRNAseq) have been used to characterise the myeloid cells in the healthy, injured and regenerating kidney (23)(24)(25) and other organs (26)(27)(28)(29)(30). However, while macrophages may mediate regression of fibrosis (31), the macrophage heterogeneity during regression of fibrosis in the kidney remains uncertain.…”

Section: Introductionmentioning

confidence: 99%

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Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease

Conway

O’Sullivan

Cairns

et al. 2020

Preprint

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Short running title: Myeloid cell heterogeneity in kidney injury and repair Corresponding authors: Bryan Conway (Bryan.Conway@ed.ac.uk), Eoin O'Sullivan (Eoin.osullivan@ed.ac.uk), Tamir Chandra (Tamir.Chandra@ed.ac.uk) Laura Denby (Laura.Denby@ed.ac.uk) Word Count = 4810 Number of figures = 8 2 AbstractThe kidney has a limited capacity to repair following injury, however, the endogenous reparative pathways are not well understood. Here we employ integrated droplet-and platebased scRNA-seq in the murine reversible unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single cell level during renal injury and resolution of fibrosis.We generate a comprehensive catalogue of the changes induced during injury and repair, revealing significant myeloid cell heterogeneity, which would not have been identifiable by conventional flow cytometry. We identify new markers for the myeloid populations within the kidney as well as identification of novel subsets including an Arg1 + monocyte population specific to early injury and a Mmp12 + macrophage subset exclusive to repair. Finally, using paired blood exchange to track circulating immune cells, we confirm that monocytes are recruited to the kidney early after injury and are the source of Ccr2 + macrophages that accumulate in late injury. Our data demonstrate the utility of complementary technologies to identify novel myeloid subtypes that may represent therapeutic targets to inhibit progression or promote regression of kidney disease. Methods Animal modelsAll protocols and surgical procedures were approved by the Animal Ethics Committee, University of Edinburgh. Animal experiments were conducted in accordance with the Animals Scientific Procedures Act UK 1986, under Home Office project licenses 70/8093 and 70/8867. Reversible unilateral ureteric obstruction model (R-UUO)The R-UUO model was performed as previously described (33). Briefly, 8 week old male C57BL/6JOlaHsd mice (Enviago) underwent laparotomy and the left ureter was isolated and the distal portion was ligated twice with 6/O black braided silk suture close to the bladder. In

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Section: Early Accumulation Of Ly6c2 + and Arg1 + Monocytes Followingmentioning

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease

Conway

O’Sullivan

Cairns

et al. 2020

Preprint

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“…These data suggest that the Irf5-expressing inflammatory macrophages stimulate Pkd1-deficient tubular epithelium by secreting IL-6 to drive p-STAT3-dependent cyst progression ( Figure 6). With the recent discovery of a candidate resident macrophage population in humans (46), our data indicate that using ASOs may be an effective means to reduce cytokine production in these cells, reduce kidney inflammation, and slow cystic growth in human patients.…”

Section: Discussionmentioning

confidence: 92%

Interferon Regulatory Factor‐5 in Resident Macrophage Promotes Polycystic Kidney Disease

et al. 2020

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Background Autosomal dominant polycystic kidney disease is caused by genetic mutations in PKD1 or PKD2. Macrophages and their associated inflammatory cytokines promote cyst progression; however, transcription factors within macrophages that control cytokine production and cystic disease are unknown.Methods In these studies, we used conditional Pkd1 mice to test the hypothesis that macrophage-localized interferon regulatory factor-5 (IRF5), a transcription factor associated with production of cyst-promoting cytokines (TNFa, IL-6), is required for accelerated cyst progression in a unilateral nephrectomy (1K) model. Analyses of quantitative real-time PCR (qRT-PCR) and flow-cytometry data 3 weeks post nephrectomy, a time point before the onset of severe cystogenesis, indicate an accumulation of inflammatory infiltrating and resident macrophages in 1K Pkd1 mice compared with controls. qRT-PCR data from FACS cells at this time demonstrate that macrophages from 1K Pkd1 mice have increased expression of Irf5 compared with controls. To determine the importance of macrophage-localized Irf5 in cyst progression, we injected scrambled or IRF5 antisense oligonucleotide (ASO) in 1K Pkd1 mice and analyzed the effect on macrophage numbers, cytokine production, and renal cystogenesis 6 weeks post nephrectomy.Results Analyses of qRT-PCR and IRF5 ASO treatment significantly reduced macrophage numbers, Irf5 expression in resident-but not infiltrating-macrophages, and the severity of cystic disease. In addition, IRF5 ASO treatment in 1K Pkd1 mice reduced Il6 expression in resident macrophages, which was correlated with reduced STAT3 phosphorylation and downstream p-STAT3 target gene expression.Conclusions These data suggest that Irf5 promotes inflammatory cytokine production in resident macrophages resulting in accelerated cystogenesis.

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“…We examined expression patterns of FCER1G and TYROBP in several publicly available single cell RNASeq datasets (Figure 4): (A) renal resident macrophages across species (GSE128993; human n = 2,868, mouse n = 3,013, rat n = 3,935, pig n = 4,671) 45 , (B) mouse CX3CR1-derived macrophage from atherosclerotic aorta (GSE123587; n = 5,355) 43 , (C) mouse inflammatory airway macrophages (GSE120000; n = 1,142) 42 , and (D) mouse dissociated whole lung tissue (GSE111664; n = 41,898) 44 . We computed the percentage of single cell sample shows high-high expression patterns with respect to both FCER1G and TYROBP.…”

Section: Resultsmentioning

confidence: 99%

“…To validate our results on single cell RNASeq data we use following datasets: mouse inflammatory airway macrophages (GSE120000; n = 1,142) 42 , mouse CX3CR1-derived macrophage from atherosclerotic aorta (GSE123587; n = 5,355) 43 , mouse dissociated whole lung tissue (GSE111664; n = 41,898) 44 , and renal resident macrophages across species (GSE128993; human n = 2,868, mouse n = 3,013, rat n = 3,935, pig n = 4,671) 45 .…”

Section: Methodsmentioning

confidence: 99%

Computational approach to identifying universal macrophage biomarker

Dang

Taheri

Das

et al. 2019

Preprint

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ABSTRACTMacrophages are a type of white blood cell, of the immune system, that engulfs and digests cellular debris, cancer cells, and anything else that does not have the type of proteins specific to healthy body cells on its surface. Understanding gene expression dynamics in macrophages are crucial for studying human diseases. Recent advances in high-throughput technologies have enabled the collection of immense amounts of biological data. A reliable marker of macrophage is essential to study their function. Traditional approaches use a number of markers that may have tissue specific expression patterns. To identify universal biomarker of macrophage, we used a previously published computational approach called BECC (Boolean Equivalent Correlated Clusters) that was originally used to identify universal cell cycle genes. We performed BECC analysis on a seed gene CD14, a known macrophage marker. FCER1G and TYROBP were among the top candidates which were validated as strong candidates for universal biomarkers for macrophages in human and mouse tissues. To our knowledge, such a finding is first of its kind.CONTRIBUTIONS TO THE FIELDWe have developed a computational approach to identify universal biomarkers of different entities in a biological system. We applied this approach to study macrophages and identified universal biomarkers of this particular cell type. FCER1G and TYROBP were among the top candidates which were validated as strong candidates for universal biomarkers for macrophages in human and mouse tissues. The expression patterns of TYROBP and FCER1G are found to be more homogeneous compared to currently used biomarkers such as ITGAM, EMR1 (F4/80), and CD68. Further, we demonstrated that this homogeneity extends to all the tissues currently profiled in the public domain in multiple species including human and mouse. FCER1G and TYROBP expression patterns were also found to be extremely specific to macrophages found in various tissues. They are strongly co-expressed together. We believe that these two genes are the most reliable candidates of universal biomarker for macrophages.

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Single-Cell RNA Sequencing Identifies Candidate Renal Resident Macrophage Gene Expression Signatures across Species

Cited by 146 publications

References 31 publications

Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease

Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease

Interferon Regulatory Factor‐5 in Resident Macrophage Promotes Polycystic Kidney Disease

Computational approach to identifying universal macrophage biomarker

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