The DOI matches its Crossref record; 59 of the 59 references it cites were found in a registry or an index; none of the 26 sources its claims cite is checked for support yet.
Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage
Open accessPublished version of a preprint
Abstract
Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury. Using scRNA-seq analysis, Bhattacharya and colleagues identify a subset of profibrotic lung macrophages that have a gene expression signature intermediate between those of monocytes and alveolar macrophages.
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30 Own result · 20 Own interpretation · 12 Not checked yet · 10 Own method · 3 No basis given
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“the expression of these genes in SiglecF+CD11c+MHCIIhi cells at 4 weeks after injury was reduced to levels more similar to those detected in SiglecF+CD11c+ alveolar macrophages at steady-state”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
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“immunofluorescence of lung tissue indicated the expression of MAFB, a marker detected specifically in clusters C2 and C3, in CD68+ macrophages from patients with idiopathic lung fibrosis compared to healthy controls”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 and C3 markers are found in alternate lung fibrosis models
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In the paper
“The annotation scores for alveolar and interstitial macrophages were strongest at the poles of the macrophage cluster”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
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“showed three clusters of fibroblasts, cluster A, B, and C”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
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In the paper
“TdTomato-SiglecF+ alveolar macrophages (cluster C1) were detectable throughout the lung except in regions where Pdgfrb+ fibroblasts accumulated at sites of injury”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
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In the paper
“SingleR differentiated cellular subtypes such as memory and naïve B and T cells”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Reference datasets enhance annotation of scRNA-seq
What the sources say
- Not checked yetcites [20] Guilliams, M. & Malissen, B. A death notice for in-vitro-generated GM-CSF dendritic cells? Immunity 42, 988–990 (2015). source
- Not checked yetcites [21] Zheng, G. X. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017). source
- Not checked yetcites [22] Kang, H. M. et al. Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nat. Biotechnol. 36, 89–94 (2018). source
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“Human CX3CR1 mRNA was also more highly expressed in lung microarray samples from 167 patients with lung fibrosis than in samples from 50 healthy controls”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
What the sources say
- Not checked yetcites [32] Naikawadi, R. P. et al. Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis. JCI Insight 1, e86704 (2016). source
plaincorrelationaln = 167- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: Changes in expression of human orthologues in bulk lung microarray samples reflect changes in macrophage populations rather than other cell types.
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In the paper
“SingleR identified two subtypes of macrophages: alveolar and interstitial macrophages”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
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In the paper
“Conditioned media from SiglecF+CD11c+MHCIIhi macrophages (corresponding to cluster C2) sorted from wild-type mice 14 days after bleomycin injury and cultured for 2 days enhanced gap closure by 3T3 fibroblasts compared to conditioned media from SiglecF+CD11c+MHCIIlo macrophages (corresponding to cluster C1) isolated from the same mice”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
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- Rests on an assumption: The 3T3 embryonic mouse fibroblast cell line is a valid proxy for lung fibroblasts in fibrosis for assessing migration and proliferation.
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In the paper
“At baseline, TdTomato+ cells represented 6% of the live lung cells by flow cytometry, likely representing the interstitial macrophages present in steady-state lung”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
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In the paper
“Using only variable genes among the cell types increased the ability to distinguish closely related cell types.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Reference datasets enhance annotation of scRNA-seq
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In the paper
“when TdTomato+ cells sorted 14 days after bleomycin injury in tamoxifen-induced Cx3cr1-CreERT2 / Rosa26-loxp-STOP-loxp-TdTomato mice were co-cultured with 3T3 fibroblasts, Pdgf-aa blocking antibody decreased the proliferation of fibroblasts as measured by EDU uptake”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
plaindirect- Asserted: passes — The authors put it forward as true.
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- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: The 3T3 embryonic mouse fibroblast cell line is a valid proxy for lung fibroblasts in fibrosis for assessing migration and proliferation.
- Rests on an assumption: The Pdgf-aa blocking antibody specifically neutralizes Pdgf-aa without affecting other PDGF isoforms or unrelated pathways.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Antibody blockade of Pdgf-aa inhibited the enhanced gap closure seen in SiglecF+CD11c+MHCIIhi compared to SiglecF+CD11c+MHCIIlo conditioned media”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: The Pdgf-aa blocking antibody specifically neutralizes Pdgf-aa without affecting other PDGF isoforms or unrelated pathways.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In bleomycin-treated mice we observed an increase in the percentage of TdTomato+ cells among total lung live cells by flow cytometry at both day 7 (23%) and day 14 (38%) compared to baseline”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Pdgfa, Pdgf-aa, was highly and uniquely expressed in TdTomato+ cells adjacent to fibroblasts that were positive for expression of Pdgfra”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“In the lung scRNA-seq dataset, Pdgfa was uniquely expressed in the fibrosis-associated clusters C2 and C3”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“TdTomato+SiglecF+ macrophages were detected in direct contact with clusters of fibroblasts as defined by areas delimited by multiple adjacent Pdgfrb+ cells”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“This analysis indicated a gradient of cellular identity in the macrophage cluster between alveolar and interstitial macrophage”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“SingleR identified 33 of the 48 cells thought in the study to be BMDCs as macrophages”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Reference datasets enhance annotation of scRNA-seq
What the sources say
- Not checked yetcites [12] Heng, T. S. & Painter, M. W. The Immunological Genome Project Consortium. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008). source
- Not checked yetcites [15] SingleR GitHub repository, https://github.com/dviraran/SingleR .
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Pdgfa mRNA was more highly expressed by bulk RNA-seq in SiglecF+CD11c+MHCIIhi (cluster C2) macrophages than SiglecF+CD11c+MHCIIlo macrophages (cluster C1) at two weeks”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
plaincorrelational- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The number of lung macrophages was higher in the bleomycin-treated condition than at steady state”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
plaincorrelationaln = 3- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“SingleR performed similarly when applied to >50 publicly available scRNA-seq datasets”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Reference datasets enhance annotation of scRNA-seq
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“We detected a progressive increase in the expression of genes characteristic of clusters C2 and C3 in the lung lysates from tamoxifen-treated SPC-CreERT2 / Trf1fl/fl mice compared to control, with peak expression at 9 months”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 and C3 markers are found in alternate lung fibrosis models
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In the paper
“the expression of the alveolar macrophage marker SiglecF on TdTomato+ cells increased (as assessed by mean fluorescence intensity) across the time series”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“SiglecF+CD11c+MHCIIhi macrophages had higher expression of genes identified by scRNA-seq in cells from clusters C2 and C3 compared to SiglecF+CD11c+MHCIIlo cells at 2 weeks after injury”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“In three lung explants displaying such regional heterogeneity, we found that PDGF-AA+ macrophages were associated with the fibrotic rather than non-fibrotic regions”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
plaincorrelational- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: The human lung explants with regional heterogeneity are representative of idiopathic pulmonary fibrosis fibrotic foci.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The fact that TdTomato+ cells within the scar were also SiglecF+ suggests that the macrophages localizing to fibrotic zones were in a transitional state representative of cluster C2.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“indicating the acquisition of an alveolar macrophage marker by monocyte-derived macrophages across time”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“These analyses show that reference-based comparison enhances cell type annotation of scRNA-seq.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Reference datasets enhance annotation of scRNA-seq
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“suggesting the fibroblast response was dependent on Pdgf-aa secreted by SiglecF+CD11c+MHChi macrophages”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: The Pdgf-aa blocking antibody specifically neutralizes Pdgf-aa without affecting other PDGF isoforms or unrelated pathways.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Taken together, these studies support the relevance of the C2 and C3 markers identified in the bleomycin model to multiple contexts of fibrosis, mouse and human.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 and C3 markers are found in alternate lung fibrosis models
boostedcorrelationaln = 167- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: The bleomycin-induced mouse lung fibrosis model recapitulates key features of human lung fibrosis, so findings in it can inform human disease.
- Rests on an assumption: Changes in expression of human orthologues in bulk lung microarray samples reflect changes in macrophage populations rather than other cell types.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Taken together, these results show that macrophage cluster C2 localizes to the fibrotic niche.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 macrophages localize to the fibrotic niche
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“suggesting that cluster C2 was intermediate in gene expression between C1 and C3”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“These data suggest that between the 2- and 4-week time points, SiglecF+CD11c+MHCIIhi cells, which correspond to cluster C2 identified by SingleR, transition toward a steady-state alveolar macrophage profile (corresponding to cluster C1).”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Our observation that ablation of macrophages expressing Cx3cr1 decreased fibroblasts and lung collagen content after bleomycin treatment has relevance for human disease.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: The bleomycin-induced mouse lung fibrosis model recapitulates key features of human lung fibrosis, so findings in it can inform human disease.
In the paper
“Age-similar, non-diseased, normal lung tissues were procured from lungs not used by the Northern California Transplant Donor Network54.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Human Lung Tissues
What the sources say
- Not checked yetcites [54] Lachmann, A. et al. Massive mining of publicly available RNA-seq data from human and mouse. Nat. Commun. 9, 1366 (2018). source
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“during fibrogenesis CX3CR1+SiglecF+ macrophages localize to sites of Pdgfra+ and Pdgfrb+ fibroblast accumulation at sites of bleomycin-induced injury in the lung”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“In the current study, we used dissociated whole lung for single cell analysis, which enabled an unbiased view of all cellular subtypes in the lung.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“As such, SingleR could make the distinction between macrophages and dendritic cells in an unbiased fashion based on the granularity and breadth of the transcriptomic comparison.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Reference datasets enhance annotation of scRNA-seq
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“These observations indicate that the Pdgf pathway is crucial to disease pathogenesis.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
What the sources say
- Not checked yetcites [39] Richeldi, L., Collard, H. R. & Jones, M. G. Idiopathic pulmonary fibrosis. Lancet 389, 1941–1952 (2017). source
- Not checked yetcites [40] Noble, P. W., Barkauskas, C. E. & Jiang, D. Pulmonary fibrosis: patterns and perpetrators. J. Clin. Invest. 122, 2756–2762 (2012). source
- Not checked yetcites [41] Wollin, L. et al. Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. Eur. Respir. J. 45, 1434–1445 (2015). source
- Not checked yetcites [42] Olson, L. E. & Soriano, P. Increased PDGFRα activation disrupts connective tissue development and drives systemic fibrosis. Dev. Cell 16, 303–313 (2009). source
- Not checked yetcites [43] Iwayama, T. et al. PDGFRα signaling drives adipose tissue fibrosis by targeting progenitor cell plasticity. Genes Dev. 29, 1106–1119 (2015). source
- Not checked yetcites [44] Horikawa, S. et al. PDGFRα plays a crucial role in connective tissue remodeling. Sci. Rep. 5, 17948 (2015). source
- Not checked yetcites [45] Hayes, B. J. et al. Activation of platelet-derived growth factor receptor alpha contributes to liver fibrosis. PLoS ONE 9, e92925 (2014). source
- Not checked yetcites [46] Demaria, M. et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev. Cell 31, 722–733 (2014). source
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“This result indicates that CX3CR1+ macrophages were required for the expansion of the fibroblast compartment and the deposition of fibrotic scar.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
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In the paper
“Pulmonary fibrosis lung tissues were obtained at the time of lung transplantation from patients with a pathologic diagnosis of usual interstitial pneumonia and a consensus clinical diagnosis of idiopathic pulmonary fibrosis (IPF) according to available guidelines53.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Human Lung Tissues
What the sources say
- Not checked yetcites [53] Aran, D., Hu, Z. & Butte, A. J. xCell: digitally portraying the tissue cellular heterogeneity landscape. Genome Biol. 18, 220 (2017). source
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Our results implicate the CX3CR1+SiglecF+ macrophage as the source of Pdgf-aa in the fibrotic niche.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
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In the paper
“A limitation of using Cx3cr1-CreERT2 is that Cx3cr1 is expressed in both clusters C2 and C3, limiting the specificity of our studies.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Together, these results suggest the dependence of fibroblast proliferation, which is a key step in fibrogenesis38, on trophism exerted by PDGF-AA+ macrophages recruited to sites of injury.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
What the sources say
- Not checked yetcites [38] Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015). source
hedgeddirect- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: The 3T3 embryonic mouse fibroblast cell line is a valid proxy for lung fibroblasts in fibrosis for assessing migration and proliferation.
- Rests on an assumption: The Pdgf-aa blocking antibody specifically neutralizes Pdgf-aa without affecting other PDGF isoforms or unrelated pathways.
None of the sources cited for this claim have been checked yet.
In the paper
“Because macrophages and dendritic cells have similar transcriptomic profiles23–25, we repeated SingleR analysis using published RNA-seq datasets of mouse lung macrophages and dendritic cells26,27 to isolate the macrophages”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
What the sources say
- Not checked yetcites [23] Li, H. et al. Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors. Nat. Genet. 49, 708–718 (2017). source
- Not checked yetcites [24] SingleR web tool, http://comphealth.ucsf.edu/SingleR/ .
- Not checked yetcites [25] Gautier, E. L. et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat. Immunol. 13, 1118–1128 (2012). source
- Not checked yetcites [26] Hume, D. A., Mabbott, N., Raza, S. & Freeman, T. C. Can DCs be distinguished from macrophages by molecular signatures? Nat. Immunol. 14, 187–189 (2013). source
- Not checked yetcites [27] Randolph, G. & Merad, M. Reply to: “Can DCs be distinguished from macrophages by molecular signatures?”. Nat. Immunol. 14, 189–190 (2013). source
plaincorrelational- Asserted: passes — The authors put it forward as true.
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- It may state two separate claims as one. 19
- Probably not a substantial claim. 14
- The quote only partly states this claim. 1
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“C1 had higher expression of alveolar macrophage genes than cluster C2, which in turn had higher alveolar macrophage genes than cluster C3”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
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In the paper
“Clusters C2 and C3 were enriched in cells isolated from mice with bleomycin-induced fibrosis and expressed Cx3cr1, Ccr2, Mafb and MHCII genes, consistent with monocytic origin”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
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In the paper
“Cluster A showed the highest expression of cell cycle genes and was predominantly composed of cells from bleomycin-treated mice”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“We developed a computational tool for unbiased annotation as well as clustering of scRNA-seq, called SingleR (single cell recognition). SingleR assigns cellular identity for single cell transcriptomes by comparison to reference datasets of pure cell types sequenced by microarray or RNA-seq.”
- Also stated in: § Reference datasets enhance annotation of scRNA-seq · plain
“we developed a computational method called SingleR, which correlated single cell transcriptomes with reference transcriptomic datasets and improved its inferences iteratively”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Abstract
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- Also stated in: § Reference datasets enhance annotation of scRNA-seq · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“This analysis identified three distinct subclusters (C1, C2 and C3), all three detected at varying levels in both steady-state and bleomycin-treated mice”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
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In the paper
“cluster C1 was equally present in steady-state lung and bleomycin injury and corresponded to the cells that were annotated as alveolar macrophages by the lung-specific datasets”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“45% of cluster C2 cells (but only 7.3% of cluster C1 cells and 4.7% of cluster C3 cells) expressed at least 33% of cluster C1 genes and at least 33% of cluster C3 genes”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > SingleR identifies a transitional macrophage in fibrosis
plaincorrelational- Asserted: passes — The authors put it forward as true.
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The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.
In the paper
“Pdgfra was not detected in the lung scRNA-seq dataset, which may reflect the limitation of the depth of sequencing with the single-cell method”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
hedgedconditional- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“This analysis showed a significant decrease in the expression of genes associated with alveolar macrophages (cluster C1) and a significant increase in the expression of MHC II genes (cluster C2 and C3) in the human fibrosis samples compared to the healthy controls”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 and C3 markers are found in alternate lung fibrosis models
What the sources say
- Not checked yetcites [32] Naikawadi, R. P. et al. Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis. JCI Insight 1, e86704 (2016). source
plaincorrelationaln = 167- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
- Rests on an assumption: Changes in expression of human orthologues in bulk lung microarray samples reflect changes in macrophage populations rather than other cell types.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“At 21 days after bleomycin injury, bleomycin-injured, tamoxifen-induced Cx3cr1-CreERT2 / Rosa26-loxp-STOP-loxp-Diphteria Toxin A mice had significantly fewer macrophages quantified by immunofluorescent labeling of either Mertk+ or SiglecF+ cells in the lung”
- Also stated in: § Discussion · plain
“Deletion of these macrophages during the fibrotic period (days 8–21) eliminated Pdgfra+ or Pdgfrb+ fibroblasts and total lung collagen.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
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It may state two separate claims as one. The quote only partly states this claim.
- Also stated in: § Discussion · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Altogether, the filtered data contained 8,366 cells and 13,861 genes (Supplementary Table 1).”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Drop-seq and data analysis
plain- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“a significant expansion of Pdgfra+ fibroblasts after bleomycin treatment has been reported in a study analyzing the fibroblast compartment in this model of fibrosis 35”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > CX3CR1-expressing macrophages drive lung fibrosis
What the sources say
- Not checked yetcites [35] Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79–91 (2013). source
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“the results are consistent with a common genetic signature and a generalizable pattern of macrophage response during fibrogenesis”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Results > Cluster C2 and C3 markers are found in alternate lung fibrosis models
plaincorrelational- Asserted: passes — The authors put it forward as true.
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A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results also demonstrate the value of using reference-based annotation of single cell datasets in order to map the heterogeneity of canonical cell types, such as macrophages, and to enhance the differentiation of transcriptomically unique subclusters within the data.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
boosteddirect- Asserted: passes — The authors put it forward as true.
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What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“SingleR uses the 80th percentile of correlation values, to prevent misclassification due to heterogeneity in the reference samples.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Cell Type Annotation of Single Cells (SingleR)
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“All animal experiments were in accordance with protocols approved by the UCSF Institutional Animal Care and Use Committee.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Mice
plain- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“our annotation of cell states using SingleR relies on the particularities of the reference datasets”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Written informed consent was obtained from all subjects, and the study was approved by the UCSF institutional review board.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Human Lung Tissues
plain- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our analysis suggests an evolution of cluster C3 towards cluster C1, with C2 representing a transitional state.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“The ImmGen database is a large compendium of cell types from multiple organs, which greatly facilitated our clustering analysis by differential annotation.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
What the sources say
- Not checked yetcites [12] Heng, T. S. & Painter, M. W. The Immunological Genome Project Consortium. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008). source
boosteddirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“Yang et al.32 performed gene expression profiling for 167 lung tissues from idiopathic interstitial pneumonia subjects and 50 healthy controls.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Analysis of published human lung microarray datasets
What the sources say
- Not checked yetcites Yang et al.32
plain- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Our studies indicate that these lungs are physiologically and pathologically normal.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Human Lung Tissues
hedged- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper may give no reason why it holds. It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“suggesting that paracrine interactions between these macrophages and fibroblasts sustain fibroblast proliferation and tissue fibrosis”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
hedgeddirect- Asserted: passes — The authors put it forward as true.
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A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“These findings underscore the potential therapeutic significance of our functional studies in mice.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
hedged- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. It may be too vague to check against evidence.
- Rests on an assumption: The bleomycin-induced mouse lung fibrosis model recapitulates key features of human lung fibrosis, so findings in it can inform human disease.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Strain-appropriate controls were used in all experiments”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Mice
plain- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Using only variable genes increases the ability to distinguish closely related cell types.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Cell Type Annotation of Single Cells (SingleR)
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“All the samples passed the quality control parameters established by Agilent.”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Online Methods > Microarray profiling in telomere dysfunction model of lung fibrosis
plain- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“this study identified a profibrotic macrophage subpopulation that localizes to sites of fibrotic scar with activating effects on the mesenchyme”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
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A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Mechanistic studies indicated that fibroblast migration and proliferation were dependent on secretion of Pdgf-aa by SiglecF+CD11c+MHCIIhi and CX3CR1+ cells, respectively”
§ Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage > Discussion
hedgeddirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
- Rests on an assumption: The 3T3 embryonic mouse fibroblast cell line is a valid proxy for lung fibroblasts in fibrosis for assessing migration and proliferation.
- Rests on an assumption: The Pdgf-aa blocking antibody specifically neutralizes Pdgf-aa without affecting other PDGF isoforms or unrelated pathways.
The paper relies on this without support.
The bleomycin-induced mouse lung fibrosis model recapitulates key features of human lung fibrosis, so findings in it can inform human disease.
“Our observation that ablation of macrophages expressing Cx3cr1 decreased fibroblasts and lung collagen content after bleomycin treatment has relevance for human disease.”
The paper relies on this without support.
The 3T3 embryonic mouse fibroblast cell line is a valid proxy for lung fibroblasts in fibrosis for assessing migration and proliferation.
“we performed in vitro assays with 3T3 fibroblasts, an embryonic mouse fibroblast cell line.”
The paper relies on this without support.
The Pdgf-aa blocking antibody specifically neutralizes Pdgf-aa without affecting other PDGF isoforms or unrelated pathways.
“Antibody blockade of Pdgf-aa inhibited the enhanced gap closure seen in SiglecF+CD11c+MHCIIhi compared to SiglecF+CD11c+MHCIIlo conditioned media”
The paper relies on this without support.
Changes in expression of human orthologues in bulk lung microarray samples reflect changes in macrophage populations rather than other cell types.
“we performed single-sample gene set enrichment analysis (ssGSEA)31 to quantify the expression of human orthologues of genes of interest in a published microarray dataset of 167 lung samples from patients with lung fibrosis and 50 healthy controls32”
The paper relies on this without support.
The human lung explants with regional heterogeneity are representative of idiopathic pulmonary fibrosis fibrotic foci.
“In three lung explants displaying such regional heterogeneity, we found that PDGF-AA+ macrophages were associated with the fibrotic rather than non-fibrotic regions”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“The pathogenic role of Mo-AMs in lung fibrosis has been largely attributed to their activity in producing a number of pro-fibrotic mediators, such as TGF-β, PDGF-α, osteopontin (OPN), and MMPs1,3,11,13,15,18,28.”
From TREM2 promotes lung fibrosis via controlling alveolar macrophage survival and pro-fibrotic activity (2025) · cites this paper as [18]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“the M3-TAMs, actually the FABP4+ TAMs previously identified as the alveolar macrophages in the lung, were dominant in the lung metastatic osteosarcoma lesion [24].”
From Advances on immunotherapy for osteosarcoma (2024) · cites this paper as [24]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“To identify myeloid clusters, we checked the expression of canonical myeloid marker genes (Supplementary Data 3). Furthermore, we used SingleR v2.0.074 for automated annotation based on the Novershtern immune reference dataset75 from the celldex v1.8.0 package74.”
From CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis (2024) · cites this paper as [74]
Experimental — accuracy not yet measuredThe check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“To address the ambient RNA contamination SoupX version 1.5.2[54] was utilized to identify and remove ambient RNA background noise from the data.”
From Single-cell transcriptomics unveils skin cell specific antifungal immune responses and IL-1Ra- IL-1R immune evasion strategies of emerging fungal pathogen Candida auris (2024) · cites this paper as [54]
Experimental — accuracy not yet measuredIn the citing paper
“The pericellular fibrotic matrix is a remarkably dynamic environment, which exerts profound influences on cell behavior, and many of the key cell–cell and cell–matrix interactions which regulate fibrosis are mediated by members of the integrin family (noncovalent α/β heterodimers from 18 different α subunits and 8 β subunits resulting in 24 known members in humans)86,87.”
From Fibrosis: from mechanisms to medicines (2020) · cites this paper as [86]
In the citing paper
“integrins can mediate the translation of spatially-fixed extracellular signals into a wide variety of changes in cell behavior including cell adhesion, migration, proliferation, differentiation and apoptosis86,87.”
From Fibrosis: from mechanisms to medicines (2020) · cites this paper as [86]
In the citing paper
“Of key relevance to fibrosis, integrins can also potentiate signals from soluble pro-fibrogenic growth factors such as TGF-β1.”
From Fibrosis: from mechanisms to medicines (2020) · cites this paper as [86]
Citations
Cites
STAR: ultrafast universal RNA-seq aligner
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Massively parallel digital transcriptional profiling of single cells
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Self-citationFate Mapping Reveals Origins and Dynamics of Monocytes and Tissue Macrophages under Homeostasis
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Cited by
Sorted by citations
Fibrosis: from mechanisms to medicines
Henderson, N. C., Rieder, F., & Wynn, T. A. (2020) · Nature · doi:10.1038/s41586-020-2938-9 · Cited by 1,868
A single-cell atlas of the peripheral immune response in patients with severe COVID-19
Wilk, A. J., Rustagi, A., Zhao, N. Q., Roque, J., Martínez-Colón, G. J., McKechnie, J. L., Ivison, G. T., Ranganath, T., Vergara, R., Hollis, T., et al. (2020) · Nature Medicine · doi:10.1038/s41591-020-0944-y · Cited by 1,493
A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells
Cheng, S., Li, Z., Gao, R., Xing, B., Gao, Y., Yang, Y., Qin, S., Zhang, L., Ouyang, H., Du, P., et al. (2021) · Cell · doi:10.1016/j.cell.2021.01.010 · Cited by 1,601
BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection
Cao, Y., Yisimayi, A., Jian, F., Song, W., Xiao, T., Wang, L., Du, S., Wang, J., Li, Q., Chen, X., et al. (2022) · Nature · doi:10.1038/s41586-022-04980-y · Cited by 1,306
Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment
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Metadata
- Authors
- Aran, D., Looney, A. P., Liu, L., Wu, E., Fong, V., Hsu, A., Chak, S., Naikawadi, R. P., Wolters, P. J., Abate, A. R., Butte, A. J., & Bhattacharya, M.
- Affiliations
- University of California, San Francisco (US)
- Gladstone Institutes (US)
- QB3 (US)
- Corresponding author
- Bhattacharya, Mallar
- ORCID
- Venue
- Nature Immunology
- Published
- 2019-01-14
- Volume
- 20
- Issue
- 2
- Pages
- 163-172
- ISSN
- 1529-2908 (ISSN-L)
- 1529-2916
- PMC
- PMC6340744
- OpenAlex
- W2907514116
- Semantic Scholar
- a4d99547655d02c1cfa5d8c627a10248507b96f2
- Cited by
- 4,984
- References
- 56
- Citations by year
- 2026: 831 · 2025: 1024 · 2024: 966 · 2023: 856 · 2022: 662 · 2021: 394 · 2020: 181 · 2019: 63
- FWCI
- 249.29
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- cc-by-nc-nd
- www.springer.com/tdm (tdm)
- Locations
- Nature Immunology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Gladstone Institutes
- National Institutes of Health (HHSN272201200028C)
- University of California, San Francisco
- National Heart, Lung, and Blood Institute (L30 HL110167, R01HL131560, R01HL139897)
- National Institute of Allergy and Infectious Diseases (HHSN272201200028C)
- Topics
- Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Health Sciences › Medicine › Surgery › Transplantation: Methods and Outcomes
- Keywords
- pulmonary fibrosis, macrophage heterogeneity, bleomycin-induced lung fibrosis, idiopathic pulmonary fibrosis, monocyte-derived macrophages, alveolar macrophages, CX3CR1, fibrotic niche, macrophage activation
- MeSH terms
- CX3C Chemokine Receptor 1, Animals, Bleomycin, Cells, Cultured, Disease Models, Animal, Female, Humans, Lung, Macrophage Activation, Male, Antigens, Differentiation, Myelomonocytic, Up-Regulation, Macrophages, Alveolar, Sequence Analysis, RNA, Gene Expression Profiling, Mice, Idiopathic Pulmonary Fibrosis, Single-Cell Analysis, Sialic Acid Binding Immunoglobulin-like Lectins
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
A language model drew the claims from the paper on 2026-10-06, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-10-06 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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