Coal dust exposure triggers heterogeneity of transcriptional profiles in mouse pneumoconiosis and Vitamin D remedies

Mu, Min; Li, Bing; Zou, Yuanjie; Wang, Wenyang; Cao, Hangbing; Zhang, Yajun; Sun, Qixian; Chen, Haoming; Ge, Deyong; Tao, Huihui; Hu, Dong; Yuan, Liang; Tao, **nrong; Wang, Jianhua

doi:10.1186/s12989-022-00449-y

Coal dust exposure triggers heterogeneity of transcriptional profiles in mouse pneumoconiosis and Vitamin D remedies

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Published: 20 January 2022

Volume 19, article number 7, (2022)
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Coal dust exposure triggers heterogeneity of transcriptional profiles in mouse pneumoconiosis and Vitamin D remedies

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Min Mu^1,2,3,4^na1,
Bing Li³^na1,
Yuanjie Zou³,
Wenyang Wang³,
Hangbing Cao³,
Yajun Zhang^1,2,3,4,
Qixian Sun³,
Haoming Chen³,
Deyong Ge^1,2,3,4,
Huihui Tao^1,2,3,4,
Dong Hu^1,2,3,4,
Liang Yuan^1,2,3,4,
**nrong Tao^1,2,3,4 &
…
Jianhua Wang^1,5

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Abstract

Background

Coal dust particles (CDP), an inevitable by-product of coal mining for the environment, mainly causes coal workers’ pneumoconiosis (CWP). Long-term exposure to coal dust leads to a complex alternation of biological processes during regeneration and repair in the healing lung. However, the cellular and complete molecular changes associated with pulmonary homeostasis caused by respiratory coal dust particles remain unclear.

Methods

This study mainly investigated the pulmonary toxicity of respirable-sized CDP in mice using unbiased single-cell RNA sequencing. CDP (< 5 μm) collected from the coal mine was analyzed by Scanning Electron Microscope (SEM) and Mass Spectrometer. In addition, western blotting, Elisa, QPCR was used to detect gene expression at mRNA or protein levels. Pathological analysis including HE staining, Masson staining, immunohistochemistry, and immunofluorescence staining were performed to characterize the structure and functional alternation in the pneumoconiosis mouse and verify the reliability of single-cell sequencing results.

Results

SEM image and Mass Spectrometer analysis showed that coal dust particles generated during coal mine production have been crushed and screened with a diameter of less than 5 µm and contained less than 10% silica. Alveolar structure and pulmonary microenvironment were destroyed, inflammatory and death (apoptosis, autophagy, and necrosis) pathways were activated, leading to pneumoconiosis in post 9 months coal dust stimulation. A distinct abnormally increased alveolar type 2 epithelial cell (AT2) were classified with a highly active state but reduced the antimicrobial-related protein expression of LYZ and Chia1 after CDP exposure. Beclin1, LC3B, LAMP2, TGF-ß, and MLPH were up-regulated induced by CDP, promoting autophagy and pulmonary fibrosis. A new subset of macrophages with M2-type polarization double expressed MLPH + /CD206 + was found in mice having pneumoconiosis but markedly decreased after the Vitamin D treatment. Activated MLPH + /CD206 + M2 macrophages secreted TGF-β1 and are sensitive to Vitamin D treatment.

Conclusions

This is the first study to reconstruct the pathologic progression and transcriptome pattern of coal pneumoconiosis in mice. Coal dust had obvious toxic effects on lung epithelial cells and macrophages and eventually induced pulmonary fibrosis. CDP-induced M2-type macrophages could be inhibited by VD, which may be related to the alleviation of the pulmonary fibrosis process.

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Introduction

Coal remains the primary source of electricity (60%) and iron production (40%) globally [1]. Coal dust hazards in coal mining have been studied continuously by the WHO (World Health Organization) and governments of many countries. As of 2018, pneumoconiosis accounted for 90% of occupational diseases reported in China's occupational health status reports, with 200 million people still at risk of occupational health exposure [2]. The rates of pneumoconiosis in Australia have started rising [3]. Many studies have clearly shown that coal particles have cytotoxicity, pro-inflammatory, pro-fibrotic properties, and a complex progress mechanism [4,5,6]. The most extensive study on pneumoconiosis in the U.S. revealed that respirable dust from coal miners caused progressive massive fibrosis (PMF) even after dust exposure has ceased [7, 8]. Mine Safety and Health Administration (MSHA) issued new regulations [9] for respirable coal dust concentration limit (from 2.0 to 1.5 mg/ m³). However, the prevalence rate continued to increase, with some regions such as the central Appalachian area seeing a sixfold increase [10]. There is no effective treatment for coal workers' pneumoconiosis.

Continued exposure to high concentrations of coal dust may lead to an inability to remove accumulated dust particles, resulting in structural damage to the alveoli [11]. Alveolar epithelial cells are the primary functional unit for O₂ exchange and the first point of contact for inhaled particles [11]. Successful alveolar epithelial repair and functional recovery require the proliferation and differentiation of alveolar type 2 stem/progenitor cells (AT2). During the regeneration process, alveolar epithelial cells have been reported to have dysplasia or developmental delays in repair [12], which is associated with many chronic lung diseases [13]. Commonly, inflammatory monocytes and pulmonary macrophages are the keys to lung repair and fibrosis regulators. Lung macrophages in healthy adults are divided into mononuclear mesenchymal macrophages and tissue-resident macrophages that develop from embryonic precursors. Abnormal tissue niches can lead to the development of abnormally differentiated tissue-resident macrophages [14]. Alveolar macrophages (AMs), the central target cells of coal dust during phagocytosis and clearance, are related to oxidative stress and the activation of the innate immune system [15]. Exogenous pathways regulate apoptosis and the release of fibrotic factors from AMs. TNF-α, IL 6, and IL 1β are recognized pro-inflammatory factors released by macrophages [Full size image

To further predict the toxic damage mechanism in CMP lungs, we studied the signaling correspondence between epithelia and macrophages. After coal dust treatment, these cells co-located in space and actively signaled to each other. There were significant positive and negative signal states in macrophages, indicating that the cell state transition under coal dust exposure was highly regulated. Specifically, the TNF pathway showed rich signaling interactions among all four states, with CD74 receptors expressed primarily by macrophages. In addition, the dominantly ligand CD44 was contributing to dermal TGF signaling, which promotes granuloma formation by stimulating the in situ proliferation of mononuclear cells through autocrine and paracrine signaling [52]. Furthermore, macrophages secreted SPP1 to multiple cell surface receptors, consistent with the known role of physiological and pathological processes, including wound healing, inflammation, tumorigenesis, and ischemia. In addition, epithelial cells were the major ligand source for another important Notch signaling pathway that primarily expresses autocrine Notch1 and Notch2 in CMP.

The Cell Phone DB analysis also predicted that this dominant macrophage notch signaling was supplemented by a minor epithelial-derived Nothch2 ligand paracrine signaling, promoting macrophage M2 polarization, dependent on the interaction with CD47 and mediated by intracellular signaling through SHP-1 [53, 54]. Our analysis showed solid intercellular communication between macrophages and epithelial cells, including the innate immune system signaling members SIRPA and CD47, and Notch signaling ligands Jag1, gag2, Tgfb2, and Fam3c (Fig. 7D). These results further emphasize the indispensable roles of these well-defined pathways during coal dust lung specification. In addition, we observed robust ligand-receptor pairs within the epithelial cells and macrophage population, including SSP1, TGF-b, and CD74, indicating a robust autocrine relationship at this stage (Fig. 7C). However, some of these ligand-receptor pairs differed between epithelial cells and macrophages, suggesting heterogeneity in coal dust lung cells, as illustrated in our DEG analysis.

The infiltrated M2-type alveolar macrophages with CD206⁺ and MLPH⁺ subset after the long-term coal exposure were significantly decreased after Vitamin D treatment

Next, we focused on markers of specialized biological features of macrophages. Using scRNA-Seq, the marker gene expression of macrophage clusters was shown in Additional file 4: Fig. S4 and Additional file 8: Table S7. The specialized M2 phenotypes expressing MLPH markers were found, further illustrating the specific M2 subset (Fig. 8B). Finally, alveolar macrophages in CMP lungs were polarized into the M2-type subset (Fig. 8A), and a fraction of this population was identified with double CD206⁺MLPH⁺ expression in vivo and in vitro. In addition, the scRNA-Seq showed that 9-month coal-dust exposed lung tissues had increased M2 cells and relatively decreased M1 cells compared to control groups; however, this trend was reversed in the VD₃-treated group (Fig. 8C).

Here, we focused on CD86, Mrc1, and MLPH for two reasons. First, CD68 and Mrc1 represent two different identities of the classical macrophage classification of M1 and M2 [55]. Second, scRNA-Seq data suggested that the MLPH expression was explicitly increased in alveolar macrophages at 9-month CMP, but only increased in M2 cells. Using selective marker genes (CD86 for M1 and Mrc1 for M2), we were able to confirm the predicted emergence of MLPH⁺ macrophages and upregulated in M2 cells only in CWP lungs (Fig. 8B). Vitamin D exerts its beneficial effects on many macrophage components, modulating phagocytic activity and cytokine production [56]. To validate that VD₃ accurately regulated gene expression in specific macrophage populations, we performed in situ immunofluorescent staining (Fig. 8D) and western blot (Fig. 8E, F) of the samples from the same tissues used for single-cell RNA-Seq analysis. The result showed that the MLPH expressed in CMP lungs increased significantly more than in the control lungs but markedly decreased after the Vitamin D treatment. Furthermore, the qPCR and ELISA analysis revealed that coal-exposed macrophages had increased TGF-β expression and secretion but decreased after the VD₃ treatment (Fig. 8G, H). TGF-β plays a critical role in lung inflammation and is a recognized indicator of fibrosis. Our results suggest that VD₃ may be a potential alternative treatment for alleviating pulmonary fibrosis caused by coal dust. These results also support the feasibility of the supplement of the VD₃ in patients with coal pneumoconiosis.

Discussions

Coal dust-induced alveolar structural damage and repair are major challenges in lung disease. Although it has been reported that AT2 epithelial cells have significant regeneration capacity, this regeneration process leads to abnormal or stunted repair [12, 57]. Herein, we provide new insights into the single-cell analysis. A new population of HAAS emerged in the long-term coal-exposed mouse lungs, which lost antibacterial pathways, including CHI3L1, APOC1, CXCL15, IL33, and LYZ1, and played an important role in pulmonary immunity. IL-33 is a significant driver of cellular differentiation and lung maturation, produced mainly in AT2 cells [58], which binds uniquely to IL1rl1 in adjacent basophils of lung tissue. The proportion of AT2 epithelial cells was small in normal lungs, but there was an abnormal increase after coal dust exposure. Furthermore, newborn AT2 cells produce more disease-associated factors, such as IL33. IL-33 specifically primes basophils and mast cells and affects AM maturation toward M2-type polarization [59, 60]. IL33 is also a marker of repair promotion and is expressed in AT2 during lung development and maturation [61]. It is also a significant factor that regulates hypoxia-induced lung injury and can mediate hypoxia-inducible factors [62]. Fate-map** studies indicate a clear lineage relationship between AT2 and HAAS. Therefore, we amended the hierarchy of alveolar epithelial cells involving these convertible cell states. Specific gene expression signatures demonstrate that HAAS gradually loses AT2 characteristics and represents a unique convertible population. The HAAS of the human lung has been previously identified and is especially apparent in the fibrotic regions of IPF lungs. Regarding human HAAS cells, HAAS-like cells in the present murine lungs were characterized by enrichment genes related to cellular senescence, Notch signaling, and WNT-regulated genes, all of which are linked to lung fibrosis [25, 63]. In contrast, we found a high expression of TRP63, KRT5, and COL1A1 in the epithelial cells of mice with coal pneumoconiosis; these were previously found only in human HAAS cells.

We found that AT2 cells undergo broad stimulation and deformation after coal dust entering the alveoli, causing them ready for DNA damage, a feature of most lung diseases with pulmonary fibrosis [12]. Previous studies have revealed that respirable particles lead to DNA damage once they move or remain in the pulmonary interstitium [64,65,66]. Our in vivo and in vitro study suggests that mouse AT2 cells undergo extensive stimulation and deformation during exposure to coal dust particles. Therefore, this specific AT2 cell state induced by the coal dust implies DNA damage in lung diseases.

The lungs efficiently match air and blood within the vast area of the alveolar surface, ensuring oxygen intake and delivery. Coal dust induced-hypoxia is a crucial driver of Notch signaling activation in epithelial stem/progenitor cells. It has previously been reported that hypoxia can induce Notch activity in vitro [67]. Our single-cell sequencing analysis showed that coal pneumoconiosis activates Notch activity. Using CellphoneDB, we found that anoxic/notch-activated signals in the epithelium and macrophages communicate with a group of genes controlling motility and invasion. Nevertheless, taken together, these data paint a pneumoconiosis epithelium and macrophage picture suggestive of more preventative, coactive, and reparative roles on their part, crucial in maintaining lung function and homeostasis. Future studies are required to characterize the potential of alveolar progenitors to drive a regenerative alveolar process and decode the immune microenvironment that favors AT2 proliferation.

A combination of single-cell transcriptome sequencing and immunofluorescence studies confirmed that disease-relevant MLPH⁺ macrophages are connected by a branched lineage that proceeds from coal dust-induced M2 polarization. We found that the homeostatic alveolar macrophages in mice rarely contained the MLPH⁺ subset but expressed highly CD86. However, coal exposed-alveolar macrophages had a large proportion of the CD206⁺ MLPH⁺ double staining subset, which may ultimately lead to pulmonary fibrosis. Interestingly, MLPH is involved in intracellular substance transport and becomes an upstream complex of TGF-β signaling. In addition, we deconstructed the VD3 supplement by scRNA-Seq as a comprehensive model and analyzed the effects of VD3 on macrophages to the subset level. Our scRNA-Seq data suggest that the coal-induced macrophage M2 polarization is suppressed by VD₃ supplementation, which defines VD₃ as a key to homeostatic M1/M2 of murine macrophages in a manner that involves other activities but does not exclude anti-fibrosis. This important function of VD₃ in macrophage therapy may benefit from their hormone activity alone or their ability to coordinate both paracellular and transcellular transport activity across the other cells.

Taken together, we uncovered a highly active AT2 state (HAAS), an abnormal increase that spans between AT2 and AT1. Notable, we further identified coal pneumoconiosis macrophage niches and clarified multiple functional enrichment pathways for alveolar macrophages, through which activated alveolar macrophages facilitate two-way communication with epithelial cells. Our results indicated M2-type macrophage polarization in long-term coal-exposed mouse lungs and disclosed an M2-macrophage subset with MLPH⁺CD206⁺ expression that significantly decreased after the administration of the Vitamin D₃ treatment. These findings provide potential alternative targets for CWP in the future. This study was based on a mouse model of coal pneumoconiosis. Next, we will validate the molecular mechanism discovered by single cell sequencing in human pneumoconiosis samples.

Conclusions

This study successfully established a reliable coal pneumoconiosis mouse model to study the cytotoxicity of respiratory coal dust particles to lung epithelial cells and macrophages in coal mines. After 9 months of coal dust stimulation, pulmonary inflammation and pulmonary fibrosis were significantly induced. Single-cell transcriptome sequencing technology allows the characterization of whole lung cells and molecules and provides a more comprehensive picture of coal-dust-induced lung cell changes and possible signal communication than traditional mechanism studies. Our study is the first to show that chronic coal dust stimulation can lead to substantial heterogeneity of epithelial cells and macrophages, and induce cellular inflammatory and death pathways, thereby promoting the progression of pulmonary fibrosis. Among them, HAAS, the highly active state of epithelial cells with no antibacterial ability, suggested the repair imbalance of lung epithelial cells. In addition, a subset of M2-type macrophages generated by the polarization of M2 macrophages induced by coal dust could be inhibited by VD, which may be related to the alleviation of the pulmonary fibrosis process by VD.

Availability of data and materials

All data related to this study are publicly available upon reasonable request to the corresponding author.

Abbreviations

AMs:: Alveolar macrophages
AP:: Alkaline phosphatase
AT1:: Alveolar type 1 cells
AT2:: Alveolar type 2 cells
BMDMs:: Bone marrow-derived macrophages
CCP3:: Cleaved caspase 3
CMP:: Coal pneumoconiosis in mice
CWP:: Coal worker’s pneumoconiosis
DEGs:: Differentially expressed genes
GEMs:: Gel beads in emulsions
GO:: Gene Ontology
GSEA:: Gene set enrichment analysis
GSVA:: Gene set variation analysis
HAAS:: Highly active AT2 state
HE:: Hematoxylin and eosin
IHC:: Immunohistochemistry
PBST:: Phosphate buffered saline with Tween
BSA:: Bovine serum albumin
ATP:: Adenosine triphosphate
AAT2:: Actived alveolar epithelial cell type 2
CDPs:: Coal dust particles
FN:: Fibronectin
RFs:: Repair related factor
BEC:: Blood endothelial cells
ExCM:: Extracellular matrix
IM:: Interstitial macrophages
KEGG:: Kyoto Encyclopedia of Genes and Genomes
KRT5:: Keratin 5
M1:: Macrophage type 1
M2:: Macrophage type 2
MSHA:: Mine Safety and Health Administration
NS:: Normal saline
PBS:: Phosphate-buffered saline
PCA:: Principal component analysis
PI:: Propidium iodide
PMF:: Progressive massive fibrosis
PVDF:: Polyvinylidene difluoride
SCRNA-SEQ:: Single-cell RNA sequencing
scRNAseq:: Single-cell transcriptomics
SDEGs:: Significantly DEGs
SPC:: Surfactant protein C
tSNE:: T-distributed stochastic neighbor embedding
qPCR:: Quantitative real-time PCR
UMI:: Unique molecular identifier

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Acknowledgements

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Funding

This study was supported by the Anhui University Science Research Project (No. KJ2019ZD13, No. KJ2019A0131) and The University Synergy Innovation Program of Anhui Province (GXXT-2021-077).

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Min Mu and Bing Li have contributed equally to this work.

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Key Laboratory of Industrial Dust Control and Occupational Health of the Ministry of Education, Anhui University of Science and Technology, Huainan, China
Min Mu, Yajun Zhang, Deyong Ge, Huihui Tao, Dong Hu, Liang Yuan, **nrong Tao & Jianhua Wang
Key Laboratory of Industrial Dust Deep Reduction and Occupational Health and Safety of Anhui Higher Education Institutes, Anhui University of Science and Technology, Huainan, China
Min Mu, Yajun Zhang, Deyong Ge, Huihui Tao, Dong Hu, Liang Yuan & **nrong Tao
School of Medicine, Department of Medical Frontier Experimental Center, Anhui University of Science and Technology, 168 Taifeng Road, Huainan, China
Min Mu, Bing Li, Yuanjie Zou, Wenyang Wang, Hangbing Cao, Yajun Zhang, Qixian Sun, Haoming Chen, Deyong Ge, Huihui Tao, Dong Hu, Liang Yuan & **nrong Tao
Anhui Province Engineering Laboratory of Occupational Health and Safety, Anhui University of Science and Technology, Huainan, China
Min Mu, Yajun Zhang, Deyong Ge, Huihui Tao, Dong Hu, Liang Yuan & **nrong Tao
Cancer Institute, Fudan University Shanghai Cancer Center, Fudan University, Shanghai, China
Jianhua Wang

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Min Mu
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Bing Li
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Yuanjie Zou
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Wenyang Wang
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Hangbing Cao
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Yajun Zhang
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Qixian Sun
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Haoming Chen
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Deyong Ge
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Huihui Tao
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Dong Hu
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Liang Yuan
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**nrong Tao
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Jianhua Wang
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Contributions

Manuscript writing: MM, BL, WYW, HBC, QXS; Formal analysis: YJZ, HMC, DYG, Conceptualization, Methodology: JHW, XRT, YL; Editing: HHT, DH. All authors read and approved the final manuscript. We thank OE Biotech Co., Ltd (Shanghai, China) for providing single-cell RNA-seq, **aohua Yao for assistance with bioinformatics analysis.

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Correspondence to Liang Yuan, **nrong Tao or Jianhua Wang.

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The Biomedical Research Ethics Committee of Anhui University of Science and Technology approved our study (No. 2020006).

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1

. Figure S1. Macrophage annotation and identification. (A) Visualized transcriptome cluster by tSNE. (B) Expression levels of the indicated genes projected onto tSNE in Clasters10,11,17. (C) Overview of violin plot for expression of critical lineage-associated genes by macrophages (number of cells in each cluster displayed in B).

Additional file 2

. Figure S2. Distribution of two subsets of functional macrophages types across tSNE plot. Macrophages expressed pro-inflammatory (CD207⁺, IL1β⁺, TNF⁺, IL6⁺) and anti-inflammatory (IL10⁺, APOE⁺, CD163⁺, C1qb⁺) genes.

Additional file 3

. Figure S3. IHC analysis of CD206, Beclin1, LC3B, LAMP2 in coal dust-exposed lung. Data values represent (Mean ± SEM) obtained from two independent experiments in triplicate assays. The graph showed the quantification of intensity when comparing the sections from the coal group with the control ones (*p < 0.05, *p < 0.01, *p < 0.001): scale bar, 50 μm.

Additional file 4

. Figure S4. The heat map showed the top 10 markers of macrophage.

Additional file 5

. Figure S5. Pulmonary score and Lung function test of mice exposed to coal dust for nine months. (A) Pathology score performed by Roderick J. Pathology classification standard of lung injury at three, six, nine-months. (B) Pulmonary fibrosis score performed by Ashcroft Criteria for grading lung fibrosis at three, six, nine -months. (C) Body weight recorded throughout the nine months. (D) The main indexes of lung function are Penh: enhanced pause; Mv: minute volume; Av: accumulated volume; EF50: expiratory flow 50%; PIF: peak inspiratory flow; PEF: peak expiratory flow; F: frequency; Rpef: ratio of time to peak expiratory flow (*p < 0.05, **p < 0.01, ***p < 0.001).

Additional file 6

. Figure S6. Toxic effects of coal dust on MLE-12 cells. Causing 50% inhibition of cell survival (CC50) of coal dust was measured in MLE-12 cells treated with different dose (A) and different time point at 200 μg/ml (B) (*P < 0.05, **P < 0.01, ***P < 0.001, Treatment vs Control).

Additional file 7

. Advanced analytical methods.

Additional file 8

. Raw datasets.

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Mu, M., Li, B., Zou, Y. et al. Coal dust exposure triggers heterogeneity of transcriptional profiles in mouse pneumoconiosis and Vitamin D remedies. Part Fibre Toxicol 19, 7 (2022). https://doi.org/10.1186/s12989-022-00449-y

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Received: 30 September 2021
Accepted: 13 January 2022
Published: 20 January 2022
DOI: https://doi.org/10.1186/s12989-022-00449-y

Coal dust exposure triggers heterogeneity of transcriptional profiles in mouse pneumoconiosis and Vitamin D remedies

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The infiltrated M2-type alveolar macrophages with CD206+ and MLPH+ subset after the long-term coal exposure were significantly decreased after Vitamin D treatment

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The infiltrated M2-type alveolar macrophages with CD206⁺ and MLPH⁺ subset after the long-term coal exposure were significantly decreased after Vitamin D treatment