3.1 Successful establishment of coal mice pneumoconiosis by exposure to coal dust particles.
By exposing mice to coal dust twice-weekly for four weeks, we established a pathological phenotype of coal pneumoconiosis in these mice. Using this model, we studied the severity of coal dust lung damage at three, six, and nine months post-exposure, mainly reflected in the injury and repair of alveolar structures (Fig. 2A). In the third month, coal dust was embedded in the spaces between lung tissues, and cell proliferation resulted in alveolar stroma thickening. The coal dust particle has been phagocytosed by macrophages and aggregated into a ball, with lymphocytes proliferating into nodules in the sixth month. Finally, in the ninth month, the coal dust particles were again dispersed in the alveolar area, and the alveolar structure was destroyed and discontinuous. The significant increase in the number of AT2 cells and the activation of repair functions resulted in the extracellular matrix deposition. Epithelial wounds repaired by infiltrating immune cells are shown in Fig. 2B, as illustrated by the diagram.
These results suggested that this technique was a reliable method for modeling CMP, as it produced an asymmetrical distribution of coal dust on both sides of the lungs. In addition, the inflammatory injuries in the lungs presented a trend of severe, moderate, and mild from the hilum to the lung margin (Fig. 2C1-2C4). These three levels presented obvious boundaries (as delineated by the red, yellow, and green lines), and the proportions of affected areas were 17%, 47%, and 36%, respectively (Fig. 2D). The progression of pulmonary fibrosis in these mice at three, six, and nine months was demonstrated by Masson staining (Fig. 2E). Experimental coal pneumoconiosis in the mice had been latent for an extended period of six months before the formation of significant fibrosis. However, nine months after coal dust exposure, there was significant interstitial pulmonary fibrosis with a darker toluidine blue margin, suggesting an idiopathic centripetal fibrosis-like progression. The collagenous volume fraction (CVF), which reflects fibrosis progression, was quantitatively assessed, as was the alveolar volume fraction, an indicator for lung function in mice. There was a significant increase in CVF in nine months after coal exposure (p<0.001) (Fig. 2F). With the increase of CVF in the development of fibrosis, the alveolar volume fraction (AVF) of the mice decreased (Fig. 2G), indicating a decline in respiratory function. At nine months, mice exposed to coal dust were subject to spontaneous death, a disease outcome associated with fibrosis progression.
3.2 Classification of lung cells based on cell-type specific marker genes.
According to the experimental flow chart (Fig. 3A), single-cell suspensions of 9-month-exposed murine pulmonary cells were prepared. We isolated and sequenced 50,156 cells from whole lung cell suspensions of four male mice, including two vehicle control mice and two coal-exposed mice. We first cataloged mouse lung cell types in an unbiased manner using droplet-based scRNA-Seq on the 10× Genomics platform and performed analyses using a single-tube protocol with unique transcript counting through barcoding with UMIs. Then, the effects of UMI and mitochondrial gene content were examined (Fig. 3B). The clustering of cells was not affected by UMI or the mitochondrial gene content. Besides, genes positively correlated with mitochondria-encoded proteins were found associated with solute transport rather than cellular stress responses. After quality filtering, ~50 million unique transcripts were obtained from 42,252 cells, in which more than 1,303 genes could be detected. Of these, 24,094 cells (48%) originated from coal pneumoconiosis-infected lungs and 26,062 from control lungs (Table S1). We performed dimensionality reduction with t-SNE subspace alignment, followed by an unsupervised clustering assay (Fig. 3C). To characterize the heterogeneity of coal exposed-pulmonary cells at a higher resolution, we focused on seven clusters from 12,855 cells of coal-exposed and control lungs (Fig. 3D). 19 distinct cell clusters consisting of as few as 1,186 cells to 15,665 cells per cluster were classified. The representative markers identified 19 clusters, including epithelial cells, endothelial cells, fibroblast cells, macrophages, monocytes, neutrophils, and lymphocytes (Fig. 3C&E). All the clusters included cells from both coal dust-treated and untreated lungs, except for cluster 19, which included cells mostly from coal dust-treated lungs (Fig. S1A). Two superclusters were categorized and focused: one consisted of clusters 13, 14, 16, and 18 with higher Krt18 and EPCAM expression, and the other consisted of clusters 10, 11, and 17 with higher CD14 and CD68 expression (Fig. S1B&C). Cluster 10 with the expression of CD14 and CD300E was recognized as proliferating cells. Clusters 11 and 17 expressed macrophage markers, such as CD14 and CD68. Cluster 18 expressed epithelial cell markers, such as ITGA1 and SCNN1G, and the highest level of Aqp4 (Fig. 3E). The cell-type composition distribution of each mouse was observed to differ substantially (Fig. 3F). As expected, immune cells, such as lymphocytes and macrophages, were the dominant cell type for coal pneumoconiosis. 3.3 Intrinsic epithelial cell subpopulations underlying coal pneumoconiosis injury.
We detected 2,863 epithelial cells, including both epithelial and stromal cells. All the cells were re-analyzed, yielding 12 clusters. As expected, given the hypervascular nature of the lungs, the epithelial cells were less abundant (963 cells) in the coal pneumoconiosis group (Fig. 4A). Next, we attempted to identify marker genes for each cluster and assign them to known epithelial cell types. In many cases, the unbiased cluster identifier was a known cell-type-specific marker, such as surfactant protein C (SPC) for AT2 cells, Hopx for alveolar type I cells (AT1), and keratin 5 (KRT5) for basal cells (Fig. 4E). Notably, we identified a great number of additional markers, followed by some of the known markers. One set of 48 Clara cells was found in coal pneumoconiosis samples (cluster 10; marker genes Scgb1a1 and MUC5b), as well as three sets of lymphatic epithelial cells (PDPN+); two mainly were coal pneumoconiosis-derived (clusters 4 and 5; AQP3+ and AQP1+) and one controlled lung-derived (cluster 3; SCNN1G). To identify differentially expressed genes (DEGs) that may play an essential role in the defense response to coal dust in murine lungs, the CWP group (Table S2) that inhaled coal dust was compared to the vehicle control group. 793 DEGs were identified using the standard FDR of <0.01 and fold-change of 1.5, of which 330 were upregulated and 463 were downregulated (Fig. 4C). The downregulated genes included those involved in the heat shock protein family (Hspa1b, Hsp90aa1, and Hspa1a), calcium-regulated actin-modulating protein (Gsn, Mgp, and LIMCH1), and intercellular adhesion (Thbs1 and Vcam). The distribution of DEGs is illustrated using a volcano plot (Fig. 3C), where the red color represents elevated expression and blue represents decreasing expression levels. To visualize the gene expression profiles of the top 20 genes, we generated a heatmap using a ring diagram (Fig. 4B). To further tell the function of DEGs, 793 DEGs from mice with or without coal dust exposure were subjected to GO annotation (Fig. 4D). The results demonstrated that 5,269 GO terms were annotated, including 3,711 biological processes (9,543 unigenes), 618 cell components (5,164 unigenes), and 940 molecular functions (3,993 unigenes). Most of the differentially expressed genes were mainly involved in the biological processes of cells. The DEGs in this study were mainly involved in cellular processes (GO:0098869), molecular function (GO: GO:0032981), ribosomal function (GO:1990932 and GO: GO:0006412), and ATP biosynthetic processes (GO:0006754), all of which indicate that numerous physiological and biochemical alternation in the epithelial cells during coal dust inhalation into the lung, an important role in the coal dust defense response. According to the epithelial subtype identification results, we classified the epithelial cells into nine types. Compared with the control group, coal pneumoconiosis type II epithelial cells were more and had a significant difference in dimensionality reduction clustering, which was defined as a highly active AT2 state (HAAS) (Fig. 4E), and focused on the reprogramming of AT2 epithelial cells (cluster 2 and cluster 9). Next, a direct comparison of HAAS and normal AT2 epithelial cells was performed. We found that the antimicrobial-related protein expression of LYZ and Chia1 was missing in the AT2 epithelial cells of mice with coal pneumoconiosis (Fig. 4F). Remarkably, the total read counts were 7- to 8- fold higher in HAAS than in normal AT2 epithelial cell clusters, accounting for 48% of the total coal pneumoconiosis epithelial cells (Table S3). Using immunohistochemical analysis of independent coal pneumoconiosis samples for CHIL1, SFTPC, and HBEGF markers of clusters 2 and 9, we confirmed the presence of these cells as separate cellular entities, which were enriched in coal pneumoconiosis tissues (Fig. 4G). The analysis of hallmark gene expression signatures highlighted that, while the two-coal pneumoconiosis epithelial cell clusters showed some differences, most changes were between the control lung and coal pneumoconiosis-derived AT2 epithelial cells (Fig. 4H). In short, these results indicate that coal pneumoconiosis epithelial cells are remodeled to increase the number of new AT2 epithelial cells, thus contributing to coal pneumoconiosis regeneration and the repair of coal dust-injured epithelial tissue. Finally, to map cell types, states, and transitions during the regeneration of dynamic biological processes in coal pneumoconiosis, we performed a pseudotime analysis of the epithelial cells using Monocle. The data suggested two diverging cell fates, starting at clusters 6, 8, and 9, progressing toward clusters 1, 5, and 7 at one end, and cluster 2 at the other, with cluster 4 being a transitioning state spreading along the axis (Fig. 4I).
3.4 Coal dust induces murine AT2 cell apoptosis and reprogramming.
To verify the toxicity of coal dust in vitro, we used the mouse AT2 epithelial cell line MLE-12. MLE-12 cells grown on polylysine-coated slides in 24-well-plates were supplemented with a complete culture medium containing 10% FBS before they were exposed to 0 µg/mL, 200 µg/mL, 400 µg/mL, and 800 µg/mL coal dust for 24 h. Meanwhile, we analyzed cell apoptosis using an annexin V/PI apoptosis detection kit under confocal laser microscopy. FITC-conjugated annexin V and propidium iodide (PI) staining were used to discriminate between early apoptotic and late apoptotic/necrotic cells (Fig. 5A). The MLE-12 cells treated with 200 µg/mL had the highest number of green-colored apoptotic cells after 24 h post-exposure relative to the 400 µg/mL or 800 µg/mL groups, indicating that AT2 epithelial cells were sensitive to the stimulation of coal dust at a low dose. Conversely, cells treated with 400 µg/mL had the highest number of red-colored late apoptotic/necrotic cells, indicating that the limit of the lethal concentration of coal dust in AT2 cells was 400 µg/mL (Fig. 5B). Additionally, we verified AT2 apoptosis in vitro using the immunofluorescent staining of anti-caspase3 antibody (green fluorescence) and found that CCP3 positive AT2 cells in the coal dust group had obvious nuclear pyknosis and fragmentation (arrowhead) (Fig. 5C).
We believe that epithelial cells are the first line of defense (as opposed to immune cells), and any damage to them causes pro-inflammatory factor release, which initiates the body's immune response. Following the in vitro experiment of CWP epithelial cells, the apoptosis/necrosis pathways were further analyzed. The proliferated AT2 cells were apoptotic with a higher signal of regulation of DNA fragments or necrosis relative to the control cells. Furthermore, newborn AT2 cells showed high sensitivity to the tumor necrosis factor response. (Fig. 5D). GSEA was performed on the DEGs of epithelial cells. Nine-month-old coal-exposed AT2 epithelial cells exhibited declined outside-related transcription levels after mechanical stimulation. Moreover, the mitochondrial transport, protein coupling, and intracellular ATP increased (Fig. 5E), suggesting that energy metabolism disorder is a critical link in the toxic process of coal dust that leads to cell death.
AT2 epithelial cells are a few essential cells, accounting for only 5% of the total lung cells, but they can proliferate and differentiate into AT1 epithelial cells, which repair alveolar structures. After the coal dust treatment, the alveolar type II cells proliferated significantly, indicating that their repair function was activated. However, our transcriptomes disclosed that proliferated AT2 showed functional heterogeneity, with reduced antibacterial activity, functional loss, and abnormal proliferation. Thus, the space-occupying lesions of coal dust in the lung interstitium may have caused abnormal processes or initiated their reprogramming. Nevertheless, the time-series analysis suggested that the newly generated AT2 epithelial cells were still highly active, which may be critical in inducing lung consolidation.
3.5 Alveolar macrophages show rheostatic phenotypes and M2 polarization in mice with coal pneumoconiosis.
Mononuclear macrophages in the lung play a critical role in maintaining tissue homeostasis and controlling the inflammatory response to dust inhaled from the environment and lung disease pathogenesis[36]. To define the transcriptional landscape of lung-resident macrophages, we partitioned 3,582 transcriptionally distinct mononuclear macrophages into 11 clusters, which were shown in the coal dust and control groups (Fig. 6A). The cell-type composition of macrophages differed substantially in mice with coal pneumoconiosis (Fig. 6B&D). In all clusters, essential marker genes were highly expressed, while markers of other cell types were minimally expressed, except for OGN genes (Fig. S2). A dissection of the heterogeneous origins of mouse alveolar macrophages revealed that these were macrophages, monocytes, and dendritic cells (Fig. 6A, bottom). The mixed profile of alveolar macrophages (AMs) represents phenotypic plasticity in response to inhaled coal dust. Here, two types of macrophages were detected in the normal mouse lung. It has been reported that AMs highly express the MARCO, FABP4, and MCEMP1 genes. The other type, which consists of interstitial macrophages, is derived from circulating monocytes. These cells are functionally different from tissue-resident macrophages, recruiting and expressing profibrotic genes during lung fibrosis[37]. Next, the AM type in lung tissues, including anti-inflammatory AM (C2 and 8, APOE+, CD163+, and C1qb+), pro-inflammatory AM (C10, IL1β+, and C6 IL6+), and actively cycling AM expressing anti-inflammatory markers were found. Moreover, as expected, we confirmed the decline of macrophage type 1 (M1)/M2 in mice with 9-month (advanced-stage) coal pneumoconiosis, indicating the activation of adaptive anti-inflammatory repair in response to long-term coal dust exposure (Fig. 6C & Fig. S2). Consistently, the lungs of mice with coal pneumoconiosis were strongly enriched in Macs (anti- and pro-inflammatory macrophages in clusters 1, 3, and 8). In addition, both normal lungs and those infected with coal pneumoconiosis contained clusters of C1qb+ macrophages (C2).
To investigate the functional role of coal dust-induced changes at the transcription levels in macrophages, we identified 395 significantly differentially expressed genes (SDEGs), which included 266 downregulated and 129 upregulated genes in the coal group relative to the control group (Fig. 6E, Table S4). Herein, we marked the top 20 upregulated and downregulated genes between the coal and control groups according to the log2FC in Table S2. Significantly downregulated genes included ATP2b1, Thbs1, Ninj1, Lpar6, Lfitm, Scgb1a1, Mgp, CD14, and Cxcl3. To further study the complex behavior of CMP macrophages, a pathway enrichment analysis of DEGs was applied to understand better the biological function of a gene and its interaction with others. The differential genes in the macrophages were found to be involved in 37 subclasses of metabolic pathways in six broad categories. Next, the data analysis by the metabolic pathway database KEGG was performed (Fig. 6F). A high number of genes were found among these signaling pathways, including the immune system (51 genes), infectious diseases (48 genes), signal transduction (38 genes), cancers (29 genes), and cardiovascular diseases (18 genes). Metabolic pathways are involved in signal transduction during environmental changes.
Meanwhile, there were four pathways associated with immune and disease resistance, including signaling molecules and interaction (36 genes), transport and catabolism (24 genes), cell growth and death (19 genes), and cellular community–eukaryotes (12 genes). These pathways are involved in all aspects of mammalian immunization. In short, the experimental KEGG annotation pathway enrichment analyses for macrophages based on lineage-specific genes identified by scRNA-Seq strongly highlighted immune-induced regulatory pathways in the pathogenesis of coal pneumoconiosis in mice. This study’s genes and pathways associated with the immune system, signal transduction, and disease processes were similar to those previously reported for particle-induced pneumoconiosis.[38, 39, 40] Thus, studies on immune-related genes and pathways identification here can advance our understanding of the molecular immune mechanisms in coal dust-activated CMP.
KEGG pathway enriched and analyzed all important signal transduction pathways and biochemical metabolic pathways regulated by DEGs [41]. SDEGs of coal group/control group were annotated by 196 KEGG pathways, and the first 20 enrichment pathways are shown in Fig. 6G (Table S5 in details). We demonstrated six significantly enriched KEGG pathways, including the phagosome, tuberculosis, cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, and Salmonella infection pathways. The most significant enrichment signal is the cytokine - cytokine receptor interaction pathway, including 18 SDEGs such as interferon-gamma receptor 1 (Ifngr1), Ccl17, Ccl24, Ccl4, Ccl6, Ccl9, Ccr5, Cxcl1, Cxcl10, Cxcl13, Cxcl2, Cxcl3, Cxcr4, and more. In addition, we confirmed that autophagosome markers LAMP2, LC3B and Beclin1, which are related to phagocytic pathways, were mostly co-located or adjacent to M2 macrophages that phagocytic coal dust particles (Fig. S3). The single-cell analysis-based dissection uncovered macrophage phenotype changes linked to functional state transitions in the 9-month coal-exposed mouse lungs.
The transcriptome of macrophages reveals changes responsible for immune defense and tissue repair. To reveal some of the pathways that might contribute to coal pneumoconiosis formation in macrophages, GO term analysis was performed on the DEGs of the CMP transcriptome to compare the results to the control group. The top 22 most significantly upregulated and downregulated transcripts are listed in Fig. 6H. Pathway analysis revealed that the most highly upregulated biological processes involved wound healing, the spread of epidermal cells (GO0035313), the positive regulation of the apoptotic process (GO0043065), epithelial cell differentiation (GO0030855), and collagen catabolic processes (GO003057). The repressed biological processes included processing in the regulation of l-kappaB kinase/NF-kappaB signaling (GO0043122), defense response to bacteria (GO0002526), cell adhesion (GO0030593), and defense response (GO0009611).
3.6 The epithelial crosstalk with macrophage in mice with coal pneumoconiosis is predicted using ligand-receptor interaction analysis.
As we have successfully outlined the fate of epithelial cells and macrophages and delineated the molecular characteristics of different cell populations, we then infer that intercellular communication is mediated by a common ligand-receptor database in advanced coal dust lung disease. CellPhoneDB was developed by Efremova et al [35]. to predict the key signaling events between the spatially co-located cell groups. The CellPhoneDB analysis performed on different epithelial cell types, including endothelial cells, fibroblasts, macrophages, neutrophils, and lymphocytes, showed that macrophages were the dominant communication hubs that secrete and reverse signals via 262 and 316 ligand-receptor pairs (Fig. 7A&E). We classified possible ligand-receptor pairs in different cell populations, after comparing ligand-receptor pairs with cell-specific genes (Table S6-1&6-2). Regarding ligand-receptor pairs (epithelium and macrophage specification) in the control and CMP groups, we found a stronger interaction relationship between the epithelial cells and macrophages in CMP (Fig. 7B&C).
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 [42]. 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 CellPhoneDB 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 [43, 44]. 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.
3.7 The infiltrated M2-type alveolar macrophages with CD206 + and MLPH+ subset after the long-term coal exposure were greatly 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 (Fig. S4, Table S7), 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 [45]. 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 up-regulated in M2 cells only in CWP lungs (Fig. 8B). Vitamin D exerts its beneficial effects on many macrophage components, modulating both phagocytic activity and cytokine production [46]. 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. 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.