Sub-chronic real-ambient PM exposure induces chronic lung injuries in C57BL/6J mice
To mimic a real-ambient PM exposure scenario, we constructed a PM exposure system located in Shijiazhuang, China as described previously [5], and first conducted whole-body PM inhalation experiments in C57BL/6J male mice from November 27th, 2019 to March 26th, 2020 (Fig. 1A). The mean concentration of PM2.5 in ambient air and PM exposure chambers was 123.52 μg/m3 and 73.94 μg/m3, respectively, over a period of 16 weeks (Fig. 1B, Additional file 1: Table S1). To address the molecular mechanisms underlying the transition from acute pulmonary inflammation to chronic fibrotic changes, we sacrificed mice on days 11 (N = 10), 15 (N = 10), 23 (N = 10), 56 (8 weeks) (N = 20), and 112 (16 weeks) (N = 20), respectively, as representative time points of acute and sub-chronic PM exposures (Fig. 1A). In addition, 69 organic chemical components and 43 trace elements and metal species from collected PM2.5 were quantitatively analyzed (Additional file 1: Tables S2–S6). In summary, this exposure system mimics human exposure to the extent possible and the mice in the exposure chambers received a sustained high concentration of PM exposure.
Consistent with previous observation [5], exposure to PM resulted in pathological changes. Acute inflammation was profound at 56 days (8 weeks) in mice following real-ambient PM exposure, while mild inflammation was present in mice placed in chambers installed with three layers of HEPA filters (Fig. 2A). As expected, chronic lung injuries were remarkable following 16-week PM exposure. Moreover, the chronic lung injuries were characterized by chronic inflammatory infiltrates, thickened alveolar septa with gentle fibrotic changes, isolated alveolar septa with gentle knot-like formations, moderate peri-bronchiolar fibrosis and pleural plaques due to excessive collagen deposition, as observed by Masson's trichrome and Sirius red staining (N = 8) (Fig. 2B, Additional file 1: Fig. S1). The acute lung injury (ALI) scores from H&E examination corroborated a more severe acute lung inflammation following 8-week versus 16-week exposure (Fig. 2C). The relative mRNA expression of pro-inflammatory cytokines, including IL-1β, IL-6, and IL-17A and anti-inflammatory cytokines such as IL-10 was 1.05- ~ 1.92-fold higher in mouse lung following 8-week PM exposure (N = 5) (all P < 0.05) (Additional file 1: Fig. S2A–F). The degree of fibrosis significantly increased in mouse lung tissue after 16-week PM exposure compared to the control group (P < 0.001) based on the increase in collagen content (%) and Ashcroft score [15] (Fig. 2D, E, Additional file 1: Table S7). In line with the pathological findings, we found that the content of hydroxyproline (Hyp) in lung tissue was 2.25-fold higher following 16-week exposure compared to that in control group (P < 0.001), while we failed to observe significant changes in lung tissue following 8-week PM exposure (N = 3) (Fig. 2F). In addition, the content of type 1 collagen in lung tissue only significantly increased in lung tissue of mice experiencing 16-week PM exposure (N = 3) (Additional file 1: Fig. S2G). Moreover, the expression of fibrosis-related genes, including Acta2, Tgfb1, Col1a1, Fibronectin and S100a4 were significantly increased in mouse lung tissues only after 16-week PM exposure (N = 5) (all P < 0.05) (Fig. 2G, H, Additional file 1: Fig. S2H–J). Collectively, the pathological fibrotic changes and relative biochemical indicators revealed the occurrence of initiation of mild pleural and interstitial pulmonary fibrosis accompanied with chronic inflammatory infiltrations following 16-week PM exposure. Here, we established a time-course animal model under real-ambient PM exposure, which provided us an ideal scenario to address mechanisms which trigger the transition from acute inflammation to chronic lung injuries and initiation of lung fibrosis.
ScRNA-seq analysis reveals immune dysregulations and activation of IL-17A signaling pathway associated with PM-induced pulmonary fibrosis
To clarify the interactions between immune cells and stromal cells in response to sub-chronic PM exposure, we conducted scRNA-seq on whole lung samples isolated from control and sub-chronic PM exposure groups (N = 3) at the end of the 16-week exposure (Fig. 3A). With aggregation and the quality control of pre-processing, we obtained datasets of mouse lungs containing 26,745 and 26,532 cell profiles in control and PM exposure group, respectively (Additional file 1: Table S8). To characterize the whole lung cell atlas, we performed canonical correlation analysis (CCA) to integrate datasets with unsupervised clustering and visualization by Uniform Manifold Approximation and Projection (UMAP) (Fig. 3B). Using the online databases including PanglaodB and single-cell Mouse Cell Atlas (scMCA) available for annotations of specific cell markers, we were able to identify 11 major types of cells that fell into 13 cell clusters (Fig. 3C, D). Particularly, the immune cells, including alveolar macrophages (AMs, clusters 0 and 5), neutrophils (clusters 1 and 8), B cells (cluster 2), T cells (cluster 3), monocytes-derived cells (monocytes, cluster 4), NK cells (cluster 6) and plasmocytes (cluster 12) were the major cell populations in the lung tissue. As shown in Fig. 3D, we identified that pulmonary stromal cells were comprised mainly of fibroblasts (cluster 7), Clara cells (cluster 9), type 2 alveolar epithelial cells (AT2, cluster 10), and type 1 alveolar epithelial cells (AT1, cluster 11) (detailed information of markers were listed in Additional file 1: Table S9). Notably, following 16-week PM exposure, the number of major immune cells, including AMs, B cells, T cells, NK cells, and epithelial cells, decreased significantly, while the number of neutrophils, monocytes, and fibroblasts increased by 1.92-, 1.15-, and 2.96-fold, respectively, compared to the control group (Fig. 3E). Meanwhile, consistent with the scRNA-seq results, the number and proportion of total T cells significantly decreased (P < 0.001), but interstitial macrophages significantly increased (P < 0.001), which were confirmed by flow cytometry assay (Additional file 1: Fig. S3A). The differentially expressed genes (DEGs) analysis was conducted in each cell cluster (Additional file 2). We found all cell clusters in PM-exposed mouse lungs ubiquitously expressing higher levels of chronic inflammation markers, S100a8 and S100a9. The elevated expression of specific genes in relation to the synthesis of extracellular matrix (ECM) and tissue remodeling, including Fn1, Mmp9, Col1a1, and Col3a1 only appeared in three cell clusters including neutrophils, monocytes, and fibroblasts (Fig. 3F). Taken together, a significant increase in the number of fibroblasts and upregulation of profibrotic gene expression characterize the initial state of chronic fibrosis. Importantly, the decreased population of most immune cells implicates that immune dysregulation might be critical at the initial stage of pulmonary fibrosis upon sub-chronic PM exposure.
To identify the essential cytokines or chemotaxis factors that trigger the initiation of pulmonary fibrosis, we performed analysis on DEGs in each of the cell cluster using the GO Biological Process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment programs (detailed information was listed in Additional file 3, Additional file 4). Notably, most of the DEGs were abundantly expressed in neutrophils, monocytes, and fibroblasts with an overlapping annotation of IL-17 signaling pathway (Fig. 4A, Additional file 1: Fig. S3B). The activation of IL-17 pathway was also identified using the Ingenuity Pathway Analysis (IPA) software (Additional file 1: Fig. S3C, D). Moreover, the IL-17 pathway was significantly enriched in various cell clusters (P < 0.05), indicating that the perturbation of IL-17 signaling was ubiquitous in pulmonary stroma (Fig. 4B). With the searching terms in GOBP program, the leukocyte chemotaxis and neutrophil migration in monocytes and fibroblasts, and the response to interferon-α/β/γ and collagen metabolic process in neutrophils, we showed that perturbation of IL-17 signaling pathway in these cell clusters might act as a mediator for recruiting immune cells in chronic inflammatory microenvironment upon sub-chronic PM exposure (Additional file 1: Fig. S4A–C). Taken together, we speculate that the activation of IL-17 signaling pathway might be involved in the development of PM-induced pulmonary fibrosis.
IL-17 signaling pathway activation mediates PM-induced pulmonary fibrosis
To analyze cellular interactions based on scRNA-seq data, we focused on the top 20 significant changes in category ‘cytokines’ and discovered the enhanced interaction of Il17a and Il17rc within T cells, which might be responsible for the increased number of Th17 cells and secretion of IL-17A (Additional file 1: Fig. S5A, B and Additional file 5). As shown in Fig. 4C and Additional file 1: Table S1, the respective cumulative exposure burden of PM2.5 at 5 time-points increased, while the mean concentration varied. Next, we examined the level of IL-17A, which was formally regarded as IL-17, in mouse lung tissues isolated from different time points of T1, T2, T3, 8-week, and 16-week PM exposure (N = 5). No significant difference was observed in the secretion level of IL-17A at T1, T2, and T3 time points with less cumulative PM exposure burden. Even though the mean concentration of PM2.5 two days before the end of 16-week PM exposure was much lower than the time point of 8-week exposure group, the secretion of IL-17A gradually increased, starting at the end of 8-week throughout the end of 16-week exposure, showing a significant difference from the control group (Additional file 1: Fig. S5C). As the cumulative PM exposure burden increased over three time points (T3, 8-week and 16-week), the relative mRNA expression of IL-17A was significantly upregulated by 25.48%, 48.04%, and 72.84% in lung tissues (all P < 0.01), respectively (Fig. 4D). Importantly, we showed that the slightly increased IL-17A was produced by T cells and the specific receptor IL-17RA was distributed in most of the immune cells including AMs, neutrophils, monocytes, etc. (Additional file 1: Fig. S5D). In addition to elevated mRNA expression of IL-17A, the expression of IL-17 signaling downstream genes including p65, IL-1β, IL-6, S100a9, Cox2, and Ccr2 were only significantly upregulated at the time point of 16-week PM exposure (P < 0.05) (Additional file 1: Figs. S2B, D, S5E–H). Taken together, these findings reveal that the gradually rising of IL-17A occurs concomitantly with an increasing cumulative burden of PM exposure, implying that IL-17A plays an important role in the onset of pulmonary fibrosis.
To further assess the fundamental role of IL-17A in mediating PM-induced pulmonary fibrosis, homozygous IL-17A−/− mice and wildtype (WT) littermate (20 mice/group) were subjected to 8-week and 16-week PM exposure in real-ambient PM exposure system in the winter season of 2020. For quality control, we verified that the target exons were successfully knocked out and IL-17A expression was significantly decreased by 87% in IL-17A−/− mouse lung (P < 0.001) (N = 5) (Additional file 1: Fig. S6A–C and Additional file 6). The level of IL-17A mRNA remained unchanged in splenic T cells derived from IL-17A−/− mice after anti-CD3/anti-CD28 co-activation and the secretion level of IL-17A was undetectable in the supernatant (Additional file 1: Fig. S6D, E). As for WT mice, the mean content of IL-17A was 1.58 times higher than in the control group (P < 0.001) following 16-week PM exposure, while no significant difference was observed after 8-week exposure (N = 5) (Fig. 4E). Concomitant to the significant decrease in IL-17A secretion in IL-17A−/− mice following PM exposure (N = 3) (Fig. 4E), we observed a 38.25% decline in acute inflammation based on the ALI score following 8-week PM exposure (N = 8), suggesting efficient attenuation of lung inflammation induced by PM exposure (Fig. 5A, B, Additional file 1: Table S10). The collagen content (%) and Ashcroft score in IL-17A−/− mice following 16-week PM exposure were decreased by 27.22% and 43.01% (both P < 0.001), respectively (N = 8) (Fig. 5C–E, Additional file 1: Table S10). Meanwhile, we observed a 39.80% decrease in Hyp content in lung tissue of IL-17A−/− mice following 16-week PM exposure compared to WT mice (Additional file 1: Fig. S6F). Collectively, depletion of IL-17A could efficiently attenuate chronic lung injuries induced by sub-chronic PM exposure.
It has been demonstrated that elevated TGF-β contributes to fibrogenesis in multiple organs [16]. To clarify the interplay between IL-17A and TGF-β in the development of progressive fibrosis, we collected mouse lung tissues from WT and IL-17A−/− mice after 8-week or 16-week PM exposure. As described above, significantly increased secretion of IL-17A was noted in the lungs of WT mice following 16-week PM exposure compared to the control group (P < 0.001). The extent of increased IL-17A was correlated with the levels TGF-β in mouse lung after 16-week PM exposure, indicating that IL-17A and TGF-β might be cooperating in mediating pulmonary chronic inflammation. Notably, the secretion of TGF-β decreased by 20.9% or 52.0% in IL-17A−/− mice after 8-week or 16-week PM exposure, respectively (Fig. 5F), indicating a significant role of IL-17 in regulating TGF-β secretion. Taken together, we reveal that activation of IL-17 pathway is indispensable in the course of the development of pulmonary fibrosis upon sub-chronic PM exposure through regulation of TGF-β signaling cascade.
IL-17 signaling is involved in recruitment of immunosuppressive MDSCs
To further address the impact of the perturbation of IL-17 signaling on immune dysregulation, firstly we elaborated the immune dysfunctions by analyzing the scRNA-seq dataset. Of the top 20 significant pairs of ligand-receptor that regulate the cell–cell interaction, the cytotoxic T lymphocyte activation-4 (Ctla4) was activated by enhanced ligand-receptor pairs Cd86-Ctla4 between neutrophils and T cells, or Cd80-Ctla4 between monocytes and T cells, indicating that the differentiation of immunosuppressive regulatory T cells (Tregs) might be mediating chronic inflammation upon PM exposure (Fig. 6A, Additional file 5).
Next, we analyzed the specific markers for extracted subsets in neutrophils or monocytes cluster and revealed that myeloid-derived suppressor cells (MDSCs) were involved in regulation of immune response by suppressing T cells upon PM exposure. Two subtypes of MDSCs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and monocytic myeloid-derived suppressor cells (M-MDSCs) share the same myeloid progenitors of neutrophils and monocytes. With specific markers, we annotated cluster 0 and cluster 1 as monocyte progenitors or immature monocytes, cluster 2 as dendritic cells, and cluster 3 as interstitial macrophages (Fig. 6B, detailed information was provided in Additional file 1: Table S11). The pseudotime trajectory analysis and pseudotime score calculated by Monocle package proposed a chronological order of differentiation from cluster 0 into cluster 1 (State 1), followed by dendritic cells (State 2) and interstitial macrophages (State 3) as two branches of terminal states (Fig. 6C, D). With respect to the neutrophil subset, cluster 1 in the state of the lowest pseudotime scores increased in response to PM exposure (Fig. 6E, F and the detailed information of top 10 markers and cellular proportions in different groups were provided in Additional file 1: Table S12). Moreover, gene expression of Wfdc17, Arg2, Il-1β, and Ifitm1, which have been implicated in immunosuppressive effects, was greatly upregulated in this sub-cluster of neutrophils (Fig. 6G). The pseudotime analysis revealed that sub-clusters in the early and transitional states within neutrophilic and monocytic lineages exhibited enhanced immunosuppressive activity, which might be carried out by MDSCs.
In addition, we performed flow cytometry assay to distinguish the subtypes of MDSCs from mouse lungs and bone marrows isolated at the end of 8-week and 16-week PM exposure (N = 4). As shown in Fig. 7A and Additional file 1: Fig. S7A, the markers CD11b+Gr1(Ly6G/Ly6C)+, CD11b+Ly6C−Ly6Ghi, and CD11b+Ly6C+Ly6Gint were used for labeling total MDSCs, PMN-MDSCs, and M-MDSCs, respectively. We found a significant increase in the proportion of MDSCs in viable cells by 81.37%, respectively, from mouse bone marrow especially following 16-week PM exposure (P < 0.001) (Fig. 7B). Moreover, there was an increasing trend in number of MDSCs recruited into mouse lung interstitium with the increase of exposure time (P < 0.001) (Fig. 7C). The proportion of both PMN-MDSCs and M-MDSCs subsets were significantly increased in bone marrows and lung tissues of mice undergoing 16-week PM exposure (P < 0.05). However, the significant difference appeared in M-MDSCs subset only in bone marrow following 8-week PM exposure (P < 0.05) (Additional file 1: Fig. S7B–G). To further address whether IL-17A was critical for MDSCs recruitment, we examined the amounts of MDSCs in IL-17A−/− mice following real-ambient PM exposure. As shown in Fig. 7B–D, the number of MDSCs significantly declined by 22.76% in mouse bone marrow, by 38.23% in mouse spleens, and by 70.27% in mouse lungs (all P < 0.05) of IL-17A−/− mice following 8-week and 16-week PM exposure compared to those in WT mice, indicating that the recruitment of MDSCs in lung might be regulated by IL-17A signaling. These results suggest that IL-17 signaling activation is prerequisite for recruitment of MDSCs in lung upon PM exposure, leading to alterations in the microenvironment and favoring the development of chronic inflammation.
Furthermore, the immunosuppressive effect of recruited MDSCs was examined by ex vivo proliferation functional assay. The results showed a 17.62% and 5.12% suppression by MDSCs derived from WT mouse bone marrow and lungs after exposure to PM for 16 weeks compared to the controls (all P < 0.05), indicating that chronic inflammation induced by PM exposure conferred an immunosuppressive state carried out by MDSCs. Notably, the immunosuppressive activities of both bone marrow and lung MDSCs were significantly attenuated (18.48% and 6.15% in bone marrow and lungs, respectively) in IL-17A−/− mice following 16-week PM exposure compared to WT littermates (all P < 0.001) (Fig. 7E, F). Corroborating the in vitro observations of enhanced immunosuppressive activity of MDSCs, we found that the proliferations of total T cells in lung tissue were significantly suppressed after 8-week or 16-week PM exposure (both P < 0.01) (Additional file 1: Fig. S3A). In parallel, by conducting in vivo MDSCs depletion experiment, we noticed that the decline of MDSCs attenuated the transition from acute to chronic lung injury, demonstrating the potential profibrotic role of MDSCs in the progression of pulmonary fibrosis (Additional file 1: Fig. S8A–E). Collectively, we conclude that the increased recruitment of MDSCs by activation of IL-17A in response to PM exposure exhibited enhanced immunosuppressive potential that might be involved in initiation of fibrosis through elevating TGF-β production.
Activation of IL-17A pathway triggers the initiation of pulmonary fibrosis through altering microenvironment comprising of excessive TGF-β secretion and defective macrophages functions
Notably, we showed that enhanced immunosuppressive activity of MDSCs was related to higher level of profibrotic factor TGF-β in a coculture system engaging MDSCs and T cells, where MDSCs were isolated from mice receiving 16-week PM exposure compared to the coculture model with MDSCs isolated from the control mice or T cells (P < 0.05). Additionally, concomitant with the weaker immunosuppressive activity of MDSCs derived from bone marrow and lung of IL-17A−/− mice, there was a 56.45% and 39.70% decrease in the secreted TGF-β in supernatant of the coculture system (P < 0.001) (Fig. 7G, H). These findings enhanced the notion that increased IL-17A participates in generating and recruiting immunosuppressive MDSCs which might be responsible for regulating of TGF-β secretion in the lung microenvironment. Key events in the initiation of pulmonary fibrosis include fibroblast activation, increasing synthesis of collagen, and impeded ECM degradation. We analyzed a unique cluster of fibroblast subset, containing fibrocytes derived from bone marrows that expressed abundantly higher levels of Ccl2 and Fn1 in mouse lungs in PM exposed group (Additional file 1: Fig. S9A–C). Upregulation of Ccl2 in lung induced by one of the immunosuppressive cytokines IL-10 has been previously demonstrated to mediate the recruitment of fibrocytes [17] and promote the differentiation of activated fibroblasts and myofibroblasts especially under the microenvironment with excessive TGF-β [18]. Notably, these activated fibroblasts were able to proliferate and synthesize collagen. To further clarify the role of increasing interstitial macrophages involved in mediating immunosuppression, we conducted informatics analysis on the scRNA-seq data and perform ex vivo functional studies. We found that the increased population of interstitial macrophages partially originated from M-MDSCs in response to PM exposure. The upregulated DEGs with M2 profibrotic annotation, including S100a9/S100a8, S100a4, Chil3 (encoding YM-1), and Arg1 were increased in mouse interstitial macrophages following 16-week PM exposure, suggesting the potential defective macrophage functions (Additional file 1: Fig. S9D–G). Collectively, these findings indicate that MDSCs recruited by activated IL-17A might work in synchrony with the activation of fibroblasts to perturb the balance between collagen synthesis and clearance, leading to the initiation of pulmonary fibrosis in response to PM exposure.