ZC3H4 regulates infiltrating monocytes, attenuating pulmonary fibrosis through IL-10

Silica promotes an infiltrating phenotype in monocytes

First, we used single-cell sequencing (sc-Seq) technology to examine whole lungs of mice in the 7-d saline group, 7-d SiO2 model group, 56-d saline group and 56-d SiO2 model group by digestion analysis, and all of the cell classifications in the mouse lung were obtained (Fig. 1A) by R language analysis. A 7-day mouse model of silicosis indicates acute exposure to SiO2, when the lungs are mainly in an inflammatory phase; in a 56-day mouse model of silicon lung, the lungs are chronically exposed to SiO2, mainly in the fibrosis phase [16, 17]. The number of monocytes in the 7-d SiO2 model group was not significantly different from that in the 7-d saline group, while the number of macrophages in the 7-d SiO2 model group was elevated. The number of monocytes in the 56-d SiO2 model group was significantly lower than that in the 56-d saline group, while the number of macrophages was elevated in the 56-d SiO2 model group (Fig. 1B, C). After pseudotime analysis, the increased macrophages were shown to be transdifferentiated from monocytes (Fig. 1D). Many studies have shown that alveolar macrophages play an important role in the process of fibroblast activation and the development of silicosis [18, 19]. As macrophage precursor cells, monocytes are recruited in large numbers during silicosis, but their effects are still unclear. To determine whether SiO2 affects the monocyte phenotype, the THP-1 cell line was exposed to SiO2 (100 µg/ml), and phenotypic changes were assessed. Interestingly, the immunoblotting results (Fig. 1E, F) showed that SiO2 upregulated CCR2 but not integrin subunit alpha X (ITGAX, CD11C, Fig. 1E, G), adhesion G protein-coupled receptor E1 (ADGRE1, F4/80, Fig. 1E) or CD163 molecule (Cd163, Additional file 1: Fig. S1A, B), suggesting that the infiltrating monocyte phenotype was increased. This finding was further confirmed by immunostaining analysis of CCR2 in THP-1 cells (Fig. 1H; Additional file 1: Fig. S1C) compared with that in the control groups, indicating that transdifferentiation from monocytes to macrophages was blocked, which was contrary to the sc-Seq results. Therefore, the role of infiltrating monocytes in the process of fibrosis is worth exploring.

Fig. 1figure 1

Silica promotes an infiltrating phenotype in monocytes. A Visualization of major classes of cells using t-SNE. Dots, individual cells; color, cell types. One mouse was used for each group. B Cell numbers and relative proportions of monocytes and macrophages are shown as pie charts at 7 d and 56 d. C As shown in the images, the monocyte numbers were decreased or showed no obvious difference in the SiO2 group compared with the NC group, while macrophage numbers increased in the SiO2 group. D Cell trajectory analysis of monocytes and macrophages and pseudotime analysis. E Representative Western blot showing that SiO2 induced CCR2 expression in a time-dependent manner in THP-1 cells. F Densitometric analyses of CCR2 levels from five independent experiments; *P < 0.05 compared with the 0 h group. G Densitometric analyses of ITGAX levels in five independent experiments. H Representative immunocytochemical staining images showing that SiO2 induced CCR2 expression in THP-1 cells. Scale bar, 100 μm

Monocytes play a negative role in fibroblast activation, migration and viability after silica treatment

Lung fibroblasts, which are the direct effector cells of pulmonary fibrosis, gradually transform into myofibroblasts during the progression of silicosis [20]. To determine whether infiltrating monocytes affect fibroblast activation, we treated the HPF-a cell line with conditioned medium monocytes treated with SiO2 (CM) or PBS (Con). The immunoblotting results demonstrated that compared with Con, CM inhibited the expression of the fibroblast activation marker proteins COL1A1 and ACTA2 (Fig. 2A–C). To determine whether infiltrating monocytes affect fibroblast viability, HPF-a cells were cultured in CM or Con. As indicated by the gel contraction assay results, the CM groups exhibited less cell viability than the Con group (Fig. 2D, E). Moreover, the CCK-8 assay results showed that CM inhibited fibroblast viability compared with that in the control group (Fig. 2F), confirming the gel contraction assay results. Considerable evidence has suggested that pulmonary fibroblast migration is one main cause of pulmonary fibrosis. We examined whether infiltrating monocytes affected fibroblast migration. The results of the scratch assay (Fig. 2G, H) showed that CM inhibited the migration of HPF-a cells. Taken together, these results suggested that monocyte exposure to SiO2 exerted a protective effect against fibrosis.

Fig. 2figure 2

Monocytes play a negative role in fibroblast activation, migration and viability after silica treatment. A Representative Western blot showing the effect of CM on the upregulation of COL1A1 and ACTA2 in fibroblasts. B Densitometric analyses of COL1A1 levels in three independent experiments; *P < 0.05 vs. the control group. C Densitometric analyses of ACTA2 levels in three independent experiments; *P < 0.05 vs. the control group. D Representative images of the gel contraction assay of fibroblasts treated with CM. E Gel contraction assay results demonstrating the conditioned medium-induced decrease in fibroblasts; *P < 0.05 vs. the control group. F CCK-8 assay results showing that CM attenuated fibroblast viability; *P < 0.05 vs. the corresponding time point in the control group, n = 3. G Representative images of a scratch assay showing that the migration of fibroblasts was attenuated by CM. Scale bar, 20 μm. H Quantification of the scratch gap distance in three independent experiments; *P < 0.05 vs. the corresponding time point in the control group

Increased IL-8 expression is not a key factor that affects fibroblast activation, viability or migration

To further investigate which cytokines influence the functions of fibroblasts, we examined several factors. First, we measured the classic cytokine TGF-β1 [21, 22] and found that the mRNA level was not changed after SiO2 stimulation (Fig. 3A). Then, we measured 12 inflammation-related cytokines by ELISA and found an increase in IL-8 expression (Fig. 3B). To confirm whether IL-8 was increased by SiO2, we measured its mRNA and protein expression. The results showed that IL-8 mRNA expression (Fig. 3C) and IL-8 protein levels increased (Fig. 3D) in the THP-1 cell line after SiO2 stimulation. To identify whether IL-8 was the main factor in CM that affected fibroblasts, we added IL-8 to normal medium. Interestingly, both ACTA2 and COL1A1 were significantly increased after IL-8 treatment (Fig. 3E–G). In addition, IL-8 also promoted fibroblast viability (Figure S2A). The 2D scratch assay was used to evaluate migration, and IL-8 promoted cell migration, as expected (Fig. 3H, I).

Fig. 3figure 3

Increased IL-8 expression is not a key factor that affects fibroblast activation, viability and migration. A RT‒qPCR analysis showing that SiO2 stimulation had no effect on TGFB1 expression. B SiO2 induced the expression of 12 inflammatory factors in THP-1 cells. C RT‒qPCR analysis showing that cxcl8 expression was increased in THP-1 cells in response to SiO2 stimulation (n = 5); *P < 0.05 vs. the 0 h group. D ELISA analysis showing that IL-8 protein expression was increased in THP-1 cells in response to SiO2 stimulation (n = 3); *P < 0.05 vs. the 0 h group. E Representative Western blot showing the effect of IL-8 on the upregulation of COL1A1 and ACTA2 in fibroblasts. F Densitometric analyses of COL1A1 protein expression levels in three independent experiments; *P < 0.05 vs. the control group. G Densitometric analyses of ACTA2 protein expression levels in three independent experiments; *P < 0.05 vs. the control group. H Representative images from the scratch assay showing that the migration of fibroblasts was increased by IL-8. Scale bar, 20 μm. I Quantification of the scratch gap distance in three independent experiments; *P < 0.05 vs. the corresponding time point in the control group

Decreased IL-10 release is a key factor that affects fibroblast activation, viability and migration

After examining the effect of IL-8, we found that it was inconsistent with the effect of CM; thus, we reviewed the previous data and shifted our focus to the decreased fibrosis factor IL-10 [19, 23, 24] (Fig. 3B). We analyzed the differentially expressed genes in monocyte populations by single-cell sequencing and found that the expression of IL-10 receptors was decreased in the model group (Fig. 4A). To confirm that the change in IL-10 was induced by SiO2, we measured IL-10 protein levels and found a time-dependent decrease in response to SiO2 stimulation (Fig. 4B). To further determine the role of IL-10, we added IL-10 to CM. As expected, the fibroblast activation marker proteins ACTA2 and COL1A1 were both restored after the addition of IL-10 to CM (Fig. 4C–E). In addition, IL-10 promoted the migration of HPF-a cells compared with those treated with CM (Fig. 4F, G). In addition, the CCK-8 assay (Fig. 4H) and gel contraction assay (Fig. 4I, J) results indicated that IL-10 could abrogate the negative effects of CM on HPF-a cell viability.

Fig. 4figure 4

Decreased IL-10 release is a key factor that affects fibroblast activation, viability and migration. A IL10ra in the NS group and the SiO2 group is shown in the heatmap of the monocyte cluster. B ELISA analysis showing that SiO2 decreased IL-10 protein expression in THP-1 cells (n = 5); *P < 0.05 vs. the 0 h group. C Representative Western blot showing the effect of CM and IL-10 on the specific upregulation of COL1A1 and ACTA2 in fibroblasts. D Densitometric analyses of ACTA2 levels in three independent experiments; *P < 0.05 vs. the control group, *P < 0.05 vs. the CM group. E Densitometric analyses of COL1A1 levels in three independent experiments; *P < 0.05 vs. the control group, *P < 0.05 vs. the CM group. F Representative images from the scratch assay showing that the migration of fibroblasts was increased by CM and IL-10. Scale bar, 20 μm. G Quantification of the scratch gap distance in three independent experiments; *P < 0.05 vs. the corresponding time point in the control group. H CCK-8 assay results showing that fibroblast viability was increased by CM and IL-10; *P < 0.05 vs. the corresponding time point in the control group, #P < 0.05 vs. the corresponding time point in the CM group, n = 3. I Representative images of gel contraction assays showing fibroblasts treated with CM and IL-10. J Gel contraction assay results demonstrating the decrease in fibroblast viability induced by CM and IL-10; *P < 0.05 vs. the control group, #P < 0.05 vs. the CM group, n = 3

Autophagy is involved in the silica-induced reduction in IL-10 release by monocytes

Based on these results, we showed that the cytokine that affected fibroblast function was IL-10, but how silica regulated the release of IL-10 by THP-1 cells was unclear. To determine what affected the expression of IL-10, we first measured the mRNA level of IL-10. Unexpectedly, the results demonstrated that IL-10 mRNA levels did not show significant changes (Fig. 5A; Additional file 1: Fig. S3A). Based on a literature review, we initially thought that a posttranslational modification might regulate IL-10 expression. First, we were concerned that endoplasmic reticulum (ER) stress could control the release of cytokines to a certain extent [25, 26]. To verify our hypothesis, we used Western blotting to analyze ER stress markers, but no significant difference was found in the expression of the marker proteins HSPA5 and DDIT3 (Additional file 1: Fig. S3B–D). Then, we moved to the autophagy pathway, which is highly associated with cytokine release [27, 28]. We obtained a string map (Fig. 5B) and bubble map (Fig. 5C) through biological information technology and found that IL-10 played an important role in autophagy signaling pathways. To explore whether autophagy regulated the expression of IL-10, we first measured the expression of autophagy markers. The immunoblotting results showed upregulation of the markers MAP1LC3B, BECN1 and ATG5 (Fig. 5D, E). Furthermore, after using the autophagy inhibitor 3-MA to block autophagy, the SiO2-induced decrease in IL-10 release was significantly inhibited, and the IL-10 protein level was restored (Fig. 5F). Moreover, after treatment with the autophagy agonist rapamycin, IL-10 showed a further decline in the SiO2 treatment group (Fig. 5G). Notably, the level of IL-10 decreased in the vehicle group after drug administration, which may be due to the nonspecific effect of the drug on cell viability (Additional file 1: Fig. S4A, B).

Fig. 5figure 5

Autophagy is involved in the silica-induced reduction in IL-10 release by monocytes. A RT‒qPCR analysis showing that SiO2 stimulation had no effect on cxcl10 expression. B, C As shown in the string map and bubble map, IL-10 plays an important role in autophagy signaling pathways. D Representative Western blot showing the effect of SiO2 on the upregulation of MAP1LC3B, ATG5 and BECN1 in THP-1 cells. E Densitometric analyses of MAP1LC3B levels in five independent experiments; *P < 0.05 vs. the 0 h group. Densitometric analyses of ATG5 levels in five independent experiments; *P < 0.05 vs. the 0 h group. Densitometric analyses of BECN1 levels in five independent experiments; *P < 0.05 vs. the 0 h group. F ELISA analysis showing that the SiO2-induced reduction in IL-10 protein release by THP-1 cells was reversed by the autophagy blocker 3-MA (n = 5), *P < 0.05 vs. the corresponding group in the control group. #P < 0.05 vs. the con group and the SiO2 group. G ELISA analysis showing that the SiO2-induced reduction in IL-10 protein release by THP-1 cells was promoted by the autophagy agonist rapamycin (n = 5), *P < 0.05 vs. the corresponding group in the control group. #P < 0.05 vs. the con group and the SiO2 group

ZC3H4 regulates the silica-induced release of IL-10 by monocytes

Previous studies in our laboratory have shown that the zinc finger proteins MCPIP1 (ZC3H12A) and ZC3H4 play important roles in the process of fibrosis caused by the inflammatory response in macrophages [19, 29]. Whether the zinc finger protein ZC3H4 is involved in the effect of monocytes on fibrosis is unclear. To identify whether ZC3H4 is involved in monocytes, we first measured ZC3H4 protein levels. The immunoblotting results showed that ZC3H4 was significantly increased after SiO2 treatment (Fig. 6A, B). Immunostaining also confirmed this effect (Fig. 6C and Figure S5A). CRISPR/Cas9 technology (Fig. 6D, E) was used to knock down the ZC3H4 protein (NIC). Moreover, ZC3H4-NIC upregulated the IL-10 expression level to that of the control group in the presence of SiO2 (Fig. 6F).

Fig. 6figure 6

ZC3H4 is involved in regulating the silica-induced release of IL-10 by monocytes. A Representative Western blot showing that SiO2 induced ZC3H4 expression in a time-dependent manner in THP-1 cells. B Densitometric analyses of ZC3H4 protein expression levels in five independent experiments; *P < 0.05 vs. the 0 h group. C Representative immunocytochemical staining images showing that SiO2 induced ZC3H4 expression in THP-1 cells. Scale bar, 100 μm. D Representative Western blot showing that ZC3H4 protein was knocked down in THP-1 cells after plasmid transfection. E Densitometric analyses of ZC3H4 protein expression levels in five independent experiments; *P < 0.05 vs. the corresponding group in the control group. #P < 0.05 vs. the Con-NIC+ ZC3H4-NIC− group and the SiO2 group. F ELISA analysis showing that knocking down the ZC3H4 protein increased IL-10 protein release by THP-1 cells. *P < 0.05 vs. the corresponding group and the control group. #P < 0.05 vs. the Con-NIC+ ZC3H4-NIC− group and the SiO2 group

ZC3H4 regulates IL-10 release through autophagic processes

Based on these findings, the results showed that ZC3H4 and autophagy could both regulate the expression of IL-10. According to the literature, associations exist between various zinc finger proteins and autophagy. To verify the relationship between ZC3H4 and autophagy, we knocked down ZC3H4 and observed changes in autophagy marker proteins. The results suggested that the autophagy-related proteins MAP1LC3B, BECN1, and ATG5 were all decreased in the SiO2 group (Fig. 7A–C). To further validate this result, THP-1 cells were transduced with dual fluorescent mRFP-GFP-MAP1LC3 adenovirus to detect autophagy by monitoring autophagosome formation in real time with fluorescence microscopy. mRFP was used to label and track LC3, whereas GFP fluorescence is sensitive to acidic conditions; thus, GFP fluorescence will be quenched when a lysosome and an autophagosome form an autolysosome. Controls that were not SiO2-stimulated had almost no obvious autophagic flux (Additional file 1: Fig. S5D, F). SiO2 significantly induced autophagic flux, and this effect of SiO2 was attenuated by knocking down ZC3H4 (Fig. 7D, F). Moreover, after knocking down ZC3H4, rapamycin reduced the expression of IL-10 under SiO2 stimulation compared with the effect of no rapamycin.

Fig. 7figure 7

ZC3H4 regulates IL-10 release through autophagic processes. A Representative Western blot showing that knocking down ZC3H4 downregulated MAP1LC3B, ATG5 and BECN1 expression levels in THP-1 cells treated with SiO2. B Densitometric analyses of MAP1LC3B protein expression levels in five independent experiments; *P < 0.05 vs. the corresponding group and the control group. #P < 0.05 vs. the Con-NIC+ ZC3H4-NIC− group and the SiO2 group. C Densitometric analysis of BECN1 protein expression levels in five independent experiments; *P < 0.05 vs. the corresponding group and the control group. #P < 0.05 vs. the Con-NIC+ ZC3H4-NIC− group and the SiO2 group. D Densitometric analysis of ATG5 protein expression levels in five independent experiments; *P < 0.05 vs. the corresponding group and the control group. #P < 0.05 vs. the Con-NIC + ZC3H4-NIC- group and the SiO2 group. E Representative images of the fluorescence map showing that autophagy was attenuated in THP-1 cells stimulated with SiO2 and ZC3H4 protein knockdown. Scale bar, 80 μm. F Quantification of autophagy levels in THP-1 cells treated with SiO2 after ZC3H4 protein knockdown; *P < 0.05 vs. the corresponding color in the CON-NIC group. G Quantification of autolysosomes and autophagosomes in THP-1 cells treated with SiO2 after ZC3H4 protein knockdown; *P < 0.05 vs. the corresponding color in the CON-NIC group. H ELISA analysis showing that the autophagy agonist rapamycin decreased IL-10 protein release in THP-1 cells treated with SiO2 after ZC3H4 protein knockdown. ***P < 0.001 vs. the Con-NIC+ Rapa− group. ###P < 0.001 vs. the NIC+ Rapa− group

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