Aging accelerates APAP-ALI and liver inflammation
We firstly examined the effect of aging on APAP-ALI. The results showed that aging significantly aggravated liver injury induced by APAP, manifested as increased serum ALT and AST contents (Fig. 1A) and increased necrotic area shown by H&E staining (Fig. 1B). Meanwhile, the positive rate of TUNEL staining was significantly increased in liver tissue sections from aged mice (Fig. 1C). In addition, increased MΦ infiltration was found in the liver of aged mice after APAP-ALI (Fig. 1D). Consistent with this, aging promoted the expression of pro-inflammatory cytokines, such as SASP components (Il1b, Il6, Tnfa), and decreased the expression of anti-inflammatory cytokine Il10 in liver (Fig. 1E). The severity of APAP-induced liver injury was dose-dependent. In the survival rate study, the mortality of aged mice was significantly increased under a higher dose of APAP (500 mg/kg) (Fig. 1F). The above results indicate that aging greatly exacerbates APAP-ALI, accompanied by increased infiltration of MΦs and expression of pro-inflammatory cytokines.
Bmp9 Promotes Hepatocyte Injury And Senescence
RT-qPCR and immunoblotting detection revealed that the expression of Bmp9 and Cebpa both increased in liver tissue from aged mice (Fig. 2A, B). Since BMP9 is a secreted protein and enriched in aged liver, we used mouse BMP9 recombinant protein (Rm-BMP9) to investigate the effect of BMP9 on APAP-treated hepatocytes. With the prolongation of APAP treatment time in vitro, the ratio of hepatocyte death in the addition of Rm-BMP9 was significantly higher than that in APAP treatment alone, indicating that BMP9 might have a direct role in promoting hepatocyte injury in APAP-ALI (Fig. 2C). We further explored the mechanism by which BMP9 aggravates hepatocyte injury in vitro. Increased phosphorylation of SMAD1/5/9, representing activation of BMPs signaling, suggests that BMP9 might activate downstream signaling cascades in APAP-induced hepatocyte injury (Fig. 2D). Additionally, activation of SMAD1/5/9 was increased in the liver of aged mice, whereas phosphorylation of SMAD1/5/9 was decreased in the liver of aged Bmp9 knockout (Bmp9−/−) mice (Figure S1A).
Autophagy-related 3 (ATG3, E2-like enzyme activity) and ATG7 (E1-like enzyme activity) are key molecules in the process of autophagy, mediating the transformation of microtubule-associated protein 1 light chain 3 II (MAP1LC3 II/LC3 II) and the maturation of autophagosomes (17, 18). Noteworthy, Rm-BMP9 down-regulated ATG3 and ATG7 expression, and significantly decreased the transformation of LC3 II in APAP-injured hepatocytes, which was consistent with the accumulation of SQSTM1/p62 (Fig. 2E).
Intrahepatic BMP9 increases with aging, but the effect of increased BMP9 on hepatocyte senescence is still unclear. By using etoposide to induce hepatocyte senescence, we found that Rm-BMP9 induces higher levels of cellular senescence (Fig. 2F).
The above results illustrate the aggravating effect of BMP9 on aging-associated APAP-ALI in vitro, and then we further investigated the effect of BMP9 on APAP-ALI by injecting Rm-BMP9 into mice in vivo. Serum ALT/AST levels (Fig. 2G) and H&E staining (Fig. 2H) exhibited a significantly increased APAP-ALI, and the positive rate of TUNEL staining was increased (Figure S1B). Then we found that Rm-BMP9 promoted the expression of Ilb, Il6 and Tnfa in liver (Figure S1C). Consistent with the results obtained from exogenous BMP9 supplementation, Bmp9 deletion reduced liver injury in both young and aged mice (Fig. 2I, J). Additionally, the positive rates of TUNEL staining in the livers from young and aged mice treated by APAP were significantly decreased in Bmp9−/− groups (Figure S1D). RT-qPCR assays revealed that Bmp9 deletion reduced the expression of pro-inflammatory factors (Ilb, Il6 and Tnfa) in liver tissue (Figure S1E).
These results suggest that BMP9 inhibits hepatocyte autophagy through ATG3 and ATG7, thereby participating in the regulation of APAP-ALI, and high levels of BMP9 protein associated with aging may be an important factor in the aggravation of APAP-ALI during aging.
The expression of BMP9 is regulated by C/EBPα in vitro
Given the controversy over which cell type express BMP9 in previous studies, to visualize the expression and distribution of BMP9 in aged liver, we performed immunofluorescence (IF) staining for F4/80 and BMP9 in liver tissues from young and aged mice. In the absence of liver injury, it was observed that BMP9 was mainly localized to F4/80-positive cells, and BMP9 expression was higher in aged mouse liver (Fig. 3A). When APAP-ALI occurs, BMP9 accumulates in the injured area, and the expression of BMP9 localized to F4/80 positive cells increases, and the increase of BMP9 can also be found in hepatocytes (Fig. 3A). Then, we isolated hepatocytes and hepatic MΦs from young and aged mice. BMP9 expression was dramatically up-regulated in both hepatocytes and hepatic MΦs from aged group (Fig. 3B, C). Notably, we also detected increased C/EBPα, and decreased ATG3, ATG7 in isolated hepatocytes and hepatic MΦs from aged mice (Fig. 3B, C).
C/EBPα is an important transcription factor which determines multiple physiological functions. High expression of C/EBPα and BMP9 was detected in both senescent hepatocytes and MΦs, however, it remains unclear whether BMP9 is regulated by C/EBPα. Concomitant with the overexpression (OE) of Cebpa, BMP9 expression was increased in immortalized murine bone marrow-derived MΦ (iBMDM) cell line (Fig. 3D). Similarly, inhibition of Cebpa in iBMDMs by siRNA reduced BMP9 expression (Fig. 3E). BMDMs derived from young and aged mice were then stimulated with the APAP-treated AML-12 cell supernatant (Sup) to mimic DAMPs signaling induced by hepatocyte death. Moreover, enhanced Cebpa expression promoted iNOS expression and decreased CD206 expression in iBMDMs after stimulation by the Sup (Fig. 3F). Following Cebpa overexpression, mRNA expression of Il1b, Il6, and Tnfa induced by the Sup were increased in iBMDMs (Fig. 3G). In addition, Cebpa overexpression also increased BMP9 in AML-12 cells (Fig. 3H), and Cebpa inhibition reduced BMP9 expression correspondingly (Fig. 3I).
These results suggest that the expression of BMP9 is driven by C/EBPα signaling in MΦs and hepatocytes during aging, which might be a key intrinsic factor in aggravated APAP-ALI associated with aging.
The C/ebpα-bmp9 Axis In The Liver Promotes Apap-ali Progression
Given the link between C/EBPα and BMP9, the effect of C/EBPα on BMP9 and APAP-ALI in vivo was investigated by overexpressing Cebpa using AAV8. Both mRNA and protein expression of Bmp9 were up-regulated after Cebpa overexpression in the liver after 2 weeks of Cebpa AAV8 injection via the tail vein (Figure S2A, B). Notably, IF and IHC confirmed successful overexpression of Cebpa (Figure S2C). After the successful overexpression of Cebpa, the APAP-ALI model was established. Increased serum ALT and AST levels suggest that Cebpa overexpression aggravates APAP-ALI (Fig. 4A), paralleling H&E staining images (Fig. 4B), and increased the positive rate of TUNEL staining (Fig. 4C). Noteworthy, Cebpa overexpression induced mRNA expression of Il1b, Il6 and Tnfa in the liver (Fig. 4D). Moreover, we also detected increased of Cxcl1, Cxcl13 and Mcp1, and decreased Arg1 and Il10 (Figure S3A, B). In the survival rate study, we increased the dose of APAP administration (500 mg/kg), and the results showed that Cebpa overexpression in the liver significantly increased the mortality of mice treated by APAP (Figure S3C).
IHC staining of liver sections showed that the expression of BMP9 was up-regulated after overexpression of Cebpa (Fig. 4E). Meanwhile, phosphorylated SMAD1/5/9 was increased (Fig. 4F). These results indicate that Cebpa overexpression promoted BMP9 production and activated BMPs signaling. Next, we investigated potential proteins that control APAP-ALI progression in the liver following Cebpa overexpression. By immunoblotting, we further confirmed that Cebpa overexpression was successful, and the expression of BMP9 and phosphorylated SMAD1/5/9 was up-regulated (Fig. 4G). Furthermore, we detected a downregulation of ATG3 and ATG7 in the liver, accompanied by blockade of autophagy (Fig. 4H).
IF staining of liver sections for F4/80 and BMP9 revealed that Cebpa overexpression increased the expression of BMP9 localized to F4/80-positive cells in the liver (Fig. 4I). Similar to what was observed in aged livers, increased expression of BMP9 in non-F4/80-positive cells was also seen in livers after APAP treatment, suggesting the diversity and complexity of BMP9 sources in the liver (Fig. 4I).
These above results suggest that the expression of BMP9 in the liver is regulated by C/EBPα. Excessive C/EBPα leads to excessive production of BMP9. BMP9 inhibits the autophagy process by downregulating the expression of ATG3 and ATG7 in liver cells, thereby promoting the aggravation of liver inflammation and injury.
Bmp9 Inhibits Atg3 And Atg7, And Thereby Blocks Autophagy In MΦs
In the above results, we confirmed that BMP9 is abundantly expressed in hepatic MΦs and directly aggravates APAP-induced injury by inhibiting hepatocyte autophagy. Rapamycin (RAPA) has been extensively reported to activate autophagy and reduce senescence (19, 20). Therefore, we investigated the roles of aging and BMP9 on MΦs themselves with RAPA treatment. The conversion of LC3 II was reduced while BMP9 was highly expressed in primary hepatic MΦs from aged mice with RAPA or/and bafilomycin (Ba) treatment (Figure S4). Next, by transfecting Ad-mCherry-GFP-LC3 adenovirus and expressing mCherry-CFP-tagged LC3 in BMDMs, we confirmed that autophagy in aged mouse-derived MΦs was hindered compared with the young group (Fig. 5A). Dual-color IF staining for LC3 and BMP9 was then performed on BMDMs derived from aged mice, and we found that BMP9 increased while LC3 decreased in cells from aged mice through high-resolution images (Fig. 5B). Furthermore, the mCherry puncta were enhanced with RAPA treatment after Bmp9 deletion (Fig. 5C).
Autophagy was restrained in the short term after exposure to the above-mentioned Sup, which may be an important factor for MΦs to appear pro-inflammatory during the acute phase of liver injury (Fig. 5D, E). After BMP9 depletion, ATG3 and ATG7 expression in MΦs was up-regulated, LC3 II conversion was increased, and p62 was decreased, indicating an activated autophagy condition (Fig. 5D). Additionally, the Rm-BMP9 addition resulted in downregulated ATG3 and ATG7, decreased LC3 II conversion, and increased p62 accumulation in MΦs (Fig. 5E).
In previous studies, the decline of autophagy in MΦ promoted its M1-type polarization during ALI, manifesting as a pro-inflammatory and pro-injury effect (21). Importantly, BMP9 can also directly promote APAP-induced hepatocyte death, thus the aging-associated excess accumulation of BMP9 may be an essential factor in the aggravation of liver injury.
Bmp9 Regulates Senescence And Immunophenotype Of MΦs
After etoposide (1 µg/ml) treatment for 2 days, the mRNA levels of p21 and p53 in iBMDMs increased, as well as the expressions of SASP components Il1b, Il6 and Tnfa were up-regulated (Figure S5A). Similar to BMP9 promoting etoposide-induced hepatocyte senescence, BMP9 also promotes etoposide-induced senescence phenotype in iBMDMs (Figure S5B). In hepatic MΦs isolated from Bmp9−/− mice, Il1b, Il6, Tnfa and iNos were reduced after etoposide treatment alone and/or plus APAP-treated AML-12 cell supernatant (Figure S5C).
Based on these results, we isolated primary hepatic MΦs from young WT, Bmp9−/− mice, and aged WT, Bmp9−/− mice. SA-β-gal staining revealed that Bmp9 deletion reduced MΦ senescence phenotype (Fig. 6A). Next, by treating these young and aged cells with Sup, the expression of Il1b, Il6 and Tnfa were decreased in the Bmp9−/− group (Fig. 6B). Moreover, the cytokines (IL-1β, IL-6, TNF-α and IL-10) in the supernatants of cells with different treatment were detected by ELISA. The results showed that Bmp9 deletion reduced the secretion of IL-1β, IL-6 and TNF-α (pro-inflammatory) and increased the production of IL-10 (inflammation suppression and injury repair), while exogenous Rm-BMP9 significantly increased the secretion of IL-1β, IL-6 and TNF-α, and reduced the concentration of IL-10 (Fig. 6C). The expression and intracellular distribution of iNOS and CD206 were then detected by IF staining, and the results showed that aging promoted iNOS production and decreased CD206, and excess BMP9 further enhanced iNOS production and reduced CD206, while inhibiting Bmp9 expression reversed this phenomenon (Figure S6A).
To further study the effect of MΦ-derived BMP9 on APAP-ALI, an F4/80-specific Bmp9 overexpression AAV8 virus was constructed (AAV8-F4/80-Bmp9) (22). APAP-ALI was established after 2 weeks of tail vein injection of AAV8-F4/80-Bmp9 as mentioned above. IF staining of isolated hepatic MΦs showed that BMP9 expression in AAV8-F4/80-Bmp9 group was significantly up-regulated, confirming the successful overexpression of MΦ-specific BMP9 (Figure S6B). As shown by serum ALT/AST levels and H&E staining, APAP-ALI was significantly increased in the group of F4/80-specific Bmp9 overexpression (Fig. 6D, E). Additionally, the positive rates of TUNEL staining in the livers from F4/80-specific Bmp9 overexpression mice treated by APAP were significantly increased (Fig. 6F). Meanwhile, the mRNA expression of Il1b, Il6 and Tnfa were up-regulated in the livers from F4/80-specific Bmp9 overexpression mice (Fig. 6G).
These data suggest that BMP9 also plays a key role in the senescence process of MΦs, and MΦ-derived BMP9 prominently drives the progression of APAP-ALI.