Exposure to ETS, a mixture of tobacco smoke from the side-stream of cigarettes and the exhaled smoke from active smokers, has shown correlations with asthma and COPD development. However, no clinical evidence showed how ETS exposure contributes to fibrogenesis development (12, 32). Recent animal studies have described that CS exacerbates the fibrotic progression induced by different causes such as bleomycin, lipopolysaccharides (LPS), polyhexamethylene guanidine, and influenza A virus (IAV) (3, 6, 7, 33, 34). CS exposure followed by bleomycin administration showed significantly increased hydroxyproline and collagen deposition compared to bleomycin alone (3, 6). CS exposure also augments acute inflammatory responses induced by LPS treatment to fibrotic lesion formation, with increased levels of TGFβ, αSMA, and collagens (34). Here, we found that ETS exposure exacerbates collagen overexpression and activation of C3 complement signaling induced by bleomycin in relatively younger ages. After chronologically aging, ETS exposure showed no effects on bleomycin-induced collagen content upregulation. Our results showed that ETS exposure could exaggerate collagen biosynthesis and modification through MMPs and LOX.
Tobacco smoke is a well-known risk factor for COPD/emphysema development, usually with decreased elastance, increased compliance, and airspace enlargement. Previously, we have shown that main-stream CS exposure induced increased compliance, mean linear intercept (Lm), and inflammatory cell-flux infiltration (13). In this study, we have noticed slightly increased compliance, decreased elastance after ETS exposure without significance, and limited airspace enlargement compared to air control. Since ETS is considered a low dose of tobacco smoke, a longer exposure duration and more robust tobacco-smoke exposure might be needed to develop emphysema. After bleomycin administration, increased elastance and decreased compliance were observed, which are the hallmarks of fibrotic progression. Interestingly, slightly decreased elastance and increased compliance were noticed in the ETS+BLM group compared to Air+BLM, which might be the combined effects of tobacco smoke exposure and bleomycin, which showed as a phenomenon of combined pulmonary fibrosis and emphysema (CPFE). It has been confirmed clinically that patients with CPFE have intermediated value of lung mechanics parameters between patients with emphysema and fibrosis (35). A similar trend of dysregulated lung mechanics was found in both young and old, which showed that ETS exposure and bleomycin administration-induced lung injury is age-independent. We also found increased collagen deposition in the lung lesion area caused by bleomycin, while no overall collagen deposition was promoted after ETS exposure, which does not concur with the previous study (6). Subtypes of collagen: COL4A1 and COL5A1 recorded significantly increased RNA expression levels in ETS+BLM compared to the Air+ETS group, and non-significant increased COL1A1, COL1A2, and COL4A2 were found as well in the ETS+BLM group compared to Air+BLM. Besides, our study also showed further increased protein levels of COL1A1 in males rather than females in the ETS+BLM group compared to the Air+BLM group, which partially agreed that males are more vulnerable during fibrogenesis (36). A recent study showed that CS exposure aggravated the collagen expression induced by IVA infection (7). The same study has shown CS exposure exaggerated the IAV infection-activated fibroblast to myofibroblast differentiation with increased TGFβ and αSMA (7). Another study also showed CS exposure mediated fibrotic progression and exacerbation might occur through TGFβ/Smad2 signaling pathway (6). Our study has further confirmed that collagen dynamic was also one of the possible pathways contributing to fibrogenesis exacerbation due to ETS exposure. Although we noticed increased gene levels of TGFβ and its receptors, no significant difference was identified between Air+BLM and ETS+BLM groups. More studies are required to understand how tobacco smoke exposure contributes to fibrogenesis development.
Our gene expression results and pathway analysis show further activation of ECM degradation, collagen biosynthesis and modification, and ECM synthesis. We, therefore, focused on MMPs and lysyl oxidases, which are the enzymes responsible for ECM remodeling. Increased protein abundance of MMP2 was found either in lung fibrosis animal models or fibrotic clinical samples (37). Our results showed increased gene and protein levels of MMP2 which agreed with the previous study, and our results further illuminated that ETS exposure further exaggerated active MMP2 protein levels in females, and no significant upregulation in Air+BLM compared to the Air+PBS group. However, the upregulation of MMP2 was found in males treated with bleomycin regardless of ETS or Air exposure. It has been well-stated that female has better survival during fibrogenesis than male, and our results contribute to this statement that upregulated MMP2 during fibrogenesis is only found in males. ETS exposure could be one of the factors in helping develop fibrotic progression in females (36). Another enzyme tested in this study is lysyl oxidase, a copper-dependent amine oxidase that helps to stabilize the ECM structure via crosslinking collagen and elastin fibers (38). Upregulation of lysyl oxidase has been identified from the fibrotic lesion in lung fibrosis patients and in bleomycin-induced lung fibrotic injury (39). Upregulated lysyl oxidases during fibrogenesis drive severe crosslinking of collagen and elastin fibers, preventing degradation of fibrous matrix and contributing to irreversible scarring (40). In this study, we first identified that fibrogenesis exacerbation induced by ETS exposure could be through lysyl oxidase-mediated abnormal collagen dynamics. Although the gene expression levels of LOX, LOXL1, and LOXL2 showed no significant difference between the ETS+BLM and Air+BLM groups, a significantly increased protein abundance of LOX was found especially in the fibrotic lesion area in the ETS+BLM group compared to Air+BLM group. More importantly, ETS exposure impacts bleomycin-induced upregulated protein abundance of active LOX in males only, whereas no difference is identified in females. Our results of lysyl oxidase differentiated expression in different groups in a sex-dependent manner contribute to the statement that males showed worse lung injury during fibrotic progress than females (36). Other than sex-dependent manner, we also studied whether aging affects ETS exposure on bleomycin-induced fibrogenesis. Our results show no significant alternation of most of the fibrotic markers except for active MMP2. Our results showed that ETS exposure has no significant impact on the exacerbation of fibrotic progression, while ETS exposure augments collagen synthesis and modification during bleomycin-induced fibrogenesis in relatively younger mice. Since COPD and IPF are both senescence/aging-related diseases (41, 42), we have customized a senescence-focused NanoString transcriptomic panel to understand the effect of ETS exposure and bleomycin-induced cellular senescence. During cellular senescence, molecular checkpoint systems responsible for DNA damage, cell cycle, apoptosis, and other routine cellular functions are disrupted and finally become irreversible (42, 43). After bleomycin administration, our results showed increased gene expressions of different cyclin-dependent kinase inhibitors (CDKN1A, CDKN2B, and CDKN2C). A non-significant further increased CDKN1A was found in ETS+BLM compared to the Air+BLM group. The protein encoded by CDKN1A is p21, which can cause cell cycle arrest by inhibiting CDK2 and CDK4 (44). Either CS exposure or bleomycin alone has been reported to increase CDKN1A in the alveolar epithelium (15). Our result agreed with the previous study and further showed that ETS exposure could exacerbate bleomycin-induced CDKN1A upregulation. Another SASP gene, SERPINE1, showed upregulation after bleomycin administration and a further upward trend was found in the ETS+BLM group compared to the Air+BLM group. It has shown that the upregulation of SERPINE1 induced the phosphorylation of p53 and increased the protein level of p21, which activated p53-p21-Rb-associated cell cycle repression (45). Our results confirmed that exacerbated upregulation of SERPINE1 by ETS could augment the senescence process induced by bleomycin.
Besides the CDKN family and SERPINE1, the transcriptomic panel also showed irregular activation of the complement system, which plays an important role during cellular senescence and aging (46). It has been shown that complement C3 level is positively correlated with age, and inhibition of C3 could prevent senescence progression in the renal system (47). Multiple studies focus on complement system development in emphysema and IPF (48, 49). Removing C3 or C3A receptor (C3AR) helped prevent inflammation and airspace enlargement caused by chronic CS exposure (48). In the same study, increased protein levels of C3 and C3AR were increased after chronic CS exposure (48). However, our results showed no change in the protein level of C3A and gene expression of C3AR after ETS exposure. Longer exposure duration and higher doses of ETS should be considered to activate C3 and C3AR. Another study showed that an increased protein abundance of C3A was found after bleomycin treatment (49). Increased protein abundance of C3A and C5A protein drives the expression of FN1 and αSMA in lung fibroblast, which are the hallmarks of fibrogenesis (49). In the same study, blocking C3AR attenuated lung injury and upregulated collagens induced by bleomycin treatment (49). Our results agreed with the increased expression of C3 and C3AR, and we further identified that ETS exposure exacerbates the bleomycin-induced activation of C3AR. Besides, the protein abundance of C3A showed a significant increased and non-significant increased trend for C3B and C3C in ETS+BLM compared to Air+BLM group in male mice, whereas females either showed no difference or decreased trend. Our results in complement components again confirmed that males were more vulnerable during fibrogenesis than females. However, limited SASP markers were dysregulated significantly, adjusting the duration post-BLM might help emphasize the exacerbation of SASP by ETS exposure.
As mentioned before, fibrogenesis is an aging disease usually occurring in elders. However, based on our normalized gene expressions of collagen, lysyl oxidase, and proteases, older mice did not show significant dysregulation levels than younger mice in most fibrotic markers. It has been reported that the bleomycin-induced fibrosis model serves better in the old mice, in which the chronological aging could delay fibroblast-dominated repair capacity, hence inducing persistent scarring and ECM deposition, whereas young mice showed recovery in lung scarring (50, 51). Bleomycin treatment for 14 days might not be enough for mimicking the fibrogenesis progression in humans since it is a chronic lung disease. A longer duration post-BLM (28 days) in old mice could help produce persistent scar tissue in the lungs, which is the pathological phenomenon of lung fibrogenesis. Besides, removing ETS-induced overexpression of p16, one of the senescence markers, did not help resolve the lung injury induced by bleomycin, indicating that targeting p16 might be efficient for emphysema development, but not fibrotic progression (52).
In conclusion, we showed that ETS exposure exacerbated bleomycin-induced overexpression of collagen subtypes, lysyl oxidase, and C3A-receptor signaling. Removing p16 high-expressed cells did not help alleviate the lung injury induced by bleomycin. More importantly, our results showed that male mice were more susceptible than females during fibrogenesis exacerbation. Our study provided potential signaling that ETS exposure not only activated the TGFβ/SMAD2 pathway but also C3A-receptor signaling, which might be the potential reason for exacerbated abnormal collagens dynamics during fibrogenesis.