Environmental tobacco smoke exposure exaggerates bleomycin- induced collagen overexpression during pulmonary fibrogenesis

Environmental tobacco smoke (ETS) is known to cause lung inflammatory and injurious responses. Smoke exposure is associated with the pathobiology related to lung fibrosis, whereas the mechanism by which ETS exposure augments lung fibrogenesis is unclear. We hypothesized that ETS exposure could exacerbate fibrotic responses via collagen dynamic dysregulation and complement activation. C57BL/6J and p16–3MR mice were exposed to ETS followed by bleomycin administration. ETS exposure exacerbated bleomycin-induced collagen and lysyl oxidase overexpression in the fibrotic lesion. ETS exposure also led to augmented bleomycin-induced upregulation of C3 and C3AR, which are pro-fibrotic markers. Moreover, overexpressed collagens and C3 levels were highly significant in males than females. The old mice (17 months old) were exposed to ETS and treated with bleomycin to induce fibrogenesis, since fibrogenesis is an aging-associated disease. Fewer gene and protein dysregulations trends were identified between ETS exposure with the bleomycin group and the bleomycin alone group in old mice. Based on our findings, we suggested that ETS exposure increases the risk of developing severe lung fibrotic responses via collagen overexpression and lysyl oxidase-mediated collagen stabilization in the fibrotic lesion. ETS exposure also potentially affected the complement system activation induced by bleomycin. Further, male mice were more susceptible than females during fibrogenesis exacerbation.


Introduction
Tobacco smoke is not only a global public health concern (1), but also one of the major risk factor involved in chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary brosis (IPF) (2,3).IPF is a chronic interstitial lung disease with unknown causes (4), and lung brosis is usually considered a chronic in ammatory disease with excessive burdens of permanent scar formation in the lungs (5).The initial stage of brogenesis starts with an in ammatory response and activated wound healing process, which contributes to temporary extracellular matrix (ECM) deposition.However, chronic in ammation results in dysregulated repair and continuously accumulated ECM, which could turn into permanent scars.
Tobacco smoke exposure can be considered active smoking exposure or passive smoking exposure.ETS is a mixture of side-stream cigarette smoke (CS) and main-stream CS exhaled by active smokers.It is well known that both CS and ETS could induce a signi cant in ammatory response, abnormal respiratory syndromes, and CS exposure aggravates brotic progression (6-9).CS exposure exacerbates the collagen deposition and α-smooth muscle actin (αSMA) expression induced by in uenza virus (IAV) infection in primary pulmonary broblast (7).Pre-expose to CS also contributes to augmented collagen deposition induced by bleomycin in mouse lungs (6).One possible mechanism is the TGFβ/Smad2 pathway, which was activated by CS extract (CSE) treatment in lung broblasts and pleural mesothelial cells (6).Except for the animal model, several studies have shown that ex-smoker with IPF exhibit increased and more severe cellularity in the alveolar space (10).ETS exposure showed increased risks of asthma and COPD development (11,12).Our previous study showed that CS exposure could activate epithelial-to-mesenchymal transition and induce collagen deposition in the lungs (13), while no study investigated the role of ETS in brotic progression.More importantly, lung brosis is usually considered as a senescence/aging-associated disease and CS exposure is also well-known to induce premature senescence and aging (14,15).ECM is formed with extracellular proteins as basal material to support cells in the three-dimension microenvironment.The dynamic production and degradation of ECM are involved in multiple disease mechanisms and overexpression of ECM is commonly observed in brotic progression.The major ECM proteins are collagens, bronectin, and elastin.Among collagens, types 1 and 3 are the major brous collagens to form ECM in alveoli and type 4 majorly exists in the basal membrane for supporting epithelium (16,17).Enzymes, such as lysyl oxidase (LOX) and MMPs are involved in ECM posttranslational modi cation.LOX families are copper-based amino-oxidases that help to crosslink brous ECM proteins i.e. collagens and elastin, which can increase the strength of ECM and prevent the degradation of brous components (18)(19)(20).Increased expression of LOX is usually found in IPF patients compared to healthy control, as well as the small airway epithelium from smokers compared to nonsmokers (21,22).
Here, we hypothesized that sub-chronic exposure to ETS augments brogenesis induced by bleomycin (BLM) administration via collagen dynamic dysregulation.C57BL/6J and p16-3MR mice were exposed to ETS for 30 days and followed by bleomycin administration for 14 days.The duration of ETS exposure and post-bleomycin administration is based on previous publications to avoid emphysema development induced by CS exposure and recovery of scaring injury after 21 days post-bleomycin (13,(23)(24)(25)(26)(27)(28)(29).To determine the age-related effects during ETS-induced brotic progression, young (4-5 months) and old (15-17 months) mice were used.

Animals and treatments
Young (4-5 months) and old (15-17 months) groups of C57BL/6 and p16-3MR (male and female) mice were housed in a 12-hour-light/12-hour-dark cycle in the inhalation core facility at the University of Rochester.First, both young and old groups of each strain were exposed to ETS for a total of 30 days, 5 days/week, via the Teague machine with 100 total particular matter (TPM, mg/m 3 ).Air-exposed control groups of mice were housed separately.After 6 weeks of exposure, bleomycin (BLM) (1.5U/kg) or PBS (50µl) were oropharyngeal administered.Mice were sacri ced after 14 days.During the sacri ce, pentobarbital (100mg/kg, intraperitoneal (i.p.) injection) was given, lung mechanics were measured during the sacri ce, and lung lobes were then snap-frozen for protein/RNA analysis.Lung lobes in ated with 1% low-melting agarose were used for histological analysis.Ganciclovir (GCV, 25mg/kg) was given to p16-3MR mice via intraperitoneal (i.p.) injection of every alternative day for a total of 8 times post-BLM.

Lung mechanics measurement
Lung mechanics (compliance, resistance, and elastance) were determined with the FlexiVent device (Scireq, Montreal, QC, Canada).Mice were rst anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg).After a tracheotomy, an 18-gauge cannula was introduced into the exposed trachea and attached to a computer-controlled rodent ventilator (FlexiVent; Scireq).Following the initial ventilation, compliance, resistance, and elastance were measured by a computer-generated program at 3cmH 2 O positive.The impedance of the equipment and tracheal tube was removed with appropriate calibration.These measurements were repeated 3 times for each animal (30).

RNA isolation and NanoString measurement
Snap-frozen lungs were mechanically homogenized with QIAzol reagent, then each sample was mixed with chloroform and vortexed vigorously for 10 sec.The samples were centrifuged at 20,000g for 15 min at 4°C, after that, the aqueous phase was moved to new RNase-free tubes and mixed with isopropanol for 2 hr at -20°C.The mixtures were spun down at 20,000g for 15 min at 4°C, and the supernatant was removed.RNA pellets were washed with 75% EtOH and then centrifuged at 20,000g for 15 min at 4°C.Discarded the EtOH and re-suspended RNA with RNase-free water.All of the RNA samples were cleaned up by RNeasy Plus Mini Kit following the manufacturer's protocol.RNA samples were kept at − 80°C until analysis.RNA samples were quanti ed using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies).A total of 100ng of RNA samples were used for NanoString analysis.We have used the nCounter Fibrosis Panel (Cat# XT-CSO-MFIB2-12, NanoString Technologies, Inc.) in this study.Brie y, the hybridization reaction mix was prepared by RNA samples, reporter codeset, and capture codeset.The mixtures were incubated at 65°C for 16 hrs and stored at 4°C until further pro ling.NanoString running cartridge was used to load all the sample mixtures and performed gene expression pro ling via nCounter SPRINT Pro ler (NanoString Technologies, Inc.).After pro ling, gene expression results were analyzed by nSolver 4.0 software, and reported as normalized count for data presentation and statistical analysis.
Raw RCC les were uploaded to ROSALIND for advanced analysis and generated the signaling pathway scores as directed enrichment scores with at least 10% change, and p ≤ 0.05 were considered as signi cantly altered.

H&E staining
The H&E staining has been performed as previously described (31).Brie y, lung sections (5 µm) were depara nized by xylene 3 times, 5 min each, and rehydrated with 100%, 95%, and 70% ethanol.Next, slides were quickly washed with water, and stained with hematoxylin for 1 min.Then sections were washed with water and then blued with 0.1% ammonia water for 10 s.Sections were washed with water, then soaked in Eosin for 1 min.Sections were washed with 95% ethanol for 1 min, then dehydrated with 95% and 100% ethanol, 2 times, 3 min each, and xylene, 3 times, 5 min each.Finally, slides were mounted in Permount mounting media (UN1294, Fisher Chemical) for microscopy.

Gomori's Trichrome staining
Gomori's trichrome staining has been used to quantify collagen deposition in the lungs from different experimental groups.Lung sections (5 µm) were depara nized, rehydrated, and nally rinsed with tap water.Sections were then subjected to Gomori's trichrome staining (Thermo Fisher Scienti c kit; Cat# 87020).In brief, sections were placed in Bouin's solution at 56°C for 1h, and then washed with water until the yellow color is removed.Next, sections were immersed in Working Weigert's Iron Hematoxylin Stain for 10 min and washed with water for 10 min.After, the sections were soaked in Trichrome Stain for 15 min and immediately after in acetic acid 1% for 1 minute.The sections were rinsed with water for 30 sec and dehydrated.Lastly, the slides were mounted by Permount (UN1294, Fisher Chemical) for microscopy observation.For each sample, 10 pictures (20x magni cation) have been taken by Nikon microscopy (Eclipse Ni-U) and the amount of collagen has been quanti ed by Colour Deconvolution via FIJI ImageJ.

Immunohistochemistry (IHC) staining
The staining protocol has been performed as previously described (13).Lung sections (5 µm) were depara nized and rehydrated, and then the antigens were unmasked with antigen retrieval solution (10x) (S1699; Dako).Sections were blocked with 10% normal goat serum and then incubated overnight at 4°C with primary antibodies: anti-Collagen 1α1 (1:100, NBP1-30054, Novus Biologicals); anti-Collagen 4α1 (1:100, ab227616, Abcam) and anti-LOX (1:50, ab174316, Abcam).Then, slides were soaked in 0.3% hydrogen peroxide for 15 min and washed with TBS.Goat anti-Rabbit Secondary antibody (1:1000, ab6721; Abcam) was applied to the section at RT for 1 hour.Slides were developed with DAB Quanto Chromogen and Substrate (TA-125-QHDX; Thermo Fisher Scienti c) and then counter-stained with hematoxylin.Finally, the slides were dehydrated and mounted for further analysis.These markers have been evaluated in sequential sections and for each sample, 10 pictures (20x magni cation) have been taken and compared between groups.The positive staining has been quanti ed using Colour Deconvolution via FIJI ImageJ.

Statistical analysis
All the data were analyzed through one-way ANOVA with Šidák correction using GraphPad Prism software (version 9.0, GraphPad, San Diego, CA, USA).Results were presented as mean ± SEM, and p < 0.05 was considered signi cant.

Study Approval
The animal experiments performed in this study was following the standards from the United States Animal Welfare Act, NIH, and protocol approved by the Animal Research Committee of the University of Rochester.

Results
Bleomycin-induced lung injury and impaired lung mechanics were not affected by ETS exposure Mice were exposed to ETS for 30 days and then BLM was administered oropharyngeal to evaluate how ETS exposure contributes to BLM-induced lung injury (Fig. 1A).Signi cant body weight declines were noticed after 7 days post-BLM injury in either young or old mice (Fig. 1B).ETS exposure only (ETS + PBS) did not show body weight alternation compared to the Air + PBS group, and ETS exposure did not affect the body weight reduction induced by BLM administration (ETS + BLM) in either young or old mice (Fig. 1B).H&E-stained lung sections showed similar lung injury between Air + BLM and ETS + BLM groups in both young and old mice.Limited airspace enlargement was seen in the ETS + PBS group compared to the Air + PBS group (Figure S1).There was no difference in BLM-induced lung injury between either young and old mice, or male or female mice (Figure S1).We also performed the Trichrome staining to determine dysregulated collagen deposition among different conditions.BLM (Air + BLM and ETS + BLM)-induced signi cant collagen deposition compared to Air + PBS or ETS + PBS groups, and ETS exposure did not affect the collagen deposition in the injured areas from either young or old mice (Fig. 1C).ETS exposure only (ETS + PBS) showed no altered collagen amount compared to the Air + PBS group in both young and old mice (Fig. 1C).The altered collagen deposition induced post-BLM did not show either age or sexbased differences (Figure S2).
Lung mechanics (Resistance, Compliance, and Elastance) were measured on day 14 post-BLM administration.There was no signi cant alteration in resistance, while signi cantly decreased elastance and increased compliance was found in ETS + PBS compared to the Air + PBS group (Fig. 1D).BLM (Air + BLM) signi cantly increased the resistance and elastance and decreased compliance compared to the control group (Air + PBS).The decreased trend of resistance and elastance, and increased trend of compliance were found in the ETS + BLM group compared to Air + BLM group (Fig. 1D).Similar trends of lung mechanics were observed in both young and old mice (Fig. 1D).

ETS exposure exacerbates bleomycin-induced abnormal collagen biosynthesis and modi cation
To further determine the role of ETS exposure in BLM-induced lung injury and dysregulated repair in young mice, RNA was isolated from lung homogenates prepared from young mice, and brotic genes were measured via NanoString pro ling (Fig. 2).BLM administration, either after ETS (ETS + BLM) or Air (Air + BLM) exposure, showed increased gene expression of collagens.Sub-chronic ETS exposure followed by BLM administration (ETS + BLM) augmented the upregulated transcription levels of COL1A1, COL1A2, COL4A1, COL4A2, and COL5A3.Gene expression levels of COL4A1 and COL5A3 showed a signi cant increase in ETS + BLM compared to Air + BLM, while COL1A1, COL1A2, and COL4A2 showed non-signi cant increase trends (Fig. 2).Other types of collagens, such as COL3A1, COL5A1, COL14A1, and COL16A1, were upregulated post-BLM with no signi cant difference after Air or ETS exposure.Other ECM genes, such as ELN and FN1 showed upregulated gene expression levels after BLM, while no signi cant difference was noticed between ETS + BLM and Air + BLM groups.The transcription levels of lysyl oxidase and MMPs were also tested.Signi cantly increased gene expression levels of LOX, LOXL1, LOXL2, and LOXL4 were found in Air + BLM and ETS + BLM compared to the Air + PBS group.There was no difference between Air + BLM and ETS + BLM groups (Fig. 2).A similar trend was noticed in gene expression of MMPs MMP2, MMP12, MMP13, and MMP14 was signi cantly upregulated in Air + BLM and ETS + BLM groups compared to the Air + PBS group, while no signi cant difference between Air + BLM and ETS + BLM (Figure S3).The MMPs inhibitor, TIMP1, showed increased gene expression levels in Air + BLM and ETS + BLM groups compared to either Air + PBS or ETS + PBS groups (Fig. 2).One of the well-known brogenesis regulators, TGFβ signaling, was also tested in this study.Gene expression levels of TGFB1, TGFB1I1, TGFBR1, and TGFBR2 were signi cantly increased post-BLM with either air (Air + BLM) or ETS (ETS + BLM) exposure compared to the respective PBS groups (Air + PBS and ETS + PBS) (Figure S3).
Based on the dysregulated gene list, pathway analysis was performed to determine how ETS affects the lung injury induced by BLM (Table 1).BLM administration showed signi cant upregulation in ECM degradation, collagen biosynthesis & modi cation, ECM synthesis, and PDGF signaling, all exacerbated by ETS exposure (Table 1).Besides, myo broblast regulation and Senescence-Associated Secretory Phenotype (SASP) increased slightly after BLM administration, while ETS exposure with BLM exaggeratedly activated the signaling.Since the collagen biosynthesis and modi cation were activated by BLM administration and augmented in ETS exposure with BLM, protein expressions of COL1A1, COL4A1, LOX, and MMP2 were measured.Protein abundances of COL1A1, LOX, activated LOX, and activated MMP2 were upregulated after BLM administration, and ETS exposure showed no difference in total protein levels identi ed from immunoblot (Fig. 3A).Notably, the protein abundance of COL1A1 in males showed a signi cant increase in ETS + BLM compared to Air + BLM groups, while females showed a decreased trend (Figure S4).The signi cantly increased protein level of activated MMP2 was found in females after ETS exposure and BLM compared to Air + PBS, while no difference between Air + BLM and ETS + BLM groups was found in males (Figure S4).To determine the protein abundance in the lesion area, IHC staining was performed.
The protein expressions of COL1A1, COL4A1, and LOX were increased after BLM administration following either air (Air + BLM) or ETS (ETS + BLM) exposure compared to Air + PBS or ETS + PBS respectively.The protein level of LOX in the lesion areas was signi cantly increased in the ETS + BLM group compared to Air + BLM, while COL4A1 and COL1A1 showed non-signi cant increased protein expression in the injury area of the ETS + BLM group compared to Air + BLM group (Fig. 3B).

ETS exposure augmented cellular senescence induced by bleomycin treatment
Based on the signalling pathway analysis, senescence-associated secretory phenotype (SASP) was slightly activated after BLM administration (BLM + PBS), and ETS exposure followed by BLM (ETS + BLM) showed a further increase (Table 1).More importantly, ETS exposure only (ETS + PBS) showed a higher activation level than the BLM only group (Air + BLM) (Table 1).To further understand the dysregulated gene and protein expression levels, a customized NanoString panel focused on senescence genes was used to determine the dysregulated transcript levels.Gene expression levels of complement components (C1QA, C1QB, C1QC, C3AR1, and C5AR1) were upregulated after BLM administration either with air (Air + BLM) or ETS exposure (ETS + BLM) (Fig. 4A), while only C3AR1 showed a signi cant difference between ETS + BLM and Air + BLM group.The gene levels of cyclin-dependent kinase inhibitors (CDKN1A, CDKN2B, CDKN2C, CDKN2D, CDKN1B, and CDKN1C) were tested as well.Increased CDKN1A and CDKN2B gene expression levels were found in Air + BLM and ETS + BLM groups compared to Air + PBS group, and ETS exposure with BLM (ETS + BLM) showed further increased gene expression of CDKN1A when compared to Air + BLM group without signi cant difference (Fig. 4A).Other SASP markers, such as SERPINE1, showed signi cantly increased transcript levels in ETS + BLM compared to either Air + PBS or ETS + PBS group, whereas the Air + BLM group showed an increased trend compared to Air + PBS without signi cance.The Sirtuin family genes, SIRT1 and SIRT3, were also tested in this study, and gene expression levels were found signi cantly downregulated after BLM administration either with Air (Air + BLM) or ETS (ETS + BLM) exposure (Fig. 4A).
We found a signi cant difference in the gene expression of C3AR1 between the ETS + BLM and Air + BLM groups.Then, we tested the protein abundance of C3 to con rm the activation of the complement system.A signi cant increase of C3 alpha chain (C3A) was seen after ETS exposure and BLM compared to Air + PBS or Air + BLM groups, and there is no change between Air + PBS and Air + BLM groups (Fig. 4B and Figure S5).A signi cantly increased protein level of C3A was identi ed in males, and no alternation of C3A protein abundance was identi ed in females (Figure S5).Increased protein abundances of C3 alpha chain fragment (C3B) and C3 alpha chain fragment 2 (C3C) were also signi cantly upregulated in Air + BLM and ETS + BLM groups compared to the Air + PBS group.ETS + BLM group showed further increased protein abundance of C3B and C3C compared to Air + BLM, but without signi cant difference (Fig. 4B), and similar trends were found in both males and females (Figure S5).Interestingly, the gene expression of C3 showed a higher basal level in the old mice than in the young mice.However, a lower basal transcript level was detected in C3AR1 gene expression (Figure S6).Despite the different basal levels of gene abundance of C3 and C3AR1, the dysregulated trend under ETS exposure post-BLM showed similarly between young and old mice (Figure S6).

ETS exposure does not affect bleomycin-induced brotic progression after chronologically aging
To determine the impact of ETS exposure on lung brotic progression after chronological aging, 18 months old mice were used in this study.Increased gene expression levels of collagens (COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, COL5A1, and COL5A3) were observed after BLM administration, and ETS exposure did not affect the transcript levels (Fig. 4A).Similar to the gene levels of collagens, gene expressions of lysyl oxidase (LOX, LOXL1, and LOXL2) and MMP2 showed upregulation in both Air + BLM and ETS + BLM groups compared to Air + PBS, whereas no alternation between Air + BLM and ETS + BLM groups (Fig. 5A).Other ECM genes, such as FN1, ELN, SERPINE1, and VIM, showed upregulated transcript levels after BLM administration and no alternation between Air + BLM and ETS + BLM groups (Fig. 5A).
Similar to young mice treated with BLM, gene expression levels of MMPs (MMP2, MMP3, MMP8, MMP12, MMP13, and MMP14) were upregulated after BLM administration regardless followed by Air (Air + BLM) or ETS (ETS + BLM) exposure (Figure S7).In old mice, both TIMP1 and TIMP2 gene levels were upregulated after BLM administration, and there was no signi cant difference between Air + BLM and ETS + BLM groups (Figure S7).The gene levels of TGFβ and its receptors (TGFB1, TGFB1I1, TGFBR1, and TGFBR2) showed upregulation in Air + BLM and ETS + BLM group compared to either Air + PBS or ETS + PBS groups, and there was no signi cant difference between Air + BLM and ETS + BLM groups (Figure S7).
Besides the gene expression measurement, protein abundances were also tested from lung homogenates (Fig. 5B).The increased protein level of COL1A1 was found in Air + BLM, ETS + PBS, and ETS + BLM groups compared to the Air + PBS group, while there was no signi cant difference among the groups (Fig. 5B).Protein abundance of LOX and activated LOX were increased after BLM administration, and ETS exposure did not alter the BLM-induced protein levels upregulation (Fig. 5B).Increased protein abundance of activated MMP2 was found after BLM and ETS exposure followed by BLM (ETS + BLM) augmented the activated MMP2 protein level compared to the Air + BLM group (Fig. 5B).When normalizing the gene expression levels from young and old mice, similar trends of differential gene expression after ETS exposure post-BLM for all different subtypes of collagens (Figure S8-S9).Most of the collagens gene dysregulation trends are similar between young and old mice under different conditions, while transcript levels of COL3A1, COL5A1, and COL14A1, showed further upregulation post-BLM in old mice compared to young mice.However, the basal gene expression of COL4A1 showed lower in old mice despite the dysregulation trends are similar between young and old mice (Figure S8).Unlike the gene expression of collagens, the transcript levels of lysyl oxidases and proteases showed decreased basal levels in older mice, although the dysregulation trend is comparable between young and old mice (Figure S8-S9).

Removing of p16 positive cells did not recover bleomycininduced bodyweight decline and impaired lung function
Since the ETS exposure augmented premature senescence induced by BLM, we also utilized p16-3MR mice which can remove the p16 overexpressing senescent cells with the help of ganciclovir (GCV).GCV was given to individual mice during BLM administration after ETS exposure on alternative days (Fig. 6A).After dosing GCV, there is no difference in body weight decline in either young or old mice, and there is no difference in lung mechanics (resistance, compliance, and elastance) among different experimental groups in either young or old mice (Fig. 6B-C).Administration of GCV showed non-signi cantly decreased p16 expressed cells in Air + BLM + GCV and ETS + BLM + GCV groups compared to Air + BLM and ETS + BLM groups respectively, in young mice (Fig. 6D).However, there was no difference of p16 expressed cells in Air + BLM and Air + BLM + GCV groups in old mice, and a decreased trend was found in ETS + BLM + GCV compared to ETS + BLM groups, without signi cance (Fig. 6D).

Discussion
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 brogenesis development (12,32).Recent animal studies have described that CS exacerbates the brotic progression induced by different causes such as bleomycin, lipopolysaccharides (LPS), polyhexamethylene guanidine, and in uenza A virus (IAV) (3,6,7,33,34).CS exposure followed by bleomycin administration showed signi cantly increased hydroxyproline and collagen deposition compared to bleomycin alone (3,6).CS exposure also augments acute in ammatory responses induced by LPS treatment to brotic 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 modi cation 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 in ammatory cell-ux in ltration (13).In this study, we have noticed slightly increased compliance, decreased elastance after ETS exposure without signi cance, 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 brotic 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 brosis and emphysema (CPFE).It has been con rmed clinically that patients with CPFE have intermediated value of lung mechanics parameters between patients with emphysema and brosis (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 signi cantly increased RNA expression levels in ETS+BLM compared to the Air+ETS group, and nonsigni cant 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 brogenesis (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 broblast to myo broblast differentiation with increased TGFβ and αSMA (7).Another study also showed CS exposure mediated brotic progression and exacerbation might occur through TGFβ/Smad2 signaling pathway (6).Our study has further con rmed that collagen dynamic was also one of the possible pathways contributing to brogenesis exacerbation due to ETS exposure.Although we noticed increased gene levels of TGFβ and its receptors, no signi cant difference was identi ed between Air+BLM and ETS+BLM groups.More studies are required to understand how tobacco smoke exposure contributes to brogenesis development.
Our gene expression results and pathway analysis show further activation of ECM degradation, collagen biosynthesis and modi cation, 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 brosis animal models or brotic 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 signi cant 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 brogenesis than male, and our results contribute to this statement that upregulated MMP2 during brogenesis is only found in males.ETS exposure could be one of the factors in helping develop brotic 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 bers (38).Upregulation of lysyl oxidase has been identi ed from the brotic lesion in lung brosis patients and in bleomycin-induced lung brotic injury (39).Upregulated lysyl oxidases during brogenesis drive severe crosslinking of collagen and elastin bers, preventing degradation of brous matrix and contributing to irreversible scarring (40).In this study, we rst identi ed that brogenesis 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 signi cant difference between the ETS+BLM and Air+BLM groups, a signi cantly increased protein abundance of LOX was found especially in the brotic 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 identi ed 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 brotic progress than females (36).Other than sex-dependent manner, we also studied whether aging affects ETS exposure on bleomycin-induced brogenesis.Our results show no signi cant alternation of most of the brotic markers except for active MMP2.Our results showed that ETS exposure has no signi cant impact on the exacerbation of brotic progression, while ETS exposure augments collagen synthesis and modi cation during bleomycininduced brogenesis 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 nally 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-signi cant 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 con rmed 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 in ammation 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 broblast, which are the hallmarks of brogenesis (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 identi ed that ETS exposure exacerbates the bleomycin-induced activation of C3AR.Besides, the protein abundance of C3A showed a signi cant increased and non-signi cant 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 con rmed that males were more vulnerable during brogenesis than females.However, limited SASP markers were dysregulated signi cantly, adjusting the duration post-BLM might help emphasize the exacerbation of SASP by ETS exposure.
As mentioned before, brogenesis 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 signi cant dysregulation levels than younger mice in most brotic markers.It has been reported that the bleomycin-induced brosis model serves better in the old mice, in which the chronological aging could delay broblast-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 brogenesis 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 brogenesis.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 e cient for emphysema development, but not brotic 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 brogenesis 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 brogenesis.

Declarations
Figures    Figure 5 ETS exposure does not affect on bleomycin-induced brotic progression after chronological aging.

Table 1
Dysregulated pathways after ETS exposure and bleomycin administration