Malondialdehyde-Acetaldehyde Extracellular Matrix Protein Adducts Attenuate Unfolded Protein Response During Alcohol and Smoking-Induced Pancreatitis.

To investigate the molecular players driving chronic alcohol and smoke-induced RAP and CP, we generated the Lieber-DeCarli ethanol-fed and chronic smoke-exposed RAP model ( Figure 1A ). Chronic alcohol and cigarette smoke-exposed (combination hereafter) animals recapitulated RAP pathology with marked acinar dropout, fibrosis, ECM deposition ( Figures 1B and S1A ), and high inflammatory response ( Figures S1B and S1C ). Quantitative-RT-PCR analysis showed a significant increase in the expression of ECM markers such as SPARC, FAP, and Col1A2 in the combination group ( Figure 1C ). TUNEL staining further validated acinar cell death in the combination group, especially adjacent to the fibrotic areas ( Figures 1D , S1D , and S1E ). Next, we performed an unbiased tandem mass tag (TMT)-based quantitative proteomic analysis on the mouse pancreata from different treatment groups. The proteomic PCA analysis demonstrated a distinctive proteomic signature with the most frequent and abundant proteomic alterations occurring in the combination group (56.5%), followed by smoke-exposed (21%) and alcohol-fed (17%) exposed mice ( Figure 1E and S1F ). The volcano plots analysis using log10 (p-value) vs. log2 (fold change) demonstrated a unique proteomic signature in combination ( Figure 1I ) compared to cerulein ( Figure 1F ), alcohol ( Figure 1G ), or smoke ( Figure 1H ) exposed animals. Top differentially altered proteins from the proteome analysis were also evaluated at the transcript levels in different treatment groups ( Figure 2A ). The Sord, Arf1, Reg3b, and Snx3 showed a significant modulation in their transcript levels after cerulein treatment. The difference in the transcripts and proteomic analysis of proteins like Sord and Arf1 in cerulein-treated animals might be due to their protein stability during pancreatic insult. The expression of Sord, Arf1, and Snx3 further changed in the combination group compared to cerulein alone. IHC and western blot analyses showed high Arf1, and Sord expression in the acinar cells and lymphovascular area, respectively, in various treatment groups, which validated the proteomics analysis ( Figures 2B - 2D and S2 ). Next, the gene ontology (GO) analysis on differentially expressed proteins suggested that the oxidation-reduction process, protein localization, and toxin metabolic processes were among the top pathways modulated in the combination group ( Figure 2E ). Based on this, we evaluated the expression of oxidative stress markers, 4HNA, MDA, and MAA adducts in pancreatic tissues. The IF analysis showed the presence of 4HNE and MAA adducts in the pancreata of cerulein and combination groups ( Figures 2F and 3A ). The unstable aldehyde and MDA adducts generate the hybrid MAA adducts. 30 Interestingly, the MAA adducts were predominantly localized in the pancreatic interstitial sites explicitly in the combination group, indicating the presence of MAA adducts on ECM ( Figure 2F ). Collectively, this data suggests that exposure to alcohol and smoking significantly alters pancreatic proteome and generates toxic aldehyde adducts, including 4HNE and MAA. The presence of MAA adducts in interstitial regions, coinciding with fibrotic areas intrigued us to examine their presence on ECM proteins. Interestingly, there was a significant colocalization of MAA adducts with Col1A1 in the combination group, which was predominantly present in the areas of substantial acinar cell loss ( Figures 3A , 3B , and S3A ). Next, we queried proteomics data for the gene set enrichment analysis (GSEA) ( Figure 3C ) and MsigDB analysis for significantly altered pathways ( Figure 3D ). We observed a significantly reduced UPR in the combination group compared to cerulein alone. We did not observe a similar alteration in the UPR pathway in cerulein vs. alcohol or smoke-exposed groups ( Figure S3B ). An increase in the expression of CHOP and cytoskeleton-linking membrane protein 63 31 (CLIMP63, marker of ER expansion upon stress) in pancreatic tissues further suggested ER stress and apoptosis due to attenuated UPR in the combination group ( Figures 3E and 3F ). Additionally, increased expression of inositol-requiring protein1 alpha (IRE1α) and CHOP in pancreatic tissues in the cerulein and combination group further validated the presence of increased ER stress ( Figure 3G ). The ER stress and UPR are implicated in pancreatitis. 17 , 32 However, no study so far has reported the role of adducted ECM in acinar cell death and predisposition to subsequent insults. Further, the presence of MAA adducted ECM close to acinar cell death led us to investigate the contribution of adducted ECM to ER stress and loss of pancreatic parenchyma. We isolated the primary acini, cultured them on collagen-coated and MAA-adducted collagen-coated plates, and treated them with ethanol and CSE to investigate the impact of MAA adducts on acinar cell viability ( Figures 3H and S3C ). The significant decrease in acinar cell viability ( Figures 3I and S3D ) and increased PARP cleavage ( Figure 3J ) in the combination group in the presence of MAA-adducts suggested that MAA-adducted ECM alone can significantly predispose acinar cells to ethanol and smoke exposure-mediated cell death. As aldehyde adducts are detected by scavenger receptors (SRs), we analyzed their expression in the pancreas using the genotype-tissues expression portal (GTEx, https://gtexportal.org , Broad Institute, MIT). The SRs, SCARB2, SR-A1, and STAB1, are expressed in the normal pancreas ( Figure S3E ). To identify the underlying mechanism of the MAA-adducted ECM in acinar cell biology, we investigated the modulation in three arms of UPR. There was a significant decrease in the XBP1 transcript, a primary downstream regulator of the IRE1α UPR sensor, in the pancreatic tissues in the combination group ( Figure 4A ). XBP1 splicing (sXBP1) is a central event in adaptive UPR, and a reduction in its expression or splicing results in severe pancreatic damage. 16 , 32 Remarkably, acinar cells cultured on MAA-adducted collagen showed a drastic reduction in XBP1 expression with no evident splicing in the presence of ethanol and CSE ( Figure 4B ), suggesting attenuation of UPR by MAA-adducts. Further, these samples showed increased phospho-PERK, phospho-eIF2α, and CHOP levels, indicating ER stress-induced cytotoxicity ( Figures 4C and 4D ). Next, we analyzed the expression of known regulators of XBP-1, like ATF6 and HNF4A. There was a significant decrease in the expression of ATF6 and HNF4A on the MAA-adducted collagen, which might be responsible for the reduced XBP-1 expression in the combination group ( Figure 4E ). Further analysis of sXBP1 target genes required for ER protein folding and degradation, EDEM1 (endoplasmic reticulum degradation enhancing alpha-mannosidase like protein 1) and ERDj4 (endoplasmic reticulum DnaJ 4) demonstrated a significant decrease in the presence of MAA-adducts compared to normal collagen ( Figure 4F ). IF analysis on the pancreatic tissue ( Figure 4G ) and immunoblotting analysis on primary acinar cells cultured on MAA-adducted collagen further indicated decreased expression of EDEMI, and ERp57, respectively, which may result in the accumulation of unfolded proteins and ER stress in the combination group ( Figure 4H ). Collectively, MAA-ECM adducts attenuate XBP1-mediated UPR response and activate PERK pathway and CHOP expression resulting in acinar cell death in the presence of alcohol and smoke exposure ( Figure 4I ). Next, we investigated the effect of alcohol and smoke cessation on pancreatic recovery and the fate of MAA adducts using the recovery/cessation model ( Figure 5A ). We found a significant decrease in the expression of ECM proteins in cerulein-treated mice pancreata upon recovery ( Figure 5B and S4A ). Similarly, the levels of αSMA decreased in cerulein-treated animals after 2-weeks of recovery ( Figures 5C and 5D ). However, levels of α-SMA remained high in the pancreatic tissues in the continuous (continued alcohol and smoke exposure for additional 2-weeks) group, and a non-significant decrease in the cessation (alcohol and smoke abstinence for 2-weeks) groups ( Figures 5C and 5D ). Furthermore, the expression of other stromal markers and ECM proteins such as FAP and Col1A1 remained high in the pancreatic tissues from continuous and cessation groups ( Figure 5E ). Along the same lines, we investigated the presence of MAA adducts on ECM in continuous and cessation groups. Surprisingly, the MAA adducts on ECM protein remained significantly high in both the continuous and cessation groups ( Figures 5F and 5G ), suggesting a prolonged predisposition to pancreatic insults in the alcohol and smoke-exposed animals. Considering the presence of MAA-ECM adducts, we next analyzed the expression of ER stress markers in the pancreata of alcohol, smoke, combination, and continuous and cessation groups. Unlike alcohol and smoking ( Figure S4B ), the pancreatic tissues of continuous and cessation groups showed a significant presence of stable MAA-Col1A1 adducts and significantly high expression of CHOP, and CLIMP63, indicating persistent ER stress ( Figures 5H and 5I ). The GSEA from the downregulated proteins in proteomic analysis in the combination group suggested significant involvement of cell cycle regulation ( Figure 3D and S5A ). Further analysis demonstrated that the acinar cells cultured on MAA-adducted collagen showed a reduction in the expression of various CDKs (CDK7, CDK5, and CDK4) and cell cycle-associated proteins (ROCK2 and NEK7) in the presence of ethanol and CSE ( Figures 6A , and 6B ). The prolonged Ki67 nuclear positivity, even after 2-weeks, in the continued and cessation pancreatic tissues indicated an ongoing but slower recovery of acinar cells compared to complete recovery in the cerulein-treated pancreata ( Figures 6C , 6D , and S6 ). The β-catenin localizes to the pancreatic acini plasma membrane and translocate to the cytoplasm during regeneration. 33 , 34 There was increased plasma membrane localization of β-catenin in acinar cells in cerulein and combination groups. However, unlike cerulein-treated mice with predominant cytoplasmic β-catenin expression, the combination-exposed pancreata showed plasma membrane localization suggesting impaired cell cycle and ADM ( Figure 6E ). The TFs, Mist1 and Hes1, master regulators of pancreatic regeneration, were significantly downregulated in acinar cells cultured on MAA-adducted collagen in the presence of ethanol and CSE ( Figure 6F ). In addition, fibroblast growth factor 7 (Fgf7), a marker of acinar cell regeneration, showed a 50-fold increase in the acinar cells cultured on the collagen-coated compared to no change on MAA-adducted collagen in the presence of ethanol and CSE ( Figure 6G ). These findings suggest a significant impairment in pancreatic repair and regeneration by the stable MAA adducts in the combination group. Collectively, alcohol and smoking lead to the generation of stable ECM-MAA adducts, which decrease XBP1 expression leading to unresolved ER stress and a decrease in the expression of proteins involved in the recovery and regeneration of damaged pancreatic parenchyma ( Figure 6H ). Histopathological examination of pancreata from CP patients showed increased fibrosis and acinar cell loss during combined alcohol and smoke exposure ( Figure 7A ). The IF analysis on resected pancreatic tissues from the CP patient with a history of alcohol and smoking demonstrated the presence of stable MAA adducts on ECM protein, Col2A1 ( Figures 7B , and 7C ). In line with our findings in murine models, TUNEL staining on CP tissues showed increased acinar cell death along the ECM tracts in the patients with alcohol consumption and smoking ( Figure 7D ), suggesting the contribution of MAA-adducted ECM in the replacement of pancreatic parenchyma along the fibrotic area and worsening of the CP pathology. Next, patients with an alcohol and smoking combination showed higher expression of ER stress marker, CHOP ( Figures 7E and 7F ) and CLIMP63 ( Figures 7E and 7G ) in the resected pancreatic tissues, unlike alcohol or smoke exposure ( Figure S5B ), corroborating our findings in murine models demonstrating impaired UPR due to stable MAA-ECM adducts in RAP.

The RAP murine model, along with alcohol and smoke exposure, was generated using 8-10-week old healthy C57BL/6 mice fed on isocaloric Lieber-Decarli control or ethanol diet (control diet: protein 151 kcal/L, fat 359 kcal/L, carbohydrate 490 kcal/L; ethanol diet: protein 151 kcal/L, fat 359 kcal/L, carbohydrate 135 kcal/L, and ethanol 355 kcal/L). 23 The animals were smoke-exposed using a smoking chamber (Teague TE-10C, Davis, California, United States, at 150mg TSP/m 3 ) and standard cigarettes from University of Kentucky Reference Cigarette, 3R4F, Lexington, Kentucky, United States. 24 The pancreatic injury was induced by intraperitoneal cerulein (75ug per kg body weight) injections (8 injections per day, 3-times a week for 4-weeks). Saline was used as a control. After 4-weeks, animals were euthanized, and pancreatic tissues were isolated for further analysis. A pathologist assessed the extent of pancreatic damage and disease pathology. Recovery model: To demonstrate the effect of alcohol and smoking cessation on pancreatic regeneration and recovery, after 4-weeks of exposure, animals were either continued (continuous group) or abstained from (cessation group) smoking or ethanol feeding for additional 2-weeks without cerulein treatment. After 2-weeks, animals were euthanized, and pancreatic tissues were used for further analysis. All animal protocols were approved (#17-057-08FC) by the Institutional Animal Care and Use Committees of UNMC. The proteomic analysis was performed as described previously. 25 Peptide Preparation: Frozen tissues were ground and lysed with RIPA lysis buffer. Protein samples were trypsin digested (2.5 ug Trypsin) overnight at 37°C. TMT Tagging: The peptides were incubated with anhydrous acetonitrile dissolved TMT reagents (ThermoFisher Scientific, Rockford IL, TMT10 Plex Ref 90110 Lot UC279034) for 1 hour at room temperature. The reaction was quenched and fractionated with Pierce High pH Reversed-Phase Peptide Fractionation Kit (ThermoFisher Scientific, Cat: 84868). LC-MS/MS and Protein Identification: The peptides were analyzed by LC-MS/MS on a Dionex Nano Ultimate 3000 RSLCnano system (ThermoFisher Scientific) coupled to an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific). Database searching: The mass spectra were extracted using Proteome Discoverer (2.1.1.21). See supplementary methods and key resource table for details. Pancreatic acinar cell culture was performed as described previously. 26 Briefly, the pancreata were minced finely and subjected to collagenase P digestion for 15 minutes at 37°C. Enzymatic activity was neutralized by Hank’s balanced salt solution (HBSS) containing 5% serum, and the suspension was passed through 100 μm nylon filters and loaded on 30% HBSS to obtain primary acinar cells. Acinar cells were cultured in Waymouth's culture medium containing 5% FBS, dexamethasone (1μg/ml), soybean trypsin inhibitor (100μg/ml), penicillin, and streptomycin overnight. The next day, cells were treated with alcohol (50mmol/L), cigarette smoke extract (CSE, 40μg/ml, 3R4F; University of Kentucky), or in combination for the indicated time. RNA isolation from primary acinar cells and pancreatic tissues, cDNA synthesis, and qRT-PCR analysis were performed by a previously published protocol. 27 Briefly, 1.0μg of total RNA was reverse transcribed using iScript cDNA synthesis kit as per the manufacturer's instruction. The qRT-PCR was performed using CFX connect real-time system (Bio-Rad). The data were normalized with actin, and fold change was calculated as 2^ΔΔCT. Immunohistochemistry (IHC) and immunofluorescence (IF) on mice and human pancreatic tissues were performed as described previously. 28 Tissue sections baked overnight at 58°C were deparaffinized by submerging twice in xylene, followed by sequential rehydration in graded ethanol. Sections were then treated with 3% hydrogen peroxide in 50 percent methanol to quench endogenous peroxidase. Antigens were retrieved using 0.05M citrate buffer (pH 6.0), and tissue sections were blocked using 2.5% horse serum (ImmPRESS Universal antibody kit; Vector Laboratories, Burlingame, CA) for 1h by incubation with the desired anti-4HNE, -MAA 29 and other primary antibodies overnight at 4 0 C. The following day, after washing the slides 3-times with 1 X phosphate buffer saline (PBS), tissues were incubated with a secondary antibody cocktail (anti-rabbit/anti-mouse secondary antibodies; ImmPRESS Universal antibody kit; Vector Laboratories) for 1h. Slides were then washed 3-times with 1 X PBS developed using chromogen, 3, 3’-diaminobenzidine (DAB substrate kit; Vector Laboratories). Nuclei were counterstained with hematoxylin, and slides were further processed for pathology evaluation by dehydration and mounting with permount (Vector Laboratories). For IF analysis, instead of using HRP-labeled antibodies, tissues were treated with appropriate fluorophore-labeled secondary antibodies for Ih in the dark. Nuclei were counterstained withVectashield containing DAPI nuclear stain. MAA-collagen adducts were prepared by incubating the collagen-coated plates with 2mM MDA in the presence of 1.0mM acetaldehyde in 0.1M phosphate buffer, pH 7.2, containing 2mM diethylenetriaminepentaacetic acid and 2mM phytic acid at 37°C for 3-days, followed by extensive washing of the plate before plating cells. Staining was performed following VitroVivo Pico-Sirius red staining kit (Cat No. VB-3017). Xylene deparaffinized tissue slides were rehydrated using graded ethanol (100%, 95%, 70%) and rinsed with distilled water three times for 2 minutes each. First, the nuclei were stained by Wiegert's hematoxylin, and slides were washed with running water followed by Pico-Sirius red solution for 1h. slides were then washed with acidified water followed by distilled water and dehydrated sequentially with 95% and 100% ethanol and three xylene changes. Dried slides were mounted using permount and analyzed for pathology. Primary acinar cells were grown either on collagen or MAA-adducted collagen plates were treated with ethanol (50mmol/L), CSE (40μg/ml) or combination (ethanol and CSE) for indicated time and lysed by 1X RIPA lysis buffer containing 50mM Tris pH 8.0, 150mM NaCl, 0.5% Triton X-100, 2mM Na 3 VO4, 2mM NaF, 0.1mM PMSF and 1X protease inhibitor cocktail. Lysates were precleared by high-speed centrifugation at 18000g for 30 minutes at 4 0 C, quantified, and loaded on SDS PAGE. For Immunoblotting, 40μg of cell lysate was separated by SDS-PAGE and transferred to the PVDF membrane to analyze the protein expression. Histology scores were calculated by the intensity and percentage area covered. Densitometry on immunoblots was done using ImageJ1.52s software and normalized with respective actin. Student's t-test was used to quantify the results from three independent experiments. For more than three samples, ANOVA was performed to calculate the significance. Pairwise comparisons were made, and significance was calculated after the Bonferroni correction. Bioinformatics analysis was performed by considering altered proteins with FDR<0.05 for pathway or gene ontology analysis. P values less than 0.05 ( P<0.05 ) were considered statistically significant. Quantification of confocal data from patient and mice tissues was done by ZEN 2.3 SP1 black edition and ImageJ1.52s software.

The lack of transcriptomic or proteomic analysis for the alterations caused by smoking and alcohol, two epidemiologically established risk factors in pancreatitis, limits identifying the major drivers or pathways associated with its pathology. 35 Further, the effect of alcohol and smoke cessation on recovery and regeneration of the pancreas is poorly understood. Therefore, we generated a RAP murine model with chronic alcohol-feeding and smoke exposure along with 2-weeks of cessation or continuous exposure to alcohol and cigarette smoke. Our proteomic analysis showed that the alcohol, smoking, and the combination group modulate the expression of unique proteins during pancreatic insult, but, interestingly, these changes converge on the common pathways involved in stress management. Our bioinformatics analysis of the significantly altered proteins in various treatment groups also demonstrated the increase in toxic metabolites and decreased expression of UPR mediators in the combination group. Acinar cells metabolize alcohol, leading to free aldehyde adducts. 36 These aldehydes form stable DNA, lipid, and protein adducts that modulate their functional attributes. 37 The acetaldehyde pool generated by alcohol metabolism also affects duodenal cholecystokinin secretion 38 , which may add to the severity of pancreatitis. Studies have demonstrated that intracellular adducts in the acinar cells are a measure of oxidative stress. 39 Our study, for the first time, demonstrated the presence of stable MAA adducts on the ECM proteins in the pancreas during RAP in the combination group and CP patients with a history of alcohol and smoking. Our studies have further revealed the significant role of MAA-ECM adducts in reducing the acinar cell viability by decreasing the expression of XBP1 in the presence of alcohol and smoking, thus providing the mechanistic insights into the acinar cell death along the ECM tract in murine models and patient samples. A previous study has also demonstrated that ethanol feeding in Xbp1 deficient mice upregulates the proapoptotic signals due to defects in protein disulfide isomerase-mediated protein folding in ER 32 . Alcohol has also been shown to significantly accelerate the development of CP in the murine model carrying CP patient associated mutation, N256K in the carboxypeptidase A1(CPA1), due to the misfolding of mutant protein in the ER. 40 The aldehyde adducts, including MAA adducts, act through SRs. 41 The normal pancreas expresses multiple SRs like SCARB2, SR-A1, and STAB1. Future studies will be directed to delineate the contribution of these unexplored receptors and their signaling in acinar cell biology and pancreatitis. It will be interesting to evaluate the role of SRs in trypsinogen activation, ER stress, and autophagy during pancreatitis. In addition, the inhibition of SRs signaling through p38MAPK, JNK, Fak/Rac, and NF-kB pathways 42 may significantly reduce the severity of RAP and slow its progression to CP as the MAA adducted ECM also affects the acinar cell recovery and regeneration. Studies in other pathologies have demonstrated the role of MAA adducts in the inflammatory response and detected anti-MAA antibodies in serum. 43 , 44 Likewise, the stable MAA-ECM adducts in pancreatitis may facilitate chronic inflammation leading to a fibroinflammatory response by facilitating the crosstalk between different cellular compartments, including macrophages, PSCs, and acinar cells. Macrophages express SRs, and MAA-adducted proteins stimulate the secretion of proinflammatory cytokines like TNF-α, and IL-6. 30 TNF-α promotes the proliferation of PSC 45 ; therefore, setting up a feed-forward loop for the development of fibrosis and deposition of ECM proteins. The crosstalk between PSCs and alternatively activated macrophages through IL4/IL13 has also been shown to increase fibrosis during CP. 18 In addition, aryl hydrocarbon receptor agonist-mediated activation of T-cells secrete IL22, which is involved in increased fibrosis and ECM deposition 19 , suggesting that MAA-adducted ECM can further facilitate immune cell and PSC crosstalk leading to fibrotic response during RAP. Although pancreatic and hepatic stellate cells (HSCs) have the same morphological and functional characteristics 46 , the exocrine part during recurrent pancreatic damage can create a unique milieu of proteolytic enzymes and cytokines that modulate the PSC’s biology very differently from HSCs. Similarly, the endocrine compartment is also impacted by reactive aldehyde species. The AA adducts detected on insulin impair its biological activity and contribute to endocrine insufficiency and β-cell dysfunction. 47 Therefore, the unique microenvironment of the pancreas may respond differently to adducted ECM and confer distinct and underappreciated pathobiology. Our study has also demonstrated the presence of stable MAA-ECM adducts even in the alcohol and smoking cessation group, providing a plausible mechanistic explanation for the slower recovery in the patients with a history of alcohol and smoking. In the continuous group, the extent of MAA adducts remains significantly higher even in the absence of the pancreatic insult (no cerulein for 2-weeks), which may lead to chronic inflammation, progressive fibrosis, and loss of pancreatic parenchyma due to MAA-ECM adduct mediated acinar cell death. Therefore, targeting MAA-ECM-mediated cellular crosstalk may help to reduce the severity and progression of RAP. Here, we propose SRs as novel targets to counter the acinar cell loss and fibrosis during RAP and CP. A recent molecular docking study has identified novel inhibitors for SR-A1, expressed in pancreas and macrophages, including Rhein, Tannic acid, and Danthron, 48 which will be evaluated in our future studies. The present study also has diagnostic implications, as these adducts are also present on the secreted molecules; future efforts will be directed to correlate the serum levels of adducted molecules and antibodies against MAA adducts with the imaging modalities to develop the diagnostic markers to predict the severity of RAP and CP.

Acute pancreatitis (AP) is a necro-inflammatory disorder of the pancreas, 1 which accounts for most hospital admissions among all gastrointestinal disorders in the United States. 2 Alcohol misuse, smoking, and biliary obstruction are the epidemiologically established risk factors for AP. Alcohol contributes to 25%–35% of AP cases. 3 The continuous alcohol misuse in 34% of patients results in recurrent acute pancreatitis (RAP) compared to patients who abstained from alcohol, and 26% of the patients with recurrent episodes progress to chronic pancreatitis (CP). 3 Alcohol misuse is often associated with smoking, which is a long-established independent risk factor for AP and CP. 4 However, the combination of heavy drinking and smoking can further increase the risk of AP up to four times compared to non-smokers. 3 Further, the onset of pancreatitis was observed 5 years earlier in smokers with alcohol pancreatitis than in nonsmokers, suggesting a strong synergism between smoking and alcohol consumption. 5 , 6 Alcohol is metabolized by aldehyde dehydrogenase (ADH) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1), expressed by pancreatic acinar cells. 7 - 9 Alcohol metabolism creates oxidative stress in pancreatic acinar cells, increasing their susceptibility to subsequent insults. 10 , 11 In addition, alcohol increases the number of autophagosomes; however, impaired lysosomal biogenesis leads to insufficient autophagy and promotes alcohol-induced pancreatitis. 12 The tobacco-specific constituents like 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) bind to nicotinic acetylcholine receptors (nAChRs) expressed on the acinar cells and induce zymogen activation leading to acinar cells injury 13 , which in combination with alcohol increases the severity of the pancreatic insult. The loss of pancreatic parenchyma mechanistically involves alcohol-induced endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Pancreatic acinar cells have significantly high biosynthetic activity and are vulnerable to accumulating unfolded proteins and ER stress. 14 Among three important pathways of UPR, alcohol activates the most conserved inositol requiring enzyme 1 (IRE-1) arm, which splices XBP1 in the cytoplasm. The spliced XBP1 translocates to the nucleus, acts as a transcription factor (TF), and increases the expression of chaperone proteins as an adaptive response. 15 The prolonged UPR and dysfunctional XBP1 response activate protein kinase RNA-like ER kinases (PERK), which leads to C/EBP homologous protein (CHOP) upregulation and acinar cell death. 16 , 17 Smoking has been shown to decrease the expression of XBP1 through an unknown mechanism, leading to an additive effect to alcohol misuse and acinar cell death. The fibroinflammatory response in CP involves the activation of the pancreatic stellate cells (PSCs) predominantly by the secreted cytokines from the infiltrating immune cells. Studies have shown the contribution of alternatively activated macrophages and T-cells in pancreatic fibrosis. 18 , 19 Concurrently, cigarette smoke contains aryl hydrocarbon ligands such as nicotine and NNK, which activate aryl hydrocarbon receptor signaling in pancreatic acinar and stellate cells resulting in increased oxidative stress lymphocyte activation and extracellular matrix (ECM) deposition. 19 , 20 The ECM deposition is associated with the loss of pancreatic parenchyma and pancreatic insufficiency in CP patients. 21 , 22 Nevertheless, the mechanisms and players involved in the RAP, progressive loss of pancreatic parenchyma, and contribution of alcohol and smoking cessation to pancreatic regeneration remain poorly understood. To address these gaps, we generated a murine model of RAP with chronic alcohol and smoke exposure and performed an unbiased quantitative proteomic analysis on the murine pancreata. Our analyses identified novel and previously known molecules associated with the severity of pancreatitis. Interestingly, subsequent analysis led to the identification of stable malondialdehyde-acetaldehyde (MAA) adducts on the deposited ECM proteins in the pancreata of the RAP murine model and CP patients. No study has so far examined the role of MAA-ECM adducts in acinar cell biology and fibroinflammatory response during RAP or CP.