Combined repetitive inhalant endotoxin and collagen-induced arthritis drive inflammatory lung disease and arthritis severity in a testosterone-dependent manner.

Respiratory-related diseases are a leading cause of death in rheumatoid arthritis (RA) and are disproportionately higher in men, which may be attributable to environmental risk factors. Animal studies have demonstrated potentiated autoimmunity, arthritis, and profibrotic/inflammatory lung disease with a combination of airborne exposures and collagen-induced arthritis (CIA). This study aimed to determine whether hormone-dependent differences explained these observations. Arthritis-prone male intact and castrated DBA/1J mice received intranasal inhalation of lipopolysaccharide (LPS) daily for 5 wk and CIA induction. Arthritis scores and serum pentraxin-2 levels were increased in castrated versus intact mice. In contrast, airway cell influx, lung tissue infiltrates, and lung levels of proinflammatory and profibrotic markers (C5a, IL-33, and matrix metalloproteinases) were reduced in castrated versus intact mice. CIA + LPS-induced lung histopathology changes and the expression of lung autoantigens including malondialdehyde acetaldehyde (MAA)- and citrulline (CIT)-modified proteins and vimentin were reduced in castrated animals. There were no differences in serum anti-MAA or anti-CIT protein antibody (ACPA) levels or serum pentraxin levels between groups. Testosterone replacement led to a reversal of several lung inflammatory/profibrotic endpoints noted earlier in castrated male CIA + LPS-treated mice with testosterone supplementation promoting neutrophil influx, MAA expression, and TNF-α, IL-6, and MMP-9. These findings imply that testosterone contributes to lung and arthritis inflammatory responses following CIA + LPS coexposure, but not to systemic autoantibody responses. The CIA + LPS model provides a paradigm for investigations focused on the mechanistic underpinnings for epidemiologic and phenotypic sex differences in RA-related lung disease.NEW & NOTEWORTHY Our study shows that testosterone acts as a key immunomodulatory hormone contributing to critical features of rheumatoid arthritis (RA)-associated lung disease in the setting of airborne endotoxin (lipopolysaccharide; LPS) exposures and concomitant arthritis induction in mice. The exaggerated airway inflammation observed following combined exposures in male mice was accompanied by increases in profibrotic mediators, netosis, and increased expression of lung autoantigens, all relevant to the pathogenesis of lung disease in arthritis.

Targeting transitioning lung monocytes/macrophages as treatment strategies in lung disease related to environmental exposures.

BACKGROUND: Environmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear. METHODS: CCR2 RFP+ mice were intratracheally instilled with high concentration ODE (25%), LPS (10 µg), or gram-positive peptidoglycan (PGN, 100 µg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey'smultiple comparison) and nonparametric statistical (Kruskal-Wallis, Dunn's multiple comparison) tests were used following Shapiro-Wilk testing for normality. RESULTS: Intratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2+ CD11cintCD11bhi monocytes/macrophages and both CCR2+ and CCR2- CD11c-CD11bhi monocytes at 48 h. There were also increases in CCR2+ CD4+ and CD8+ T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11cintCD11bhi cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11cintCD11bhi monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome. CONCLUSION: Highly concentrated environmental/occupational exposures induced the recruitment of CCR2+ and CCR2- transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.

Serum alarmins and the risk of incident interstitial lung disease in rheumatoid arthritis.

OBJECTIVES: To quantify associations of serum alarmins with risk of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). METHODS: Using serum collected at enrolment, three alarmins (interleukin [IL]-33, thymic stromal lymphopoietin [TSLP], and IL-25) were measured in a multicentre prospective RA cohort. ILD was classified using systematic medical record review. Cross-sectional associations of log-transformed (IL-33, TSLP) or quartile (IL-25) values with RA-ILD at enrolment (prevalent RA-ILD) were examined using logistic regression, while associations with incident RA-ILD developing after enrolment were examined using Cox proportional hazards. Covariates in multivariate models included age, sex, race, smoking status, RA disease activity score, and anti-cyclic citrullinated antibody positivity. RESULTS: Of 2,835 study participants, 115 participants (4.1%) had prevalent RA-ILD at baseline and an additional 146 (5.1%) developed incident ILD. There were no associations between serum alarmin concentrations and prevalent ILD in unadjusted or adjusted logistic regression models. In contrast, there was a significant inverse association between IL-33 concentration and the risk of developing incident RA-ILD in unadjusted (HR 0.73 per log-fold increase; 95% CI 0.57-0.95; p= 0.018) and adjusted (HR 0.77; 95% CI 0.59-1.00, p= 0.047) models. No significant associations of TSLP or IL-25 with incident ILD were observed. CONCLUSIONS: In this study, we observed a significant inverse association between serum IL-33 concentration and the risk of developing incident RA-ILD, but no associations with prevalent ILD. Additional investigation is required to better understand the mechanisms driving this relationship and how serum alarmin IL-33 assessment might contribute to clinical risk stratification in patients with RA.

Elevations in adipocytokines and mortality in rheumatoid arthritis.

OBJECTIVES: This study assessed whether circulating levels of adiponectin and leptin are associated with higher mortality in patients with RA. METHODS: Participants were adults from the Veterans Affairs RA Registry. Adipokines and inflammatory cytokines were measured as part of a multi-analyte panel on banked serum at enrolment. Dates and causes of death were derived from the Corporate Data Warehouse and the National Death Index. Covariates were derived from medical record, biorepository and registry databases. Multivariable Cox proportional hazard models evaluated associations between biomarkers and all-cause and cause-specific mortality. RESULTS: A total of 2583 participants were included. Higher adiponectin levels were associated with older age, male sex, white race, lower BMI, autoantibody seropositivity, radiographic damage, longer disease duration, prednisone use and osteoporosis. Higher adiponectin concentrations were also associated with higher levels of inflammatory cytokines but not higher disease activity at enrolment. Leptin was primarily associated with greater BMI and comorbidity. The highest quartile of adiponectin (vs lowest quartile) was associated with higher all-cause mortality [hazard ratio (HR): 1.46 (95% CI: 1.11, 1.93), P = 0.009] and higher cardiovascular mortality [HR: 1.85 (95% CI: 1.24, 2.75), P = 0.003], after accounting for covariates. Higher leptin levels were also associated with greater all-cause and cancer mortality. CONCLUSIONS: Elevations in adipokines are associated with age, BMI, comorbidity and severe disease features in RA and independently predict early death. Associations between adiponectin and inflammatory cytokines support the hypothesis that chronic subclinical inflammation promotes metabolic changes that drive elevations in adipokines and yield adverse health outcomes.

Increased susceptibility to organic dust exposure-induced inflammatory lung disease with enhanced rheumatoid arthritis-associated autoantigen expression in HLA-DR4 transgenic mice.

Immunogenetic as well as environmental and occupational exposures have been linked to the development of rheumatoid arthritis (RA), RA-associated lung disease, and other primary lung disorders. Importantly, various inhalants can trigger post-translational protein modifications, resulting in lung autoantigen expression capable of stimulating pro-inflammatory and/or pro-fibrotic immune responses. To further elucidate gene-environment interactions contributing to pathologic lung inflammation, we exploited an established model of organic dust extract (ODE) exposure with and without collagen-induced arthritis (CIA) in C57BL/6 wild type (WT) versus HLA-DR4 transgenic mice. ODE-induced airway infiltration driven by neutrophils was significantly increased in DR4 versus WT mice, with corresponding increases in bronchoalveolar lavage fluid (BALF) levels of TNF-⍺, IL-6, and IL-33. Lung histopathology demonstrated increased number of ectopic lymphoid aggregates comprised of T and B cells following ODE exposure in DR4 mice. ODE also induced citrullination, malondialdehyde acetaldehyde (MAA) modification, and vimentin expression that co-localized with MAA and was enhanced in DR4 mice. Serum and BALF anti-MAA antibodies were strikingly increased in ODE-treated DR4 mice. Coupling ODE exposure with Type II collagen immunization (CIA) resulted in similarly augmented pro-inflammatory lung profiles in DR4 mice (relative to WT mice) that was accompanied by a profound increase in infiltrating lung CD4+ and CD8+ T cells as well as CD19+CD11b+ autoimmune B cells. Neither modeling strategy induced significant arthritis. These findings support a model in which environmental insults trigger enhanced post-translational protein modification and lung inflammation sharing immunopathological features with RA-associated lung disease in the selected immunogenetic background of HLA-DR4 mice.

Disease activity, cytokines, chemokines and the risk of incident diabetes in rheumatoid arthritis.

PURPOSE: Rheumatoid arthritis (RA) is associated with a higher risk of diabetes mellitus (DM). Our aim was to determine associations between inflammatory disease activity (including evaluation of specific cytokines and chemokines) and incident DM. METHODS: Participants were adults with physician-confirmed RA from Veteran's Affairs Rheumatoid Arthritis Registry. Disease activity and clinical assessments occur longitudinally as part of clinical care. Thirty cytokines and chemokines were measured in banked serum obtained at the time of enrolment. Cytokine/chemokine values were log-adjusted and standardised (per SD). Incident DM was defined based on validated algorithms using diagnostic codes and medications. Multivariable Cox proportional hazard models evaluated associations between clinical factors and incident DM. Independent associations between cytokines/chemokines and incident DM were assessed adjusting for age, sex, race, smoking, body mass index (BMI) and medication use at baseline. RESULTS: Among 1866 patients with RA without prevalent DM at enrolment, there were 130 incident cases over 9223 person-years of follow-up. High Disease Activity Score (DAS28)-C reactive protein (CRP), obese BMI, older age and male sex were associated with greater risk for incident DM while current smoking and methotrexate use were protective. Patients using methotrexate were at lower risk. Several cytokines/chemokines evaluated were independently associated (per 1 SD) with DM incidence including interleukin(IL)-1, IL-6 and select macrophage-derived cytokines/chemokines (HR range 1.11-1.26). These associations were independent of the DAS28-CRP. CONCLUSIONS: Higher disease activity and elevated levels of cytokines/chemokines are associated with a higher risk of incident DM in patients with RA. Future study may help to determine if targeted treatments in at-risk individuals could prevent the development of DM.

Malondialdehyde-Acetaldehyde Modified (MAA) Proteins Differentially Effect the Inflammatory Response in Macrophage, Endothelial Cells and Animal Models of Cardiovascular Disease.

Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.

MyD88 regulates a prolonged adaptation response to environmental dust exposure-induced lung disease.

BACKGROUND: Environmental organic dust exposures enriched in Toll-like receptor (TLR) agonists can reduce allergic asthma development but are associated with occupational asthma and chronic bronchitis. The TLR adaptor protein myeloid differentiation factor88 (MyD88) is fundamental in regulating acute inflammatory responses to organic dust extract (ODE), yet its role in repetitive exposures is unknown and could inform future strategies. METHODS: Wild-type (WT) and MyD88 knockout (KO) mice were exposed intranasally to ODE or saline daily for 3 weeks (repetitive exposure). Repetitively exposed animals were also subsequently rested with no treatments for 4 weeks followed by single rechallenge with saline/ODE. RESULTS: Repetitive ODE exposure induced neutrophil influx and release of pro-inflammatory cytokines and chemokines were profoundly reduced in MyD88 KO mice. In comparison, ODE-induced cellular aggregates, B cells, mast cell infiltrates and serum IgE levels remained elevated in KO mice and mucous cell metaplasia was increased. Expression of ODE-induced tight junction protein(s) was also MyD88-dependent. Following recovery and then rechallenge with ODE, inflammatory mediators, but not neutrophil influx, was reduced in WT mice pretreated with ODE coincident with increased expression of IL-33 and IL-10, suggesting an adaptation response. Repetitively exposed MyD88 KO mice lacked inflammatory responsiveness upon ODE rechallenge. CONCLUSIONS: MyD88 is essential in mediating the classic airway inflammatory response to repetitive ODE, but targeting MyD88 does not reduce mucous cell metaplasia, lymphocyte influx, or IgE responsiveness. TLR-enriched dust exposures induce a prolonged adaptation response that is largely MyD88-independent. These findings demonstrate the complex role of MyD88-dependent signaling during acute vs. chronic organic dust exposures.

Liver tissue metabolically transformed by alcohol induces immune recognition of liver self-proteins but not in vivo inflammation.

Precision-cut liver slices (PCLSs) provide a novel model for studies of alcoholic liver disease (ALD). This is relevant, as in vivo ethanol exposure does not appear to generate significant liver damage in ethanol-fed mice, except in the National Institute on Alcohol Abuse and Alcoholism binge model of ALD. Previous studies have shown that the two metabolites of ethanol consumption, malondialdhyde (MDA) and acetaldehyde (AA), combine to form MDA-AA (MAA) adducts, which have been correlated with the development and progression of ALD. In this study, murine PCLSs were incubated with ethanol and examined for the production of MAA adducts. PCLSs were homogenized, and homogenates were injected into C57BL/6 mice. PCLSs from control-, pair-, and ethanol-fed animals served as targets in in situ cytotoxic assays using primed T cells from mice hyperimmunized with control or ethanol-exposed PCLS homogenates. A CD45.1/CD45.2 passive-transfer model was used to determine whether T cells from the spleens of mice hyperimmunized with PCLS ethanol-exposed homogenates trafficked to the liver. PCLSs incubated with ethanol generated MAA-modified proteins in situ. Cytotoxic (CD8+) T cells from immunized mice killed naïve PCLSs from control- and pair-fed mice in vitro, a response that was blunted in PCLSs from ethanol-fed mice. Furthermore, CD45.1 CD8+ T cells from hyperimmunized mice trafficked to the liver but did not initiate liver damage. This study demonstrates that exposure to liver tissue damaged by ethanol mediates robust immune responses to well-characterized alcohol metabolites and native liver proteins in vitro. Moreover, although these proinflammatory T cells traffic to the liver, these responses appear to be dampened in vivo by locally acting pathways. NEW & NOTEWORTHY This study shows that the metabolites of ethanol and lipid breakdown produce malondialdehyde-acetaldehyde adducts in the precision-cut liver slice model system. Additionally, precision-cut liver slices exposed to ethanol and harboring malondialdehyde-acetaldehyde adducts generate liver-specific antibody and T cell responses in the spleens of naïve mice that could traffic to the liver.

N-Acetyl Cysteine Treatment Restores Early Phase Fracture Healing in Ethanol-Fed Rats.

BACKGROUND: Fracture healing in alcoholics is delayed and often associated with infections resulting in prolonged rehabilitation. It has been reported that binge drinking of alcohol increases oxidative stress and delays fracture healing in rats, which is prevented by treatment with the antioxidant n-acetyl cysteine (NAC). Oxidative stress is a significant factor in pathologies of various organs resulting from chronic alcoholism. Therefore, we hypothesize that treatment with NAC reduces oxidative stress and restores fracture healing in chronic alcoholics. METHODS: Rats (10 months old) were pair-fed the Lieber-DeCarli ethanol (EtOH) diet or control diet for 16 weeks. A closed fracture was performed and rats allowed to recover for 72 hours. Rats were divided into 4 groups-control, control + NAC, EtOH, and EtOH + NAC-and injected intraperitoneally with 200 mg/kg of NAC daily for 3 days. Serum and bone fracture callus homogenates were collected and assayed for traditional markers of inflammation, oxidative stress, and bone regeneration. RESULTS: The oxidative stress marker malondialdehyde (MDA) was increased in both serum and bone tissue in EtOH-fed animals compared to controls. NAC treatment significantly (p < 0.01) reduced MDA to near normal levels and dramatically increased the index of antioxidant efficacy (catalase/MDA ratio) (p < 0.01). Inflammatory markers tumor necrosis factor-α, interferon-γ, and interleukin-6 were significantly decreased in serum and callus following NAC treatment. NAC treatment reduced EtOH-induced bone resorption as evidenced by significant decreases in C-telopeptide of type-I-collagen levels (p < 0.05) and band-5 tartrate-resistant acid phosphatase levels in the tissue (p < 0.001). CONCLUSIONS: Oxidative stress and excessive inflammation are involved in the inhibition of fracture healing by EtOH. In this study, early short-term treatment of EtOH-fed animals with the antioxidant NAC reduced oxidative stress and normalized the innate immune response to fracture in the early phase of fracture healing, thereby restoring the normal onset of bone regeneration.

Novel Antioxidant Properties of Doxycycline.

Doxycycline (DOX), a derivative of tetracycline, is a broad-spectrum antibiotic that exhibits a number of therapeutic activities in addition to its antibacterial properties. For example, DOX has been used in the management of a number of diseases characterized by chronic inflammation. One potential mechanism by which DOX inhibits the progression of these diseases is by reducing oxidative stress, thereby inhibiting subsequent lipid peroxidation and inflammatory responses. Herein, we tested the hypothesis that DOX directly scavenges reactive oxygen species (ROS) and inhibits the formation of redox-mediated malondialdehyde-acetaldehyde (MAA) protein adducts. Using a cell-free system, we demonstrated that DOX scavenged reactive oxygen species (ROS) produced during the formation of MAA-adducts and inhibits the formation of MAA-protein adducts. To determine whether DOX scavenges specific ROS, we examined the ability of DOX to directly scavenge superoxide and hydrogen peroxide. Using electron paramagnetic resonance (EPR) spectroscopy, we found that DOX directly scavenged superoxide, but not hydrogen peroxide. Additionally, we found that DOX inhibits MAA-induced activation of Nrf2, a redox-sensitive transcription factor. Together, these findings demonstrate the under-recognized direct antioxidant property of DOX that may help to explain its therapeutic potential in the treatment of conditions characterized by chronic inflammation and increased oxidative stress.

Enrichment of malondialdehyde-acetaldehyde antibody in the rheumatoid arthritis joint.

Objective: To characterize the expression of malondialdehdye-acetaldehyde (MAA) adducts and anti-MAA antibody in articular tissues and serum of patients with RA. Methods: Paired sera and SF were examined from 29 RA and 13 OA patients. Anti-MAA antibody, RF, ACPA and total immunoglobulin were quantified. SF-serum measures were compared within and between disease groups. The presence and co-localization of MAA, citrulline and select leukocyte antigens in RA and OA synovial tissues were examined using immunohistochemistry. Results: Circulating and SF anti-MAA antibody concentrations were higher in RA vs OA by 1.5- to 5-fold. IgG (P < 0.001), IgM (P = 0.006) and IgA (P = 0.036) anti-MAA antibodies were higher in paired RA SF than serum, differences not observed for total immunoglobulin, RF or ACPA. In RA synovial tissues, co-localization of MAA with citrulline and CD19+ or CD27+ B cells was demonstrated and was much higher in magnitude than MAA or citrulline co-localization with T cells, monocytes, macrophages or dendritic cells (P < 0.01). Conclusion: Anti-MAA antibodies are present in higher concentrations in the RA joint compared with sera, a finding not observed for other disease-related autoantibodies. Co-localization of MAA and citrulline with mature B cells, coupled with the local enrichment of anti-MAA immune responses, implicates MAA-adduct formation in local autoantibody production.

A role for B cells in organic dust induced lung inflammation.

BACKGROUND: Agriculture organic dust exposures induce lung disease with lymphoid aggregates comprised of both T and B cells. The precise role of B cells in mediating lung inflammation is unknown, yet might be relevant given the emerging role of B cells in obstructive pulmonary disease and associated autoimmunity. METHODS: Using an established animal model, C57BL/6 wild-type (WT) and B-cell receptor (BCR) knock-out (KO) mice were repetitively treated with intranasal inhalation of swine confinement organic dust extract (ODE) daily for 3 weeks and lavage fluid, lung tissues, and serum were collected. RESULTS: ODE-induced neutrophil influx in lavage fluid was not reduced in BCR KO animals, but there was reduction in TNF-α, IL-6, CXCL1, and CXCL2 release. ODE-induced lymphoid aggregates failed to develop in BCR KO mice. There was a decrease in ODE-induced lung tissue CD11c+CD11b+ exudative macrophages and compensatory increase in CD8+ T cells in lavage fluid of BCR KO animals. Compared to saline, there was an expansion of conventional B2-, innate B1 (CD19+CD11b+CD5+/-)-, and memory (CD19+CD273+/-CD73+/-) B cells following ODE exposure in WT mice. Autoreactive responses including serum IgG anti-citrullinated protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) autoantibodies were increased in ODE treated WT mice as compared to saline control. B cells and serum immunoglobulins were not detected in BCR KO animals. CONCLUSIONS: Lung tissue staining for citrullinated and MAA modified proteins were increased in ODE-treated WT animals, but not BCR KO mice. These studies show that agriculture organic dust induced lung inflammation is dependent upon B cells, and dust exposure induces an autoreactive response.

Malondialdehyde-Acetaldehyde (MAA) Protein Adducts Are Found Exclusively in the Lungs of Smokers with Alcohol Use Disorders and Are Associated with Systemic Anti-MAA Antibodies.

BACKGROUND: Malondialdehyde (MDA) and acetaldehyde (AA) exist following ethanol metabolism and tobacco pyrolysis. As such, lungs of individuals with alcohol use disorders (AUDs) are a target for the effects of combined alcohol and cigarette smoke metabolites. MDA and AA form a stable protein adduct, malondialdehyde-acetaldehyde (MAA) adduct, known to be immunogenic, profibrotic, and proinflammatory. MAA adduct is the dominant epitope in anti-MAA antibody formation. We hypothesized that MAA-adducted protein forms in lungs of those who both abuse alcohol and smoke cigarettes, and that this would be associated with systemically elevated anti-MAA antibodies. METHODS: Four groups were established: AUD subjects who smoked cigarettes (+AUD/+smoke), smokers without AUD (-AUD/+smoke), AUD without smoke (+AUD/-smoke), and non-AUD/nonsmokers (-AUD/-smoke). RESULTS: We observed a significant increase in MAA adducts in lung cells of +AUD/+smoke versus -AUD/-smoke. No significant increase in MAA adducts was observed in -AUD/+smoke or in +AUD/-smoke compared to -AUD/-smoke. Serum from +AUD/+smoke had significantly increased levels of circulating anti-MAA IgA antibodies. After 1 week of alcohol that MAA-adducted protein is formed in the lungs of those who smoke cigarettes and abuse alcohol, leading to a subsequent increase in serum IgA antibodies. CONCLUSIONS: MAA-adducted proteins could play a role in pneumonia and other diseases of the lung in the setting of AUD and smoking.

Increased inflammation and disease activity among current cigarette smokers with rheumatoid arthritis: a cross-sectional analysis of US veterans.

OBJECTIVES: Cigarette smoking is a major risk factor for RA and has been associated with increased disease severity and lower rates of disease remission. We hypothesized that inflammation and disease activity would be associated with smoking status and this would be related to levels of ACPA. METHODS: RA patients from the Veterans Affairs RA registry were studied (n = 1466): 76.9% anti-CCP2 positive, 89% male, median age 63 years (interquartile range 57-72), median disease duration 8.45 years (interquartile range 2.8-18). Baseline serum samples were evaluated for levels of anti-CCP2, RF, 19 distinct ACPAs and 17 cytokines. Smoking status at baseline was recorded as current, former or never. The association of smoking status with cytokines, autoantibodies and disease activity (DAS28) was evaluated. RESULTS: Among anti-CCP-positive RA patients, RA-associated cytokines (false-discovery rates q < 0.1%) and DAS28 (P < 0.01) were higher in current smokers compared with former or never smokers. DAS28 and cytokine levels were similar between former and never smokers. In contrast, ACPA concentrations were higher among both current and former smokers compared with never smokers, and levels of ACPA were not associated with DAS28 or cytokine levels. CONCLUSION: Among anti-CCP2-positive RA patients, current smoking status is associated with elevations in pro-inflammatory cytokines and increased RA disease activity. Similar levels of inflammation and disease activity among former and never smokers suggests that the detrimental effects of smoking could be ameliorated through tobacco cessation. The effect of tobacco cessation on RA disease activity should be evaluated prospectively.

Antibodies against malondialdehyde-acetaldehyde adducts can help identify patients with abdominal aortic aneurysm.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a pathologic dilation of the aorta. Inflammation of the aortic wall has been shown to be involved in AAA formation. Malondialdehyde-acetaldehyde (MAA) adducts are MAA/protein hybrids with immunogenic, proinflammatory, and profibrotic properties. Levels of MAA adducts are elevated in patients with coronary artery disease; however, the role of MAA adducts in AAA is unclear. We hypothesize that levels of circulating antibodies against MAA adducts are increased in patients with AAA. METHODS: Plasma samples were collected from mice and patients with AAA and control patients with atherosclerosis but not AAA. AAA was induced in mice by a standard CaCl2 protocol, with matching sham mice. Plasma levels of anti-MAA antibodies were quantified by enzyme-linked immunosorbent assay. RESULTS: Patients with AAA exhibited higher levels of immunoglobulin G and immunoglobulin A anti-MAA antibody subtypes (P = .049 and .026, respectively) compared with control patients. Conversely, immunoglobulin M anti-MAA antibodies in AAA patients were lower compared with control patients (P = .018). In CaCl2-treated mice, immunoglobulin G anti-MAA antibodies were elevated after AAA formation (P = .006). CONCLUSIONS: The pattern of anti-MAA antibodies is able to distinguish between patients with AAA and patients with atherosclerosis but no AAA. These results demonstrate that MAA adducts are associated with AAA and suggest that they may play a role in either initiating or propagating chronic inflammation in AAA.

Aldehyde dehydrogenase 2 deficiency ameliorates alcoholic fatty liver but worsens liver inflammation and fibrosis in mice.

UNLABELLED: Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40-50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. In the present study, wild-type and ALDH2(-/-) mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4 ) treatment, and liver injury was assessed. Compared with wild-type mice, ethanol-fed ALDH2(-/-) mice had higher levels of malondialdehyde-acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic interleukin (IL)-6 expression but surprisingly lower levels of steatosis and serum alanine aminotransferase (ALT). Higher IL-6 levels were also detected in ethanol-treated precision-cut liver slices from ALDH2(-/-) mice and in Kupffer cells isolated from ethanol-fed ALDH2(-/-) mice than those levels in wild-type mice. In vitro incubation with MAA enhanced the lipopolysaccharide (LPS)-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2(-/-) mice than in wild-type mice. An additional deletion of hepatic STAT3 increased steatosis and hepatocellular damage in ALDH2(-/-) mice. Finally, ethanol-fed ALDH2(-/-) mice were more prone to CCl4 -induced liver inflammation and fibrosis than ethanol-fed wild-type mice. CONCLUSION: ALDH2(-/-) mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis by way of MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that alcohol, by way of acetaldehyde and its associated adducts, stimulates hepatic inflammation and fibrosis independent from causing hepatocyte death, and that ALDH2-deficient individuals may be resistant to steatosis and blood ALT elevation, but are prone to liver inflammation and fibrosis following alcohol consumption.

Age Impacts Pulmonary Inflammation and Systemic Bone Response to Inhaled Organic Dust Exposure.

Agricultural workers have high rates of airway and skeletal health disease. Studies recently demonstrated that inhaled agricultural organic dust extract (ODE)-induced airway injury is associated with bone deterioration in an animal model. However, the effect of age in governing these responses to organic dusts is unclear, but might be important in future approaches. Young (7-9 wk) and older (12-14,o) male C57BL/6 mice received intranasal (i.n.) inhalation exposure to ODE from swine confinement facilities once or daily for 3 wk. Acute ODE-induced neutrophil influx and cytokine and chemokine (tumor necrosis factor [TNF]-α, interleukin [IL]-6, keratinocyte chemoattractant [CXCL1], macrophage inflammatory protein-2 [CXCL2]) airway production were reduced in older compared to young mice. Repetitive ODE treatment, however, increased lymphocyte recruitment and alveolar compartment histopathologic inflammatory changes in older mice. Whole lung cell infiltrate analysis revealed that young, but not older, mice repetitively treated with ODE demonstrated an elevated CD4:CD8 lymphocyte response. Acute inhalant ODE exposure resulted in a 4-fold and 1.5-fold rise in blood neutrophils in young and older mice, respectively. Serum IL-6 and CXCL1 levels were elevated in young and older mice i.n. exposed once to ODE, with increased CXCL1 levels in younger compared to older mice. Although older mice displayed reduced bone measurements compared to younger mice, younger rodents demonstrated ODE-induced decrease in bone mineral density, bone volume, and bone microarchitecture quality as determined by computed tomography (CT) analysis. Collectively, age impacts the airway injury and systemic inflammatory and bone loss response to inhalant ODE, suggesting an altered and enhanced immunologic response in younger as compared to older counterparts.

Precision-cut liver slices from diet-induced obese rats exposed to ethanol are susceptible to oxidative stress and increased fatty acid synthesis.

Oxidative stress from fat accumulation in the liver has many deleterious effects. Many believe that there is a second hit that causes relatively benign fat accumulation to transform into liver failure. Therefore, we evaluated the effects of ethanol on ex vivo precision-cut liver slice cultures (PCLS) from rats fed a high-fat diet resulting in fatty liver. Age-matched male Sprague-Dawley rats were fed either high-fat (obese) (45% calories from fat, 4.73 kcal/g) or control diet for 13 mo. PCLS were prepared, incubated with 25 mM ethanol for 24, 48, and 72 h, harvested, and evaluated for ethanol metabolism, triglyceride production, oxidative stress, and cytokine expression. Ethanol metabolism and acetaldehyde production decreased in PCLS from obese rats compared with age-matched controls (AMC). Increased triglyceride and smooth muscle actin production was observed in PCLS from obese rats compared with AMC, which further increased following ethanol incubation. Lipid peroxidation, measured by thiobarbituric acid reactive substances assay, increased in response to ethanol, whereas GSH and heme oxygenase I levels were decreased. TNF-α and IL-6 levels were increased in the PCLS from obese rats and increased further with ethanol incubation. Diet-induced fatty liver increases the susceptibility of the liver to toxins such as ethanol, possibly by the increased oxidative stress and cytokine production. These findings support the concept that the development of fatty liver sensitizes the liver to the effects of ethanol and leads to the start of liver failure, necrosis, and eventually cirrhosis.

Associations between periodontitis and serum anti-malondialdehyde-acetaldehyde antibody concentrations in rheumatoid arthritis: A case-control study.

BACKGROUND: Malondialdehyde-acetaldehyde (MAA) adducts lead to generation of anti-MAA autoantibodies and have been independently identified in inflamed periodontal and rheumatoid arthritis (RA) tissues. This study evaluates serum samples from RA cases and osteoarthritis (OA) controls to quantify associations between periodontal clinical measures, alveolar bone loss (ABL), and anti-Porphyromonas gingivalis, anti-Prevotella intermedia, and anti-Fusobacterium nucleatum antibody concentrations with anti-MAA antibody concentrations. METHODS: Participants (n = 284 RA cases, n = 330 OA controls) underwent periodontal clinical assessments and ABL measurements. Serum immunoglobulin (Ig) A, IgG, and IgM anti-MAA and serum IgG antibacterial antibody concentrations were quantified by enzyme-linked immunosorbent assay (ELISA). Analyses utilized simple linear regression and multivariable adjusted models. RESULTS: No significant associations of periodontal clinical measures with serum anti-MAA were found. Moderate (p = 0.038 and p = 0.036, respectively) and high ABL (p = 0.012 and p = 0.014, respectively) in RA cases (but not in OA) were positively associated with IgG and IgM anti-MAA. Anti-P. gingivalis and anti-P. intermedia antibody concentrations were positively associated with IgA (p = 0.001 for both), IgG (p = 0.007 and p = 0.034, respectively), and IgM anti-MAA antibody concentrations (p < 0.001 and p = 0.020, respectively), while anti-F. nucleatum was positively associated with IgG anti-MAA (p = 0.042), findings that were similar across groups. CONCLUSIONS: A positive association was demonstrated between ABL and serum IgG and IgM anti-MAA antibody concentrations that was unique to RA and not observed in OA. Serum anti-P. gingivalis, anti-P. intermedia, and anti-F. nucleatum antibody concentrations displayed significant associations with anti-MAA antibody in both groups. These findings suggest MAA may play a role in the interrelationship between the periodontium and RA.