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.

Citrullinated and malondialdehyde-acetaldehyde modified fibrinogen activates macrophages and promotes an aggressive synovial fibroblast phenotype in patients with rheumatoid arthritis.

Objective: Post-translational protein modifications with malondialdehyde-acetaldehyde (MAA) and citrulline (CIT) are implicated in the pathogenesis of rheumatoid arthritis (RA). Although precise mechanisms have not been elucidated, macrophage-fibroblast interactions have been proposed to play a central role in the development and progression of RA. The purpose of our study was to evaluate the downstream effects of macrophage released soluble mediators, following stimulation with fibrinogen (FIB) modified antigens, on human fibroblast-like synoviocytes (HFLS). Methods: PMA-treated U-937 monocytes (Mϕ) and macrophage-differentiated peripheral blood mononuclear cells (MP) were stimulated with FIB, FIB-MAA, FIB-CIT, or FIB-MAA-CIT. HFLS-RA cells were stimulated directly with FIB antigens or with supernatants (SN) from macrophages (Mϕ-SN or MP-SN) stimulated with FIB antigens. Genes associated with an aggressive HFLS phenotype, extracellular matrix proteins, and activated signaling pathways were evaluated. Results: HFLS-RA cells treated with Mϕ-SNFIB-CIT and Mϕ-SNFIB-MAA-CIT demonstrated significant increases in mRNA expression of genes associated with an aggressive phenotype at 24-h as compared to direct stimulation with the same antigens. Similar results were obtained using MP-SN. Cellular morphology was altered and protein expression of vimentin (p<0.0001 vs. Mϕ-SNFIB) and type II collagen (p<0.0001) were significantly increased in HFLS-RA cells treated with any of the Mϕ-SN generated following stimulation with modified antigens. Phosphorylation of JNK, Erk1/2, and Akt were increased most substantially in HFLS-RA treated with Mϕ-SNFIB-MAA-CIT (p<0.05 vs Mϕ-SNFIB). These and other data suggested the presence of PDGF-BB in Mϕ-SN. Mϕ-SNFIB-MAA-CIT contained the highest concentration of PDGF-BB (p<0.0001 vs. Mϕ-SNFIB) followed by Mϕ-SNFIB-CIT then Mϕ-SNFIB-MAA. HFLS-RA cells treated with PDGF-BB showed similar cellular morphology to the Mϕ-SN generated following stimulation with modified FIB, as well as the increased expression of vimentin, type II collagen, and the phosphorylation of JNK, Erk1/2 and Akt signaling molecules. Conclusion: Together, these findings support the hypothesis that in response to MAA-modified and/or citrullinated fibrinogen, macrophages release soluble factors including PDGF-BB that induce fibroblast activation and promote an aggressive fibroblast phenotype. These cellular responses were most robust following macrophage activation with dually modified fibrinogen, compared to single modification alone, providing novel insights into the combined role of multiple post-translational protein modifications in the development of RA.

Peptidyl arginine deiminase expression and macrophage polarization following stimulation with citrullinated and malondialdehyde-acetaldehyde modified fibrinogen.

OBJECTIVE: Post-translational modifications of extracellular matrix proteins such as fibrinogen may lead to tolerance loss and have been implicated in rheumatoid arthritis (RA) pathogenesis. The purpose of this study was to determine whether fibrinogen (FIB) modified with citrulline (CIT), malondialdehyde-acetaldehyde (MAA) or both leads to altered macrophage polarization, peptidyl arginine deiminase (PAD) expression, or production of citrullinated proteins. METHODS: PMA-treated U-937 cells (M0 cells) were stimulated with MAA, CIT or MAA-CIT modified FIB. Macrophage (M1/M2) phenotypes were evaluated by flow cytometry, RT-PCR, and ELISA. PAD enzyme expression and protein citrullination was evaluated using RT-PCR and Western Blot. RESULTS: Flow cytometry revealed that M0 macrophages stimulated with FIB-MAA-CIT resulted in mixed M1/M2 phenotypes as demonstrated by cell surface expression and mRNA levels of CD14, CD192, CD163, and CD206 (p < 0.001 vs. others), and the release of IL-18, IP-10, CCL22, and IL-13 (p < 0.001 vs. others). While FIB-MAA treated M0 cells demonstrated a mixed M1/M2 phenotype, cytokine and cell surface markers differed from FIB-MAA-CIT. Finally, M0 cells treated with FIB-CIT demonstrated markers and cytokines consistent with only the M1-like phenotype. Exposure of M0 cells to FIB-MAA-CIT (at 48 h) and FIB-MAA (at 24 h) led to increased mRNA expression and protein expression of PAD2 (p < 0.001) with increased protein citrullination. CONCLUSION: These findings suggest that MAA-modification and citrullination of FIB, in isolation or combination, yield specific effects on macrophage polarization, PAD expression and citrullination that ultimately may induce inflammatory and fibrotic responses associated 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.

Identification of Immunoglobulin G Autoantibody Against Malondialdehyde-Acetaldehyde Adducts as a Novel Serological Biomarker for Ulcerative Colitis.

INTRODUCTION: Inflammatory bowel disease (IBD) is associated with immune responses with oxidative stress wherein high levels of malondialdehyde result in the formation of a highly stable and immunogenic malondialdehyde-acetaldehyde adduct (MAA). Thus, this study evaluated the status of MAA and anti-MAA antibody isotypes in IBD and their potential as novel serological biomarkers for differentiating ulcerative colitis (UC) from Crohn's disease (CD). METHODS: Levels of MAA and anti-MAA antibodies were examined in patients with IBD (171), non-IBD gastrointestinal diseases (77), and controls (83) from 2 independent cohorts using immunohistochemistry and enzyme-linked immunosorbent assay. Receiver operating characteristic curves and Youden cutoff index from logistic regression were used to determine the sensitivity and specificity. RESULTS: The MAA and blood immunoglobulin G (IgG) anti-MAA antibody levels were significantly elevated in IBD compared with non-IBD patients (P = 0.0008) or controls (P = 0.02). Interestingly, patients with UC showed higher levels of IgG anti-MAA (P < 0.0001) than patients with CD including those with colonic CD (P = 0.0067). The odds ratio by logistic regression analysis predicted stronger association of IgG anti-MAA antibody with UC than CD. Subsequent analysis showed that IgG anti-MAA antibody levels could accurately identify (P = 0.0004) UC in the adult cohort with a sensitivity of 75.3% and a specificity of 71.4% and an area under the curve of 0.8072 (0.7121-0.9024). The pediatric cohort also showed an area under the curve of 0.8801 (0.7988-0.9614) and precisely distinguished (P < 0.0001) UC with sensitivity (95.8%) and specificity (72.3%). DISCUSSION: Circulating IgG anti-MAA antibody levels can serve as a novel, noninvasive, and highly sensitive test to identify patients with UC and possibly differentiate them from patients with CD.

Associations between an expanded autoantibody profile and treatment responses to biologic therapies in patients with rheumatoid arthritis.

BACKGROUND: Although biologics represent a major advance in rheumatoid arthritis (RA), many patients fail to achieve adequate responses to these agents. We examined whether combined positivity to three well-characterized autoantibodies predicts treatment response among RA patients initiating biologics. METHODS: The study included biologic-naïve patients initiating anti-TNF treatment, biologic-exposed patients switching to rituximab or tocilizumab, and patients (biologic naïve or exposed) initiating abatacept. Rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP) antibody, and IgG antibodies to malondialdehyde-acetaldehyde (MAA) were measured using banked enrollment serum. The relationship between the number of autoantibodies positive (0-3) and treatment response (absolute improvement in 28-joint Disease Activity Score [DAS28-CRP] or improvement > 1.2) at 6 months was examined using multivariable linear and logistic regression. RESULTS: Of 1,229 patients initiating biologics, 79% were women; 89% were Caucasian. The number of baseline RA-related autoantibodies positive was associated with improved treatment response in a dose-dependent fashion. Compared to patients seronegative for all autoantibodies, adjusting for covariates, those positive for all three were more than twice (OR 2.35; 95% CI 1.57-3.51) as likely to achieve DAS28 improvement > 1.2 units. Associations of autoantibody positivity with biologic treatment response were strongest for anti-CCP antibody, persisted in analyses limited to biologic naïve patients, and did not appear to differ markedly among different agents examined. CONCLUSION: An expanded autoantibody profile appears to significantly predict RA treatment response to biologic treatment in a dose-dependent fashion. Incorporating these serologic profiles with additional biomarkers or other informative patient characteristics could provide an opportunity to personalize RA management.

Surface nano-modification by ion beam-assisted deposition alters the expression of osteogenic genes in osteoblasts.

Biomaterials with enhanced biocompatibility are favored in implant studies to improve the outcomes of total joint replacement surgeries. This study tested the hypothesis that nano-structured surfaces for orthopedic applications, produced by the ion beam-assisted deposition method, would enhance osteointegration by altering the expression of bone-associated genes in osteoblasts. The ion beam-assisted deposition technique was employed to deposit nano-films on glass or titanium substrates. The effects of the ion beam-assisted deposition produced surfaces on the human osteosarcoma cell line SAOS-2 at the molecular level were investigated by assays of adhesion, proliferation, differentiation, and apoptosis on coated surfaces versus uncoated cobalt-chrome, as the control. Ion beam-assisted deposition nano-coatings enhanced bone-associated gene expression at initial cell adhesion, proliferation, and differentiation compared to cobalt-chrome surfaces as assessed by polymerase chain reaction techniques. Increased cell proliferation was observed using a nuclear cell proliferation-associated antigen. Moreover, enhanced cell differentiation was determined by alkaline phosphatase activity, an indicator of bone formation. In addition, programmed cell death assessed by annexin V staining and flow cytometry was lower on nano-surfaces compared to cobalt-chrome surfaces. Overall, the results indicate that nano-coated surfaces produced by the ion beam-assisted deposition technique for use on implants were superior to orthopedic grade cobalt-chrome in supporting bone cell adhesion, proliferation, and differentiation and reducing apoptosis. Thus, surface properties altered by the ion beam-assisted deposition technique should enhance bone formation and increase the biocompatibility of bone cell-associated surfaces.

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.

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.

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.

Unique antibody responses to malondialdehyde-acetaldehyde (MAA)-protein adducts predict coronary artery disease.

Malondialdehyde-acetaldehyde adducts (MAA) have been implicated in atherosclerosis. The purpose of this study was to investigate the role of MAA in atherosclerotic disease. Serum samples from controls (n = 82) and patients with; non-obstructive coronary artery disease (CAD), (n = 40), acute myocardial infarction (AMI) (n = 42), or coronary artery bypass graft (CABG) surgery due to obstructive multi-vessel CAD (n = 72), were collected and tested for antibody isotypes to MAA-modifed human serum albumin (MAA-HSA). CAD patients had elevated relative levels of IgG and IgA anti-MAA, compared to control patients (p<0.001). AMI patients had a significantly increased relative levels of circulating IgG anti-MAA-HSA antibodies as compared to stable angina (p<0.03) or CABG patients (p<0.003). CABG patients had significantly increased relative levels of circulating IgA anti-MAA-HSA antibodies as compared to non-obstructive CAD (p<0.001) and AMI patients (p<0.001). Additionally, MAA-modified proteins were detected in the tissue of human AMI lesions. In conclusion, the IgM, IgG and IgA anti-MAA-HSA antibody isotypes are differentially and significantly associated with non-obstructive CAD, AMI, or obstructive multi-vessel CAD and may serve as biomarkers of atherosclerotic disease.

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats.

Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.