Induction of Osteoarthritis‐like Pathologic Changes by Chronic Alcohol Consumption in an Experimental Mouse Model

Osteoarthritis (OA) is characterized by a slow and progressive deterioration of articular cartilage. OA likely arises from a combination of systemic (genetics, age, environmental factors) and local factors (abnormal joint loading, overuse or trauma) working in concert to create a condition with definable morphologic and clinical characteristics. Several risk factors for OA have been previously identified, including genetic predisposition, obesity, diabetes, hypertension, hyperuricemia, previous trauma, and aging (1). However, due in large part to an inability to control for confounding factors, the underlying pathogenesis and causative factors responsible for initiation and progression of the disease remain largely unknown. There is a growing body of evidence correlating progression of OA with an upregulation of inflammatory processes (2). Oxidative stress elicited by reactive oxygen species (ROS) further disturbs cartilage homeostasis and promotes catabolism via induction of cell death, breakdown of matrix proteoglycans (PGs), upregulation of latent matrix-degrading enzyme production, inhibition of extracellular matrix (ECM) synthesis, and oxidation of intracellular and extracellular molecules (3). Thus, environmental factors that promote oxidative stress and inflammatory states could potentially act as a risk factor for OA. Alcohol consumption could be one potential risk factor because: [1] chronic alcohol consumption, highly common in Western and industrial societies, generates reactive oxygen species (ROS), leading to systemic and tissue oxidative stress in human and rodents, and [2] alcohol is capable of inducing pro-inflammatory states in multiple organs such as liver, heart, central nervous system, and pancreas (4, 5). Several studies have previously attempted to elucidate a relationship between alcohol consumption and inflammatory arthritis such as rheumatoid arthritis, with conflicting results (6, 7). However, despite recent evidence demonstrating the importance of oxidative stress and pro-inflammatory states in the development and progression of degenerative joint disease, the impact of alcohol consumption on OA has not yet been studied. The findings of the present study suggest that chronic alcohol exposure may increase susceptibility to the development and/or progression of OA. Using a validated in vivo model of chronic alcohol treatment, we show that chronic alcohol consumption increases PG loss in both knee and shoulder joints of mice, stimulates multiple inflammatory, catabolic, and anti-anabolic mediators involved in cartilage. In our experimental protocol, young adult male (aged 7–9 weeks) C57BL/6 mice were provided ad libitum access to an alcohol-diet (i.e., the Nanji diet), containing 4.5% (v/v) ethanol (29% ethanol-derived calories) or an isocaloric alcohol-free control diet for 8 weeks (n=14–16 per group). All animal protocols and practices were reviewed and approved in advance by the Institutional Animal Care and Use Committees at Rush University and Northwestern University. Following eight weeks of alcohol-containing diet or the control diet, mice were euthanized and joint sections were collected, fixed, paraffin-embedded, and stained with Safranin-O to assess cartilage structure and matrix proteoglycan (PG) content. The alcohol diet is a slight modification of the well-validated Leiber-DiCarli diet in which the fat source comes from fish oil and consumption of this diet in BL6 mice has been shown to produce blood alcohol levels that are in the low to moderate level (8). Our group, and others, have successfully used this alcohol diet to induce a variety of alcohol pathologies including colon cancer, intestinal hyperpermeability, endotoxemia, and liver pathology in rodents (8–10). Serum alcohol level in the alcohol-fed mice at the time of euthanasia was ~3mg/dL and these mice did not show any overt behavioral abnormalities during our experimental protocol. Histological examination of knee joints of control diet-fed mice demonstrated normal architecture of articular cartilage with intense Safranin-O staining. In contrast, knee joints of alcohol-fed mice displayed OA-like characteristics, with increases in PG loss represented by Safranin-O staining and mild fibrillation (Figure 1A). These results were quantified using an OARSI semi-quantitative scoring system. Alcohol-fed mice scored significantly higher (1.3±0.67) than control mice (0.3±0.27; P < 0.05), indicating more severe arthritic changes in knee joints of alcohol-fed mice compared to control (Figure 1A, lower panel). Similar results were found in shoulder joints (Figure 1B), as Safranin-O staining revealed a decrease in gross PG content and an irregular cartilage surface in shoulder joints of alcohol-fed mice compared to the control group. We applied the OARSI scoring to quantify pathological changes in shoulder, and found significantly higher OARSI scores for alcohol-fed mice (0.75±0.28) compared to control mice (0.12±0.25; P < 0.05) (Figure 1B, lower panel). Interestingly, the intervertebral discs showed no pathological changes in alcohol-fed mice compared to control mice (Figure 1C). Figure 1 Chronic ethanol consumption induces pathological changes in articular cartilage Our results demonstrate a pathologic role of alcohol on specific catabolic and anti-anabolic mediators in knee joints (Supplemental Figure 1) that may increase susceptibility of OA induction. In our study, phospho protein kinase C δ (pPKCδ), pNF-κB, and pERK1/2 were significantly increased in knee joints of alcohol-fed compared to control mice (Supplemental Figure 1A, P < 0.05), suggesting that chronic alcohol consumption stimulates these catabolic signaling pathways, which may result in subsequent production of cartilage destructive enzymes. Hypertrophic marker RUNX2, as well as a key cartilage destructive enzymes matrix metalloprotease- 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), were significantly increased in knee joints of alcohol-fed mice compared to the control mice (Supplemental Figure 1B; P < 0.05). In addition to induction of catabolic effects, we observed that chronic alcohol consumption resulted in a striking reduction of anabolic and anti-inflammatory mediators in articular chondrocytes as assessed by immunohistochemistry (Supplemental Figure 1C, P < 0.05). These molecules include tissue inhibitor of metalloproteinase-3 (TIMP-3), SOX-9, high-mobility group protein-2 (HMGB2) and suppressor of cytokine signaling-2 (SOCS-2), molecules that were significantly decreased in OA cartilage and associated with both cartilage protection and repair. These findings clearly demonstrate that chronic alcohol consumption augments catabolic signaling pathways and suppresses anabolic, reparative, and anti-inflammatory activity in mouse knee articular chondrocytes. We also performed micro-computed tomography (uCT) to determine whether any pathological changes in bone pathology after chronic alcohol treatment were evident. However, we did not find significant bone pathology in the knee-joints of alcohol-fed mice from our experimental protocol (data not shown). To our knowledge, these findings provide the first evidence that chronic alcohol consumption may be a novel risk factor for the development of OA. The pathology associated with chronic alcohol consumption is multi-factorial, with diverse consequences in different cell types through direct toxic effects as well as indirect effects of specific metabolites, oxidative stress, immunologic/inflammatory processes. It is well-established that alcohol consumption causes endotoxemia that is thought to contribute to alcohol liver damage (11). Indeed, we have shown that alcohol consumption promotes intestinal hyperpermeability and endotoxemia in rodents including BL6 mice (8) phenomenon that are also observed in human alcoholics (12). Future studies are necessary to evaluate the contribution of intestinal barrier dysfunction and endotoxemia in our studies. There are, however, several limitations of this study that need to be mentioned. First, we have addressed the effects of chronic alcohol consumption in an in vivo mouse model of the knee joint, which makes it difficult to generalize results across species to human joint tissue. Clinical studies are necessary to confirm our data to associate chronic alcoholism and joint pathology. Second, the current study reports the potent catabolic activity of chronic alcohol in cartilage homeostasis via PKCδ, and MAPK signaling, but a detailed understanding of the specific cell signaling pathways and molecular mechanisms underlying these findings remains to be elucidated. Finally, although these results have significant potential clinical relevance, several aspects of the underlying mechanisms remain unknown. Further studies will facilitate a better understanding of the multiple, complex effects stimulated by alcohol in joint tissue.