IL-6 promotes tumor growth through immune evasion but is dispensable for cachexia.

Various cytokines have been implicated in cancer cachexia. One such cytokine is IL-6, deemed as a key cachectic factor in mice inoculated with colon carcinoma 26 (C26) cells, a widely used cancer cachexia model. Here we tested the causal role of IL-6 in cancer cachexia by knocking out the IL-6 gene in C26 cells. We found that the growth of IL-6 KO tumors was dramatically delayed. More strikingly, while IL-6 KO tumors eventually reached the similar size as wild-type tumors, cachexia still took place, despite no elevation in circulating IL-6. In addition, the knockout of leukemia inhibitory factor (LIF), another IL-6 family cytokine proposed as a cachectic factor in the model, also affected tumor growth but not cachexia. We further showed an increase in the infiltration of immune cell population in the IL-6 KO tumors compared with wild-type controls and the defective IL-6 KO tumor growth was rescued in immunodeficient mice while cachexia was not. Thus, IL-6 promotes tumor growth by facilitating immune evasion but is dispensable for cachexia.

Exploiting common aspects of obesity and cancer cachexia for future therapeutic strategies.

Obesity and cancer cachexia are diseases at opposite ends of the BMI. However, despite the apparent dichotomy, these pathologies share some common underlying mechanisms that lead to profound metabolic perturbations. Insulin resistance, adipose tissue lipolysis, skeletal muscle atrophy and systemic inflammation are key players in both diseases. Several strategies for pharmacological treatments have been employed in obesity and cancer cachexia but demonstrated only limited effects. Therefore, there is still a need to develop novel, more effective strategies. In this review we summarize existing therapies and discuss potential novel strategies that could arise by bridging common aspects between obesity and cachexia. We discuss the potential role of macrophage manipulation and the modulation of inflammation by targeting Nuclear Receptors (NRs) as potential novel therapeutic strategies.

Antibody-mediated inhibition of GDF15-GFRAL activity reverses cancer cachexia in mice.

Cancer cachexia is a highly prevalent condition associated with poor quality of life and reduced survival1. Tumor-induced perturbations in the endocrine, immune and nervous systems drive anorexia and catabolic changes in adipose tissue and skeletal muscle, hallmarks of cancer cachexia2-4. However, the molecular mechanisms driving cachexia remain poorly defined, and there are currently no approved drugs for the condition. Elevation in circulating growth differentiation factor 15 (GDF15) correlates with cachexia and reduced survival in patients with cancer5-8, and a GDNF family receptor alpha like (GFRAL)-Ret proto-oncogene (RET) signaling complex in brainstem neurons that mediates GDF15-induced weight loss in mice has recently been described9-12. Here we report a therapeutic antagonistic monoclonal antibody, 3P10, that targets GFRAL and inhibits RET signaling by preventing the GDF15-driven interaction of RET with GFRAL on the cell surface. Treatment with 3P10 reverses excessive lipid oxidation in tumor-bearing mice and prevents cancer cachexia, even under calorie-restricted conditions. Mechanistically, activation of the GFRAL-RET pathway induces expression of genes involved in lipid metabolism in adipose tissues, and both peripheral chemical sympathectomy and loss of adipose triglyceride lipase protect mice from GDF15-induced weight loss. These data uncover a peripheral sympathetic axis by which GDF15 elicits a lipolytic response in adipose tissue independently of anorexia, leading to reduced adipose and muscle mass and function in tumor-bearing mice.

Oxidation of HMGB1 Is a Dynamically Regulated Process in Physiological and Pathological Conditions.

Acute inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens or cell damage, and is essential for immune defense and proper healing. However, unresolved inflammation can lead to chronic disorders, including cancer and fibrosis. The High Mobility Group Box 1 (HMGB1) protein is a Damage-Associated Molecular Pattern (DAMP) molecule that orchestrates key events in inflammation by switching among mutually exclusive redox states. Fully reduced HMGB1 (frHMGB1) supports immune cell recruitment and tissue regeneration, while the isoform containing a disulphide bond (dsHMGB1) promotes secretion of inflammatory mediators by immune cells. Although it has been suggested that the tissue itself determines the redox state of the extracellular space and of released HMGB1, the dynamics of HMGB1 oxidation in health and disease are unknown. In the present work, we analyzed the expression of HMGB1 redox isoforms in different inflammatory conditions in skeletal muscle, from acute injury to muscle wasting, in tumor microenvironment, in spleen, and in liver after drug intoxication. Our results reveal that the redox modulation of HMGB1 is tissue-specific, with high expression of dsHMGB1 in normal spleen and liver and very low in muscle, where it appears after acute damage. Similarly, dsHMGB1 is highly expressed in the tumor microenvironment while it is absent in cachectic muscles from the same tumor-bearing mice. These findings emphasize the accurate and dynamic regulation of HMGB1 redox state, with the presence of dsHMGB1 tightly associated with leukocyte infiltration. Accordingly, we identified circulating, infiltrating, and resident leukocytes as reservoirs and transporters of dsHMGB1 in tissue and tumor microenvironment, demonstrating that the redox state of HMGB1 is controlled at both tissue and cell levels. Overall, our data point out that HMGB1 oxidation is a timely and spatially regulated process in physiological and pathological conditions. This precise modulation might play key roles to finetune inflammatory and regenerative processes.

Non-coding RNA and immune-checkpoint inhibitors: friends or foes?

Non-coding RNAs (ncRNAs) are an abundant component of the human transcriptome. Their biological role, however, remains incompletely understood. Nevertheless, ncRNAs are highly associated with cancer development and progression due to their ability to modulate gene expression, protein translation and growth pathways. Immune-checkpoint inhibitors (ICIs) are considered one of the most promising and highly effective therapeutic approaches for cancer treatment. ICIs are monoclonal antibodies targeting immune checkpoints such as CTLA-4, PD-1 and PD-L1 signalling pathways that stimulate T cell cytotoxicity and can result in tumor growth suppression. This Review will summarize existing knowledge regarding ncRNAs and their role in cancer and ICI therapy. In addition, we will discuss potential mechanisms by which ncRNAs may influence ICI treatment outcomes.

IL-1R Regulates Disease Tolerance and Cachexia in Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular parasite that establishes life-long infection in a wide range of hosts, including humans and rodents. To establish a chronic infection, pathogens often exploit the trade-off between resistance mechanisms, which promote inflammation and kill microbes, and tolerance mechanisms, which mitigate inflammatory stress. Signaling through the type I IL-1R has recently been shown to control disease tolerance pathways in endotoxemia and Salmonella infection. However, the role of the IL-1 axis in T. gondii infection is unclear. In this study we show that IL-1R-/- mice can control T. gondii burden throughout infection. Compared with wild-type mice, IL-1R-/- mice have more severe liver and adipose tissue pathology during acute infection, consistent with a role in acute disease tolerance. Surprisingly, IL-1R-/- mice had better long-term survival than wild-type mice during chronic infection. This was due to the ability of IL-1R-/- mice to recover from cachexia, an immune-metabolic disease of muscle wasting that impairs fitness of wild-type mice. Together, our data indicate a role for IL-1R as a regulator of host homeostasis and point to cachexia as a cost of long-term reliance on IL-1-mediated tolerance mechanisms.

Macrophages potentiate STAT3 signaling in skeletal muscles and regulate pancreatic cancer cachexia.

Incidence of cachexia is highly prevalent in pancreatic ductal adenocarcinoma (PDAC); advanced disease stage directly correlates with decreased muscle and fat mass in PDAC patients. The pancreatic tumor microenvironment is central to the release of systemic factors that govern lipolysis, proteolysis, and muscle and fat degeneration leading to the cachectic phenotype in cancer patients. The current study explores the role of macrophages, a key immunosuppressive player in the pancreatic tumor microenvironment, in regulating cancer cachexia. We observed a negative correlation between CD163-positive macrophage infiltration and muscle-fiber cross sectional area in human PDAC patients. To investigate the role of macrophages in myodegeneration, we utilized conditioned media transplant assays and orthotopic models of PDAC-induced cachexia in immune-competent mice with and without macrophage depletion. We observed that macrophage-derived conditioned medium, in combination with tumor cell-conditioned medium, promoted muscle atrophy through STAT3 signaling. Furthermore, macrophage depletion attenuated systemic inflammation and muscle wasting in pancreatic tumor-bearing mice. Targeting macrophage-mediated STAT3 activation or macrophage-derived interleukin-1 alpha or interleukin-6 diminished myofiber atrophy. Taken together, the current study identified the critical association between macrophages and cachexia phenotype in pancreatic cancer.

Consumption of latex from Euphorbia tirucalli L. promotes a reduction of tumor growth and cachexia, and immunomodulation in Walker 256 tumor-bearing rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia tirucalli L. is an African plant that grows well in Brazil. Individuals diagnosed with cancer frequently consume latex from E. tirucalli, dissolved in drinking water. In vitro studies confirm the antitumor potential of E. tirucalli latex, but in vivo evaluations are scarce. AIM OF THE STUDY: To evaluate the effect of intake of an aqueous solution of E. tirucalli latex on tumor growth, cachexia, and immune response in Walker 256 tumor-bearing rats. MATERIALS AND METHODS: Latex from E. tirucalli was collected and analyzed by LC-MS. Sixty male Wistar rats (age, 90 days) were randomly divided into four groups: C, control group (without tumor); W, Walker 256 tumor-bearing group; SW1, W animals but treated with 25 µL latex/mL water; and SW2, W animals but treated with 50 µL latex/mL water. Animals received 1 mL of latex solution once a day by gavage. After 15 d, animals were euthanized, tumor mass was determined, and glucose and triacylglycerol serum levels were measured by using commercial kits. Change in the body weight during tumor development was calculated, and proliferation capacity of tumor cells was assessed by the Alamar Blue assay. Phagocytosis and superoxide anion production by peritoneal macrophages and circulating neutrophils were analyzed by enzymatic and colorimetric assays. Data are analyzed by one-way ANOVA followed by Tukey's post-hoc test, with the significance level set at 5%. RESULTS: The analysis of the latex revealed the presence of triterpenes. The ingestion of the latex aqueous solution promoted 40% and 60% reduction of the tumor mass in SW1 and SW2 groups, respectively (p < 0.05). The proliferative capacity of tumor cells from SW2 group was 76% lower than that of cells from W group (p < 0.0001). Animals treated with latex gained, on average, 20 g (SW1) and 8 g (SW2) weight. Glucose and triacylglycerol serum levels in SW1 and SW2 animals were similar to those in C group rats. Peritoneal macrophages and blood neutrophils from SW1 and SW2 animals produced 30-40% less superoxide anions than those from W group animals (p < 0.05), but neutrophils from SW2 group showed an increased phagocytic capacity (20%, vs. W group). CONCLUSIONS: E. tirucalli latex, administered orally for 15 d, efficiently reduced tumor growth and cachexia in Walker 256 tumor-bearing rats. Decreased tumor cell proliferative capacity was one of the mechanisms involved in this effect. Further, the data suggest immunomodulatory properties of E. tirucalli latex. The results agree with folk data on the antitumor effect of latex ingestion, indicating that it may be useful as an adjunct in the treatment of cancer patients. For this, further in vivo studies in animal and human models need to be conducted.

Microglia in the hypothalamus respond to tumor-derived factors and are protective against cachexia during pancreatic cancer.

Microglia in the mediobasal hypothalamus (MBH) respond to inflammatory stimuli and metabolic perturbations to mediate body composition. This concept is well studied in the context of high fat diet induced obesity (HFDO), yet has not been investigated in the context of cachexia, a devastating metabolic syndrome characterized by anorexia, fatigue, and muscle catabolism. We show that microglia accumulate specifically in the MBH early in pancreatic ductal adenocarcinoma (PDAC)-associated cachexia and assume an activated morphology. Furthermore, we observe astrogliosis in the MBH and hippocampus concurrent with cachexia initiation. We next show that circulating immune cells resembling macrophages infiltrate the MBH. PDAC-derived factors induced microglia to express a transcriptional profile in vitro that was distinct from that induced by lipopolysaccharide (LPS). Microglia depletion through CSF1-R antagonism resulted in accelerated cachexia onset and increased anorexia, fatigue, and muscle catabolism during PDAC. This corresponded with increased hypothalamic-pituitary-adrenal (HPA) axis activation. CSF1-R antagonism had little effect on inflammatory response in the circulation, liver, or tumor. These findings demonstrate that microglia are protective against PDAC cachexia and provide mechanistic insight into this function.

Exercise as an anti-inflammatory therapy for cancer cachexia: a focus on interleukin-6 regulation.

Cancer cachexia is a complicated disorder of extreme, progressive skeletal muscle wasting. It is directed by metabolic alterations and systemic inflammation dysregulation. Numerous studies have demonstrated that increased systemic inflammation promotes this type of cachexia and have suggested that cytokines are implicated in the skeletal muscle loss. Exercise is firmly established as an anti-inflammatory therapy that can attenuate or even reverse the process of muscle wasting in cancer cachexia. The interleukin IL-6 is generally considered to be a key player in the development of the microenvironment of malignancy; it promotes tumor growth and metastasis by acting as a bridge between chronic inflammation and cancerous tissue and it also induces skeletal muscle atrophy and protein breakdown. Paradoxically, a beneficial role for IL-6 has also been identified recently, and that is its status as a "founding member" of the myokine class of proteins. Skeletal muscle is an important source of circulating IL-6 in people who participate in exercise training. IL-6 acts as an anti-inflammatory myokine by inhibiting TNFα and improving glucose uptake through the stimulation of AMPK signaling. This review discusses the action of IL-6 in skeletal muscle tissue dysfunction and the role of IL-6 as an "exercise factor" that modulates the immune system. This review also sheds light on the main considerations related to the treatment of muscle wasting in cancer cachexia.

Cachexia induced by Yoshida ascites hepatoma in Wistar rats is not associated with inflammatory response in the spleen or brain.

Recent data indicate that peripheral, as well as hypothalamic pro-inflammatory cytokines play an important role in the development of cancer cachexia. However, there are only a few studies simultaneously investigating the expression of inflammatory molecules in both the periphery and hypothalamic structures in animal models of cancer cachexia. Therefore, using the Yoshida ascites hepatoma rat's model of cancer cachexia we investigated the gene expression of inflammatory markers in the spleen along with the paraventricular and arcuate nuclei, two hypothalamic structures that are involved in regulating energy balance. In addition, we investigated the effect of intracerebroventricular administration of PS-1145 dihydrochloride (an Ikß inhibitor) on the expression of selected inflammatory molecules in these hypothalamic nuclei and spleen. We observed significantly reduced food intake in tumor-bearing rats. Moreover, we found significantly decreased expression of IL-6 in the spleen as well as decreased NF-κB in the paraventricular nucleus of rats with Yoshida ascites hepatoma. Similarly, expression of TNF-α, IL-1ß, NF-κB, and COX-2 in the arcuate nucleus was significantly reduced in tumor-bearing rats. Administration of PS-1145 dihydrochloride reduced only the gene expression of COX-2 in the hypothalamus. Based on our findings, we suggest that the growing Yoshida ascites hepatoma decreased food intake by mechanical compression of the gut and therefore this model is not suitable for investigation of the inflammation-related mechanisms of cancer cachexia development.

Systemic inflammation in colorectal cancer: Underlying factors, effects, and prognostic significance.

Systemic inflammation is a marker of poor prognosis preoperatively present in around 20%-40% of colorectal cancer patients. The hallmarks of systemic inflammation include an increased production of proinflammatory cytokines and acute phase proteins that enter the circulation. While the low-level systemic inflammation is often clinically silent, its consequences are many and may ultimately lead to chronic cancer-associated wasting, cachexia. In this review, we discuss the pathogenesis of cancer-related systemic inflammation, explore the role of systemic inflammation in promoting cancer growth, escaping antitumor defense, and shifting metabolic pathways, and how these changes are related to less favorable outcome.

From cachexia to obesity: the role of host metabolism in cancer immunotherapy.

PURPOSE OF REVIEW: Currently, several clinical trials in cancer therapy have demonstrated the success of immunomodulatory therapies. However, only a variable fraction of patients actually benefit from these treatments. The understanding of key mechanisms behind this response heterogeneity is one of the major unmet need and intense research field in immuno-oncology. This review will discuss the host metabolic dysfunctions derived from cachexia or obesity that can affect the response to cancer immunotherapy. RECENT FINDINGS: Preclinical studies demonstrated that chronic inflammation, nutritional intake impairment and endocrine dysfunction may affect anticancer innate and adaptive immunity, both in cachexia and obesity. New emerging clinical findings have highlighted the impact of metabolic biomarkers in predicting response to immune checkpoint inhibitors in cancer patients. SUMMARY: Patient's weight and inflammatory status could be relevant in the clinical decision-making process before starting cancer immunotherapy and for an effective patient selection and stratification in future clinical trials employing this class of anticancer agents.

Evaluation of the impact of cachexia on clinical outcomes in aggressive lymphoma.

Cancer cachexia is a state of involuntary weight loss and altered body composition triggered by an underlying malignancy. We sought to correlate measures of cachexia with clinical outcomes in aggressive lymphomas and to identify biological pathways involved in the cachexia phenotype for possible druggable targets. Radiographic measures of cachexia were collected in a retrospective cohort of 109 patients with aggressive B-cell lymphoma and followed for clinical outcome. We found males with sarcopenia had reduced progression-free survival (5·4 vs. 72·3 months, P < 0·0005) and overall survival (OS; 30·2 months vs. not reached, NR, P = 0·02); males with adipopenia also had decreased OS (21·6 months vs. NR, P = 0·04). A trend for increased OS was observed in female sarcopenics only (32·8 months vs. NR, P = 0·08). Additionally, we analysed a prospective cohort of 14 patients for differences in circulating molecular targets involved in various biological pathways. There was a significant correlation with cachexia for reduced serum levels of mediators within the glucose utilization [insulin -like growth factor (IGF)-binding protein 6, P = 0·04; IGF-1, P = 0·02], inflammation (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells; LIGHT, P = 0·005), and energy intake/expenditure (leptin, P = 0·004). We conclude that cachexia in patients with aggressive lymphomas has sex-specific prognostic utility and correlates with measurable changes in metabolism and immune function.

Reduced lung cancer burden by selective immunomodulators elicits improvements in muscle proteolysis and strength in cachectic mice.

Identification of to what extent tumor burden influences muscle mass independently of specific treatments for cancer-cachexia remains to be elucidated. We hypothesized that reduced tumor burden by selective treatment of tumor with immunomodulators may exert beneficial effects on muscle wasting and function in mice. Body and muscle weight, grip strength, physical activity, muscle morphometry, apoptotic nuclei, troponin-I systemic levels, interleukin-6, proteolytic markers, and tyrosine release, and apoptosis markers were determined in diaphragm and gastrocnemius muscles of lung cancer (LP07 adenocarcinoma cells) mice (BALB/c) treated with monoclonal antibodies (mAbs), against immune check-points and pathways (CD-137, cytotoxic T-lymphocyte associated protein-4, programed cell death-1, and CD-19; N = 10/group). Nontreated lung cancer cachectic mice were the controls. T and B cell numbers and macrophages were counted in tumors of both mouse groups. Compared to nontreated cachectic mice, in the mAbs-treated animals, T cells increased, no differences in B cells or macrophages, the variables final body weight, body weight and grip strength gains significantly improved. In diaphragm and gastrocnemius of mAbs-treated cachectic mice, number of apoptotic nuclei, tyrosine release, proteolysis, and apoptosis markers significantly decreased compared to nontreated cachectic mice. Systemic levels of troponin-I significantly decreased in treated cachectic mice compared to nontreated animals. We conclude that reduced tumor burden as a result of selective treatment of the lung cancer cells with immunomodulators elicits per se beneficial effects on muscle mass loss through attenuation of several biological mechanisms that lead to increased protein breakdown and apoptosis, which translated into significant improvements in limb muscle strength but not in physical activity parameters.

Toxoplasma gondii infection triggers chronic cachexia and sustained commensal dysbiosis in mice.

Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive lean weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness, they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role in sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute to chronic cachexia development, pathology and potential reversal.

Contribution of STAT3 to Inflammatory and Fibrotic Diseases and Prospects for its Targeting for Treatment.

Signal transducer and activator of transcription (STAT) 3 plays a central role in the host response to injury. It is activated rapidly within cells by many cytokines, most notably those in the IL-6 family, leading to pro-proliferative and pro-survival programs that assist the host in regaining homeostasis. With persistent activation, however, chronic inflammation and fibrosis ensue, leading to a number of debilitating diseases. This review summarizes advances in our understanding of the role of STAT3 and its targeting in diseases marked by chronic inflammation and/or fibrosis with a focus on those with the largest unmet medical need.

Doxorubicin triggers splenic contraction and irreversible dysregulation of COX and LOX that alters the inflammation-resolution program in the myocardium.

Doxorubicin (DOX) is a widely used drug for cancer treatment as a chemotherapeutic agent. However, the cellular and integrative mechanism of DOX-induced immunometabolism is unclear. Two-month-old male C57BL/6J mice were divided into high- and low-dose DOX-treated groups with a maintained saline control group. The first group was injected with a high dose of DOX (H-DOX; 15 mg·kg-1·wk-1), and the second group was injected with 7.5 mg·kg-1·wk-1 as a latent low dose of DOX (LL-DOX). H-DOX treatment led to complete mortality in 2 wk and 70% survival in the LL-DOX group compared with the saline control group. Therefore, an additional group of mice was injected with an acute high dose of DOX (AH-DOX) and euthanized at 24 h to compare with LL-DOX and saline control groups. The LL-DOX and AH-DOX groups showed obvious apoptosis and dysfunctional and structural changes in cardiac tissue. Splenic contraction was evident in AH-DOX- and LL-DOX-treated mice, indicating the systems-wide impact of DOX on integrative organs of the spleen, which is essential for cardiac homeostasis and repair. DOX dysregulated splenic-enriched immune-sensitive lipoxygenase and cyclooxygenase in the spleen and left ventricle compared with the saline control group. As a result, lipoxygenase-dependent D- and E-series resolvin precursors, such as 16HDoHE, 4HDoHE, and 12-HEPE, as well as cyclooxygenase-mediated PG species (PGD2, PGE2, and 6-keto-PG2α) were decreased in the left ventricle, suggestive of defective immunometabolism. Both AH-DOX and LL-DOX induced splenic contraction and expansion of red pulp with decreased CD169+ metallophilic macrophages. AH-DOX intoxicated macrophages in the spleen by depleting CD169+ cells in the acute setting and sustained the splenic macrophage loss in the chronic phase in the LL-DOX group. Thus, DOX triggers a vicious cycle of splenocardiac cachexia to facilitate defective immunometabolism and irreversible macrophage toxicity and thereby impaired the inflammation-resolution program. NEW & NOTEWORTHY Doxorubicin (DOX) triggered splenic mass loss and decreased CD169 with germinal center contraction in acute and chronic exposure. Cardiac toxicity of DOX is marked with dysregulation of immunometabolism and thereby impaired resolution of inflammation. DOX suppressed physiological levels of cytokines and chemokines with signs of splenocardiac cachexia.

Allylpyrocatechol attenuates methotrexate-induced hepatotoxicity in a collagen-induced model of arthritis.

The cornerstone of treatment for rheumatoid arthritis is low dose methotrexate (MTX), but its use is limited by concerns regarding its potential for hepatotoxicity. Allylpyrocatechol (APC), a phytoconstituent sourced from leaves of Piper betle demonstrated antioxidant, anti-inflammatory, and antiarthritic properties. The present study aimed to evaluate the combined effect of APC and MTX on limiting progression of lipopolysaccharide accelerated collagen-induced arthritis, along with reduction of MTX-induced hepatic damage. A collagen-induced arthritis (CIA) model was established by immunising Sprague-Dawley rats with bovine collagen type II (CII) and lipopolysaccharide, followed by a booster dose of CII on day 15. Rats from days 11-27 were administered APC (20 mg/kg), methotrexate (1.5 mg/kg), or a combination of MTX and APC. The combinatorial therapy of APC and MTX significantly improved the parameters of arthritis as evident from the reduction in paw oedema and arthritic score and was endorsed by radiological and histopathological changes. This combination prevented the rise in levels of proinflammatory cytokines, tumour necrosis factor (TNF-α), and interleukin 6 (IL-6). Furthermore, unlike MTX-monotherapy, the APC-MTX combination decreased the associated cachexia, splenomegaly, and oxidative stress. Importantly, the hepatic damage mediated by MTX monotherapy was effectively attenuated by the inclusion of APC. Taken together, antioxidants such as APC when combined with MTX not only potentiated the antiarthritic effect but importantly alleviated the MTX-induced hepatic damage, thus endorsing its effectiveness in preventing progression of articular diseases such as rheumatoid arthritis.