Cardiotonic Steroids as a Potential Novel Approach for Immunomodulation in Inflammatory Bowel Disease

Abstract

Inflammatory bowel disease (IBD) is a chronic condition that significantly impairs the quality of life of millions of individuals. The pathogenesis of IBD is closely linked to dysbiosis of microbiota and the activation of various inflammatory pathways, which are characterized by elevated levels of activated immune cells, such as neutrophils and lymphocytes. While several therapeutic options, including corticosteroids and biologic agents, are available for the treatment of IBD, their efficacy remains limited. Consequently, the development of novel therapies is essential. In this context, cardiotonic steroids, a class of drugs traditionally known for their effects on the cardiovascular system, have gained attention due to their potential immunomodulatory properties. Thus, this review aims to explore the emerging therapeutic potential of cardiotonic steroids in the treatment of IBD.

1. Introduction

Inflammatory bowel disease (IBD) is divided into Crohn’s disease and ulcerative colitis—two distinct, variable, and disabling conditions characterized by chronic inflammation, recurrent flare-ups, and a wide range of debilitating symptoms. IBD can be defined as a non-infectious, chronic, and immune-mediated inflammatory disorder that predominantly targets the digestive tract [1,2]. This condition significantly impairs patients’ quality of life due to the corresponding immune dysregulation and sustained inflammatory dysfunction [3]. Several risk factors may contribute to the development of IBD, although none are definitive predictors [4,5].

Although symptoms of IBD most commonly manifest in individuals during their thirties and forties, the condition is also increasingly diagnosed in older adults [4,5]. The incidence of IBD varies substantially across geographic regions and age groups. According to Caron et al. [6], annual incidence rates range from 23.7 to 39.8 per 100,000 in Oceania, 10.5 to 46.1 per 100,000 individuals in Europe, 7.3 to 30.2 per 100,000 in North America, 1.4 to 1.5 per 100,000 in Asia and the Middle East, and 0.2 to 3.7 per 100,000 in South America. Projections estimate that by 2030, the prevalence of IBD could reach several million individuals in certain countries, including over 3.5 million in the United States, more than 700,000 in the United Kingdom, approximately 815,200 in Germany, and 283,930 in Australia [7].

Currently, there is no single standardized tool for diagnosing IBD [8]. Diagnosis typically requires a comprehensive combination of clinical, radiologic, endoscopic, and histopathologic assessments [9]. Over the past decade, multiple biologic agents and small-molecule therapies have become available to induce and maintain remission in patients with IBD [10]. Aiming to treat IBD, various therapies have been developed based on the modulation of these factors. Most existing treatments often involve immunosuppressant, nonsteroidal, anti-inflammatory, and steroidal drugs. These therapies regulate inflammatory signals, one of the central points of the IBD pathophysiology [10,11,12]. The drug and the treatment vary depending on the severity of the disease. For example, aminosalicylates (e.g., mesalazine and sulfasalazine) are used to manage mild-to-moderate disease [13,14,15]. Topical delivery (e.g., rectal suppositories and enema formulations) are used to treat patients with left-sided disease [16]. Oral formulations can be combined with topical formulations to control disease of the transverse and ascending colon [17]. For severe ulcerative colitis, high-dose oral corticosteroids (e.g., prednisone) are usually prescribed until symptoms stop. For severe and fulminant cases, intravenous corticosteroids are treatment mainstays [17].

In selected patients, severe ulcerative colitis may be refractory to aminosalicylates and corticosteroids [18]. Under these circumstances, janus kinase inhibitors (e.g., tofacitinib), integrin antagonists (e.g., vedolizumab and etrolizumab), cyclosporine, and anti-tumor necrosis factor (TNF) drugs (e.g., infliximab, golimumab, and adalimumab) can be used. However, among them, cyclosporine has more adverse effects [15,19,20,21]. Furthermore, antibiotics should be considered during immunosuppressive drug use [17]. Depending on disease severity and the presence of complications such as strictures or fistulas, surgical intervention may be necessary. Surgical procedures may range from segmental resection to total colectomy [22]. Vieujean et al. [23] fully reviewed the therapeutic options for IBD. Although this therapeutic arsenal is used, it is associated with numerous side effects and limited efficacy, highlighting the need for the development of new therapeutic strategies.

2. Immunological Insights into IBD

Current evidence suggests that IBD results from a multifactorial interplay between genetic predispositions and environmental influences, ultimately leading to compromised intestinal barrier integrity, dysregulated immune responses, and alterations in gut microbial composition (Figure 1) [24]. The intestinal microbiota serves as a dynamic biological barrier that enhances innate immunity, complementing the mechanical and chemical defenses of the gastrointestinal tract. Disruption of this balance may trigger inflammatory responses and contribute to the onset of various diseases [25]. Although establishing a direct causal relationship between the intestinal microbiota and IBD in humans remains challenging, the interaction between host and microbiota is critical for maintaining immune homeostasis [26].

Aberrant immune responses in IBD are associated with dysregulation of both innate and adaptive immunity [27]. In this regard, innate immune cells such as neutrophils accumulate in inflamed mucosa due to intestinal barrier disruption. They are the most abundant immune cells in human blood and are rapidly recruited to sites of infection or inflammation [27,28]. Their transmigration across the epithelium correlates with disease severity and disruption of crypt architecture in both IBD and experimental colitis [29].

Neutrophils eliminate pathogens through phagocytosis, production of reactive oxygen species (ROS), release of cytotoxic granules (e.g., myeloperoxidase, defensins, lysozyme, neutrophil elastase, proteases, and hydrolases), degranulation, and formation of neutrophil extracellular traps (NETs) [27,30,31]. NET accumulation has been linked to increased TNF-α and interleukin (IL)-1β via the ERK1/2 signaling pathway, exacerbating inflammation in IBD [32]. Neutrophil infiltration also promotes inflammation by generating ROS that damage the epithelial barrier and releasing cytokines such as IL-8, TNF-α, IL-22, and leukotriene B4, which recruit additional neutrophils and monocytes [33,34,35]. Conversely, reducing NET formation can protect against colitis by suppressing pro-inflammatory mediators [32,36]. CD177⁺ neutrophils confer protection in IBD by exerting potent antimicrobial activity while producing lower levels of pro-inflammatory cytokines (e.g., IL-6, IL-17A, IFN-γ) and higher levels of IL-22 and transforming growth factor-β (TGF-β), promoting tissue repair [28,37].

Additionally, in IBD, macrophages exhibit elevated expression of TNF-α, IL-1β, IL-6, IL-12, IL-23, and inducible nitric oxide synthase (iNOS) [38,39,40]. The recruitment of inflammatory C-C chemokine receptor type 2 (CCR2)⁺ monocytes to intestinal tissue contributes to disease severity; notably, CCR2 deficiency ameliorates dextran Sulfate Sodium (DSS)-induced colitis in mice [41]. Moreover, circulating dendritic cells migrate to secondary lymphoid organs and inflamed tissues, where they secrete IL-6, IL-8, and TNF-α, and promote pro-inflammatory T cell phenotypes [42]. Dendritic cell-derived cytokines also modulate epithelial barrier integrity, cell proliferation, and apoptosis via NF-κB2 signaling [43]. In murine DSS-induced colitis, increased mTOR activity correlates with epithelial proliferation in inflamed areas [44,45]. Notably, loss of mTOR signaling in dendritic cells impairs IL-10 production by conventional type 2 dendritic cells, heightening susceptibility to colitis [46,47].

Conventionally, Crohn’s disease is characterized by Th1-mediated immune response, and ulcerative colitis is characterized by Th2-mediated immune response. Furthermore, the Th17-mediated immune response should also be considered, contributing to intestinal inflammation in both forms of IBD, particularly in Crohn’s disease [48,49,50,51,52]. In contrast, Treg cells have tolerizing and anti-inflammatory properties, maintaining self-tolerance, and preventing autoimmunity. Specifically, this maintenance encompasses both a state of tolerance toward self-antigens and the ability to trigger antimicrobial defenses. For this, a few mechanisms act together, including reciprocal regulation of pro-inflammatory effector CD4⁺ T cells and tolerizing anti-inflammatory effects of Treg cells [52,53,54].

Th17 cytokines, such as IL-17A, IL-17F, IL-21, and IL-22, have a dual effect on IBD, attenuating or increasing effects in the gut, as shown in experimental colitis models [55]. For example, adoptive transfer of IL-17A-deficient naïve CD4⁺ T cells to mice with recombination activating gene-1 deficiency results in more severe colitis with higher expression of genes encoding Th1-type cytokines in colon tissue [56]. On the other hand, neutralization of both IL-17A and IL-17F ameliorated colitis at early administration, but administration of IL-17A or IL-17F did not [57]. Nevertheless, secukinumab, a specificIL-17A blocker, exacerbated disease activity in patients with Crohn’s disease [58,59]. Conversely, vidofludimus, an IL-17A and IL-17F blocker, ameliorated both steroid-dependent Crohn’s disease and ulcerative colitis [60]. For a more comprehensive review of IBD immunology, see Uhlig and Powrie [61].

3. Cardiotonic Steroids as Potential New Therapeutics in IBD Treatment

As described earlier, the pathogenesis of IBD involves multiple factors. One of them is gut microbiota dysbiosis, which is marked by an increase in harmful bacteria and a reduction in beneficial probiotics. Another factor is the exacerbation of inflammation, facilitated by excessive secretion of pro-inflammatory cytokines, including TNF-α and IL-1β. In addition, there is an increase in intestinal levels of reactive oxygen species, including hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH), and superoxide anions (•O²⁻)) [62,63,64]. The pleiotropic mechanisms associated with this disease open up opportunities for the development of multiple targeted therapeutic strategies.

Given the ongoing search for new therapies to treat chronic diseases with limited treatment options, drug repurposing has emerged as a promising strategy. This approach can reduce development time, lower costs, increase clinical success rates, and enable the exploration of novel therapeutic pathways [2]. In this context, cardiotonic steroids have gained attention. They are known to inhibit Na⁺/K⁺-ATPase and to promote a positive inotropic effect [65]. Although these steroids were initially used to treat heart failure and arrhythmias [66], several biological activities have been reported, including anti-inflammatory activity [67,68,69,70]. For that, cardiotonic steroid modulates the immune system mainly by affecting signal transduction [69,71].

Digoxin, an archetypal cardiotonic steroid, is still used for treatment of patients with atrial fibrillation and heart failure in low doses, as reviewed by Fender et al. [72] and Whayne [73]. It was shown that this molecule is used to suppress Th17 cell differentiation by antagonizing RORγt activity [74,75]. Moreover, digoxin also reduced the severity of experimental autoimmune encephalomyelitis in mice [74,75] and experimental arthritis in mice [76]. More recently, it has been shown that digoxin suppresses Th17-related cytokines (IL-17A and IL-17F) in colonic mucosa in murine T cell transfer-induced colitis [77]. Additionally, modulating T cell proliferation may represent a potential strategy for attenuating T cell-mediated pathogenesis. Notably, ouabain—a cardiotonic steroid previously used clinically for the treatment of heart failure—has been shown to influence T cell proliferation, possibly through the downregulation IL-2 production [78].

Ouabain has also been reported to modulate neutrophil function. Studies have shown that this molecule reduces neutrophil migration both in vitro and in vivo in response to various inflammatory stimuli, including zymosan, ovalbumin, and Leishmania spp. [67,68,69,79]. The mechanisms underlying this inhibition appear to involve a reduction in CD18 integrin expression and inhibition of the p38 mitogen-activated protein kinase protein. In addition, marinobufagenin, a cardiotonic bufadienolide steroid, also inhibits neutrophil migration into the peritoneal cavity [70]. Given the well-established role of neutrophils in IBD pathogenesis, therapeutic strategies aimed at modulating their activity may represent a promising avenue for treatment [80].

Beyond their effects on cell migration, cardiotonic steroids can also reduce levels of pro-inflammatory cytokines, such as TNF and IL-1β, many of which are associated with IBD [81]. Furthermore, recent studies have shown that a synthetic cardiotonic steroid, γ-benzylidene digoxin 8, upregulates the production of IL-10, an anti-inflammatory cytokine, in peritoneal macrophages [82]. The regulation of various cytokines is linked to the NF-κB and mitogen-activated protein kinase signaling pathways, both of which have been shown to be modulated by cardiotonic steroids. Ouabain has also been shown to reduce vascular permeability induced by inflammatory stimuli [67]. In addition, ouabain modulates tight junctions and gap junctional communication in epithelial cells [83,84]. Based on these findings, it is conceivable that similar mechanisms might contribute to the stabilization of the intestinal epithelial barrier. Although direct evidence in the context of gut epithelium remains limited, this hypothesis warrants further investigation, particularly in the setting of IBD, where epithelial dysfunction plays a central role. Notably, several immunomodulatory effects of cardiotonic steroids have been described in the peritoneal cavity [67,70,79], and, although direct evidence remains to be fully established, this may suggest a potential anti-inflammatory action in intestinal inflammatory conditions, warranting further investigation.

It is noteworthy that the anti-inflammatory and immunomodulatory effects of cardiotonic steroids, particularly ouabain, are not limited to peritoneal inflammation. Ouabain has demonstrated significant anti-inflammatory activity within the central nervous system, notably in the rat hippocampus, where it reduced messenger ribonucleic acid expression levels of iNOS and IL-1β [85] and has also been shown to modulate retinal neuroinflammation [86]. In addition, ouabain exerts protective effects in pulmonary inflammation by reducing cytokine production, inhibiting leukocyte migration, and attenuating tissue remodeling in the lungs [68,87]. Collectively, these findings reinforce the anti-inflammatory potential of cardiotonic steroids and highlight their possible application in the treatment of other inflammatory conditions.

A potential concern regarding the use of cardiotonic steroids is the possibility of dose-dependent side effects. While this is a valid point, it is important to note that many of the immunomodulatory effects of these compounds have been observed at very low doses and concentrations—often insufficient to inhibit Na⁺/K⁺-ATPase, which is primarily responsible for the more severe adverse effects [88]. Moreover, the application of advanced technologies in the development of novel pharmaceutical formulations can help mitigate adverse effects and enhance therapeutic efficacy [89].

As highlighted in a recent review by Snelson et al. [90], disruption of the heart–gut axis contributes to systemic inflammation and is closely linked to the pathophysiology of heart failure. This dynamic connection indicates that chronic inflammatory diseases of the gut, such as IBD, may exacerbate cardiac dysfunction. In this context, the knowledge about the immunomodulatory properties of cardiac glycosides, such as ouabain [67,68,69,70], could offer a therapeutic advantage beyond their classical cardiovascular effects. While these agents are not currently used in the management of IBD, their ability to modulate inflammatory pathways may provide not only local but also systemic benefits, potentially attenuating inflammatory drivers of heart–gut axis dysfunction.

4. The Gut Microbiota Plays an Important Role in Cardiotonic Steroid Bioavailability

IBD development has been linked to inflammation induced by certain intestinal microbiota, such as Clostridium difficile [91]; species from Clostridium cluster XIVa, IV, and XVIII [92,93]; colibactin-producing Escherichia coli; and Fusobacterium nucleatum [54]. Furthermore, the microbiota also influences the bioavailability of oral drugs, including digoxin [94]. Digoxin can be converted into cardioinactive forms—digoxin reduction products (DRPs)—such as dihydrodigoxin, in which the single double bond in the lactone ring of digoxin is reduced [95,96]. DRP has low affinity for the Na⁺/K⁺-ATPase. Moreover, cardiac tissue has low rate of DRPs, and DRPs can be rapidly excreted, resulting in significantly lower cardiotonic therapeutic efficacy compared to digoxin [95]. Since 1981, when the effect of gut microbiota on digoxin inactivation was first seen [96], several bacteria have been associated with this phenomenon, including Eggerthella lenta [95]. The inactivation of digoxin by E. lenta was discovered in 2013 [97]. In this context, two proteins were predicted using sequence homology and secondary structure predictions. The first, cardiac glycoside reductase (CGR) 1, exhibits homology with the NapC/NirT family of cytochrome c reductases. The second, CGR2, shares structural similarity with fumarate reductases, suggesting a potential role in redox reactions linked to digoxin metabolism [98]. The function of CGR1 is to transfer electrons from quinones to its associated terminal electron reductase partner. For this, CGR1, anchoring CGR2 to the membrane, forms a complex with CGR2, facilitating electron transfer through a flavin adenine dinucleotide redox-dependent mechanism. The digoxin-binding site is present in the CGR2 protein and contains negatively charged polar amino acids and non-polar hydrophobic residues [99]. Some molecules, such as arginine, can inhibit the conversion of digoxin to inactive forms [95,97], required for the growth of E. lenta [100]. Therefore, a high-arginine diet may be beneficial, improving digoxin bioavailability [101]. In addition, the gut microbiota can also activate Th17 cells, which can be inhibited by digoxin. For example, human gut E. lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor RORγt. Although Th17 activation varies across E. lenta strains, the activation is attributable to CGR2 that induces IL-17A [102].

5. Final Considerations and Future Directions

Considering the multiple pathways involved in the pathogenesis of IBD and the pressing need for new therapies to address this complex inflammatory condition (Figure 2), the investigation of cardiotonic steroids, a group of molecules with pleiotropic immunomodulatory effects, emerges as a plausible and promising strategy in the search for novel IBD treatments.

Thus, to advance this approach with a focus on drug repurposing, several strategies can be considered. These include the analysis of electronic medical records and public databases to identify retrospective evidence supporting the efficacy of cardiotonic steroids in IBD; the assessment of the cost-effectiveness of this potential treatment—particularly relevant given that drugs such as digoxin are already in therapeutic use and may offer a financially viable option; and the careful selection of molecular targets and disease biomarkers to evaluate and monitor therapeutic responses.

We also emphasize that the use of cardiotonic steroids in the treatment of IBD remains a hypothesis-generating consideration, and further preclinical and clinical studies are necessary to determine their potential translational applicability in this context.