Introduction

Chemotherapy is a commonly prescribed treatment for cancer, however it is associated with serious adverse effects that influence patient well-being and clinical outcomes [1]. Affecting the entire alimentary tract (AT) from mouth to anus [2], mucositis presents with symptoms such as mouth ulcers, intractable vomiting, diarrhoea and pain, which are complicated by weight loss and infectious events such as sepsis [2]. Mucositis affects almost half of all patients receiving standard dose chemotherapy and 100% of those receiving high-dose chemotherapy or total body irradiation [3]. These patients are at a greater risk of treatment complications requiring significant supportive care methods to be adopted, thus adding to the growing economic burden of cancer therapy [4]. Although occurring in response to most anti-cancer agents, including newer targeted agents, mucositis is most problematic following 5-fluorouracil (5-FU) and irinotecan (CPT-11), with rates of severe mucositis exceeding 15% [2], thus limiting their widespread clinical implementation.

Although the mechanisms underpinning mucositis remains poorly understood [5], it is thought to involve interactions between the resident microflora, epithelial barrier function and the mucosal immune system [6]. This physiological balance requires a basal activation state of pattern recognition receptors, known as Toll-like receptors (TLRs), which limit inflammatory responses in intestinal epithelial cells (IEC) [7]. Conversely, TLRs are essential for triggering an appropriate immune response to infection [8], which manifests as inflammation initially. This is currently proposed to underpin a number of gastrointestinal (GI) diseases including inflammatory bowel disease (IBD), and as such is a working hypothesis for the damage that occurs in the GIT following chemotherapy. Damage to the cells by chemotherapy releases danger signals that result in multiple inflammatory events [5] causing activation of TLRs, especially TLR4 [1]. This elicits upregulation and release of inflammatory cytokines such as interleukin-1β, interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) via the transcription factor, nuclear factor-κB (NF-κB) [9]. IL-6 is considered a hallmark of mucositis [10] and a variety of other inflammatory disorders [11], due to its pro-inflammatory properties [12]. Although association between TLR4-mediated IL-6 production and mucositis has been established [13] it is unclear if the protective effects of TLR4 deletion act through the blockade of IL-6 production directly. This review will therefore outline current evidence for the potential involvement of the TLR4/IL-6 signalling pathway in GI mucositis development.

Mucositis

Clinical and economic impact

Although able to affect any part of the AT, mucositis is often separated into oral mucositis (OM) and gastrointestinal mucositis (GIM) [14]. Both OM and GIM are thought to stem from common underlying mechanisms, underpinned by irreversible DNA damage, both histopathological and symptomatic differences do exists owing to the unique specialities of these distinctly different parts of the AT. OM often presents as erythematous and ulcerative lesions of the oral mucosa observed in patients with cancer being treated with chemotherapy or radiotherapy of the oral region [15]. These impact daily activities such as speaking, eating and drinking, and predispose to oral infections. In the large majority of cases, however, GIM is considered more impactful to the patient given the overriding symptom of diarrhoea +/− rectal bleeding, with not only impacts on patient comfort but also predisposition to infection, dehydration, weight loss, pain and kidney failure. Even in its most mild form, diarrhoea renders people housebound due to embarrassment, pain, fatigue, dehydration and simply the fear of needing to defecate suddenly. This can exacerbate social pressures, preventing active participation in the workforce (or school for younger patients), and lead to psychological distress and personal economic burden. In severe cases, diarrhoea is not adequately managed in the outpatient setting, requiring hospitalisation and extensive supportive care measure to prevent infectious complications and severe dehydration. The persistence of these symptoms often requires dose reductions, delays or complete cessation of treatment, thus impacting on the oncological outcomes for the patient [2, 13].

Economically, the management of mucositis remains a significant burden at the personal, societal and governmental level. Although the true impact of diarrhoea is difficult to quantify, conservative estimates suggest that for each diarrhoea-related episode there are an additional AUD1300 Medicare-related costs [16]. This estimate does not consider lost income due to time off work, lost productivity of carers and the use of complementary medicines; thus, the associated financial toxicity is likely much greater [17, 18].

Pathogenesis

The pathogenesis of mucositis (both OM and GIM) comprises a complex five-phase model proposed by Sonis [9, 19]. Direct DNA damage and subsequent cell death initiates a cascade of events, resulting in the production of reactive oxygen species (ROS), damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), including secretory products, high mobility group box 1 (HMGB1) protein, DNA and RNA from bacteria, virus and fungi, and lipopolysaccharides (LPS) from Gram-negative bacteria [20]. DAMPs and PAMPs serve to activate TLRs [9], leading to primary damage response characterised by activation of transcription factor, NF-κB [10]. NF-κB activation is a key driver of mucositis development [9]. This is followed by a signal amplification phase, with a pro-inflammatory ‘storm’ of cytokines such as IL-6, TNF-α and IL-1β [10], which cause enhanced tissue damage, leading to the phase of ulceration that is the clinically relevant stage, as it is paralleled by major symptoms. This is supported by evidence of an increase in tissue level of NF-κB and inflammatory cytokines prior to histological changes in the mucosa of chemotherapy-treated rats [21]. Healing is the final phase which occurs spontaneously upon cessation of the chemotherapy treatment; however, residual changes within the mucosa remain [9]. The whole process results in marked histological changes in the GI mucosa. Histological findings of mucositis involve characteristic apoptotic bodies, villous blunting, and crypt degeneration throughout the GI mucosa; as seen in the jejunum and colon of rats treated with irinotecan [22]. Restoration of the mucosa in the GIT of the rats was evident 120 h after irinotecan administration, indicated by the return of architectural integrity [22].

Intestinal barrier dysfunction and bacterial translocation

Although not directly addressed in the current model of mucositis, a growing body of evidence now indicates that the epithelial barrier properties are critical in the severity of mucositis [23, 24]. This is particularly important for the development of GIM, given the critical role of the epithelial barrier in maintaining ion/water movement and preventing bacterial translocation. Marked abnormalities in intestinal permeability have been observed clinically in patients receiving myeloablative treatment [25] and high-dose chemotherapy [26]. This leads to altered fluid movement through leak-flux mechanisms, thus contributing to diarrhoea development [27]. This breakdown of the mucosal barrier also enables pathogens to penetrate the epithelium [22]; resulting in bacterial translocation which can predispose to systemic infection and secondary complications [28]. Guabiraba et al. [29] reported that irinotecan induces direct epithelial cell damage by modulating the release of IL-33. IL-33, in turn, stimulates chemokine production leading to tissue damage and bacterial translocation. This concept has been studied further in animal models, with bacteria found in the mesenteric lymph nodes of chemotherapy-treated rats [22]. Further evidence supporting the role of bacterial translocation in the pathogenesis of GIM is provided by an iatrogenic cohort study, in which elevated levels of LPS were found in the peripheral circulation of patients with haematological malignancies, signifying bacterial invasion [30]. Escherichia coli, a Gram-negative organism commonly resident in the lower intestine, was most frequently isolated from the blood of bacteraemic patients [31].

Gut microbiome and mucositis

About 100 trillion microorganisms [32] inhabit the GIT, existing in a finely tuned balance of commensal, pathobiont and pathogenic species. The microbiome has been increasingly considered for its role in the development of GI mucositis due to its ability to modify the host’s local and systemic immune system [33] and metabolise certain chemotherapeutic agents [5, 34]. As such, significant research efforts have been directed towards characterising the changes that occur in the microbiome following chemotherapy.

It is now understood that chemotherapy causes dysbiotic changes in the microbiome with a shift from β-glucuronidase inhibiting Bifidobacterium spp. towards β-glucuronidase producing Salmonella spp. and E. coli, corresponding with diarrhoea [35]. Studies have shown that lactobacillus spp. decreases 12–48 h after treatment with irinotecan, while E. coli increases as measured by PCR [36]; these changes in commensal bacteria correlate with irinotecan-induced diarrhoea [12]. Although the specific species involved in symptom generation is unclear, there is uniformity in the overall shift in the microbiome that occurs following chemotherapy, dominated by Gram-negative pathogenic species in both preclinical [37] and clinical settings [38, 39]. This is also accompanied by an overall loss in species diversity, enabling the proliferation and colonisation of opportunistic pathogens. Mechanistically, however, the causal relationship between gut dysbiosis and symptom generation remains difficult to achieve. Research using germ-free mice, or antibiotic-induced depletion of the microbiome, has produced conflicting results. In the setting of irinotecan-induced diarrhoea, germ-free mice have shown significantly improved diarrhoea compared to their wild-type counterparts [34]. Importantly, this protection was reversed following re-colonisation of the germ-free mice. However, clinically, the use of prophylactic antibiotics is typically associated with worsened mucositis severity [13, 40].

Further research on the key role of intestinal dysbiosis in chemotherapy-induced mucositis led to the concept that normalisation of intestinal homeostasis could be an appropriate strategy to reduce chemo-toxicity [41], with the obvious investigation of probiotics [41]. Evidence in human [42, 43] and animal [44] studies suggests that probiotics have a role in the prevention of mucositis and they rarely cause sepsis [45]. A randomised clinical study by Valta and co-workers [42] showed that colorectal cancer patients treated with 5-fluorouracil-based regimens who received Lactobacillus preparations presented with diarrhoea less often. A small placebo-controlled trial of B. breve strain Yakult in children undergoing chemotherapy also demonstrated that the treatment group experienced fewer episodes of fever compared with controls [43]. Bowen and colleagues found that VSL#3, which contains a mixture of Streptococcus thermophilus, Bifidobacterium breve, B. longum, B. infantis, Lactobacillus paracasei, L. delbrueckii subsp. bulgaricus, L. acidophilus and L. plantarum, reduced diarrhoea and weight loss in irinotecan-treated rats and inhibited intestinal apoptosis [44]. However, two recent meta-analyses only reported beneficial results for prophylactic probiotics for radiation-induced GIM, with unclear support in the setting of chemotherapy [46, 47]. This likely reflects the heterogeneity that exists in various chemotherapy regimens, agents and doses; all of which are likely to involve different microbial phenotypes. Nonetheless, there is clear evidence for the correlative involvement of the microbiome in the development of chemotherapy-induced GIM, and as such TLRs are considered critical in its symptomology [23, 48].

Toll-like receptors in the GI tract

Recent research has acknowledged an emerging role for pattern recognition receptors (PRR) [8, 49] such as TLRs, in the setting of chemotherapy-induced mucositis [13]. Typically expressed on the plasma and intracellular membranes of innate immune cells, epithelial cells, endothelial cells and cancer cells [20], TLRs play a key role in a number of inflammatory disorders affecting the GIT [50]. This is also the case for mucositis [51], with increasing evidence implicating various members of the TLR family in its pathogenesis. Among 13 known members of the TLR family, 10 are known to be present in humans [32] of which TLR4 is of particular importance in the setting of chemotherapy-induced mucositis, specifically irinotecan-induced mucositis [48].

Activation of TLR4 and its co-receptors CD-14 and MD-2 [5] by the toxic metabolite of irinotecan, SN-38, and LPS [1], involves recruitment and activation of adapter protein myeloid differentiation protein (MyD88) [49, 50]; leading to initiation of an inflammatory cascade involving NF-κB and IL-6 [9, 10]. Excessive activation of TLR4 has been suggested to exacerbate the production of inflammatory cytokines leading to a vicious cycle of tissue destruction through increased inflammation [5]. It is this process that is thought to underpin mucositis [30]; however, this hypothesis remains predominantly based on preclinical research with only anecdotal clinical evidence supporting a role for TLR4.

TLR4 and mucositis

Tight control of basal TLR4 signalling in the healthy gut is essential to maintain intestinal homeostasis and physiological state [5]. Extensive data exist that imply paradoxical roles for TLR4 in different inflammatory-based pathologies including inflammatory bowel disease. Expression of TLR4 is found to increase in the gut during peak injury and undetectable during healing [13]. However, intact TLR4 signalling through MyD88 is important in limiting bacterial translocation, with increased sepsis seen in TLR4 knockout mice compared to wild-type mice [50].

In the setting of chemotherapy-induced mucositis, substantial anecdotal evidence exists suggesting a role for TLR4, yet few studies have directly assessed TLR4-dependent mechanisms. [52] reported a significant increase in TLR4 mRNA expression in the small intestine of methotrexate-treated rats during the acute phase of gut toxicity. These changes coincided with a pro-inflammatory setting and increased permeability [52]. In addition, studies indicate upregulated TLR4 mRNA, NF kB, IL-1β and IL-6 [10] levels in the colon of dark agouti rats following irinotecan [4, 53]. Some studies suggest that TLR4 deletion has a protective role in the setting of chemotherapy-induced mucositis [13]. This concept is supported by an experiment where TLR4 knockout mice had significant reductions in diarrhoea, weight loss [54] and intestinal apoptosis as compared to wild-type mice after irinotecan administration [13]. Conversely, pre-treatment with the TLR4 agonist, LPS, has shown to be protective against radiotherapy-induced intestinal injury in mice [55].

TLR4-mediated protective effects seem to be limited to acute insults, with TLR4 deficiency exacerbating chronic inflammatory diseases [13]. Furthermore, previous studies have shown that a TLR4 knockout mouse model was associated with increased breast tumour growth during treatment with oxaliplatin and doxorubicin [54]. In addition, patients with a TLR4 genetic deficiency had an increased incidence of metastases 5 years after surgery for breast cancer [54]. Pre-clinical trials have also been conducted that for the first time indicate that a non-specific TLR4 antagonist, naloxone, is not effective in reducing irinotecan-induced mucositis [54]. In another study involving a model of dextran sulphate sodium- (DSS) induced colitis, mice deficient in TLR4 or MyD88 exhibited diminished tissue repair [51]. Hence, these contradictory studies strongly suggest that activation of TLRs could be a double-edged sword that can be either beneficial or detrimental to intestinal integrity [28], with the efficacy of TLR targeted interventions depending on the setting (e.g. health vs cancer) and chronicity of symptoms. It also suggests that pharmacological inhibition of TLR4 may not be the best approach in the prevention of GI toxicity. Instead, it may be more beneficial to target a product of TLR4 signalling that confers intestinal protection and resistance to toxicity. Based on previous findings in the field of GIM, we suggest that this may be achieved through targeting IL-6.

TLR4/IL-6 interactions and their relevance to mucositis

IL-6 is an important cytokine in the TLR4-mediated inflammatory pathway of mucositis, particularly affecting the gut [49]. Being a member of a trio of cytokines of acute inflammation (IL-1β, TNF-α, IL-6) [56]; it is unique in having both pro-inflammatory and anti-inflammatory properties [56]. The primary source of IL-6 is monocytes, tissue macrophages at the site of acute inflammation and T cells at the site of chronic inflammation [56]. Although IL-1β and TNF-α also constitute stimulus to produce IL-6 through activation of NF-κB [56], TLR4 activation is one of the earliest events leading to IL-6 production [57].

The anti-inflammatory action of IL-6 is mediated via a classical (cis) signalling pathway and pro-inflammatory property is mediated via a trans signalling pathway [56]. In cis-signalling, binding of IL-6 to IL-6R recruits a membrane-bound glycoprotein 130 (gp130) co-receptor that initiates downstream signalling, including Janus kinases (JAKs) and finally phosphorylation and activation of STAT3 [49]. STAT3 further releases IL-6 directly and indirectly via a TLR4-mediated pathway (Fig. 1). Hepatocytes and some immune cells have IL-6R and thus respond to cis-signalling [56]. This pathway promotes growth differentiation, regeneration and induction of apoptosis in neutrophils that resolves acute inflammation [56]. Alternatively, IL-6 can function through a pathway known as trans-signalling by binding to the soluble form of IL-6R (sIL-6R). This complex also interacts with gp130 and activates the same intracellular pathways as cis-signalling [49]. This pathway is responsible for the pro-inflammatory property of IL-6 as gp130 is present on all body cells; it causes accumulation of T cells resistant to apoptosis, migration and accumulation of inflammatory cells and production of TNF-α and IL-1β [56].

Fig. 1
figure 1

Pathways for IL-6 production. Direct IL-6 production occurs through the activation of a receptor formed by two distinct subunits, an alpha subunit for ligand specificity and glycoprotein (GP) 130. Binding of IL-6 to its receptor (IL-6R) initiates cellular events including JAK kinases, which when phosphorylated, activates signal transducers and activators of transcription-3 (STAT3). This then translocates to the nucleus resulting in the production of IL-6. Indirect IL-6 production is mediated through innate immune receptor, Toll-like receptor 4 (TLR4). TLR4 is expressed on glial, immune and intestinal epithelial cells with bacterial lipopolysaccharide (LPS) as its primary ligand. Upon LPS binding, TLR4 undergoes a conformational change resulting in the recruitment of TIR domains containing adaptor molecules. In particular, myeloid differentiation primary response (MyD)88-dependent signalling results in nuclear translocation of nuclear factor kappa B (NF-κB) and the subsequent production of IL-6 [57]

Full size image

The same mechanism underlies a variety of inflammatory disorders like inflammatory bowel disease (IBD) and rheumatoid arthritis [49], where IL-6 trans-signalling is critically involved in the maintenance of a disease state [56]. This concept is further supported by findings where high expression of IL-6 mRNA was seen in inflamed mucosa of IBD patients as compared to healthy controls and even in normal mucosa adjacent to inflamed mucosa [11]. The same trans-signalling is believed to be involved in the pathogenesis of chemotherapy-induced mucositis [56]. Logan et al. [10, 53] found elevated levels of NF-κB and inflammatory cytokines, TNF-α, IL-1β and IL-6 in serum and AT (both buccal mucosa and intestinal tissue) of rodents after administration of irinotecan, 5-fluorouracil and methotrexate. Similar findings were seen in adults receiving chemotherapy [21] and children having treatment for acute lymphoblastic leukemia, where an increase in blood levels of IL-6 and TNF-α was seen compared to healthy controls, 72 h after administration of methotrexate [58].

Keeping in view these findings, one can suggest that polymorphonuclear inflammatory cells recruited by IL-6 might be a relevant component of chemotherapy-induced GIM. Also, this inflammatory reaction of IL-6 seems to be mediated through TLR4 activation [57]. This concept is further supported by a study in which expression of intestinal IL-6 was significantly reduced in TLR4 knockout mice as compared to wild type, 6 h after irinotecan administration [13]. In fact, TLR4 null mice completely lacked an IL-6 response, supporting the hypothesis that TLR4-dependent NF-κB activation is required for the release of IL-6 from macrophages [59]. Stunted IL-6 production has also been seen in MyD88-deficient mice [60] and TLR4-null macrophages [61], implicating TLR4 in the regulation of IL-6. Hence, IL-6 is recognised as a pivotal component of GIM through its pro-inflammatory properties and TLR4 constitutes a major pathway for its production.

Important considerations for systemic IL-6 blockade

We have outlined a strong body of evidence supporting investigation into IL-6 blockade for the prevention of GIM. However, this approach must be considered in the larger setting of supportive oncology. IL-6 is not exclusively linked to mucosal injury in the gastrointestinal tract. Certainly, there are roles for IL-6 in immunological cell death, critical to the efficacy of some newer targeted agents and immunotherapies. However, more importantly, IL-6 is also considered critical in the response to potentially life-threatening infections that underpin febrile neutropaenia. On one hand, this presents as a promising avenue in which to explore, with the opportunity to simultaneously manage febrile neutropaenia associated with GIM (also termed febrile mucositis [62]). However systemic IL-7 blockade may also mask this febrile response to infection, thus preventing rapid detection of infection in neutropaenia patients [30]. As such, targeting systemic production of IL-6 to alleviate mucositis must be approached with caution, and preclinical models must adequately incorporate measures of infection and neutropaenia in their analyses.

Conclusion

Chemotherapy-induced mucositis, affecting the GIT, is a debilitating side effect of chemotherapy which limits the therapeutic efficiency of cancer treatment. The pathobiology of GIM is complex involving innate immune responses, pharmacokinetics modulation and the gut microbiome. Data increasingly points towards TLR4 as a key regulator of GIM, particularly induced by irinotecan. However, pharmacologically targeting TLR4 and genetic inhibition have presented challenges regarding tumour growth and the inability to overcome chronic insults. Given that TLR4-null mice lack an IL-6 response, it is plausible that protection from GIM is mediated through this pathway. As such, we suggest that IL-6 may be a more promising therapeutic target to prevent or reduce the severity of GIM caused by chemotherapy and should form the basis of new research models.