Intestinal mucositis represents a common complication and dose-limiting toxicity of many anticancer drugs. The overall incidence of mucositis varies and frequencies up to 89% have been reported for patients undergoing bone marrow transplantation who were receiving high-dose chemotherapy . The condition may affect the entire alimentary tract and cause a wide range of symptoms, including severe pain, mouth ulceration, abdominal bloating, nausea/vomiting, diarrhea/constipation and malnutrition with weight loss. Furthermore, patients with gastrointestinal mucositis are at increased risk of infection and episodes of bleeding . Costs of treatment increase significantly with mucositis severity . Unscheduled chemotherapy dosage and timing adjustments because of poor treatment tolerance are common clinical practice, but may negatively affect cancer outcome. Thus, chemotherapy-mediated gastrointestinal toxicity can have a severe adverse impact on the quality of life, morbidity and mortality of cancer patients. Although practice guidelines are available to help clinicians in the selection of effective management strategies , there is so far no therapeutic intervention that successfully prevents or treats all symptoms of disease.
The precise pathophysiology underlying chemotherapy-induced intestinal mucositis has not been clarified in detail yet. Current understanding suggests the culmination of a dynamic five-phase sequence of complex inflammatory events that are initiated by direct or indirect processes of chemotherapy-induced damages to crypt cells and cells in the underlying mucosal tissue . In brief, mucosal injury by chemotherapy and drug metabolites induce DNA damage, excessive reactive oxygen species (ROS) production and apoptosis. Endogenous danger signals are released by injured cells, which imbalance mucosal homeostasis by triggering aberrant innate immune activation of nuclear factor (NF)-κB (and other transcription factors). A proinflammatory ‘storm’ of cytokines and matrix metalloproteinases accelerates intestinal mucosal damage and barrier dysfunction. Gut bacteria may colonize ulcers and translocate, resulting into expansion of specific mucosal immune cells that further exaggerate destructive inflammatory responses. Finally, mucosal healing spontaneously occurs upon cessation of chemotherapy. Recent findings [6▪] suggest that type 3 innate lymphoid cells are required for complete mucosal regeneration and restitutio ad integrum by shielding small intestinal Lgr5+ stem cells and progenitors from the adverse effects of chemotherapeutic insults via STAT3–IL-22 signaling.
Growing evidence implies that multidirectional interactions between gut microbiota and the host innate immune system may influence the development and progression of chemotherapy-induced intestinal inflammation. Within the healthy gastrointestinal tract, the microbial community (microbiota) has a mutually beneficial relationship with the host. Although the resident intestinal microbiota helps to maintain mucosal barrier homeostasis through limited inflammatory and accelerated healing responses, immune cells of the intestinal mucosa influence the commensal composition and function . Metabolic products of anaerobic bacterial fermentation, such as butyrate, may exert anti-inflammatory effects, which protect against chemotherapy-induced mucosal damage . During chemotherapy exposure, severe disruption of this fine-tuned symbiotic partnership may occur, resulting in loss of commensal tolerance and inappropriate immune responses with increased barrier permeability, which perpetuate mucositis.
Commensal microbiota affect anticancer drug metabolism by multiple mechanisms [9▪▪]. The gut microbiota may have impact on pharmacology and host (de)toxification of drugs and related metabolites by modifying delivery, absorption and availability and altering biotransformation through induction of host drug-metabolizing enzymes and transporters [10▪▪], thus either increasing or decreasing toxicity of cancer chemotherapy. In addition, chemotherapy drugs may compromise the protective colonic mucus layer, impairing antimicrobial host defense mechanisms and thus diminishing bacterial clearance . Furthermore, xenobiotics may directly shape the bacterial community structure and activity. Chemotherapeutic agents can reduce the number and diversity of microbiota  and cause dysbiosis , an alteration in the composition of the microbiota, which allows rare microbial species to overgrow, turning the gut microbiota into a disease-accelerating entity that may promote aberrant innate immune signaling in the intestinal mucosa during the development of gastrointestinal mucositis.
Conserved structures from gut microbes, so-called pathogen-associated molecular patterns (PAMPs) are recognized by Toll-like receptors (TLRs), a class of innate pattern recognition receptors (PRRs) present in various cell types of the intestinal mucosa. TLRs also sense danger signals from dead or injured host cells. Upon activation, TLRs recruit diverse adaptor proteins (MyD88, TRIP, TIRAP/MAL, TRAM and SARM). Subsequent transcriptional activation of TLR target genes encoding pro-inflammatory and anti-inflammatory cytokines and chemokines, effector molecules and type I interferons initiate the activation of antigen-specific and nonspecific adaptive immune responses . Recent findings, mainly derived from murine colitis models, have shown that the net outcome of TLR signaling in the gut is entirely context-dependent and cell/tissue-type specific . Genotoxic stress induced by chemotherapeutic agents elicits different changes in TLR expression that are cell line-specific and damage-specific . Paradoxically, TLRs may mediate both protective and destructive responses in the intestinal mucosa. Tight control of basal TLR signaling in the healthy gut is essential to avoid otherwise deleterious activation and ensure rapid tissue repair. Failure of control deregulates basal TLR signaling that imbalances commensal-dependent mucosal barrier homeostasis, facilitating inflammatory injury. This review focuses on recent advances in our understanding of the ambivalent microbial-innate immune influences via distinct TLRs in the pathophysiology of chemotherapy-induced intestinal mucositis.
TOLL-LIKE RECEPTOR 2-MEDIATED MUCOSAL PROTECTION AGAINST INTESTINAL CHEMOTOXICITY
TLR2, a member of the TLR family, recognizes conserved molecular patterns associated with both gram-negative and gram-positive bacteria, including lipopeptides/proteins. In general, triacylated lipopeptides are recognized by TLR2/TLR1, whereas diacylated lipopeptides use TLR2/TLR6 heteromers for signaling. TLR2 is differentially expressed by many distinct cell types throughout the healthy gastrointestinal tract, including enterocytes, goblet cells and different subsets of lamina propria mononuclear cells . Recent reports have identified several TLR2-dependent mechanisms that promote intestinal epithelial cell (IEC) wound healing (barrier integrity, survival, cell-cell communication and restitution) and limit proinflammatory immune responses during acute and chronic murine colitis [17,18]. The probiotic Lactobacillus rhamnosus GG, which may modulate immune functions through direct activation of TLR2, has been shown to protect mice against radiation-induced or chemotherapy-induced small intestinal IEC injury by enhancing crypt survival through COX-2-dependent  and Nrf2-dependent [20▪] signaling pathways.
The ABC transporter P-glycoprotein (P-gp; ABCB1 or MDR1) actively pumps a broad range of chemically diverse compounds out of the cell, protecting the intestinal mucosa against xenobiotics and metabolites, including chemotherapy drugs. Absence of drug-transporting P-gp can result in cellular accumulation of xenobiotics and increased toxicities with severe side-effects . Gut bacteria may modulate host P-gp function . Stimulation of TLR2 induces sustained P-gp synthesis and activity in monocytes and macrophages of the lamina propria, thus essentially reducing drug-mediated cell death [23▪]. Absence of TLR2 signaling (by either genetic deletion or depletion of the indigenous gut microbiota by antibiotics) dramatically aggravates chemotherapy-induced intestinal mucositis, which correlates with loss of P-gp in the inflamed mucosa. However, oral supplementation with a specific TLR2 agonist during chemotherapy preserves P-gp expression and limits genotoxic stress-induced damage in the small intestine. Taken together, this recent study [23▪] implies that commensal-mediated innate immune modulation via TLR2 signaling may accelerate host detoxification by instructing cells to expel harmful chemotherapy drugs, thus critically controlling the severity of cancer therapy-induced mucosal damage in the gastrointestinal tract.
Future studies will need to investigate whether manipulating TLR2 may indeed represent a useful therapeutic approach in order to ameliorate side-effects in the gut during high-dose chemotherapy. So far, only one specific chemotherapy drug (methotrexate – MTX) has been tested in this context [23▪]. It remains to be shown whether TLR2 signaling could be efficiently targeted for the prevention and treatment of intestinal mucositis induced by other chemotherapy drugs that are P-gp substrates. TLR2 may also regulate the expression of many other ATP-dependent drug transporters in a tissue-specific manner. For instance, activation of constitutive androstane receptor (CAR), a xenosensor that induces the ABCC2 and ABCC3 genes, is inhibited by TLR2 signaling in the liver , while activation of pregnane X receptor (PXR), another xenosensor which is involved in transcriptional activation of ABCB1/MDR1, may increase gene expression of TLR2 (and other PRRs) in vascular endothelial cells . These findings suggest that multidirectional signaling pathways between drug metabolism and innate immunity exist. However, the exact signaling mechanisms via TLR2 (and other TLRs) remain to be determined.
An additional concern has arisen: induction of P-gp represents the principal mechanism underlying multidrug resistance, which is a key cause of treatment failure in cancer . TLR2 is expressed on many types of cancer cells. Thus, excessively activated TLR2 could lead to an undesirable dose reduction of the anticancer drug via P-gp, which would hamper therapeutic effectiveness and compromise survival outcome. It will be pivotal to rule out the possibility that using TLR2 agonists in the treatment of chemotherapy-induced intestinal mucositis may drive manifestation of multidrug resistance and subsequent tumor progression.
TOLL-LIKE RECEPTOR 4-MEDIATED MUCOSAL DESTRUCTION IN INTESTINAL CHEMOTOXICITY
Cancer chemotherapy drugs exert cytotoxic and pro-immunogenic effects in normal and malignant cells. Dead or injured cells release numerous endogenous damage-associated molecular patterns (DAMPs), such as HMGB1 , ROS  and uric acid , which induce sustained innate immune activation, significantly contributing to uncontrolled mucosal inflammation during repeating cycles of chemotherapy treatment. The main host receptor that senses DAMPs and responds to tissue damage is TLR4. In addition, TLR4 is the major receptor for lipopolysaccharide (LPS) recognition, which requires the presence of accessory receptors (MD-2, CD14 and LPS-binding-protein). Abnormal or deficient TLR4 signaling may impair the antibacterial host response, facilitating colonization and invasion of the intestinal mucosa by gut bacteria . Acute endotoxemia, experimentally induced by systemic administration of LPS, has been shown to exacerbate MTX-induced intestinal mucositis in mice . Gene expression of TLR4 is significantly upregulated in the inflamed small intestinal mucosa after exposure to the chemotherapeutic agent MTX [23▪,31]. Sterile injury resulting from chemotherapy may prime mucosal immune cells to hypersensitivity through upregulation of TLR4 mediated by augmented release of DAMPs and production of proinflammatory cytokines, like IFNγ . Excessive activation of TLR4 may further enhance mucosal production of inflammatory cytokines/chemokines, proteases and ROS, leading to a vicious cycle that causes self-tissue destruction through increased inflammation. Lack of signaling via PXR [33▪] or TLR2 [23▪] may also lead to mucosal hyperresponsiveness to LPS via TLR4, implying that defects in xenobiotic defense via ABCB1/MDR1 P-gp signaling may be involved in triggering compromised tolerance to LPS. Mice deficient in the essential TLR4-coreceptor MD-2 [23▪] or mice that harbour a defective TLR4 (C3H/HeJ)  are indeed resistant to MTX-induced gastrointestinal damage. Collectively, these data suggest that aberrant innate immune signaling via MD-2/TLR4 is required for the severity of chemotherapy-induced intestinal damage, at least in the case of MTX.
So far, only indirect proof exists that aberrant TLR4 signaling may also be involved in the pathogenesis of mucositis induced by other chemotherapy drugs. CPT-11 (irinotecan) represents a first-line chemotherapeutic drug for metastatic colorectal cancer . TLR4 signaling has been linked to the IL-33/ST2 pathway. TLR4 activation increases production of interleukin-33  and inhibition of interleukin-33 attenuates CPT-11-mediated mucositis [36▪]. CPT-11 causes a loss in mucosal TLR4 protein expression in the jejunum 4 days after administration , which potentially delays mucosal healing after chemotherapy . It will be essential to use mice deficient in TLR4 to dissect functionally the precise molecular mechanisms of cause and effect in CPT-11-mediated toxic damage in the gut. In addition, it has been shown that the mRNA levels of TLR9 and MyD88 are increased in the intestinal mucosa after CPT-11 exposure . Genetic deletion of TLR9 and MyD88 seems to decrease CPT-11-mediated toxic injury in the small intestine , but baseline villus length was already lower in untreated TLR9-knockout compared with untreated wild-type (WT) mice, which makes interpretation difficult.
Chemotherapy drugs can cause persistent nociceptive pain associated with intestinal mucositis. It has recently been hypothesized that neuropathy and gastrointestinal toxicity may be systemically linked via a common TLR4-dependent pathway [40▪]. LPS or DAMPs may trigger peripheral hyperalgesia via TLR4 [41,42]. The chemotherapeutic agent paclitaxel directly binds TLR4 and shares its downstream signaling pathway . Local toxicities of paclitaxel include peripheral neuropathy and mucositis. Pharmacological inhibition of TLR4 reduces paclitaxel-induced behavioural hypersensitivity, preventing the increase of TRPV1-mediated capsaicin responses [44▪]. Future studies must demonstrate whether TLR4 blockade also alleviates paclitaxel-induced intestinal mucositis (and other side-effects, such as fatigue or hypersensitivity reactions).
INVOLVEMENT OF OTHER PATTERN RECOGNITION RECEPTORS IN INTESTINAL CHEMOTOXICITY
The functional effects of innate immune signaling seem to be chemotherapy drug-dependent. It is likely that each chemotherapy drug differentially modulates host immunity and microbiota function. Only little evidence exists whether (in addition to TLRs) other PRRs, such as NOD-like receptors (NLRs), may also be involved in the pathophysiology of gastrointestinal chemotoxicity.
Doxorubicin represents an example of a chemotherapy drug that impairs commensal-mucosal homeostasis by subverting the innate immune system on multiple levels. Doxorubicin-induced massive oxidative stress causes severe side-effects, including cardiomyopathy and intestinal mucositis. It affects gut microbiota-related metabolism  and reversely, soil microbes, such as environmental Actinomycetes, are capable to degrade and inactivate this anticancer drug . Doxorubicin induces rapid and profound bacterial community shifts in cancer patients, with, for example, increases in Gemella haemolysans and Streptococcus parasanguinis in the oral cavity after chemotherapy . Of note, Gemella species has been associated with cardiovascular disease  and S. parasanguinis with mucositis and gastrointestinal symptoms , respectively. Although the TLRs involved in functional recognition have not yet been defined, it is possible that PAMPs of G. haemolysans and S. parasanguinis may activate pro-inflammatory and anti-inflammatory responses via TLR2, TLR4 and TLR9 signaling, as shown for other Streptococcus species . Of note, mice that are deficient in TLR2 or TLR9 seem to be protected against doxorubicin-mediated cardiac dysfunction  and intestinal mucosal damage . Interestingly, NOD2 stimulation by muramyl dipeptide, a peptidoglycan motif common to all bacteria (including Streptococcus species), preserves intestinal stem cells against doxorubicin-mediated damage [53▪]. Future studies must address whether aberrant expansion of G. haemolysans and S. parasanguinis is indeed a common feature in doxorubicin-induced tissue damage and whether these species are functionally the real culprits that launch gut tissue-destructive host responses and drive detrimental side-effects via TLR2 and/or TLR9. Because doxorubicin itself is derived from Streptomyces bacterium and Streptomyces peucetius var. caesius, it may even act as a direct TLR2/TLR9 ligand, but proof has not been provided yet.
TOLL-LIKE RECEPTOR GENOTYPES ASSOCIATED WITH CHEMOTOXICITY
Variations in TLR genes may impair host–microbial communication. TLR gene dysfunction may alter ligand recognition and signaling pathways, modify commensal community structure and impair mucosal immune tolerance. The presence of the TLR2-Arg753Gln and TLR4-Asp299Gly polymorphisms has been associated with increased incidence of infections in patients with acute myeloid leukemia undergoing induction chemotherapy . Yet, it remains to be proven whether selected candidate TLR gene variants may indeed influence an individual's capacity to process different chemotherapeutic drugs. A preliminary report [55▪] suggests that the TLR2-1350T>C polymorphism may help to predict the potential risk of severe gastrointestinal toxicity in response to 5-fluorouracil-based chemotherapies. Based on recent research in mice, as outlined above [23▪], loss-of-function deletion of the TLR2 gene may significantly alter a key step of xenobiotic metabolism which may maximize the adverse effects of distinct chemotherapy drugs (P-gp substrates). In-vitro experiments have recently suggested that the TLR2-R753Q polymorphism impairs intestinal epithelial restitution  and gap junctional intercellular communication , which could delay mucosal healing after the conclusion of the anticancer treatment. In contrast, the more common gain-of-function TLR4-D299G gene variant may promote an auto-inflammatory microenvironment  with enhanced bacterial colonization , which would increase susceptibility to pathogenic infections  in cancer patients and aggravate chemotherapy-induced intestinal mucositis. However, chemotherapy-induced genotoxicity probably represents a multigenic and multifactorial trait . It remains to be shown in large studies with genome-wide approaches whether genetic testing of TLRs may indeed be useful (in combination with other genetic factors, such as NOD2 polymorphisms ) to unambiguously identify cancer patients with an increased risk for chemotoxic side-effects in the gastrointestinal tract. TLR polymorphisms with rare variant alleles alone may only point towards possible inter-individual variability in the sensitivity to certain chemotherapeutic drugs and associated complications in some cancer patients, but may not represent a valid genetic biomarker for overall risk stratification.
GUT MICROBIOTA DEPLETION BY ANTIBIOTICS MODULATES INTESTINAL CHEMOTOXICITY
Chemotherapy-induced toxic damage to the intestinal mucosal barrier increases the risk of infection for cancer patients by facilitating bacterial translocation and systemic inflammatory responses. As mentioned, chemotherapy may alter the gut microbiota composition favouring dysbiosis. For instance, imbalances in taxonomic composition and metabolic capacity in the gut microbial community have been associated with intestinal inflammation in lymphoma patients receiving high-dose combination chemotherapy [62▪]. Certain facultative-pathogenic species may especially prevail in seriously ill, hospitalized and immune-compromised cancer patients. To avoid life-threatening infections, antibiotics are routinely administered to patients during anticancer therapy. Antibiotic prophylaxis may be advantageous to prevent intestinal mucositis induced by a number of chemotherapeutic drugs in which gut bacterial enzymes generate highly toxic metabolites. For instance, bacterial β-glucuronidase deconjugates SN-38G to the toxic metabolite SN-38, which is responsible for CPT-11-mediated intestinal damage . Accordingly, mice that are germ-free  or ablated from gut microbiota by antibiotics  are protected against CPT-11-induced intestinal mucositis.
However, antibiotics strip away not only the ‘bad’ bacteria, but also the ‘good’ ones. Antibiotics broadly deplete many populations of beneficial commensal microbiota (at least in mice), thus reducing beneficial TLR signaling and negatively affecting downstream regulation of innate host defenses. The surviving antibiotic-resistant microbes may deregulate mucosal homeostasis by exhibiting enhanced inflammatory potential. Ablation of gut microbiota using antibiotics reduces host–microbial interactions, leading to a decline in immune gene expression and decrease in T lymphocyte numbers in the lamina propria as well as inhibition of mitochondrial gene expression and increase in IEC death [66▪]. Thus, antibiotic treatment may promote susceptibility to chemotherapy-induced intestinal mucositis in mice that are otherwise chemotoxicity-resistant [23▪]. Yet, oral supplementation of a TLR2 ligand rescues these mice from exacerbated DNA damage-associated mucosal inflammation induced by chemotherapy after antibiotic treatment [23▪]. In addition, two recent studies suggested that gut microbiota depletion by antibiotics compromises the antitumor efficacy of chemotherapy with cyclophosphamide or platinum [67,68]. Chemotherapy-induced barrier dysfunction allows microbes to gain entry into deeper host tissues and circulating TLR ligands may provide essential adjuvant antitumor effects . Oral administration of selective intestinal microbes, such as Bifidobacterium or Bacteroides, seems to improve efficacy of cancer immunotherapy with antibodies that block immune inhibitory pathways, specifically the CTLA-4 and PD-L1 axes, in mice models of melanoma [70▪,71▪]. Taken together, antibiotics may put patients who are being treated for cancer at increased risk of gastrointestinal toxicity and chemotherapy inefficacy. Future studies will need to investigate whether modulating microbial activities by, for example, probiotic supplementation during antibiotic treatment may alleviate mucosal chemotoxicity and boost drug efficacy via TLR signaling. So far, large, controlled, randomized, double-blinded clinical trials using probiotics in cancer patients are lacking.
The gut microbiota, which represents a rich source of TLR ligands, may modify xenobiotic metabolism of chemotherapy drugs, resulting in their activation or inactivation. The outcome of TLR-mediated innate immune responses during chemotherapy exposure appears to be ambivalent – depending on the pharmacology and toxicology of individual chemotherapeutic agents, distinct changes in gut microbial community structure and function as well as dynamic cellular kinetics of induction and resolution phases during genotoxic inflammation. Therefore, targeting microbial–TLR interactions for preventing or treating chemotherapy-induced gastrointestinal toxicity will be a major challenge. Future studies must investigate in more detail whether manipulating the delicate balance between gut microbiota and host immune responses by either monotherapy or combinations of different TLR agonists and antagonists may be indeed useful to limit the toxic side-effects of complex chemotherapy regimens, accelerate mucosal tissue regeneration and improve the anticancer treatment response.
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Conflicts of interest
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REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
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62▪. Montassier E, Gastinne T, Vangay P, et al. Chemotherapy
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This study performed high-throughput DNA-sequencing analysis from faecal samples before and after chemotherapy in lymphoma patients receiving myeloablative conditioning treatment prior to hematopoietic stem cell transplantation (no antibiotics). The findings imply that dysbiosis is associated with chemotherapy-induced intestinal mucositis. For instance, the authors demonstrated a decrease in anti-inflammatory Bifidobacterium, but an increase in proinflammatory Citrobacter after chemotherapy.
63. Wallace BD, Wang H, Lane KT, et al. Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science 2010; 330:831–835.
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66▪. Morgun A, Dzutsev A, Dong X, et al. Uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks. Gut 2015; 64:1732–1743.
Cancer patients receiving chemotherapy are often treated with antibiotics. Here, the authors found that general intestinal alterations resulting from antibiotic treatment in mice may be explained by three major influences: microbiota depletion, direct effects on host mucosa (repression in mitochondrial gene expression) and effects of microbiota shifts (overgrowth of antibiotic-resistant species).
67. Iida N, Dzutsev A, Stewart CA, et al. Commensal bacteria
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68. Viaud S, Saccheri F, Mignot G, et al. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science 2013; 342:971–976.
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See annotation to 71.
71▪. Vetizou M, Pitt JM, Daillere R, et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 2015; 350:1079–1084.
Additional proof that the gut microbiota may influence the efficacy of cancer therapy by promoting antitumor immune responses in the host.