Introduction

Inflammation of the intestinal tract can result in acute or chronic manifestations of intestinal diseases; it may cause irritation, exposure to bacteria, and a dysregulation of the homeostatic balance. This leads to a range of debilitating symptoms that may affect patients' quality of life. Current treatment modalities used for intestinal inflammation are associated with a range of disadvantages including poor efficacy and unwanted side effects. The incidence rates for intestinal inflammation have been steadily increasing around the world for the last 50 years with an increased prevalence most notable in newly industrialized nations [1, 2]. Factors such as cell types, immunological abnormalities, tissue specificity and genetic/environmental factors are involved in the pathogenesis of intestinal inflammation.

Toll-like receptors (TLRs) are type 1 transmembrane proteins belonging to the wider family of pattern recognition receptors, and are responsible for the recognition of a variety of molecular signals, including endogenous damage and pathogen-associated signals, damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), respectively. Both immune (dendritic cells, monocytes, mast cells, macrophages) and non-immune cells (fibroblasts, epithelial cells) express these pattern recognition receptors [3]. The pattern recognition receptor-ligand binding between DAMPs and PAMPs prompts a downstream signalling cascade, which results in the recruitment of leukocytes [3]. TLRs activate downstream signalling pathways, which originates from the toll-interleukin receptor (TIR) domain–containing adaptor proteins such as myeloid differentiation primary-response protein 88 (MyD88), TIR-domain–containing adaptor protein (TIRAP), TIR domain–containing adaptor protein inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) [4]. These adaptor molecules are essential to produce inflammatory cytokines, type 1 interferons, chemokines and co-stimulatory molecules. At present, 13 human TLRs have been identified and are located on various cellular compartments (including extracellular membrane, endosome and golgi apparatus) with each TLR responding to specific stimuli [4, 5].

In a healthy intestinal tract, enterocytes coexist with luminal and mucosa-associated commensal bacteria without the initiation of inflammatory responses. However, the exact mechanism behind bacterial tolerance within the intestinal tract is still largely unknown. TLRs play a pivotal role in immune tolerance to intestinal microbes [6]. These immune tolerance and responses are organized by Peyer’s patches, mesenteric lymph nodes and the lamina propria [7]. These lymphoid organs are populated with dendritic cells, which produce interleukin 10 (IL-10) in turn, transforming T cells into transforming growth factor-β (TGF-β) [7]. The production of these cytokines leads to immune tolerance and homeostasis, as well as unnecessary inflammation [7]. This suggests that TLR4 signalling has an effect not only on immune responses but also on the balance of the intestinal microbial ecosystem [8, 9].

In contrast, during conditions of stress (such as disease) in the intestinal tract, inflammatory cytokines are released from enterocytes and mucosal immune cells responding to the stimulation of TLRs [10]. This leads to apoptosis and reduced proliferation of enterocytes, which in turn promotes translocation of bacteria into the lamina propria, exacerbating intestinal inflammation [10]. One of the most well-characterized TLRs is TLR4, which has been shown to be involved in homeostasis, apoptosis, intestinal inflammation and inflammatory bowel disease [11]. The focus of this review will therefore be based on the role of TLR4 antagonism in inflammatory conditions with the purpose of generating a hypothesis to support the use of TLR4 antagonists in intestinal inflammation.

Toll-like receptor 4 activation and signalling

In 1997, toll proteins in Drosophila were discovered to mediate protection against fungal infections [12]. Toll proteins in Drosophila were activated by fungi and Gram-positive bacteria, which do not contain lipopolysaccharide (LPS). They do, however, trigger a toxic shock response that is similarly induced by LPS [12]. This then led to research focusing on the now established TLR4-LPS signalling cascade. This early work also suggests a much broader role of TLR in homeostasis, tissue repair and immune defence [13].

TLR4 is an intra- and extracellular receptor expressed on endosomes and cytoplasmic membranes, which recognizes PAMPs (flagellin and LPS) and DAMPS (calprotectin, S100A8/9 HMGB1 and HSP70) through its co-receptors MD2 and CD14 [14, 15]. In addition, TLR4 has recently shown to be activated by certain pharmacological agents, including chemotherapeutic agents (paclitaxel). TLR4 is located on many different cell types (endothelial cells, lymphocytes, cardiac myocytes and glial cells) throughout the body [16,17,18]. In the intestine, TLR4 is expressed on antigen-presenting cells such as macrophages and dendritic cells, and on enterocytes and lymphocytes [19]. TLR4 consists of leucine-rich repeats (LRRs) with a horseshoe-like shape made up of 839 amino acids. The complex ligand specificity of the TLR4/MD2 complex is composed of two antiparallel β sheets, which form a large hydrophobic pocket in MD2 [20]. LPS is able to bind to this hydrophobic pocket through its lipid chains, which are completely buried in the MD2 hydrophobic pocket [20]. However, one of these lipid chains is partially exposed to the outer surface, which allows some interaction with TLR4 [20]. These hydrophilic and hydrophobic interactions between LPS and the TLR4/MD2 complex mediate the dimerization of extracellular domains in the TLR4, thus triggering a downstream signalling cascade leading to the release of pro-inflammatory cytokines [20]. A study by Abreu et al. [21] discovered that increases in TLR4 expression alone would not result in a reaction from LPS without the accompanying expression of MD2. In the study, they challenged different intestinal epithelial cell lines (Caco-2, T84, HT-29) with LPS and found that a decreased expression of TLR4 and MD2 correlated with intestinal epithelial protection against pro-inflammatory gene expression in response to bacterial LPS. It was concluded that careful regulation of both TLR4 and MD2 is necessary to maintain homeostasis in the intestinal tract due to it being continuously exposed to high concentrations of bacteria.

Upon stimulation, TLR4 will activate two signalling pathways, the TRIF-dependent pathway (Fig. 1) and the MyD88-dependent pathway (Fig. 2). In the TRIF-dependent pathway, TLR4 heterodimers recruit TRAM, which is needed to activate TRIF, resulting in the binding of TRIF with TNF receptor–associated factor 3 (TRAF3) and TRAF6 for binding with RIP, a receptor-interacting serine-threonine kinase 1 protein. Subsequently, this leads to the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The TRIF-activated pathway leads to the activation of interferon regulatory transcription factor 3 (IRF3) by TANK-binding kinase 1 (TBK1) and inhibitor of NF-κB-kinase complex stimulation (IKK), which results in the production of type 1 interferons and anti-inflammatory cytokines (such as IL-10).

Fig. 1
figure 1

Pathogen-associated molecular pattern toll-like receptor 4 signalling pathway in an enterocyte. LPS Lipopolysaccharide, TLR Toll-like receptor, TIRAP TIR domain–containing adaptor protein, TRAM TRIF-related adaptor molecule, MyD88 Myeloid differentiation primary-response protein 88, IKK Inhibitor of NF-κB-kinase complex, TRIF TIR -domain–containing adaptor protein inducing interferon-β, TBK1 TANK-binding kinase 1, NF-κB Nuclear factor-kappaB, IRF3 Interferon regulatory transcription factor 3

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Fig. 2
figure 2

Toll-like receptor 4 activation by damage-associated molecular patterns from tissue damage leads to a downstream signalling pathway, which induces inflammatory gene expression. DAMPs Damage-associated molecular patterns, TLR Toll-like receptor, TRAM TRIF-related adaptor molecule, TRIF TIR -domain–containing adaptor protein inducing interferon-β, TIRAP TIR domain–containing adaptor protein, MyD88 Myeloid differentiation primary-response protein 88, IKK Inhibitor of NF-κB-kinase complex, IRF3 Interferon regulatory transcription factor 3, NF-κB Nuclear factor-kappaB, IL Interleukin

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In the MyD88 signalling pathway, TLR4 heterodimers will bind to MyD88, which results in the formation of IRAK (interleukin 1 receptor–associated kinases) and TRAF6 complexes [14]. Formation of IRAK and TRAF6 complexes leads to a downstream signalling cascade. Various other complexes such as TAK1, TAB1/2/3, MAP kinases and IκB will be phosphorylated or activated to allow the translocation of NF-κB into the nucleus, ultimately driving the transcription of cytokine genes (such as TNFs, ILs and chemokines) to regulate pro-inflammatory responses [14, 22].

Dysregulation of TLR4 signalling has been linked to the development of a variety of inflammatory diseases. Studies have investigated functional genetic variants of TLR4 and their impact on LPS signalling response. A study by Hold et al. found that cells carrying TLR4 D299G and T399I variants, when stimulated with LPS, had a sixfold lower expression of NF-κB compared to wild-type TLR4 [23]. Ferwerda et al. demonstrated that patients carrying a variant at position 299 (Gly) but not at position 399 (Ile) had a stronger pro-inflammatory cytokine response with increased TNF-α levels in whole blood samples when stimulated with LPS compared to patients carrying wild-type TLR4 alleles at both positions [24]. Weinstein et al. showed that the same TLR4 variants in patients with acute ischemic stroke are associated with worse neurological outcomes and alterations in systemic markers of inflammation [25]. This dramatic difference in cytokine expression caused by dysregulation of TLR4 signalling due to genetic polymorphisms will affect a person’s ability to respond to LPS leading to a dysregulated immune response to infection. Considering that TLR4 downstream signalling plays a pathological role in inflammation, using antagonists or inhibitors to target TLR4 signalling may be beneficial in treating inflammatory disorders.

TLR4-mediated intestinal inflammation

An important component of immunity and host-microbial interactions in the intestinal tract is the recognition of DAMPs, PAMPs and endogenous ligands by TLR4 expressed on the enterocytes and antigen-presenting cells. Any imbalance from this interaction may contribute to the pathogenesis of inflammation within the intestinal tract [26]. There has also been substantial evidence indicating the involvement of TLR4 in intestinal inflammatory diseases such as ulcerative colitis (UC), Crohn’s disease (CD) and intestinal mucositis (IM). It was reported that, in the colonic mucosa of patients with UC and CD, a significant increase in TLR4 mRNA and protein expression was observed compared to healthy controls [27, 28]. However, this may be due to the increased influx of TLR4 expressing innate immune cells. There is also mounting evidence that TLR4 polymorphism is associated with the development of UC and CD, whereby the allele frequencies of the TLR4 Asp299Gly polymorphism were discovered to be significantly higher in UC and CD patients [29].

There have also been several studies using animal models of acute intestinal inflammation. TLR4 expression is strongly upregulated in animal models induced with colitis [30]. Animal models with TLR4 knocked-out were observed to be protected from colitis or colon tumorigenesis by preventing the downstream signalling pathways that induce colitis [30]. For example, TLR4 knockout (KO) mice induced with acute colitis had a decrease in COX-2 expression, prostaglandin production and NF-κB signalling, which lead to a significant reduction of acute inflammatory cells and therefore significantly reduced acute inflammation in the intestinal tract [31]. Other studies have observed an increase in pathogenic E. coli and a decrease in beneficial intestinal microbiomes (Bifidobacterium spp. and Lactobacillus spp.) in a DSS-induced mouse model, which may be associated with an increase in the TLR4 expression observed in the colon of the animals [8], while another study using TLR4 KO mice showed a reduction in pathogenic E. coli compared to the DSS-induced wild-type mice, which showed a tenfold increase in pathogenic E. coli [9]. These TLR4-deficient mice also displayed reduced disease activity index and histopathological scoring.

It therefore stands to reason that, by inhibiting TLR4, a protective effect from intestinal inflammation will be induced. Recent research has shown that, by inhibiting TLR4 using antagonists such as paeoniflorin, monoclonal antibodies and CRX-526, DSS-induced intestinal inflammation was attenuated with a significant reduction in disease activity and histopathological scoring [32,33,34]. However, other studies have discovered that there was no protective effect observed in the clinical symptoms and histology scores when blocking TLR4 during chronic intestinal inflammation [35] despite the opposite results observed during acute intestinal inflammation [31], most likely due to the low involvement of innate immune cells in chronic compared to acute inflammation [36].

TLR4 antagonists as a potential therapeutic alternative for treatment of intestinal inflammation

Inflammatory bowel disease (IBD) (consisting of UC and CD) is known as a non-specific, chronic gastrointestinal inflammatory disorder [37], with periods of disease activation and remission, and in some cases progressive disease [38]. It is considered an autoimmune disease due to the combined effect of genetic factors and abnormal immune responses to the intestinal bacteria and other foreign substances [37]. In addition, one of the most serious complications that IBD patients encounter is colorectal cancer, which accounts for increased mortality rates associated with UC [39]. The severity of inflammation in the intestinal tract also correlates with the risk of colorectal cancer in patients with IBD [39].

Severe intestinal inflammation leads to a range of debilitating symptoms that significantly affects patient quality of life. Current treatment modalities for intestinal inflammation are associated with a range of disadvantages including poor efficacy and unwanted side effects. The TLR4 signalling cascade also plays an important role in intestinal inflammation, with its extracellular and intracellular components being attractive therapeutic targets for the treatment of both acute and chronic intestinal inflammation. The link between intestinal inflammation and colon cancer also offers the possibility of identifying and developing novel ways to prevent cancer.

The incidence rates for IBD have been steadily increasing around the world for the last 50 years with the majority of cases occurring in westernized and industrialized countries [1, 40]. IBD is a chronic and lifelong condition that has no cure and requires a lifetime of care. It has a significant effect on patient quality of life [41, 42]. However, UC and CD are clinically distinct diseases and known to have different anatomical, clinical and histological features [43].

Presently, the etiology and pathogenesis for IBD remain largely unknown [43], with a plausible hypothesis for the etiology of IBD being the unregulated activation of both the body’s innate and adaptive immune systems, potentially in response to resident gut microbes. This immune response may be mediated by the innate immune receptor TLR4 in response to luminal antigens (fungi, bacteria) in the intestinal tract [43]. Treatment approaches include aminosalicylates, corticosteroids and antibiotics [44, 45].

As previously stated, patients with IBD are at higher risk of developing colorectal cancer, and a common complication of cancer treatment is intestinal mucositis (IM), occurring in 40% of patients who receive a standard dose of chemotherapy and 100% of patients who are receiving high doses of chemotherapy [46, 47]. IM is the ulceration and inflammation of the mucosa in the intestinal tract caused by chemotherapy and radiation for cancer, and is an acute form of intestinal inflammation [48].

Unfortunately, the gastrointestinal tract is particularly susceptible to the devastating effects of chemotherapy and radiation. TLR4 is the main receptor that detects DAMPs and responds to tissue damage in the intestinal tract [49]. The cytotoxic effects of chemotherapy and radiation on both normal and malignant cells cause the release of DAMPs. This produces a sustained innate immune activation, which develops into the mucosal inflammation seen in patients with repeating cycles of chemotherapy and radiation treatments [49]. While mucositis has been recognized as a major dose-limiting toxicity for decades, there is yet to be an effective treatment to manage intestinal inflammation. Pre-clinical studies have focused on inhibition of inflammation via multiple mechanisms, or accelerating healing with growth factors [50].

IM has a significant impact on the quality of life of patients whereby the average number of days a patient suffering from IM needs to be hospitalized is 3 times more than the 4 days required by patients not suffering from IM [51]. This increased length of stay increases the strain on hospital resources [51]. Patients also suffer from symptoms such as vomiting, abdominal pain and severe diarrhea. In certain cases, the symptoms of IM will cause patients to require a dose reduction, delay or even discontinuation of their regimen, which will affect the patient’s survival [52]. To date, studies have shown a link between TLR4/MD2 signalling and the development of IBD and IM [34, 53]. TLR4 antagonists show potential as therapeutic agents in both the settings. However, the majority of studies have focused mainly on sepsis models as well as diseases and infections unrelated to the development of intestinal inflammation, leaving a significant gap in the literature.

TLR4 is overexpressed in both UC (fold increase: 2.33) and CD (fold increase: 1.71) [27, 54, 55]. In IBD, abnormal signal transmission mediated by the upregulation of TLR4 promotes the sustained release of inflammatory cytokines (IL-6, TNF-α). This, in turn, develops and persists as intestinal inflammation. Only low levels of TLR4 and MD2 are expressed on the intestinal epithelium and very little was known about their regulation on intestinal epithelial cells. However, it was established that, during inflammation, expression of both TLR4 and MD2 is increased. It was later discovered by Abreu et al. [56] that the expression of TLR4 and MD2 in the intestinal tract is also regulated by immune-mediated signals. There was an increase in TLR4/MD2 expression when the intestinal epithelial cell lines (T84, HT-29) were exposed to pro-inflammatory cytokines (IFN-γ, TNF-α) highlighting the potential link between the innate and adaptive immune systems in intestinal epithelial cells only in response to pathogenic organisms. Another study by Ungaro et al. has also shown that the inflammation in IBD is decreased in TLR4-deficient mice [35]. However, the study also found that TLR4-deficient mice were unable to undergo mucosal healing and demonstrated decreased epithelial cell proliferation [35]. This shows that TLR4 serves as a mediator for both mucosal healing and inflammation in the intestinal tract.

A similar pattern can also be observed in IM. A study by Wardill et al. has shown that genetic deletion of TLR4 from mice was able to improve chemotherapy-induced gut toxicity and pain [53]. TLR4 KO mice had reduced diarrhea and weight loss compared to wild-type mice [53]. The TLR4 KO mice also exhibited a muted inflammatory response, with no significant increase in IL-1β, IL-6 or TNF-α, compared to their wild-type counterparts [53].

These studies point out the critical role of TLR4 in regulating inflammation in the intestinal tract, and by targeting and inhibiting TLR4, the outcome of intestinal inflammation and its consequence may be prevented. However, careful selection of TLR4 elimination vs. selective or temporary inhibition as a therapeutic is needed since TLR4 has beneficial effects for mucosal healing and homeostasis.

TLR4 antagonists

Targeting TLR4 could represent a potential approach to regulate immune responses and treat inflammation. However, any potential therapeutic agent must be able to block the harmful effects of TLR4 activation without negatively affecting the host’s defence functions. Currently, many different antagonists are being investigated for their potential in managing inflammatory-based diseases and settings, summarized in Table 1.

Naturally occurring

The first naturally occurring TLR4 antagonist discovered was from a photosynthetic Gram-negative bacterium that was non-pathogenic, known as Rhodobacter sphaeroides [57]. The LPS produced from this bacterium, known as Rhodobacter sphaeroides lipid A (RsDPLA), was non-toxic towards murine and human cells and was able to compete with toxic LPS for binding sites. RsDPLA was also able to interact with the TLR4/MD2 complex found in rodents and humans with antagonistic effects [58]. Further in vitro and in vivo studies on the LPS produced by Rhodobacter sphaeroides and other bacteria/cyanobacteria have shown potent antagonistic activity of this type of LPS in murine and human cells as well as preventing endotoxic shock in mice.

Additionally, traditional Asian medicines produced from plants, including curcumin, turmeric and a variety of herbs, provide a rich and natural source of molecules which are being investigated for bio-activities that act as TLR4 antagonists [59, 60]. The modulation of TLR4 using herbal extracts promoted a large area of research to determine their pharmacological potential. It was found that certain bio-actives from bacteria or plants had a positive relationship against sepsis and septic shock [61,62,63]. These bio-actives were also discovered to have positive relationships against inflammatory diseases such as Alzheimer’s, arthritis and inflammatory bowel diseases [35, 64, 65]. A summary of research conducted on some of these naturally sourced TLR4 antagonists can be found in Table 1. Although the main focus of this study is on the therapeutic potentials of TLR4 antagonists on intestinal inflammation, there are a limited number of studies, which used intestinal inflammation as a disease model.

Table 1 Summary of the effect of natural TLR4 antagonists in previous pre-clinical in vitro, in vivo and in silico studies
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Synthetic

Although there are many plant-based products capable of targeting and inhibiting TLR4 in vitro and in vivo in both rodent and human models, these do not possess the necessary stability and target specificity to be considered a potential therapeutic option compared to products and molecules extracted from microorganisms [59], which was why the molecules produced from microorganisms such as Rhodobacter sphaeroides have been used as a model to create synthetic antagonists. RsDPLA was used to design the synthetic TLR4 antagonists eritoran (E5564) and E5531 [80, 81]. E5531 was a first-generation lipid A analogue synthesized as part of a program to develop therapeutic agents for septic shock [82], while eritoran (E5564) is a second-generation lipid A analogue designed for the same purpose but was found to be more potent in its anti-endotoxin effects, longer lasting and easier to manufacture compared to E5531 [83]. Studies on eritoran have shown a positive effect against sepsis [83] and other inflammatory conditions [84, 85]. This led to the development of other synthetic analogues such as TAK-242 and FP7 with antagonistic effects on the TLR4/MD2 complex to treat various inflammatory diseases such as neuroinflammation and influenza infections. Although the main focus of this study is on the therapeutic potentials of TLR4 antagonists on intestinal inflammation, there are a limited number of studies, which used intestinal inflammation as a disease model. Therefore, studies which encompass different inflammatory diseases have been included and summarized in Table 2 in order to show the potential broader anti-inflammatory effects of TLR4 antagonism.

Table 2 Summary of the effect of synthetic TLR4 antagonists in pre-clinical in vitro and in vivo research studies
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The most well-known TLR4 antagonist to enter the clinical phase was eritoran, followed by TAK-242; and although many synthetic TLR4 antagonists have been developed and studied, very few have actually made it into clinical trials, due to the limited evidence currently available. Table 3 summarises only the TLR4 antagonists that have been, or are, undergoing clinical trials in different inflammatory disease models. However, this will allow for a broader view of using TLR4 antagonists in inflammatory diseases to support its use in intestinal inflammation.

Table 3 Summary of TLR4 antagonists used in clinical trials
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Conclusions

Both IBD and IM have significant effects on a patient’s quality of life as well as economic and social burdens [51, 99, 100]. While the pathophysiology for chronic intestinal inflammation remains unknown, previous research has identified that TLR4 signalling in the intestinal tract is a critical regulator of intestinal immune homeostasis. The use of a TLR4 antagonist has potential as a novel therapeutic for IBD and IM patients whose disease pathogenesis relies heavily on TLR4 signalling. Previous studies have shown that inhibiting LPS-induced TLR4 stimulation with antagonists can reduce intestinal inflammation in animal models [101]. Regardless of how promising TLR4 antagonists are in the treatment of intestinal inflammation, there are still challenges in bioavailability and delivery. Nonetheless, anti-TLR4 therapies present a promising alternative for future innovative treatments for both IBD and IM. In the future, there is a need for tissue specific studies looking into these anti-TLR4 therapies in order to mimic the therapeutic setting of IBD and IM.