Send Orders for Reprints to [email protected] Current Pharmaceutical Design, 2014, 20, 000-000 1 Gut Inflammation: Current Update on Pathophysiology, Molecular Mechanism and Pharmacological Treatment Modalities Gyires K.*, Tóth V.E. and Zádori Z.S. Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4., 1089 Budapest, Hungary Abstract: Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory condition of the gastrointestinal tract. The two main forms of IBD are Crohn's disease and ulcerative colitis. According to the recent concept the disease is caused by a combination of factors, including genetics, immune dysregulation, barrier dysfunction and the change in microbial flora. Environmental factors, such as changes in diet, antibiotic use, smoking or improved domestic hygiene (e.g. eradication of intestinal helminths) probably contribute to the development and increased prevalence of IBD. Dysregulation of mucosal immunity in IBD causes an overproduction of inflammatory cy- tokines which resulted in uncontrolled intestinal inflammation. Based on extensive research over the last decade, besides the conventional therapy, there are several novel pathways and specific targets, on which focus new therapeutics. New therapeutics aim 1./ to correct ge- netic susceptibility by stimulating NOD2 expression, TLR3 signaling or inhibition of TLR4 pathway, 2./ to restore the immune dysregu- lation by inhibition of pro-inflammatory cytokines (TNF-(cid:1), IL-6, IL-13, IL-17, IL-18, IL-21), Th1 polarisation (IL-2, IL-12, IL-23, IFN- (cid:3)), T-cell activation, leukocyte adhesion, as well as by immunostimulation (GM-CSF, G-CSF) and anti-inflammatory cytokines (IL-10, IL-11, IFN-(cid:2)-1a), 3./ to restore mucosal barrier function and stimulate mucosal healing by different growth factors, such as GH, EGF, KGF, TGF-(cid:2), VEGF, 4./ to restore the normal bacterial flora by antibiotics, probiotics. However, in spite of these numerous potential tar- gets, the true value and clinical significance of most of the new biologics and molecules are not clear yet. Keywords: Inflammatory bowel disease, colitis, microbiota, pattern recognition receptors, pro-inflammatory cytokines. 1. INTRODUCTION As regards the clinical symptoms, body weight loss and fever Inflammatory bowel disease (IBD) is a chronic and relapsing are more common in CD. Ulcerations, granulomas, and bowel fistu- las are characteristic for CD, in contrast, UC affects the mucosa in a inflammatory condition of the gastrointestinal (GI) tract. The two continuous manner. Moreover, smoking was found to be protective main forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC), though other forms are also known, which are also classified against UC and might improve its course, but seems to increase the risk of developing CD and worsens its course [13]. as not typical IBD (e.g. collagenous colitis, lymphocytic colitis). The cause of IBD is not exactly known. The recent consensus is On the other hand, extra-intestinal manifestations (liver prob- lems, arthritis, skin manifestations and eye problems) can develop that IBDs are initiated and perpetuated by an impaired immune response against the gut microbiota in genetically susceptible indi- in both CD and UC. Some patients have an extra-intestinal manifes- viduals [1, 2] and the disease is caused by a combination of factors, tation as their first symptom of the disease, while they still have only mild gastrointestinal manifestation, or none at all. Anemia is including genetics, immune dysregulation, barrier dysfunction, change in microbial flora and environmental influences (see re- the most prevalent extraintestinal complication of both IBDs [14, views [3-7]). 15]. Though UC and CD share some common clinical symptoms, The chronic inflammation of the gut causes wide-ranging clini- cal symptoms in both forms, like nausea, diarrhea (which is often the two diseases possess very distinct features. First of all, the loca- porridge-like in CD, while mucus-like with blood in UC) or tion of the inflammation is different; CD can develop at any part of the intestine, though most of the cases are localized at the terminal abdominal pain [16, 17]. ileum. In contrast, in UC the inflammatory process is restricted to UC or CD patients have increased risk for colorectal carcinoma the colon and the rectum. Moreover, the pathological changes in (CRC). Patients with UC and Crohn’s ileocolitis have an elevated CD affect the whole bowel wall and manifested as transmural le- risk of developing colon cancer, while patients with CD and enteri- sions, while in UC the inflammation is restricted to the mucosa tis have an elevated risk of developing small-bowel cancer [18, 19]. (epithelial lining of the gut). Also differences in immunological The cumulative risk for developing colorectal cancer was 8% at 22 response of intestinal mucosa have been described. CD is associ- years from onset of symptoms for Crohn’s colitis and 7% at 20 ated with the activation of types 1 and 17 T-helper (Th) cells in years from onset of symptoms for UC, as it accounts for one in six response to interleukin (IL)-12, IL-18, IL-23 and transforming of all deaths in IBD patients [20]. growth factor (cid:2) (TGF-(cid:2)), and activation of these cells results in The high incidence and prevalence of IBD (worldwide inci- increased secretion of the pro-inflammatory cytokines IL-2, IL-17, dence of UC and CD varies between 0.5-24.5 and 0.1-16 individu- interferon (IFN)-(cid:3) and TNF-(cid:1) [8, 9]. In patients with UC the mu- als per 100.000 inhabitants, respectively) [21], and the costs of the cosal inflammation of the colon is mainly associated with a Th2 cell long-term and only symptomatic treatment of the patients place a activation mediated by IL-4, IL-5 and IL-13 that results in an in- significant burden on the healthcare system: the expenses exceed creased level of IL-13 [10, 11]. However, in both cases T-cells are 1.7 billion dollars per year in the United States [22], and are in also activated by direct contact with antigens [12]. similar range in European countries. Although in the last decade our knowledge about the pathome- *Address correspondence to this author at the Department of Pharmacology chanism of IBDs greatly expanded (which is also clearly demon- and Pharmacotherapy, Semmelweis University, Nagyvárad tér 4., 1089, strated by the continuously rising number of publications in this Budapest, Hungary; Tel: 36-1-210-4416; Fax: 36-1-210-4412; field), and several important milestones have been achieved, dis- E-mail: [email protected] 1381-6128/14 $58.00+.00 © 2014 Bentham Science Publishers 2 Current Pharmaceutical Design, 2014, Vol. 20, No. 00 Gyires et al. tinction between causing events and secondary consequences is still Hungarian [48] or New Zealand patients [49, 50]. Nevertheless, challenging. meta-analyses have provided evidences for an association between The aim of this review is to shortly summarize the current D299G and IBD [49, 50]. Similar discrepancies were observed with the T399I SNP, because significantly increased allele and carrier knowledge and newest findings in the pathomechanism of IBD as well as to overview some of the therapeutic targets and strategies frequencies for this mutation were observed in patients with UC in (convential and novel ones) for the treatment of IBD. a German cohort [44], while other groups could not demonstrate such association [45, 47, 49, 50]. 2. PATHOMECHANISM The importance of PRR mutations in the development of IBD is 2.1. Genetic Susceptibility further supported by the findings that a TLR9 polymorphism (- 1237T/C) was significantly higher in patients with CD [50], while The familial aggregation of IBD has already been observed polymorphism in the CARD9 gene (rs10870077), which encodes an several decades ago and studies conducted on twins also confirmed adaptor molecule of PRR signaling, was associated with both CD the importance of hereditary factors in the pathogenesis of IBD and UC [51, 52]. (especially for CD), though they also highlighted the role of envi- ronmental trigger factors [23-27]. Beside PRR genes GWASs have identified several other sus- ceptibility genes, which has led to better understanding of the path- Genome wide association studies (GWASs) performed during omechanism. The identification of IBD associated polymorphisms recent years provided better insight into the genetic background of in autophagy-related 16-like 1 (ATG16L1) and immunity-related IBD. They revealed 163 genomic susceptibility loci associated with GTPase family M protein (IRGM) genes has revealed that impaired IBD so far, 110 with both disease phenotypes, and further 30 and autophagy, and the consequent defects in innate immune responses 23 associated selectively either with CD or UC, respectively [28, to intracellular pathogens may be critical components of the chronic 29]. The considerable overlap of susceptibility loci in CD and UC inflammation in IBD [53-56]. IBD associated alterations in X-box- indicates that these two IBD phenotypes share several common binding protein 1 (XBP1) and orosomucoid-like 3 (ORMDL3) factors in their pathogenesis. genes imply that changes in the unfolded protein response (UPR) Although the exact functional role of several IBD susceptibility and the failure to manage endoplasmic reticulum stress may also genes still remains to be established, many of them are associated contribute to the pathogenesis, for example due to increased apop- with host immune functions, including both innate and adaptive tosis of Paneth cells [57, 58]. Mutations in the mucin genes (e.g. immunity. MUC1, MUC19) or in the prostaglandin receptor EP4 gene The first susceptibility gene identified for CD was NOD2/ (PTGER4) can lead to impaired mucosal barrier functions [51, 59], CARD15, which brought the role of innate immunity and pattern while genetic variations in the IL18RAP [52], IL23R [60, 61], recognition receptors (PRRs) in the pathogenesis of IBD to the fore. STAT3 [62] or SMAD3 [51] genes highlight the importance of As described in the next section, PRRs play an essential role in the failures in the adaptive immune responses in IBD. host microbial interaction by sensing conserved microbial structures (pathogen-associated molecular patterns, PAMPs). Binding of 2.2. Immune Dysregulation PAMPs results in the activation of multiple signaling pathways The intestinal mucosa is continuously exposed to a vast number including nuclear factor-(cid:1)B (NF-(cid:1)B) and mitogen activated protein of antigens (both dietary and microbial), which are recognized by kinases (MAPKs), which in turn induce the production of inflam- the mucosal immune system. Under normal circumstances it distin- matory mediators and also initiate multiple cellular processes, in- guishes between beneficial and pathogenic microbes - it tolerates cluding cell proliferation and differentiation [30-32]. NOD2, a normal commensal bacteria, while eliminates invading pathogens. member of the nucleotide-binding oligomerization domain (NOD)- Today it is widely accepted that abnormal immune regulation is a like receptor (NLR) family, recognizes muramyl dipeptide (MDP), key factor in the pathomechanism of IBD and alterations in both a component of peptidoglycan (PGN) in nearly all bacteria [30]. innate and adaptive immunity have been observed. This section The three most common NOD2 mutations, a frame-shift insertion shortly overviews the key players of the immune system and their mutation (3020insC) and two missense mutations (R702W and contribution to IBD. For more comprehensive recent immunologi- G908R) result in impaired recognition of MDP and in loss of NF- cal reviews see e.g. [63-66]. (cid:1)B activation in response to lipopolysaccharide (LPS) and PGN The pattern recognition receptors (PPRs) play a key role in the [33-36]. Several groups confirmed the association of NOD2 with bacteria-host interaction. These innate immune receptors are ex- CD, but interestingly, no such connection was found in Japanese pressed by different cells of the intestinal mucosa (like dendritic individuals [37]. This is in line with other findings (see below) cells (DCs), macrophages and intestinal epithelial cells (IECs)), and indicating that genetic determinants can differ significantly between recognize conserved microbial structures (PAMPs). Probably the populations. best-characterized family of PRRs are the Toll-like receptors In the last 2 decades several other PRR genes have been associ- (TLRs). This family comprises 13 members in mammals, ten in ated with IBD. A british group reported that a complex inser- humans (TLR1-10) and 12 in mice (TLR1-9, TLR11-13) [67]. Most tion/deletion polymorphism in NOD1/CARD4 (+32656) may con- TLRs (TLR1, 2, 4, 5, 6 and 11) are localized on the cell surface, tribute to the development of IBD and can result earlier onset and where they recognize mainly bacterial cell wall components (LPS extra-intestinal manifestations [38], but other groups could not re- of Gram negative bacteria by TLR4, lipoproteins from Gram- produce these findings in German [39], Scottish and Swedish pa- positive bacteria by TLR1, 2 and 6, flagellin by TLR5), while some tients [40]. members in this family are localized intracellularly in the en- Single nucleotide polymorphisms (SNPs) in Toll-like receptor dosomes (TLR3, 7, 8 and 9) and recognize viral or bacterial nucleic (TLR) TLR1 and TLR2 genes (R80T and R753Q) were found to acids [68, 69]. increase the risk of pancolitis in UC, but did not increase the sus- The family of NOD-like receptors (NLRs) includes 23 members ceptibility to disease development [41]. in humans and 34 in mice [31, 69]. These cytoplasmic receptors Several studies have been conducted to identify the role of the contain a leucine-rich repeats (LRR) domain, which senses bacterial TLR4 gene in the pathogenesis of IBD, but similarly to NOD2, ligands, a central NOD domain (also called NATCH domain) re- substantial heterogeneity was found between populations. The quired for activation and an N-terminal effector domain that medi- D299G SNP was associated with IBD in Belgian [42], German [43, ates interactions with other signaling proteins. Based on the effector 44], Greek [45] or Australian [46], but not in Southern Italian [47], domain five subfamilies can be distinguished, these are NLRA (also called CIITA, which contains an acidic domain), NLRB (or NAIP, Gut Inflammation: Current Update on Pathophysiology Current Pharmaceutical Design, 2014, Vol. 20, No. 00 3 which contains baculovirus inhibitor repeats (BIR)), NLRC (or also likely to improve mucosal healing. Decreased epithelial prolif- NOD, which possesses a caspase recruitment domain (CARD)), eration was found in TLR4 deficient mice [96] and antibody di- NLRP (or NALP, contains a pyrin domain (PYD)) and NLRX (con- rected against TLR4 impaired mucosal healing due to reduced ex- tains an unidentified domain) [30, 69, 70]. pression of cyclooxygenase-2, prostaglandin E2 (PGE2) and am- Further PRRs are the retinoic acid-inducible gene-I (RIG-I)-like phiregulin [97]. Beside TLRs also NLRs are able to modulate the receptors (RLRs), like RIG-I, MDA5 and LGP-2, which are also regeneration of the IECs. NLRP3 (also known as NALP3 or cryopyrin), which is one of the best-characterized NLRPs, recruits intracellularly localized and sense primarily viral RNAs [71], and the C-type lectin receptors (CLRs), including Dectin-1, Dectin-2, ASC (apoptosis-associated speck-like protein) and pro-caspase-1 mannose receptor, C-type lectin receptor DC-SIGN and Mincle, into a large protein complex (inflammasome) to mediate the secre- tion of IL-1(cid:2) and IL-18 [66]. Zaki et al. [98] demonstrated that which play an essential role in antifungal immunity [72, 73]. NLRP3-, ASC- and caspase-1 KO mice are highly susceptible to As mentioned above, PRRs are expressed by various cells in the dextran sulfate sodium (DSS) colitis, which is due to the decreased intestinal mucosa and their activation modulates inflammatory maturation and secretion of IL-18, and the consequent reduction of processes at various levels. At first, they regulate the barrier func- epithelial regeneration. tion of the mucosa, which is the front line of defense against intes- Beside regulating mucosal barrier functions, PRRs on DCs and tinal pathogens. The damage of IECs and their barrier function leads to an increased penetration of the microbes in the gut wall, macrophages are key factors in innate immunity. which in turn activates immune cells and causes inflammation. DCs express a wide range of PRRs and interpret microbial pat- Several studies demonstrate barrier disturbance in both animal coli- terns to direct other immune cells towards immunity or tolerance. tis models and in patients with CD and UC [74-77]. In IBD IEC They are able to sample luminal antigens directly by forming tran- permeability increases both transcellularly (in which TNF-(cid:1) has a sepithelial dendrites [99]. Upon encounter with pathogens, DCs major role) [78] and paracellularly via junctional complexes. For undergo rapid maturation characterized by upregulated expression instance, an impaired tight junction sealing due to upregulation of of major histocompatibility complex (MHC) and co-stimulatory claudin 2 and downregulation of claudin 5, claudin 8 and occludin molecules (like CD80, CD86, CD40) and production of pro- was reported in patients with active CD [79]. PRRs are involved in inflammatory cytokines (IL-1, IL-6, IL-12, IL-23, TNF-(cid:1)). Then the modulation of epithelial integrity. Activation of TLR2 enhances they migrate to the draining lymph nodes, where promote the pro- the transepithelial resistance in vitro (through redistribution of the liferation and differentiation of naïve CD4 T-cells to Th1, Th2 or tight junction protein ZO-1) and increases tight junction-associated Th17 subsets. On the other hand, DCs are also important for the IEC barrier integrity in vivo [80, 81], and recent evidence suggest maintenance of homeostasis and tolerance against the commensal that NOD2 potentiates the TLR2-induced improvement of mucosal microbes via the production of anti-inflammatory cytokines such as barrier [82]. Beside TLR2 also TLR9 has been shown to enhance IL-10 and TGF-(cid:2) and the production of tolerogenic regulatory T transepithelial resistance [83], while the action of TLR4 is still not (Treg) cells [63, 100, 101]. clear, since both improvement and disruption of the mucosal barrier Several studies demonstrate the active involvement of DC in have been described upon stimulation with LPS [82, 84]. the pathogenesis of IBD. The number of DCs expressing the matu- PRRs are also able to enhance mucosal barrier functions via ration markers CD80, CD83, CD86 and CD40 is elevated in CD stimulating the secretion of antimicrobial peptides (AMPs), like and UC [102-105]. DCs express also higher levels of TLR2 and cathelicidin and defensins. These peptides are secreted into the gut TLR4 [102] and accordingly, show exaggerated response to LPS in lumen by leukocytes and epithelial cells and regulate host microbial IBD [106]. This may result in false recognition of commensal bac- interaction and the composition of the commensal microbiota [74, teria and induction of pro-inflammatory immune responses. Beside 85]. The expression of (cid:1)-defensins by Paneth cells is associated secreting higher amounts of pro-inflammatory cytokines, DCs are with NOD2 signaling [86] and decreased (cid:1)-defensin production was also less able to induce tolerogenic Foxp3+ Treg cells in CD [107]. observed in CD patients with NOD2 mutations [87, 88] although it The role of intestinal macrophages in IBD has also been inten- has also been raised that reduced (cid:1)-defensin production is only the sively studied [63]. Similarly to DCs, macrophages also present consequence, and not the cause of the inflammation [89]. Beside antigens to T-cells and induce their differentiation to pro- or anti- regulating the (cid:1)-defensin production, PRRs stimulate also the secre- inflammatory subsets [108, 109]. M1 macrophages produce pro- tion of other AMPs. The activation of TLR4- and TLR2 increased inflammatory cytokines TNF-(cid:1), IL-12 and IL-23, and promote a (cid:2)-defensin-2 expression by human IECs [90] and the expression of polarized Th1 response, while M2 macrophages are characterized cathelicidin by mucosal macrophages was connected with TLR9 by production of IL-10 [109]. Thus, M2 macrophages may have signaling in mice [91]. Taking the manifold effects of PRRs on important role in maintaining intestinal homeostasis and alterations AMP production into consideration, it is not unexpected that PRR in the levels of macrophages and their cytokines can contribute to signaling can also shape the structure of the gut microbial commu- the pathomechanism of colitis in animal models and in IBD. Ac- nity. For example in TLR2 KO mice the proportion of Firmicutes cordingly, Smith et al. [110] found different cytokine profiles re- and Proteobacteria was significantly higher and lower, respec- leased by macrophages in healthy controls and CD patients after tively, in the gut microbiota [92], while NLRP6 deficiency was stimulation with heat-killed Escherichia coli or with the TLR2 associated with increased representation of Prevotellaceae [93]. ligand Pam CSK . 3 4 Hence, not only microbes can influence the host immune response It is noteworthy, that Kamada et al. [111] observed the infiltra- via PRRs, but vice versa, PRRs can also influence the make up of tion of unique CD14+ intestinal macrophages in the mucosa of CD the microbiota and control the load of commensal bacteria. patients at both inflamed and non-inflamed sites. These macro- The healing of the intestinal mucosa in case of epithelial injury phages produced larger amounts of pro-inflammatory cytokines, is essential to restore barrier functions. The controlled migration, such as IL-6, IL-23 and TNF-(cid:1), than typical intestinal resident proliferation and functional differentiation of IECs is regulated by macrophages, and the authors raised the possibility that CD14+ various growth factors (including epithelial growth factor (EGF), macrophages may play a key role in the predominance of Th1 im- TGF-(cid:1), TGF-(cid:2) and fibroblast growth factor (FGF)), chemokines, mune response found in CD [111]. regulatory cytokines (e.g. IL-6, IL-22) and trefoil peptides [94]. The role of macrophage migration inhibitory factor (MIF), an Several results suggest that epithelial restitution is influenced by other pro-inflammatory cytokine originating from both T-cells, PRRs. TLR2 induces gap junctional intercellular communication innate immune cells and epithelial and endothelial cells in the path- via connexin-43, and controls IEC restitution during acute and omechanism of IBD is emerging [112, 113]. MIF promotes the chronic inflammatory damage [95]. Similarly to TLR2, TLR4 is 4 Current Pharmaceutical Design, 2014, Vol. 20, No. 00 Gyires et al. recognition of LPS and Gram-negative bacteria by upregulating the In the last decades considerable efforts have been made to iden- basal expression of TLR4 in the macrophages, thus serves as a key tify a specific pathogen in IBD, and some bacteria (e.g. Mycobacte- factor in the initiation of innate immune response [114]. The release rium avium spp. paratuberculosis or adherent-invasive Escherichia of MIF stimulates the release of inflammatory cytokines, potenti- coli (AIEC)) have been proposed as causative agents, mainly in CD ates the recruitment of neutrophils to the inflammatory site and [138-140]. Moreover, various clinical studies were conducted to triggers metalloprotease (e.g. MMP-13) expression, leading to in- analyze the potential therapeutic effect of different antibacterial flammation and tissue damage [112]. The expression of MIF was regimens (usually with the agents clarithromycin, metronidazole, increased in DSS-induced colitis and administration of anti-MIF ciprofloxacin or rifaximin) (see section 3.4.), but the results are antibody significantly improved the DSS-induced symptoms [115]. conflicting [141, 142]. Now it is generally assumed, that instead of Moreover, the levels of MIF in the sera of UC patients were signifi- one (or few) distinct pathogen(s) an altered composition of gut cantly higher [116], which suggests that anti-MIF therapy may be a flora, resulting in dysbiosis, and an overactive immune response new therapeutic approach in IBD. Since increased MIF-expression may lead to chronic intestinal inflammation [143]. is also associated with tumorigenesis [117], it is tempting to specu- It is well established that the microbiota in IBD patients differs late that inhibition of MIF may also reduce the risk of colon cancer significantly from that of healthy people. One main difference is the related to chronic inflammation. decreased representation of the Firmicutes phylum. A reduction of As depicted above, the activation of PRRs expressed on innate the Clostridium leptum and coccoides groups (also known as Clos- immune cells and the consequent release of inflammatory cytokines tridium cluster IV and XIVa, respectively), and in particular the results in different T-cell pattern. In CD mainly the Th1 cytokines decreased amount of Faecalibacterium prausnitzii was observed by (IL-12, TNF-(cid:1), IFN-(cid:3)), while in UC predominantly Th2-associated several groups [144-148]. Indigenous Clostridia may possess anti- cytokines (like IL-5 and IL-13) are dominating [118-120], and rec- inflammatory properties by inducing IL-10 expressing Foxp3+ ognition of the importance of these cytokines led to the develop- CD4+ Treg cells in the colon [149]. Moreover, the loss of these ment of anti-cytokine biologic agents in the therapy of IBD. How- bacteria may result in a decreased butyrate production, which is one ever, it has to be emphasized that the cytokine profile of these dis- of the most important bacterial products in the gut and exerts vari- eases is much more complex and even individual cytokines may ous anti-inflammatory effects via regulating the migration of neu- possess diverse or opposing action in different clinical and immu- trophils, inhibiting the production of pro-inflammatory cytokines nological settings [64]. and increasing the expression of tight junction proteins in colon epithelia [136, 150]. Furthermore, the recent discovery of the Th17 lineage has raised a new paradigm. Th17 cells differentiate from naïve CD4 T- Another important genus in this phylum is the Lactobacillus, cells, which process is induced and regulated by various cytokines, which contains several probiotic strains. Some of them (e.g. the like IL-1(cid:2), IL-6, IL-21, IL-23 and TGF-(cid:2) [121]. It has been re- Lactobacillus rhamnosus GG strain) may possess beneficial effects vealed that Th17 cells, producing IL-17 and other pro-inflammatory in IBD [151] and recent evidences indicate that Lactobacillus sali- cytokines play an essential role in the development of colitis in both varius Ls33 is able to inhibit experimentally induced colitis in a mice and humans [122-124]. NOD2-dependent manner [152]. Furthermore, von Schillde et al. identified a Lactobacillus paracasei prtP-encoded protease named The activation of PRRs (both TLRs and NLRs) by the intestinal microbiota is essential for the development of Th17 cells, which is lactocepin, which degrades the pro-inflammatory chemokine IP-10 clearly demonstrated by the marked reduction of the Th17 cell (interferon gamma-induced protein 10) and consequently alleviates colonic inflammation [153]. Thus, a reduced amount of Lactobacil- number in germ-free mice [125, 126]. Accordingly, stimulation of TLR5 on lamina propria DCs promoted the differentiation of Th17 lus may contribute to the pathogenesis of IBD. Indeed, such altera- cells [127], while TLR9-deficient mice had decreased number of tions in the microbiota were observed by several authors [93, 145], though increased amounts [144] or no change of Lactobacillus lev- lamina propria Th17 cells [128]. Moreover, the stimulation of DCs with MDP has been shown to enhance NOD2-mediated production els [147] have also been reported. of IL-1(cid:2) and IL-23, which in turn promoted the IL-17 production Alterations in the Bacteroidetes phylum are also likely to con- by memory T-cells [129]. These results highlight the importance of tribute to the chronic inflammation. An increased representation of PRRs also for adaptive immunity. In addition, although originally the Prevotellaceae has been documented in IBD patients [154, 155] PRRs were thought to regulate innate immunity and only indirectly and recently also in the intestines of mice with an impaired innate the adaptive responses, it turned out that also T- and B-cells express immunity [93]. It is assumed that these bacteria are colitogenic via PRRs, and TLR or NLR agonists are able to directly influence their producing sulfatases, that degrade mucus oligosaccharides and functions [32, 130, 131]. disrupt mucosal barrier function [156]. In summary, disruption of the mucosal barrier, increased and In contrast, Bacteroides fragilis is supposed to exert anti- altered activation of DCs and macrophages, impaired balance be- inflammatory effects. Its Polysaccharide A (PSA) component has tween pro- and anti-inflammatory cytokines and polarisation of the been shown to inhibit Helicobacter hepaticus-induced experimental adaptive immune response towards the effector T-cells all contrib- colitis in mice [157] and to directly induce the anti-inflammatory ute to the pathomechanism of IBD, and PRRs are an important link function of Foxp3+ Treg cells by acting on TLR2 [158]. Although between the participants of this complex system. early observations suggested a higher abundance of Bacteroides fragilis in IBD patients [159], recent studies found lower levels of 2.3. The Microbiota this commensal [146, 147]. The human gut is inhabited by ~ 100 trillion bacteria, which can Similarly to Lactobacilli, Bifidobacteria from the Actinobacte- consist of more than 1000 species overall and at least 160 species in ria phylum also contain several probiotic strains and exert anti- each individual [132]. This indigenous bacterial community (the inflammatory effects. Bifidobacteria had beneficial effect in both microbiota) is influenced by several factors (like diet, age or health animal models [160] and in patients with UC and CD [161-163], status) and varies between individuals, although mainly only at the while lower count of Bifidobacteria was measured in IBD [147]. levels of strains and species, while most of the bacteria are mem- One potential protective mechanism is the activation of Tregs, as it bers of the Bacteroidetes and Firmicutes phyla [133, 134]. The has been observed in the case of Clostridia and B. fragilis (see microbiota plays a fundamental role in energy metabolism and im- above). The probiotic Bifidobacterium breve induced IL-10- munity, however, its role has also been implicated in several dis- producing Treg type 1 cells by activating intestinal CD103+ den- eases, e.g. obesity, insulin resistance and IBDs (recently reviewed dritic cells via the TLR2/MyD88 pathway [164]. Another strain, e.g. by [135-137]). Gut Inflammation: Current Update on Pathophysiology Current Pharmaceutical Design, 2014, Vol. 20, No. 00 5 Bifidobacterium lactis significantly decreased the colonic expres- tive), are given in mild disease, whereas more effective (but poten- sion of various pro-inflammatory, dendritic and T-cell markers, like tially more toxic) agents are given in severe disease or in patients IL-6, TNF-(cid:1), COX-2, CD40-L or IFN-(cid:3) in mice [160]. who are unresponsive to first-line therapy. Common conventional medications currently start with 5-aminosalicylic acid drugs, In summary, there is growing evidence that an imbalance be- tween colitogenic (e.g. AIEC) and tolerogenic bacteria (like Clos- corticosteroids (prednisolone, methylprednisolone, budesonide) for tridia, Lactobacilli and Bifidobacteria) has a major role in the path- UC as well as CD. Immunosuppressive agents such as azathioprine and 6-mercaptopurine were shown to be effective in both CD and omechanism of IBD. Colitogenic bacteria can damage the epithelial barrier functions either directly or via producing toxins. For in- UC, while methotrexate proved to be effective as a steroid-sparing stance AIEC strain LF82 disrupts the tight junction protein zonula agent in CD. On the other hand, cyclosporine induced a pronounced therapeutic effect in severe, active UC [182, 183, 184]. occludens-1 [165] and through binding to the cell adhesion mole- cule CEACAM 6 can lead to abnormal expression of claudin 2 A milestone in the therapy of CD was the introduction of in- [166], while enteropathogenic Escherichia coli (EPEC) induces fliximab, the first monoclonal antibody against TNF-(cid:1), proved to be epithelial cell apoptosis by producing a bacterial toxin called cycle effective in induction of remission in CD patients who had been inhibiting factor [167]. Both results in an increased intestinal per- refractory to other therapeutic agents [185]. Later, it was demon- meability, which permits the penetration of luminal antigens and strated that both adalimumab, and certolizumab, monoclonal anti- microbes, that can stimulate pro-inflammatory responses. bodies against TNF-(cid:1), maintained the clinical remission [186, 187]. Surprisingly, etanercept (another anti-TNF-(cid:1) agent, that fuses the However, the altered microbial flora can also induce inflamma- TNF receptor to the constant end of the IgG1 antibody) failed to tion indirectly, via reduced production of anti-inflammatory bacte- rial metabolites, like butyrate and other short-chain fatty acids (see exert similar beneficial effect in CD [183, 188]. above) [136] or via an altered metabolism of bile acids. Primary The more effective biological therapies are usually considered bile acids cholic acid and chenodeoxycholic acid are transformed as a last option and only in case of refractory diseases, because they by intestinal bacteria to their secondary forms, deoxycholic acid often cause severe adverse effects [189, 190]. This strategy is rec- and lithocholic acid, which can disrupt intestinal barrier functions ommended by current guidelines [191]. [168, 169]. The recent results of Duboc et al. provide direct evi- However, the natural course of the disease is not likely to be dence of the connection between dysbiosis, bile acid dysmetabolism modified by conventional treatment [192]. Since anti-TNF-(cid:1) ther- and chronic colonic inflammation [144]. They found a marked de- apy can induce and maintain clinical remission and mucosal heal- crease in bacteria of the Firmicutes phylum and altered levels of ing, early administration of anti-TNF-(cid:1) biological agents may pre- secondary and conjugated bile acids in the faeces of patients with vent late complications [193]. Consequently, the question has been IBD. raised recently: whether to maintain or to reverse the traditional therapeutic pyramid [194]. On the other hand, it also has to be kept 2.4. Environmental Factors in mind that majority of CDs have benign course and immune Environmental factors may also play a role in the pathogenesis modulators and biologics have severe adverse effects, that may of IBD. As mentioned earlier, the composition of the microbiota is result in increased risk of infections and malignant diseases substantially influenced by diet and other life style factors [170, (lymphoma) [195]. Consequently, treatment of the patients, who 171], and accordingly, dietary changes may contribute to the path- have a mild, benign course of the disease with highly potent omechanism of IBD. Indeed, it was demonstrated that consumption biologics, such as TNF-(cid:1) antagonists is not a good therapeutic of a diet high in saturated (milk derived)-fat promoted colitis in IL- choice, because of the risk of adverse effects. 10 knock out mice [172]. The triggering factor was presumably the Beside the side effects of anti-TNF-(cid:1) agents, additional prob- increased taurine-conjugation of bile acids, which increased the lems have been raised: among the primary responders only a third availability of organic sulfur and the amount of the sulfite-reducing of patients will maintain remission after 1 year [196], and the ther- microbe Bilophila wadsworthia, which in turn induced Th1 immune apy is often limited by a loss of efficacy. Therefore, finding novel responses. Moreover, Kim et al. [173] reported that high fat diet targets and the development of novel therapeutic strategies became altered the microbiota leading to an increased lumenal LPS content an urgent need. in the colon, which increased intestinal permeability and induced inflammation through a TLR4 signaling pathway. The strategy to find new targets is based on the main pathologi- cal alterations that characterize IBD. Since the disease is caused by In addition, vitamin D deficiency [174], as well as active and a combination of factors, including genetic susceptibility, immune passive tobacco smoking [13, 175], air pollution [176] or improved dysregulation, barrier dysfunction, and the change in microbial domestic hygiene and sanitation [177] are additional factors that flora, the new therapeutics aim to correct or restore 1./ the genetic may modify the homeostasis of the intestinal mucosa. The observa- alterations, 2./ the immune dysregulation, 3./ the barrier dysfunction tions, that IBD is common in Western countries, while uncommon and mucosal resistance 4./ the altered composition of gut flora in less developed areas, raised the intriguing hypothesis that im- (Fig. 1). proved hygiene and the consequent loss of routine exposure to parasitic worms (helminths) may play an important role in the path- 3.1. Correction of Genetic Alterations omechanism of IBD [178]. Under normal, healthy condition the intestinal mucus layer Helminth infections may have several beneficial effects on the prevents exposure of IECs to luminal bacteria. Several mechanisms immune system (reviewed by [179, 180]). They induce the forma- are involved in protection of intestinal epithelium against luminal tion of regulatory DCs, regulatory Treg cells and the production of microbiota, e.g. defensins (secreted by Paneth cells) and the pro- anti-inflammatory cytokines (IL-10, TGF-(cid:2)), while decreasing the duction of immunoglobulin A (IgA). PRRs are involved in the pro- formation of effector T-cells and pro-inflammatory cytokines (like duction of cytokines necessary for the development of immunity IFN-(cid:3), IL-17 or IL-12/23). Moreover, they seem to alter the compo- and have crucial role in innate microbial sensing by IECs, DCs and sition of the microbiota and promote the growth of probiotic Lacto- macrophages [30]. Their activation initiates NF-(cid:4)B and MAPK bacillaceae [181]. signaling pathways resulting in the production of pro-inflammatory cytokines and antimicrobial peptides [5, 197]. In addition, auto- 3. THERAPY phagy has a crucial role in the maintenance of intracellular homeo- The traditional therapeutic concept of CD is based on the so- stasis preventing abnormal accumulation of protein aggregates, called "step-up" approach: less toxic drugs (but often less effect- intracellular components, such as organelles, apoptotic bodies, and 6 Current Pharmaceutical Design, 2014, Vol. 20, No. 00 Gyires et al. Fig. (1). Therapeutic approaches in IBD targeting different factors (genetic variations, immune dysregulation, barrier dysfunction and intestinal microbiota alterations), that are involved in the pathomechanism of IBD according to the recent consensus. microbes [198]. One of the key proteins involved in the execution various inflammatory diseases. However, their real role in IBD of the autophagic process is ATG16L1 [199]. remains to be clarified. Though a large number of genetic loci have been found to be 3.2. Restoration of Immune Dysregulation associated with CD, the polymorphism of two genes, NOD2/ CARD15 [200] and ATG16L1 seem to have particular importance Today it is widely accepted that abnormal immune regulation is in the development of the disease [201]. While in healthy subjects a key factor in the pathomechanism of IBD and alterations in both ATG16L1 encodes threonine at amino acid position 300 innate and adaptive immunity have been observed. Dysregulation of (ATG16L1*300T), ATG16L1 encoding alanine (ATG16L1*300A) mucosal immunity in IBD leads to an overproduction of inflamma- instead of threonine at the same position increases the risk of the tory cytokines and trafficking of effector leukocytes into the intesti- development of CD, due to impairement in bacterial capture by nal mucosa, thus resulting in an uncontrolled intestinal inflamma- autophagy [202]. tion. Under homeostatic condition there is a balance between regu- latory (Treg) and effector T-cells (Th1, Th2 and Th17). Mucosal Moreover, patients with CD showed decreased expression of inflammation is induced either by an increase in the effector T-cell mucosal TLR3 and increased expression of TLR4, which results in population and an increased production of pro-inflammatory cyto- downstream release of inflammatory modulators, for example TNF- kines (such as TNF-(cid:1), IFN-(cid:3), IL-1, IL-6, IL-12, IL-17, IL-23) or a (cid:1) and IL-1 [203]. Consequently, stimulation of the TLR3 or NOD2 reduced function of Treg cells and decreased level of anti- pathway may represent a new approach of the therapy of IBD. Ex- inflammatory cytokines (e.g. TGF-(cid:2), IL-4, IL-10 or IL-11) pro- perimental data suggest that activation of TLR3 (e.g. by synthetic duced by different immune cells located in the lamina propria of the viral RNA) or NOD2 were effective for prevention of DSS-induced intestinal mucosa. Cytokines may therefore be targets for IBD ther- acute colitis in the mouse [204-206], which implies that stimulation apy [211]. Moreover, elimination of intestinal inflammation may be of TLR3 or NOD2 signaling may represent a new, successful thera- achieved either by reduction of effector T-cell populations or by peutic strategy for the treatment of CD in those patients who show increasing regulatory T-cell activity [5, 212]. reduced expression of TLR3 or carry NOD2 mutations [207]. On the other hand, blocking TLR signaling may represent another ap- Several reviews have been published recently on the potential proach for IBD treatment, because TLR2 and TLR4 are up- therapeutic agents in IBD that target the immune dysregulation [7, regulated in IBD [208]. Experimental data suggest that TLR4 12, 188, 190, 211, 213-218]. blockade decreased inflammation in DSS-induced colitis in mice, Restoration of the immune dysregulation may be achieved by but also interfered with colonic mucosal healing, since anti-TLR4 several mechanisms, such as inhibition of 1./ pro-inflammatory antibody treatment during recovery from DSS colitis resulted in cytokines, 2./ Th1 polarisation and proliferation, 3./ T-cell stimula- defective mucosal healing (as described above) [209]. tion (anti-CD3 therapy), 4./ cell adhesion, as well as 5./ by immune Attempts have been made to develop new TLR4 signaling in- stimulation (Granulocyte Macrophage Colony Stimulating Factor) hibitors. Recently, arylidene malonate derivatives were found to /GM-CSF)/ and Granulocyte Colony Stimulating Factor /G-CSF/ suppress LPS-induced production of NF-(cid:4)B, TNF-(cid:1), IL-1(cid:2) and and 6./ by increase of anti-inflammatory cytokines. nitric oxide [210], suggesting its potential therapeutic value for Gut Inflammation: Current Update on Pathophysiology Current Pharmaceutical Design, 2014, Vol. 20, No. 00 7 3.2.1. Inhibition of Pro-inflammatory Cytokines Novel TNF-(cid:1) inhibitors have been developed, such as golimu- 3.2.1.1. Inhibitors of TNF-(cid:1) mab, dersalazine, HMPL-004 and ozoralizumab (ATN-103). These compounds are in various phases of the clinical trial process, and TNF-(cid:1) is a fundamental mediator of this abnormal immune their real therapeutic values have to be determined [12]. In addition, response. TNF-(cid:1) has two forms, a transmembrane and soluble form, recently a vaccine against TNF-(cid:1) has been developed (TNF-(cid:1) ki- and its action is mediated by 2 receptors, TNF-R1 (TNF receptor noid, Debio-01512), as a new mechanism for inhibition of TNF-(cid:1) type 1, CD120a or p55/60) and TNF-R2 (TNF receptor type 2, [196], and phase I/II clinical trials in patients with moderate to se- CD120b or p75/80) [219] resulting in complex and differential vere CD were found to be promising [235, 236]. actions. While the pro-inflammatory effects of TNF-(cid:1) are mediated 3.2.1.2. Inhibition of IL-6 by TNF-R1, the immunoregulatory functions are independent from this receptor. Consequently, selective inhibitors of TNF-R1 that IL-6 has a fundamental role in immune regulation and inflam- reduce the pro-inflammatory function of TNF-(cid:1) without affecting mation. The IL-6 receptor (IL-6R) system has both a membrane- its immunoregulatory effects, may represent a new therapeutic ap- bound (IL-6R) and a soluble form (sIL-6R). Increased serum con- proach of IBD. centrations of IL-6 and sIL-6R have been shown to correlate to In the last 15 years, biological agents targeting TNF-(cid:1) have clinical activity of CD, and animal models have strongly suggested significantly improved the therapy of IBD refractory to conven- the therapeutic potential of anti-IL-6R monoclonal antibody. For example, anti-IL-6R monoclonal antibody reduced the symptoms of tional drugs. The efficacy of this therapy alone reflects the plei- otropic effects of TNF-(cid:1). colitis in Th1 cell-mediated murine colitis model. Similarly, block- ade of sIL-6R in vivo by a newly designed gp130-Fc fusion protein Infliximab, a monoclonal chimeric antibody, targeting human resulted in reduction of colitis activity and induction of apoptosis, TNF-(cid:1), became the first monoclonal antibody available for the indicating that sIL-6R suppresses mucosal T-cell apoptosis. Ac- treatment of CD and UC. The potency of this agent in moderate-to- cordingly, it was shown recently that IL-6 induces the anti- severe CD and UC has been one of the most important advances in apoptotic genes BCL2 and BCL-XL [237]. These results suggest the treatment of IBD. the therapeutic potential of anti-IL-6R monoclonal antibody in CD Infliximab also induces T-cell apoptosis, that contributes to its [237, 238]. therapeutic effect [220]. Namely, in the gut, there is a tight control Tocilizumab (also known as MRA) is a humanized IL-6 recep- of activation and expansion of T-cells. T-cell expansion is limited tor antibody, recognizes both the membrane-bound and the soluble by apoptosis, and T-cell resistance to apoptosis with consequent T- form of IL-6R and specifically blocks IL-6-induced actions [239]. cell expansion was observed in patients with IBD, which may con- Tocilizumab is expected to ameliorate the autoimmune inflamma- tribute to the pathomechanism [221]. Hence, it may be raised that tory diseases characterized by IL-6 overproduction and has been induction of apoptosis in T-cells and other effector cells may have developed as a therapeutic agent for rheumatoid arthritis (see re- therapeutic importance in the treatment of IBD and pro-apoptotic view [240]). Tocilizumab treatment of patients with active CD in- signaling might be a target for drug development [5]. duced reduction of the disease activity index compared to the pla- It has been shown that some anti-TNF-(cid:1) agents induce apopto- cebo group, however, remission of the disease was registered only sis in monocytes and lymphocytes both in vitro and in vivo [222, at very low proportion of the patients [241]. The beneficial effect of 223]. Consequently, apoptosis-induction seems to be an important tocilizumab was confirmed by Nishimoto [242]. part of the therapeutic action of TNF-(cid:1) antagonists and differences 3.2.1.3. Inhibition of IL-13 in their apoptotic efficacy might contribute to the differences found in their clinical efficacy. In addition to infliximab, several other IL-13 has been shown to be pathogenic in IBD, particularly in UC. It impairs the function of the epithelial barrier and also causes therapeutic agents that are effective in the treatment of IBD, includ- apoptosis of epithelial cells [10, 11, 243, 244]. IL-13 overexpres- ing corticosteroids, sulfasalazine, azathioprine, 6-mercaptopurine and anti-IL-12 antibody, induce apoptosis of activated T-cells [222, sion in the inflamed mucosa is particularly characteristic for UC and IL-13 is considered as the major effector cytokine in UC [245]. 224-226]. Adalimumab, a fully human IgG1 monoclonal antibody to TNF- The anti-IL-13 antibody anrukinzumab (IMA-638) as well as tralokinumab (CAT-354), a fully human anti-IL-13 antibody are (cid:1), was found to be effective in patients with CD refractory to conventional therapy and in patients with an attenuated response to under clinical studies (phase IIa) in patients with mild to moderate infliximab [211, 227, 228]. Recent study showed that adalimumab UC. QAX576, another fully human antibody against IL-13, is in phase I/II trials in patients with CD, the final results are pending could induce and maintain clinical remission in patients with mod- erate-to-severe UC as well, who did not have a satisfactory re- [246, 247]. sponse to steroids or immunosuppressive agents [229]. 3.2.1.4. Inhibition of Mitogen Activated Protein Kinases (MAPKs) Certolizumab and certolizumab pegol (its PEGylated Fab' Expression of pro-inflammatory cytokines, which are critical in fragment with increased plasma half-life) are humanized TNF-(cid:1) the pathogenesis of IBD, is regulated by one or more MAPK path- monoclonal antibodies. Majority of the human studies demonstrated ways. Accordingly, activation of several MAPK members was their effectiveness in maintenance of response and remission in CD found in biopsies from the inflamed mucosa of CD patients [248]. [230, 231]. However, Sandborn et al. failed to confirm the effec- CNI-1493 (semapimod), inhibitor of JNK/p38MAP kinases, tiveness of certoluzimab after 6 weeks treatment [232]. showed significant clinical improvement of severe CD, confirming Etanercept is a genetically engineered fusion protein consisting the potential role of inflammatory MAPKs in the pathogenesis of of two recombinant human TNF p75 receptors linked to an Fc por- CD [249]. In contrast, a highly potent inhibitor of p38 MAPK, tion of human IgG1 fragment. It was found to be ineffective for the BIRB 796 (doramapimod) was studied in chronic active CD in a treatment of patients with moderate to severe CD in the same dose multicenter, multinational trial with placebo control, and the results range that was effective in rheumatoid arthritis [233]. failed to show evidence for clinical efficacy [250]. CDP571, an immunoglobulin G4 humanized monoclonal anti- Concerns with the use of MAPK inhibitors have been raised, TNF-(cid:1) antibody showed a slight and short lived reduction in clini- for example inhibition of p38 and 42/44 MAPK reduces normal cal activity of UC [234]. It proved to be less effective than inflixi- bactericidal activity of neutrophils [251]. Moreover, ubiquitous mab and further clinical development of CDP571 for the treatment presence of the MAPK pathways and their involvement in several of CD has been discontinued (see review [194]). processes suggests that MAPK inhibition should be selective for an isoform, rather than general. 8 Current Pharmaceutical Design, 2014, Vol. 20, No. 00 Gyires et al. 3.2.1.5. New Avenues Against Pro-inflammatory Cytokines or with decreased Th1-mediated inflammatory cytokines at the site of Downstream Signaling Pathways disease [265]. Currently, several novel agents have been developed to target Ustekinumab, and briakinumab are human monoclonal antibod- either pro-inflammatory cytokines or downstream signaling path- ies against IL-12 and IL-23, both targeting the p40 subunit. Usteki- ways. Pro-inflammatory cytokines IL-17, IL-18 and IL-21 were numab significantly increased the rates of response and remission found to be elevated in the inflamed intestinal mucosa of patients as maintenance therapy, and it may be particularly useful in patients with IBD [252-254]. Monoclonal antibodies developed against who previously did not respond to anti-TNF therapy [266]. Briaki- these cytokines are currently in phase I/II clinical trials in patients numab, however, in patients with moderate to severe CD was not with CD. Unexpectedly, secukinumab (AIN 457) targeting IL-17 in effective for the induction or maintenance of remission ([267]. double-blind, placebo controlled study in patients with CD wors- Apilimod is an inhibitor of the transcription of IL-12 and IL-23. ened the disease compared with placebo [255]. In contrast, inhibitor In a randomised controlled trial apilimod failed to induce signifi- of the release of IL-17, vidofludimus (4SC-101/SC12267) showed cantly greater effect than placebo treatment [268]. beneficial effect in a single-arm, open-label study, indicating that it 3.2.2.3. Interferon-(cid:1) may be useful in maintaining clinical remission both in patients with CD and UC [256]. Further investigation is needed to clarify IFN-(cid:3) was found to be elevated in all genetic animal models of the modulatory role of IL-17 in IBD. IBD and seems to have determining role in the development of Th1 responses. If the target of IBD therapy is to reduce Th1 responses, On the other hand, gut inflammation can also be restricted by then inhibition of IFN-(cid:3) represents one potential therapeutic ap- blocking the downstream signaling pathways mediated by cytoki- proach. nes. Signaling molecules that interact with cytokine receptors are the Janus kinases (JAK), JAK1, JAK2 and JAK3, which play a Fontolizumab, a humanized monoclonal antibody to IFN-(cid:3) was fundamental role in the development and differentiation of immune shown to have a beneficial effect on disease activity [12, 211, 269]. cells. An other study suggested that though a strong clinical response was not induced by fontolizumab, a significant decrease in C-reactive Tofacitinib, inhibitor of JAK3 was developed recently [257], protein levels was observed. Further studies are necessary to deter- but various clinical efficacies have been experienced. A multicen- mine its efficacy [270]. tre, double-blind, placebo controlled study with tofacitinib in pa- tients with moderate to severe active CD showed no clinically sig- 3.2.3. Inhibition of T-cell Stimulation nificant response following 4 weeks of treatment compared with Since intestinal inflammation may be resulted by an increased placebo, but in patients with moderate to severe UC it showed im- activity of the effector T-cell population with excessive inflamma- provement in both clinical response and remission rates [258, 259]. tory responses and CD3 is required for T-cell activation, drugs that 3.2.2. Inhibition of TH1 Polarisation and Proliferation target T-cell activation may exert therapeutic effect in IBD. 3.2.2.1. Inhibition of IL-2 Visilizumab, a humanized anti-CD3 monoclonal antibody was demonstrated to induce apoptosis in activated T-cells selectively, Inhibition of the binding of IL-2 to the IL-2 receptor results in and enhance the production of IL-10, a potent anti-inflammatory inhibition of the growth, proliferation and differentiation of T-cells cytokine [271]. It proved to be effective against T-cell transfer coli- to 'effector' T-cells. tis [272]. The results of an open-label phase I human study with Basiliximab (chimeric monoclonal antibody) and daclizumab visilizumab in patients with severe corticosteroid-refractory UC (humanized monoclonal antibody) are targeted against the (cid:1)-chain suggested that it may be clinically beneficial [273]. of the IL-2 receptor (CD25), and inhibit the binding of IL-2 to the Another study confirmed this beneficial effect of visilizumab in IL-2 receptor. However, daclizumab failed to induce a significant a pilot randomized phase I/II study. Visilizumab improved both action in UC [260]. Neither basiliximab increased the effect of cor- symptomatic and clinical responses in severe, steroid-refractory ticosteroids in the induction of remission in patients with corticos- UC, though, all patients experienced adverse reactions [274]. How- teroid-resistant moderate to severe UC [261]. ever, in a randomised, double-blind, placebo-controlled study vis- 3.2.2.2. Inhibition of IL-12 and IL-23 ilizumab was not effective for severe, corticosteroid-refractory UC CD is associated with an enhanced Th1 cytokine response, [275]. The same conclusion was drawn by Wood [276]. which results in increased production of IL-12, a pro-inflammatory CD40 is also involved in T-cell activation. Humanized mono- cytokine, that stimulates the production of IFN-(cid:3) and TNF-(cid:1). IL-12 clonal antibody against CD40L has been developed, however in a is also involved in the differentiation of naïve T-cells into Th1 cells. phase II trial for CD due to side effect (thromboembolism) the trial It is a heterodimeric protein with 2 subunits: the p35 and p40. The was halted [277]. structurally similar cytokine IL-23 (its p40 subunit is identical to 3.2.4. Inhibition of Cell Adhesion IL-12p40, and its p19 subunit shows certain similarities to IL-12 p35) has also an important role in intestinal inflammation, in con- Lymphocyte trafficking to the gut is a basically important step junction with IL-6 and TGF-(cid:2)1. IL-23 stimulates naïve CD4+ T in the initiation and maintenance of intestinal inflammation in pa- cells to differentiate to Th17 cells. The highly aggressive immune tients with IBD. Alpha 4 integrin, a cell-surface glycoprotein in- response together with IL-12/IL-23 could have a determining role volved in the adhesion, migration and activation of immune cells, is in initiation and perpetuation of chronic intestinal inflammation in expressed on most of the lymphocytes, and combined with either a CD [262, 263]. (cid:2)1 subunit (that interacts predominantly with the endothelial ligands, vascular cellular adhesion molecule 1 /VCAM-1/) or a (cid:2)7 Recently p40 peptide-based vaccines have been developed. subunit (that interacts predominantly with the mucosal addressin Pretreatment of rats with the vaccines induced specific antibodies to cellular adhesion molecule 1 (Mad-CAM-1)) [278]. The interaction IL-12 and IL-23, which was associated with improvement of intes- between (cid:1)4(cid:2)7 integrin and Mad-CAM-1 is important in mediating tinal inflammation and fibrosis, indicating that the vaccine may leukocyte homing to gut mucosa [279]. Several therapeutic agents provide a potential approach for the long-term treatment of CD have been developed that specifically target the (cid:1)4(cid:2)7 subunit of [264]. integrin. A human study showed that treatment with a human mono- Natalizumab, a humanized monoclonal antibody against the cell clonal antibody against IL-12 may induce clinical responses and adhesion molecule (cid:1)4-integrin subunit, was proved to be effective remissions in patients with active CD. This treatment is associated in experimental colitis both in the mouse and the rat [280, 281]. Gut Inflammation: Current Update on Pathophysiology Current Pharmaceutical Design, 2014, Vol. 20, No. 00 9 Moreover, the effect of natalizumab has been studied also in hu- tions in IL-10 and IL-10 receptor (IL-10R) in patients with very mans, and it was shown to block the adhesion and migration of early onset IBD was observed [299]. white blood cells into the gut and to reduce chronic inflammation However, Buruiana et al. [300] on the basis of a systematic associated with CD [282]. Reviews based on relevant literature and review of the literature concluded that IL-10 does not appear to meta-analysis of the controlled trials of natalizumab suggested that provide any benefit for the treatment of active CD. the therapy was superior to placebo in inducing remission of CD 3.2.6.2. IL-11 [283, 284]. However, natalizumab therapy is associated with an increased risk of reactivation of latent John Cunningham (JC) virus, IL-11 besides its thrombocytopoietic properties improves the which causes the potentially fatal progressive multifocal leukoen- mucosal barrier function and inhibits the inflammatory reaction by cephalopathy (PML). The risk to develop PML is increased by the reducing expression of NF-(cid:3)B and in turn IL-1, TNF-(cid:1) and other presence of anti-JC virus antibodies, and previous or concomitant proinflammatory peptides [301]. Therefore IL-11 was examined on treatment with immunosuppressive (IF-(cid:2)1 or azathioprine) agents experimental colitis in the rat induced by trinitrobenzene sulfonic [285]. acid (TNBS), where IL-11 exerted protective effect [302]. Recent findings confirmed the beneficial action of IL-11: administration of Vedolizumab (MNL-0002) is gut-specific, (cid:1)4(cid:2)7-integrin- exogenous IL-11 was found to be protective against lethal colitis in neutralizing monoclonal antibody, that appears to lack systemic TLR2-deficient mice (TLR2 is involved in maintaining epithelial effects. It was particularly effective in UC and also in CD, indicat- barrier function) [303]. However, in a human study recombinant ing that vedolizumab might be therapeutic option for the treatment human IL-11 was less effective than prednisolone in the treatment of therapy-refractory patients [286]. A recent review evaluated the of CD [304]. safety and efficacy of vedolizumab for the treatment of CD, and it was concluded that vedolizumab is an effective and well-tolerated 3.2.6.3. Interferon-(cid:1)-1a (IFN-(cid:1)-1a) drug [287]. Though it does not increase the risk of infection, it was Data of the literature reflect conflicting results on the effect of demonstrated that vedolizumab may reduce the number of Treg IFN-(cid:2)-1a in IBD. Some studies indicate that IFN-(cid:2)-1a exerts a cells and consequently their suppressive effect on colonic inflam- therapeutic effect, while others found that IFN-(cid:2)-1a did not produce mation (see review [288]). a significant therapeutic action compared with placebo. Recent Alicaforsen (ISIS 2302) is an antisense oligodeoxynucleotide to clinical trial in patients with CD came to the conclusion that there ICAM-1 [289]. Experimentally, it inhibited the DSS-induced colitis was no difference between the effects of the administration of IFN- both in mice and rats [289, 290], but in human studies both im- (cid:2)-1a and placebo [305, 306]. provement of clinical symptoms and lack of effect have been shown in CD (see reviews [12, 211, 291, 292]). In patients with active UC 3.3. Restoration of Barrier Dysfunction and Stimulation of Mu- cosal Healing and Resistance alicaforsen enemas induced a beneficial effect, and the drug was well tolerated [291]. Repeated intestinal epithelial damage and the consequent dis- 3.2.5. Immune Stimulation ruption of the intestinal barrier function is a key mechanism of IBD [3, 6]. Namely, the alteration of intestinal barrier function may re- Though granulocyte macrophage colony stimulating factor sult in translocation of commensal bacteria into the intestinal wall, (GM-CSF) and granulocyte colony stimulating factor (G-CSF) are leading to uncontrolled T-cell activation and inflammation. Damage hematopoietic growth factors, they stimulate cells of the innate of barrier integrity, with increased antigen and bacterial uptake is immune system (neutrophils, macrophages and DCs). They have believed to be important in the pathophysiology of CD [4]. also been shown to be produced within Paneth cells of intestinal An intact barrier function of the intestinal epithelium prevents mucosa, and their receptors are expressed within IECs [293]. translocation of commensal bacteria into the mucosa. Consequently, Moreover, GM-CSF has been demonstrated to promote prolifera- tion within these cells [294]. though mucosal healing has been considered as a sign of complete healing of gut inflammation, it should be emphasized that mucosal GM-CSF has been shown to reduce DSS-induced colitis and healing can be considered as an initial step in suppression of in- decrease the levels of pro-inflammatory cytokines (TNF-(cid:1) and IL- flammation [307]. In a systematic review Neurath and Travis [307] 1(cid:2)) in colonic tissue samples [295, 296]. These results were con- analyzed the influence of conventional therapeutic agents as well as firmed recently; GM-CSF decreased the DSS-induced colitis and the biologics on the healing of intestinal mucosal injury in IBD, and the expression of pro-inflammatory cytokines. In addition, the dura- they concluded that while corticosteroids have little or no positive tion of ulcer healing was shorter and epithelial regeneration was effects on induction/maintenance of mucosal healing in CD (but facilitated in GM-CSF-treated mice [295]. Based on these preclini- induced healing in patients with UC), azathioprine, in a lesser ex- cal results, a potential role for GM-CSF in the therapy of patients tent methotrexate, as well as natalizumab, infliximab, adalimumab with IBD has been raised. and certolizumab pegol could induce mucosal healing [307]. Sargramostim (recombinant human GM-CSF) and filgrastim Following injury of intestinal mucosa, IECs migrate into the (recombinant human G- CSF) have been examined in several hu- damaged area to restore barrier integrity [298, 308], followed by man studies and majority of them suggested a significant improve- proliferation of IECs to correct the epithelial defect. Finally, differ- ment and remission of CD ([297] and reviews [12, 298]). However, entiation of IECs is necessary to restore mucosal barrier and epithe- sargramostim in a phase III multicentre double-blind, placebo con- lial function. These consecutive events (restitution, proliferation trolled study in patients with active CD failed to induce a signifi- and differentiation) are mediated by regulatory proteins (such as cant difference in clinical efficacy compared to placebo. Further chemokines), defensins, as well as by growth factors [298]. Growth human studies are necessary to reveal their role in the treatment of factors have crucial role in cell restitution/proliferation/ differentia- IBD [211]. tion, and in angiogenesis. Moreover, TGF-(cid:2) inhibits the differentia- 3.2.6. Anti-inflammatory Cytokines tion of naïve T-cells to Th1/Th2 subtypes. 3.2.6.1. IL-10 3.3.1. Growth Hormone (GH) IL-10 reduces the production of pro-inflammatory cytokines IL- GH, a regulatory peptide that stimulates aminoacid and electro- 1(cid:1), IL-6 and TNF-(cid:1), downregulates and controls the acute inflam- lyte uptake by the intestine, decreases intestinal permeability and mation and thereby may improve the course of IBD. IL-10 poly- stimulates collagen synthesis by induction of the expression of insu- morphisms have been shown to be associated with IBD and muta- lin-like growth factor (IGF). IGF was shown to promote epithelial repair in intestinal inflammation in preclinical animal models. For 10 Current Pharmaceutical Design, 2014, Vol. 20, No. 00 Gyires et al. example, recombinant human growth hormone induced protective factor (PIGF), has also been suggested to be a marker of pathologic effects in TNBS-induced colitis [309]. A preliminary study showed angiogenesis and may play a critical role in pathogenesis of UC. that human growth factor was effective in CD, it improved the Accordingly, inhibition of pathologic angiogenesis by either anti- clinical symptoms (the number of liquid/soft stools, severity of VEGF or anti-PIGF, was demonstrated to be a new approach to abdominal pain and overall well being) and a statistically signifi- attenuate UC [321, 322]. cant increase in circulating levels of IGF-1 was observed [310]. The role of additional growth factors, such as glucagon-like Further, placebo controlled human studies should be performed to peptide or trefoil factors have been raised to be involved in healing determine the real place of growth factor in the therapy of IBD. and restoration of mucosal integrity in IBD [298, 323]. Some of 3.3.2. Epidermal Growth Factor (EGF) them have been studied in clinical trials or experimental colitis Human recombinant EGF was shown to be effective in experi- models and their potential role in the treatment of IBD remains to be further analyzed. mental colitis induced by TNBS given prophylactically, before the induction of colitis [311]. In UC patients EGF enemas administered However, it should keep in mind that both UC and CD are as- parallel with mesalamine resulted in a significant improvement of sociated with dysplastic lesions and with increased risk of the de- symptoms [312], however, the effect of EGF enema alone (with velopment of colorectal cancer [324]. Since growth factors may placebo) has not been studied. induce stimulation of dysplastic tissues, growth factor therapy rep- resents a potential risk for induction of malignant alterations in 3.3.3. Keratinocyte Growth Factor (KGF) these patients [325]. KGF-1 and -2 are ligands of the fibroblast growth factor (FGF) In contrast, the recent results of Dube et al. [326] suggested that family. KGFs may have role in wound healing and maintaining epithelial homeostasis. Expression of KGF-1 was found to be up- EGF receptors by modulating epithelial regeneration and reducing inflammation might limit the subsequent tumorgenesis. Namely, regulated in patients with active CD and UC, indicating that KGF since chronic inflammation increases the risk of the development of may have role in the pathomechanism of IBD [313]. cancer in IBD patients, disrupting the cycle of inflammation and In animal models, KGF-2 proved to be protective against DSS- epithelial injury may result in reduction of cancer risk. The authors induced colitis given both prophylactically and therapeutically, both showed that inactivation of the EGF-receptors accelerated the pro- the clinical as well as the histological symptoms were improved gression of colorectal tumors in mouse models of colitis, which significantly [314, 315]. However, human recombinant KGF-2 suggests that therapy with EGF may reduce long-term cancer risk. failed to improve symptoms in patients with moderate to severe colitis, when compared with placebo. The contradictory results 3.4. Normalization of the Altered Composition of Microbial gained from animal experiments and human studies were explained Flora by miscalculation in estimating the target dosage in humans [316]. Convincing evidence from both animal models and clinical However, a recent study confirmed the previous finding; KGF-2 observations indicate that the microbiota is the most probable factor (and EGF) did not show efficacy in phase II trials concerning pa- that initiates chronic inflammation in CD. In accordance with this tients with either CD or UC (see review [298]). concept, antibiotics are one potential therapy in the treatment of 3.3.4. Transforming Growth Factor (cid:1) (TGF-(cid:1)) active CD and UC. Moreover, it was shown that intestinal bacteria The TGF-(cid:2) superfamily in the intestinal epithelium controls cell are necessary for the development of experimental colitis in the proliferation, differentiation, apoptosis, as well as the activation mouse [225, 327]. state of differentiated cell types. The TGF-(cid:2) superfamily can further Bacterial adhesion and invasion into the intestinal mucosa may be divided into subfamilies and within the gut these include the be particularly important in the development of intestinal mucosal TGF-(cid:2) and bone morphogenetic protein (BMP) subfamilies. TGF-(cid:2) inflammation, e.g., as in the case of Escherichia coli [328]. Though superfamily ligands appear to facilitate the maintenance of normal several pathogens have been supposed to be involved in the devel- epithelial homeostasis. Dysfunction of TGF-(cid:2)/BMP signaling opment of IBD (see section 2.3.), none of them have been proven to within the intestinal epithelium has been raised to be involved in the have a causal role, rather, microbial antigens that are present in the pathogenesis of human colitis [317]. intestinal lumen under normal conditions seem to drive intestinal Recent preclinical studies demonstrated the role of BMP-7 both inflammation [213]. as a prophylactic and a therapeutic agent in experimental colitis The microbiota is involved in both beneficial and deleterious induced by TNBS in the rat. Cytokine analysis of treated rats processes in the intestinal tract. Epithelial cells may act as a mu- showed a reduced mRNA expression of pro-inflammatory cytoki- cosal protective and antimicrobial defensive system. Antimicrobial nes, like IL-6, TNF-(cid:1), and ICAM-1 and decreased expression of peptides, such as defensins, released by IECs and Paneth cells, play pro-fibrogenic cytokines (TGF-(cid:2)1) [318]. an essential role in host defense. Clinical studies showed reduced 3.3.5. Vascular Endothelial Growth Factor (VEGF) expression of both (cid:1)- and (cid:2)-defensins and the consequent reduced killing of certain microorganisms by the intestinal mucosa of pa- VEGF has basic role in the regulation of angiogenic processes tients with CD [329]. These findings indicate that defensin defi- during both development and in pathologic conditions (inflamma- ciency should be corrected in patients with CD. The nuclear recep- tion or tumorgenesis). Six mammalian VEGF family ligands tor peroxisome proliferator-activated receptor gamma (PPAR-(cid:3)) (VEGF-A - VEGA-F) and 3 distinct receptors (VEGFR-1, VEGFR- plays an essential role in intestinal homeostasis. PPAR-(cid:3) was 2 and VEGFR-3) have been described. In rats, anti-VEGF antibody shown to function as an antimicrobial factor by maintaining consti- was investigated in colitis induced by iodoacetamide given intra- tutive epithelial expression of a subset of (cid:2)-defensin in the colon. colonically, and the results showed markedly less severe colitis as Defective killing of several intestinal microbiota in colonic mucosa well as significant reductions in pro-inflammatory cytokines com- of PPAR-(cid:3) mutant animals, for example Candida albicans, Bacter- pared with controls. The beneficial effect of anti-VEGF antibody on oides fragilis, Enterococcus faecalis and Escherichia coli was dem- colitis may be due to its moderating effects on vascular permeabil- onstrated recently [330]. Furthermore, the PPAR-(cid:3) agonist rosigli- ity and inflammatory cell recruitment [319]. Further studies showed tazone is a potent inducer of a subset of (cid:2)-defensin in mouse colon. that the levels of VEGF-A and VEGFR-2 were increased in patients Accordingly, rosiglitazone was shown to be efficacious in mild-to- with IBD as well as in DSS-treated mice. Furthermore, overexpres- moderate UC [331]. This finding raises a new potential mechanism sion of VEGF-A in mice worsened DSS-induced colitis, whereas for the improvement of gut barrier function in CD. administration of a soluble VEGF receptor led to decreased colonic inflammation [320]. Another angiogenic factor, placental growth