Pathogenesis Of Inflammatory Bowel Disease Biology Essay


Inflammatory disorders of the bowel comprise of Crohns disease and ulcerative colitis. Both of these are chronic and relapsing conditions. Crohn's disease is a granulomatous inflammation affecting the full thickness of the bowel wall, therefore known as a transmural inflammatory bowel disease. This condition can affect any part of the gastrointestinal tract from mouth to anus and the most common site of disease is at the terminal ileum with discontinuous or patchy distribution. Approximately 5-7 in 100,000 people in the UK suffer from Crohn's disease and the incidence of this is increasing (1). Currently, there is no cure for this chronic inflammatory disorder, but the mortality rate of it is relatively low. However, it could still cause a small increase in risk of small bowel and colorectal carcinoma (2). While ulcerative colitis is a non-granulomatous, diffuse superficial inflammation sited at the colorectal area. It is characterised by relapses and remissions. Symptoms of Ulcerative colitis can resolve by it-self but usually requires treatment to go into remission. (3) This condition affects about 10 in 100,000 people in the UK. Though, incidence in developing countries and warmer countries are lower. It is also found that ulcerative colitis increases the risk of colorectal cancer substantially (1).

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As the name suggests, Inflammatory Bowel disease (IBD), the underlying problem of this condition is inflammation within the gastrointestinal tract (4). Idiopathic IBD normally affects immunocompetent patients with symptoms caused by cytokine-driven (non-infectious) inflammation. In the case of Crohn's disease, symptoms are due to excess production of IL-12/IL-23 and IFN-γ/IL-17. While excessive IL-13 production can cause symptoms of ulcerative colitis (5). Hence, the treatment for IBD is primarily to reduce the chronic inflammation using drugs like Aminosalicyclates, Corticosteroids, Methotrexate, Cyclosporin, and Anti-tumour necrosis factor (anti-TNF) therapy (6). Of course, there is a need to include other therapies like anti-diarrhoeals and nutritional supplements (B12 vitamin) to relief the symptoms of IBD (6). Also, surgeries might cure ulcerative colitis by removing the pathological part of bowel, but, it cannot cure Crohn's disease as it can only treat bowel blockages and fistula complications (7). Despite the abundant treatment options, the exact cure for IBD is still not found. This is simply because up till now, the exact etiology and pathogenesis of Crohn's and ulcerative colitis remain unknown. Though, several hypotheses have been suggested and it may also be a combination of all the hypotheses (1). Genetic, infectious, immunological and psychological factors have been implicated to influence this idiopathic disease (8). The hypotheses made for both Crohn's and ulcerative colitis are the same with the exception of smoking which unusually is related to a decreased risk in ulcerative colitis (1).

One of the hypotheses made is that there is an excessive immunological response to normal gut flora due to dysregulation of mucosal immune system. It is either due to defective mucosal effector T cells that overact upon normal gut flora or under-acting regulatory T cells that suppress immune system (9). Duchmann and colleagues had conducted an experiment on IBD some 15 years ago (10). Studies showed that lamina propia mononuclear cells (LPMC) derived from the inflamed IBD tissue exhibit intense stimulation when cultured with autologous or heterologous microflora, causing extensive proliferation of LPMC after the co-culture (10). This proliferation can then be inhibited by anti-MHC class II, suggesting that this process is driven by antigen (10). On the other hand, cells of normal gut tissue (adjacent non-inflamed intestine areas of the same IBD patient), LMPC or peripheral blood mononuclear cells (PBMC), only respond to heterologous microflora (10). This suggests that IBD patients are lack of tolerance to antigens of autologous gut flora. But, it is also possible that these results are secondary to dysregulation of the mucosal which causes the increase exposure of autologous microflora and hence increase reactivity (9).

Similar observations of immune response hyper-reactivity to normal gut flora are found in murine models with underlying genetic defects, such as IL-2 or IL-10 deficiency. These defieciency causes abnormal effector or regulatory T cells responses (11). IL-2 is usually produced in the body during immune response (12), when antigens are presented. It drives differentiation and proliferation of T cells (13) (14). Also, IL-2 is necessary for T cell development in the thymus especially for the maturation of regulatory T cells (Treg cells) (15). As Treg cells leave the thymus, it prevents other T cells from acting on self-antigens by preventing these responding cells to produce IL-2 (16). In other words, it prevents autoimmune diseases, which in this case, IBD. Hence, in case of IL-2 deficient, there will be less maturation of Treg cells, causing an increase in self-reacting T cells. IL-10 on the other hand is a potent immunoregulator. It can inhibit synthesis of pro-inflammatory cytokines like IL-2 and TNF-alpha by mast cells and Th1 cells (17). It also potently suppresses the antigen presenting capacity. So, IL-10 deficiency would promote inflammation (17). However, when the experiment was done in a germ-free environment, on the same knockout mice, gut inflammation did not take place (9). This study further proves that presence of normal gut commensals is required for mucosal inflammation leading to IBD.

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The next question to the first hypothesis of excessive immune responses is that, is there a genetic influence to it? Hence, a study involving innate immune responses, inducing inflammation was carried out on animal models. It is well established that microbial components such as surface membrane and endosomal TRLs, intracytoplasmic members of the nucleotide-binding oligomerization domain containing (NOD) and Pyrin families of proteins are required to stimulate abnormal inflammatory responses (18) (19). This experiment involves STAT-3 gene deficient mice, whereby STAT-3 is essential for IL-10 signalling (20). Since, IL-10 deficiency promotes inflammation, the mice exhibit spontaneous enterocolitis similar to human IBD. These mice were then crossed with TLR-4 deficient mice. Toll-like receptors (TLRs) induce genes related to immune responses (21). Each TLR is responsible for recognition of specific pathogen component; in this case, TLR-4 is responsible for lipopolysaccharides (LPS) (22). Interestingly, The STAT-3/TLR-4 double deficient mice manifested far lesser colitis and IFN-γ production (20). This indicates that microflora derived LPS is needed to trigger inflammatory processes by Th1 cells. This study shows that certain human genes affecting innate immune responses will lead to IBD. Therefore identification of these genes may as well find a cure for IBD.

Crohn's disease is a chronic inflammatory disorder which is thought to be an effect of environmental factors in a genetically predisposed individual (23). The susceptible gene for Crohn's disease identified is the caspase recruitment domain protein 15 (CARD15) genes, which is related to the innate immune response. This gene encodes for NOD2 mapped at chromosome 16q12.This mutation will cause mucosal immune defects which supports the first hypothesis mentioned. Mutation of this gene was found in 10%-20% of Caucasians with Crohn's disease. Arg702Trp, Gly908Arg, and frameshift Leu 1007 are the 3 types of NOD2 mutants which accounts for 82% of all the Crohn-associated CARD15 mutation (24) (23). NOD1 and NOD2 have important roles in innate immunity as sensors of microbial components (19). AS expected, patients with homozygous mutation will have substantial increased in risk of developing Crohn's (20 to 40-times), while heterozygous patient have lesser increase in risk (2 to 4 times ) (19).The microbial ligand is a peptide fragment (muramyl dipeptide), derived from peptidoglycan which is present in almost all bacterial walls. Recognition and activation of NOD2 involves many components. Since NOD2 is expressed in antigen presenting cells (APCs) and Paneth cells (epithelial cells at the base of small intestine crypts), dysfunction or dysregulation of either cell types would lead to Crohn's disease (9). NOD2 has a leucine-rich repeat (LRR) domain which acts as the microbial recognition unit and an NOD domain which is crucial for activation of a NOD2 molecule after recognising the microbial ligand. Activation of the NOD2 molecule will then activate the next molecule known as the receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK) (25). The pathway then continues to activate NF-κB, a key pro-inflammatory transcription factor (26). The NF-κB will then trigger the Th1 responses. Since mutation of the CARD15 gene is found related to Crohn's patients, it will definitely cause a decrease in NF-κB levels which should cause a deficient NF-κB dependant Th1 responses. However, it was also known that there were excessive Th1 responses in Crohn's disease. Therefore, studies have been conducted by Watanabe, et al. using CARD15 knock-out mice. This study reported that intact or normal NOD signals would inhibit Toll-like receptor-2 driven NF-κB activation. Hence, in case of Crohn's disease with decreased NOD2 deficiency, enhance the toll-like receptor-2 activation and hence the increase in Th1 responses (26).

Although NOD2 deficient is key factor that increase Crohn's susceptibility, NOD2 deficient mice did not express spontaneous colitis when they have the normal population of gut microflora. However, when the mice are introduced to a specific antigen that is recognised by the T cells, inflammation took place. In this study, it was reported that the NOD2-deficient APCs produced increased IL-12 levels in the presence of ovalbumin (OVA) peptide and peptidoglycan or a recombinant E. Coli that expresses OVA (ECOVA). It also increases the IFNγ responses from co-cultured OVA-specific CD4 Tcells. Hence, when ECOVA is introduced intrarectally, the mice developed colitis associated with the expansion of OVA-specific CD4 T cells, producing IFNγ (27). As mentioned before, Toll-like receptor (TLR) function is suppressed by NOD2 deficient. So, colitis is highly dependent on the TLR2 function. In other words, NOD2 deficient patients would become susceptible to colitis due to an increase in TLR2 responses (27). Further studies reported that mice with NOD2 and TLR2 deficiency did not express colitis characteristics, which indicates that colitis is caused y by the unmodulated TLR2 responses. Hence, NOD2 deficiency predisposes IBD but only with the presence of a second defect which is an excessive immune response to microflora antigens (9) (27).

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Several studies on animal models with impairment of mechanisms to deal with massive antigen challenge presented by gut microflora had been carried out. One of these studies was done on the spontaneously colitic mouse stain, C3H/HeJBir, in which these mice developed spontaneous colitis under certain environment, and remits later (28). These C3H/HeJBir mice have dyregulated toll-like receptor 4 (TLR4) signalling. Hence, they do not respond to the major TLR ligand, lipopolysaccharides (LPS). LPS inhibits other TLR ligands, so, development of colitis might be due to a loss of inhibitory effect that LPS have. The study on these mice had shown that the mice produce antibodies (B cells) in respond to very limited number of bacterial antigens. This is also why the overall response is restricted and most of the antibodies acted on antigens of Enterobacteriaceae and Enterococcus species only (less than 1% of the microflora) (29). Studies were also done on these mice to determine the T cells reactivity towards antigens. It was reported that the T cell responded strongly towards antigens of enteric microflora which mediates chronic inflammatory bowel disease, but not towards epithelial or food antigens (30). Generally, many antigens are involved while the immune response is somehow being restricted in inflammation. The possible explanation to this might be that "epitope spreading" (primary autoimmune respond to a dominant autoantigen) is the means in which the immune system responded to gut microflora during inflammation (31). Epitope, an antigenic determinant, is a localised region on the surface of an antigen that is able to elicit an immune response and to combine with a specific antibody to counter that response (32). Epitope spreading is a way in which autoimmunity occur using the molecular mimicry mechanism. The autoreactive T cells are activated by self epitopes released secondary to pathogen-specific T cell-mediated bystander damage. T cells respond to less dominant epitopes and are activated due to the release of other antigens secondary to the destruction of the homologous immunodominant sequenced pathogen. Therefore, inflammation induced by specific pathogens that triggers Th1 (pro-inflammatory) responses enables inflammation within a genetically susceptible host to persist (33). Epitope spreading can also be due to targeted antigens being linked intracellularly as members of a complex self antigen. This causes an autoimmune response, triggered by an exogenous antigen (34). By using the serological expression cloning technique, it provides evidence to support this possibility and the existence of dominant autoantigen. As a result, flagellins were identified as a form of immunodominant antigen (about 25%) that stimulates pathogenic intestinal immune reactions in genetically susceptible hosts (35). Besides, flagellins when transferred to an immunocompromised host causing colitis. Also, the fact that anti-flagellin antibody is present in Crohn's patients (but not in the case of UC) further support the point that flagellin is a dominant antigen in IBD (35) (36). Though, the anti-flagellin response seems to be dominant just because flagellin is a powerful antigen that rapidly assumes prominence. Plus, the dysregulated immune system still responds to many other bacterial antigens (37).

In summary, NOD2/CARD15 mutation which is associated with an increase in mucosal innate immune responses towards gut microflora proves that mucosal hyperreactiveness towards normal gut commensals is a cause of IBD. At least, this is true in the case of Crohn's disease (9). Studies had also shown that defects in the innate immunity would cause adaptive immunity to facilitate the response towards certain specific microflora. It would be beneficial if further studies are done on patients or murine models without NOD2 mutations and to have an insight whether or not similar mechanisms applies (9).

The second major hypothesis is that the occurrence of IBD is due to presence of abnormal microflora which could cause change in composition of gut microflora and/or deranged epithelial function causes pathological responses from normal mucosal immune system (9). In other words, this is a problem with the microflora rather than the mucosal immune system, unlike the first hypothesis mentioned. There has been a debate as to whether IBD symptoms are caused by low-grade infection which leads to inflammation or caused by the defective epithelial barrier which enables non-pathologic organisms' proliferation, evoking inflammation (9).

Several studies had been carried out to prove that IBD symptoms are caused by low-grade infection, using conventional culture techniques and demonstrated larger amounts of microflora associated with the mucosa in IBD patients as compared to the control group. Swidsinki et al. obtained biopsy tissue from IBD patients and compared it with the control group (38). But, there is no evidence of bacteria penetrating the lamina propia and it is also found that the non-inflammed mucosa has more mucosa-associated bacteria than the inflamed areas (38). Similar findings were also reported by Darfeuille Michaud et al. on a study on Crohn's patients. This time, a pathogen-like invasive E.Coli was found to be associated with 20%-40% of the ileal mucosa, while only 6% of the mucosa in the control group is associated with it (39). In contrast, only 4% of the colonic biopsy from Crohn's patients and the control group is associated with the invasive bacteria, compared with 12% in Ulcerative Colitis patients (39). In other words, this study shows that IBD is gut-flora specific, which in this case, the pathogen-like invasive E.Coli. However, a more recent study by Martin et al. doubted Michaud's study. It reported that much larger amount of mucosa-adherent bacteria can be found in most Crohn's patients (80%) and 40% of the control subjects and these adherent bacteria are not limited to ileum only (40). Hence, it shows that larger amount of mucosal bacteria may not be the cause of IBD.

Studies were then carried out using molecular techniques like PCR and ribosomal DNA analysis to understand the microbiota of IBD gut mucosa (41). As a result, no known extracellular or intracellular organisms were found. Furthermore, no consistent DNA sequence, phylogenic group or subgroup was found associated with inflammed tissues compared to normal tissues of the same IBD patient (42). Colonic biopsies also show a variety of different microflora present within guts of different individual. For example, Crohn's patients had an increase in the Ruminococcus gnavus subgroup with a decrease in the Clostridium leptum and Prevotella nigrescens subgroups (42). Each patient expresses a distinct bacterial profile that cannot be generalized for all IBD patients. Hence, these differences may just reflect individual variation rather than disease association (42). Hence, the conclusion made was that IBD is not caused by invasive bacteria specifically on the site of lesions. In other words, it contradicts with the previous studies above and do not support the presence of specific, pathogenic organisms in IBD patients. However, dysbiosis is said to be present (42).

Next, we will investigate evidence proving that IBD symptoms are caused by defective epithelial barrier, which allows excessive interaction between normal mucosal immune system and normal microflora. In a study lead by Wehkamp J,. et al. reported reduced Paneth cell α-defensins and colonic epithelial β-defensins in Crohn's disease patients (43). Paneth cells (PC) are cells at the base of intestinal crypts which contribute to the maintenance of gastrointestinal barrier especially when exposed to bacteria antigens (44). Hence, it is the major source of antimicrobial peptides which include human α-defensins (HD) (43). Defensins are cysteine-rich cationic proteins which are active against bacteria, fungi and many viruses. It plays an important role in maintaining microbial balance in the intestinal lumen (45). They tested on the hypothesis, reduce in α-defensins would compromise mucosal host defences and predispose to Crohn's disease. It was shown that there is a 50% decrease in HD5 expression in Crohn's patients (patients without NOD2 mutation) as compared to the control subjects. Interestingly, the other Paneth cell antibacterial factors were expressed at the same level or higher levels. Though, the decrease in α-defensin level was not dependant on the degree of inflammation or severity of the disease and it was not observed in either colon of Crohn's patients or Ulcerative Colitis. This simply suggests that there is a specific and primary defect in α-defensin production which characterises ileal Crohn's disease (43). Similar studies were carried out on human β-defensin (HBD) expression of UC patients, Crohn's patients and the control subjects. HBD2 and HBD3 are strongly correlated. They were increased exclusively in Ulcerative Colitis but not in Crohn's disease (46). Expression of both inducible defensins is enhanced by inflammation. This suggests that Crohn's disease is lack of β-defensin induction. The missing induction of HBD2 and HBD3 may affect the mucosal barrier function predisposing to bacteria invasion (46). It was also reported that there is a genetic factor to influencing the β-defensin induction which may lead to Crohn's disease. The DNA copy number of β-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within healthy population (47). The results showed that in colonic Crohn's patients, the median HBD2 copy number is lesser than ileal Crohn's disease and Ulcerative Colitis. This suggests that low β-defensin-gene copy number may be the cause of reduced β-defensin induction in colonic Crohn's disease (47).

As mentioned above, nucleotide-binding oligomerization domain containing 2 (NOD2) is an intracellular patern recognition receptor which is expressed by cells of innate immune system to identify pathogen-associated molecule pattern (PAMP) (48) (49). NOD2 is required for expression of subgroup of intestinal anti-microbial peptides known as cryptdins (50). It was found that NOD2 might be involved in α-defensin production and it is involved in protecting epithelial cells from bacterial infection (51). Hence, mutation of the NOD2 gene located in chromosome 16 will affect α-defensin levels in Crohn's patients. One form of mutation is the frameshift mutation at Leu1007. These patients present with lower levels of HD5 as compared to patients without NOD2 mutation. But, there was no significant reduction in HD6 observed (50). Another 2 forms of mutation are at Arg702Trp and Gly908Arg. These patients exhibit similar levels of HD5 as compared to patients without NOD2 mutation. Therefore, not all NOD2 mutation affects α-defensin levels and the frameshift mutation doesn't affect all types of α-defensins (50).

It is also important to understand that a gross disruption of epithelial barrier can lead to gut inflammation. Hermiston and Gordon studied on adult chimeric mice, transfected with dominant negative N-cadherin mutant (NCAD-delta) which leads to leaky tight junctions (52). Cadherins are cell adhesive molecules and are essential for normal development. This analysis reports expression of NCAD delta along the entire crypt-villus axis, producing features resembling Crohn's disease. Interestingly, the lamina propria of epithelial cells expressing this gene developed severe inflammation, and not in areas without the gene. It was concluded that NCAD delta disturbed the proliferation, migration and death cycle in crypts that may lead to adenomas (52). In a normal gut tissue, gut microflora have extensive contact with the immune system in several ways without causing inflammation. Firstly, lamina propria dendritic cells (DC) which is an important part of the innate and adaptive immunity acting upon intestinal microflora, were found to form transepithelial dendrites. The formation of transepithelial dendrites is dependent on chemokine receptors (CX3CR1) to sample luminal antigens directly. In other words, CX3CR1, which controls host interaction with the normal microflora or pathogenic bacteria, regulates the immunological tolerance and the immune response (53). Although, intestinal pathogens are rapidly killed by macrophages, intestinal DCs can retain some of the pathogens to selectively induce IgA response (54). These IgAs will then be preferentially taken up by M cells and transported to the underlying Payer's patches (gut-associated lymphoid tissue). Hence this is another way of unimpeded organism entry into the Payer's patches and mesenteric lymph nodes (55). Lastly, it was found that the probiotic bacteria (bacteria with anti-inflammatory properties, thought to benefit the host) induced the expansion of Treg cells. The experiment was done on murine models whereby probiotic bacteria were introduced into rectum of these mice. It is therefore another way of luminal organisms interacting with the gut mucosal immune system to a lesser extent (56). Despite the interactions, why inflammation did not take place? It is likely that the invasion of small amount of commensal bacteria during these interactions is not sufficient to stimulate any innate immune response causing inflammation. While a gross loss of tight junctions in the case of mice expressing dominant negative N-cadherin (NCAD-delta) gene, allows entry of large number of normal commensals, and is sufficient to induce inflammation (52).

Our 2nd hypothesis that changes in microflora composition with normal mucosal immune system is does not seem to be supported. In other words, disruption of α-defensin production or change in commensal composition which cause excessive exposure of the immune system to gut commensals without immune system defects is not likely to be the hypothesis (9). This not the case because, firstly, as mentioned above, the mucosal immune system is in contact with gut flora and there is not clear evidence that additional bacteria load would favour inflammation (unless invasion of lamina propria prior onset of inflammation) (9). Secondly, murine models, lack of Paneth cell function do not exhibit gut inflammation spontaneously (57). This is clearly seen in the studies of mice with cystic fibrosis. These mice also present with thickened mucus layer in the intestinal crypts due to reduced fluid secretion (58). Due to blocked intestinal crypts and trapped Paneth cell products (forexample, α-defensins that cannot reach luminal bacteria), bacteria overgrow in the gut lumen. Though, these mice did not present with any inflammation (58). This suggests that the disruption of gut flora composition and deranged epithelial function with normal immune responses will not cause IBD.

Although a defect of α-defensin alone is not likely to cause Crohn's disease, this does not mean that epithelial barrier function defects have no contribution towards IBD inflammation. This can be explained by the fact that NOD2 mutation is observed more in ileal-Crohn's disease patients as this mutation is associated with α-defensin defects (9). It is also found via genetic studies on murine models that factors that affect the epithelial barrier function seemed to bring microflora closer to the defected mucosal immune system and act more profoundly than α-defensin defects. It was reported that the multiple drug resistance (mdr) gene, mdr1a knock-out mice are susceptible to severe and spontaneous gut inflammation which is similar to that of human IBD (59). Besides, it is also found that genetic mutation in cell membrane organic anion transporter 1 and 2 (OCTN) will further increase the risk of Crohn's disease if it co-exists with a NOD2 gene defect. Defects of the OCTN gene could lead to altered bacteria processing causing impairment of epithelial barrier functions. This would then expose a normal or defected immune system to excess gut microflora.

In conclusion, as mentioned above, IBD is genetically predetermined with one or more defects that allow mucosal immune system overreacting on normal gut constituents. These defects are enhanced by genetically determined gut epithelial barrier functions which further the interaction between the mucosal immune system and gut microflora (9). However, no matter what is the pathogenesis or cause of IBD inflammation, the process of the disease still come to the same immunopathology pathway at the end, either Th1 cell- mediated inflammation as in Crohn's disease or Th2 cell-mediated inflammation as in Ulcerative Colitis. Hence, no matter what is the fundamental basis of IBD, IBD can be treated if the therapy used can stop this final common pathway (9).

Existing therapies such as corticosteroid and immunosuppressant aimed to reduce abnormal inflammation. These treatments worked on secretion of cytokines and elaboration of neutrophils without considering the importance of T-cells in contributing to the gut inflammation. There are also newer therapies that act against TNFα and α-integrin molecules (eg. Infliximab, Natalizumab). These will reduce inflammation by eliminating specific inflammatory cytokines or reduce the accumulation of cells at the inflamed areas. It showed positive results in treating some groups of IBD patients, but significant complications such as fatal infections were reported (60) (61). All these therapies, conventional or new, they all allow intestinal tissues to heal and relief the symptoms of IBD. When symptoms are under control, medications are used to prevent relapses and also maintain remission (8). Hence, the benign drugs (the least harmful drugs) or drugs taken for a short period of time such as corticosteroids and aminosalicylates will be prescribed first. If the symptoms of IBD are not being relieved, drugs of higher steps like immune modifying agents (eg. Infliximab, Prednisone, Budesonide) will be used (8). As mentioned before, up till now, there is no one drug that completely cease IBD inflammation and work without any worrying side effects. So, there is still room for researches in order to reach something specific for IBD treatment or something with more promising outcomes. And this is believed to be achieved if we can stop one of the T-helper cell mediated inflammation.

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