Interplay of commensal and pathogenic bacteria, genetic mutations, and immunoregulatory defects in the pathogenesis of inflammatory bowel diseases


R. Balfour Sartor, UNC Department of Medicine/GI, CB #7032, Room 7309, Biomolecular Building, Chapel Hill, NC 27599 7032, USA.
(fax: (919) 843 6899; e-mail:


Enteric microbiota can contribute to Crohn’s disease and ulcerative colitis in several ways. Pathogenic or functionally altered commensal bacteria with increased mucosal adherence, invasion and intracellular persistence can activate pathogenic T cells and chronic intestinal inflammation. Compositional changes in intestinal microbiota can lead to decreased protective and increased aggressive species. Genetic polymorphisms resulting in increased mucosal permeability, decreased microbial killing, ineffective clearance of bacteria, biased TH1 and TH17 immune responses and loss of immunological tolerance are probably key contributors to IBD. Future therapies for these heterogeneous diseases should be individualized based on the patient-specific subset.


Crohn’s disease and ulcerative colitis are chronic, relapsing, immunologically mediated inflammatory bowel diseases (IBD) with unknown aetiologies. Although microbial pathogens have been postulated to cause these disorders, it now appears that commensal enteric bacteria, some with increased virulence, cause overly aggressive T cell responses and chronic inflammation in the setting of genetic polymorphisms that regulate mucosal barrier function, innate microbial killing, and immune responses [1, 2]. Abnormal microbial composition and host–microbial interactions in IBD have been elucidated in experimental rodent models, translational research, clinical trials and research [3, 4]. This review briefly summarizes four aetiological theories of the manner in which commensal enteric bacteria or pathogens induce and perpetuate chronic intestinal inflammation and IBD in susceptible individuals (Fig. 1).

Figure 1.

 Proposed mechanisms by which bacteria and fungi induce chronic immune-mediated inflammation and injury of the intestines. (a) Pathogenic bacteria. A traditional pathogen or functional alterations in commensal bacteria, including enhanced epithelial adherence, invasion, resistance to killing by phagocytes or acquisition of virulence factors, can result in increased stimulation of innate and adaptive immune responses. (b) Abnormal microbial composition. Decreased concentrations of bacteria that produce butyrate and other short-chain fatty acids (SCFA) compromise epithelial barrier integrity. Meanwhile, overgrowth of aggressive commensal microbial species increases the number of adjuvants and antigens (Ag) that induce pathogenic immune responses or increase production of toxic metabolites such as hydrogen sulphide (H2S) that block colonocyte utilization of butyrate and increase mucosal permeability. (c) Defective host containment of commensal bacteria. Increased mucosal permeability can result in overwhelming exposure of bacterial TLR ligands and antigens that activate pathogenic innate and T cell immune responses. Defective secretion of antimicrobial peptides or secretory IgA (sIgA) can lead to mucosal bacterial overgrowth. Defective killing of phagocytosed bacteria can lead to persistent intracellular bacteria and ineffective clearance of bacterial antigens. (d) Defective host immunoregulation. Antigen-presenting cells and epithelial cells overproduce cytokines due to ineffective downregulation, which results in TH1 and TH17 differentiation and inflammation. Dysfunction of regulatory T cells (T-reg) leads to decreased secretion of IL-10 and TGF-β, and loss of immunological tolerance to microbial antigens (an overly aggressive T cell response.) Figure reprinted from Gastroenterology (Ref. 1) with approval of the publisher.

Microbial pathogens

Mycobacterium avium subspecies paratuberculosis (MAP) is an obligate intracellular pathogen that has been intensely investigated as an aetiological agent of Crohn’s disease due to the similarities between granulomatous ileocolitis and intestinal tuberculosis that were first noted in the early 1900s [5, 6] (Fig. 1a). Around the same time, MAP was implicated as the cause of Johne’s disease, a wasting disease seen in cattle and other ruminants marked by granulomatous enterocolitis and watery diarrhoea. The histopathological similarities noted between Johne’s disease in ruminants and Crohn’s disease in humans led to focused attempts at culturing MAP from affected tissues. However, MAP is slow-growing and fastidious, and hence difficult to culture. It was not until 1984 that is was cultured from Crohn’s disease-affected intestinal tissue [7]. The DNA insertion sequence of MAP, IS900, was subsequently detected in relatively high numbers of Crohn’s patients compared to ulcerative colitis patients and controls [8].

These findings were particularly concerning because MAP has been isolated from tap water and milk, although a recent epidemiological report failed to support exposure through drinking water or contaminated milk [9]. Yet its potential as a source of zoonotic infection exists, with widespread MAP infections in the dairy herds of Europe, North America and Australia. Although many studies report increased recovery of MAP in Crohn’s disease tissues compared to inflammatory or normal control tissues, the detection rate in Crohn’s disease ranges from 0% to 100% [1] which, along with its detection in healthy control tissue samples, casts doubt on the significance of its association with Crohn’s disease. Furthermore, a well-designed, 2-year prospective trial of the antimycobacterial regimen of clarithromycin, rifabutin and clofazamine failed to show a sustained response in Crohn’s disease patients [10]. It is likely that MAP is not the causative agent of Crohn’s disease, but rather that this ubiquitous environmental agent selectively colonizes the ulcerated mucosa of Crohn’s disease patients. However, it is possible that MAP might opportunistically infect a certain subset of genetically susceptible Crohn’s patients with intracellular bacterial killing defects caused by ATG 16L1, NOD2 or NCF 4 polymorphisms.

Commensal bacteria that undergo functional alterations might contribute to the pathogenesis of IBD. Escherichia coli are commensal aerobic Gram-negative bacteria that play an important role in maintaining normal intestinal homeostasis. Modifications of luminal bacteria concentrations, including E. coli, have been observed in Crohn’s disease patients [11], and E. coli have been isolated in greater numbers in the neoterminal ileum [12, 13] and mesenteric lymph nodes [14, 15] of patients with Crohn’s disease compared to controls. Escherichia coli DNA is found in 80% of microdissected granulomas of Crohn’s disease patients. Antibody titres to E. coli have been found to be higher in serum of Crohn’s disease patients than in controls [16], and high levels of antibodies against E. coli outer membrane protein C (Omp-C) are present in 37–55% of patients with Crohn’s disease, but in less than 5% of controls [17].

A unique, potentially pathogenic group of E. coli associated with Crohn’s disease mucosa has been identified. Adherent invasive E. coli (AIEC) were recovered from 65% of chronically inflamed ileal resections and 36% of mucosal biopsies of the neoterminal ileum of patients with early postresection recurrent Crohn’s disease and 22% of endoscopically normal ileal Crohn’s disease biopsies in contrast to 3.7% of colonic biopsies from the same patients and 6% of normal control ileal biopsies [18]. AIEC do not possess any of the known genetic invasive determinants described for enteroinvasive or enteropathogenic E. coli (EPEC) and Shigella strains [13, 19]. AIEC is unique in that its invasion depends on functioning host cell microtubules as well as actin microfilaments, as opposed to being dependent on functioning actin microfilaments alone, as is the case with Yersina enterocolitica, Listeria monocytogenes and Shigella flexneri. Type 1 pilus-mediated adherence, a mechanism which is distinct from that of (EPEC), plays a critical role in AIEC’s invasive ability by inducing membrane extensions [20]. AIEC pili adhere to CEACAM 6 on the apical surface of intestinal epithelial cells (IECs), particularly in the ileum, which AIEC selectively colonizes in Crohn’s disease.

Adherent invasive E. coli also persist within epithelial cells and macrophages, induce secretion of large amounts of tumour necrosis factor (TNF) and other cytokines, but do not induce cell death of infected macrophages [21]. Recovered E. coli strains from ileal Crohn’s disease patients do not produce toxins, but do express virulence factors that are closely related to avian and uropathic E. coli strains [22].

Functional alterations such as toxin production in other commensal bacterial species and their roles in IBD are being investigated. Enterotoxigenic Bacteroides fragilis has been identified in 19.3% of stool specimens from patients with active IBD versus 2.9% of controls [23]. Bacteroides species are responsible for >60% of the biofilm mass in patients with IBD [24]. Clostridium difficile toxin can reactivate quiescent IBD [25]. Enterococcus faecalis, which can generate superoxide and acquire virulence factors, induces chronic progressive IBD in interleukin-10-deficient mice [26]. Broad-range bacterial 16s rDNA PCR allows for amplification and identification of most bacterial phylotypes in a microbial community, but does not differentiate amongst strains or identify functional changes. Therefore, virulence factors, toxin production and the reciprocal regulation of bacterial and host epithelial genes must be investigated by other methods.

Dysbiosis of commensal enteric microbiota

The commensal intestinal microbiota is critical to the healthy host, as it modulates intestinal development, maintains a healthy intestinal pH, promotes immune homeostasis, and enhances metabolism of drugs, hormones and carcinogens (Fig. 1b). Alterations in the composition of the indigenous intestinal microbiota could play a role in the aetiology of IBD by altering dominant antigens or metabolic function of the gut bacteria. The composition of the intestinal microbiota in patients with IBD and in healthy subjects has been assessed using molecular methods such as 16S rDNA sequence analysis and PCR [11]. Past studies are limited, however, in statistical power and most have used faeces as a representative specimen type. However, the faecal microbiota differ from the mucosal microbiota, and it is more likely that the gut wall microbes are involved in the pathogenesis of IBD [24, 27]. Supporting this concept, colon biopsy specimens from patients without IBD are nearly free of bacteria when they are washed of faecal contents, in contrast to high bacterial concentrations from IBD colon biopsy specimens [27]. Intestinal mucosal bacteria have been found to be at concentrations greater than 109 mL−1 in 90–95% of IBD patients, 95% of patients with self-limiting colitis, 65% of IBS patients and 35% of healthy controls [24]. Of note, Crohn’s disease occurs in intestinal segments with the highest bacterial concentrations.

Although bacterial concentrations are increased in IBD, microbial diversity is diminished, particularly in patients with active disease. An increase in Enterobacteriaceae, including E. coli, is seen in IBD, along with a decrease in Firmicutes phyla members compared to controls [11]. No consistent differences have been demonstrated in the probiotic genera Lactobacillus and Bifidobacterium [28]. Recent bacterial surveys confirm previous reports of compositional shifts in IBD-associated gastrointestinal microbial communities, although there have been some results from fluorescence in situ hybridization studies, including reports of increased Bacteroides species in IBD, that are inconsistent with previous studies [24, 27]. Almost all studies have shown an increase in E. coli in faecal and mucosal microbiota in patients with Crohn’s disease, particularly in tissues within granulomas and surrounding apthous ulcers and fistulas [11, 22, 27, 29, 30]. Meanwhile, no consensus has emerged regarding differences between intestinal microbiota in patients with Crohn’s disease versus ulcerative colitis, or in patients with active versus inactive disease [11, 21, 22, 27, 29].

When comparing intestinal microbiota of patients with IBD versus that of controls, trends are seen that could influence the pathogenesis of IBD. First, there is a decreased ratio of protective commensal bacterial species compared to aggressive species in patients with IBD. Decreases in Clostridium XIVa and IV groups within the Lachnospiraceae family largely account for decreased numbers of the phyla Firmicutes seen with IBD. Clostridia species, along with Bacteroides species, which also have been found to be decreased in IBD in many studies, produce butyrate and other short-chain fatty acids that are important sources of energy for colonic epithelial cells, can enhance epithelial barrier integrity and modulate the intestinal immune responses. Short-chain fatty acid concentrations are diminished in faecal extracts of IBD patients. Meanwhile, overgrowth of aggressive commensal microbial species is seen in IBD patients. An increase in sulphate-reducing bacterial species could enhance hydrogen sulphide production, which blocks colonocyte utilization of butyrate [31]. The sulphate-reducing bacteria Desulfobrio piger has been isolated in 55% of patients with IBD compared to 12% of healthy individuals [32]. Feeding sulphated polysaccharides such as carrageenan, but not unsulphated polysaccharides, to guinea-pigs or rabbits, can induce experimental colitis [33]. Furthermore, mesalamine, a medication commonly used to treat ulcerative colitis, lowers sulphide concentrations [34]. Finally, Candida albicans stimulates anti-Saccharomyces cerevisiae antibodies (ASCA), a serological marker of Crohn’s disease, suggesting that this fungus could serve as an antigenic stimulant in Crohn’s disease [35]. Candida can attach to and invade IECs and activate T cell responses. Microbes profoundly influence gut metabolism by interacting with metabolic pathways. Further studies should address the correlation between quantitative changes in enteric microbial composition and luminal metabolic alterations.

Host genetic defects in containing commensal microbiota

Increased mucosal permeability is a feature of IBD, although it is unclear whether this is a primary pathogenic process or secondary to inflammation (Fig. 1c). Mucosal permeability is influenced by several factors. The first is the integrity of the epithelial cell layer and the basement membrane. Molecularly this can be compromised by downregulating tight junction components Claudins 5 and 6, upregulating pore-forming Claudin 2 [36], which can be accomplished by TNF and IL-13, or increasing epithelial apoptosis, which has been achieved in mice by blocking nuclear factor kappa-B (NFκB) signalling via IEC-targeted deletion of IKKy (NEMO). A primary role for epithelial barrier function is demonstrated by the focal intestinal inflammation seen with experimentally altering tight junctions or blocking NFκB signalling [37, 38]. The surface mucus layer also impacts mucosal permeability, as demonstrated by spontaneous colitis in Muc-2-deficient mice [39], and increased dextran sulphate sodium-induced colitis in intestinal trefoil factor-deficient mice [40]. Finally, autonomic nervous system function affects epithelial permeability, as demonstrated by mice that develop fulminant jejuno-ileitis following ablation of enteric glial cells [41].

There are several IBD-associated genetic mutations that affect the epithelial barrier. Both the multidrug resistance-1 (mdr-1) gene that encodes p-glycoprotein, which acts as a transmembrane efflux pump, and OCTN 1, 2, an organic cation (carnitine) transporter in the IBD 5 gene complex, are postulated to clear microbial xenotoxins that permeate epithelial cells. Carnitine is essential for transport of long-chain fatty acids from the cytosol into the mitochondria for beta oxidation [42], and defects within this system might lead to impaired pathogen killing by oxidation burst-mediated mechanisms [43]. OCTN gene variants are associated with Crohn’s disease and ulcerative colitis [44, 45], whilst mdr-1 polymorphisms are associated with ulcerative colitis [46]. Increased mucosal permeability during preclinical inflammation and lack of bone marrow transplant regulation of ileitis suggest a primary mucosal barrier dysfunction in Samp-1/YIT and mdr-1-deficient mice [47].

Defective microbial killing is also seen in Crohn’s disease patients. Defective antimicrobial peptide production is seen, involving alpha defensin 5 in ileal disease [48], and human beta defensin 2 copy numbers in the colon [49], with functional abnormalities in killing E. coli, E. faecalis and Bacteroides vulgatus [50]. NOD 2 polymorphisms are associated with decreased alpha defensin production by Paneth cells [48] and defective clearance of intracellular pathogens by colonic epithelial cells [51]. Mutations in two additional Crohn’s related genes that regulate intracellular bacterial killing, ATG 16L1 and NCF 2, further implicate defective innate immune responses in Crohn’s disease [52, 53]. ATG 16L1 mediates autophagy, which is one mechanism of intracellular bacterial killing and processing. NCF 2 regulates NADPH-dependent production of antimicrobial reactive oxygen metabolites in phagolysosomes. TLR signalling through Myd 88 plays a role in bacterial clearance, with defective epithelial repair, neutrophil recruitment and adaptive immune responses, severe colitis and bacteraemia in Myd 88-deficient mice infected with Citrobacter rodentium [54]. Patients with primary functional defects in neutrophil killing exhibit a Crohn’s disease-like phenotype, revealing the critical role of bacterial killing by phagocytic cells in the intestine [55].

Intestinal mucosal homeostasis depends on the interplay of a relatively impermeable epithelial cell layer, secretion of luminal antimicrobial peptides, extrusion of xenotoxins, rapid repair of epithelial defects, and phagocytosis and killing of bacteria that translocate across the epithelial barrier. Defects in any of these processes can result in increased microbial antigenic exposure, pathogenic T-cell responses and chronic intestinal inflammation.

Defective immunoregulation

The bowel is the largest immunological organ of the body, with continuous interaction between the mucosal immune system and the intestinal flora (Fig. 1d). It appears that an aberrant mucosal immune response is involved in the initiation and perpetuation of IBD [1, 2]. The host senses bacterial stimuli and discerns pathogens from commensals via pattern recognition receptors (PRRs) that recognize specific molecular patterns of pathogens. Toll-like receptors (TLRs) and nucleotide oligomerization domains (NODs) are receptors that stimulate signalling cascades such as the NFκB pathway, which results in proinflammatory cytokine production and induction of co-stimulatory signals that initiate effector adaptive immune response. TLRs are key receptors of the innate immune system, and activation of epithelial NFκB by commensal bacterial TLR ligands is a critical component of mucosal homeostasis and contributes to normal mucosal barrier function. TLRs are carefully regulated to mute proinflammatory responses towards mutualistic organisms in healthy individuals [37]. NODs are cytosolic PRRs involved in intracellular recognition of bacterial products such as peptidoglycan (PGN) derived from Gram-positive bacteria [56].

Lipopolysaccharide (LPS), derived from the outer envelope of Gram-negative bacteria, is a major inducer of the inflammatory response to these bacteria. LPS signalling is mainly mediated through TLR4 activation of NFκB. Colonic epithelial cells express relatively low levels of cell-surface TLRs, resulting in their relative hyporesponsiveness to bacterial LPS. Polymorphisms in TLRs have been linked to Crohn’s disease [57, 58], and immunofluorescence studies reveal that epithelial TLR expression is markedly upregulated in IBD [59]. TLR4, for example, is induced by proinflammatory cytokines and is highly expressed in IECs, resident macrophages and dendritic cells in active IBD [59, 60]. The functional variant Asp299Gly of TLR4 is associated with IBD and increased susceptibility to Gram-negative infections [57]. Disrupted TLR4 signalling could engender an inappropriate innate and adaptive immune response necessary to eradicate pathogens, which would result in severe inflammation. Polymorphisms of TLRs 1, 2 and 6 are associated with more extensive disease localization in colonic Crohn’s disease and ulcerative colitis [61], although they are not associated with increased incidence of IBD. Ulcerative colitis patients have an association between a TLR7 variant and the prevalence of pANCA antibodies, which crossreact with enteric bacterial antigens [62, 63]. A polymorphism in the CD14 promoter gene, which regulates LPS binding, is associated with Crohn’s disease and steroid use in ulcerative colitis [64, 65], and co-existence of CD14 and TLR4 mutated alleles is higher in Crohn’s disease patients than in ulcerative colitis patients or healthy subjects [66]. Blockade of bacterial signalling through NFκB in IECs potentiates chemically induced colitis in TLR4 and TLR9-deficient mice [67, 68].

The most convincing argument for Crohn’s disease to be an intestinal immunodeficiency comes from the discovery of the first susceptibility gene on chromosome 16q, NOD2 [69, 70]. Intracellular NOD2 induces the NFκB pathway when it recognizes bacterial muramyl dipeptide [71]. Intestinal epithelial expression of NOD2 appears to serve a protective function [51, 72]. The NOD2 variants Gly908Arg, Arg702Trp and Leu1007fsinsC are associated with defective NFκB activation by muramyl peptide. These polymorphisms have been associated with young age at onset, fibrostenosing disease and ileal involvement of Crohn’s disease [73].

Biased TH-1 and TH17 immune responses also play a role in the pathogenesis of Crohn’s disease [2, 74]. In Crohn’s disease, macrophages, mucosal dendritic cells and B lymphocytes overproduce cytokines, including IL-6, IL-12 and IL-23, leading to TH1 and TH17 differentiation and inflammation. Immunologic tolerance to bacterial antigens is primarily mediated by IL-10 and transforming growth factor-β (TGF-β), produced by regulatory T cells. Deletion of IL-10, IL-10 receptor, TGF-β or IL-2, which is the ligand for CD25, induces colitis [74, 75]. In Crohn’s disease, Gp 96 stress protein, an innate tolerogenic molecule, is decreased [76]. Crohn’s disease and ulcerative colitis patients exhibit T cell [77] and serological responses [78, 79] to a wide variety of microbial antigens, including ASCA, E. coli (Omp-C), Pseudomonas (I2) and flagellin (cBIR) [62], and fail to develop oral tolerance [80]. The most effective treatments for Crohn’s disease, including infliximab, 6-mercaptopurine, azathioprine and anti-IL-12 p40 antibodies, are directed against TH1/TH17 responses.

Further investigation is critical to clarifying the complex interaction pathways between pathogenic and commensal mucosal bacterial flora and the host immune response. Developing individualized immunomodulating therapies depends on characterizing specific immune defects in a given patient.


The microbiota of the distal ileum and colon are complex, metabolically active and interact with intestinal epithelial and immune cells. Alterations in the composition and metabolic profile of these microbes in IBD are being elucidated with molecular techniques. Increased exposure to commensal enteric bacteria, and possibly fungi, in individuals with genetic defects that result in increased mucosal permeability, decreased microbial killing and/or impaired immune responses, can provide constant antigenic stimuli for pathogenic adaptive immune responses. Some microbes, including E. coli, undergo functional alterations that lead to increased epithelial adherence, invasion and persistence, and may opportunistically cause disease in a host with genetic defects, such as a NOD2 polymorphism with accompanying decreased secretion of antimicrobial defensins or bacterial killing. Other functionally altered bacteria, including those that are toxigenic, sulphate-reducing or defective in producing short-chain fatty acids, have been associated with IBD. One aspect of IBD that makes it difficult to study is that Crohn’s disease and ulcerative colitis consist of heterogeneous groups of distinct diseases with microbial, phenotypic, genetic and immunological diversity.

Future studies should focus on better characterizing the bacterial and fungal constituents of the human microbiome in individuals both with and without IBD, and the metabolic alterations in IBD. Functional changes in the microbiota in IBD patients need to be clarified using genomic, metabolomic and proteomic profiling, in coordination with molecular microbiological techniques. The most effective therapy will involve individualizing selection of antibiotics, probiotics and prebiotics to treat specific IBD subsets, based on patient-specific microbial composition, dominant microbial antigens and host genetic polymorphisms. Replacing antimicrobial peptides in appropriate individuals is one example of a targeted treatment that could be offered. Individualized treatment should lead to improved therapeutic responses in patients with IBD.

Conflict of interest statement

No conflict of interest was declared.