Crohn’s Disease: Myobacterium Avium Paratuberculosis and Molecular Mimicry

Abstract

Crohn’s disease (CD) is a T-cell mediated complex autoimmune inflammatory bowel disease (IBD) that arises in genetically predisposed individuals as a result of dysregulation of mucosal immune responses to enteric bacteria. CD is characterised by ulceration and chronic inflammation that can arise in any region of the gastrointestinal tract, accompanied by other systemic abnormalities. The intestinal lesions are associated with a transmural inflammatory infiltrate often accompanied by granuloma formation (1).

IBDs are associated with a range of extra-intestinal manifestations (EIMs) that may be even more serious than the underlying intestinal disease, and in some cases may be the first presenting symptoms of the disorder. The most common extraintestinal organ systems involved in IBD are musculo-skeletal, dermatologic and ocular. As nearly every organ system in the body may potentially be involved, some believe that IBD is a systemic disorder with primarily gastrointestinal manifestations (2).

The mechanisms responsible for IBDs may include genetic susceptibility, impaired self-recognition, antigenic display of determinants homologous to auto-epitopes, and immuno-pathogenic auto-antibodies targeted against organ-specific cellular antigens shared by both the intestinal tissues and other organs (2).

The incidence of CD has grown over the past 50 years, with an estimated prevalence of 375 CD cases per 100,000 individuals living in the UK (subjects born in 1970). This increase results from unidentified environmental factors or lifestyle changes, or combination of both (3). Since both the distal ileum and colon contain very high concentrations of intestinal bacteria, compared to the stomach, jejunum, duodenum and proximal ileum, it would not be surprising to discover that luminal bacteria play an important role in CD pathogenesis (4).  The most convincing evidence for this was demonstrated by Swidsinski et al.(5) (figure 1-2).

Defence dysfunction

A population of intestinal stem cells is responsible for maintenance and epithelial cell turnover in the intestinal tract. These stem cells produce four types of epithelial cells: columnar (for absorption), goblet (for secretion of mucin and protection of luminal mucosal layer), neuro-endocrine (hormone-releasing), and most importantly, Paneth cells. The principal function of Paneth cells is to keep the crypts of small intestine and ascending colon sterile by secreting antimicrobial alpha defensins HD5 and HD6, as well as other antimicrobial molecules (4).  The function of these stem cells is regulated by several important signalling pathways. Dysregulation in these pathways leads to development of malignancies within the gut (4). The Wnt signalling pathway regulates differentiation of stem cells into Paneth cells.  TCF4, the transcription factor responsible for this, forms an intracellular complex with beta catenin when Wnt pathway is activated. The complex translocates to the nucleus, from which TCF4 controls the expression of several downstream genes, including Paneth cell alpha defensins (4).

Within the gastrointestinal tract, the defensins help control the composition and concentration of colonising bacteria, and provide protection against water-borne and food-borne pathogens (6). Wehkamp et al. (6) reported that in ileal CD a decreased expression of TCF4 leads to a reduced expression of both HD5 and HD6 (figure 3-4), compared to patients with strictly colonic CD, who had normal TCF4 and alpha defensin levels. The reduction in Paneth cell alpha defensins is TCF4-dependent and causes weakening of enteric antimicrobial defense (6).

In the colon, a different group of defensins is expressed: HBD1, HBD2 and HBD3 beta defensins. Colonic CD is associated with an attenuated secretion of several classes of antimicrobials, most importantly beta defensins. An HBD2 gene coding for an important beta defensin, with less than 4 copy numbers per diploid genome is associated with a reduced mucosal HBD2 mRNA expression (6). A lower HBD2 gene copy number in the beta defensin locus puts one at risk of developing colonic CD by promoting a reduced beta defensin expression, which leads to the weakening of antibacterial barrier in colonic mucosa (7) (figure 5).

Genetics of CD

Genetic contribution appears to be an important factor due to high disease concordance rates in identical twins and a 10-25-fold increased predisposition in relatives of individuals with CD (Mathew, 2008). The five firmly established CD susceptibility loci are NOD2, IBD5 locus on chromosome 5q31, IL23R, ATG16L1 and the gene desert on chromosome 5p13.1(3). NOD2 protein is predominantly expressed in monocytes and macrophages, but is also found in intestinal epithelial cells, Paneth cells and neutrophils. All CD-associated polymorphisms occur in its region of leucine-rich repeats (8). NOD2 is a pattern recognition receptor that recognises and gets activated by muramyl dipeptide (MDP), a constituent of peptidoglycan found in both Gram-positive and Gram-negative bacteria. Recognition of MDP by NOD2 results in activation of NF-kB which orchestrates an inflammatory response. In the NOD2 sequence variants associated with disease risk, the ability of NOD2 to activate NF-kB in human cells is diminished, suggesting that loss of function in the innate immune response allows increased bacterial survival and proliferation, and development of chronic inflammation (3).  An interleukin IL-23 is known to activate Th17 T cells that secrete IL-17 and induce chronic inflammation. A gain of IL-23 function is likely to promote pathogenesis, while an impaired IL-23R signalling may compromise barrier function, resulting in increased intestinal permeability characteristic of CD (3).

Autophagy is a process of recycling of cellular components delivered in double-membrane bound vesicles in the lysosome. If invading microbes are not cleared promptly they may cause infection and inflammation (9). Enterocyte apoptosis is elevated in inflamed areas of CD, which may be pathogenically important by impairing the integrity of epithelial barrier defense, thus allowing direct exposure of the immune system to potential luminal pathogens (10). Autophagy is involved in innate immune response by removing intracellular microbes, and also has a role in adaptive immunity by eliminating intracellular proteins via MHC class II antigen processing pathway (Mathew, 2008). Coding for an autophagy protein, ATG16L1 gene, as well as a second essential autophagy protein ATG5, are essential for the functions of Paneth cells which secrete granules containing antimicrobial peptides. The protein that the ATG16L1 gene encodes is called ATG16 autophagy related 16-like 1 protein. Paneth cells deficient in this protein as well as in the autophagy protein 5 (ATG5) have major defects in granule exocytosis pathways. ATG16L1 protein-deficient Paneth cells also display a gain-of-function which includes upregulated expression of  genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism,  of acute phase reactants, and of two immuno-regulatory cytokines, leptin and adiponectin, known to directly co-ordinate the magnitude of intestinal injury responses (11). Cadwell et al. (11) concluded that deficiencies in autophagy undermine the ability of the body to respond to intracellular pathogens in CD. Impaired clearance of pathogens and apoptotic debris causes accumulation of late apoptotic as well as secondary necrotic material, which establish a pro-inflammatory milieu that undermines immune tolerance (9).

Associations with MAP

Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). MAC occur widely in the environment, and colonise healthy animal and human intestine without causing any pathologies unless the host is compromised (12). However, unlike other MAC, MAP is a pathogen and is known to cause chronic inflammation of the gut in various animals, sometimes following a state of dormancy after initial colonisation of the intestine years earlier (12).  MAP is a common pathogen that humans often come into contact with through intake of water, cows’ milk and food, and variations in innate immune responses are likely to contribute to differences in susceptibility to MAP infections, since not all people who come into contact with MAP go on to develop CD.

MAP in CD is present at low concentration, is coated with methylated and acetylated fucose, and characterised by an absence of conventional mycobacterial cell wall often allowing it to remain undetected (12). Individuals with MAP infection are at 17 times greater risk of developing inflammatory bowel disease than people without MAP infection (13).

CD-associated susceptibility genes regulate innate immune responses, defence provided by the mucosal barrier, and killing and elimination of pathogens. Mutations in these genes can lead to changes in intestinal epithelial barrier function that enhance exposure of the mucosal immune system to bacterial components and subsequently lead to chronic relapsing inflammation (14).

Since the initial description of CD, it was believed that pathogenic organisms play an important role in disease development. Many different bacterial species have been implicated in pathogenesis of CD, but MAP appears to be one of the strongest candidates. Over the past two decades much work has been done to investigate this link, with some research groups strongly supporting the hypothesis, and others refuting it.

The hypothesis that MAP is the causative agent in CD has been around for about eight decades, and with advances in molecular techniques evidence to support this association is becoming more abundant. The supporters of the Mycobacterial theory argue that MAP which causes Johne’s disease in cattle may also be a causative agent in CD, with the dysregulated immune responses being a secondary phenomenon, while its critics maintain that MAP is a secondary invader in an already established defective environment, rather than a causal factor (15). CD is a multifactorial disease, but the consensus is that for the development of chronic intestinal mucosal inflammation, interaction of the innate immune system with the luminal microbes must occur (16). Despite the possible association between MAP and CD, relatively little is known about the interaction of MAP and the innate immune system (16).  A study supporting the hypothesis that MAP may be a causative agent of CD was provided by Naser et al. (17), who detected viable MAP in peripheral blood in a greater proportion of individuals with CD than in controls (figure 6).

The recognition of microbe-associated molecular patterns by pathogen recognition receptors (PRR) is essential for initiation and development of innate immune responses, which act as the first line of defense against the invading bacteria (16). A study by Ferwerda et al. (16) has demonstrated that pattern-recognition receptors TLR2, TLR4 and NOD2 are involved in recognition of MAP by the innate immune system.

Feller et al. (15) demonstrated that MAP DNA was much more prevalent in intestinal tissue samples of indviduals with CD than in controls, as detected by PCR. Also, the prevalence of antibodies against MAP determinants appeared to be higher in CD samples than in controls, as tested by enzyme-linked immuno-sorbent assay (ELISA). This evidence agrees with the proposed hypothesis linking MAP and CD; however, this data neither supports nor disproves the theory that MAP may be a causative agent in CD. Bull et al. (18) detected IS900 MAP DNA in ileocolonic mucosal biopsy specimens in CD patients (figure 7) and concluded that the detection frequency of MAP DNA in CD individuals is highly significant and thus implicates this chronic enteric microbe in CD causation.

The most irrefutable evidence to support a theory which claims that a certain microbe is causing a disease is a long-term remission of clinical symptoms and an altered natural history of disease subsequent to elimination of infection-causing pathogen through the use of antibiotic agents (19).
In a recent study by Selby et al. (20) the controversial theory of the association between MAP and CD suffered a serious setback. Selby et al. (20) used a combination of antibiotic therapy with clarithromycin, rifabutin and clofazimine, administered together with corticosteroids, for up to two years to see if MAP is indeed a causative agent of CD. This study does not support the mycobacterial hypothesis because no long-term advantage in CD was observed after two year treatment with antibiotics (figure 8) (20).

MAP has characteristics that make it resistant to treatment with many potentially effective antibiotics, with clarithromycin and azithromycin being the most effective medicines for management of MAP (19).

Results of a meta-analysis by Borgaonkar et al. (21) indicate that mycobacterial therapy might still be effective for maintaining a state of remission in CD patients following a course of corticosteroids combined with anti-mycobacterial therapy to bring about remission.

Arguments against a role of MAP in CD stem from epidemiologic evidence, which seems to go against the hypothesis. First, farmers and those living in rural areas should in theory be at greater risk of CD due to contact with livestock carrying disease-associated pathogen. Second, environmental conditions such as poor sanitation appear to protect against CD, rather than favour transmission of infection. Third, detection of MAP in CD patients is neither bacterium- nor disease-specific, in that other types of bacterial DNA are also present in the granulomas of intestinal CD (19). Nonetheless, MAP still remains a popular candidate for several reasons: it causes inflammation of the intestine in cattle and primates, it has been detected in the intestinal tissues and blood samples of many CD patients, antibodies to MAP are often disease-associated, and in some cases anti-mycobacterial therapy was found to ameliorate the disease (19).

CD and molecular mimicry

Molecular mimicry is defined as sharing of the molecules’ linear amino acid sequences or their conformational fits by molecules from unrelated genes or by their protein products (Oldstone, 1998). The causes of autoimmune disease are still not fully understood. Molecular mimicry is one of the most popular yet controversial theories attempting to explain the causation of autoimmune disease, with hundreds of published studies supporting the hypothesis, but the evidence in cases of human disease still remains circumstantial (22).

Under normal circumstances, autoimmunity is prevented in two ways: through negative selection of self-reactive T lymphocytes in the thymus resulting in apoptosis, as well as making the immune cells tolerant to self antigens through the process of tolerisation in the periphery via clonal deletion or induction of anergy and non-responsiveness (23). However, not all auto-reactive T cells are deleted in the thymus or the periphery, or tolerised against auto-antigens, possibly because they were not presented with the antigen properly, or because the level of antigen presented was not sufficient to activate them (24).

In autoimmunity this tolerance to auto-antigens is broken, allowing the potentially auto-reactive T and B cells to get activated and attack own tissues as a result of cross-reactivity between the self-antigen and the mimicking infectious pathogen.  The development of autoimmunity is controlled both by cellular (T cell) responses and antibody (B cell) responses. It is likely that auto-reactive B cells escape deletion by similar mechanisms to those that allow auto-reactive T cells to survive (24). Most B cell responses require the assistance of T helper cells.  Cross-reactivity has been observed at both B and T cell level, and both cross-reactive antibodies and T cell responses may contribute to the development of autoimmune disease (25).

According to Davies (22), the most compelling evidence is support of molecular mimicry theory as a cause of autoimmune disease includes: (1) cross-reactivity of auto-reactive T cells from individuals with autoimmune disease, (2) proof that B cells are capable of presenting autoantigens to naïve autoreactive T cells, and (3) demonstration that immune responses against an original instigating antigenic determinant can spread to new determinants via mechanism of epitope spreading. These fundamental factors would be essential for molecular mimicry to contribute to the development of autoimmune disease.

Although infectious agents and molecular mimicry are suspected to have an important role in autoimmune disease pathogenesis, direct evidence is extremely hard to obtain because often the pathogen initiating the autoimmune process may be eliminated by the host immune response well before first clinical symptoms appear, thus making it impossible to isolate (26). Therefore, a ‘hit-and-run’ scenario would take place whereby an initiated autoimmune reaction would continue against the cross-reactive self-antigen (26).

An important study by Polymeros et al. (27) suggested a link between MAP and antibody self-reactivity. The study shows the presence of disease-specific cross-reactive responses in individuals with CD with significant reactivity found against MAP glycosyl transferase d (gsd) and human gastrointestinal glutathionine peroxidase (GPg; figure 9) homologues in 30% of CD patients, and MAP alkylohydroperoxidase C (ahp C) and human tumour over expressed protein (TOG) in 20% of CD patients. However, it is important to appreciate that molecular mimicry is unlikely to be the sole cause of autoimmunity, but is rather an important component of a complex orchestra of factors responsible for the onset and development of an autoimmune disease.

There are several non-specific mechanisms initiated by infection other than molecular mimicry that can result in the breakdown of immunologic tolerance:

1. tissue damage and cell necrosis can uncover cryptic epitopes of auto-antigens, causing reactivation of quiescent auto-reactive T cells (28).

2. up-regulation of MHC and co-stimulatory molecules on antigen-presenting cells, resulting in inappropriate activation of T cells (23).

3. non-specific T cell activation may take place following binding of microbial and other proteins termed super antigens (23).

Molecular mimicry however is a mechanism that directly utilises specific immune responses to break tolerance to host’s auto-antigens.

There are three forms of molecular mimicry:

1. Amino acid sequence identity shared between a host protein and a pathogen protein molecule, recognised by a T cell or a B cell (antibody). In such diseases, the mimicking pathogen succeeds in breaking immunologic tolerance by being similar enough or identical to cryptic (hidden, as opposed to dominant) auto-epitoes, yet different enough in terms of its other polypeptide sequences to break tolerance and elicit an immune response (figure 10).

2. Mimicry occurs between structurally similar molecules, which do not necessarily need to share amino acid sequence identity.

3. Recognition by one monoclonal antibody of completely dissimilar structures. This is likely to be due to both the structural properties of the microbial epitope and the antibody molecule configurations, which may make structural rearrangements to accommodate several different antigenic structures.

Conclusion

It is difficult to prove a causative link between CD, MAP infection and molecular mimicry because, as with any autoimmune disease, the symptoms may persist long after the pathogen has been eliminated or the pathogen may be gone before the clinical symptoms of the disease appear.

It is not impossible that molecular mimicry is just an epiphenomenon, occurring as a consequence of the primary phenomenon, which is  an immune response towards a persistent infectious agent resulting in autoimmune disease (23). Mimicry may also arise as a consequence of an alteration of host antigenic determinants during tissue injury and the generation of neoepitopes with subsequent production of autoantibodies and T cell responses, making them the symptoms of tissue damage, not the cause of it (23).  Tissue injury also contributes to epitope speading.

Proving that molecular mimicry is the cause of autoimmunity still remains an unaccomplished mission. While all individuals are exposed to infection at some point in life, how can we explain the fact that only a small minority develop autoimmune disease? Although molecular mimicry still remains to be proven as the pathologic agent in autoimmunity, it offers many researchers an attractive explanation uniting the current concepts of immune responses towards the invading pathogen with ideas of autoimmunity.

A mechanism able to break the host’s natural immunologic tolerance -molecular mimicry may only be a component cause in autoimmunity. However, the presence of homologous sequences in a pathogen in not necessarily sufficient to result in pathogenesis per se, and more profound evidence in humans will be required to ascertain the role of molecular mimicry in the causation of CD.

References

1. Shanahan F. Crohn’s disease. Lancet 2002 Jan 5;359(9300):62-9.
2. Ardizzone S, Puttini PS, Cassinotti A, Porro GB. Extraintestinal manifestations of inflammatory bowel disease. Dig Liver Dis 2008 Jul;40 Suppl 2:S253-S259.
3. Mathew CG. New links to the pathogenesis of Crohn disease provided by genome-wide association scans. Nat Rev Genet 2008 Jan;9(1):9-14.
4. Gersemann M, Wehkamp J, Fellermann K, Stange EF. Crohn’s disease–defect in innate defence. World J Gastroenterol 2008 Sep 28;14(36):5499-503.
5. Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, et al. Mucosal flora in inflammatory bowel disease. Gastroenterology 2002 Jan;122(1):44-54.
6. Wehkamp J, Koslowski M, Wang G, Stange EF. Barrier dysfunction due to distinct defensin deficiencies in small intestinal and colonic Crohn’s disease. Mucosal Immunol 2008 Nov;1 Suppl 1:S67-S74.
7. Fellermann K, Stange DE, Schaeffeler E, Schmalzl H, Wehkamp J, Bevins CL, et al. A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon. Am J Hum Genet 2006 Sep;79(3):439-48.
8. Marks DJ, Segal AW. Innate immunity in inflammatory bowel disease: a disease hypothesis. J
9. Schulze C, Munoz LE, Franz S, Sarter K, Chaurio RA, Gaipl US, et al. Clearance deficiency–a potential link between infections and autoimmunity. Autoimmun Rev 2008 Oct;8(1):5-8.
10. Di SA, Ciccocioppo R, Luinetti O, Ricevuti L, Morera R, Cifone MG, et al. Increased enterocyte apoptosis in inflamed areas of Crohn’s disease. Dis Colon Rectum 2003 Nov;46(11):1498-507.
11. Cadwell K, Liu JY, Brown SL, Miyoshi H, Loh J, Lennerz JK, et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 2008 Nov 13;456(7219):259-63.
12. Hermon-Taylor J. Protagonist. Mycobacterium avium subspecies paratuberculosis is a cause of Crohn’s disease. Gut 2001 Dec;49(6):755-6.
13. Scanu AM, Bull TJ, Cannas S, Sanderson JD, Sechi LA, Dettori G, et al. Mycobacterium avium subspecies paratuberculosis infection in cases of irritable bowel syndrome and comparison with Crohn’s disease and Johne’s disease: common neural and immune pathogenicities. J Clin Microbiol 2007 Dec;45(12):3883-90.
14. Pineton de CG, Colombel JF, Poulain D, rfeuille-Michaud A. Pathogenic agents in inflammatory bowel diseases. Curr Opin Gastroenterol 2008 Jul;24(4):440-7.
15. Feller M, Huwiler K, Stephan R, Altpeter E, Shang A, Furrer H, et al. Mycobacterium avium subspecies paratuberculosis and Crohn’s disease: a systematic review and meta-analysis. Lancet Infect Dis 2007 Sep;7(9):607-13.
16. Ferwerda G, Kullberg BJ, de Jong DJ, Girardin SE, Langenberg DM, van CR, et al. Mycobacterium paratuberculosis is recognized by Toll-like receptors and NOD2. J Leukoc Biol 2007 Oct;82(4):1011-8.
17. Naser SA, Ghobrial G, Romero C, Valentine JF. Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet 2004 Sep 18;364(9439):1039-44.
18. Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, et al. Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn’s disease. J Clin Microbiol 2003 Jul;41(7):2915-23.
19. Mendoza JL, Lana R, az-Rubio M. Mycobacterium avium subspecies paratuberculosis and its relationship with Crohn’s disease. World J Gastroenterol 2009 Jan 28;15(4):417-22.
20. Selby W, Pavli P, Crotty B, Florin T, Radford-Smith G, Gibson P, et al. Two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for Crohn’s disease. Gastroenterology 2007 Jun;132(7):2313-9.
21. Borgaonkar MR, MacIntosh DG, Fardy JM. A meta-analysis of antimycobacterial therapy for Crohn’s disease. Am J Gastroenterol 2000 Mar;95(3):725-9.
22. Davies JM. Introduction: Epitope mimicry as a component cause of autoimmune disease. Cell Mol Life Sci 2000 Apr;57(4):523-6.
23. Albert LJ, Inman RD. Molecular mimicry and autoimmunity. N Engl J Med 1999 Dec 30;341(27):2068-74.
24. Goodnow CC. Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. Proc Natl Acad Sci U S A 1996 Mar 19;93(6):2264-71.
25. Davies JM. Molecular mimicry: can epitope mimicry induce autoimmune disease? Immunol Cell Biol 1997 Apr;75(2):113-26.
26. Oldstone MB. Molecular mimicry and immune-mediated diseases. FASEB J 1998 Oct;12(13):1255-65.
27. Polymeros D, Bogdanos DP, Day R, Arioli D, Vergani D, Forbes A. Does cross-reactivity between mycobacterium avium paratuberculosis and human intestinal antigens characterize Crohn’s disease? Gastroenterology 2006 Jul;131(1):85-96.
28. Horwitz MS, Bradley LM, Harbertson J, Krahl T, Lee J, Sarvetnick N. Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry. Nat Med 1998 Jul;4(7):781-5.