Causation of Crohn’s disease by Mycobacterium avium subspecies paratuberculosis
Hermon-Taylor J
Can J Gastroenterol. 2000 Jun;14(6):521-39.

Is Crohn’s disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne’s disease
Greenstein RJ
Lancet Infect Dis. 2003 Aug;3(8):507-14.

Title: A Common Genetic Fingerprint in Leprosy and Crohn’s Disease?
Authors: Erwin Schurr, Ph.D. and Philippe Gros, Ph.D.
Source: New England Journal of Medicine: Editorial: December 16, 2009

The cause-and-effect relationship between severe infections and death suggests that microbial pathogens are evolutionary sculptors of the genome. However, the genetic component of susceptibility to infections in the general population is complex and heterogeneous and is modulated by environmental factors such as determinants of microbial virulence. Thus, it is a challenge to identify specific genetic effects in human populations. Availability of the human genome sequence, combined with knowledge of genetic variation, has facilitated the genomewide association study, a powerful approach to detecting genetic associations. In this issue of the Journal, Zhang and colleagues¹ describe a genomewide association study of leprosy, a bacterial disease.

Leprosy manifests with a broad pathologic spectrum. At one end is the localized paucibacillary form, characterized by a small number of hypopigmented, anesthetic skin lesions; at the other end is the disseminated multibacillary form, involving numerous skin lesions with a high bacillary load. Paucibacillary infection is associated with immune responses mediated by type 1 helper T (Th1) cells (involving the production of interferon-γ and interleukin-2) that promote granuloma formation and limit bacterial replication and dissemination. The multibacillary form, in contrast, is associated with the Th1 polarization of the immune response (and the production of interleukin-4 and interleukin-10), which promotes uncontrolled bacterial replication and more severe pathology.

Population studies and studies of twins have established that there is a genetic component to susceptibility to leprosy.^2,3 Linkage and association studies have implicated variants of the HLA-DR region, PARK2 (encoding parkin), LTA (encoding lymphotoxin alpha), and chromosome 10p13 in conferring susceptibility to leprosy in independent populations.³

Zhang and colleagues describe the results of genomewide scanning in persons with paucibacillary or multibacillary forms of leprosy from eastern or southern China. The authors compared the prevalence of each genetic marker — in this case, each single-nucleotide polymorphism (SNP), which is currently the marker typically used in genomewide association studies — in 706 case patients and 1225 unaffected persons. A total of 93 SNPs were shown to have a significant association with leprosy. These SNPs were then tested in three replication sets of more than 3000 patients and nearly 6000 controls from eastern or southern China. The data implicate CCDC12 (the gene encoding coiled-coil domain containing 122), C13orf31 (encoding chromosome 13 open reading frame 31), NOD2 (encoding nucleotide-binding oligomerization domain containing 2), TNFSF15 (encoding tumor necrosis factor [ligand] superfamily member 15), RIP2K (encoding receptor-interacting serine–threonine kinase 2), and the HLA-DR–DQ locus. Several of the proteins encoded by these genes are involved in microbial sensing and in the early innate immune and inflammatory responses. NOD2 recognizes a component of the mycobacterial wall, and stimulation of NOD2 results in the recruitment of RIPK2 and indirectly prompts the activation of the transcriptional regulator nuclear factor κB (NF-ΚB)⁴ — which in turn activates the transcription of genes encoding proinflammatory cytokines including TNFSF15. On the surface of phagocytes, HLA-DR molecules present bacterial antigens to CD4+ T cells to initiate Th1-cell polarization.

Human genetic studies have implicated both IL12B (the gene encoding interleukin-12β) and NOD2 in increased susceptibility to mycobacterial disease, and mouse mutants lacking either Nod2, Ripk2, or Infg (encoding interferon-γ) are highly susceptible to tuberculosis.^5,6,7 The fact that such genes are now implicated through a genomewide association study of leprosy not only validates this approach to studying the disease, but also raises interest in the newly implicated genes (e.g., CCDC12 and C13orf31), the functions of which are not known.

Another interesting aspect of the study is that variation in some of the implicated genes is known to be associated with bowel inflammatory conditions. A frame-shift mutation in NOD2 has been identified as a strong susceptibility factor for Crohn’s disease; additional NOD2 mutations have been discovered not only in persons with Crohn’s disease but also in those with Blau’s syndrome and in those with early-onset sarcoidosis.⁸ Likewise, variants of TNFSF15 and IL12B have been associated with Crohn’s disease.⁹ These findings are consistent with studies of mouse models that have also established a role for Nod2, Ripk2, and Nfkb in intestinal homeostasis and colitis.^10,11 Together, these studies establish a strong genetic and functional link between susceptibility to leprosy and predisposition to Crohn’s disease.

Although the results described by Zhang and colleagues are exciting, additional experiments are required to validate and refine their conclusions. Genomewide association studies are fairly crude. For example, a regulatory SNP in the LTA gene, previously identified as a leprosy risk factor,¹² was not tested in the platform used by Zhang and colleagues. Moreover, linkage disequilibrium (a state in which genetic markers — typically those in close physical proximity — are more likely than not to be inherited together) can extend over large intervals, with the main genetic effect located within a region that is a considerable distance from the SNPs showing the disease association. As known SNPs increase in number, the dissection of local effects of linkage disequilibrium will become more accurate, and testing for the presence of associations in groups of persons of differing ancestries will shed light on the extent to which these findings of Zhang and colleagues apply to other populations.

How do we move from P values to understanding pathogenesis and response to infection and, finally, to clinical outcomes? Although genomewide association studies rarely identify causative genetic lesions, they do point to specific genes and biologic pathways that can be targeted for pharmacologic intervention, irrespective of the mechanism underlying genetic susceptibility. A particularly attractive aspect of the study by Zhang and colleagues is the apparently narrow focus of the genetic control, which highlights early antigen sensing and signaling in the pathogenesis of both leprosy and Crohn’s disease. It is tempting to speculate that these common genetic signatures support, albeit indirectly, the proposal that a proportion of Crohn’s disease cases may have a mycobacterial cause.^13,14 Irrespective of its strength, such a link may broaden the therapeutic treatment options for both diseases.

In comparison with the study by Zhang and colleagues, genomewide association studies of susceptibility to malaria¹⁵ and to infection with the human immunodeficiency virus¹⁶ suggest that the contribution of common genetic variants is more limited. Why would this be so? The genomic variability of Mycobacterium leprae isolates is very small, and M. leprae has undergone substantial reductive evolution, possibly through adaptation to its human host. This may suggest that larger host genetic effects in infectious disease reflect decreased pathogen variability. In this view, pathogens with greater genetic variability, such as M. tuberculosis, will give rise to a more complex genetic architecture of host susceptibility.

An innovative study at the Research Institute of the MUHC has brought us closer to an explanation for Crohn’s disease

Montreal, July 9th 2009 – Twenty-five per cent of Crohn’s disease patients have a mutation in what is called the NOD2 gene, but it is not precisely known how this mutation influences the disease. The latest study by Dr. Marcel Behr, of the Research Institute of the MUHC and McGill University, has provided new insight into how this might occur. The study will be published on July 9th in the Journal of Experimental Medicine.

When the NOD2 gene functions normally, it codes for a receptor that will recognize invading bacteria and then trigger the immune response. This study demonstrates that the NOD2 receptor preferentially recognizes a peptide called N-glycolyl-MDP, which is only found in a specific family of bacteria called mycobacteria. When mycobacteria invade the human body, they cause an immediate and very strong immune response via the NOD2 receptor.

“Now that we have a better understanding of the normal role of NOD2, we think that a mutation in this gene prevents mycobacteria from being properly recognized by the immune system,” explained Dr. Behr. “If mycobacteria are not recognized, the body cannot effectively fight them off and then becomes persistently infected.”

Researchers were already aware of the relationship between mycobacteria and Crohn’s disease, but they did not know whether the presence of bacteria was a cause or a consequence of the disease. This new discovery associates the predisposition for Crohn’s disease with both the NOD2 mutation and the presence of mycobacteria, but researchers must still determine the precise combination of these factors to understand how the disease develops.

More research is required to establish a complete explanation. From this, it is expected that new therapeutic approaches that fight the cause of Crohn’s disease may be developed

http://www.ncbi.nlm.nih.gov/pubmed/19074813

PURPOSE: NOD1 plays an important role in host defense and recognizes the minimal component of bacterial cell walls, meso-diaminopimelic acid (iE-DAP). Polymorphisms in NOD1 are associated with autoinflammatory diseases characterized by uveitis such as Crohn’s disease and sarcoidosis. NOD1 is homologous to NOD2, which is responsible for an autosomal dominant form of uveitis. Nonetheless, the role of NOD1 in intraocular inflammation has not been explored. The induction of uveitis by iE-DAP in mice and the potential contribution of interleukin (IL)-1beta were investigated. METHODS: BALB/c mice or mice deficient in caspase-1 or IL-1R1 and their congenic controls were injected intravitreally with iE-DAP or saline. The time course, dose response, and contribution of IL-1beta to ocular inflammation were quantified by intravital video microscopy, histology, and immunohistochemistry. NOD1 and IL-1beta were measured in eye tissue by immunoblotting and ELISA. RESULTS: NOD1 protein is expressed in the eye and promotes ocular inflammation in a dose- and time-dependent fashion. The authors previously defined the role of IL-1beta in NOD2 uveitis and tested whether NOD1 and NOD2 used similar mechanisms. Treatment with iE-DAP significantly increased IL-1beta, which was caspase-1 dependent. However, in contrast to NOD2, caspase-1 and IL-1R1 were essential mediators of iE-DAP-induced uveitis, suggesting that NOD1 and NOD2 induce ocular inflammation by distinct mechanisms involving IL-1beta. CONCLUSIONS: These findings demonstrate that NOD1 is expressed within the eye and that its activation results in uveitis in an IL-1beta-dependent mechanism. Characterizing the differences between NOD1 and NOD2 responses may provide insight into the pathogenesis of uveitis.

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Association between CARD15/NOD2 gene polymorphisms and paratuberculosis infection in cattle
Pinedo, P.J., Buergelt, C.D., Donovan, G.A., Melendez, P., Morel, L., Wud, R., Langaee, T.Y., Rae, D.O. (2009)
Veterinary Microbiology, 134, 346-352
http://www.ncbi.nlm.nih.gov/pubmed/18926647

Paratuberculosis represents a major problem in farmed ruminants and at the present is considered a potential zoonosis. The disease is caused by Mycobacterium avium subsp. paratuberculosis, and susceptibility to infection is suspected to have a genetic component. Caspase recruitment domain 15 (CARD15) gene encodes for a cytosolic protein implicated in bacterial recognition during innate immunity. Crohn’s disease (CD) is an idiopathic inflammatory bowel disease in humans comparable in many features to bovine paratuberculosis involving an abnormal mucosal immune response. The association between mutations in the CARD15 gene and increased risk of Crohn’s disease has been described. The objective of this candidate gene case-control study was to characterize the distribution of three polymorphisms in the bovine CARD15 gene and test their association with paratuberculosis infection in cattle. Three previously reported single nucleotide polymorphisms (E2[-32] intron 1; 2197/C733R and 3020/Q1007L) were screened for the study population (431 adult cows). The statistical analysis resulted in significant differences in allelic frequencies between cases and controls for SNP2197/C733R (P < 0.001), indicating a significant association between infection and variant allele. In the analysis of genotypes, a significant association was also found between SNP2197/ C733R and infection status (P < 0.0001); cows with the heterozygous genotype were 3.35 times more likely to be infected than cows with the reference genotype (P = 0.01). Results suggest a role for CARD 15 gene in the susceptibility of cattle to paratuberculosis infection. These data contribute to the understanding of paratuberculosis, suggest new similarities with Crohn’s disease and provide new information for the control of bovine paratuberculosis.

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Abnormalities in the handling of intracellular bacteria in Crohn’s disease: a link between infectious etiology and host genetic susceptibility.
Glasser AL, Darfeuille-Michaud A.
Arch Immunol Ther Exp (Warsz). 2008 Jul-Aug;56(4):237-44.
http://www.ncbi.nlm.nih.gov/pubmed/18726145

The etiology of Crohn’s disease (CD) is still poorly understood, but recent advances have highlighted the importance of the innate immune system and the critical relationship between the gut flora and the intestinal mucosa. Several combinations of genetic factors predisposing to CD have been described, with the most significant replicable associations including genes for intracellular receptors of bacterial cell walls (NOD2/CARD15) and for bacterial clearance and antigen processing via autophagy (ATG16L1 and IRGM). One theoretical link between susceptibility genes NOD2/CARD15, ATG16L1, and IRGM is that CD is primarily induced by the presence of a dysfunctional immunological response to persistent infection by intracellular bacterial pathogens such as Mycobacterium avium subspecies paratuberculosis or adherent-invasive Escherichia coli, both first- rank candidates on the basis of host genetic susceptibility, which concerns impaired functions in the defense against intracellular bacteria.

http://aem.asm.org/cgi/content/abstract/AEM.00981-08v1
http://www.ncbi.nlm.nih.gov/pubmed/18676709

Antibacterial activities of 18 naturally-occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols and other plant extracts) against three strains of Mycobacterium avium subspecies paratuberculosis (Map), a bovine isolate NCTC 8578, a raw milk isolate 806R and a human isolate ATCC 43015, were evaluated using a macrobroth susceptibility testing method. Map was grown in 4 ml Middlebrook 7H9 broth containing 10% OADC, 0.05% Tween 80 (or 0.2% glycerol) and 2 microg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in optical density (OD) of the cultures at 600 nm as a measure of CFUs was recorded at intervals over an incubation period of 42 days at 37 degrees C. Six of the compounds were found to inhibit the growth of Map. The most effective compound was trans-cinnamaldehyde with a minimum inhibitory concentration (MIC) of 25.9 microg/ml, then cinnamon oil (26.2 microg/ml), oregano (68.2 microg/ml), carvacrol (72.2 microg/ml), 2,5-dihydroxybenzaldehyde (74 microg/ml), and 2- hydroxy-5-methoxybenzaldehyde (90.4 microg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three Map strains studied. Possible mechanisms of the antimicrobial effects are discussed.

As with previous ICP, the number of papers presented on the relationship between Crohn’s Disease and Mycobacterium Paratuberculosis has grown, as knowledge of this complex organism and its potential to cause disease in humans, has grown.

The list of 9ICP presentations in the “Public Health” segment of the Colloquium are listed here

http://www.paratuberculosis.org/pubs/proc9/section6.htm

As acceptance of the relationship between MAP and CD grows, related papers are now being presented outside of the dedicated “Public Health” segment. The following paper discusses how the pathogenetic mechanisms of paratuberculosis in Johnes Disease and Crohn’s Disease can be compared in order to increase understanding of both; it was presented in the “Pathogenesis and Immunology” segment.

Role of Mycobacterium avium subsp. paratuberculosis in the pathogenesis of Crohn’s disease
http://www.paratuberculosis.org/pubs/proc9/abst4a.htm

Also of interest is the growing evidence that Mycobacterium avium subsp paratuberculosis may be a cause of Type-1 diabetes.

Association of Mycobacterium avium subsp. paratuberculosis with Type-1 diabetes, a possible trigger
http://www.paratuberculosis.org/pubs/proc9/abst181f_o3.htm

Paratuberculosis and Type I Diabetes -Mapping the TRIGR-
http://www.paratuberculosis.org/pubs/proc8/abst3b_o20.htm

http://www.paratuberculosis.org/pubs/proc9/abst182f_o6.htm

As noted by the authors, “These data may partially explain the paradoxical response of Crohn’s disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs i.e. they do not worsen with anti-inflammatory treatment as would be expected with a microbial etiologic pathogen.”
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The in vitro susceptibility of human and bovine-origin Mycobacterium paratuberculosis to the thiopurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) were established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against M. paratuberculosis; isolates from Crohn’s disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of Mycobacterium avium, used as controls, were generally resistant to both AZA and 6-MP even at high concentrations (>=64.0 microg/mL). Among rapidly growing mycobacteria, M. phlei was susceptible to 6-MP and AZA whereas M. smegmatis strains were not. AZA and 6-MP limited the growth of, but did not kill, M. paratuberculosis in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-amino- salycilic acid (mesalamine)) had little or no antibacterial activity against M. paratuberculosis. The conventional antibiotics azithromycin and ciprofloxacin (CPX) used as control drugs were bactericidal for M. paratuberculosis, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of M. paratuberculosis to 6- MP and CPX resulted in significantly higher CFUs as compared to use of CPX alone. These data may partially explain the paradoxical response of Crohn’s disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs i.e. they do not worsen with anti-inflammatory treatment as would be expected with a microbial etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohn’s disease: azathioprine drugs may confound interpretation of data on therapeutic responses both antibiotic-treated and control groups.

I note that one of the gene patterns associated with Crohn’s relates to “autophagy”, an innate immunity mechanism which the body uses to defend itself against intracellular bacterial pathogens.

http://en.wikipedia.org/wiki/Autophagy

“”"
The study has also confirmed the importance of a process known as autophagy in the development of Crohn’s disease. Autophagy, or “self eating”, is responsible for clearing unwanted material, such as bacteria, from within cells. The may be key to the interaction of gut bacteria in health and in inflammatory bowel disease and could have clinical significance in the future.
“”"

http://www.medicalnewstoday.com/medicalnews.php?newsid=73420

“”"
One of the newly identified genes, called ATG16L1, has been thought to be required for autophagy, a process that leads to programmed cell death and is involved in the process of inflammation. When the research team used RNA interference to suppress the gene’s activity in bacterially infected cells, decreased molecular action associated with autophagy confirmed that the process depends on ATG16L1 activity.
“”"

http://www.sciencedaily.com/releases/2007/04/070415160159.htm

Autophagy is one of the most important defenses against the class of intracellular bacterial pathogens known as mycobacteria. One of the most studied organisms in relation to autophagy is mycobacterium tuberculosis

Autophagy in Immune Defense Against Mycobacterium tuberculosis
http://www.landesbioscience.com/journals/autophagy/article/2830

Mycobacterium avium subspecies paratuberculosis (MAP) is another intracellular obligate pathogen with which autophagy would be a key defense mechanism.

So it is no surprise to me that recent results indicate a defective autophagy process in Crohn’s Disease; this lends further weight to the theory that Mycobacerium paratuberculosis is a cause of Crohn’s Disease.

Current thinking among researchers in the field is that MAP itself is mostly not the culprit in the tissue damage. MAP’s role is to initiate an inflammatory response in the bowel, through some unknown process/antigen/inflammatory-pathway. This immune response results in inflammation of bowel tissue, whereby the cells of the intestine physically separate, to allow immune cells, primarily macrophages and CD4 + CD8 T-cells, to reach the site of infection. This leads to “leaky gut” syndrome , which permits the contents of the bowel (most accurately described as being similar to the contents of a sewer), to leak through the bowel wall, resulting in a *massive* inflammatory response against the many microbes present in the fecal stream.

At this point, the inflammatory process becomes self re-inforcing, with consequent runaway production of inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-alpha), various Interleukins, Interleukin-antagonists, etc.

In some patients, this results in granuloma formation, as the massively overstimulated immune system tries to contain the original MAP infection, present inside macrophages, which are unable to kill the “phagocytosed” (i.e. “eaten”) MAP, which have immune evasion techniques to avoid being killed by macrophages. The infected macrophage goes into overdrive, producing copious quantities of inflammatory cytokines, which cause it to be surrounded by a layer of CD4 T-cells, and then another layer of CD8 T-cells: a granuloma is formed, which contains the mycobacterial infection and cuts off the flow of inflammatory cytokines, but also has the unfortunate consequence of causing significant permanent scarring, thus leading to stenosis/narrowing of the bowel, and eventually strictures.

In other patients, for reasons unknown (although possibly related to the failure of the Th1 inflammatory response), this granuloma formation does not take place. Instead, the flood of antigens from the bowel contents infects the bowel “transmurally”, i.e. through the entire thickness of the bowel, leading to all kinds of scarring, and in some unfortunates, the formation of fistulas, as well as all manner of secondary infections. It is this sub-group of patients that responds best to treatment with wide-spectrum antobiotics, since it is secondary microbial infections that are primarily responsible for the tissue damage, with these secondary non-mycobacterial microbes being susceptible to non-mycobacterial antibiotic treatment.

Crohn’s disease, Mycobacteria, and NOD2
http://infection.thelancet.com/journal/vol4/iss3/full/laid.4.3.reflection_and_reaction.28813.1

The researchers describe the case of a man who was diagnosed as having Crohn’s Disease. He tested positive (by PCR) for mycobacterium avium paratuberculosis (MAP) infection, was treated with an anti-paratuberculosis antibiotic regimen, and his health improved (after a period of suffering from a “flu-like syndrome”, a common problem in CD when treated with anti-paratuberculosis antibiotics).

More interestingly, he also tested positive for NOD2, an identified “IBD susceptibility gene”, which relates to host defences against bacterial infection. As the researchers note: ” …. this man has evidence of typically defined Crohn’s disease with a Crohn’s disease susceptibility mutation, but also has evidence of human MAP disease. Applying the principle of Occam’s razor, the most parsimonious explanation in a patient without other illness is that MAP infection in a genetically susceptible host resulted in the Crohn’s disease phenotype.”

The researchers conclude their paper with this statement: “We believe that this case illustrates a potential conceptual approach to Crohn’s disease aetiology, which involves a tandem search for bacterial trigger and host susceptibility. The proportion of Crohn’s disease cases potentially attributable to MAP and the clinical/epidemiological consequence of MAP exposure among human beings are the focus of continuing studies.”

More research needs to take this approach. Seeking genetic susceptibilities or genetic flaws, in isolation from the actual agent which exploits the susceptibility or flaw, is a tragic waste of vital research resources, time and money.