The Crohnie

From: McGill University Press Release: Toward an explanation for Crohn’s disease?

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

NOD1 expression in the eye and functional contribution to IL-1beta-dependent ocular inflammation in mice.
Rosenzweig HL, Galster KT, Planck SR, Rosenbaum JT.
Casey Eye Institute, Oregon Health & Science University, Portland,
Oregon 97239, USA

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.

So, it seems that variations in the NOD2/CARD15 gene make cattle more susceptible to infection with Mycobacterium avium subspecies paratuberculosis, as well as making humans more susceptible to Crohn’s Disease.

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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.

The abstracts from the Ninth International Colloquium on Paratuberculosis (9ICP), which I attended in Tsukuba, Japan, have been published online.

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

It appears that some Azathioprine and 6-MP, which are used for the treatment of Crohn’s Disease, and whose mechanism of action is currently unknown, have antibiotic activity against Mycobacterium avium subspecies paratuberculosis (MAP).

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.

An interesting new piece of research about the genetics of Crohn’s Disease Diabetes and other diseases.
http://www.nature.com/nature/journal/v447/n7145/pdf/nature05911.pdf

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.

I’ve just returned from Copenhagen, Denmark, where I attended the 8th International Colloquium on Paratuberculosis (at considerable personal expense: Copenhagen is EXPENSIVE!)

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.

I want to draw your attention to a recent paper in The Lancet, by a group of researchers from McGill University, Montreal, Canada.

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.

I want to draw your attention to a recent epidemiological study from Wisconsin, which studied the epidemiology of both Crohn’s Disease, and IBD in general, in children in the state of Wisconsin. You can read an abstract of the study here

Epidemiologic and clinical characteristics of children with newly diagnosed inflammatory bowel disease in Wisconsin: a statewide population-based study.
http://www.ncbi.nlm.nih.gov/pubmed/14571234

Generally, the results of this study cast doubt on the belief that Crohn’s Disease and IBD are caused by a “genetic predisposition” or abnormality.

The most important points to note are

1. The researchers found that incidence rates of IBD were equal among all racial and ethnic groups. This casts doubt on the perceived wisdom that certain racial or ethnic groups are more “genetically susceptible” to Crohn’s Disease and IBD.

2. 89% of the new (incidence) cases of IBD described were non-familial, meaning that there was no family history of IBD in 89% of cases. This further casts doubt on “genetic susceptibility”.

3. There were no difference in the incidence rates between rural and urban areas.

4. The researchers found that there were more than twice as many cases of Crohn’s Disease as Ulcerative Colitis.

The researchers conclude their summary with this statement:

“This study provides novel, prospective, and comprehensive information on pediatric IBD incidence within the United States. The surprisingly high incidence of pediatric IBD, the predominance of Crohn’s disease over ulcerative colitis, the low frequency of patients with a family history, the equal distribution of IBD among all racial and ethnic groups, and the lack of a modulatory effect of urbanization on IBD incidence collectively suggest that the clinical spectrum of IBD is still evolving and point to environmental factors contributing to the pathogenesis.”

The conclusion that “environmental factors contribute to the pathogenesis” is an important one.

That there was a “surprisingly high incidence of pediatric IBD” mirrors results from neighbouring Minnesota, where Mayo Clinic researchs noted in 1998 that, in Minnesota, “No pediatric cases of Crohn’s disease were diagnosed before 1954, but 17% of patients diagnosed in the last two decades of [our] study period were under the age of 20 years.”

Crohn’s Disease in Olmsted County, Minnesota, 1940-1993: Incidence, Prevalence, and Survival
http://crohn.ie/archive/research/epidem/olmsted.htm

Researchers at the University of Minnesota, in conjunction with researchers from the United States Department of Agriculture, have completed their work on the sequencing of the genome of MAP. Their press release can be found here

http://www.johnes.org/handouts/files/UM_news_release.pdf

The availability of a new wealth of information about MAP, and its relationship with other closely-related disease-causing mycobacteria, including

Mycobacterium leprae
http://www.pasteur.fr/actu/presse/press/leprosy.html

Mycobacterium tuberculosis
http://genolist.pasteur.fr/TubercuList/
http://www.sanger.ac.uk/Projects/M_tuberculosis/

Mycobacterium bovis
http://www.sanger.ac.uk/Projects/M_bovis/

Mycobacterium marinum
http://www.sanger.ac.uk/Projects/M_marinum/

will hopefully begin the vitally important process of developing new diagnostics for detecting MAP infection in humans, and developing a deeper understanding of the relationship between such infection and Crohn’s Disease.

We can further hope that knowledge from the Tuberculosis and Leprosy research fields will enrich the spectrum of anti-MAP treatments currently available.