Crohn's: An Autoimmune or Bacterial-Related Disease?

In 1996 geneticist Gilles Thomas and colleagues at the INSERM, the French Institute for Health and Medical Research in Paris, reported that IBD1, a region on the long arm of chromosome 16, was a Crohn's disease susceptibility locus.1 Five years later, in a second study, they announced that they had pinpointed the exact gene. The researchers contended that the connection between Crohn's and NOD2, the implicated gene located in IBD1, provided evidence to support the hypothesis that the disease is not strictly autoimmune and that a bacterial pathogen is involved. Another group, led by pathologist Gabriel Nunez, University of Michigan and gastroenterologist Judy Cho, University of Chicago, came to the same conclusion.2,3 Both identified a mutant NOD2 gene as a risk factor, which increases susceptibility to Crohn's disease. They also found that this mutation accounts for the condition in about 15 percent of Crohn's patients. They discussed their findings at the recent 2001 Digestive Disease Week conference in Atlanta. This research, while not uncovering the etiology of Crohn's, which affects between 500,000 and 1 million Americans, strengthens the argument that environmental factors play a larger role than scientists previously thought. However, other work is showing that the case for an autoimmune basis is still strong, and Thomas' discovery apparently has not discouraged that line of thinking. Crohn's, which normally affects the ileum and causes, among other problems, abdominal pain, diarrhea, and sometimes fistulas, continues to prove itself a complex disease. Nevertheless, the NOD2 discovery is welcome. "[It] gives us new insights into the disease and reaffirms what we have found in experimental models," says Charles O. Elson, chief of the division of gastroenterology, University of Alabama at Birmingham, and chair of the National Scientific Advisory Committee for the Crohn's and Colitis Foundation of America. Dennis Lang, a program officer in the division of microbiology and infectious diseases, National Institute of Allergy and Infectious Diseases (NIAID), agrees: "This discovery points to a new target for research. It's certainly not a cure-all, but it's another piece in the puzzle." NOD2 encodes the protein NOD2, which is a member of the Nod-1/Apaf-1 superfamily of apoptosis regulators. Nunez and Cho believe that NOD2 might help to reveal the missing link between genes and bacteria that interact to cause Crohn's. NOD2, expressed in monocytes, activates the nuclear factor NF * B pathway. The mutations--one frameshift and two missense--affect either the leucine-rich repeat (LRR) domain or the adjacent region in NOD2. In humans, the LRR domain of wild-type NOD2 is required for the recognition of bacterial lipopolysaccharides (LPS). An error in that domain, the result of a frameshift mutation in NOD2, produces a truncated protein that is unable to activate NF-kB in response to LPS. Presumably, a deficit in sensing bacteria results in an exaggerated inflammatory response from the adaptive immune system, Nunez says. Having one mutant copy doubles the risk of CD; having two copies increases the risk 20-fold, says Cho. Examining the Complexities Decades ago, based on what researchers knew, Crohn's appeared to be an autoimmune disease. "An autoimmune etiology was given for lack of a better explanation," Elson says. "In the past eight years, we have been experimenting with animal models, especially mice, and using genetic manipulation to... induce colitis." When kept in a germ-free environment, continues Elson, induced mutants do not develop colitis. The current hypothesis, in place before Thomas' work, is that Crohn's is caused by "an abnormal reactivity to constituents of the bacterial flora in genetically predisposed hosts," says Elson. "NOD2 fits this hypothesis very nicely. It supports the idea that there is a response to bacteria and supports the evidence we have found in animal models." But some researchers are not abandoning the notion that Crohn's is an autoimmune disease. Kevin Becker, scientist in the Gerontology Research Unit, National Institute on Aging, says, "An unknown antigen--either environmental, dietary, viral, or bacterial--triggers an initial immune response in the gut, which eventually results in tissue destruction of that organ." By comparing data from genome scan experiments, Becker identified 18 clusters of non-MHC candidate loci connecting several autoimmune and inflammatory diseases and immune disorders, including multiple sclerosis, asthma, psoriasis, and type I diabetes.4 IBD1 falls into the chromosome 16 susceptibility cluster. Polymorphisms in the vitamin D receptor gene, located in the chromosome 12 cluster, have been associated not only with Crohn's, but also with other autoimmune diseases not cited in Becker's study, such as Graves' disease,5 systemic lupus erythematosus (SLE),6 and rheumatoid arthritis.7 A locus on chromosome 1p that demonstrates epistasis--a phenomenon in which one gene alters the phenotypic expression of another--with IBD1 falls into another of the susceptibility clusters.8 But most Crohn's researchers, such as Stephen James, divisional deputy director of digestive diseases and nutrition, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), now are reluctant to classify Crohn's disease as strictly autoimmune: "The term [autoimmune] is sometimes used to describe a grab-bag of inflammatory diseases with unknown causes. Crohn's disease is not autoimmune in the same sense, say, that lupus is." Compounding the issue even further, recent research involving the major histocompatibility complex (MHC) indicates that another immunodeficiency also might be at work. In 1999, researchers at University of California at Los Angeles designated an area in the primary MHC region on chromosome 6, which encodes the human leucocyte-associated antigens (HLA) class II alleles, as IBD3. (The original location of IBD2 on chromosome 12 is still under debate.) This region, which contributes an estimated 10 to 33 percent of the total genetic risk to development of Crohn's disease,9 also codes for the tumor necrosis factor (TNF), a cytokine that promotes inflammation. Mice engineered to overproduce TNF suffer from severe intestinal inflammation similar to that experienced with Crohn's, which suggests that aberrant TNF plays a role in the disease.10 Many Crohn's patients respond well to injections with an anti-TNF antibody called infliximab,11 but ultimately the goal is to find, and possibly correct, the genetic error in TNF production. James predicts, "In five or 10 years, the treatments that we will be using will be quite different. I hesitate to call them gene therapies, but it could be something similar." The End Is Not Near With possible loci on as many as nine chromosomes, no one is predicting a clear answer in the near future. According to Nunez, "This work has changed how we think about Crohn's disease. We need to do more genetic analysis. Only about 20 percent of Crohn's cases are familial; we need to look at all the sporadic cases." The NIDDK hopes to help: In May, the NIDDK Advisory Council approved the concept of an Inflammatory Bowel Disease Genetic Research Consortium. James explains, "We expect that Crohn's disease, like so many other disorders, is caused by multiple gene interactions. We're working on a proposal to fund a group of investigators to coordinate research." The proposal, which is still waiting for final approvals, could be published as early as next year, according to James. While some are looking into the genetics, others continue discussing the search for the antigen. Becker remarks, "What's the antigen? We don't know. There are lots of suspects floating around, but no definite bad guy." Adds Elson, "Over the years there have been several candidates, including Mycobacterium paratuberculosis and measles, but none of those has ever stood the test of time." Nunez agrees, "In all the studies done so far, many types of bacteria have been present. We need to identify which bacteria are at work in animals models." Although understanding the NOD2 association won't result in a new medication or treatment immediately, Lang hopes that continued research on this gene eventually will lead to "alternate ways to treat or prevent the disease. This is a hopeful promise." Elson also predicts, "The race is on--a number of labs want to get to work on this." Maria Anderson (missria99@aol.com) is a freelance science writer in Athens, Ga. References 1. J.P. Hugot et al., "Mapping of a susceptibility locus for Crohn's disease on chromosome 16," Nature, 379:821-3, 1996. 2. J.P. Hugot et al., "Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease," Nature, 411:599-603, May 31, 2001. 3. Y. Ogura et al., "A frameshift mutation in Nod2 associated with susceptibility to Crohn's disease, "Nature, 411:603-6, May 31, 2001. 4. K.G. Becker et al., "Clustering of non-major histocompatibility complex susceptibility candidate loci in human autoimmune diseases," Proceedings of the National Academy of Sciences, USA, 95:9979-84, 1998. 5. Y. Ban et al., "Vitamin D receptor gene polymorphism is associated with Graves' disease n the Japanese population," Journal of Clinical Endocrinology and Metabolism, 85[12]:4639-43, Dec. 2000. 6. Y. Ozaki et al., "Vitamin-D receptor genotype and renal disorder in Japanese patients with systemic lupus erythematosus," Nephron, 85[1]:86-91, May 2000. 7. T. Fukazawa et al., "Association of vitamin D receptor gene polymorphism with multiple sclerosis in Japanese," Journal of the Neurological Sciences, 166[1]:47-52, 1999. 8. J. Cho et al., "Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: Evidence for epistasis between 1p and IBD1," Proceedings of the National Academy of Sciences, 95:7502-7, 1998. 9. H. Yang et al., "Linkage of Crohn's disease to the major histocompatibility complex region is detected by multiple non-parametric analyses," Gut, 44[4]:519-26, 1999. 10. M.F. Neurath et al., "Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice," European Journal of Immunology, 27[7]:1743-50, 1997. 11. F.J. Baert et al., "Tumor necrosis factor alpha antibody (infliximab) therapy profoundly down-regulates the inflammation in Crohn's ileocolitis," Gastroenterology, 116[1]:22-8, 1999. Debating Crohn's pathogenesis Researchers are using genetics to do more than just identify susceptibility loci, and the DNA they're studying isn't necessarily that of Homo sapiens. For more than 25 years, physicians and scientists have debated the relationship between Crohn's and Mycobacterium avium subspecies paratuberculosis (MAP). MAP can stay dormant for years, and it's difficult to grow in solid culture, which makes it almost impossible to locate and study. But MAP has been proven to cause Johne's disease, a gastrointestinal affliction in animals similar to human Crohn's. Cases of MAP-induced Johne's have been identified in ruminants, such as sheep and cows, and four subhuman primates. In the early 1990s, researchers identified a DNA element known as IS900 in the MAP genome. This multicopy insertion element has been detected in fecal samples and intestinal tissue cultures using nested primer PCR, a highly sensitive form of PCR. Several studies have used this MAP-specific technique to compare the amount of MAP present in tissue samples from Crohn's, ulcerative colitis (UC), and control patients. Some researchers report that up to two-thirds of all Crohn's disease patients have some form of the bacteria in their systems. John Hermon-Taylor, department of surgery chairman, St. George's Hospital Medical School, London, has studied the bacterium since 1985. He recently received funding from the British Medical Research Council--after 25 grant proposals were rejected between 1991 and 1999. He believes that MAP contributes to between 50 and 70 percent of all Crohn's cases. "I am certain that paratuberculosis is present in the guts of people with Crohn's disease. [But] the correct methods haven't been used to detect and study it. It's a tough bug." Hermon-Taylor says that he has treated 150 Crohn's cases with a combination of drugs, including rifabutin and clarithromycin, which are more active against MAP. About 70 percent of these cases, he says, went into remission after the intestine was healed. Others are not so easily convinced. Herbert J. Van Kruiningen, a pathobiology professor, University of Connecticut, refuted the idea that MAP is related to Crohn's due to lack of evidence.1 He explains, "There are parts of the world where Johne's disease does not occur--Sweden and western Australia--and where Crohn's disease nevertheless occurs with a prevalence equal to that of other parts." Instead, he has studied other possible bacterial pathogens, including Streptococcus, Escherichia coli, and Listeria,2 and he is currently working on familial clustering in French families based on those pathogens. In the past decade, 18 studies have addressed the connection between MAP and Crohn's; half reported finding traces of MAP in Crohn's patients. In response to a 1998 report from the National Institute of Allergy and Infectious Diseases (NIAID) entitled "Crohn's Disease--Is There a Microbial Etiology? Recommendations for a Research Agenda," the federal government funded a project at the University of Maryland to compare the microbe contents of gut tissue samples from Crohn's and control patients. "We're not convinced that paratuberculosis is the culprit," explains Dennis Lang, program officer at NIAID. "We're trying to keep an open mind and look for any possible microbes." NIAID hopes to review more grant applications for disease research this fall. Hermon-Taylor is optimistic: "Pretending [a threat] is not there has a bad track record in public health history. The evidence [to date] has justified the need for a long-term controlled study of this [connection between MAP and Crohn's]. Hopefully we're moving out of the period of stagnation." --Maria Anderson References 1. H.J. Van Kruiningen, "Lack of support for a common etiology in Johne's disease of animals and Crohn's disease in humans," Inflammatory Bowel Diseases, 5[3]:183-91, 1999. 2. Y. Liu, et al., "Immunocytochemical evidence of Listeria, Escherichia coli and Streptococcus antigens in Crohn's disease," Gastroenterology, 108[5]:1396-1404, 1995.

Crohn's: An Autoimmune or Bacterial-Related Disease?

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