Doi:10.1016/s0140-6736(03)15024-6

Hypothesis
Crohn’s disease: the cold chain hypothesis Jean-Pierre Hugot, Corinne Alberti, Dominique Berrebi, Edouard Bingen, Jean-Pierre Cézard Crohn’s disease is the result of an abnormal immune response of the gut mucosa triggered by one or more
environmental risk factors in people with predisposing gene variations, including CARD15 mutations. Epidemiological
data allow assessment of familial environmental risk factors related to western lifestyle, diet, bacteria, and domestic
hygiene. All findings point to refrigeration as a potential risk factor for Crohn’s disease. Furthermore, cold-chain
development paralleled the outbreak of Crohn’s disease during the 20th century. The cold chain hypothesis suggests
that psychrotrophic bacteria such as Yersinia spp and Listeria spp contribute to the disease. These bacteria have been
identified in Crohn’s disease lesions and we discuss their pathogenic properties with respect to our knowledge of the
disease. From a molecular perspective, we postulate that the disease is a result of a defect in host recognition by
pathogenic bacterial components that usually escape the immune response (eg, Yop molecules), which results in an
excessive host response to these bacteria.
Crohn’s disease is characterised by a relapsing disease has been positively associated with good standards inflammatory process in the digestive tract.1 Since its first of domestic hygiene (such as hot running water),9–11 and description in 1913, the cause of Crohn’s disease is still that environmental risk factors contribute to familial largely unknown, although it is recognised to be a aggregations of the disease.12 Because inflammation complex trait that is the result of an interaction between occurs in the digestive tract, antigens present in the gut environmental and genetic risk factors. Although some lumen, including components of food and intestinal genetic factors are known,2,3 uncertainty still exists around bacteria, have been implicated in the development of environmental factors, except for cigarette smoking.4 Crohn’s disease. However, no specific dietary component Knowledge of causative environmental factors is crucial has been identified, suggesting that many foods contain if disease mechanisms are to be understood and a specific putative contaminants. Several infectious agents have treatment developed. We postulate that Crohn’s disease is been proposed, including species of Mycobacterium, the result of an excessive response to pathogenic Listeria, and Yersinia, and Escherichia coli, but none has psychrotrophic bacteria in some genetically predisposed been proven as a causative factor. However, the link people. Such bacteria can exist and grow at temperatures between bacterial components and Crohn’s disease was between –1ºC and 10ºC, and we believe that the advent of lent strong support by the discovery that mutations in domestic refrigeration contributed to the outbreak of CARD15, a gene involved in innate immunity, Crohn’s disease in the 20th century.
In view of the observation that Crohn’s disease is linked to a familial environmental risk factor related to modern Crohn’s disease is common in Europe and North western lifestyles, domestic hygiene, diet, and infectious America, where incidence has risen in the second half of agents, we propose a specific candidate for the the 20th century.5 Thus, environmental factors, probably development of Crohn’s disease: the refrigerator.
related to the modern occidental way of life, might have a Domestic refrigeration began with the advent of ice role in Crohn’s disease.6–8 From the first to the second half containers in the 19th century. The first refrigerating of the 20th century, many crucial changes occurred with machines were built around 1875, and during the first respect to food, housing, transport, leisure, and clothing.
part of the 20th century, refrigeration methods were more Thus, to tease out the relevant causative risk factors for widespread in the USA than in other countries.13 The first Crohn’s disease from the mesh of inter-related variables is domestic refrigerator was developed by Kelvinator in 1918 in the USA. It became more and more popular and It is beyond the scope of this paper to discuss in 1921, 5000 refrigerators were produced, with the yearly epidemiological data in detail, but of note is that Crohn’s production rate rising to 75 000 in 1925, 850 000 in1930, and 1 700 000 in 1935. By 1937, 49% of In 1930s’ Europe, only wealthy families had Laboratoire de Génétique des Maladies Inflammatoires de refrigerators, and ownership became more common only l’Intestin, Projet Avenir INSERM, Fondation Jean Dausset CEPH, after World War II with improvements in living standards, Paris, France (J P Hugot MD); Department of Paediatric and with the supply of electricity to homes. Even in 1958, Gastroenterology (J-P Hugot, Prof J-P Cézard), Unité d’Epidémiologie only 10% of French and 12% of British families had a Clinique, Department of Public Health (C Alberti MD), EA3102, refrigerator—ownership was as low as 2% in Spain, the Department of Pathology (D Berrebi MD), and EA3105, Department USSR, and Japan. The European exception was Sweden, of Microbiology (Prof E Bingen), Hôpital Robert Debré, Assistance where at the same time just over half of families had a refrigerator developed by Electrolux.
Correspondence to: Dr Jean-Pierre Hugot, Service de Although difficult to define precisely, population-based Gastroentérologie, Hopital Robert Debré, 48 Boulevard Sérurier, data suggest that the increase in prevalence of Crohn’s disease was in the 1940s or before in the USA,8 in the 1950s (e-mail: jean-pierre.hugot@rdb.ap-hop-paris.fr) or before in Sweden,14,15 in the 1960s in the UK,6,7 and later THE LANCET • Vol 362 • December 13, 2003 • www.thelancet.com For personal use. Only reproduce with permission from The Lancet publishing Group.
in southern Europe. These examples show temporal and bacterial detection. In accordance with this point of view, geographical coincidences between the development of the yersinia was not a predominant microorganism in biopsy refrigerator and the outbreak of Crohn’s disease. Of course, samples from ten patients with the disease,21 lending the cold theory for the development of Crohn’s disease support to the theory of a chronic bacterial infestation that is cannot be limited to the domestic refrigerator, which is the last part of the cold chain. The cold chain is much more Because data from microbiological studies are compatible complex: of the 520 kg of food eaten every year per person with the cold chain hypothesis, we have further developed in France, 320 kg are, at some time, conserved at a low the theory of chronic infestation by psychrotrophic bacteria.
temperature. Furthermore, external and complex factorscould affect the quality of the cold chain including machine maintenance procedures, food conservation habits, and the Crohn’s disease lesions occur predominantly where there is a high density of lymphoid follicles, in the small bowel We are aware that the suggested association between the where the follicles are grouped to form Peyer patches, and cold chain and Crohn’s disease needs confirmation by more in the colon where they are isolated. In the small intestine, detailed analyses before being retained. Additionally, even lesions are more frequent in the distal ileum where Peyer if firmly established, temporal and geographical patches are most common. In addition to this spatial coincidences cannot prove a causal relation by themselves, relationship, a temporal relationship has also been and we cannot rule out the possibility that the cold chain is suggested by Van Kruiningen and colleagues.26 Peyer a confounding risk factor that occurred in parrallel with the patches develop from birth to age 10–15 years, and then undergo involution. Of note is that the age-dependentincidence curve for Crohn’s disease is roughly parallel to the number of Peyer patches with a peak in the third decade If our hypothesis is valid, it points to the existence of of life. A delay of 5–10 years is then seen between the bacteria capable of surviving or developing at low development of Peyer patches and the occurrence of symptoms. Such a delay is compatible with chronic psychrotrophic bacteria, have optimum growth at infection. Conversely, the ileal location of Crohn’s disease temperatures higher than 30ºC but are able to grow, at a is less frequent in elderly people after the involution curve slower rate, at temperatures between –1ºC and 10ºC—ie, of Peyer patches.27 In the colon, by contrast with the small the temperature inside refrigerators.
intestine, the number of lymphoid follicles is less subject to The most frequently encountered psychrotrophic variation. This observation can account for the fact that colonic Crohn’s disease can occur at any age. Furthermore, monocytogenes, Yersinia enterocolitica, Clostridium botulinum aphthoid ulcerations (thought to be the first lesions in and Bacillus cereus.16 The pathogenicity of B cereus and development of Crohn’s disease) are centred on lymphoid C botulinum is mainly related to toxin production. To our knowledge, these bacteria have not been studied in Crohn’s Together, these data suggest that lesions initially develop disease. L monocytogenes is a gram-positive bacillus that on lymphoid follicles and accord with a theory of specific induces acute diarrhoea. It has been identified in Crohn’s infection. Many infectious agents exhibit a specific tropism disease lesions by immunohistochemical analysis, but not by for M cells including Salmonella spp, Shigella spp, PCR.17–21 Y enterocolitica is an ubiquitous gram-negative M paratuberculosis, E coli, Vibrio cholerae, Campylobacter bacillus that has been detected in the digestive tract of many jejuni, reovirus, poliovirus, HIV, and the psychrotrophic wild and domestic animals. In addition to Y enterocolitica, bacteria Y enterocolitica and L monocytogenes.
less virulent species of the Yersinia genus are also Yersinia spp are able to produce an invasin, which allows encountered in food, but their pathogenic role is less clear.
M cell penetration. This specific tropism for M cells makes Yersinia species are commonly found in humans and in yersinia pathogenic for the lymphoid follicles but not for foods such as milk, beef, pork, chicken, sausages, other parts of the mucosa. In mice infected with yersinia, vesicles appear on the lymphoid follicles, which are People with Crohn’s disease have been tested for the progressively destroyed with micro-abscess formations.29 presence of Yersinia with conventional methods. Sera from Inflammation of the gut is discontinuous and only 30–50% patients were reactive to Y pseudotuberculosis more often than were control sera in one study,23 but most studies In human beings, Yersinia spp are able to induce a clinical report negative results. More interestingly, with PCR- ileitis or ileocolitis, and a mesenteric adenolymphitis, most specific primers, Yersinia spp were detected by two often in children and young adults. They might also induce independent groups in 63% (13/19) and 31% (17/54) of reactive arthritis and erythema nodosa. Histological analysis people with Crohn’s disease, respectively.24,25 Intestinal shows that intestine and lymph-node lesions can exhibit samples were positive for Y enterocolitica, Y pseudo- granuloma formations. All these findings are common to tuberculosis, or both. The clinical and histological features of Crohn’s disease and yersiniosis, explaining that these two the yersinia-positive cases were indistinguishable from disorders have to be classically differentiated during the unlikely, since median follow-up in the two study groups Additional features are relevant for Crohn’s disease. Like was 9 and 10 years, respectively. Thus, the data clearly Helicobacter pylori, yersinia produces a urease that is known indicate that Yersinia spp can exist in lesions associated with to be essential in gastritis formation, a finding which could Crohn’s disease. Moreover, the large number of yersinia- explain the high frequency of focally enhanced gastritis in positive cases and the absence of specific clinical or Crohn’s disease. Y enterolitica produces a heat-shock protein histological features suggest that a large proportion of that might cause diarrhoea and autoimmune colitis in people with the disease have chronic contact with the animal models.30 The bacteria has been implicated in spondylarthropathies, which are known to be associated However, Yersinia spp might be present at only very low with Crohn’s disease. Finally, antibiotics active against rates in Crohn’s disease lesions, in view of the negative Yersinia spp such as ciprofloxacin have a limited effect in results from studies that use conventional methods for THE LANCET • Vol 362 • December 13, 2003 • www.thelancet.com For personal use. Only reproduce with permission from The Lancet publishing Group.
inhibition of the NF-␬B pathway in wild CARD15 mutated patients, inducing a variations have been reported for bothyersinia infections and relapses of to predispose people to Crohn’sdisease.2,3 CARD15 encodes for a protein that isable to induce NF-␬B activation (andpotentially apoptosis), after recog- inheritance at the CARD15 locus,which is characterised by a dosage Links between Crohn’s disease, the cold chain, and psychrotrophic bacteria effect of the Crohn’s disease muta-tions.2 However, the model does not accord with the increased NF-␬B activation noted in The three main mutations of the CARD15 gene associated with Crohn’s disease in white people (G702R, R908W, Of note is that CARD15 is also mutated in another and 1007FS) have probably occurred only recently in granulomatous condition characterised by an inflammation human history. There were large outbreaks of plague in of the eyes, joints, and skin but not the digestive tract— Europe that occurred periodically between the 6th and the Blau syndrome.38 In this mendelian dominant trait, a gain- 14th centuries.32 Thus, in the Middle Ages, CARD15 of-function model has been established with an excess of mutations might have provided carriers with a selective advantage during plague outbreaks and it could be To relate the two disorders because of a common postulated that mutation carriers have a more intense activation of the CARD15 pathway that induces an reaction against Yersinia spp.
inflammation with granulomas is tempting. Several With time, Y pestis disappeared—probably because it bacteria are usually able to inhibit NF-␬B activation was replaced in Europe by less pathogenic strains such as through virulence factors. We can, thus, postulate that Y pseudotuberculosis (and later Y enterocolitica), which Crohn’s disease mutations might induce not only a loss of probably induced a cross-immunisation to Y pestis in NF-␬B activation by the peptidoglycan, but also a loss of rodents carrying the plague agent.32 However, the intense NF-␬B inhibition in the presence of bacterial agents of reaction against Yersinia spp developed by mutation virulence. As a result, hosts carrying CARD15 mutations carriers might have become inadequate for these less should have a more intense reaction toward virulent virulent strains and the intestinal site of the infection, which is usually characterised by an immune tolerance.
The virulence factors of the Yersinia species are mainly Y enterocolitica was isolated for the first time in 1934 in carried by a plasmid (pYV) encoding for 12 secreted Yop the USA, 2 years after the complete description of the and Ysc proteins forming the secretory apparatus type III.
regional enteritis by Burrill B Crohn, Leon Ginzburg, and Some of the Yop proteins are involved in the inhibition of Gordon D Oppenheimer. In Europe, Y enterocolitica phagocytosis and NF-␬B activation. NF-␬B inhibition has infections increased after the 1960s—the same time as the been shown via YopJ/P, which is able to interfere with IKK Crohn’s disease outbreak.32 Because Crohn’s disease is a proteins.39,40 However, the mechanism of inhibition is not complex genetic trait, progressive population exposure to completely understood, Yop proteins might also interact Yersinia spp might have allowed more and more genetically physically with CARD15 to induce an inhibition of the predisposed people to be affected. Interestingly, the NF ␬B by an alternative pathway. This NF-␬B inhibition present stabilisation of the disease incidence in North could be due to YopJ/P or other molecules, including America and in some European countries suggests that YopM which is characterised, like CARD15, by leucine almost all people who are genetically at risk are now rich repeats (LRRs). A similar model can also be proposed THE LANCET • Vol 362 • December 13, 2003 • www.thelancet.com For personal use. Only reproduce with permission from The Lancet publishing Group.
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