In contrast to the relatively weak genetic effects identified in the GWAS (Wellcome Trust Case Control Consortium, 2007
), a common major phenotypic abnormality becomes apparent in CD when the innate immune system is stressed in vivo. This is the result of a severely impaired acute inflammatory response in these individuals (Marks et al., 2006
). We have suggested that the intensity of the inflammatory response in the whole population follows a normal Gaussian distribution. Individuals at the extreme lower tail of this spectrum, as a consequence of the additive effect of many gene variants, are at risk of CD. NOD2, and potentially other systems, provide a compensatory boost to acute inflammation, which is protective in this setting (Marks and Segal, 2008
These studies provide the first demonstration in CD that there is a profound defect in the recruitment of neutrophils after the introduction of E. coli
into the tissues and that the subsequent clearance of these organisms is grossly delayed in this condition. This bacterium was used in these studies because it is the major aerobic component of the bowel flora and present in those regions of the gut affected by CD (Finegold, 1969
). E. coli
have been implicated in the pathogenesis of CD (Rhodes, 2007
), have been found within macrophages in Crohn's tissue (Liu et al., 1995
), and have also been cultured from draining mesenteric lymph nodes (Ambrose et al., 1984b
). Granulomas removed from CD tissue have been shown by PCR to contain E. coli
DNA (Ryan et al., 2004
), and an ileal CD E. coli
isolate (LF82) was capable of inducing granuloma formation in vitro (Meconi et al., 2007
). In addition, we had previously shown that there was an important abnormality of acute inflammation in response to E. coli
in CD (Marks et al., 2006
The observed abnormalities in CD did not occur as a consequence of the inflammatory process, drug therapy, or bowel ulceration. All the patients were in remission and most were not on therapy, with a minority receiving 5-aminosalicylic acid–containing drugs. Bacterial clearance was normal in the control group of subjects with UC, who were also in remission and on 5-aminosalicylic acid or no treatment.
The rate at which neutrophils accumulate at sites of bacterial ingress into the tissues is crucial to the final outcome. It has previously been demonstrated that in a three-dimensional tissue matrix, bacterial phagocytosis is directly related to neutrophil numbers (Li et al., 2004
). In the absence of adequate neutrophil recruitment, monocytes and macrophages might function in a containing role, such that material remaining after inoculation is surrounded and only slowly degraded (Issekutz et al., 1981
). This is supported by the recovery of bacterial products from Crohn's granulomata (Ryan et al., 2004
The consequence of the delayed neutrophil accumulation is that foreign fecal material is not cleared adequately from the tissues, leading to granulomatous inflammation. Congenital monogenic innate immunodeficiencies, in which neutrophil dysfunction predominates, frequently manifest bowel inflammation that is indistinguishable from CD (Rahman et al., 2008
). These disorders are also associated with major defects in the clearance of foreign material caused by failure of neutrophils to accumulate in sufficient numbers (congenital neutropenias and leukocyte adhesion deficiency), poor digestion within excessively acidic phagolysosomes (chronic granulomatous disease and glycogen storage disease-1b), and impaired vesicle trafficking and fusion of the granules and phagosomes (Chediak-Higashi and Hermansky-Pudlak syndromes). The genes underlying these disorders have not been detected by GWAS of CD because of their extreme rarity.
In the absence of a monogenic lesion in the immune system, CD patients might also be unduly susceptible to bacterial infection, although the necessary large-scale studies have not been performed (Kyle, 1980
; Ambrose et al., 1984a
; Kaplan et al., 2007
). It becomes more difficult to assess the incidence of disease-specific infection once a diagnosis of CD has been made because of confounding factors including surgery, malnutrition, immunosuppressant therapy, and bowel-related complications such as abscesses and fistulae. Another consideration is bacterial load. Most acute infections originate from a small number of inoculating organisms, which even the dampened immunity in CD appears to be able to control. Our findings of the relationship between bacterial dose and clearance are important in this respect because they demonstrate that CD patients can deal efficiently with small numbers of organisms in the tissues but that the clearance mechanisms are overwhelmed by a large bolus of bacteria. CD occurs almost exclusively in the terminal ileum and colon, which respectively contain ~108
bacteria/ml, higher than the 105
bacteria/ml or less in other regions of the small bowel (Farthing, 2004
). Reflux through the ileocaecal valve carries caecal contents into the terminal ileum, elevating the microbial levels in this region (Malbert, 2005
). After surgery, recurrence is seen in ~80% of cases, and where small bowel has been resected, recurrence generally occurs in the small bowel just proximal to the anastomosis. This indicates that recurrence is highly dependent on the proximity to the large bowel contents rather than a specific abnormality associated with the terminal ileum (Cameron et al., 1992
). Therefore, the large bowel and adjacent ileum provide a unique environment in the body that contains a massive bacterial load, which can gain instant access to the tissues if the mucosal barrier is breeched.
The detrimental effect of fecal content in the tissue might not primarily result from the infectious nature of the organisms, as many are not particularly virulent, but by the quantity of organic material that must be removed before resolution can occur. Difficulty in clearing exogenous organic material could also account for the false-positive results in CD to Kveim tests, which were conducted to diagnose sarcoidosis by injecting a preparation of the spleen of a patient into the skin, which was subsequently examined for a granulomatous response (Mitchell, 1971
Clearly, the retention of fecal material in the tissues of the wall of the bowel will have major local and systemic inflammatory, immunological, and constitutional consequences. Many of the immunological studies conducted on Crohn's patients, such as those implicating adaptive immunity in local tissue damage (Xavier and Podolsky, 2007
), might be measuring secondary immunological responses to this foreign material.
We then turned our attention to identifying the mechanism underlying impaired neutrophil accumulation. CD is caused by the complex interplay of many genes coupled with bacterial exposure, which makes it difficult, if not impossible, to identify specific molecular lesions common to these patients, and GWASs clearly indicate that there are no dominant causative genes. However, the characteristic pathological features of the condition are suggestive of a common cellular pathology produced by the various additive molecular aetiologies. The neutrophils themselves appear functionally normal (Moráin et al., 1981
), and their diminished migration to acute inflammatory sites in the skin or bowel has been attributed to defective local cytokine production (Marks et al., 2006
). These mediators are primarily secreted by resident tissue macrophages (Medzhitov, 2008
), which, in the bowel, are derived and continually replenished from peripheral blood monocytes (Smythies et al., 2006
). We therefore measured the secretion of a panel of inflammatory cytokines and chemokines by macrophages in response to stimulation with E. coli
or TLR ligands and observed the production of dramatically low levels of proinflammatory cytokines in cells from CD patients. In our previous study, we found local levels of IL-8 and IL-1β to be low after trauma to the skin and bowel (Marks et al., 2006
). In the current study, we have found that macrophages secrete normal amounts of IL-8 after stimulation with bacteria. This discrepancy between tissue levels and isolated macrophages in response to E. coli
could lie in the type of cells producing IL-8 in skin windows and bowel biopsies, and/or in a different agonist. Neutrophils themselves produce copious amounts of IL-8 and IL-1β, so the lower levels in skin windows and bowel biopsies in CD are consistent with the reduced recruitment at these sites (Fujishima et al., 1993
; Greten et al., 2007
). In the study by Marks et al. (2006)
, the release of IL-8 by macrophages from CD patients was diminished after stimulation with wound fluid, TNF, and C5a but unaltered after stimulation with the TLR4 ligand lipopolysaccaride. IL-8 release is normal after bacterial stimulation (whole E. coli
as well as TLR2 stimulation), so the lower levels in the in vivo study could be the result of activation of macrophages by nonmicrobial agonists.
To explain the cause of impaired cytokine secretion by macrophages from CD patients, we examined the profiles of RNA expression against a chip containing fragments of genes representing the whole human transcriptome. The first important observation was that macrophages from patients with colonic or ileal CD and UC exhibited distinct expression profiles when compared with HC, identifying these as three separate disease clusters. Second, levels of mRNA encoding proinflammatory cytokines were normal in unstimulated and HkEc stimulated macrophages, providing evidence that the defective secretion was not at the level of gene transcription.
A possible mechanism responsible for diminished proinflammatory cytokine release was provided by examining the abnormally expressed genes common to both types of CD after HkEc stimulation. Approximately half of the genes had some association with cellular secretory systems, suggesting that impaired macrophage secretion is responsible for the diminished proinflammatory cytokine release. The failure of a secretory system would provide a generic mechanism for the impaired release of several functionally related cytokines. Hermansky-Pudlak syndrome and Chediak-Higashi disease, both of which are associated with noninfectious granulomatous enterocolitis, are the result of primary abnormalities in the cytoskeletal transport of vesicles (Rahman et al., 2008
). These findings are particularly compelling given the recent descriptions of variants in autophagy genes which are associated with CD susceptibility (Hampe et al., 2007
; Parkes et al., 2007
; Rioux et al., 2007
). Autophagy involves vesicle trafficking in the formation of double-membrane vesicles that deliver cytoplasmic material to the lysosomes (Levine and Deretic, 2007
). Additionally, autophagy is required to kill and remove intracellular organisms in macrophages, so its dysfunction is consistent with the theme we have developed: that CD results from a failure to digest and remove microbial and other foreign material.
Interestingly, there was no other direct relationship between the collections of genes identified in this study in macrophages and the polymorphisms highlighted by the previous GWAS (Wellcome Trust Case Control Consortium, 2007
; Barrett et al., 2008
), with the exception of HLA-G and SPTLC2, which were up-regulated and down-regulated, respectively, in colonic CD (Table S3). This is not entirely unexpected because association studies interrogate the whole genome in a nonselective manner, whereas we have focused on RNA expression in a single cell type, which plays a pivotal role in inflammation, in resting cells and those after activation with a naturally occurring agonist.
Several microarray expression studies have previously been performed in CD and UC. However, their experimental approach differed greatly from ours, as did the results they obtained. This is not surprising in view of the very diverse cell populations and variable states of stimulation in their samples. Those on blood cells were performed on freshly isolated unstimulated mixed cell populations consisting of lymphocytes and monocytes with granulocyte contamination (Mannick et al., 2004
; Burczynski et al., 2006
). The majority used bowel biopsies, in which a wide spectrum of cells is present in noninflamed tissue. These are further supplemented by innate and adaptive immune cells when inflammation is present (Lawrance et al., 2001
; Costello et al., 2005
; Hughes, 2005
To understand the failure of macrophages to secrete normal quantities of proinflammatory cytokines in CD, we used TNF as a model, because this molecule has been extensively studied and experimental tools are readily available. We found that gene transcription and mRNA stability are normal in CD macrophages. Bref-A prevents vesicular movement between the ER and the Golgi, resulting in accumulation of proteins within the former. Stimulation in the presence of Bref-A revealed normal cytokine synthesis by CD macrophages. Normal synthesis coupled with deficient secretion and low intracellular levels of the cytokines indicate accelerated degradation. The accumulation of normal amounts of TNF and other proinflammatory cytokines after the addition of lysosomal inhibitors to CD cells indicates that this aberrant breakdown occurs in the lysosomal compartment.
The observation that macrophage secretion of TNF in CD is abnormally low might at first seem paradoxical, given the therapeutic efficacy of TNF blockade in many of these patients (van Deventer, 1999
). However, we are comparing two temporally distinct events, acute inflammation over a time course of hours with chronic inflammation over weeks to months. The acute inflammatory response, dependent on the acute release of proinflammatory cytokines, is important for the clearance of bacteria and fecal material from the tissues, which takes place over a few hours. The lack of TNF after the administration of therapeutic antagonists for the treatment of other chronic inflammatory diseases has been reported to precipitate the development of CD (Charach et al., 2008
; Yazisiz et al., 2008
Failure to clear foreign material from the tissues induces a chronic granulomatous reaction around the retained organic material over a much longer time frame. The accumulation of macrophages and T lymphocytes in these granulomata leads to local tissue damage and constitutional symptoms through the sustained secretion of cytokines (Bazzoni and Beutler, 1996
). Even if the net production of cytokines by each cell were lower than normal, the overall number of cells is so great that damaging concentrations are produced. In this setting, drugs directed against TNF can prove beneficial by direct cytokine blockade, by inducing leukocyte apoptosis, and by stimulating the effect of Treg
cells, which suppress inflammation (Wong et al., 2008
). A clear example of the dichotomous effect of TNF is provided by the dextran sodium sulfate (DSS) bowel inflammation model in TNF knock-out mice. Contrary to initial expectations, mice deficient in this cytokine are more susceptible to the induction of bowel inflammation by DSS, whereas TNF inhibition is effective in ameliorating established DSS colitis in wild-type animals (Naito et al., 2003
The relationship of impaired cytokine secretion to disordered packaging and vesicle transport, rather than defects in their production or stability, provides a novel insight into the mechanisms underlying the pathogenesis of CD. Similar mechanisms could lead to abnormalities in other relevant cell types, such as the Paneth cell (Cadwell et al., 2008
). A basic abnormality in macrophage biology could also explain extraintestinal manifestations of CD, such as arthritis, and lesions in the eyes, skin, lungs, and other tissues (Ephgrave, 2007
). There may be many other diseases that present with exuberant granulomatous inflammation as a result of an underlying failure of acute inflammation and innate immunity, leading to defective clearance of the initiating agent.