Epithelial-to-Mesenchymal Transition

Epithelial-to-mesenchymal transition refers to a mechanism by which epithelial cells lose their essential epithelial-defining properties while gaining the qualities of mesenchymal cells. EMT, which is associated with the ability to migrate and to penetrate into adjacent layers, may be involved in the pathogenesis of CD fistulas. It is believed that the phenomenon of EMT may be a driving force behind the formation of CD-associated fistulas.

The phenomenon of EMT is found in most CD-associated fistulas. It is speculated that EMT, which promotes epithelial integrity and reestablishes the mucosal barrier, might be an attempt of the intestinal nonimmune cells to close the mucosal defects and might play an important role in the formation of Crohn’s fistula. This has been supported by the positive staining for markers of EMT in perianal, enterocutaneous, enteroenteric, and enterovesical fistulas. However, EMT may be a general histologic feature showed by CD and non-CD conditions. CD fistulas are associated with myofibroblast-like cells/transitional cells that express cytokeratin 8 and 20. Recent data also provide evidence for a role of Dickkopf-homolog-1 (DKK-1), an important factor in the regulation of cell migration, in the pathogenesis of CD-associated perianal fistulas. It is speculated that, in CD, the reduced ability of inflammation-modified myofibroblasts to close ulcers and deep tissue defects can lead to epithelial cells to undergo EMT. Therefore, EMT, which can be mediated by several factors, plays an important role in the formation of CD-related fistula/abscess.

Matrix Metalloproteinases

MMPs are a number of enzymes that degrade all components of the extracellular matrix, and increased MMP activity may lead to immune-mediated tissue injury including the pathological features seen in CD. In Crohn’s fistulas, strong MMP-3 expression is detected independent in mononuclear cells and fibroblasts of the stage of inflammation, suggesting that increased synthesis and release of MMP-3 play an important role in immune-mediated tissue injury in CD. Increased expression of MMP-9 is also found in granulocytes and fibroblasts in CD fistulas. MMP-3 and MMP-9 are markedly upregulated in Crohn’s fistulas and may contribute to fistula formation through the degradation of the extracellular matrix, activation of T cell and tumor necrosis factor (TNF)-α, and promoting extravasation of neutrophils in areas with acute inflammation. In addition, MMP-13 protein expression is detectable in lamina propria fibroblasts from untreated CD fistula but is almost absent in lamina propria fibroblasts from nonfistula. Other MMPs, such as MMP-1, MMP-7, and MMP-10, are only weakly expressed or not detectable around CD fistulas. Interestingly, activated MMP-2 was only detectable in CD fistulas. These observations imply a critical role for matrix remodeling enzymes in the pathogenesis of CD fistula.

Cytokines in Fistula Formation

Cytokine profiles differ in subpopulations of patients with CD fistulas. In patients with CD, elevated levels of serum TNF-α and interleukin (IL)-6 were found to be correlated with perianal fistulas, active intestinal CD without perianal manifestations, and perianal complications after restorative proctocolectomy. Upregulated expression of TNF-α and IL-12 was shown to be related to the presence of fistulizing complications in patients with CD. The increased expression in TNF-α and IL-12 as well as IL-6 in those tissue specimens implies the involvement of these cytokines in the pathogenesis of penetrating complications in CD. Clinically anti-TNF therapy has shown to be effective in reducing fistula drainage and in helping healing of the fistula in patients with CD.

IL-13 is detected in CD fistula tracts. While transforming growth factor (TGF)-β, a key mediator of EMT, is capable of inducing IL-13 secretion in patients with fistulizing CD, suggesting that TGF-β and IL-13 play a synergistic role in the pathogenesis of fistulas. The interaction between these cytokines can be associated with EMT, the increased expression of TGF-β, and the function of fibroblasts, leading to the formation of Crohn’s fistula/abscess. Indeed, blocking or inhibition of cytokines including TNF-α and IL-13 represents a novel therapeutic approach for fistula treatment in CD.

Genetic Factors in Fistula Formation

In recent years, more than 200 genetic polymorphisms have been found to be associated with increased risk for (or protect from) the development of CD. Association between certain genetic polymorphisms and fistulizing CD has been reported. The carriage of the variant alleles for PRDM1 (PR domain containing 1, with ZNF domain), ATG16L1 (autophagy-related 16-like 1), PRDM1, or any NOD2 variant is associated with a higher risk for penetrating CD, whereas the carriage of the IL-23R rs11465804 variant is related to a decreased risk for penetrating CD. Alleles for IL-23R, LOC441108, PRDM1, NOD2 polymorphisms were also found to be associated with increased risk for developing a fistulizing phenotype A C-allele at the CDKAL1 (CDK5 regulatory subunit–associated protein 1-like 1) rs6908425 variant, the NOD2 variant rs72796353, the TNFSF15 (tumor necrosis factor superfamily of proteins member 15) rs4574921 CC genotype, and the absence of NOD2 variants were independently associated with perianal fistulas. Various state-of-the-art technologies might allow for the identification of genetic-based treatment strategies for CD-associated fistula.

Intestinal Microbiota and Crohn’s Fistula

The literature on microbiota composition within fistula tracts and the potential role of intestinal microbiota in the development of fistula in CD is limited. It was previously reported that infection is not a major contribution to the persistence of fistulas. However, the important role for bacteria to be part of pathological processes leading to the formation of fistulas has been recognized. It is recently reported that the bacterial cell wall muramyl dipeptide can stimulate the expression of factors including TNF-α and TGF-β, which are associated with EMT and invasion in intestinal epithelial cells, suggesting that luminal bacteria may play an important role in the process of fistula formation via the onset of EMT. Indeed, this is supported by the fact that treatment with antibiotics is effective for fistula in a group of patients. Therefore the investigation in microbiota compositions and their role in the development of CD-associated fistula are warranted.

The Role of Stricture in Crohn’s Fistula

In most CD patients, fistula results from persistent mucosal and transmural inflammation and stricture. Indeed, common pathological features of Crohn’s fistula include chronic inflammation, a granulomatous reaction, and fibrosis. For the natural history of fistulizing CD, there is a proposed rule: “no inflammation, no stricture; no stricture, no fistula; and no fistula, no abscess.” However, the underlying mechanisms are less known. EMT plays an important role in tissue remodeling, tissue fibrosis, and wound repair. EMT, which can lead to tissue fibrosis and the subsequent pathological processes of intestinal stricture, can occur when there is a perpetual and chronic bowel inflammation. If a superficial tissue defect of the mucosa caused by inflammation and ulcer cannot be closed by intestinal fibroblasts, epithelial cells may migrate toward the defect and undergo EMT, which is associated with the formation of fistula in CD.

The Role of Mesentery in CD Fistula

Crohn’s disease is a chronic transmural and systemic disease, and its current theories in pathogenetic pathways are mainly based on the “outside-to-inside” model, that is, the sequence of mucosal, mesentery, and then extraintestinal manifestations. Recently, it is noted that mesenteric structures including lymphatics, fat, and nerves play a crucial role in the pathogenesis of CD, and an “inside-out” model is proposed. However, similar to the “outside-to-inside” model, this “inside-out” model may not explain whole pathogenesis of CD. Therefore the axis may be bidirectional. Once inflammation extends beyond the intestinal wall, an internal fistula and associated abscess can be formed.

The macroscopic features of the mesentery adjacent to the intestinal segments in CD include hypertrophied adipose tissue and mesenteric thickening. Adipose tissue, which is currently being recognized as an endocrine organ, plays an important role in the process of intestinal inflammation through secretion of adipokines and cytokines. Adiponectin is one of the adipokines produced by mesenteric adipose tissue, with its concentration in hypertrophied mesenteric adipose tissue in CD patients with an internal fistula being significantly lower than those without an internal fistula. Adiponectin is reported to have the antiinflammation ability. Indeed, TNF-α treatment of adipocytes is associated with an increase in adiponectin production. In CD with internal fistulas, low adiponectin concentrations may aggravate the inflammation and increase the risk for the formation internal fistula formation. The role of mesentery in Crohn’s fistula may be more than we have known, the exact mechanisms of adipose tissue, lymphatics, and nerves that cause a fistula in CD remain unknown, and further studies are needed to identify these mechanisms.