LAS FINANZAS INTERNACIONALES

A number of the transgenic mice with abrogated NF-κB signalling have a defined gastrointestinal phenotype. Mice with constitutive deletion of NF-κB1 demonstrate increased colonic proliferation. They have longer colonic crypts in comparison to wild-type animals (31 cells per hemicrypt c.f. 22) and significantly longer proliferating zones, as shown by proliferating cell nuclear antigen (PCNA) and Bromodeoxyuridine (BrdU) immunohistochemistry (248). Mice with enterocyte specific deletion of IKK-γ develop microscopic foci of colitis within the first couple of weeks of postnatal life. These areas are associated with breakdown of mucosal integrity and translocation of bacteria into the lamina propria of the intestine, resulting in recruitment of neutrophils, dendritic cells and CD4+ T cells. Over time, this colitis progresses to affect the entire colon. This phenotype is recapitulated in mice with the intestine specific double knockout of IKK-α and IKK-β. This suggests that it is a truly NF-κB signalling derived effect of NEMO, rather than an effect of disturbing the IKK complex stoichiometry, and furthermore reveals a degree of redundancy within the IKK complex. Deletion of IKK-β in the intestine results in markedly reduced classical pathway NF-κB activity, whilst deletion of IKK-γ results in the complete abrogation of this signalling, presumably accounting for the more severe phenotype that is observed in animals with deletion of IKK-γ (229).

p100-Δc mice were generated by the targeted disruption of the NF-κB2 gene. A stop codon and the SV40 promoter were inserted at codon 451. This generates a constitutively active gene that produces truncated NF-κB2 which is functionally similar to the p52 protein. Animals with this transgene have a shortened life expectancy, with 50% dying by postnatal day 25. This early mortality was associated with markedly abnormal gastric morphology and histology. Stomachs of 2 week old mice were significantly smaller than wild-type animals, with a reduction in luminal contents and thickened antral mucosa and hyperkeratosis in the forestomach (256).

86 Unfortunately, cellular differentiation and corpus specific changes have not been published in connection with this model.

In addition to the baseline phenotypes described above, a number of animal models with abrogated NF-κB signalling show altered responses to a variety of gastrointestinal stimuli. Mice with abrogated classical pathway signalling through either intestinal epithelial cell specific deletion of IKKβ (227) or constitutive deletion of NF-κB1 (247) demonstrate increased susceptibility to γ-irradiation induced intestinal epithelial apoptosis. In mice with intestinal epithelial cell specific IKKβ deletion, this is observed in the colon, and is associated with an up-regulation of phosphorylated p53. In addition, unlike wild-type mice, radiation induced apoptosis could not be rescued by pre-treatment with lipopolysaccharide (227). In NF-κB1 null mice, increased apoptosis and lethality was observed: the LD50 at 7 days for wild- type mice was a dose of 13.12Gy, whilst for NF-κB1 null mice, the required dose was 7.75Gy (247). Similarly, abrogation of intestinal epithelial cell IKK β results in significantly worse pathology in response to instillation of Clostridium difficile toxin A than is seen in wild-type mice. These animals had increased mucosal fluid secretion and also had significantly more epithelial cell apoptosis than their littermate controls (240).

Classical pathway NF-κB signalling does not, however, universally protect the gastrointestinal mucosa from injury. In mice with intestinal epithelial cell specific deletion of IKKβ, acute dextran sodium sulphate induced colitis is exacerbated, but when these animals are crossed with IL10 null mice that develop spontaneous colitis (when housed in normal animal house conditions), the colitis that develops is unchanged. If animals with myeloid specific IKKβ deletion are crossed with the IL10 knockout mouse, the colitis that is induced is ameliorated (242). These observations suggest a protective role for intestinal epithelial NF-κB signalling in acute colitis, but a role in the perseverance of chronic colitis for myeloid derived classical pathway NF-κB signalling.

NF-κB deletion also has an impact on gut helminth infestations. Deletion of both NF- κB1 and NF-κB2 leads to persistence of infestation with Trichuris muris, whilst wild-

87 type mice and c-Rel null mice effectively clear such an acute infestation (251). The underlying cause for the failure to clear Trichuris in both NF-κB1 and NF-κB2 null mice is a defective Th2 response to the infection, however the defect in this response is more profound in NF-κB1 null than NF-κB2 null mice, as anti-IL-12 antibody therapy can restore a normal Th2 response and induce helminth expulsion in the NF-κB2 null mouse, whilst similar therapy fails in NF-κB1 null animals (251).

A further model of gastrointestinal disease that has been studied in these animals is an intestinal ischaemia-reperfusion model. Mice with intestine specific IKK-β deletion underwent laparotomy during which the superior mesenteric artery was clamped for 30 minutes before being released. Animals were administered fluid resuscitation and allowed to recover before being culled at 4 hours. In this model, wild-type animals developed a marked Th1 type inflammatory response as typified by elevated TNF-α transcription; they also developed multiple organ dysfunction, typified by massive inflammatory infiltrate into the lungs. In mice with intestine specific IKK-β deletion, the TNF-α response was significantly attenuated and pulmonary pathology was markedly less severe. However, in the intestine, wild-type animals developed only mild oedema, whilst in mice with intestine specific IKK-β deletion, there was severe epithelial sloughing and higher apoptotic indices (243). These data demonstrate both the role of local NF-κB signalling in rescuing the gastrointestinal phenotype, and also the contribution of systemic effects of such signalling to the multiple organ dysfunction syndrome.