Danilo Millimaggi

Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease

Background and aims: Epithelium derived interleukin (IL)-15 signalling via IL-15Rα is critical for the development, activation, and survival of intraepithelial lymphocytes (IEL). We aimed to better understand the IL-15 driven effects on IEL underlying mucosal damage and lymphomagenesis in coeliac disease (CD). Methods: Enterocytes, IEL, and lamina propria mononuclear cells (LPMC) were isolated from 46 patients with uncomplicated CD (25 untreated and 21 treated) and 22 controls. IL-15 and IL-15Rα expression were determined by immunoblotting. Secretion of IL-15, interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), and granzyme B into cell culture supernatants was assessed by ELISA. The ability of IL-15 to regulate IEL proliferation, perforin/granzyme dependent cytotoxicity, and apoptosis was tested by adding different combinations of IL-15, IL-15 blocking antibody, or chloroquine to IEL cultured alone or with Caco-2 cells as target. IL-15 mucosal levels were also determined by ELISA in five patients with complicated CD (two ulcerative jejunoileites, one refractory sprue, and two enteropathy associated T cell lymphomas) tested for T cell receptor γ chain clonality. Results: IL-15 was overexpressed in untreated CD enterocytes and LPMC, and in the mucosa of complicated CD patients and uncomplicated untreated CD patients, where its levels correlated with the degree of mucosal damage. Enterocytes from untreated, but not treated, CD patients and controls secreted IL-15. Untreated CD IEL, characterised by higher IL-15Rα expression, showed increased proliferation, production of IFN-γ and TNF-α, and perforin/granzyme dependent cytotoxicity, and a decreased propensity to apoptosis in response to IL-15. Conclusions: Our findings suggest that IL-15 plays a crucial role in the generation of epithelial damage in active CD. Its promotion of IEL survival in CD may predispose to the emergence of T cell clonal proliferations. Blocking IL-15, by suppressing uncontrolled IEL activation and survival, has the potential to provide new therapeutic tools to prevent tissue damage and lymphomagenesis in CD.

Defective mucosal T cell death is sustainably reverted by infliximab in a caspase dependent pathway in Crohn’s disease

Background and aims: To verify whether targeting defective mucosal T cell death underlies the sustained therapeutic benefit of infliximab in Crohn’s disease, we explored its in vivo proapoptotic effect after 10 weeks of treatment, and its in vitro killing activity on lamina propria T cells (LPT) and peripheral blood T cells (PBT), both isolated from Crohn’s disease patients. Methods: Endoscopic intestinal biopsies were collected from 10 Crohn’s disease patients (six steroid refractory and four fistulising) before and after three consecutive infusions of infliximab, administered at week 0, 2, and 6 in a single intravenous dose (5 mg/kg), and from 10 subjects who proved to have functional diarrhoea. Apoptosis was determined in vivo by TUNEL assay, and in vitro by fluorescein isothiocyanate-annexin V/propidium iodide staining on LPT and PBT from Crohn’s disease patients cultured with infliximab. The effect of the broad caspase inhibitor Z-VAD-FMK and the neutralising anti-Fas antibody ZB4 was tested in vitro on LPT and PBT treated with infliximab. Caspase-3 activity was determined by immunoblotting. Results: In Crohn’s disease patients, infliximab treatment induced a sustained LPT apoptosis, still evident four weeks after the last infusion. In vitro infliximab induced death of LPT from Crohn’s disease patients occurred via apoptosis rather than necrosis. LPT showed a higher susceptibility to infliximab induced apoptosis than PBT in Crohn’s disease patients. The signalling pathway underlying the restoration of infliximab induced LPT apoptosis occurred via the caspase pathway but not Fas-Fas ligand interaction in Crohn’s disease. Conclusions: These findings demonstrate that apoptosis is the major mechanism by which infliximab exerts its killing activity on LPT in Crohn’s disease. The sustained LPT proapoptotic action of infliximab, which extends far beyond its circulating half life, may be responsible for the sustained remission induced in Crohn’s disease patients by infliximab retreatment.

Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis whereas both populations are active in Fas based cytotoxicity in coeliac disease

BACKGROUND Lamina propria (LPLs) and intraepithelial (IELs) lymphocytes are markedly increased in coeliac mucosa, and are thought to play a crucial role in the generation of villous atrophy in coeliac disease (CD). However, the mechanisms by which they mediate the killing of enterocytes in this condition are still poorly characterised. AIM We investigated Fas mediated cytotoxicity and apoptosis of both LPLs and IELs, isolated from 10 untreated coeliac patients, 10 coeliac patients on a gluten free diet, and 10 biopsied controls. METHODS Fas and Fas ligand expression were assessed by flow cytometry and immunocytochemistry. Lymphocyte cytotoxicity against Fas expressing Jurkat cells was determined by the Jam test. The effect of the antagonist ZB4 anti-Fas antibody on apoptotic activity exerted by coeliac lymphocytes against enterocytes was analysed. Lymphocyte apoptosis was assessed by oligonucleosome ELISA. RESULTS LPLs and IELs showed increased apoptotic activity and higher levels of Fas ligand expression in untreated CD compared with treated CD patients and controls. Enterocyte apoptosis observed after coculturing coeliac lymphocytes and enterocytes in the presence of ZB4 antibody was reduced. In active CD, LPLs manifested increased apoptosis whereas IELs showed decreased apoptosis. CONCLUSIONS Our results support the involvement of the Fas/Fas ligand system in CD associated enterocyte apoptosis. Increased LPL apoptosis is likely to downregulate mucosal inflammation whereas decreased IEL apoptosis could be responsible for autoimmune and malignant complications of CD.

Apoptosis and peripheral blood lymphocyte depletion in coeliac disease

In coeliac disease (CD) immunological abnormalities are not confined to the small bowel and it has been suggested that changes in peripheral blood lymphocytes (PBL), such as lymphopenia and increased T‐cell activation, may predispose to malignant or autoimmune complications of this condition. In the light of the recent findings about the Fas–Fas ligand (FasL) system in regulating lymphocyte homeostasis, the aim of the present study was to investigate peripheral lymphocyte Fas‐mediated apoptosis in CD to establish whether the homeostatic role of apoptosis in peripheral T‐cell selection is maintained. Moreover, because a soluble form of Fas has been described to be functionally implicated in the Fas signalling system, suggesting a relationship between some disorders and soluble Fas function, we measured levels of soluble Fas in sera of coeliac patients and analysed the relationship between these levels and the proportions of apoptotic and Fas+ PBL to further explore the function of the Fas–FasL pathway in this condition. Finally, we evaluated whether the increased prevalence of anticardiolipin antibodies, recently described in CD, could be related to PBL apoptosis in this condition. We demonstrated an increased apoptosis and higher levels of Fas and FasL expression in PBL isolated from untreated coeliac patients when compared to treated coeliac patients and controls. In addition, low levels of soluble Fas and a significant positive correlation between anticardiolipin antibodies and PBL apoptosis were found in untreated CD. Then, our results showed an increased susceptibility of PBL to undergo Fas‐mediated apoptosis in active CD. This increased apoptosis could be responsible for both lymphopenia and immunogenic exposure of phospholipids with subsequent production of autoantibodies.

Isolation and characterization of circulating tissue transglutaminase- specific T cells in coeliac disease

Tissue transglutaminase (TG2) was identified as the humoral autoantigen in coeliac disease, but whether it can also serve as T cell autoantigen is still unknown. We aimed, therefore, to firstly explore the presence of TG2-specific T cells in peripheral blood of ten adult patients (four active, i.e. carrying both serological and histological features of the disease; four treated, i.e. with proven mucosal recovery and disappearance of specific antibodies after an adequate period of gluten free diet; and two potential coeliacs, i.e. carrying the serological stigmata of the disease, but not the intestinal lesions), and four healthy controls (two carrying the HLA-DQ2 haplotype of susceptibility to the disease), and secondly to carry out a detailed in vitro characterization of the isolated antigen-specific T cells. T cell lines were first established by means of weekly stimulation with human recombinant TG2 followed by generation of T cell clones through distribution of T cells on plates at one cell/well limiting dilution and further rounds of stimulation. Antigen specificity and HLA-DQ2 restriction were both assessed by evaluating the proliferative response to TG2 in the absence and presence of human sera blocking HLA-DQ2 molecules, after exclusion of impurities in the antigen preparation. Immune phenotyping of T cell clones was performed by flow cytometry, and the expression of IL-1β, IL-4, IL-6, IL-10, IL-12, TGF-β, IFN-γ and TNF-α was determined by ELISA assay on the supernatants of these clones. A total of 91 T cell clones were isolated from the three HLA-DQ2-positive, active patients, but none from the other patients and controls. The immune phenotyping showed that the majority of them (85.7%) were CD3/CD4+ and only a small percentage (14.3%) were CD3/CD8+, all carried the TCR αβ, and had a memory phenotype. The cytokine profile showed high levels of IFN-γ and IL-6 that, together with the absence of IL-4, placed these T cell clones in the T helper type 1-like category. Further in vitro analysis was carried out on 32/91 CD4+ clones and showed a specific and dose-dependent proliferative response towards TG2 and an HLA-DQ2 restriction. Finally, when incubating duodenal mucosal specimens of treated patients with the supernatant of TG2-specific T cell clones, characteristic disease lesions were found, indicating a role for TG2-specific cellular immune response in the pathogenesis of coeliac disease.