Digestive and Liver Disease - Volume 48 - Supplement 2 - February 2016

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Abstracts of the 22nd National Congress of Digestive Diseases / Digestive and Liver Disease 48S2 (2016) e67–e231 e105
in the regulation of gut immune homeostasis. Still, unrestrained
iNKT cell activity has been implicated in the pathogenesis of
ulcerative colitis (UC), one of the two major forms of Inflammatory
Bowel Diseases (IBD) in humans. Up to now, functional analyses of
human intestinal iNKT cells under steady state and in IBD have been
hampered by a low extraction rate from intestinal specimens, due
to the relative rarity of these cells in the gut mucosa with respect to
other immune cells.
Material and methods: To evaluate the function of intestinal iNKT
cells in IBD patients, we developed two novel methods to generate
iNKT cell lines and clones from human intestinal surgical specimens.
iNKT cells were isolated from lamina propria mononuclear cells
(LPMC) with a 9-color multiparametric FACS-sorting. For the
generation of iNKT cell lines, sorted cells were restimulated ex-
vivo with phyto-hemoagglutinin (PHA) and allogeneic irradiated
peripheral blood mononuclear cells (PBMC), which were used as
feeder cells in the presence of human interleukin (IL)-2. After 3 to
4 weeks iNKT cells could be used for phenotypical and functional
analyses. For the generation of human iNKT cell clones, we
adapted the protocol of cloning by limiting dilution used to clone
conventional T cells from peripheral blood Libero, T cell
protocols, Springer ed.). iNKT cells were sorted from LPMC with a
9-color multiparametric FACS-sorting before cloning. After 15 days
clones were harvested and checked for growth and specificity.
Results: With these two approaches we succeeded to generate 5
iNKT cell lines, 1 from healthy intestine, 1 from a CD patient, 2 from
UC patients and 1 from peripheral blood as internal control. iNKT
lineage was confirmed by the expression of specific iNKT surface
markers. Intestinal iNKT cells were able to produce both regulatory
and inflammatory cytokines, including IL13, IFNg, IL10 and IL17. A
series of in vitro antigen presentation assays with exogenous and
endogenous lipid antigens was performed. By the limiting dilution
protocol, 250 iNKT cell clones were generated from one UC patient.
50 clones out of 250 were further characterized phenotypically and
functionally. 20 out of 50 were used for in vitro antigen presentation
assays with exogenous and endogenous lipid antigens.
Conclusions: To our knowledge, this is the first report of the
generation of iNKT cell lines and clones from human surgical
intestinal specimens. iNKT cells lines and clones display a different
phenotypic and functional profile according to the tissue (PB vs gut)
and to the inflammatory status of the specimen (healthy vs IBD).
They secrete a different array of cytokines and are activated in vitro
by both endogenous and exogenous lipid antigens. These results
will help to shed light regarding the role of iNKT cells in intestinal
inflammation.
OC.09.6
PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA
AGONIST REDUCES THE DEVELOPMENT OF NECROTIZING
ENTEROCOLITIS IN A NEONATAL RAT MODEL
Corsini I.*2, Polvani S.1, Tarocchi M.1, Tempesti S.1, Marroncini G.1,
Generoso M.2, Bresci C.2, Gozzini E.2, Bianconi T.2, Galli A.1, Dani C.2
1Department of experimental and clinical biomedical sciences,
University of Florence, Florence, Italy, 2Neonatal Intensive Care Unit,
Careggi University Hospital, Florence, Italy
Background and aim: Necrotizing enterocolitis (NEC) is the most
common neonatal gastrointestinal emergency in preterm infants.
Intestinal mucosa and innate immunity play the major role in
the pathogenesis of NEC. Factors affecting innate immunity and
acting as inflammatory regulators, such as the nuclear peroxisome
proliferator-activated receptors (PPARs), could be crucial.
We hypothesized that the PPARg agonist Pioglitazone (PIO) might be
effective in preventing the development of NEC.
Material and methods: We studied a rat model in which NEC was
induced using the following protocol: newborn rats were formula
fed and stressed twice daily with hypoxia by breathing 100% nitrogen
gas in a closed chamber for 60 s, followed by 10 min exposure to 4°C
temperature.
Rat pups were divided into two groups: the treatment group (TG;
n=30) received enteral PIO (daily dose 10 mg/kg/die divided on six
aliquotes) and the control group (n=30) which was not. Animals were
sacrificed 96h after birth. NEC was diagnosed evaluating histological
ileum changes, and its severity was scored with a standard method.
Moreover, we measured levels of RNA of 6 cytokines: IL-4, IL-12, IL-
6, IL-10, INF-g and TNF-a using the qPCR technology. Deposition of
extracellular matrix in kidney was tested by Sirius Red staining.
Results: Body rats weights gradually decreased in both groups but
the decrease was higher in the CG starting from 48 h (p<0.01) to 96
h (p<0.01).
The occurrence of NEC was significantly higher (p<0.001) in the CG
(18/30; 60%) than in the TG (5/30; 16.7%) and was paralleled by a
higher NEC score (p<0.001) in the CG than in the TG. Pioglitazone
treatment significantly reduced the local intestinal inflammation:
pro-inflammatory IL-12 and INF-g mRNA levels were significantly
lower in the TG than in the CG (p=0.02 and 0.04, respectively);
conversely, anti-inflammatory IL-4 mRNA level was significantly
higher in the TG than in the CG (p=0.02). Finally, extracellular matrix
deposition in the kidney was reduced in TG rats compared to CG
rats, confirming the overall health improvement in treated rats and
the protecting effects of PIO.
Conclusions: Our results demonstrate for the first time that
administration of PIO reduces the incidence and severity of NEC in a
neonatal rat animal model of the disease.
OC.09.7
INHIBITION OF THE MITOCHONDRIAL ATP SYNTHASE PROMOTES
T CELL APOPTOSIS AND SUPPRESSES INTESTINAL INFLAMMATION
Monteleone I.*1, Franchi L. 2, Marafini I.1, Zorzi F.2, Di Fusco D.1,
Laudisi F.1, Dinallo V.1, Pallone F.1, Opipari A.2, Glick G.2,
Monteleone G.1
1Cattedra di Gastroenterologia, dipartimento di Medicina dei Sistemi,
Università Tor Vergata, Roma, Italy, 2University of Michigan, AnnArbor,
MI, United States
Background and aim: Defective T cell apoptosis occurs in
Crohn’s disease (CD) thus contributing to sustain the pathogenic
inflammatory response. Indeed, drugs that enhance T cell apoptosis
(i.e. anti-TNF antibodies and azathioprine) attenuate the CD-
associated tissue damaging immune response. We have recently
developed an inhibitor of the mitochondrial ATP synthase (ATPase),
which increases superoxide formation and consequently induces
caspase-dependent and independent T cell apoptosis. This study
was aimed at evaluating the ability of such a compound to induce
apoptosis in CD lamina propria (LP) T cells and to inhibit T cell-
mediated colitis in mice.
Material and methods: Apoptosis and markers of T cell
differentiation were analyzed in CD LP T cells either left untreated or
treated with the ATPase inhibitor by flow cytometry. The therapeutic
effect of the ATPase inhibitor was evaluated in mice with acute or
chronic 2,4,6-trinitrobenzene-sulfonic acid (TNBS)-induced colitis
or chronic adoptive T cell-transfer colitis.
Results: The ATPase inhibitor promoted CD LP T cell apoptosis
even in cells isolated from CD patients resistant to Infliximab. The
ATPase-driven apoptosis was associated with reduction of the
percentages of T cells expressing Th1/Th17-related markers and no
change in Foxp3-expressing regulatory T cells. Mice given orally the
ATPase inhibitor showed a marked increase of LP T cell death, down-