Alessandra Vitale

T300A Variant of Autophagy ATG16L1 Gene is Associated with Decreased Antigen Sampling and Processing by Dendritic Cells in Pediatric Crohn?s Disease

Background:The single-nucleotide polymorphism T300A of ATG16L1, a Crohns disease (CD)–associated gene, is responsible for decreased autophagy. This study aimed to investigate the effects of this single-nucleotide polymorphism on the uptake and processing of antigens by dendritic cells (DCs) and the interaction between DC and intestinal epithelium in pediatric patients with CD. Methods:Pediatric patients who homozygously carry either the protective (wild type, n = 7) or risk allele (risk, n = 13) of ATG16L1, as well as heterozygous patients (het, n = 13) were enrolled. The monocyte-derived DC were analyzed for phenotype, antigen sampling, and processing by flow cytometry, whereas the capability of DC to form transepithelial protrusions was determined by confocal microscopy. Results:DC generated from wild type patients showed higher bacteria sampling and antigen processing compared with risk patients. Additionally, after exposure to either bacteria particles or the antigen DQ-ovalbumin, wild type DC showed a significant increase in the expression of the HLA-DR and CD86 when compared with risk DC. Interestingly, also het patients showed an impairment in bacteria uptake and expression of activation marker when compared with the wild type. In the Caco2/DC coculture, the formation of transepithelial protrusions were less numerous in risk DC compared with wild type and the antigen uptake decreased. Conclusions:DC of pediatric patients with CD carrying the T300A allele showed a marked impairment of antigen uptake and processing and defective interactions between DC and intestinal epithelium. Collectively, our results suggest that an autophagy defect is associated with an impairment of intestinal innate immunity in pediatric CD.

Cytokine production profile in intestinal mucosa of paediatric inflammatory bowel disease

In the recent years, the incidence of inflammatory bowel disease (IBD) has dramatically increased in young subjects, however, the pathogenesis of paediatric IBD is poorly investigated. In this study we aimed to evaluate the cytokine pattern and the phenotype of cytokine producing cells in the intestinal mucosa of paediatric patients affected by Crohn’s disease (CD) or ulcerative colitis (UC) and of non-IBD healthy controls (HC). Cytokine (IL-15, TNF-α, INF-γ) production was analyzed at basal condition and after mitogen stimulation either intracellularly by flow cytometry or in intestinal cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). A higher frequency of enterocytes (EpCam+ cells) was observed in UC patients compared to CD or HC. An expansion of enterocytes producing IL-15 and TNF-α were found in IBD patients compared to HC. A marked expression of IL-15 in the intestinal epithelium of IBD patients was further confirmed by immunohistochemistry. Myeloid dendritic (CD11c+) cells producing TNF-α and INF-γ were increased in IBD biopsies. Unexpectedly, only after a strong mitogen stimulus, as phytohaemagglutinin, the frequency of CD3+ cells producing IFN-γ was increased in IBD compared to control intestinal mucosa. Interestingly, functional studies performed on organ cultures of intestinal biopsies with neutralizing anti-IL-15 monoclonal antibody showed a marked reduction of mononuclear cell activation, proliferation of crypt enterocytes, as well as a reduction of TNF-α release in organ culture supernatants. In conclusion, we found that in the gut mucosa of IBD children both enterocytes and dendritic cells produce proinflammatory cytokines. The over-expression of IL-15 by enterocytes in IBD intestine and the reduced IBD inflammation by IL-15 blockage suggests that this cytokine could be a therapeutic target in IBD.

Bifidobacteria Enhance Antigen Sampling and Processing by Dendritic Cells in Pediatric Inflammatory Bowel Disease.

Abstract:Bifidobacteria have been reported to reduce inflammation and contribute to intestinal homeostasis. However, the interaction between these bacteria and the gut immune system remains largely unknown. Because of the central role played by dendritic cells (DCs) in immune responses, we examined in vitro the effects of a Bifidobacteria mixture (probiotic) on DC functionality from children with inflammatory bowel disease. DCs obtained from peripheral blood monocytes of patients with Crohns disease (CD), ulcerative colitis, and noninflammatory bowel disease controls (HC) were incubated with fluorochrome-conjugated particles of Escherichia coli or DQ-Ovalbumin (DQ-OVA) after a pretreatment with the probiotic, to evaluate DC phenotype, antigen sampling and processing. Moreover, cell supernatants were collected to measure tumor necrosis factor alpha, interferon gamma, interleukin 17, and interleukin 10 production by enzyme-linked immunosorbent assay. DCs from CD children showed a higher bacteria particles uptake and DQ-OVA processing after incubation with the probiotic; in contrast, DC from both ulcerative colitis and HC showed no significant changes. Moreover, a marked tumor necrosis factor alpha release was observed in DC from CD after exposure to E. coli particles, whereas the probiotic did not affect the production of this proinflammatory cytokine. In conclusion, the Bifidobacteria significantly improved the antigen uptake and processing by DCs from patients with CD, which are known to present an impaired autophagic functionality, whereas, in DCs from ulcerative colitis and HC, no prominent effect of probiotic mixture was observed. This improvement of antigen sampling and processing could partially solve the impairment of intestinal innate immunity and reduce uncontrolled microorganism growth in the intestine of children with inflammatory bowel disease.

The DMT1 IVS4+44C>A polymorphism and the risk of iron deficiency anemia in children with celiac disease.

Background Iron deficiency anemia in celiac disease is related to impaired duodenal mucosal uptake, due to villous atrophy. Iron enters the enterocytes through an apical divalent metal transporter, DMT1. Different DMT1 transcripts have been identified, depending on the presence of an iron-responsive element that allows DMT1 up-regulation during iron starvation. An intronic DMT1 polymorphism, IVS4+44C>A, has been associated with metal toxicity, and the CC-carriers show high iron levels. Aims This study investigates the association between DMT1 IVS4+44C>A and anemia in a cohort of 387 Italian celiac children, and the functional role of the polymorphism. Methods and results By association analysis, we found that DMT1 IVS4+44-AA genotype confers a four-fold risk of developing anemia, despite of atrophy degree. By analysis of mRNA from gastroesophageal biopsies, we found that total DMT1 is significantly upregulated in presence of mild, but not severe, atrophy, independently from IVS4+44C>A variant, and in normal but not in atrophic CC-biopsies. Moreover, we found that A-allele is associated to preferential expression of the DMT1 transcripts lacking the iron-responsive element, thus limiting the DMT1 overexpression that normally occurs to respond to iron starvation. Discussion Possibly, the IVS4+44-AA-related dysregulation of the iron-induced changes in DMT1 expression is not able to impair iron absorption in physiological condition. However, if exacerbated by the concomitant massive loss of functional absorbing tissue paralleling worsened stages of villus atrophy, it might be ineffective in counteracting iron deficiency, despite of DMT1 overexpression. Conclusion We suggest, for the first time, that celiac disease may unmask the contribution of the DMT1 IVS4+44C>A polymorphism to the risk of anemia.