Dottorato in TECNOLOGIE BIOMEDICHE INNOVATIVE IN MEDICINA CLINICA

Titolo della tesi: Tregs function and gut microbiota diversity in preterm neonates at high risk of NEC: identification of early markers of disease.

Background: Preterm neonates suffer from intestinal immaturity which can impair the readiness of the gastro-intestinal tract to receive enteral nutrition, potentially leading to the occurence of the necrotizing enterocolitis (NEC). The early identification of those newborns at high risk of intestinal impairment is still challenging for neonatologists. Despite multifactorial pathogenesis, dysregulation of inflammatory immune response and early gut microbiota establishment may play a crucial role in NEC. Regulatory T cells (Tregs) are involved in immune tolerance early in life and gut microbiota plays a crucial role in intestinal maturation, in preterm newborns. We aimed to investigate the predicting role of Tregs in developing NEC in neonates at high risk and to verify the hypothesis that early gut microflora composition may influence intestinal maturation of preterm newborns. Methods: We studied six newborns with a diagnosis of NEC (cases) in comparison with 52 controls (without NEC). We further classified controls as Intolerant controls and Tolerant controls according with the degree of readiness of the gastro-intestinal tract to receive enteral nutrition. We analyzed the blood frequency of Tregs at three time points: 0–3 (T0), 7–10 (T1), and 27–30 (T2) days after birth by flow cytometry. To define the modalities of gut microbiota establishment and its relationship with intestinal maturation, we analyzed bacterial composition of stool samples, collected early in life, of 30 preterm newborns classified as Intolerant or Tolerant. We analyzed the alpha-diversity in terms of Pielou-evenness of gut microbiota. Results: We observed, at T0, a significantly lower frequency of Tregs in NEC cases (p < 0.001) compared with both Intolerant (p < 0.01) and Tolerant controls (p < 0.01). Multivariate analysis reported that the occurrence of NEC was independently influenced by Treg frequency at birth (ß 2.98; p = 0.039). The Pielou-evenness index was significantly (p < 0.05) increased in Intolerant compared with Tolerant newborns. Data corrected for cofounding variables confirmed that the occurrence of gut maturation was independently influenced by Pielou-evenness at birth (ß 2.48; p = 0.016). A lower bacterial diversity very early in life is associated with increased feeding tolerance in preterm newborns. The abundance analysis showed that neonates not ready to receive enteral nutrition show, after birth, increased abundance of Protebacteria, Lachnospiracae, Enterobacter and Acinetobacter. Conclusions: Tregs frequency and features in the peripheral blood of preterm neonates, early in life, may contribute to identifying neonates at high risk of developing NEC. A lower alpha-diversity evenness, in the first days of life, may facilitate the seeding of pioneer bacteria that in turn drive a healthy microbial colonization during neonatal life.