Reduction in regulatory T cells in preterm newborns is associated with necrotizing enterocolitis

Abstract

Background: Despite multifactorial pathogenesis, dysregulation of inflammatory immune response may play a crucial role in necrotizing enterocolitis (NEC). Regulatory T cells (Tregs) are involved in immune tolerance early in life. We aimed to investigate the predicting role of Tregs in developing NEC in neonates at high risk.

Methods: We studied six newborns with a diagnosis of NEC (cases) in comparison with 52 controls (without NEC). We further classified controls as neonates with feeding intolerance (FI) and neonates without it (FeedTol). The rate of female and male neonates (sex defined as a biological attribute) was similar. We analyzed the blood frequency of Tregs (not overall numbers) at three time points: 0-3 (T0), 7-10 (T1), and 27-30 (T2) days after birth by flow cytometry. Neonates' sex was defined based on the inspection of external genitalia at birth.

Results: We observed, at T0, a significantly lower frequency of Tregs in NEC cases (p < 0.001) compared with both FI (p < 0.01) and FeedTol 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).

Conclusion: 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.

Impact: Regulatory T cells may play a pivotal role in regulating the immune response in early life. Reduction of Tregs in early life could predispose preterm newborns to necrotizing enterocolitis. Early markers of necrotizing enterocolitis are still lacking. We demonstrated a predicting role of assessment of regulatory T cells in the diagnosis of this gastrointestinal emergency. Early identification of newborns at high risk of necrotizing enterocolitis through measurement of regulatory T cells may guide clinicians in the management of preterm newborns in order to reduce the development of this severe condition.

Fig. 1. Neonates who develop NEC are characterized by a lower frequency of Tregs and actTregs as compared to control neonates with (FI) or without (FeedTol) feeding intolerance, at early and late time points post birth.

a, b Representative plots (a) and cumulative analysis (b) showing the percentages of Tregs (CD127^low CD25^high) in gated CD4⁺ cells, estimated at different time points in peripheral blood of preterm neonates who develop NEC (n = 6) and in controls with feeding intolerance (FI, n = 19) or tolerance (FeedTol, n = 28). Blood samples were collected from each neonate at 0–3 (T0), 7–10 (T1), or 28–31 (T2) days post birth. c, d Representative plots (c) and cumulative analysis (d) showing the percentages of CD45RA^lowFOXP3^high actTregs (heavy thickness square) in gated Tregs (identified as CD127^low CD25^high CD4 T cells), in the same samples as above. Numbers indicate the percentages of each subset. *p < 0.05, **p < 0.01, by Mann–Whitney test, between subgroups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by Wilcoxon matched-paired test, between time points within each subgroup. In each analysis, only data from neonates whose samples were available at least at both T0 and T1 were included.

Fig. 2. In neonates with NEC, Tregs are less proliferative and actTregs express a lower level of FOXP3 compared to controls with feeding intolerance, at 7–10 days post birth.

a, b Representative plots (a) and cumulative analysis (b) showing the percentages of Ki67⁺ cells in gated CD127^low CD25^high Tregs from the peripheral blood of preterm neonates with NEC (n = 6), feeding intolerance (FI, n = 19) or feeding tolerance (FeedTol, n = 28), collected at 0–3 (T0), 7–10 (T1), or 28–31 (T2) days post birth. Numbers indicate the percentages of Ki67⁺ cells. c, d Representative histograms (c) and cumulative analysis (d) showing FOXP3 expression in actTreg population in the same samples as above. Numbers in the histogram plots indicate the gMFI (geometric mean fluorescence intensity) of FOXP3 expression in gated CD45RA^low FOXP3^high actTregs in the neonate subgroups as indicated in the legends. *p < 0.05, by Mann–Whitney test. In each analysis, only data from neonates whose samples were available at least at both T0 and T1 were included.