Type 2 and type 3 innate lymphoid cells at the maternal-fetal interface: implications in preterm birth

Abstract

Background: Preterm birth (PTB) is one of the major causes of neonatal morbidity and mortality worldwide. It is commonly accepted that the act of giving birth is the final step in a proinflammatory signaling cascade, orchestrated by an intrauterine milieu coupled to hormonal cues. Consequently, the inflammatory process plays a pivotal role during the pathogenesis of human labor, both in term and preterm deliveries. The ability of innate lymphoid cells (ILCs) to act as pro-inflammatory mediators arose the interest to study their role in normal and pathological pregnancies. The aim of this work was to analyze the relative frequencies of ILCs subsets in pregnancy and the levels of IL-4, IL-17, IL-22, and IFN-γ as inflammatory mediators. Accordingly, we hypothesized that changes in the proportions of ILCs subpopulations could be related to preterm birth.

Methods: We analyzed 15 full-term delivery samples and six preterm delivery samples. In the full-term group (FTB) peripheral blood was taken during routine blood analysis, on 3 occasions: 1st, 2nd and 3rd trimester. After delivery, peripheral blood, cord blood and placenta were collected. In PTB group, peripheral blood samples were obtained on two occasions: before and 24 h after treatment with progesterone. We used flow cytometry to analyze ILCs in maternal peripheral blood, placenta, and cord blood samples. Maternal peripheral blood and cord blood samples were analyzed by enzyme-linked immunosorbent assay for IL-4, IL-17, IL-22, and IFN-γ plasma levels at the time of labor.

Results: We observed significantly increased relative frequencies of ILC2 and ILC3 in the decidua, as well as an increase of ILC2 in cord blood samples in PTB group, compared to FTB samples. We also found a decrease in IFN-γ in peripheral blood samples of the PTB group, suggesting a functional withdrawal. Additionally, IL-4, IL-17, IL-22 levels were similar in PTB and FTB groups, denoting a relevant role in mediating labor.

Conclusion: Our results suggest that ILC2 and ILC3 play a role in PTB by mediating an inflammatory response. Further work is necessary to evaluate the importance of ILCs in the regulation of labor.

Keywords: Inflammation; Innate immune response; Innate lymphoid cells; Preterm birth; Preterm labor.

Fig. 1

Graphic displaying the relative percentage of the different ILC populations in Peripheral Blood samples in pregnant women in the 1st (n = 24), 2nd (n = 18) and 3rd (n = 18)

Fig. 2

Graphic displaying the relative percentage of the different ILC populations in Peripheral Blood samples in the moment of labor in full term birth (FTB, n = 15) compared to preterm birth (PTB, n = 6)

Fig. 3

Graphic displaying the relative percentage of the different ILC populations in Peripheral Blood samples before (BPgr) or after the administration of Progesterone (24 h Pgr) in women who delivered PTB

Fig. 4

Decidual and cord blood ILCs in FTB and PTB. a Graphic displaying the relative percentage of the different ILC populations in full term birth (FTB, n = 15) compared to preterm birth (PTB, n = 6), in human decidua. b Graphic displaying the relative percentage of the different ILC populations in FTB (n = 15) compared to PTB (n = 5) in cord blood samples. Multiple t-student tests where used for statistical analysis with a 95% confidence interval, p-value * p < 0.05; ** p < 0.01 (two tailed)

Fig. 5

Enzyme-Linked Immunosorbent Assay (ELISA) in cord blood and maternal peripheral blood in FTB and PTB. a Graphic displaying IL-17 plasmatic concentrations in cord blood FTB (n = 15) and PTB (n = 5), as well as, plasmatic concentrations of IL-17 in maternal Peripheral Blood (PB) (n = 6). b Graphic displaying IL-22 plasmatic concentrations in cord blood FTB (n = 15) and PTB (n = 6), as well as, plasmatic concentrations of IL-22 in maternal Peripheral Blood (PB) (n = 6). c Graphic displaying Ifn-γ plasmatic concentrations in cord blood FTB (n = 15) and PTB (n = 6), as well as, plasmatic concentrations of Ifn-γ in maternal Peripheral Blood (PB). d Graphic displaying IL-4 plasmatic concentrations in cord blood FTB (n = 15) and PTB (n = 6), as well as, plasmatic concentrations of IL-4 in maternal Peripheral Blood (PB). Student’s t-tests were used for statistical analysis with a 95% confidence interval. A statistical significant decrease in Ifn-γ plasma concentration was found, in peripheral blood samples in women with PTB

Fig. 6

Gating strategy for identification of ILC3 subpopulations. a Identification of lymphocyte population. b Gating of CD45 + CD3-cells. c Selection Lin-CD127+ cells (d). isolating CD161+ cells. e Gating ILC3 cells as CRTH2- CD117+. f Discrimination between ILC3 NCR+ and ILC3 NCR- based on NKp44 expression (Data analyzed in FlowJo®)

Fig. 7

Full minus one (FMO) for CRTH2 (a) and FMO for CD161 (b)