Opposing roles of inter-α-trypsin inhibitor heavy chain 4 in recurrent pregnancy loss

Abstract

Background: The mechanism behind an increased risk of recurrent pregnancy loss (RPL) remains largely unknown. In our previous study, we identified that inter-α-trypsin inhibitor heavy chain 4 (ITI-H4) is highly expressed at a modified molecular weight of 36 kDa in serum derived from RPL patients. Yet, the precise molecular mechanism and pathways by which the short form of ITI-H4 carries out its function remain obscure.

Methods: Human sera and peripheral blood mononucleated cells (PBMCs) were collected from patients and normal controls to compare the expression levels of ITI-H4 and plasma kallikrein (KLKB1). Flow cytometric assay was performed to measure inflammatory markers in sera and culture supernatants. Furthermore, to investigate the functions of the two isoforms of ITI-H4, we performed migration, invasion, and proliferation assays.

Findings: In the current study, we showed that ITI-H4 as a biomarker of RPL could be regulated by KLKB1 through the IL-6 signaling cascade, indicating a novel regulatory system for inflammation in RPL. In addition, our study indicates that the two isoforms of ITI-H4 possess opposing functions on immune response, trophoblast invasion, and monocytes migration or proliferation.

Interpretation: The ITI-H4 (∆N⁶⁸⁸) might be a crucial inflammatory factor which contributes to the pathogenesis of RPL. Moreover, it is expected that this study would give some insights into potential functional mechanisms underlying RPL. FUND: This study was supported by the Ministry of Health & Welfare of the Republic of Korea (HI18C0378) through the Korea Health Industry Development Institute.

Keywords: Early pregnancy loss; ITI-H4; Immune tolerance; Migration and invasion; Plasma kallikrein.

Graphical abstract

Fig. 1

Elevated expression of ITI-H4 and KLKB1 in PBMCs of RPL patients. (A) Relative mRNA expression of ITI-H4 and KLKB1 in PBMCs of 10 RPL patients and 10 controls. (B) Relative mRNA expression of inflammatory factors (TNF-α, IL-6, IL-4, IL-10, IL-13, IL-1β, IFN-γ, and IL-2) in PBMCs of RPL patients and controls. Data are presented as a means ± SD. *, P < .05; **, P < .01. (C) KLKB1 activity assay identified an elevation of KLKB1 activity with the cleaved KLKB1 activity was also found to be reduced in patients with RPL when compared to controls. Data are presented as a means± SD. *, P < .05; **, P < .01.

Fig. 2

IL-6 regulates the expression of ITI-H4 and KLKB1 in human monocytes. (A) THP1 cells time-dependently treated with 20 ng/mL of IL-6 were lysed and the long isoform of ITI-H4 and the ITI-H4 (∆N⁶⁸⁸), and KLKB1 protein levels were detected by Western blotting. (B)- (D) Quantitative density of gel bands for ITI-H4 and KLKB1. Each experiment was repeated three times.

Fig. 3

ITI-H4 expression is induced by IL-6 in a STAT- and MAPK dependent manner. (A) THP1 cells were incubated for the indicated time points with IL-6. Then, ERK, STAT3, and JNK activation were assessed for total and phosphorylated signaling proteins by Western blot analysis. β-actin was used as a control. pERK, phospho-ERK; pSTAT3, phospho-STAT3; and pJNK, phospho-JNK. (B)-(D) the curves represent a means± SD of density of gel bands determined by Western blotting. (E) Cells were preincubated with SP600125 (a JNK inhibitor), PD98059 (an ERK inhibitor), or LLL12 (a STAT3 inhibitor) before treatment with IL-6. ITI-H4 expression is also shown. (F) ITI-H4 expression level was normalized to the one of β-actin. All statistical data are presented as a means (n = 3_, *, P < .05).

Fig. 4

Knockdown of ITI-H4 regulates the expressions of inflammatory factors in human placental choriocarcinoma cells. (A) Relative mRNA expression of ITI-H4. (B) Relative mRNA expression of inflammatory factors after ITI-H4 siRNA treatment in human placental choriocarcinoma cells. Data are presented as a means± SD (n = 3). *, P < .05.

Fig. 5

The related signaling pathway by which ITI-H4 and KLKB1 overexpression regulates the inflammatory factors in human placental choriocarcinoma cells. (A) Relative mRNA expression of inflammatory factors after transfection with the ITI-H4 (∆N⁶⁸⁸), the long isoform of ITI-H4, and ITI-H4 (∆BKD). (B) The ratios of Th1/Th2 cytokines are indexed by TNF-α/IL-10 and IFN-γ/IL-10, and data were presented as a means with SD. (C) Flow cytometric assay of cytokines expressions (IFN-γ, TNF-α, and IL-4) in the supernatants of transfected cell cultures. (D) Relative mRNA expression of inflammatory factors after KLKB1 and KLKB1 (S578A) transfection. (E) Relative mRNA expression of inflammatory factors after the long isoform of ITI-H4 and KLKB1 expression vector co-transfection, or the long isoform of ITI-H4 and KLKB1 (S578A) co-transfection. (F) Phosphorylation of STAT3, ERK, and JNK after the ITI-H4 (∆N⁶⁸⁸) overexpression by Western blotting. (G) Relative mRNA expression of inflammatory factors (TNF-α, IL-6, IL-1β, IFN-γ, and IL-2) after the ITI-H4 (∆N⁶⁸⁸) expression vector transfection and inhibitor treatments of specific pathways (SP600125, JNK phosphorylation inhibitor; PD98059, ERK phosphorylation inhibitor; and LLL12, STAT3 phosphorylation inhibitor) (n = 4).(H) Western blotting analysis of ERK, phosphorylated ERK, STAT3, and phosphorylated STAT3 expression in treated JEG-3 cells (n = 3). Data are presented as a means± SD. *, P < .05 versus the ITI-H4 (∆N⁶⁸⁸) expression vector transfection (Flag-ITI-H4 (∆N⁶⁸⁸). Data are presented as a means± SD. *, P < .05; **, P < .01; ***, P < .001.

Fig. 5

The related signaling pathway by which ITI-H4 and KLKB1 overexpression regulates the inflammatory factors in human placental choriocarcinoma cells. (A) Relative mRNA expression of inflammatory factors after transfection with the ITI-H4 (∆N⁶⁸⁸), the long isoform of ITI-H4, and ITI-H4 (∆BKD). (B) The ratios of Th1/Th2 cytokines are indexed by TNF-α/IL-10 and IFN-γ/IL-10, and data were presented as a means with SD. (C) Flow cytometric assay of cytokines expressions (IFN-γ, TNF-α, and IL-4) in the supernatants of transfected cell cultures. (D) Relative mRNA expression of inflammatory factors after KLKB1 and KLKB1 (S578A) transfection. (E) Relative mRNA expression of inflammatory factors after the long isoform of ITI-H4 and KLKB1 expression vector co-transfection, or the long isoform of ITI-H4 and KLKB1 (S578A) co-transfection. (F) Phosphorylation of STAT3, ERK, and JNK after the ITI-H4 (∆N⁶⁸⁸) overexpression by Western blotting. (G) Relative mRNA expression of inflammatory factors (TNF-α, IL-6, IL-1β, IFN-γ, and IL-2) after the ITI-H4 (∆N⁶⁸⁸) expression vector transfection and inhibitor treatments of specific pathways (SP600125, JNK phosphorylation inhibitor; PD98059, ERK phosphorylation inhibitor; and LLL12, STAT3 phosphorylation inhibitor) (n = 4).(H) Western blotting analysis of ERK, phosphorylated ERK, STAT3, and phosphorylated STAT3 expression in treated JEG-3 cells (n = 3). Data are presented as a means± SD. *, P < .05 versus the ITI-H4 (∆N⁶⁸⁸) expression vector transfection (Flag-ITI-H4 (∆N⁶⁸⁸). Data are presented as a means± SD. *, P < .05; **, P < .01; ***, P < .001.

Fig. 6

The ITI-H4 (∆N⁶⁸⁸) affects invasion of the human choriocarcinoma cells and human macrophage migration. (A) Invasion assay. JEG-3 cells transfected with respective constructs and kept in the medium containing G418 (400 μg/mL), seeded in Matrigel-coated invasion plates and incubated for 48 h. (B), (D) The invading cells on the underside of the membrane were enumerated by using an inverted microscope in 10 random fields (10 × 10). Experiments were performed in triplicate and the results are represented as a means of invasion cell numbers. n = 3, *, P < .05; **, P < .01; ns, not significant. (C) JEG-3 cells 48 h after transfection with siControl, siITI-H4, siKLKB1 or siITI-H4/siKLKB1, were analyzed using a Matrigel-coated Transwell. Cells successfully invaded into the Matrigel were quantified 48 h after plating. (E) The transwell assay showed that ITI-H4 overexpression at 36 h promoted human macrophage migration. (F) Data are presented as a means ± SD (N = 3). *, P < .05, **, P < .01, versus control vector transfection (Flag group).

Fig. 7

Cell proliferation assay and Co-culture with human choriocarcinoma cells and serum derived from RPL patients or normal groups. (A), (B) Human monocytic cells were transfected with respective constructs and cell proliferation was assessed by cell counting kit-8 assay over 72 h. A means ± SD of a triplicate experiment is shown. (C) Human monocytic cells were transfected with ITI-H4 or KLKB1 siRNA. At three different time points after transfection cell proliferation ratio was analyzed using live cell staining buffer (CCK-8, cell counting kit-8), then read using 450 nm filter. *, P < .05, **, P < .01, ***, P < .001. ns, not significant. Data were analyzed by a two-way analysis of variance, and significant differences were determined by Tukey-Kramer test (n = 3). (D) JEG-3 cells were cultured in DMEM supplemented with 5% FBS, 5% serum derived from RPL patients or normal groups, respectively. After incubation in 37 °C for 24 h, cell images were captured using electron microscopy, and, (E) the relative expressions of inflammatory cytokines (TNF-α, IL-4, IL-10, IL-2, IFN-γ, and IL-6) were measured. Data are presented as a means± SD (n = 3). (F) Schematic presentation of molecular mechanism for induced inflammation mediated by the increased expression of ITI-H4 (∆N⁶⁸⁸) in RPL. The long isoform of ITI-H4 is expected to be cleaved by KLKB1, and KLKB1 regulates ITI-H4, altering immune signaling factors, proliferation rates, invasion or macrophage migration. In RPL, ITI-H4 could elevate the expression of anti-inflammatory factors, while the ITI-H4 (∆N⁶⁸⁸) induced pro-inflammatory factors. Similarly, the long isoform of ITI-H4 and ITI-H4 (∆N⁶⁸⁸) possess opposing functions on immune tolerance, trophoblast invasion, and monocytes migration or proliferation.