Elsevier

Molecular Immunology

Volume 81, January 2017, Pages 76-84
Molecular Immunology

Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway

https://doi.org/10.1016/j.molimm.2016.11.019Get rights and content

Highlights

  • HuAECs reduced proliferation rate of patient-derived CD4+ T cells.

  • HuAECs reduced IL-2 released from patient-derived CD4+ T cells.

  • Overexpressed miR-101 reduced expression c-Rel and IL-2 in AKI patient-derived CD4+ T cells.

  • Overexpressed miR-101 decreased binding capacities of ‘c-Rel-NFκB’ complex on IL-2 promoter.

  • HuAECs stimulate miR-101 expression to inhibit AKI patient-derived CD4+ T cells activation.

Abstract

In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient-derived peripheral blood CD4+ T-cell activation and decreasing the ability of these cells to express and release IL-2. −Cell proliferation assay revealed that under the same culture conditions, activated AKI patient-derived CD4+ T cells had a significantly reduced proliferation rate when were co-cultured with HuAECs. And the level of IL-2 released was also significantly reduced. Western blot and qRT-PCR assays showed that the expression of c-Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR-101 in the CD4+ T cells co-cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR-101 could bind to a specific site in the c-Rel 3′ UTR and induce the post-transcriptional silencing of c-Rel. Subsequently, we over-expressed miR-101 in AKI patient-derived CD4+ T cells. The qRT-PCR and western blot assay results revealed that the expression of endogenous c-Rel was significantly reduced, while the ELISA results indicated that the level of IL-2 released was also significantly decreased. Finally, ChIP-PCR assay results showed that the miR-101-overexpressing CD4+ T-cell group and the HuAEC co-culture CD4+ T-cell group exhibited significantly decreased binding capacities between the ‘c-Rel-NFκB’ complex and the IL-2 gene promoter, and the transcriptional activity of IL-2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR-101 expression in AKI patient-derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR-101 target gene c-Rel and leading to a reduction in IL-2 expression and release.

Introduction

Acute kidney injury (AKI) refers to a sudden and sustained acute drop in kidney function. The manifestations include water, electrolyte and acid-base imbalances, azotemia, various systemic symptoms and an increase in serum creatinine to 0.5 mg/dl or more associated with little or no urine output (Allison, 2016, Lang et al., 2016, Parks and Liu, 2016, Singh, 2016, Yang and Xue, 2016, Vandenberghe et al., 2016). AKI can lead to multiple organ failure in patients, and in the immune system, AKI can cause increased white blood cell count, platelet dysfunction and persistent inflammation (Lang et al., 2016, Singh, 2016, Yang and Xue, 2016, Vandenberghe et al., 2016). In addition, in kidney, AKI not only leads to change of kidney hemodynamic and ischemia-reperfusion injury, but also formation of glomerular micro-thrombosis and kidney tubular obstruction. And, direct inflammatory factors can lead to dysfunction of vascular endothelial cells, as well as led to apoptosis or necrosis of glomerular and kidney tubular. The inflammatory cells, vascular endothelial cells and podocytes etc play such a role in AKI (Suh et al., 2013).

Human amniotic epithelial cells (HuAECs) are a group of epithelial cells with stem cell characteristics (Chen et al., 2013, Liu et al., 2012a, Liu et al., 2012b, Liu et al., 2010). HuAECs can express multiple stem-cell-specific markers, such as Oct-4, Sox-2, Nanog, REX-1 and Nestin (Chen et al., 2013, Liu et al., 2012a, Liu et al., 2012b, Liu et al., 2010). Thus, these cells can be induced to differentiate into mammalian trilaminar cells under certain conditions (Chen et al., 2013, Liu et al., 2012a, Liu et al., 2012b, Liu et al., 2010). Furthermore, HuAECs also express certain cell growth factors, such as integrin α6/β1, EGF, bFGF, NGF and leukemia inhibitory factor (LIF); therefore, the use of HuAECs as a feeder layer can effectively promote embryonic stem cell proliferation and inhibit excessive differentiation (Chen et al., 2013, Liu et al., 2012a, Liu et al., 2012b, Liu et al., 2010). Recent studies have found that HuAECs produce the soluble factors IL-10 and prostaglandin E2 (PGE2) and suppress the activation of natural killer (NK) cells and induced leukocytes, ultimately suppressing allogeneic or other related immune responses (Tan et al., 2015, Tee et al., 2013, Wang et al., 2006). However, HuAEC-mediated immune regulation is complex, and the in-depth mechanism is not clear.

Interleukin-2 (IL-2) is a protein that regulates leukocyte activation (Gong et al., 2014, Liou and Smith, 2011). IL-2 binds to the IL-2 receptor on the surface of T lymphocytes to activate immune responses (Gong et al., 2014, Liou and Smith, 2011). IL-2 is a member of the interleukin factor family, which includes interleukin signaling molecules such as IL-4, IL-7, IL-15, IL-19 and IL-21. The IL-2 protein consists of a complex with one 4-α helix and α, β, and γ chains (Gong et al., 2014, Liou and Smith, 2011). The promoter region of the IL-2 gene mediates transcriptional regulation of IL-2 mRNA and contains DNA recognition sequencesthat bind several transcription factors, including transcription factor nuclear factor (NF)-kappa B (Gong et al., 2014, Liou and Smith, 2011, Shindo et al., 2011, Visekruna et al., 2012). After stimulation, the free NFκB heterodimer is translocated into the nucleus where the NFκB subunits engage cognate κB enhancer elements and modulate the transcription of IL-2 or the CD25/IL-2 receptor alpha chain (Gong et al., 2014, Liou and Smith, 2011, Shindo et al., 2011). NFκB consists of a variety of canonical Rel-domain familydimers. Of these, only p50-p65 and p50-c-rel play an activating rolein IL-2 gene regulation (Gong et al., 2014, Gerondakis et al., 2012). However, c-rel is required for a correctly altered chromatin state across the IL-2 proximal promoter in CD3/CD28-activated primary T cells in pref-erence to other NFκB proteins (Gong et al., 2014, Liou and Smith, 2011).

microRNA (miRNA) is a special type of RNA, the length of which is approximately 21–23 base-pairs; there is no open reading frame, and miRNAs do not encode any proteins (Liu et al., 2012a, Liu et al., 2012b, Cheng et al., 2012, Xu et al., 2013). However, the pre-miRNAs are cleaved by the RNase III enzyme Dicer. Then, by binding to specific sites on target genes, miRNAs can induce AGO-mediated cleavage, ultimately silencing target gene expression (Liu et al., 2012a, Liu et al., 2012b, Cheng et al., 2012, Xu et al., 2013). miRNAs are important in vivo trans-acting factors with a widespread distribution in unicellular and multicellular organisms, and miRNA is involved in the proliferation, apoptosis and differentiation of cells, organism and tissue metabolism and development and a variety of physiological processes (Liu et al., 2012a, Liu et al., 2012b, Cheng et al., 2012, Xu et al., 2013). In this study, AKI patient-derived peripheral blood CD4+ T cells were first isolated and activated via in vitro stimulation. We then used molecular methods to determine whether the co-culture of these cells with HuAECs would weaken their subsequent activation. Additionally, we used c-Rel as a target and conducted an in-depth investigation into whether HuAECs could inhibit c-Rel and IL-2 expression by inducing the expression of endogenous miRNA in CD4+ T cells, ultimately suppressing CD4+ T cell activation.

Section snippets

Preparation of HuAECs

The HuAECs were isolated and enriched as previously described (Liu et al., 2012b). Briefly, the membrane was placed in a 250-mL flask containing DMEM medium and cut with a razor to yield 0.5–1.0 cm2 segments. The segments were digested with 0.25%trypsin-EDTA at 37 °C for 45 min. The resulting cell suspension were seeded in a six-well plate in DMEM medium supplemented with 10% fetal calf serum (PAA, Austria), penicillin (100U/mL), and glutamine (0.3 mg/ml), and incubated in a humidified tissue

HuAECs effectively inhibit the in vitro proliferation of AKI patient-derived CD4+ t cells

With 4 samples of AKI patient-derived CD4+ T cells (P) and 4 healthy human-derived CD4+ T cells (H), the flow cytometry (FCM) results showed that after in vitro co-stimulation with anti-CD3ε and anti-CD28 antibodies (Abs), the number of CD3+/CD4+ cells was significantly higher than the non-stimulated group (PBS group) and healthy human control group (Fig. 1). In order to estimate co-stimulation with anti-CD3ε Ab and anti-CD28 Ab whether influenced cell proliferation of AKI patient-derived CD4+

Discussion

The early onset of AKI may be associated with high levels of peripheral blood interleukins (IL-2, IL-6, IL-8, IL-18) as well as NFκB (Allison, 2016, Lang et al., 2016, Parks and Liu, 2016, Singh, 2016, Yang and Xue, 2016, Vandenberghe et al., 2016). Continued high levels of inflammatory factors can produce severe kidney damage. The timely removal of inflammatory factors has a positive protective effect on renal function (Allison, 2016, Lang et al., 2016, Parks and Liu, 2016, Singh, 2016, Yang

Acknowledgements

This work was supported by grant from Natural Science Foundation of China (No. 81271731), and Shanghai Municipal Health Bureau Fund (No. 20124y126), and Shanghai Natural Science Foundation of China (No. 16ZR1434000, 16ZR1437400), and project funded by China Postdoctoral Science Foundation (No. 2014M550250, 2015T80455), and Shanghai doctor degree point construction research key project (B201506), and Shanghai university of TCM foundation (2014YSN04). We declared no potential conflicts of

References (25)

  • J. Lang et al.

    Acute kidney injury

    JAAPA

    (2016)
  • G. Levy et al.

    Necrotizing enterocolitis as an adverse effect of recombinant interleukin-2 and Ch14.18 in maintenance therapy for high-risk neuroblastoma

    J. Pediatr. Hematol. Oncol.

    (2015)
  • Cited by (24)

    • Amniotic stem cells and their exosomes

      2021, Regenerative Nephrology
    View all citing articles on Scopus
    1

    These authors contributed equally to this work and shared the first authorship.

    View full text