Interleukin-33 regulates the endoplasmic reticulum stress of human myometrium via an influx of calcium during initiation of labor

  1. Li Chen
  2. Zhenzhen Song
  3. Xiaowan Cao
  4. Mingsong Fan
  5. Yan Zhou
  6. Guoying Zhang

  • Curated by eLife

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    Evaluation Summary:

    This paper addresses an interesting an important topic bearing on the initiation of labor at the end of pregnancy, invoking interleukin-33 in an alteration of Ca2+ homeostasis in uterine smooth muscle. The study implicated altered IL-33 expression in the third trimester of pregnancy in the endoplasmic reticular stress that might be involved in initiating labor.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Inflammation is currently recognized as one of the major causes of premature delivery. As a member of the interleukin-1β (IL-1β) family, interleukin-33 (IL-33) has been shown to be involved in normal pregnancy as well as a variety of pregnancy-related disorder. This study aims to investigate the potential function of IL-33 in uterine smooth muscle cells during labor.

Methods:

Myometrium samples from term pregnant (≥37 weeks gestation) women were either frozen or cells were isolated and cultured. Immunohistochemistry and western blotting were used to assess the distribution of IL-33. Cultured cells were incubated with lipopolysaccharide (LPS) to mimic inflammation as well as in the presence of 4μ8C (IRE1 inhibitor III) to block endoplasmic reticulum (ER) stress and BAPTA-AM, a calcium chelator.

Results:

LPS reduced the expression of nuclear IL-33 in a time-limited manner and induced ER stress. However, knockdown of IL-33 increased LPS-induced calcium concentration, ER stress and phosphorylation of nuclear factor kappa-B (NF-κB), and P38 mitogen-activated protein kinase (P38 MAPK). In addition, siRNA IL-33 further stimulates LPS enhanced cyclooxygenase-2 (COX-2) expression via NF-κB and p38 pathways. IL-33 expression was decreased in the nucleus with the onset of labor. LPS-induced ER stress and increased expression of the labor-associated gene, COX-2, as well as IL-6 and IL-8 in cultured myometrial cells. IL-33 also increased COX-2 expression, but after it was knocked down, the stimulating effect of LPS on calcium was enhanced. 4μ8C also inhibited the expression of COX-2 markedly. The expression of calcium channels on the membrane and intracellular free calcium ion were both increased which was accompanied by phosphorylated NF-κB and p38.

Conclusions:

These data suggest that IL-33 may be involved in the initiation of labor by leading to stress of the ER via an influx of calcium ions in human uterine smooth muscle cells.

Funding:

This study was supported by grants from the National Natural Science Foundation of China (No. 81300507).

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  1. Version published to 10.7554/elife.75072 on eLife
  2. eLife

    Evaluation Summary:

    This paper addresses an interesting an important topic bearing on the initiation of labor at the end of pregnancy, invoking interleukin-33 in an alteration of Ca2+ homeostasis in uterine smooth muscle. The study implicated altered IL-33 expression in the third trimester of pregnancy in the endoplasmic reticular stress that might be involved in initiating labor.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    The authors test a hypothesis that IL-33 plays a role in human parturition. It does so by (1) investigating changes in myometrial cell nuclear IL-33 expression during the third trimester of pregnancy. Their approach studies human myometrial cells, enhancing the clinical translatability of the present work. They demonstrate a reduced nuclear IL-33 staining with the onset of labour, further reduced by LPS. They implicate altered Ca2+ homeostasis in the actions of IL-33, and emerge with a model suggesting that IL-33 directly prevents excessive COX-2 expression in myometrial cells after LPS stimulation and it influences COX-2 expression by maintaining the severity of ER stress response.

  4. eLife

    Reviewer #2 (Public Review):

    Chen et al. investigated the potential function of IL-33 in uterine smooth muscle cells during labor. They used myometrial samples from term pregnant women and LPS to stimulate infection-induced inflammation. They found that similar to labor, LPS reduces nuclear IL-33 levels, induces ER stress, upregulates COX-2 expression via NFkappaB and MAPK p38 activation, and induces IL-8 and IL-6 release through calcium response. Transient knockdown of IL-33 appears to enhance LPS-induced ER stress, LPS-induced COX-2 expression, and NFkappaB and MAPK p38 activation. Knockdown of IL-33 also enhanced LPS-induced IL-8 and IL-6 release, and notably, IL-33 alone appears to increase IL-8 release. The data from this study supports the mechanism involved in LPS-driven inflammation, with some of these pathways being affected by reduction in IL-33.

    The conclusions of this paper are mostly supported by data, however, some aspects need to be clarified and extended.

    1. One of the aims was to investigate whether the presence of IL-33 regulates uterine smooth muscle contraction by examining changes in calcium response. Calcium response that leads to uterine contractions are often described as quick cyclic response. However, this study focuses on 6h timepoint post stimulation. The authors should demonstrate a time-course experiment to better understand the intracellular calcium changes in response to LPS treatment and/or IL-33 knockdown. Additionally, contractility experiments should be performed to determine whether IL-33 plays a role in uterine contractions.
    2. For any nuclear and cytosolic western blots, it is important to show any cross contamination.
    3. All transient knockdown of IL-33 should include controls to show sufficient reduction in IL-33 with siRNA transfection.
    4. There are multiple statements in the discussion which are not entirely supported by the data. The following statements need to be adjusted to clarify the effects of LPS or IL-33 alone on inflammatory pathways associated with labor onset, and the effects of IL-33 on LPS-driven pathways.

  5. eLife

    Reviewer #3 (Public Review):

    Employing primary myometrial cells, this study investigates molecular actions and cellular pathways regulated by interleukin-33 (IL33) a ubiquitous immune modulator that shapes type 1, type 2 and regulatory immune responses. The rational for this study is the notion that Inflammation is one of the major causes of premature delivery, a hypothesis that is not universally accepted as many investigators suggest that inflammation is a consequence of labour.

    The manuscript contains mostly appropriate methodologies, although there are some areas that at present are weak and require additional or more refined approaches.
    For example, studies on IL33 expression in human tissues have employed a small number of biopsies of limited potential. I would expect the author to use a substantive number of biopsies and calculate H-scores alongside other parameters of inflammatory pathways and develop various regression models.
    Without this crucial evidence once is left to wonder what is the rational for all follow-up studies described.
    Also, the authors need to be aware that modern approaches to quantitative PCR require multiple 'housekeeping genes and calculation of geometric means.

  6. Version published to 10.1101/2021.11.05.21265965v1 on medRxiv