Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis

Abstract

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

Keywords: ERK1/2; Hofbauer cells; IL-6; JAK/STAT; NF-κB; leptin; placenta; preeclampsia.

Figure 1

Hofbauer cells display strong IL-6 and leptin receptor expression in placenta. Representative IL-6 and leptin receptor (LEPR) immunoreactivity (upper panels) in Hofbauer cells that are immunostained with CD68 (macrophage marker) in serial sections of control placentas (lower panels). Arrows indicate IL-6 or LEPR-expressing CD68-positive Hofbauer cells. Scale bar = 30 µm. Insets represent the higher magnification of relevant areas.

Figure 2

IL-6 and LEPR immunostaining in control and preeclamptic placentas. Representative images of IL-6 and LEPR immunostaining in preeclamptic (PE) and gestational aged-matched control placentas. Hofbauer cells-specific HSCORE analysis demonstrated significantly higher IL-6 (n = 5/group) and LEPR expression in PE placentas (n = 8) vs. gestational aged-matched controls (n = 7) (83.0 ± 20.9 vs. 25.0 ± 4.5 and 63.5 ± 18.6 vs. 10.9 ± 1.0, respectively). Arrows indicate IL-6 and LEPR-expressing Hofbauer cells. Bars represent median values, * p < 0.05 vs. control (C) specimens analyzed by Mann–Whitney Rank Sum test. Scale bar = 30 µm.

Figure 3

Leptin-induced IL-6 levels in primary cultured Hofbauer cells. (A) IL-6 mRNA levels in Hofbauer cells after 6 h r-leptin treatment (10, 100, and 1000 ng/mL) by qPCR. Data represent fold change as median values; * p < 0.05 vs. control (C) vehicle-treated group (n = 3) analyzed by Kruskal–Wallis One Way ANOVA on Ranks followed by Student–Newman–Keuls multiple comparison. (B) Leptin-induced secreted IL-6 levels (n = 4 from 3 different patients) in conditioned media supernatant obtained from Hofbauer cell culture after 24 h r-leptin treatment (10, 100, and 1000 ng/mL) by ELISA. Bars represent median values; * p < 0.05 vs. control (C) vehicle-treated group analyzed by Mann–Whitney Rank Sum test. (C) Immunoblot analysis of Hofbauer cell lysates after 24 h displaying reduced IL-6 expression in 100 and 1000 ng/mL leptin-treated cells. Bars represent mean ± SEM values from IL-6 protein levels after being normalized to β-actin, analyzed by Kruskal–Wallis One Way Analysis of Variance on Ranks, followed by the Student–Newman–Keuls multiple comparison method.

Figure 4

Leptin-mediated activation of STAT3, STAT5, NF-κB p65, and ERK1/2 signaling pathways in Hofbauer cells. Representative immunoblotting of Hofbauer cells treated with either control (C) or r-leptin (10, 100, 100 ng/mL) displays phosphorylated (ph) and total (t) levels of (A) STAT5; (B) STAT3; (C) p65 NF-κB; and (D) ERK1/2 MAPK, as well as β-actin. Graphs display immunoblot densitometry readings obtained from experimental incubations of cells for phosphorylated/total forms of each protein after being normalized to β-actin. Bars represent mean ± SEM, n = 3, * p < 0.05, ** p = 0.01 or *** p = 0.001 vs. vehicle-treated control (C) analyzed by t-test.

Figure 5

Leptin-induced IL-6 mRNA expression in Hofbauer cells is primarily mediated by ERK1/2 signaling pathway. IL-6 mRNA levels in Hofbauer cells treated with 100, 250, and 1000 ng/mL r-leptin ± specific inhibitors for STAT3 (A), STAT5 (B), p65 NF-κB (C), and ERK1/2 (D) signaling pathways. Bars represent mean ± SEM, n = 3, * p < 0.05 vs. control (Cnt), analyzed by Kruskal–Wallis One Way Analysis of Variance on Ranks followed by Student–Newman–Keuls multiple comparison method, # p < 0.05 leptin with inhibitor vs. without inhibitor (A,B), ¥ p ≤ 0.001 leptin with inhibitor vs. without inhibitor (C,D) analyzed by t-test.

Figure 6

Bidirectional crosstalk between Hofbauer cells and leptin-expressing trophoblasts induces IL-6 levels in HBCs in preeclamptic placenta. Preeclamptic (PE) placentas display increased leptin expression generated primarily from syncytiotrophoblasts, extravillous trophoblasts, as well as decidual stromal cells, and leptin receptor (LEPR) levels in Hofbauer cells (HBCs). Secreted leptin (blue circle) binding to LEPR activates ERK1/2 MAPK, p65 NF-κB, and STAT 3/5 signaling pathways to induce IL-6 (brown circle) expression in HBCs, which then may further promote leptin secretion in PE placentas, which causes a vicious cycle of leptin-triggered inflammation. Upon induction by excess leptin, HBCs may serve as one of the primary sources of IL-6 production in PE women.