TLR3-Induced Placental miR-210 Down-Regulates the STAT6/Interleukin-4 Pathway

Several clinical studies have reported increased placental miR-210 expression in women with PE compared to normotensive women, but whether miR-210 plays a role in the etiology of PE is unknown. We reported that activation of TLR3 produces the PE-like symptoms of hypertension, endothelial dysfunction, and proteinuria in mice only when pregnant, but whether TLR3 activation in pregnant mice and human cytotrophoblasts (CTBs) increases miR-210 and modulates its targets related to inflammation are unknown. Placental miR-210 levels were increased significantly in pregnant mice treated with the TLR3 agonist poly I:C (P-PIC). Both HIF-1α and NF-κBp50, known to bind the miR-210 promoter and induce its expression, were also increased significantly in placentas of P-PIC mice. Target identification algorithms and gene ontology predicted STAT6 as an inflammation-related target of miR-210 and STAT6 was decreased significantly in placentas of P-PIC mice. IL-4, which is regulated by STAT6 and increases during normotensive pregnancy, failed to increase in serum of P-PIC mice. P-PIC TLR3 KO mice did not develop hypertension and placental HIF-1α, NF-κBp50, miR-210, STAT6, and IL-4 levels were unchanged. To determine the placental etiology, treatment of human CTBs with poly I:C significantly increased HIF-1α, NF-κBp50, and miR-210 levels and decreased STAT6 and IL-4 levels. Overexpression of miR-210 in CTBs decreased STAT6 and IL-4 while inhibition of miR-210 increased STAT6 and IL-4. These findings demonstrate that TLR3 activation induces placental miR-210 via HIF-1α and NF-κBp50 leading to decreased STAT6 and IL-4 levels and this may contribute to the development of PE

Placental Toll-Like Receptor 3 and Toll-Like Receptor 7/8 Activation Contributes to Preeclampsia in Humans and Mice

Preeclampsia (PE) is a pregnancy-specific hypertensive syndrome characterized by excessive maternal immune system activation, inflammation, and endothelial dysfunction. Toll-like receptor (TLR) 3 activation by double-stranded RNA (dsRNA) and TLR7/8 activation by single-stranded RNA (ssRNA) expressed by viruses and/or released from necrotic cells initiates a pro-inflammatory immune response; however it is unknown whether viral/endogenous RNA is a key initiating signal that contributes to the development of PE. We hypothesized that TLR3/7/8 activation will be evident in placentas of women with PE, and sufficient to induce PE-like symptoms in mice. Placental immunoreactivity and mRNA levels of TLR3, TLR7, and TLR8 were increased significantly in women with PE compared to normotensive women. Treatment of human trophoblasts with the TLR3 agonist polyinosine-polycytidylic acid (poly I:C), the TLR7-specific agonist imiquimod (R-837), or the TLR7/8 agonist CLO97 significantly increased TLR3/7/8 levels. Treatment of mice with poly I:C, R-837, or CLO97 caused pregnancy-dependent hypertension, endothelial dysfunction, splenomegaly, and placental inflammation. These data demonstrate that RNA-mediated activation of TLR3 and TLR7/8 plays a key role in the development of PE

HIF-1α and NF-κB induced miR-210 expression in hypertensive P-PIC mice.

<p>A. Total RNA (including small RNAs) was isolated from placentas of both P and P-PIC mice at gestational day 18. miR-210 expression was subsequently determined by qRT-PCR. qRT–PCR of snoRNA 142 was used for normalization. Results are expressed as mean+SEM for 3 independent experiments. *<i>P</i><0.05 by Student’s t-test. B. Systolic blood pressures in P and P-PIC mice were measured at gestational days 0, 13, and 17 via tail-cuff plethysmography. The results are expressed as mean+SEM with 8 mice in each group. *<i>P</i><0.05 vs. P. C and D. Immunoblot analyses using anti-HIF-1α and anti-NF-κB antibodies on cell lysates prepared from placentas of P and P-PIC mice at gestational day 18. β-actin was used as a loading control. The first lane in both immunoblots indicate the molecular weight marker. Signals from 3 independent experiments were quantified and expressed as a percentage of P. The results are expressed as mean+SEM for percentage of P for 3 independent experiments. *<i>P</i><0.05 vs. P.</p

No significant change in placental HIF-1α, NF-κB, miR-210, STAT6, and IL-4 levels in TLR3 KO P-PIC mice.

<p>A and B. Placental cell lysates from both P TLR3 KO and P-PIC TLR3 KO mice at gestational day 18 were subjected to immunoblot analyses using anti-HIF-1α and anti-NF-κB antibodies. No significant difference was noted in HIF-1α and NF-κB levels between P TLR3 KO and P-PIC TLR3 KO mice. The first lane in the immunoblot A indicates the molecular weight marker. C. Placental miR-210 levels in both P TLR3 KO and P-PIC TLR3 KO mice were determined by qRT-PCR. D. No difference was noted in placental STAT6 levels between P TLR3 KO and P-PIC TLR3 KO mice by immunoblot analysis. The first lane indicates the molecular weight marker. Because we did not observe any significant difference in STAT6 levels between WT P and TLR3 KO P mice indicating that deficiency of TLR3 in mice did not alter baseline STAT6 levels we did not include this data in Fig. 3. E. IL-4 mRNA levels were determined by qRT–PCR from placentas of both P TLR3 KO and P-PIC TLR3 KO at gestational day 18. GAPDH was used for normalization. Results are expressed as mean+SEM for 3 independent experiments. *<i>P</i><0.05 vs. P TLR3 KO.</p

Poly I:C increased HIF-1α and NF-κB which induced miR-210 leading to decreased STAT6 and IL-4 in human CTBs.

<p>A and B. CTB cells were treated with poly I:C (2 ug/ml) for 6, 24, or 48 hrs. Cell lysates were isolated and immunoblotting was performed using anti-HIF-1α and anti-NF-κB antibodies. Results are expressed as mean+SEM for 3 independent experiments. The first lane in the immunoblots in A and B indicate the molecular weight marker. C. Under the same conditions as above, miR-210 expression increased following TLR3 activation with the peak induction seen at 48 hrs as determined by qRT-PCR. D. Poly I:C treatment for 48 hrs showed the strongest down-regulation of STAT6 as determined by immunoblotting. The first and last lanes in the immunoblot indicate the molecular weight marker. E. Relative fold-change in IL-4 levels following poly I:C treatment in CTB cells. Results are expressed as mean+SEM and *<i>P</i><0.05 vs. P.</p

Placental STAT6 and IL-4 levels are decreased significantly in P-PIC mice.

<p>A. Schematic of human <i>STAT6</i> 3′-UTR sequence targeted by miR-210. The predicted seed regions by both TargetScan and miRanda algorithms are indicated. B. Placental cell lysates from both P and P-PIC mice at gestational day 18 were subjected to immunoblot analyses using anti-STAT6 antibodies. Immunoblot analysis with an anti-β-actin antibody served as a loading control. The first lane in the immunoblot indicates the molecular weight marker. C. mRNA levels of IL-4 were determined by qRT–PCR after isolation of total RNA from placentas of both P and P-PIC mice at gestational day 18. qRT–PCR of GAPDH was used for normalization. Results are expressed as mean+SEM for 3 independent experiments. *<i>P</i><0.05 vs. P.</p

Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree

Inflammation-related genes significantly (p<0.05 vs. P) altered in placentas of mice treated with a TLR3, TLR7, or TLR7/8 agonist during pregnancy.

<p>P = pregnant, P+TLR3 = pregnant+TLR3 agonist poly I:C, P+TLR7 = pregnant+TLR7 agonist R837, P+TLR7/8 = pregnant+TLR7/8 agonist CLO97. All p<0.05 vs. P, n>3 in each group.</p

TLR3/7/8 activation in pregnant mice caused PE-like symptoms.

<p>Measures of (A) urinary protein concentration, (B) maternal body weight, (C) spleen weight/body weight, (D) litter weight, and (E) number of total pups/litter and fetal demise/litter in pregnant mice treated with the TLR3 agonist poly I:C, the TLR7 agonist R837, or the TLR7/8 agonist CLO97. Results are expressed as mean + SEM and n = 9 in each group. *p<0.05 vs. P.</p