Toll-like receptor-mediated responses by placental Hofbauer cells (HBCs): a potential pro-inflammatory role for fetal M2 macrophages.

PROBLEM: Microbial-driven responses in placenta are linked with adverse pregnancy outcomes. The role of Toll-like receptor (TLR) function in Hofbauer cells (HBCs) and fetal macrophages of the placental villous core remains understudied. METHOD OF STUDY: Flow cytometry and immunohistochemistry (IHC) were used to establish the phenotype of HBCs. Regulation of cytokine secretion in response to treatment with TLR agonists and expression levels of TLRs and co-activators were compared in HBCs, placental fibroblasts (FIBs), and human umbilical vein endothelial cells (HUVECs) using ELISA and qPCR. RESULTS: Although flow cytometry and IHC revealed HBCs to be M2 (anti-inflammatory) macrophages, LPS and polyinosinic: polycytidylic acid [poly (I:C)] treatments markedly enhanced IL-6 secretion by HBCs, and expression of TLR-2, TLR-3, TLR-4, CD14, and MD-2 was the highest in HBCs. CONCLUSION: These results indicate that although HBCs are M2 macrophages, inflammatory responses are induced through TLR-3 and TLR-4 in this cell type, suggesting a role in microbial-driven placental/fetal inflammation.

Decreased levels of folate receptor-ß and reduced numbers of fetal macrophages (Hofbauer cells) in placentas from pregnancies with severe pre-eclampsia.

PROBLEM: Pre-eclampsia (PE), a pregnancy complication of unknown etiology, is a major cause of maternal and fetal mortality and morbidity. Previous studies have described placental genes that are up-regulated in expression in PE, but few studies have addressed placental gene suppression in this syndrome. METHOD OF STUDY: Gene profiling and quantitative reverse transcription PCR (qRTPCR) analyses were used to identify genes down-regulated in placentas from women with severe preterm PE compared with gestational age-matched normotensive controls with spontaneous preterm birth (sPTB). Western blotting and immunohistochemistry were used to evaluate levels and patterns of cell type-specific protein expression in PE and sPTB group placentas. RESULTS: Levels of macrophage marker [folate receptor (FR)-ß, CD163, and CD68] mRNA and FR-ß protein were significantly down-regulated in PE group placentas compared with the sPTB group. Numbers of Hofbauer cells (HBCs, fetal macrophages) and FR-ß protein in these cells were reduced in PE group placentas. CONCLUSION: Severe PE is associated with decreased placental expression of FR-ß and a reduction in the number of HBCs. Reduced placental macrophage function is likely to play a key role in the pathophysiology of PE.

Glucocorticoids enhance CD163 expression in placental Hofbauer cells.

Periplacental levels of glucocorticoid (GC) peak at parturition, and synthetic GC is administered to women at risk for preterm delivery. However, little is known concerning cell-type-specific effects of GC in placenta. Hofbauer cells (HBCs) are fetal macrophages that are located adjacent to fetal capillaries in placenta. The goal of the current study was to determine whether GC treatment altered HBC gene expression and function. Western blotting and flow cytometry revealed CD163 and folate receptor-ß (FR-ß), markers of antiinflammatory M2 macrophages, were specifically expressed by primary cultures of HBCs immunopurified from human term placentas. GC receptor mRNA and protein levels were higher in HBCs compared with placental fibroblasts. Treatment of HBCs with cortisol or dexamethasone (DEX) markedly and specifically enhanced CD163 protein and mRNA levels, whereas expression of FR-ß and CD68 were largely unresponsive to GC treatment. DEX treatment also increased hemoglobin uptake by HBCs, evidence of enhanced HBC function. The level of CD163 mRNA, but not FR-ß or CD68 mRNA, was stimulated in placental explant cultures by DEX treatment, and increased CD163/FR-ß and CD163/CD68 mRNA ratios sensitively reflected the response to GC. Maternal GC administration was associated with increased CD163/FR-ß and CD163/CD68 mRNA ratios in placentas from women with spontaneous preterm birth. In conclusion, in vitro studies indicated that GC treatment specifically up-regulated CD163 expression in HBCs and enhanced HBC function. In addition, the observed alterations in patterns of expression of macrophage marker genes associated with maternal GC administration suggest that HBCs are in vivo targets of GC action.

Focal increases of fetal macrophages in placentas from pregnancies with histological chorioamnionitis: potential role of fibroblast monocyte chemotactic protein-1.

PROBLEM: Histopathological chorioamnionitis (HCA) is caused by microbial-driven infiltration of leukocytes to the maternal-fetal interface resulting in adverse neonatal outcomes in a subset of pregnancies. The role of placental villus macrophages (i.e. Hofbauer cells, HBCs) in the pathophysiology of HCA is unelucidated. METHOD OF STUDY: The number of HBCs in human term placental villi in HCA and control groups was compared using immunohistochemistry. Levels of monocyte chemotactic protein (MCP-1) expression were measured in primary cultures of syncytioytrophoblasts (SCTs) and fibroblasts (FIBs) treated with bacterial compounds [lipopolysaccharide (LPS) and peptidoglycan] and pro-inflammatory cytokines (TNF-α and IL-1ß) using ELISA and quantitative real-time PCR. RESULTS: Immunohistochemistry revealed a focal increase in HBCs in HCA. Treatment of FIBs with LPS, IL-1ß, and TNF-α significantly increased MCP-1 mRNA and protein expression. Conversely, MCP-1 mRNA and protein levels were virtually undetectable in treated and untreated SCTs. CONCLUSION: These results demonstrate cell-type-specific regulation of MCP-1 expression in human placenta. A model is presented in which bacterial products and inflammatory cytokines initiate a fibroblast-driven cytokine cascade resulting in recruitment of fetal monocytes to placenta which focally increases levels of HBCs in pregnancies complicated by HCA.

The receptor for advanced glycation end products (RAGE) system in women with intraamniotic infection and inflammation.

OBJECTIVE: Receptor for advanced glycation end products (RAGE) is a multiligand cell-surface receptor part of the immunoglobulin superfamily with crucial roles in inflammation. S100A12/ENRAGE, a biomarker of amniotic fluid (AF) inflammation, is a ligand for RAGE. sRAGE, a competitive soluble RAGE receptor, inhibits RAGE ligands. Here we aimed to investigate the presence and changes in components of the RAGE system in women with intra-amniotic infection and inflammation. STUDY DESIGN: AF was retrieved by amniocentesis in 113 women stratified as follows: (1) positive AF culture (+AFC; GA = 27 [20-33] wk; n = 27); (2) negative AF culture (-AFC; GA = 30 [20-36] wk; n = 27); (3) second trimester control (2T-CRL; GA = 19 [15-25] wk; n = 31); (4) third trimester control (3T-CRL; GA = 36 [31-38] wk; n = 28). We used mass spectrometry (SELDI) to detect S100A12/ENRAGE in AF. sRAGE levels were measured using specific immunoassays. Placental pathology was interpreted in relationship to the presence or absence of histologic acute inflammation and immunoreactivity of S100A12/ENRAGE and RAGE. mRNA expression of S100A12/ENRAGE, sRAGE, and RAGE in amniochorion and placental villous tissue was investigated using quantitative real-time polymerase chain reaction (PCR). RESULTS: Presence of the S100A12/ENRAGE biomarker SELDI peak was confirmed in 70% of the +AFC but in only 10% of the -AFC samples (P < .001). The inflammatory biomarker was absent in all control samples. We further determined that the competitive inhibitor sRAGE is temporally regulated during gestation and that its AF levels are not influenced by the presence of either intra-amniotic infection or inflammation. Histologic choriamnionitis associated with intense staining for S100A12/ENRAGE, particularly in inflammatory cells. The immunoreactivity for extracellular domain of RAGE was localized exclusively to amnion epithelial, decidual, and extravillous trophoblast cells. Yet, acute histologic chorioamnionitis was related to increased gene expression of S100A12/ENRAGE in fetal membranes and decreased sRAGE and RAGE in the placenta. CONCLUSION: The S100A12/ENRAGE system is markedly upregulated in women with intra-amniotic infection and correlates with the degree of inflammation. Further studies remain to elucidate whether the gestational age dependence of the inhibitor molecule sRAGE may explain the higher incidence of infection-related preterm deliveries and especially rupture of the membranes at earlier gestational age.

Somatic D-loop mitochondrial DNA mutations are frequent in uterine serous carcinoma.

The mitochondria plays a role in apoptosis. Its genome is also more susceptible to mutations because of high levels of reactive oxygen species and limited repair mechanisms. The D-loop of mitochondrial DNA (mtDNA) contains essential transcription and replication elements, and mutations in this region might alter the rate of DNA replication. We examined genetic alterations in the D-loop region of mtDNA in uterine serous carcinoma (USC) samples and their paired normal adjacent endometrium. DNA was extracted after laser-capture microdissection of paraffin-embedded tissues from eight patients with USC. The entire D-loop genome was amplified using nine pairs of overlapping primers. Denatured polymerase chain reaction (PCR) products were subjected to single-strand conformation polymorphism (SSCP) analysis. Somatic mtDNA alterations were detected in five tumours (63%). Our study indicates that mtDNA D-loop sequence alterations occur at a high frequency in USC suggesting that mtDNA mutations may play a role in the development of USC.