Derek Miller

Intra-Amniotic Administration of HMGB1 Induces Spontaneous Preterm Labor and Birth

Sterile intra‐amniotic inflammation is associated with spontaneous preterm labor. Alarmins are proposed to mediate this inflammatory process. The aim of this study was to determine whether intra‐amniotic administration of an alarmin, HMGB1, could induce preterm labor/birth.

An M1-like Macrophage Polarization in Decidual Tissue during Spontaneous Preterm Labor That Is Attenuated by Rosiglitazone Treatment

Decidual macrophages are implicated in the local inflammatory response that accompanies spontaneous preterm labor/birth; however, their role is poorly understood. We hypothesized that decidual macrophages undergo a proinflammatory (M1) polarization during spontaneous preterm labor and that PPARγ activation via rosiglitazone (RSG) would attenuate the macrophage-mediated inflammatory response, preventing preterm birth. In this study, we show that: 1) decidual macrophages undergo an M1-like polarization during spontaneous term and preterm labor; 2) anti-inflammatory (M2)-like macrophages are more abundant than M1-like macrophages in decidual tissue; 3) decidual M2-like macrophages are reduced in preterm pregnancies compared with term pregnancies, regardless of the presence of labor; 4) decidual macrophages express high levels of TNF and IL-12 but low levels of peroxisome proliferator–activated receptor γ (PPARγ) during spontaneous preterm labor; 5) decidual macrophages from women who underwent spontaneous preterm labor display plasticity by M1↔M2 polarization in vitro; 6) incubation with RSG reduces the expression of TNF and IL-12 in decidual macrophages from women who underwent spontaneous preterm labor; and 7) treatment with RSG reduces the rate of LPS-induced preterm birth and improves neonatal outcomes by reducing the systemic proinflammatory response and downregulating mRNA and protein expression of NF-κB, TNF, and IL-10 in decidual and myometrial macrophages in C57BL/6J mice. In summary, we demonstrated that decidual M1-like macrophages are associated with spontaneous preterm labor and that PPARγ activation via RSG can attenuate the macrophage-mediated proinflammatory response, preventing preterm birth and improving neonatal outcomes. These findings suggest that the PPARγ pathway is a new molecular target for future preventative strategies for spontaneous preterm labor/birth.

Clinical chorioamnionitis at term VII: the amniotic fluid cellular immune response.

Abstract Objectives: 1) To characterize the cellular composition of the amniotic fluid of patients diagnosed with clinical chorioamnionitis at term, as a function of the presence or absence of microorganisms determined by cultivation techniques, and 2) to characterize the cytokine production by white blood cells present in the amniotic fluid using flow cytometry-based techniques. Materials and methods: Amniotic fluid samples from 20 women who had the diagnosis of clinical chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital Mycoplasmas). Amniotic fluid IL-6 concentrations were determined by an enzyme-linked immunosorbent assay. Amniotic fluid leukocytes were visualized by using hematoxylin and eosin staining and immunofluorescence. Immunophenotyping of surface markers and cytokines was performed in amniotic fluid leukocytes using flow cytometry. Results: 1) Neutrophils (CD45+CD15+ cells) were the most common leukocyte subset found in the amniotic fluid, followed by monocytes (CD45+CD14+ cells); other white blood cells (such as lymphocytes and natural killer cells) were scarce in the amniotic fluid; 2) the absolute counts of neutrophils and monocytes were significantly higher in patients with microorganisms found in the amniotic fluid than in those without detectable microorganisms, using cultivation techniques; 3) there was a significant correlation between the absolute counts of neutrophils and monocytes determined by flow cytometry (Spearman’s correlation=0.97; P<0.001); 4) there was a significant correlation between the absolute white blood cell count determined with a hemocytometer chamber and by flow cytometric analysis (Spearman’s correlation=0.88; P<0.001); and 5) the profile of cytokine expression differed between monocytes and neutrophils; while neutrophils predominantly produced TNF-α and MIP-1β, monocytes expressed higher levels of IL-1β and IL-1α. Conclusion: Flow cytometry analysis of the amniotic fluid of patients with intra-amniotic infection and clinical chorioamnionitis at term demonstrated that neutrophils and monocytes are the most common cells participating in the inflammatory process. We have characterized, for the first time, the differential cytokine expression by these cells in this important complication of pregnancy.

Are amniotic fluid neutrophils in women with intraamniotic infection and/or inflammation of fetal or maternal origin?

Background Neutrophils are the most abundant white blood cells found in the amniotic cavity of women with intraamniotic infection and/or inflammation. The current belief is that these neutrophils are of fetal origin. However, abundant neutrophils have been found in the amniotic fluid of women with a severe acute maternal inflammatory response but without a severe fetal inflammatory response in the placenta, suggesting that these innate immune cells can also be of maternal origin or a mixture of both fetal and maternal neutrophils. Objective We sought to investigate the origin of amniotic fluid neutrophils from women with intraamniotic infection and/or inflammation and to correlate these findings with acute histologic maternal and fetal inflammatory responses in the placenta. Study Design Amniotic fluid was collected from 15 women with suspected intraamniotic infection and/or inflammation (positive microbiological cultures and/or interleukin‐6 concentrations ≥2.6 ng/mL). Amniotic fluid neutrophils were purified by fluorescence‐activated cell sorting, DNA was extracted, and DNA fingerprinting was performed. DNA fingerprinting was also performed in the umbilical cord and maternal blood DNA. Fluorescence in situ hybridization was assayed in women with male neonates. Blinded placental histopathological evaluations were conducted. Results First, DNA fingerprinting revealed that 43% (6/14) of women who underwent a single amniocentesis had mostly fetal neutrophils in the amniotic fluid. Second, DNA fingerprinting showed that 36% (5/14) of the women who underwent a single amniocentesis had predominantly maternal neutrophils in the amniotic fluid. Third, DNA fingerprinting indicated that 21% (3/14) of the women who underwent a single amniocentesis had an evident mixture of fetal and maternal neutrophils in the amniotic fluid. Fourth, DNA fingerprinting revealed that a woman who underwent 2 amniocenteses (patient 15) had fetal neutrophils first, and as infection progressed, abundant maternal neutrophils invaded the amniotic cavity. Fifth, fluorescence in situ hybridization confirmed DNA fingerprinting results by showing that both fetal and maternal neutrophils were present in the amniotic fluid. Sixth, most of the women who had predominantly amniotic fluid neutrophils of fetal origin at the time of collection delivered extremely preterm neonates (71% [5/7]). Seventh, all of the women who had predominantly amniotic fluid neutrophils of maternal origin at the time of collection delivered term or late preterm neonates (100% [6/6]). Eighth, 2 of the women who had an evident mixture of fetal and maternal neutrophils in the amniotic fluid at the time of collection delivered extremely preterm neonates (67% [2/3]), and the third woman delivered a term neonate (33% [1/3]). Finally, most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (87% [13/15]). Conclusion Amniotic fluid neutrophils can be either predominantly of fetal or maternal origin, or a mixture of both fetal and maternal origin, in women with intraamniotic infection and/or inflammation. The findings herein provide evidence that both fetal and maternal neutrophils can invade the amniotic cavity, suggesting that both the fetus and the mother participate in the host defense mechanisms against intraamniotic infection.

Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection A New Mechanism of Host Defense

Objective: Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. Methods: Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4’,6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. Results: (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. Conclusion: NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

Inflammasome assembly in the chorioamniotic membranes during spontaneous labor at term

Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis‐associated speck‐like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase (CASP)‐1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term.

Neutrophil extracellular traps in acute chorioamnionitis: A mechanism of host defense

Neutrophil extracellular traps (NETs) were recently described as a mechanism for microbial killing in the amniotic cavity of women with intra‐amniotic infection. Such a clinical condition can result in acute chorioamnionitis, a placental lesion characterized by the infiltration of maternal neutrophils in the chorioamniotic membranes. Herein, we investigated whether these infiltrating neutrophils form NETs in the chorioamniotic membranes from women who underwent spontaneous term or preterm labor with acute chorioamnionitis.

The immunophenotype of amniotic fluid leukocytes in normal and complicated pregnancies

The immune cellular composition of amniotic fluid is poorly understood. Herein, we determined: 1) the immunophenotype of amniotic fluid immune cells during the second and third trimester in the absence of intra‐amniotic infection/inflammation; 2) whether amniotic fluid T cells and ILCs display different phenotypical characteristics to that of peripheral cells; and 3) whether the amniotic fluid immune cells are altered in women with intra‐amniotic infection/inflammation.

Innate lymphoid cells at the human maternal-fetal interface in spontaneous preterm labor

Pathological inflammation is causally linked to preterm labor and birth, the leading cause of neonatal morbidity and mortality worldwide. Our aims were to investigate whether (i) the newly described family of innate lymphoid cells (ILCs) was present at the human maternal‐fetal interface and (ii) ILC inflammatory subsets were associated with the pathological process of preterm labor.

CD71+ erythroid cells from neonates born to women with preterm labor regulate cytokine and cellular responses

Neonatal CD71+ erythroid cells are thought to have immunosuppressive functions. Recently, we demonstrated that CD71+ erythroid cells from neonates born to women who underwent spontaneous preterm labor (PTL) are reduced to levels similar to those of term neonates; yet, their functional properties are unknown. Herein, we investigated the functionality of CD71+ erythroid cells from neonates born to women who underwent spontaneous preterm or term labor. CD71+ erythroid cells from neonates born to women who underwent PTL displayed a similar mRNA profile to that of those from term neonates. The direct contact between preterm or term neonatal CD71+ erythroid cells and maternal mononuclear immune cells, but not soluble products from these cells, induced the release of proinflammatory cytokines and a reduction in the release of TGF‐β. Moreover, PTL‐derived neonatal CD71+ erythroid cells (1) modestly altered CD8+ T cell activation; (2) inhibited conventional CD4+ and CD8+ T‐cell expansion; (3) suppressed the expansion of CD8+ regulatory T cells; (4) regulated cytokine responses mounted by myeloid cells in the presence of a microbial product; and (5) indirectly modulated T‐cell cytokine responses. In conclusion, neonatal CD71+ erythroid cells regulate neonatal T‐cell and myeloid responses and their direct contact with maternal mononuclear cells induces a proinflammatory response. These findings provide insight into the biology of neonatal CD71+ erythroid cells during the physiologic and pathologic processes of labor.