Goat anti-Human IRF4 polyclonal antibody

Background and Purpose-Inflammatory mechanisms can exacerbate ischemic tissue damage and worsen clinical outcome in patients with stroke. Both alpha beta and gamma delta T cells are established mediators of tissue damage in stroke, and the role of dendritic cells (DCs) in inducing the early events of T cell activation and differentiation in stroke is not well understood. Methods-In a murine model of experimental stroke, we defined the immune phenotype of infiltrating DC subsets based on flow cytometry of surface markers, the expression of ontogenetic markers, and cytokine levels. We used conditional DC depletion, bone marrow chimeric mice, and IL-23 (interleukin-23) receptor-deficient mice to further explore the functional role of DCs. Results-We show that the ischemic brain was rapidly infiltrated by IRF4(+)/ CD172a(+) conventional type 2 DCs and that conventional type 2 DCs were the most abundant subset in comparison with all other DC subsets. Twenty-four hours after ischemia onset, conventional type 2 DCs became the major source of IL-23, promoting neutrophil infiltration by induction of IL-17 (interleukin-17) in gamma delta T cells. Functionally, the depletion of CD11c(+) cells or the genetic disruption of the IL-23 signaling abrogated both IL-17 production in gamma delta T cells and neutrophil infiltration. Interruption of the IL-23/ IL-17 cascade decreased infarct size and improved neurological outcome after stroke. Conclusions-Our results suggest a central role for interferon regulatory factor 4-positive IL-23-producing conventional DCs in the IL-17-dependent secondary tissue damage in stroke.

STUDY QUESTION: What is the role of the programmed cell death-1 (PD-1)/PD-1 ligand-1 (PD-L1) axis in macrophage polarization during early pregnancy? SUMMARY ANSWER: PD-1 signaling is a major regulator of macrophage differentiation and function, and it is critical for the success of a pregnancy. WHAT IS KNOWN ALREADY: The predominance of decidual macrophages (DMs) with an M2 phenotype is an important contributor to maternal-fetal tolerance during early pregnancy. STUDY DESIGN, SIZE, DURATION: Twenty-four women with recurrent miscarriage (RM) and 70 women undergoing elective termination of an early normal pregnancy (NP) were included. Twelve female CBA/J, four male DBA/2, and four male BALB/c mice were included and mating carried out. The 12 CBA/J pregnant mice were then categorized into three groups of four mice: healthy control group CBA/ JxBALB/c, abortion-prone pregnant group CBA/JxDBA/2 and normal pregnancies CBA/JxBALB/c treated with anti-PD-1 monoclonal antibodies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The profile of DMs, and the expression of PD-1 and PD-L1 in DMs from women with NP and RM were measured by flow cytometry. PD-L1 expression in human villi was determined by quantitative RT-PCR (qRT-PCR) and western blot. An in vitro model consisting of peripheral CD14(+) monocytes isolated from women with NP was used. The profile of differentiated macrophages and their phagocytotic activity were then measured by flow cytometry. The mRNA levels of genes potentially underlying macrophage polarization modulated by PD-1 signaling were determined by qRT-PCR. Twelve pregnant mice were included in our in vivo model and underwent different treatment. The embryo resorption rate, and macrophage profile as well as PD-1 expression in murine spleens and uterus were analyzed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with NP, women with RM had elevated percentages of M1 DMs (P < 0.01), and reduced frequencies of M2 DMs (P < 0.05), as well as decreased PD-1 protein expression (P < 0.05) in the DMs. In addition, decreased mRNA and protein levels of PD-L1 expression in placental villi were observed in women with RM (P < 0.001). Using in vitro experiments, compared to the control group, we found that PD-1 activation by recombinant human (rh) PD-L1 Fc (human PD-L1 fused to the Fc region of human IgG1) drove the differentiation of macrophages with immuno-modulatory characteristics (P < 0.01). However, PD-1 blockade promoted dominance of the M1 phenotype (P < 0.01). PD-1 polarized macrophages showed enhanced phagocytic activity (P < 0.01), which was decreased with PD-1 blockade (P < 0.001). Furthermore, PD-1 blockade promoted the expression of pro-inflammatory cytokines and interferon regulatory factor (IRF) 5 (P < 0.05), while IRF4 expression was inhibited (P < 0.05). In addition, PD-1 blockade promoted macrophage glycolysis (P < 0.01) and inhibited fatty acid oxidation (P < 0.05). The mRNA expression levels of both phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin and mitogen-activated protein kinase/extracellular signal-regulated kinase/extracellular signal-regulated kinase were upregulated (P < 0.05) with PD-1 blockade during DM metabolic reprogramming. Moreover, in vivo mice data showed that PD-1 blockade or deficiency was associated with decreased M2 percentages at the maternal-fetal interface (P < 0.05) and embryo loss (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Whether the changes in DM polarization seen in miscarriage tissues are a cause or consequence of the demise of the pregnancy still requires further investigation. In addition, conducting metabolite analysis is required to further measure bioenergetic profiles. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of the PD-1/PD-L1 axis in macrophage polarization during early pregnancy; such exploration enhances our understanding of the physiology of early pregnancy. Our study also indicates that targeting the PD-1 pathway may represent a novel therapeutic strategy to prevent pregnancy loss.