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γδ T cells represent an evolutionarily primitive T cell subset characterized by distinct T cell receptors (TCRs) and innate and adaptive immune functions. However, the presence of this T cell subset in ancient vertebrates remains unclear. In this study, γδ T cells from a zebrafish (Danio rerio) model were subjected to molecular and cellular characterizations. The constant regions of zebrafish TCR-γ (DrTRGC) and δ (DrTRDC) were initially identified. Zebrafish γδ T cells accounted for 7.7–20.5% of the total lymphocytes in spleen, head kidney, peripheral blood, skin, gill, and intestine tissues. They possess typical morphological features of lymphocytes with a surface phenotype of γ+δ+CD4−CD8+. Zebrafish γδ T cells functionally showed a potent phagocytic ability to both soluble and particulate antigens. They can also act as an antigen-presenting cell to initiate antigen (KLH)-specific CD4+ TKLH cell activation and to induce B cell proliferation and IgM production. Particularly, zebrafish γδ T cells also play a critical role in antigen-specific IgZ production in intestinal mucus. These findings demonstrated that γδ T cells had been originated as early as teleost fish, which providing valuable insights into the evolutionary history of T cell subset. It is anticipated that this study would be used as a guide to develop a zebrafish model for the cross-species investigation of γδ T cell biology.

Although epigenetic modulation is critical for a variety of cellular activities, its role in erythropoiesis remains poorly understood. Teneleven translocation (TET) molecules participate in methylcytosine (5mC) hydroxylation, which results in DNA demethylation in several biological processes. In this research, the role of TETs in erythropoiesis was investigated by using the zebrafish model, where three TET homologs were identified. These homologs share conserved structural domains with their mammalian counterparts. Zebrafish TETs mediate the conversion of 5mC to hydroxymethylcytosine (5hmC) in zebrafish embryos, and the deletion of TET2 inhibits erythropoiesis by suppressing the expression of the scl, gata-1, and cmyb genes. TET2-upregulated lineage-specific genes and erythropoiesis are closely associated with the occurrence of 5hmC and demethylation in the intermediate CpG promoters (ICPs) of scl, gata-1, cmyb, which frequently occur at specific regions or CpG sites of these ICPs. Moreover, TET2 regulates the formation and differentiation of erythroid progenitors, and deletion of TET2 leads to erythrocyte dysplasia and anemia. Here, we preliminarily proved that TET2 plays an essential role in erythrocyte development by regulating lineage-specific genes via DNA oxidative demethylation. This report is anticipated to broaden current information on hematopoiesis and pathogenesis of hematopoiesis-related diseases.

We have previously documented alterations in endogenous surfactant after lung transplantation and improved graft function in some dogs after instillation of bovine lipid extract surfactant (bLES) into the recipient. To determine the effect of bLES delivery method and timing of treatment on physiologic response and surfactant recovery, 21 canine left lung grafts were divided into four groups: (1) Treatment of the donor for 3 h with aerosolized bLES prior to graft storage (Donor Aerosol); (2) Treatment of the recipient with instilled bLES immediately after transplantation (Recipient Instilled); (3) No bLES treatment (Control); and (4) Aerosolized bLES in donors and instilled bLES in recipients (Combined Therapy). Aerosolized bLES was labeled with [3H]-dipalmitoylphosphatidylcholine (DPPC) and instilled bLES with [14C]-DPPC. Grafts were stored for 36 h, transplanted and reperfused for 6 h. The native right and transplanted left lungs were then lavaged and protein yield, surfactant aggregates, and bLES recovery were measured. After 6 h of reperfusion, PO2/FlO2 ratio was significantly better after Combined Therapy (372 +/- 52 mm Hg) than in the Recipient Instilled (117 +/- 47 mm Hg) and Control groups (87 +/- 26 mm Hg), with intermediate values in Donor Aerosol dogs (232 +/- 64 mm Hg). The recovery of donor aerosolized bLES from transplanted lungs was increased in dogs given Combined Therapy versus Donor Aerosol treatment alone (p = 0.03). Furthermore, with Combined Therapy there was an increased percentage of instilled bLES recovered from transplanted lungs compared with the Recipient Instilled group. We conclude that surfactant treatment strategies influence physiologic response and bLES recovery after prolonged lung preservation. Treatment of lung donors with exogenous surfactant prior to graft storage was associated with less severe lung injury. Combined donor and recipient bLES therapy resulted in a superior physiologic response during reperfusion in this model.

In the periphery of pancreatic ductal adenocarcinoma (PDAC), high accumulation of tumor-associated macrophages (TAMs), which exhibit M2 phenotype, has been shown to be correlated with extra-pancreatic invasion, lymph vessel invasion, lymph node involvement and shortened survival time. However, mechanisms by which tumor cells educate and reprogram TAMs remain largely unclear. The phenotype of TAMs in PDAC tissues was confirmed by immunofluoresence and confocal microscopy. Human CD14+ monocytes were incubated with recombinant human REG4 (rREG4) before being stimulated with LPS and IL-10 and IL-6 were measured with ELISA. A panel of M1 and M2 genes were measured by quantitative real-time PCR. Panc1, AsPC1 and BxPC3 cells were cultured in the conditioned medium (CM) and treated with REG4. The macrophages were infected with CREB shRNA or cultured by the CM of Panc1 cells infected with REG4 shRNA. The expression of CD163, CD206 and REG4 and the phosphorylation levels of epidermal growth factor receptor (EGFR), AKT and cAMP response element-binding protein (CREB) in cells were assessed with western blotting. Cell proliferation and invasiveness were also assessed. The rREG4 or the conditioned medium of Panc1 cells which secreted REG4 induced the polarization macrophages to M2 phenotype. Treatment of human macrophages with REG4 resulted in phosphorylation of EGFR, AKT and CREB. The latter was responsible for REG4-mediated macrophage polarization to M2. The conditioned medium of macrophages treated with rREG4 promoted the proliferation and invasion of pancreatic cancer cell lines. REG4, overexpressed in PDAC and secreted by cancer cells, promoted macrophage polarization to M2, through at least in part, activation of ERK1/2 and CREB and changed the microenvironment to facilitate cancer growth and metastasis.

Decreasing the bioavailability of serotonin (5-HT) by inhibiting its biosynthesis may represent a useful adjunctive treatment of pulmonary hypertension (PH). We assessed this hypothesis using LP533401, which inhibits the rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) expressed in the gut and lung, without inhibiting Tph2 expressed in neurons. Mice treated repeatedly with LP533401 (30-250 mg/kg per day) exhibited marked 5-HT content reductions in the gut, lungs, and blood, but not in the brain. After a single LP533401 dose (250 mg/kg), lung and gut 5-HT contents decreased by 50%, whereas blood 5-HT levels remained unchanged, suggesting gut and lung 5-HT synthesis. Treatment with the 5-HT transporter (5-HTT) inhibitor citalopram decreased 5-HT contents in the blood and lungs but not in the gut. In transgenic SM22-5-HTT+ mice, which overexpress 5-HTT in smooth muscle cells and spontaneously develop PH, 250 mg/kg per day LP533401 or 10 mg/kg per day citalopram for 21 days markedly reduced lung and blood 5-HT levels, right ventricular (RV) systolic pressure, RV hypertrophy, distal pulmonary artery muscularization, and vascular Ki67-positive cells (P < 0.001). Combined treatment with both drugs was more effective in improving PH-related hemodynamic parameters than either drug alone. LP533401 or citalopram treatment partially prevented PH development in wild-type mice exposed to chronic hypoxia. Lung and blood 5-HT levels were lower in hypoxic than in normoxic mice and decreased further after LP533401 or citalopram treatment. These results provide proof of concept that inhibiting Tph1 may represent a new therapeutic strategy for human PH.

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