Comparative Proteomic Analysis of Erythropoiesis Tissue Head Kidney Among three Antarctic Fish Species

Antarctic icefish is the only known vertebrate species that lacks oxygen-carrying hemoglobin and functional erythrocytes. To reveal the unique hematopoietic process of icefish, we used an integrated approach including tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the dynamic changes in the head kidney whole proteome of a white-blooded icefish, Chionodraco hamatus, compared to those in two other red-blooded Antarctic fish, Trematomus bernacchii and Notothenia coriiceps. Of the 4,672 identified proteins, in the Antarctic ice fish head kidney, 123 proteins were significantly up-regulated and 95 proteins were down-regulated. The functional grouping of differentially expressed proteins based on KEGG pathway analysis shows that white blood fish and red blood fish have significant differences in erythropoiesis, heme biogenesis, leucocyte and platelet cell development. The proteins involved in the hematopoietic process in icefish showed a clear trend of downregulation of erythroid lineage marker proteins and upregulation of lymphoid and megakaryocytic lineage marker proteins, including CD9, ITGB2, and MTOR, which suggests a shift in hematopoiesis in the icefish head kidney due to the loss of erythrocytes. The results of the present study not only provide basic datasets for the head kidney proteins of Antarctic fishes, but also provide important references for studies on immunity and hematopoiesis in various species.

Phenotypic Switch of Human Peritoneal Macrophages during Childhood

Introduction Human peritoneal macrophages are resident in the abdominal cavity where they support the specific microenvironmental regulation. We have previously observed a phenotypic switch of murine macrophages during infancy that was associated with a functional development. To investigate the age related changes in human peritoneal macrophages, we analyzed peritoneal macrophages of children undergoing laparoscopic procedures. Materials and Methods Immunologically healthy children who received minimally invasive surgery in our department were included in this study. In all cases, the written consent was obtained. At the beginning of laparoscopy, physiologic NaCl-solution was instilled and manually removed through the umbilical trocar to gain macrophages. Lavage cells were processed for flow cytometry analysis. CD14+ myeloid cells were monitored for specific lineage marker expression. Results A total of 21 donors (age: 7 days–18 years) were included and divided into three groups. In all age groups, 97% of myeloid cells expressed CD11b. 70% of these expressed CD14. Three subsets of CD14 cells were detected on the basis of CD14/CD16 expression (CD14 + CD16dim, CD14 + CD16inter, and CD14 + CD16high). In neonates, >80% belonged to the CD14 + CD16high subset, reducing to 30% in adolescents. In none of the cases, the M2 markers CD23 and CD25 were expressed. Conclusion This is the first study showing that lineage marker expression of peritoneal macrophages in neonates differs from that in adults. The knowledge about neonatal tissue resident macrophages might help to understand their complex interaction and to use specific macrophage properties for therapeutic approaches.

HDAC6 promotes PMA-induced megakaryocyte differentiation of K562 cells by regulating ROS levels via NOX4 and repressing Glycophorin A

The human erythroleukemia (K562) cells are considered as bipotent megakaryocyte-erythroid progenitor cells and the differentiation of these cells to megakaryocytes (MK) in the presence of phorbol 12-myristate 13-acetate (PMA) mimics in vivo differentiation of MEP (megakaryocyte-erythroid progenitor) cells in the bone marrow. Histone deacetylases (HDACs) are involved in gene suppression and their roles during the MK differentiation remains largely undefined. In the present study, we have studied the expression levels of class I and class II HDACs during phorbol 12-myristate 13-acetate (PMA)-induced differentiation of K562 cells to MK. Class IIb HDACs (HDAC6 & HDAC10) were significantly up regulated time dependently upto 4 days of PMA-induced MK differentiation along with decreased acetylation levels of H3K9 and H3K56. Pharmacological inhibition and knockdown studies of HDAC6 using tubastatin A (TubA) and shRNA-HDAC6 respectively, during MK differentiation resulted in down regulation of MK lineage marker CD61 and up regulation of erythroid lineage gene glycophorin A (GYPA). HDAC6 over expression in K562 cells showed significant up regulation of CD61, MK transcription factors (FOG1 and GATA2) and down regulation of GYPA. ChIP-PCR studies showed enrichment of HDAC6 protein on GYPA promoter during differentiation indicating GYPA gene repression by HDAC6. Further studies on elucidating the role of HDAC6 in MK differentiation clearly indicated that HDAC6 is required for the production of sustainable levels of reactive oxygen species (ROS), an important regulator of MK differentiation, via NOX4.- ROS-HDAC6 circuit. In this study, we provide the first evidence that during PMA-induced megakaryocyte differentiation of K562 cells, HDAC6 represses erythroid lineage marker gene, GYPA, and promotes the sustainable levels of ROS via NOX4 required for MK differentiation.Key pointsHDAC6 upregulated during MK differentiation is involved in sustainable production of ROS via the circuit - HDAC6-NOX4-ROS-HDAC6.HDAC6 inhibits erythroid lineage gene, GYPA, by forming a repressor complex over the promoter region.