Crotoxin Modulates Macrophage Phenotypic Reprogramming.

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 µg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 µg/animal: M1 and at 5.0 µg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 µg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Correction: Novel site-specific PEGylated L-asparaginase.

[This corrects the article DOI: 10.1371/journal.pone.0211951.].

Novel site-specific PEGylated L-asparaginase.

L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia (ALL). PEGylation refers to the covalent attachment of poly(ethylene) glycol to the protein drug and it not only reduces the immune system activation but also decreases degradation by plasmatic proteases. However, pegaspargase is randomly PEGylated and, consequently, with a high degree of polydispersity in its final formulation. In this work we developed a site-specific N-terminus PEGylation protocol for ASNase. The monoPEG-ASNase was purified by anionic followed by size exclusion chromatography to a final purity of 99%. The highest yield of monoPEG-ASNase of 42% was obtained by the protein reaction with methoxy polyethylene glycol-carboxymethyl N-hydroxysuccinimidyl ester (10kDa) in 100 mM PBS at pH 7.5 and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was found to maintain enzymatic stability for more days than ASNase, also was resistant to the plasma proteases like asparaginyl endopeptidase and cathepsin B. Additionally, monoPEG-ASNase was found to be potent against leukemic cell lines (MOLT-4 and REH) in vitro like polyPEG-ASNase. monoPEG-ASNase demonstrates its potential as a novel option for ALL treatment, being an inventive novelty that maintains the benefits of the current enzyme and solves challenges.

PLCγ2 and PKC are important to myeloid lineage commitment triggered by M-SCF and G-CSF.

Myeloid differentiation is a complex process whereby mature granulocytes or monocytes/macrophages are derived from a common myeloid progenitor through the coordinated action of hematopoietic cytokines. In this study, we explored the role of the Ca(2+)i signaling transduction pathway in the commitment of hematopoietic stem/progenitor cells to either the monocytic or granulocytic lineage in response to macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). M-CSF and G-CSF induce cell expansion and monocyte or granulocyte differentiation, respectively, without affecting the percentage of hematopoietic progenitor cells. Colony-forming units (CFUs) and flow cytometry demonstrated the involvement of phospholipase Cγ (PLCγ) and protein kinase C (PKC) in monocyte/granulocyte commitment. In addition, using flow cytometry and RNA interference, we identified PLCγ2 as the PLCγ isoform that participates in this cell expansion and differentiation. Differences in signaling elicited by M-CSF and G-CSF were observed. The M-CSF-related effects were associated with the activation of ERK1/2 and nuclear factor of activated T-cells (NFAT); the inhibition of both molecules reduced the number of colonies in a CFU assay. In contrast, using flow cytometry and confocal evaluation, we demonstrated that G-CSF activated Jak-1 and STAT-3. Additionally, the effects induced by G-CSF were also related with the participation of Ca(2+) calmodulin kinase II and the transcription factor PU.1. STAT-3 activation and the increase of PU.1 expression were sensitive to PLC inhibition by U73122. These data show that PLCγ2 and PKC are important upstream signals that regulate myelopoiesis through cytokines, and differences in M-CSF and G-CSF downstream signaling were identified.