Background Neutrophils account for 50–70% white blood cells in human blood are usually the first cell type recruited to sites of inflammation. Neutrophil extracellular traps (NETs), a web-like DNA fibers decorated with granule proteins, kill pathogens and cause tissue damage in autoimmune diseases. Although NETs have been widely reported to promote cancer metastasis, emerging evidence show anti-tumor function of neutrophils. Our previous work found that NETs induced by combined 5-FU and CB-839 treatment shrank colon tumor through Cathepsin G (CTSG) induced apoptosis. Unexpectedly, we also found that CTSG was required for combined 5-FU and CB-839 treatment induced NETs formation. In this project, we further study the role of CTSG in NETs formation induced by chemotherapy.

Methods Immunofluorescence assay was applied to detect NETs formation with Myeloperoxidase (MPO) and citrullinated histone H3 (cit-H3) as markers. Western Blotting assay was employed to detect cleaved histones. Edman sequencing was used to determined cleavage sites of histone H3 by CTSG. The Agilent QuickChange kit was utilized to introduce mutations to histone H3. TALON beads were used for purification of His-tag fused histone H3 mutants. Coomassie blue staining was applied to detect cleavage of mutant histone H3 by CTSG.

Results As citrullination and cleavage of histones are two main mechanisms to decondense chromatin DNA, we tested cit-H3 and cleaved histones in neutrophils treated with chemotherapies. We found that histone H3 was the only histones that was cleaved during NETs formation. Topoisomerase II inhibitors, Epirubicin and Daunorubicin, induced significant histone H3 cleavage and NETs formation in a cit-H3 independent manner. Epirubicin and Daunorubicin induced cleavage of histone H3 in neutrophils isolated from wild-type mouse but not CTSG-KO mouse. We cut the cleavage bands of histone H3 by CTSG for Edman sequencing and identified three potential cleavage sites. We generated and purified His-tag fused mutant histone H3 proteins and incubated them with CTSG. Mutation at Leu49 of histone H3 attenuated cleavage by CTSG.

Conclusions Epirubicin and Daunorubicin induce NETs formation by cleaving histone H3 instead of citrullinating it. CTSG is essential for cleavage of histone H3 induced by Epirubicin and Daunorubicin. Specifically, CTSG cleaves histone H3 after Leu49. These findings will provide insights into regulating NETs during chemotherapy for cancer treatments.