Extracorporeal Photopheresis Induces Netosis in Neutrophils Derived from Patients with Graft-Versus-Host Disease

Introduction

Extracorporeal photopheresis (ECP) serves as a second line treatment for patients with acute or chronic graft versus host disease (GVHD) and demonstrates efficacy in ameliorating GVHD in this setting. The mechanism by which ECP acts against GVHD has not been fully elucidated yet. Preliminary data point to an association between GVHD and increased neutrophil extracellular trap (NET) formation (NETosis) although much is unknown regarding the pathologic implication of this association. In this study we performed a preliminary assessment of the influence of ECP on NETosis among patients with GVHD.

Methods

Six patients after allogeneic transplantation treated with ECP for severe GVHD post allogeneic transplant at the Rabin Medical Center in Israel were enrolled to the study. Blood samples were obtained at 3 different time points: before an ECP cycle, immediately after the end of the ECP and 24h after the initiation of the ECP cycle. Neutrophils were obtained from whole blood samples using a percoll gradient. NETosis was assessed by measurement of neutrophil elastase activity using a commercial NETosis assay kit by Cayman Chemical and by immunofluorescence staining for DNA (DAPI (4',6-diamidino-2-phenylindole)) and for citrullinated H3 (H3Cit), a marker of NET formation. All assessments were executed in unstimulated neutrophils and in neutrophils that were stimulated with phorbol myristate acetate (PMA)(100nmol) for 4 hours.

Results

Six patients (4 males) with chronic GVHD were included in the study. The underlying hematologic disease was acute myeloid leukemia (AML) in four patients, B-cell acute lymphocytic leukemia (B-ALL) and myelodysplastic syndrome (MDS) each in one patient. ECP was executed for moderate to severe GVHD. We observed a sharp increase in the formation of NETs following treatment with ECP among all study participants. The level of neutrophil elastase activity was significantly elevated from a mean value of 2.21mU/mL (±0.6mU/mL) at baseline to a mean value of 13.82mU/mL (±5.54mU/mL) immediately after the treatment (p-value=0.0022). The mean level of neutrophil elastase activity was significantly elevated to a mean peak value of 17.35mU/mL (±10.74mU/mL) 24h following the initiation of the ECP cycle (p-value 0.0063 for the difference between the first and the last time points). Pre-incubation of the neutrophils with PMA yielded similar results, as the mean neutrophil elastase activity was 6.05mU/mL (±3.63mU/mL), 20.16mU/mL (±6.72mU/mL), and 24.57mU/mL (±13.59mU/mL) before the ECP cycle initiation, immediately after treatment, and 24h following ECP onset, respectively (Figure 1). In agreement, the expression of H3cit was also increased in neutrophils derived from patients with GVHD after ECP treatment (Figure 2).

Conclusion

Our preliminary data indicate that ECP induces NET formation among patients with GVHD. NETosis might play a role in the therapeutic effects of ECP in this setting. These initial results might set the stage for future studies in this field.

Figure 1

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Figure 1

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