The Effect of Paracrine Factors Released by Irradiated Peripheral Blood Mononuclear Cells on Neutrophil Extracellular Trap Formation

Abstract

Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation.

Keywords: PAD4; ROS; neutrophil; neutrophil extracellular traps (NETs); peripheral blood mononuclear cell secretome; secretome.

Figure 1

PBMCsec inhibits NET formation. Erythrocyte-lysed blood was treated with PBMCsec or vehicle for 20 min and subsequently stimulated with ionomycin (IM) for 2 h and analysed with flow cytometry, cytotox staining, and image stream analysis. (A) Neutrophils were identified in flow cytometry as CD66b⁺CD15⁺ cells and NET-forming neutrophils were characterized by citH3. Control, PBMCsec, or vehicle treated samples are shown in the top panel and ionomycin-activated neutrophils are shown in the bottom panel. n = 5. One representative sample is shown out of five replicates summarized in (B). (C) Extracellular DNA content was measured using cytotox staining of neutrophils after pre-treatment with PBMCsec or vehicle and subsequent activation for 2 h with (C) IM or (D) PMA. Fold change increase in relative fluorescent intensity is shown after two hours of stimulation relative to time point zero/start of stimulation/minute one after induction of NETs. (E) Visualization of IM-activated neutrophils was performed using image stream analyses. Untreated (control) and IM-PBMCsec treated neutrophils did not show citH3⁺ staining. IM and IM-vehicle treated neutrophils showed robust citH3⁺ staining of cells and additional extracellular structures (indicative for NETs). Green, citH3; magenta, CD15; purple, CD66b; n = 2. One representative sample is shown. Data are represented as individual values with mean and error bars indicate SD, one-way ANOVA and Sidak’s multiple comparisons test. * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001.

Figure 2

Isolated substance classes of PBMCsec show a synergistic effect on NET-inhibition. (A) Schematic depiction of the isolated and tested substance classes of PBMCsec. This scheme was created with BioRender.com, accessed on 2 June 2022 (B) Neutrophils were identified in flow cytometry as CD66b⁺CD15⁺ cells and NET formation was characterized by citH3+ signal. rec fractions + IM = reconstituted fractions + ionomycin. n = 6. One representative sample out of six is shown, summarized in (C) one-way ANOVA and Sidak’s multiple comparisons test. Data are represented as mean and error bars indicate SD. § = ANOVA without multiple comparison tests, p =< 0.0001; * p < 0.0332, ** p < 0.0021, **** p < 0.0001.

Figure 3

PBMCsec inhibits NET formation by a DNase-independent mode of action. (A) DNase activity was measured in a cell-free assay by co-incubation of PBMCsec or vehicle with λ-DNA. DNase I was used as positive control. n = 3, one representative sample is shown. (B) Schematic depiction of the adapted neutrophil stimulation protocol for the measurement of potential DNase activity in a cell-based assay. This scheme was created with BioRender.com, accessed on 2 June 2022 (C) Neutrophils were identified as CD66b⁺CD15⁺ cells and citH3+ signal was used to characterize NET formation. n = 3, one representative experiment is shown. (D) Statistical summary of all biological donors is shown. Data are represented as individual values with mean and error bars indicate SD. One-way ANOVA and Sidak’s multiple comparisons test. * p < 0.0332, ** p < 0.0021.

Figure 4

PBMCsec inhibits NET formation without interfering with calcium flux. (A) Ratiometric calcium flux was measured with Fura Red in neutrophils. Neutrophils were observed for approximately 30 s to record a baseline Fura Red ratio indicating homeostatic calcium flux prior to the addition of IM and subsequent analysis for a total of 120 s. (B) Statistical analysis of the percent reduction in calcium flux in PBMCsec or vehicle treated neutrophils compared to control samples is shown for the time points of stimulation (①, addition of IM) and the endpoint (②,120 s) n = 4. Error bars indicate SD. No statistically significant reduction was observed for PBMCsec or vehicle treatment.

Figure 5

PBMCsec prevents ROS production and PAD4 activity. (A) ROS production in IM stimulated and PBMCsec or vehicle treated neutrophils normalized to IM-induced ROS production. n = 5 (B) Analysis of protein levels of HO-1 and HIF-1α of isolated neutrophils stimulated with ionomycin and treated with PBMCsec or vehicle, using a proteome profiler is shown. Cell lysates of four individual donors and experiments were pooled. Error bars indicate SD of two technical replicates. (C) Enzymatic activity of PAD4 was measured in a cell-free assay upon co-incubation of PBMCsec or vehicle with the PAD4-substrate and normalized to untreated control. n = 3, data are represented as individual values with mean and error bars indicate SD, unless indicated otherwise. One-way ANOVA and Sidak’s multiple comparisons test was performed where significances are indicated. * p < 0.0332, ** p < 0.0021, *** p < 0.0002.