High-mobility group protein B1 derived mutant peptide mB Box-97 inhibits the formation of neutrophil extracellular traps

Abstract

Introduction: Neutrophil Extracellular Traps (NETs) are vital for innate immunity, playing a key role in controlling pathogen and biofilm proliferation. However, excessive NETosis is implicated in autoimmunity, inflammatory and neoplastic diseases, as well as thrombosis, stroke, and post-COVID-19 complications. Managing NETosis, therefore is a significant area of ongoing research. Herein, we have identified a peptide derived from HMGB1 that we have modified via a point mutation that is referred to as mB Box-97. In our recent study in a murine lung infection model, mB Box-97 was shown to be safe and effective at disrupting biofilms without eliciting an inflammatory response typically associated with HMGB1. Here we show that the lack of an inflammatory response of mB Box-97 is in part due to the inhibition of NETosis of which we investigated the mechanism of action.

Methods: mB Box-97's anti-NETosis activity was assessed using human neutrophils with known NET inducers PMA, LPS, or Ionomycin. Additionally, mB Box-97's binding to Protein Kinase C (PKC), in addition to downstream effects on NADPH oxidase (NOX) activation, Reactive Oxygen Species (ROS) generation and thereby NETosis were assessed.

Results: mB Box-97 significantly inhibited NETosis regardless of the type of induction pathway. Mechanistically, mB Box-97 inhibits PKC activity likely through direct binding and thereby reduced downstream activities including NOX activation, ROS production and NETosis.

Conclusions: mB Box-97 is a promising dual acting therapeutic candidate for managing NET-mediated pathologies and resolving biofilm infections. Our results reveal that PKC is a viable target for NETosis inhibition independent of NET inducer and worthy of further study. These findings pave the way for a novel class of therapeutics aimed at controlling excessive NETosis, potentially offering new treatments for a range of inflammatory and immune-related diseases.

Keywords: COVID-19; HMGB1; NET inhibition; autoimmunity; inflammation; therapeutic.

Figure 1

Only mB Box-97_syn inhibited NETosis induced by PMA. (A) Schematic depiction of the peptide constructs utilized in this study. Full-length HMGB1 is represented in brown, showcasing DNA binding domains A Box, B Box, and a C-terminal acidic tail. Various peptide constructs, indicated in gray with brown borders, illustrate amino acid positions from N-terminal to C-terminal. Construct names are denoted in white. Specific Cysteines/C and their corresponding amino acid numbers are denoted; in mB Box-97_syn, C is mutated to Serine/S at position 106. (B) NETs were induced using PMA in the presence or absence of respective peptides or recombinant HMGB1 (rHMGB1) and stained to detect DNA (magenta) and Plasma Membrane (PM) (green). NETs were visualized using confocal laser scanning microscopy (CLSM) at 20x magnification, with representative images provided for each treatment condition out of 3 independent biological replicates (n), scale bar 50 µm. (C) Formed NETs were manually quantified using ImageJ, and the percentage of NET formation was calculated relative to total PMNs in each image. Mean values of n = 5 biological replicates ± Standard error of the mean (SEM) is shown. p values (ns = p > 0.05, **p < 0.01) are from a Welch and Brown-Forsythe ANOVA followed by Dunnett’s T-test.

Figure 2

mBBox-97 inhibited both PMA- and NTHI-induced NETosis as well as the secretion of the NET-associated protein Neutrophil Elastase (NE). (A) The induction of NETs by PMA or (B) by heat-inactivated NTHI was assessed in isolated human PMNs in the presence or absence of mB Box-97_syn or B Box-97. NETs were stained for NET-associated proteins, namely Neutrophil Elastase (NE, upper panel) and Myeloperoxidase (MPO, lower panel) shown in red; DNA shown in blue and plasma membrane shown in green. Representative 63x magnification CLSM images for each treatment condition are shown out of 3 independent experiments (n), scale bar 50 µm. (C) Fluorescence of DNA released from the PMNs induced with PMA with or without the addition of mB Box-97_syn or B Box-97 was plotted as a percentage relative fluorescence compared to the untreated control group. Mean values of n = 5 biological replicates ± SEM are shown. (D) PMNs were induced to form NETs with PMA alone or PMA +/- B Box-97/mB Box-97_syn and NE released (secreted) into the media was collected. The NETs settled at the bottom of each well were treated with DNase I to liberate DNA-bound NE (DNA-bound). The concentration of NE was quantified using ELISA and presented as mean ± SEM, Mean values of n = 4 biological replicates ± SEM are shown. P values (ns = p > 0.05, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001) using from a Welch and Brown-Forsythe ANOVA followed by Dunnett’s T test.

Figure 3

Ca²⁺ ionophore-mediated NETosis was partially inhibited by mB Box-97_syn. CLSM images of the NETs induced using Ca²⁺ ionophore A23187 with or without mB Box-97_syn or B Box-97 are shown where blue represents DNA), green represents plasma membrane, and red represents NE (upper panel) or MPO (lower panel). n = 3 independent experiments, 63x magnification, scale bar 50 µm.

Figure 4

Localization of mB Box-97 within human PMNs. (A) Isolated PMNs were exposed to PMA with His-tagged mB Box-97 (mB Box-97-His) for 10, 30, 60, 120, or 180 minutes, or PMA alone for 180 min. Cells were stained to visualize DNA (gray), plasma membrane (green), and mB Box-97-His (red). NETs were visualized by CLSM, with representative 63x images being displayed, with insets at the same magnifications showing the staining for DNA, mB Box-97-His and PM. n = 3 independent experiments, scale bar 50 µm. (B) Pearson’s correlation coefficient (PCC) was calculated for the peptide either for the co-localization with PM or DNA for respective treatment time points. Mean values of n = 3 biological replicates ± SEM are shown.

Figure 5

Interaction and inhibition of PKC by mB Box-97_syn inhibits P47^phox phosphorylation and ROS generation. (A) The activity of PKC was assessed using a PKC activity assay kit in the presence of Histone H1, mB Box-97_syn, and B Box-97. Histone H1, a well-known substrate of PKC, competes with the kit provided PKC substrate peptide used in the assay. As a result, it functions as a competitive inhibitor of PKC and serves as a positive control. Mean values of n = 3 biological replicates ± SEM are shown. (B) Conditions unmodified (UM) and crosslinked with glutaraldehyde are indicated. On the left is a Western blot detecting B Box-97/mB Box-97_syn and on the right is silver-stained SDS-PAGE separating indicated complexes. PKC crosslinked with mB Box-97_syn, PKC alone and B Box-97/mB Box-97_syn are indicated. N=3. (C) ROS as a measure of Luminol luminescence from PMNs in respective conditions was plotted against incubation time. mB Box-97_syn showed a significant reduction in ROS generation compared to PMA alone or PMA + B Box-97 treatment. Mean values of n = 7 biological replicates ± SEM are shown. (D) Quantification of steady state levels of phosphorylation of p47^phox on Ser-370 in PMNs post treatment under the described conditions was assessed using Western blotting. The intensity of each band was assessed using ImageJ and the relative intensity of phosphorylated p47^phox post normalization was plotted as Arbitrary Units (AU). Mean values of n = 4 biological replicates ± SEM are shown. P values (NS >0.05, *p<0.05, **p<0.05, ***p < 0.005, ****p < 0.0001) calculated using Welch and Brown-Forsythe ANOVA followed by Dunnett’s T-test (A) or RM two-way ANOVA (C) or one-way ANOVA with Tuckey’s multiple t-test (D).

Figure 6

Neutrophil-mediated microbial killing is inactivated by mB Box-97_syn. NTHI biofilm was incubated with human neutrophils (B + N), or neutrophils treated with recombinant DNABII protein isolated from NTHI (B + N + HU_NTHI) as a positive control, mB Box-97_syn (B + N + mB Box-97_syn), or B Box-97 (B + N + B Box-97). A control consisting of NTHI biofilm without neutrophil intervention was also included against which the relative percent killing was calculated. Mean values of n = 6 biological replicates ± SEM are shown. P values (**p<0.05, ***p < 0.005, ****p < 0.0001, ns >0.05) were calculated using ordinary two-way ANOVA with Tuckey’s multiple T-test.

Figure 7

Model summarizing the effect of mB Box-97 on signaling events that lead to NETosis by human neutrophils. The cascade of events affected by mB Box-97 is depicted in a semi-transparent manner. mB Box-97 engages with PKC, thereby inducing inhibition of its enzymatic activity. Consequently, this inhibition impedes the phosphorylation process of p47^phox, resulting in the suppression of the active assembly of NOX and subsequently reducing the production of ROS. The diminished levels of ROS subsequently lead to a reduction in the release of NE and MPO, ultimately culminating in the inhibition of the formation of the NETs. Furthermore, mB Box-97 demonstrates partial inhibition of Ca²⁺-induced NETosis, plausibly mediated through the suppression of PKC activity, given the influence of Ca²⁺ on PKC functionality.