Neonatal NET-inhibitory factor inhibits macrophage extracellular trap formation

TO THE EDITOR:

Neutrophils release neutrophil extracellular traps (NETs) in response to various microbial and inflammatory stimuli. These extracellular web-like structures, composed of decondensed chromatin and antimicrobial peptides, trap and kill pathogens.¹ NET formation represents an important facet of antimicrobial defense. However, unregulated NET formation leads to inflammatory tissue damage and immunothrombosis in many disease states.^2-5 Recent reports now describe extracellular trap formation by other leukocytes.^6-8 Macrophages, with their roles in host immune defense and tissue homeostasis, may form macrophage extracellular traps (METs) in response to tissue injury and infection. METs and NETs share similar structure and composition.⁶ However, little is known about the regulators of MET formation, including factors that inhibit their release.

The discovery of specific regulatory mechanisms governing extracellular trap formation remains a central challenge to understanding and differentiating the clinical impacts of extracellular trap formation from other forms of inflammatory cell death, including pyroptosis. In 2016, we reported the discovery of neonatal NET-inhibitory factor (nNIF), a cleavage fragment of alpha-1-antitrypsin generated in utero by the placenta that prevents NET formation.^9,10 We have shown that nNIF improves survival in mouse models of sepsis and ischemic stroke.^11-13 However, whether nNIF inhibits MET formation or other innate functions of macrophage host defense remains unknown. Here, using both a murine macrophage cell line (J774A.1) and human monocyte-derived macrophages (MDMs; supplemental Figure 1), we evaluate the impact of nNIF on MET formation, pyroptosis, apoptosis, and 2 other critical macrophage functions, phagocytosis and bacterial killing.

Supplemental Materials provide methodologic details used in these in vitro experiments. We induced MET formation by incubating macrophages with the physiologic agonist lipopolysaccharide (LPS; 100 ng/mL) or a pathogenic strain of Escherichia coli (MOI 5) for 4 hours. Using a previously standardized concentration and time point, we tested the effect of nNIF by treating cells with either nNIF (1 nM) or an inactive scrambled control peptide (nNIF-scramble; 1 nM) for 1 hour before stimulation with an inflammatory agonist.¹⁰ We visualized MET formation using confocal microscopy. We assessed MET formation quantitatively using high-throughput DNA quantification and a citrullinated histone H3 (CitH3) enzyme-linked immunosorbent assay (ELISA).

Confocal imaging showed that J774 cells stimulated with LPS and MDMs stimulated with E coli released extracellular strands of DNA consistent with MET formation. Treatment with nNIF visually reduced MET formation after stimulation, whereas treatment with nNIF-scramble did not (Figure 1A-B). High-throughput DNA quantification yielded concordant results. In J774 cells, LPS stimulation induced a significant increase in supernatant extracellular DNA compared with unstimulated controls. Treatment with nNIF before LPS stimulation led to a significant reduction in supernatant extracellular DNA compared with treatment with nNIF-scramble (Figure 1C). E coli stimulation of J774 cells and MDMs induced significant increases in supernatant extracellular DNA compared with unstimulated controls in both cell types. Treatment with nNIF before E coli stimulation led to a significant reduction in supernatant extracellular DNA compared with treatment with nNIF-scramble in both cell types (Figure 1D-E). We confirmed these findings using a CitH3 ELISA as an additional quantitative measure of MET formation. E coli stimulation induced a significant increase in supernatant CitH3 concentrations in J774 cells and MDMs compared with unstimulated controls. Treatment with nNIF before E coli stimulation led to a significant reduction in supernatant CitH3 concentrations compared with treatment with nNIF-scramble in both cell types (Figure 1F-G).

We then examined the potential impact of nNIF on pyroptosis. Pyroptosis is a regulated form of inflammatory cell death observed predominantly in macrophages. Several inflammasomes mediate this process, activating caspase-1, leading to cell membrane disruption, swelling, and ultimately, the release of inflammatory cytokines, including interleukin-1β (IL-1β).^14-16 To induce pyroptosis, we primed J774 cells with LPS (100 ng/mL, 4 hours) and then stimulated with adenosine 5′ triphosphate (ATP; 3 mM, 30 minutes).¹⁷ We visualized pyroptosis using confocal microscopy and determined active caspase-1 staining. We examined pyroptosis quantitatively by measuring mean cell surface areas and mean fluorescence intensity of active caspase-1. Additionally, we measured supernatant IL-1β concentration using an IL-1β ELISA. We separately examined the impact of nNIF on apoptosis in J774 cells and found that nNIF does not alter apoptosis as assessed by annexin V staining detected using flow cytometry (supplemental Figure 2).

In cells stimulated with LPS/ATP, we visualized cell swelling consistent with pyroptosis and an increase in active caspase-1 fluorescence. On visual inspection, treatment with nNIF did not inhibit pyroptosis (Figure 2A). Quantitative assessment supported these findings. We detected no difference in mean cell surface area or mean fluorescence intensity of active caspase-1 in cells treated with nNIF or nNIF-scramble compared with cells stimulated with LPS/ATP alone (Figure 2B-C). Additionally, treatment with nNIF did not reduce supernatant IL-1β concentrations (Figure 2D).

Using the J774 cell line, we explored the impact of nNIF on macrophage phagocytosis and bacterial killing. To examine phagocytosis, we incubated cells with fluorescently labeled E coli bioparticles and calculated a phagocytic index using confocal microscopy (Figure 2E). We detected no difference in the phagocytic index of cells treated with nNIF or nNIF-scramble compared with untreated cells (Figure 2F). Next, we examined total bacterial killing of E coli using a modified bacterial killing assay.¹⁸ Cells treated with nNIF demonstrated a significant reduction in mean bacterial killing when normalized to untreated controls. In contrast, we demonstrated no difference in total bacterial killing in cells treated with nNIF-scramble. Furthermore, when normalized to untreated controls, cells treated with DNase I, which dismantles formed extracellular traps, showed significantly reduced total bacterial killing. As expected, given published observations that MET formation contributes to extracellular bacterial killing,¹⁹ we detected no difference in total bacterial killing between cells treated with nNIF and those treated with DNase I (Figure 2G).

The molecular mechanisms governing MET formation remain obscure and likely context dependent. Prior studies indicate that peptidylarginine deiminase 4 (PAD4) mediated hypercitrullination contributes to MET formation.^8,20,21 We previously demonstrated that nNIF inhibits PAD4 activity in neutrophils and in a cell-free assay.¹⁰ In this study, we did not measure intracellular citrullination. However, we found that nNIF reduced MET-associated CitH3levels in both J774 cells and MDMs. This observation suggests a similar mechanism of inhibition and builds on previous literature indicating that PAD4 activation may be a key regulating event in MET formation.

We have not fully characterized the spectrum of activity of nNIF and thus sought to evaluate the impact of nNIF on other critical macrophage functions. Emerging evidence indicates significant cross talk between extracellular trap formation and pyroptosis.^14,22-24 In this study, nNIF failed to affect caspase-1 activation or the terminal events of pyroptosis, including cell swelling or IL-1β release. Although this study is limited to the use of an immortalized cell line and MDMs, this observation aligns with our published work showing that nNIF does not reduce IL-1β levels in a mouse model of sepsis.¹¹ Importantly, nNIF did not affect phagocytosis or non-MET–dependent intracellular killing, 2 critical functions of macrophage host defense.

In this study, we primarily used the J774.1 mouse peritoneal cell line to examine the impact of nNIF on MET formation and other macrophage functions, because it may represent a first line of defense against intra-abdominal infection induced by our mouse sepsis models. To expand the focus of this initial investigation, we evaluated the impact of nNIF on MET formation in human MDMs. We acknowledge, however, the great diversity of tissue macrophages and their differential responses to pathogens and inflammatory stimuli and, thus, plan for future studies in a more diverse group of in vitro and in vivo conditions.

A mounting body of evidence supports the involvement of METs in pathological inflammation.²⁵ Here, we provide evidence that nNIF inhibits MET formation while leaving pyroptosis intact. This finding holds particular importance given the shared molecular machinery between these 2 pathways of cell death. Understanding the mechanisms by which nNIF inhibits extracellular trap formation remains an ongoing emphasis in our investigations. However, nNIF may represent a tool to unravel the mechanisms of extracellular trap formation in multiple cell types, help distinguish the clinical impacts of extracellular trap formation from other forms of inflammatory cell death, and enable the development of targeted therapies mitigating the effects of unregulated extracellular trap formation.

Acknowledgments: The authors thank Nikita Abraham and Diana Lim for graphic design expertise and figure preparation, and Matthew Rondina and Jesse Rowley for insightful discussions. The authors thank The University of Utah DNA/Peptide Synthesis and Flow Cytometry Cores. The authors also thank the Division of Neonatology, Department of Pediatrics at The University of Utah for support of the project.

This work was supported by grants from the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD093826 [C.C.Y.]), National Heart, Lung, and Blood Institute (RO1HL163019 and RO1HL160808 [R.A.C.], RO1HL167919 [A.C.P.], K08HL153953 [E.A.M.], and T32HL105321 [J.S.B.]), and the International Society on Thrombosis and Haemostasis Fundamental Grant Award (F.D.).

Contribution: J.S.B. and C.C.Y. conceived of the project and designed the experiments; J.S.B., F.D., M.J.C., C.V.d.A., and R.A.C. conducted experiments; J.S.B., F.D., A.C.P., E.A.M., R.A.C., and C.C.Y. wrote portions of the manuscript; and J.S.B. and C.C.Y. provided overall direction for the project, reviewed and analyzed data from all experiments, wrote sections of the manuscript, and edited the manuscript.

Conflict-of-interest disclosure: C.C.Y. is the author of a US patent (patent number 10,232,023 B2) held by The University of Utah for the use of neutrophil extracellular trap-inhibitory peptides for the “treatment of and prophylaxis against inflammatory disorders,” for which Peel Therapeutics, Inc holds the exclusive license. The remaining authors declare no competing financial interests.

Correspondence: Christian C. Yost, Department of Pediatrics, The University of Utah, Williams Building, 295 Chipeta Way, Salt Lake City, UT 84108; email: christian.yost@u2m2.utah.edu.