The power from within - understanding the driving forces of neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) are one of the most intriguing discoveries in immunological research of the past few years. After their first description in 2004, the number of research articles on how NETs affect immunodefense, and also how they contribute to an ever-growing number of diseases, has skyrocketed. However, tempting as it may seem to plunge into pharmaceutical approaches to tamper with NET formation, our understanding of this complex process is still incomplete. Important concepts such as the context-dependent dual functions of NETs, in that they are both inflammatory and anti-inflammatory, or the major intra- and extracellular forces driving NET formation, are only emerging. In this Review, we summarize key aspects of our current understanding of NET formation (also termed NETosis), emphasize biophysical aspects and focus on three key principles - rearrangement and destabilization of the plasma membrane and the cytoskeleton, alterations and disassembly of the nuclear envelope, and chromatin decondensation as a driving force of intracellular reorganization.

Near-Infrared Imaging of Serotonin Release from Cells with Fluorescent Nanosensors.

Serotonin is an important neurotransmitter involved in various functions of the nervous, blood, and immune system. In general, detection of small biomolecules such as serotonin in real time with high spatial and temporal resolution remains challenging with conventional sensors and methods. In this work, we designed a near-infrared (nIR) fluorescent nanosensor (NIRSer) based on fluorescent single-walled carbon nanotubes (SWCNTs) to image the release of serotonin from human blood platelets in real time. The nanosensor consists of a nonbleaching SWCNT backbone, which is fluorescent in the beneficial nIR tissue transparency window (800-1700 nm) and a serotonin binding DNA aptamer. The fluorescence of the NIRSer sensor (995 nm emission wavelength for (6,5)-SWCNTs) increases in response to serotonin by a factor up to 1.8. It detects serotonin reversibly with a dissociation constant of 301 nM ± 138 nM and a dynamic linear range in the physiologically relevant region from 100 nM to 1 µM. As a proof of principle, we detected serotonin release patterns from activated platelets on the single-cell level. Imaging of the nanosensors around and under the platelets enabled us to locate hot spots of serotonin release and quantify the time delay (≈ 21-30 s) between stimulation and release in a population of platelets, highlighting the spatiotemporal resolution of this nanosensor approach. In summary, we report a nIR fluorescent nanosensor for the neurotransmitter serotonin and show its potential for imaging of chemical communication between cells.

GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment.

Tumor tissues often contain high extracellular adenosine, promoting an immunosuppressed environment linked to mesenchymal transition and immune evasion. Here, we show that loss of the epithelial transcription factor, GRHL2, triggers NT5E/CD73 ecto-enzyme expression, augmenting the conversion of AMP to adenosine. GRHL2 binds an intronic NT5E sequence and is negatively correlated with NT5E/CD73 in breast cancer cell lines and patients. Remarkably, the increased adenosine levels triggered by GRHL2 depletion in MCF-7 breast cancer cells do not suppress but mildly increase CD8 T cell recruitment, a response mimicked by a stable adenosine analog but prevented by CD73 inhibition. Indeed, NT5E expression shows a positive rather than negative association with CD8 T cell infiltration in breast cancer patients. These findings reveal a GRHL2-regulated immune modulation mechanism in breast cancers and show that extracellular adenosine, besides its established role as a suppressor of T cell-mediated cytotoxicity, is associated with enhanced T cell recruitment.

Susceptibility of Melanoma Cells to Targeted Therapy Correlates with Protection by Blood Neutrophils.

Elevated levels of peripheral blood and tumor tissue neutrophils are associated with poorer clinical response and therapy resistance in melanoma. The underlying mechanism and the role of neutrophils in targeted therapy is still not fully understood. Serum samples of patients with advanced melanoma were collected and neutrophil-associated serum markers were measured and correlated with response to targeted therapy. Blood neutrophils from healthy donors and patients with advanced melanoma were isolated, and their phenotypes, as well as their in vitro functions, were compared. In vitro functional tests were conducted through nonadherent cocultures with melanoma cells. Protection of melanoma cell lines by neutrophils was assessed under MAPK inhibition. Blood neutrophils from advanced melanoma patients exhibited lower CD16 expression compared to healthy donors. In vitro, both healthy-donor- and patient-derived neutrophils prevented melanoma cell apoptosis upon dual MAPK inhibition. The effect depended on cell-cell contact and melanoma cell susceptibility to treatment. Interference with protease activity of neutrophils prevented melanoma cell protection during treatment in cocultures. The negative correlation between neutrophils and melanoma outcomes seems to be linked to a protumoral function of neutrophils. In vitro, neutrophils exert a direct protective effect on melanoma cells during dual MAPK inhibition. This study further hints at a crucial role of neutrophil-related protease activity in protection.

Expansion microscopy of neutrophil nuclear structure and extracellular traps.

Neutrophils are key players of the immune system and possess an arsenal of effector functions, including the ability to form and expel neutrophil extracellular traps (NETs) in a process termed NETosis. During NETosis, the nuclear DNA/chromatin expands until it fills the whole cell and is released into the extracellular space. NETs are composed of DNA decorated with histones, proteins, or peptides, and NETosis is implicated in many diseases. Resolving the structure of the nucleus in great detail is essential to understand the underlying processes, but so far, superresolution methods have not been applied. Here, we developed an expansion-microscopy-based method and determined the spatial distribution of chromatin/DNA, histone H1, and nucleophosmin with an over fourfold improved resolution (<40-50 nm) and increased information content. It allowed us to identify the punctate localization of nucleophosmin in the nucleus and histone-rich domains in NETotic cells with a size of 54-66 nm. The technique could also be applied to components of the nuclear envelope (lamins B1 and B2) and myeloperoxidase, providing a complete picture of nuclear composition and structure. In conclusion, expansion microscopy enables superresolved imaging of the highly dynamic structure of nuclei in immune cells.

Anti-citrullinated histone monoclonal antibody CIT-013, a dual action therapeutic for neutrophil extracellular trap-associated autoimmune diseases.

Neutrophil extracellular traps (NETs) contribute to the pathophysiology of multiple inflammatory and autoimmune diseases. Targeting the NETosis pathway has demonstrated significant therapeutic potency in various disease models. Here, we describe a first-in-class monoclonal antibody (CIT-013) with high affinity for citrullinated histones H2A and H4, which inhibits NETosis and reduces tissue NET burden in vivo with significant anti-inflammatory consequences. We provide a detailed understanding of the epitope selectivity of CIT-013. Detection of CIT-013 epitopes in rheumatoid arthritis (RA) synovium provides evidence that RA is an autoimmune disease with excessive citrullinated NETs that can be targeted by CIT-013. We show that CIT-013 acts upon the final stage of NETosis, binding to its chromatin epitopes when plasma membrane integrity is compromised to prevent NET release. Bivalency of CIT-013 is necessary for NETosis inhibition. In addition, we show that CIT-013 binding to NETs and netting neutrophils enhance their phagocytosis by macrophages in an Fc-dependent manner. This is confirmed using a murine neutrophilic airway inflammation model where a mouse variant of CIT-013 reduced tissue NET burden with significant anti-inflammatory consequences. CIT-013's therapeutic activity provides new insights for the development of NET antagonists and indicates the importance of a new emerging therapy for NET-driven diseases with unmet therapeutic needs.

Transport and programmed release of nanoscale cargo from cells by using NETosis.

Cells can take up nanoscale materials, which has important implications for understanding cellular functions, biocompatibility as well as biomedical applications. Controlled uptake, transport and triggered release of nanoscale cargo is one of the great challenges in biomedical applications of nanomaterials. Here, we study how human immune cells (neutrophilic granulocytes, neutrophils) take up nanomaterials and program them to release this cargo after a certain time period. For this purpose, we let neutrophils phagocytose DNA-functionalized single-walled carbon nanotubes (SWCNTs) in vitro that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling, subsequent rupture of the cellular membrane and release of the cell's whole content. By using the process of NETosis, we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation, cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release the SWCNTs. The transported and released SWCNT sensors are still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils transport and release functional nanomaterials.

Blue and Long-Wave Ultraviolet Light Induce in vitro Neutrophil Extracellular Trap (NET) Formation.

Neutrophil Extracellular Traps (NETs) are produced by neutrophilic granulocytes and consist of decondensed chromatin decorated with antimicrobial peptides. They defend the organism against intruders and are released upon various stimuli including pathogens, mediators of inflammation, or chemical triggers. NET formation is also involved in inflammatory, cardiovascular, malignant diseases, and autoimmune disorders like rheumatoid arthritis, psoriasis, or systemic lupus erythematosus (SLE). In many autoimmune diseases like SLE or dermatomyositis, light of the ultraviolet-visible (UV-VIS) spectrum is well-known to trigger and aggravate disease severity. However, the underlying connection between NET formation, light exposure, and disease exacerbation remains elusive. We studied the effect of UVA (375 nm), blue (470 nm) and green (565 nm) light on NETosis in human neutrophils ex vivo. Our results show a dose- and wavelength-dependent induction of NETosis. Light-induced NETosis depended on the generation of extracellular reactive oxygen species (ROS) induced by riboflavin excitation and its subsequent reaction with tryptophan. The light-induced NETosis required both neutrophil elastase (NE) as well as myeloperoxidase (MPO) activation and induced histone citrullination. These findings suggest that NET formation as a response to light could be the hitherto missing link between elevated susceptibility to NET formation in autoimmune patients and photosensitivity for example in SLE and dermatomyositis patients. This novel connection could provide a clue for a deeper understanding of light-sensitive diseases in general and for the development of new pharmacological strategies to avoid disease exacerbation upon light exposure.

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation.

Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes, and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well-understood. Here, we studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Young's modulus E) within the physiologically relevant range from 1 to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.

Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs).

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

Control of Integrin Affinity by Confining RGD Peptides on Fluorescent Carbon Nanotubes.

Integrins are transmembrane receptors that mediate cell-adhesion, signaling cascades and platelet-mediated blood clotting. Most integrins bind to the common short peptide Arg-Gly-Asp (RGD). The conformational freedom of the RGD motif determines how strong and to which integrins it binds. Here, we present a novel approach to tune binding constants by confining RGD peptide motifs via noncovalent adsorption of single-stranded DNA (ssDNA) anchors onto single-walled carbon nanotubes (SWCNTs). Semiconducting SWCNTs display fluorescence in the near-infrared (nIR) region and are versatile fluorescent building blocks for imaging and biosensing. The basic idea of this approach is that the DNA adsorbed on the SWCNT surface determines the conformational freedom of the RGD motif and affects binding affinities. The RGD motif was conjugated to different ssDNA sequences in both linear ssDNA-RGD and bridged ssDNA-RGD-ssDNA geometries. Molecular dynamics (MD) simulations show that the RGD motif in all the synthesized systems is mostly exposed to solvent and thus available for binding, but its flexibility depends on the exact geometry. The affinity for the human platelet integrin αIIbß3 could be modulated up to 15-fold by changing the ssDNA sequence. IC50 values varied from 309 nM for (C)20-RGD/SWCNT hybrids to 29 nM for (GT)15-RGD/SWCNT hybrids. When immobilized onto surface adhesion of epithelial cells increased 6-fold for (GT)15-RGD/SWCNTs. (GT)15-RGD/SWCNTs also increased the number of adhering human platelets by a factor of 4.8. Additionally, αIIbß3 integrins on human platelets were labeled in the nIR by incubating them with these ssDNA-peptide/SWCNT hybrids. In summary, we show that ssDNA-peptide hybrid structures noncovalently adsorb onto SWCNTs and serve as recognition units for cell surface receptors such as integrins. The DNA sequence affects the overall RGD affinity, which is a versatile and straightforward approach to tune binding affinities. In combination with the nIR fluorescence properties of SWCNTs, these new hybrid materials promise many applications in integrin targeting and bioimaging.

Chromatin swelling drives neutrophil extracellular trap release.

Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.