A fluorogenic peptide-based smartprobe for the detection of neutrophil extracellular traps and inflammation

Article abstract of DOI:10.1039/d0cc07028a

A highly sensitive optical probe for the detection of activated neutrophils and Neutrophil Extracellular Traps (NETs) is reported. It is based on a triple-quenched, super-silent tri-branched probe that generates >20 fold increase in fluorescence upon cleavage. The probe was highly specific for human neutrophil elastase, a protease that mediates a variety of inflammatory diseases, and detected NETosis and neutrophil activation in in vitro differentiated neutrophils and isolated human neutrophils. This journal is

Full text of DOI:10.1039/d0cc07028a

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A fluorogenic peptide-based smartprobe for the
detection of neutrophil extracellular traps and
inflammation†
Cite this:DOI: 10.1039/d0cc07028a
Received 22nd October 2020,
Accepted 30th November 2020
a
a
Maria R. Rios,
Gloria Garoffolo,b Giulia Rinaldi,c Alicia Megia-Fernandez,
Silvia Ferrari,b Calum T. Robb,c Adriano G. Rossi,c Maurizio Pesce
and
b
a
DOI: 10.1039/d0cc07028a
Mark Bradley
*
rsc.li/chemcomm
A highly sensitive optical probe for the detection of activated and aberrant NET formation is linked to pulmonary diseases
neutrophils and Neutrophil Extracellular Traps (NETs) is reported. like acute respiratory distress syndrome (ARDS), acute lung
It is based on a triple-quenched, super-silent tri-branched probe injury (ALI)8 and more recently, COVID-19.9 Thus, neutrophils
that generates 420 fold increase in fluorescence upon cleavage. isolated from COVID-19 patients that exhibit pneumonia-
The probe was highly specific for human neutrophil elastase, a associated ARDS are more susceptible to form NETs; a response
protease that mediates a variety of inflammatory diseases, and that correlates with severity and mortality of the disease.8 NETs
detected NETosis and neutrophil activation in in vitro differentiated forming in arteries and veins are thought to be important in
neutrophils and isolated human neutrophils.
initiating thrombosis in the vasculature, again of relevance in
the immunopathology in COVID-19 patients.10 Detection and
Neutrophils (phagocytic leukocytes) are the most abundant visualisation of NETs has therefore become a potential tool for
immune cells circulating in human blood and amongst the diagnosis of NET-associated pathologies7 however, to date, this
first cells to be recruited to inflammatory sites, providing a first has largely been based on immuno-staining of NET markers
line of immune defence against pathogens.1 However, activa- using antibodies.
tion of neutrophils is associated with many inflammatory
Activated serine proteases are present on NETs. Human
diseases and has become a cellular biomarker for a broad neutrophil elastase (hNE) is one of the four active neutrophil
range of inflammation-mediated diseases.2 Neutrophils have serine proteases. In fact, it is the most abundant serine
a wide range of defence mechanisms against invading micro- protease stored in neutrophil granules at millimolar concen-
organisms, including phagocytosis, degranulation and reactive tration. This protease is present in NETs and found bound to
oxygen species (ROS) production. In 2004, Brinkmann et al.3 DNA, the key scaffold component of NETs, due to hNE high
reported a novel process by which activated neutrophils extrude binding affinity to chromatin. hNE is thus a good target for
chromatin studded with antimicrobial proteins in the extra- detection and visualisation of NETs. Several inhibitors for hNE
cellular environment to entrap and kill invading pathogens, have been developed and some of them used clinically including
such as bacteria,3 virus4 and fungi.5 The structures formed are the inhibitor sivelestat,11 which is used to treat COVID-19
named Neutrophil Extracellular Traps (NETs).3 These extra- patients.12 When it comes to protease sensing most substrates
cellular traps are not restricted to neutrophils as they can be rely on chromogenic sensing or fluorescence, and for hNE the vast
produced by many cell types in a broad range of organisms and majority are based on the peptide sequence AAPV which is also
indeed are believed to be an evolutionary preserved defence cleavable by the closely related serine protease Proteinase 3
mechanism.6 Despite their beneficial role in host defence NETs (PR3).13 Only one highly specific sensor for hNE has been
are known to play pivotal roles in many inflammatory diseases7 validated as a preclinical fluorescent activated sensor and this
was based on the sequence PMAVVQSVP.14 Activity based probes
a EaStCHEM School of Chemistry, University of Edinburgh, David Brewster Road,
EH9 3FJ Edinburgh, UK. E-mail: Mark.Bradley@ed.ac.uk
b Tissue Engineering Research Unit, Centro Cardiologico Monzino, IRCCS,
Milan, Italy
for elastase, that bind covalently to the enzyme have been
reported for hNE, however, signal amplification is not possible
as the enzyme is generally inhibited irreversibly following the
enzymatic reaction.15 Thus, there is a need for sensing methods
that show high specificity, sensitivity and high signal-to-noise
ratios. Peptide based ‘‘dendritic’’ or multi-branched sensors offer
a promising approach to overcome these limitations as they
c Centre for Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh, UK
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc07028a
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detection and visualisation of NETs – the first molecular probe
developed that detects hNE in NETs and inflammatory induced
conditions.
Importantly this sensor provides an OFF/ON fluorescent signal
upon cleavage by hNE in activated neutrophils and NETs. The
sensor was based on a trivalent scaffold combined with FRET-
labelled peptides (Scheme 1). The trivalent structure provides
amplification of signal upon substrate (peptide) cleavage and
enhanced self-quenching capacity by linear quenching between
the fluorophores and the quenchers.19 The sequence (Glu–Glu–
IlemNle–Arg–Arg) was adapted from a peptide20 that is cleaved
specifically by hNE, where modification of methionine in the
reported sequence with norleucine provides in vivo stability. The
peptide was labelled with the fluorophore 5-carboxyfluorescein
(5-FAM) and quencher Methyl Red (MR) as the FRET pair. Methyl
red was incorporated as Fmoc-Lys(MR)-OH (4) (ESI†), avoiding the
need for orthogonal deprotection21 on the lysine, which would
complicate the synthesis of these branched peptides due to
incomplete deprotection on one or more branches. A bis-
Scheme 1 A triple-quenched, super-silent tri-branched probe (HNE-FQ),
which upon specific cleavage by human neutrophil elastase (hNE) liberates
three fluorescent molecules resulting in an increase in fluorescence.
potentially provide significantly higher signal amplification and ethylene glycol spacer was introduced between the last amino
lower background noise when compared to their linear counter- acid and the fluorophore, to increase hydrophilicity and promote
parts as well as offering high sensitivity.16 Our group previously aqueous solubility. The probe was synthesised on an amino-
reported activatable probes for serprocidins17 (hNE, PR3 and CG) methyl ChemMatrix resin functionalised with the Fmoc-Rink
and elastase18 sensing but they proved unsuccessful for NETs Amide linker. The starting trivalent isocyanate (1) was synthesized
visualisation, while their high background fluorescence, made following a previously reported procedure22 and the isocyanate
them unsuitable for sensing and in vivo applications. Herein, we coupled to the resin using DIPEA and DMAP. Removal of the
present a novel fluorogenic sensor, specific for hNE that allows Dde (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)) protecting
Fig. 1 (a) Probe synthesis started by attachment of compound 1 to a Rink-amide-ChemMatrix resin that served as a scaffold for the synthesis of the
FRET-peptide using an Fmoc/tBu solid-phase peptide synthesis strategy, with 5-carboxyfluorescein and methyl red as fluorophores and quenchers,
respectively (FRET pair). (b) Fluorescence spectrum of the probe HNE-FQ (5 mM) before (black) and after (grey) 40 minutes incubation with hNE, n = 3,
(lex/em = 495/528 nm). (c) The time-course de-quenching of probe (5 mM) following incubation with hNE (100 nM), in the presence or absence of
inhibitor Sivelestat (SIV, 100 mM) n = 3. (d) Specificity assessment of probe (5 mM) incubated in the presence of hNE (100 nM) and related serprocidins PR3
(100 nM) and Cathepsin G (300 nM) at different time points at 37 1C, n = 3.
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group with 2% hydrazine in DMF gave the scaffold (2) for the phorbol 12-myristate 13-acetate (PMA; 100 nM) and probe
synthesis of the FRET-peptides using Fmoc solid-phase peptide HNE-FQ (5 mM) for 3 h. PMA is a potent stimulator of NET
synthesis (SPPS) with DIC/Oxyma as the coupling combination with formation, since it triggers neutrophil NADPH oxidase activation
5-carboxyfluorescein capping the three peptide branches (Fig. 1a) in neutrophil activation and inflammation.24 To verify whether
(ESI†). Interestingly the use of the N-hydroxysuccinimide ester of the treatments induced NETosis, cells were treated with all-trans-
5-carboxyfluorescein diacetate in the final coupling step gave final retinoic acid (ATRA) ꢂ PMA and incubated with micrococcal
crude products that were considerably purer than those generated nuclease which disrupts and releases the DNA of NETs into the
using DIC/Oxyma/5-carboxyfluorescein (see Fig. S1, ESI†). The supernatant. DNA release was monitored by a SYTOX Orange
probe was optically silent, but rapidly de-quenched within seconds assay (ESI†). An increase in NET-associated DNA after cell
by hNE with a 20-fold increase in signal following exposure of the stimulation with PMA was observed (Fig. S4, ESI†). In order to
probe (5 mM) to the enzyme (Fig. 1b) with a KM of 2.7 ꢀ 10ꢁ6 M and visualize NETs using HNE-FQ, cells were stained with probe
a catalytic efficiency (kcat/KM) of 1.5 ꢀ 104 Mꢁ1 sꢁ1 (ESI†). Cleavage (5 mM), 40,6-diamidine-20-phenylindole dihydrochlorideI (DAPI;
specificity was confirmed by MALDI-TOF MS and LC-MS (Fig. S2 0.2 mM) and the DNA intercalating dye (SYTOX Orange; 5 mM).
and S3, ESI†), while pre-incubation with sivelestat (100 mM) resulted Morphologically, ATRA/PMA treated cells exhibited four different
in no increase of fluorescence (Fig. 1c). The closely related stages of NETosis, based on nuclear morphology and hNE
neutrophil serine proteases Cathepsin G and Proteinase 3, distribution (Fig. 2). Early stages of neutrophil activation exhib-
showed negligible cleavage demonstrating that the probe was ited a small and round nucleus with hNE colocalized with
specific for hNE (Fig. 1d). The fluorescence signal of the condensed chromatin (stage 1; yellow circles).24 At later stages,
activated probe was not affected by incubation in a range of chromatin decondensation led to spherical (stage 2; green
different biological environments (ESI†) and was stable between circles) or more ‘‘cloud-like spread’’ shapes (stage 3; red circles).
pH 6–8.5. As expected, due to the pH dependent character of In the final stage, neutrophils formed extracellular chromatin
carboxyfluorescein, fluorescence was reduced at lower pHs filaments composed by cytoplasmic granules and hNE (stage 4;
(ESI†). The human promyelocytic cell line HL-60 is a valid cell Fig. 2e).25 NETosis was induced in isolated primary human
culture model to study neutrophil differentiation, activation and neutrophils from healthy volunteers (isolated using a protocol
NETs generation.23 To validate the probe, cells were stimulated approved by the Accredited Medical Regional Ethics Committee
with all-trans-retinoic acid (ATRA, 2 mM) for 4 days to induce a (AMREC, reference number 15-HV-013)) following a similar
neutrophil-like phenotype. A quantitative nitro-blue tetrazolium procedure producing well-defined NETs (ESI†) (Fig. 3).
(NBT) assay was performed in order to assess neutrophil differ-
Activation of HL-60 neutrophils was analyzed by flow cyto-
entiation efficiency in terms of phagocytic functionality (ESI†). metry, using an antibody directed against CD11b antigen, a
For NETs formation, differentiated HL-60 cells were seeded into well-known marker of neutrophil differentiation,26 and the
8-well chamber slides coated with poly-L-lysine, and treated with probe HNE-FQ (Fig. S5, ESI†). Under these conditions, we
Fig. 2 Fluorescence microscopy images of HL-60 cells stained with DAPI (blue), HNE-FQ (green) and SYTOX orange (red). Top row left: Control HL-60
neutrophils in absence of PMA (a) blue/green channels (b) blue/red channels. Bottom row left: HL-60 neutrophils stimulated with PMA (c) blue/green
channels and (d) blue/red channels. Yellow arrows indicate cells expressing low hNE (likely at the beginning of NETosis process) while those encircled in
green, red and yellow are cells at different stages of NETosis (see text for description). (e) 3D image slice of a typical NET stained as in the panels on the
left. Arrows indicate chromatin studded with HNE-FQ, indicative of chromatin release by activated cells.
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We would like to thank Engineering and Physical Sciences
Research Council (EPSRC, UK). Interdisciplinary Research Colla-
boration grants EP/K03197X/1 and EP/R005257/1. Medical Research
Council grant MR/N02995X/1. CTR and AGR were funded by the
Medical Research Council (MR/K013386/1). GR was funded by
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Conflicts of interest
N. Vrisekoop, L. P. Leenen, M. T. Hopman, J. J. Jansen and
There are no conflicts to declare.
L. Koenderman, J. Leukocyte Biol., 2020, 1–10.
Chem. Commun.
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