Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases
Sullivan et al. Cell Death and Disease
Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases
Graeme P. Sullivan 1 3
Conor M. Henry 1 3
Danielle M. Clancy 1 3
Tazhir Mametnabiev 0 2
Ekaterina Belotcerkovskaya 0 2
Pavel Davidovich 0 2
Sylvia Sura-Trueba 0 2
Alexander V. Garabadzhiu 0 2
Seamus J. Martin 0 1 2 3
0 Cellular Biotechnology Laboratory Saint-Petersburg Technical University Moskovskii Prospekt , Saint Petersburg , Russia Edited by T. Kaufmann
1 Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College , Dublin 2 , Ireland
2 Cellular Biotechnology Laboratory Saint-Petersburg Technical University Moskovskii Prospekt , Saint Petersburg , Russia
3 Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College , Dublin 2 , Ireland
Sterile inflammation is initiated by molecules released from necrotic cells, called damage-associated molecular patterns (DAMPs). Members of the extended IL-1 cytokine family are important DAMPs, are typically only released through necrosis, and require limited proteolytic processing for activation. The IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, are expressed as inactive precursors and have been implicated as key initiators of psoriatic-type skin inflammation. We have recently found that IL-36 family cytokines are proteolytically processed and activated by the neutrophil granule-derived proteases, elastase, and cathepsin G. Inhibitors of IL-36 processing may therefore have utility as anti-inflammatory agents through suppressing activation of the latter cytokines. We have identified peptidebased pseudosubstrates for cathepsin G and elastase, based on optimal substrate cleavage motifs, which can antagonize activation of all three IL-36 family cytokines by the latter proteases. Human psoriatic skin plaques displayed elevated IL-36β processing activity that could be antagonized by peptide pseudosubstrates specific for cathepsin G. Thus, antagonists of neutrophil-derived proteases may have therapeutic potential for blocking activation of IL-36 family cytokines in inflammatory conditions such as psoriasis.
IL-1 family cytokines play major roles as initiators of
inflammation, are typically only released upon necrotic
injury, and are likely to represent canonical
dangerassociated molecular patterns (DAMPs)1–4. IL-1 family
cytokines act on multiple cell types, such as macrophages,
dendritic cells, keratinocytes, and endothelial cells lining
local blood vessels5–10. IL-36α, β, and γ are recently
described members of the IL-1 family and exhibit many of
the characteristic features of IL-1 family cytokines,
including the requirement for N-terminal processing to
release their full biological activity. As we and others have
demonstrated, removal of just a small number of residues
from the N termini of IL-36 cytokines radically increases
their biological activity11–13. This tight regulatory control
on their activity probably represents a mechanism to limit
the potential negative consequences if the activity of these
cytokines is deregulated.
IL-36α, IL-36β, and IL-36γ, which are encoded by
distinct genes, are non-conventionally secreted and it is
now well established that these cytokines are important
modulators of inflammation in barrier tissues, particularly
in skin inflammatory diseases such as psoriasis14–24.
Partial loss-of-function mutations in the IL-36 receptor
antagonist (IL-36RA) can lead to a highly debilitating
morbid form of psoriasis, termed generalized pustular
psoriasis17,18,21–23. Furthermore, analysis of skin biopsies
from individuals with the most common form of psoriasis,
psoriasis vulgaris, shows significantly increased expression
(100-fold) of all three IL-36 mRNA transcripts compared
with non-lesional skin from the same individuals, or
non-affected controls15,16. Indeed multiple lines of
evidence in vitro and in vivo confirm that deregulated
IL-36 cytokine signaling is sufficient to drive aggressive
We have recently found that the neutrophil-derived
proteases, cathepsin G and elastase, are potent
IL-36activating enzymes12. Because psoriasis plaques are
frequently associated with neutrophil infiltrates26–28, these
suggest that targeted inhibition of neutrophil granule
proteases may have significant therapeutic potential as
inhibitors of IL-36 activation in psoriasis, as well as other
inflammatory conditions characterized by neutrophil
infiltration. IL-36 cytokine or receptor neutralizing
antibody approaches are under development and are
progressing to clinical trials24,29. While systemic
antibodybased cytokine neutralization strategies targeting IL-1,
IL-17, and IL-17/23 have greatly improved therapeutic
outcomes for patients with severe plaque psoriasis, such
therapies are costly and can be associated with serious
side effects30,31. Targeted, localized inhibition of IL-36
cytokine activation in the skin, through direct application
of antagonists of IL-36 proteolytic processing, may be an
attractive and cost-effective alternative to systemic
cytokine neutralization approaches.
Here, we have identified peptide-based antagonists of
IL-36 activation based upon optimal cleavage motifs and
substrate preferences for the neutrophil granule proteases,
elastase, and cathepsin G. These pseudosubstrates exhibit
considerable potency against processing and activation of
all three IL-36 cytokines in vitro. We also demonstrate
that extracts from human psoriatic skin plaques display
elevated IL-36β processing that can be antagonized by the
latter inhibitors. Direct application of antagonists of IL-36
processing and activation to inflammatory skin lesions
may represent a novel strategy to attenuate psoriatic
Neutrophil proteases process and activate IL-36 family cytokines
Similar to other members of the IL-1 family, such as
IL-1β and IL-18, IL-36 cytokines exhibit little
proinflammatory activity as full-length proteins upon
incubation with HeLa cells stably transfected with the IL-36
receptor (Fig. 1a). However, as we have recently reported12,
IL-36 cytokines acquire potent pro-inflammatory activity
upon incubation with supernatants derived from
PMAactivated human neutrophils that contain the
granulederived proteases, elastase, proteinase-3, and cathepsin G.
As shown in Fig. 1b, HeLaIL-36R cells secreted robust
amounts of IL-6 and IL-8 upon incubation with
recombinant IL-36 cytokines that had been pre-incubated with
PMA-activated human neutrophil degranulates, which
leads to processing and activation of the latter cytokines12.
Moreover, incubation of IL-36 cytokines with purified
elastase or cathepsin G also robustly activated the latter
cytokines, with cathepsin G selectively activating IL-36β,
elastase selectively activating IL-36γ, and elastase or
cathepsin G both capable of activating IL-36α (Fig. 1c).
Figure 1d summarizes the preferences of neutrophil
proteases for processing and activation of IL-36 family
cytokines and the relevant protease cleavage sites, as recently
reported12. Thus, activated neutrophils can liberate
proteases that can promote inflammation through
extracellular processing and activation of IL-36 cytokines.
Human neutrophil degranulate preparations contain elastase and cathepsin G
It is well established that primary human neutrophils
can be induced to release the contents of their secretory
granules into the extracellular space upon activation32–34.
To establish sensitive assays for the detection of elastase
and Cat G activity (Fig. 2a), we used the synthetic
substrate peptides, FLF-sBzL (which is cleaved by cathepsin
G) and AAPV-AMC (which is cleaved by elastase). Using
these synthetic substrates, robust cathepsin G (i.e.,
FLFsBzL hydrolysis) and elastase activity (i.e., AAPV-AMC
hydrolysis) were readily detected in PMA-activated
human neutrophil degranulate preparations, as expected
(Fig. 2b). Furthermore, the latter proteases were robustly
inhibited by commercially available small molecule
inhibitors of cathepsin G, or elastase, respectively (Fig. 2b).
Using known amounts of purified cathepsin G or elastase
as calibrants (Fig. 2c), we estimated the concentration of
the latter enzymes within neutrophil degranulate
preparations prepared at 1 × 107 cells/mL to be in the range
of ~100–200 nM, respectively.
Identification of novel peptide-based antagonists of
elastase and cathepsin G
To seek novel peptide-based inhibitors of cathepsin
G- and elastase-mediated IL-36 processing and activation,
we designed a panel of tri- and tetra-peptides (Table 1),
based upon optimal recognition sequences for these
proteases35, as well as protease cleavage sites implicated in
the activation of IL-36 cytokines12. We then explored the
potential inhibitory activity of this peptide panel toward
cathepsin G and elastase. As Fig. 3a demonstrates, several
of the candidate cathepsin G inhibitory peptides exhibited
robust activity against purified cathepsin G, with
z-FLFcmk, z-EPF-cmk, and z-AFLF-cmk demonstrating the
greatest potency in this regard. As expected, none of the
latter peptides inhibited elastase activity (Fig. 3b).
Similarly, several of the candidate elastase inhibitory
peptides exhibited robust inhibitory activity against
elastase, but not cathepsin G, with z-API demonstrating
the greatest potency (Fig. 3c, d).
(see figure on previous page)
Fig. 1 IL-36 cytokines require proteolytic processing for activation. a HeLaIL-36R cells were either untreated, or were stimulated with full-length
IL-36α, IL-36β, or IL-36γ, at the indicated concentrations. After 24 h, cytokine concentrations in the culture supernatants were determined by ELISA.
Caspase-3-cleaved DEVD-IL-36γ (0.5 nM), where a caspase-3-processing motif (DEVD) was inserted into the IL-36 sequence N-terminal to the known
processing sites12,36, was used as a positive control. b HeLaIL-36R cells were stimulated either alone, or in the presence of either IL36α, β, or γ (500 pM)
that had been pre-incubated for 2 h at 37 °C with unstimulated neutrophil supernatant (Ctrl s/n), or 1:2 serial dilutions of PMA-activated neutrophil
supernatants (PMA s/n). After 24 h, cytokine concentrations in the culture supernatants were determined by ELISA. c HeLaIL-36R cells were stimulated
either alone, or with IL36α, β, or γ (500 pM) that had been pre-incubated for 2 h at 37 °C with the indicated concentrations of purified human
cathepsin G, elastase, or proteinase-3. After 24 h, cytokine concentrations in the culture supernatants were determined by ELISA. d Schematic
representation of cathepsin G, elastase, and proteinase-3 cleavage motifs within IL-36 family cytokines. Results shown are representative of at least
three independent experiments. Error bars represent the mean ±SEM of triplicate determinations from a representative experiment
Pseudosubstrates for elastase and cathepsin G can suppress IL-36 cytokine activation
Having found peptides that inhibited cathepsin G or
elastase activity in the context of synthetic substrate
peptide hydrolysis assays, we next explored the ability of
these peptides to inhibit activation of all three IL-36
cytokines by neutrophil degranulate preparations, which
contain a mixture of these proteases (Fig. 2b). To detect
IL-36 cytokine activity, we used HeLa cells stably
transfected with the IL-36 receptor36, as these cells secrete
robust amounts of IL-6 and IL-8 in response to
biologically active IL-36 (Fig. 1b, c). As Fig. 4a, b illustrate,
activation of IL-36β by neutrophil degranulate
preparations was robustly inhibited in the presence of the
cathepsin G inhibitory peptides, z-FLF-cmk, z-AFLF-cmk,
and z-EPF-cmk (Fig. 4a), with a modest effect of the same
peptides on IL-36α activation (Fig. 4b). Once again we
found that the inhibitory peptides were highly selective in
their ability to antagonize activation of specific IL-36
cytokines, as cathepsin G-specific inhibitors failed to
antagonize elastase-mediated IL-36γ activation by
neutrophil degranulates, as expected (Fig. 4c). Time course
analyses further confirmed the robust inhibitory effects of
the same panel of inhibitors against IL-36β processing by
cathepsin G (Fig. 5).
Similarly, the novel elastase-specific peptides (z-AAPV,
z-API, and z-APV) antagonized activation of IL-36γ by
the latter protease (Fig. 6a), with no effects on IL-36α or
IL-36β activation by neutrophil degranulates (Fig. 6b, c).
We then assessed the ability of the best performing
cathepsin G (z-EPF-cmk) and elastase (z-API-cmk)
inhibitors to suppress the activation of all three IL-36
family cytokines when these were simultaneously present.
As illustrated in Fig. 7, the combination of all three IL-36
cytokines exhibited robust activity in the presence of
PMA-activated neutrophil degranulates and this was
significantly attenuated by either peptide inhibitor alone.
However, the combination of elastase-specific and
cathepsin G-specific inhibitors completely blocked the
processing and activation of all three IL-36 cytokines
when present concurrently (Fig. 7), as would be likely at
many inflammatory sites.
Bi-specific pseudosubstrates abrogate IL-36β and IL-36γ
activation by neutrophil degranulates
Having identified novel antagonists of cathepsin G and
elastase activity, we next explored whether bi-specific
peptide antagonists would also be effective at targeting
both IL-36β and IL-36γ activation by activated neutrophil
degranulates. Therefore, we generated the bi-specific
peptides, z-API-EPF-cmk and z-EPF-API-cmk, and
compared their ability to block neutrophil protease-mediated
IL-36 processing. As demonstrated in Fig. 8, IL-36β
activation by neutrophil degranulate was robustly
inhibited by the cathepsin G peptide inhibitor (z-EPF-cmk),
while IL-36γ activation was robustly inhibited by the
novel elastase peptide inhibitor (z-API-cmk), as before.
Furthermore, the bi-specific peptide antagonists
z-EPFAPI-cmk and z-API-EPF-cmk were also capable of
suppressing both IL-36β and IL-36γ activation by
PMAtreated neutrophil degranulates (Fig. 8). Similarly, when
the combination of all three IL-36 cytokines was activated
simultaneously through incubation with PMA-treated
human neutrophil degranulates, the bi-specific peptides
were also highly effective at suppressing IL-36 activation
(Fig. 9). Thus, bi-specific cathepsin G and elastase peptide
antagonists may be useful in the context of inflammatory
reactions, where multiple proteases are present
Processing of IL-36 cytokines by psoriatic skin eluates can be suppressed by pseudosubstrates for neutrophil proteases
To explore the potential utility of neutrophil protease
antagonists to suppress IL-36 cytokine activation in the
skin, we used tape-stripped samples from affected skin
areas of psoriatic individuals. Skin samples were
incubated with CHAPs-containing buffer to facilitate elution
of proteins. Our previous studies have shown that eluates
from psoriatic individuals exhibit elevated levels of
cathepsin G activity, as compared to healthy controls14.
Consistent with this, we also found that psoriatic skin
samples were capable of robustly processing and
activating exogenously added IL-36β14. As can be seen from
(see figure on previous page)
Fig. 3 Identification of novel peptide-based inhibitors of cathepsin G and elastase. a Hydrolysis of the synthetic cathepsin G substrate, FLF-sBzl,
by a fixed concentration of purified cathepsin G (20 nM), in the presence or absence of the indicated concentrations of the candidate cathepsin G
peptide inhibitors. b Hydrolysis of the synthetic elastase substrate, AAPV-AMC, by a fixed concentration of purified elastase (20 nM), in the presence or
absence of the indicated concentrations of the candidate cathepsin G peptide inhibitors. c Hydrolysis of the synthetic elastase substrate, AAPV-AMC,
by a fixed concentration of purified elastase (20 nM), in the presence or absence of the indicated concentrations of the candidate elastase peptide
inhibitors. d Hydrolysis of the synthetic cathepsin G substrate, FLF-sBzl, by a fixed concentration of purified cathepsin G (20 nM), in the presence or
absence of the indicated concentrations of peptide inhibitors. Cathepsin G inhibitor I (CG Inhib I) or elastase inhibitor IV (NE Inhib IV) served as
controls. Results shown are representative of at least three independent experiments
Fig. 10a, there was no detectable cytokine activity in these
eluates when added directly to HeLaIL-36R-SEAP cells due
to the dilution factor of ~100-fold in these assays.
However, exogenously added IL-36β (but not IL-36α or
IL36γ) was processed and activated upon addition to
psoriatic skin eluates (Fig. 10b), consistent with our previous
observations12. Because cathepsin G is the main
IL-36βprocessing protease (Fig. 1d and Ref. 12), this suggested
that the cathepsin G inhibitory peptide, z-EPF-cmk,
should suppress activation of the latter. As can be seen
from Fig. 10c, this was indeed found to be the case,
whereas the elastase-selective inhibitor z-API-cmk failed
to have any effect on IL-36β-processing activity under the
same conditions. Furthermore, the bi-specific peptide
zEPF-API-cmk also exhibited activity in this assay (Fig. 10c,
top right), as did a small molecule inhibitor of cathepsin G
(Fig. 10c, bottom left).
Collectively, our data identify novel peptide-based
inhibitors of cathepsin G or elastase and demonstrate
proof-of-principle that such inhibitors might have
potential as antagonists of IL-36 cytokine activation in
disease settings, such as psoriasis, that are associated with
elevated IL-36 cytokine activity. Such inhibitors have the
advantage that they can be applied directly to active skin
plaques, rather than systemically, thereby avoiding
unwanted global suppression of neutrophil protease
activity or IL-36 cytokine activation in other locations.
Here, we report the identification of novel
monospecific and bi-specific peptides, which exhibit significant
inhibitory activity against elastase or cathepsin G, that can
antagonize the processing and activation of all IL-36
family cytokines by the latter proteases. Because elastase
and cathepsin G have also been implicated in the
processing and activation of IL-1α, IL-1β, IL-33, IL-36RA,
and other cytokines37–41, the peptides reported herein
may also suppress the activation of multiple members of
the extended IL-1 family.
IL-1 family cytokines are central players in sterile
inflammatory diseases and autoinflammatory disorders
including rheumatoid arthritis, psoriasis, gout,
neurodegenerative disorders, and atherosclerosis, as well as
systemic autoinflammatory diseases such as
cryopyrinassociated periodic syndromes (CAPS)2. Thus, targeting
of proteases responsible for the activation of multiple IL-1
family members represents an attractive therapeutic
strategy. Given the apical nature of IL-36 cytokine
signaling in inflammatory cascades and the relatively
confined expression pattern of IL-36 cytokines to skin and
barrier tissues, significant therapeutic improvement may
be achievable through targeting this cytokine subfamily,
with less of the systemic side effects associated with
treatments targeting other apical cytokines, such as TNFα.
There is now a plethora of genetic, in vitro and in vivo
evidence implicating IL-36 cytokines in the development
of inflammatory skin conditions, most notably psoriasis.
For example, transgenic overexpression of IL-36α in the
mouse leads to a psoriasis-like condition at birth and that
can be further exacerbated with the skin irritant, phorbol
acetate14,15. The most severe form of psoriasis (GPP) has
recently been shown to be an inherited disorder
associated with hypomorphic mutations (partial
loss-of-function) in IL-36RA, the endogenous countermeasure to
excessive IL-36 activity17,18,21. The observation that a
modest reduction in the function of IL-36RA is sufficient
to promote a life-threatening form of psoriasis is
indicative of the central role for IL-36 signaling in the skin.
Further studies have shown that application of a TLR7
agonist (Imiquimod) to the skin of humans and mice can
initiate psoriatic lesions that are dramatically worsened on
an IL-36RA null background, whereas
imiquimodinduced psoriasis is completely abolished in
IL-36Rdeficient mice19,25. IL-36 proteins are highly expressed in
keratinocytes and upregulated in response to IL-17, IL-22,
and TNF– cytokines that are frequently overexpressed in
psoriasis16,42,43. Related to this, it has recently been shown
that IL-36 cytokines, in particular IL-36β, can induce
robust production of pro-inflammatory cytokines (such as
IL-6 and TNF) from diverse cell types including skin
resident (Langerhans) DCs and macrophages, as well as
A characteristic feature of psoriatic inflammation is
robust neutrophil-rich cellular infiltrates26,27,44.
Neutrophils play a critical role in the first phase of the
immune response to infection or tissue damage45–47.
Fig. 4 (See legend on next page.)
While release of neutrophil proteases can exert important
protective effects during infection, if left unchecked these
proteases can cause collateral tissue damage and amplify
inflammation48–50. Thus, damage to barrier tissues, as a
result of infection or tissue trauma, that results in
neutrophil recruitment, release of neutrophil-derived
(see figure on previous page)
Fig. 4 Novel peptide-based inhibitors of cathepsin G suppress IL-36β activation by proteases contained within neutrophil degranulates.
a–c HeLaIL-36R-SEAP cells were either left untreated, or were stimulated either with recombinant IL-36α, IL-36β, or IL-36γ (500 pM) that had been
preincubated for 2 h at 37 °C, either alone, with unstimulated neutrophil supernatant (Ctrl s/n), or PMA-activated human neutrophil supernatant (PMA s/
n) in the presence or absence of a titration (10, 5, 2.5, 1, 0.5, or 0.25 μM) of candidate peptide inhibitors. After 24 h, cytokine concentrations in the
culture supernatants were determined by ELISA. Results shown are representative of at least three independent experiments. Error bars represent the
mean ±SEM of triplicate determinations from a representative experiment. Asterisk(s) indicate significance levels, ***p < .0001, **p < .001, *p < .1, by
Student’s t test
proteases and subsequent processing and activation of
IL36 cytokines, may play an important initiating role in
psoriasis. This is particularly relevant in the context of
individuals lacking endogenous buffers of IL-36
activity, such as deficiency in the IL-36R antagonist
(DITRA)17,18,21–23. Although neutrophil protease
inhibitors have not yet been approved for use in the
treatment of psoriasis, targeted depletion of neutrophils
in patients with GPP led to significant clinical
improvement, providing support for the rationale for
targeting neutrophil proteases in the context of skin
At present, biologics directed against the cytokines
TNFα, IL-17, IL-22, and IL-12/23 have been approved for
the treatment of moderate-severe plaque psoriasis by the
FDA and EMA31. However, while this approach can be
effective, systemic administration of cytokine-neutralizing
antibodies can have serious negative side effects including
increased susceptibility to opportunistic infections,
re-activation of hepatitis B/tuberculosis (TB), and risk of
development of lymphoma51,52. Indeed, a recent
meta-analysis revealed that biologics are associated with
significantly higher rates of adverse events in the short
term, precluding their use in less severe cases30.
Furthermore, systemic administration of these therapies
may fail to reach sufficient plasma concentrations to be
effective, suffering the effects of first pass metabolism. In
contrast, topical application of therapeutic peptides or
small molecule protease inhibitors to affected mucosa
may be of particular benefit in skin inflammatory
conditions, owing to their ease of application, lower cost,
localized effects, and patient familiarity with existing
topical treatments for their condition.
The primary challenges associated with peptide delivery
include overcoming difficulties with skin penetration and
maintaining peptide stability53. It is generally regarded
that molecules >400 Da are unsuitable for transdermal
delivery54. However, in psoriasis patients it has been
demonstrated that measures of transdermal molecular
trafficking are increased55,56, thus the >400 Da cutoff
may not strictly apply in this context. Furthermore, a
variety of skin penetration techniques exist to
overcome this obstacle including chemical techniques
(e.g., permeation enhancers such as alcohols, amines,
surfactants, esters, lipid-systems, or pro-drug
formulations) and physical techniques (e.g., iontophoresis,
electroporation, and microporation). Similarly,
techniques to improve peptide stability have also been
developed to overcome this limitation and include
direct structural modification (e.g., cyclization and
PEGylation), co-administration with enzyme inhibitors,
hyperglycosylation and use of carrier systems (e.g.,
liposomes, micelles, and nanoparticle carriers). Thus, viable
topical peptide antagonist delivery systems are currently
As an alternative to peptide-based antagonists of
neutrophil proteases, a number of small molecule inhibitors
of neutrophil cathepsin G or elastase/proteinase-3 have
been reported previously35,57–61. Although clinical trials
have been conducted with these compounds to explore
their potential utility in acute lung injury and
infectiondriven pulmonary inflammation, to our knowledge, none
of these inhibitors have been explored for their potential
to suppress inflammation in the skin. Although the latter
inhibitors have demonstrated limited efficacy in lung
models of infection or injury-driven lung inflammation, it
may be worth exploring their impact on psoriatic
inflammation in future studies. Potential limiting factors
relating to the failure of small molecule neutrophil
protease inhibitors to demonstrate utility in pulmonary
models of inflammation to date, may relate to issues of
poor bioavailability, poor tissue penetrance, and lack of
selectivity in vivo. However, direct application of such
inhibitors to affected areas of inflamed skin, rather than
via oral or intravenous administration, may represent a
promising approach for future investigations.
In summary, here we have identified several novel
peptide-based inhibitors of neutrophil-derived cathepsin
G or elastase that may have potential as therapeutic
modulators of IL-36 cytokine activity in inflammatory
conditions such as psoriasis.
Synthetic peptides, Ac-DEVD-AMC and suc-FLF-sBzl,
were purchased from Bachem (Germany);
Suc(oMe)AAPV-AMC was purchased from Peptanova (Germany).
Synthetic peptides, biotin-FLF-cmk, z-FLF-cmk,
z-EPFcmk, z-AFLF-cmk, z-GLF-cmk, z-GLW-cmk,
z-GLKcmk, z-AAPV, z-API, z-APL, z-APV, z-ARPV, z-RPI,
zRPL, z-RPV, z-PQR, z-DTEF, z-API-EPF-cmk,
z-EPFAPI-cmk, and -cmk derivatives thereof were synthesized
by Boston Open Labs (USA). Chemical inhibitors
Cathepsin G Inhibitor I (219372) and Elastase Inhibitor IV
(324759) were purchased from Calbiochem (UK). Purified
neutrophil-derived cathepsin G was purchased from
Calbiochem (UK). Purified neutrophil-derived elastase
was purchased from Serva (Germany). Unless otherwise
indicated, all other reagents were purchased from Sigma
HeLa cells were cultured in RPMI media (Gibco),
supplemented with 5% fetal calf serum (FCS). HeLa.
vector or HeLa.IL-36R cell lines were generated by
transfection with pCXN2.empty or pCXN2.IL-1Rrp2
(IL36R) plasmids followed by selection using G-418
antibiotic (Sigma). IL-36R over-expressing clones were
expanded from single cells. Clones were selected by
detection of responsiveness to active forms of IL-36
via ELISA. The HeLa.IL-36R.SEAP cell line was
generated by transfection with pNifty2-SEAP plasmid
(InvivoGen) followed by selection using zeocin antibiotic,
as previously described36. Clones were expanded from
single cells and tested for SEAP production. All cells were
cultured at 37 °C in a humidified atmosphere with 5%
Expression and purification of recombinant IL-36 and
Full-length IL-36α, IL-36β, and IL-36γ cytokines were
generated by cloning the human coding sequences in
frame with the poly-histidine tag sequence in the bacterial
expression vector pET45b. Protein was expressed by
addition of 600 μM IPTG to exponentially growing
cultures of BL21 RIL strain E. coli followed by incubation
for 3 h at 37 °C. Bacteria were lysed by sonication and
poly-histidine-tagged proteins were captured using
nickel-NTA agarose (Qiagen, UK), followed by elution
into PBS, pH 7.2, in the presence of 100 mM imidazole.
Modified forms of IL-36 were also generated that included
an N-terminal caspase-3-processing motif (DEVD) in the
IL-36 coding sequences36. Recombinant
poly-histidinetagged caspases -1 and -3 were also expressed and purified
as described previously62.
Protease activity assays
Reactions (50 μl final volume) were carried out
in protease reaction buffer (50 mM HEPES, pH 7.4,
75 mM NaCl, 0.1% CHAPS, 2 mM DTT) containing 50
μM Ac-DEVD-AMC, or Suc(oMe)-AAPV-AMC.
Samples were measured using an automated
fluorimeter (SPARK 10 M; TECAN) at wavelengths
of 430 nm (excitation) and 535 nm (emission). For
sucFLF-sBzl assay, substrate was diluted to a final
concentration of 300 μM in protease reaction buffer (50
mM HEPES, pH 7.4, 75 mM NaCl, 0.1% CHAPS,
DTNB 300 μM). Cathepsin G hydrolyzes the
synthetic substrate suc-FLF-sBzl with the release of the
thiobenzyl group. The free thiobenzyl group reacts
with DTNB [5,5′-dithiobis(2 nitrobenzoic acid) and
produces a chromophore (TNB), which absorbs at 430 nm.
Samples were measured by automated fluorimeter
(SPARK 10 M; TECAN).
Protease cleavage assays
Reactions (40–100 μl, final volume) were carried out in
protease reaction buffer (50 mM HEPES [pH 7.2], 75 mM
NaCl, and 0.1% CHAPS) for 2 h at 37 °C. For IL-36
bioassays, IL-36 cytokines were typically cleaved at a 50
nM concentration and subsequently diluted onto target
cells at a final concentration ranging from 0.25 to 1 nM.
Measurement of cytokines and chemokines
Cytokines and chemokines were measured from cell
culture supernatants using specific ELISA kits obtained
from R&D systems (human IL-6 and human IL-8). All
cytokine assays were carried out using triplicate samples
from each culture.
Tape strip samples from control and psoriatic skin
Healthy control volunteers (n = 6) and patients with
mild/moderate psoriasis (n = 6) and not receiving
treatment in the previous 6 months were recruited. Fixomull
(2 × 2 cm) adhesive tape strips were applied to healthy
control, uninvolved or involved psoriatic skin under firm
pressure for 10 s. The tape strips were removed gently,
placed in sterile 1.5 ml Eppendorf tubes, and eluted with
protease reaction buffer (PRB) (50 mM HEPES [pH 7.2]/
75 mM NaCl/0.1% CHAPS) under constant rotation for 1
h at 4 °C. Skin eluates were stored at −80 °C. Bio-activity
assays were conducted according to the protease cleavage
assays outlined above.
This work was supported by grants from Science Foundation Ireland (SFI 14/IA/
2622) and from the Russian government for state support of scientific research
(State Contract 14.B25.310013).
G.P.S. performed experiments, analyzed data, generated the figure panels, and
wrote the figure legends. C.M.H. and D.M.C. conducted experiments. T.M., E.B.,
P.D., S.S.-T., and A.V.G. generated reagents and provided useful discussion. S.J.
M. conceived the study, designed and analyzed experiments, supervised the
study, and wrote the manuscript with contributions from G.P.S.
Conflict of interest
The authors declare that they have no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Kono , H. & Rock , K. L. How dying cells alert the immune system to danger . Nat. Rev. Immunol . 8 , 279 - 289 ( 2008 ).
2. Lukens , J. R. , Gross , J. M. & Kanneganti , T. D. IL-1 family cytokines trigger sterile inflammatory disease . Front. Immunol . 3 , 315 ( 2012 ).
3. Martin , S. J. Cell death and inflammation: the case for IL-1 family cytokines as the canonical DAMPs of the immune system . FEBS J . 283 , 2599 - 2615 ( 2016 ).
4. Sims , J. E. & Smith , D. E. The IL-1 family: regulators of immunity . Nat. Rev. Immunol . 2 , 89 - 102 ( 2010 ).
5. Towne , J. E. , Garka , K. E. , Renshaw , B. R. , Virca , G. D. & Sims , J. E. Interleukin (IL) - 1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activatethe pathway leading to NF-kappaB and MAPKs . J. Biol. Chem . 279 , 13677 - 13688 ( 2004 ).
6. Dinarello , C. A. Immunological and inflammatory functions of the interleukin-1 family . Ann. Rev. Immunol . 27 , 519 - 550 ( 2009 ).
7. Vigne , S. et al. IL -36R ligands are potent regulators of dendritic and T cells . Blood 118 , 5813 - 5823 ( 2011 ).
8. Vigne , S. et al. IL -36 signaling amplifies Th1 responses by enhancing proliferation and Th1 polarization of naive CD4+T cells . Blood 120 , 3478 - 3487 ( 2012 ).
9. Milovanovic , M. et al. IL -33/ST2 axis in inflammation and immunopathology . Immunol. Res . 52 , 89 - 99 ( 2012 ).
10. Dietrich , D. et al. Interleukin -36 potently stimulates human M2 macrophages, Langerhans cells and keratinocytes to produce pro-inflammatory cytokines . Cytokine 84 , 88 - 98 ( 2016 ).
11. Towne , J. E. et al. Interleukin- 36 (IL-36) ligands require processing for full agonist (IL-36α, IL-36β, and IL-36γ) or antagonist (IL-36Ra) activity . J. Biol. Chem . 286 , 42594 - 42602 ( 2011 ).
12. Henry , C. M. et al. Neutrophil-derived proteases escalate inflammation via activation of IL-36 family cytokines . Cell Rep . 17 , 708 - 722 ( 2016 ).
13. Ainscough , J. S. et al. Cathepsin S is the major activator of the psoriasisassociated proinflammatory cytokine IL-36γ . Proc. Natl Acad. Sci. USA 114 , E2748 - E2757 ( 2017 ).
14. Blumberg , H. et al. Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation . J. Exp. Med . 204 , 2603 - 2614 ( 2007 ).
15. Blumberg , H. et al. IL -1RL2 and its ligands contribute to the cytokine network in psoriasis . J. Immunol . 185 , 4354 - 4362 ( 2010 ).
16. Johnston , A. et al. IL-1F5, - F6 , - F8 , and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression . J. Immunol . 186 , 2613 - 2622 ( 2011 ).
17. Marrakchi , S. et al. Interleukin-36 -receptor antagonist deficiency and generalized pustular psoriasis . N. Engl. J. Med . 365 , 620 - 628 ( 2011 ).
18. Onoufriadis , A. et al. Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis . Am. J. Hum. Genet . 89 , 432 - 437 ( 2011 ).
19. Tortola , L. et al. Psoriasiform dermatitis is driven by IL-36-mediated DCkeratinocyte crosstalk . J. Clin. Invest . 122 , 3965 - 3976 ( 2012 ).
20. Towne , J. E. & Sims , J. E. IL-36 in psoriasis . Curr. Opin. Pharmacol . 4 , 486 - 490 ( 2012 ).
21. Farooq , M. et al. Mutation analysis of the IL36RN gene in 14 Japanese patients with generalized pustular psoriasis . Hum. Mutat . 34 , 176 - 183 ( 2013 ).
22. Frey , S. et al. The novel cytokine interleukin-36α is expressed in psoriasis and rheumatoid arthritis synovium . Ann. Rheum. Dis . 72 , 1569 - 1574 ( 2013 ).
23. Kanazawa , N. , Nakamura , T. , Mikita , N. & Furukawa , F. Novel IL36RN mutation in a Japanese case of early onset generalized pustular psoriasis . J. Dermatol . 40 , 749 - 751 ( 2013 ).
24. Mahil , S. K. et al. An analysis of IL-36 signature genes and individuals with IL1RL2 knockout mutations validates IL-36 as a psoriasis therapeutic target . Sci. Transl. Med 9 , eaan2514 ( 2017 ).
25. Wu , J. K. , Siller , G. & Strutton , G. Psoriasis induced by topical imiquimod . Australas. J. Dermatol . 45 , 47 - 50 ( 2004 ).
26. Terui , T. , Ozawa , M. & Tagami , H. Role of neutrophils in induction of acute inflammation in T-cell-mediated immune dermatosis, psoriasis: a neutrophilassociated inflammation-boosting loop . Exp. Dermatol. 9 , 1 - 10 ( 2000 ).
27. Murphy , M. , Kerr , P. & Grant-Kels , J. M. The histopathologic spectrum of psoriasis . Clin. Dermatol . 25 , 524 - 528 ( 2007 ).
28. Ikeda , S. et al. Therapeutic depletion of myeloid lineage leukocytes in patients with generalized pustular psoriasis indicates a major role for neutrophils in the immunopathogenesis of psoriasis . J. Am. Acad. Dermatol . 68 , 609 - 617 ( 2013 ).
29. Ganesan , R. et al. Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies . mAbs 9 , 1143 - 1154 ( 2017 ).
30. Singh , J. A. et al. Adverse effects of biologics: a network meta-analysis and Cochrane overview . Cochrane Database Syst. Rev . 16 , CD008794 ( 2011 ).
31. Campa , M. , Mansouri , B. , Warren , R. & Menter , A. Review of biologic therapies targeting IL-23 and IL-17 for use in moderate-to-severe plaque psoriasis . Dermatol. Ther. 6 , 1 - 12 ( 2016 ).
32. Haslett , C. , Guthrie , L. A. , Kopaniak , M. M. , Johnston , R. B. & Henson , P. M. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide . Am. J. Pathol . 119 , 101 - 110 ( 1985 ).
33. Brinkmann , V. et al. Neutrophil extracellular traps kill bacteria . Science 303 , 1532 - 1535 ( 2004 ).
34. Amulic , B. , Cazalet , C. , Hayes , G. L. , Metzler , K. D. & Zychlinsky , A. Neutrophil function: from mechanisms to disease . Annu. Rev. Immunol . 30 , 459 - 489 ( 2012 ).
35. Korkmaz , B. , Horwitz , M. S. , Jenne , D. E. & Gauthier , F. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases . Pharmacol. Rev . 62 , 726 - 759 ( 2010 ).
36. Clancy , D. M. et al. Production of biologically active IL-36 family cytokines through insertion of N-terminal caspase cleavage motifs . FEBS Open Bio 6 , 338 - 348 ( 2016 ).
37. Afonina , I. S. et al. Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α . Mol. Cell 44 , 265 - 278 ( 2011 ).
38. Hazuda , D. J. , Strickler , J. , Kueppers , F. , Simon , P. L. & Young , P. R. Processing of precursor interleukin 1 beta and inflammatory disease . J. Biol. Chem . 265 , 6318 - 6322 ( 1990 ).
39. Lefrancais , E. et al. IL -33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G . Proc. Natl Acad. Sci. USA 109 , 1673 - 1678 ( 2012 ).
40. Macleod , T. et al. Neutrophil elastase-mediated proteolysis activates the antiinflammatory cytokine IL-36 Receptor antagonist . Sci. Rep . 6 , 24880 ( 2016 ).
41. Clancy , D. M. , Henry , C. M. , Sullivan , G. P. & Martin , S. J. Neutrophil extracellular traps can serve as platforms for processing and activation of IL-1 family cytokines . FEBS J . 284 , 1712 - 1725 ( 2017 ).
42. Carrier , Y. et al. Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis . J. Invest. Dermatol . 131 , 2428 - 2437 ( 2011 ).
43. Johnston , A. et al. Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation . J. Immunol . 190 , 2252 - 2262 ( 2013 ).
44. Nestle , F. O. , Kaplan , D. H. & Barker , J. Psoriasis. N. Engl . J. Med . 361 , 496 - 508 ( 2009 ).
45. Pham , C. T. Neutrophil serine proteases: specific regulators of inflammation . Nat. Rev. Immunol . 6 , 541 - 550 ( 2006 ).
46. Borregaard , N. , Sørensen , O. E. & Theilgaard-Mönch , K. Neutrophil granules: a library of innate immunity proteins . Trends Immunol . 28 , 340 - 345 ( 2007 ).
47. Kolaczkowska , E. & Kubes , P. Neutrophil recruitment and function in health and inflammation . Nat. Rev. Immunol . 13 , 159 - 175 ( 2013 ).
48. Garver , R. I. et al. Alpha 1-antitrypsin deficiency and emphysema caused by homozygous inheritance of non-expressing alpha 1-antitrypsin genes . N. Engl. J. Med . 314 , 762 - 766 ( 1986 ).
49. Hubbard , R. C. et al. Neutrophil accumulation in the lung in alpha 1-antitrypsin deficiency. Spontaneous release of leukotriene B4 by alveolar macrophages . J. Clin. Invest . 88 , 891 - 897 ( 1991 ).
50. Wiedow , O. , Wiese , F. , Streit , V. , Kalm , C. & Christophers , E. Lesional elastase activity in psoriasis, contact dermatitis, and atopic dermatitis . J. Invest. Dermatol . 99 , 306 - 309 ( 1992 ).
51. Kalb , R. E. et al. Risk of serious infection with biologic and systemic treatment of psoriasis: results from the psoriasis longitudinal assessment and registry (PSOLAR) . JAMA Dermatol . 151 , 961 - 969 ( 2015 ).
52. Sorenson , E. & Koo , J. Evidence-based adverse effects of biologic agents in the treatment of moderate-to-severe psoriasis: providing clarity to an opaque topic . J. Dermatol. Treat . 26 , 493 - 501 ( 2015 ).
53. Bruno , B. J. , Miller , G. D. & Lim , C. S. Basics and recent advance in peptide and protein drug delivery . Ther. Deliv . 4 , 1443 - 1467 ( 2014 ).
54. Cevc , G. , Schatzlein , A. & Blume , G. Transdermal drug carriers: basic properties, optimization and transfer efficiency in the case of epicutaneously applied peptides . J. Control. Release 36 , 3 - 16 ( 1995 ).
55. Hartop , P. J. , Allenby , C. F. & Prottey , C. Comparison of barrier function and lipids in psoriasis and essential fatty-acid deficient rats . Clin. Exp. Dermatol . 3 , 259 - 267 ( 1978 ).
56. Lee , Y. et al. Changes in transepidermal water loss and skin hydration according to expression of aquaporin-3 in psoriasis . Ann. Dermatol. 24 , 168 - 174 ( 2012 ).
57. Swedberg , J. E. , Li , C. L. , de Veer , S. J. , Wang , C. K. & Craik , D. J. Design of potent and selective cathepsin G inhibitors based on the sunflower trypsin inhibitor-1 scaffold . J. Med. Chem . 60 , 658 - 667 ( 2017 ).
58. Kosikowska , P. & Lesner , A. Inhibitors of cathepsin G: a patent review (2005 to present ). Expert Opin. Ther. Pat . 23 , 1611 - 1624 ( 2013 ).
59. Stevens , T. et al. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase . J. Pharmacol. Exp. Ther . 339 , 313 - 320 ( 2011 ).
60. Zeiher , B. G. , Matsuoka , S. , Kawabata , K. & Repine , J. E. Neutrophil elastase and acute lung injury: prospects for sivelestat and other neutrophil elastase inhibitors as therapeutics . Crit. Care Med . 30 , S281 - S287 ( 2002 ).
61. Tsai , Y. F. & Hwang , T. L. Neutrophil elastase inhibitors: a patent review and potential applications for inflammatory lung diseases ( 2010 - 2014 ). Expert Opin. Ther. Pat . 25 , 1145 - 1158 ( 2015 ).
62. Walsh , J. G. , Logue , S. E. , Luthi , A. & Martin , S. J. Caspase-1 promiscuity is counterbalanced by rapid inactivation of processed enzyme . J. Biol. Chem . 286 , 32513 - 32524 ( 2011 ).