MINI REVIEW
published: 26 July 2018
doi: 10.3389/fimmu.2018.01704
Inhibition of Lipopolysaccharide- and
Lipoprotein-Induced Inflammation by
Antitoxin Peptide Pep19-2.5
Lena Heinbockel 1, Günther Weindl 2, Guillermo Martinez-de-Tejada3, Wilmar Correa 4,
Susana Sanchez-Gomez 3, Sergio Bárcena-Varela3, Torsten Goldmann1, Patrick Garidel 5,
Thomas Gutsmann 4* and Klaus Brandenburg 4
1
Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany, 2 Institute of Pharmacy (Pharmacology and
Toxicology), Freie Universität Berlin, Berlin, Germany, 3 Department of Microbiology and Parasitology, Universidad de Navarra,
Pamplona, Spain, 4 Biophysics, Research Center Borstel, Borstel, Germany, 5 Martin-Luther Universität Halle-Wittenberg,
Halle, Germany
Edited by:
Rudolf Lucas,
Augusta University, United States
Reviewed by:
Vineesh Vimala Raveendran,
King Faisal Specialist Hospital &
Research Centre, Saudi Arabia
Maryna Skok,
Palladin Institute of Biochemistry
(NAS Ukraine), Ukraine
*Correspondence:
Thomas Gutsmann
tgutsmann@fz-borstel.de
Specialty section:
This article was submitted to
Inflammation,
a section of the journal
Frontiers in Immunology
Received: 12 December 2017
Accepted: 10 July 2018
Published: 26 July 2018
Citation:
Heinbockel L, Weindl G, Martinez-deTejada G, Correa W, SanchezGomez S, Bárcena-Varela S,
Goldmann T, Garidel P, Gutsmann T
and Brandenburg K (2018)
Inhibition of Lipopolysaccharideand Lipoprotein-Induced
Inflammation by Antitoxin
Peptide Pep19-2.5.
Front. Immunol. 9:1704.
doi: 10.3389/fimmu.2018.01704
The most potent cell wall-derived inflammatory toxins (“pathogenicity factors”) of Gramnegative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals via toll-like receptor
4 (TLR4) and the latter via TLR2, the physico-chemistry of these compounds exhibits
considerable similarity, an amphiphilic molecule with a polar and charged backbone and
a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the
serologically relevant structure, the inner part, the lipid A, is responsible for one of the
strongest inflammatory activities known. In the last years, we have demonstrated that
antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS
and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by
severe bacterial infections. We also showed that this anti-inflammatory activity requires
specific interactions of the peptides with LPS and LP leading to exothermic reactions
with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated
neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the threedimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several in vivo models
together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the
broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade.
Keywords: antimicrobial peptides, Pep19-2.5, sepsis, intracellular LPS signaling, endotoxin
INTRODUCTION
Peptide-based therapies for diverse applications are under investigation since many years.
Particularly, antimicrobial peptides (AMPs) are a subject of considerable research with some
particular compounds reaching clinical use. Further development, improvement, and expansion
for new microbial targets make the understanding of the underlying molecular mechanisms mandatory. AMPs have a wide range of therapeutic activities depending on their mode of action on
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the target structures. The main strategies include (i) to optimize
their direct antimicrobial activity (i.e., lowering their minimal
inhibitory and minimal bactericidal concentrations—MIC and
MBC values), (ii) to provide an immunomodulatory activity
(i.e., enhancing the human defense system), and (iii) to bind to
the responsible immune-stimulating compounds of bacteria,
lipopolysaccharide, and lipoproteins, and thus to inhibit inflammation. Approach (i) led to the investigation and development
of some antibacterial drugs, with the disadvantage that with
increasing broad-spectrum effect also the hemolytic activity and
other side effects increased. The development of approach (ii) led
to some relevant insights into immune processes, but to no successful clinical application, whereas strategy (iii) is increasingly
being considered as a highly interesting therapeutical option
and is in the focus of the present review.
Finally, a pivotal goal in the development of AMPs is to
broaden their spectrum of antibacterial and anti-inflammatory
activity while ensuring low toxicity against host cells. This balancing act has to be carefully adjusted and this will also be discussed
in this work.
ESSENTIAL ACTION OF AMPs
A critical issue in the treatment of infectious disease is the
high or even chronic inflammatory status of the patients. The
release of toxins from the bacterial cell wall due to upregulation
of immune system effectors or to an antibiotic therapy often
contributes to worsen the final outcome (1). Therefore, it is of
crucial interest during antibiotic treatment not only to kill the
bacteria but also to prevent excessive inflammation mediated by
cell debris released by lysed bacteria. Some well-known antibiotics of the polymyxin AMP family (cyclic lipopeptides active
against Gram-negative bacteria) combine a potent bactericidal
activity with the capacity to efficiently neutralize endotoxins.
Regrettably, due to their propensity to cause undesirable side
effects such as neuro- and nephrotoxicity, they are used as drugs
of last resource (2).
One of the most promising approaches for designing new
AMPs is the use of LPS-binding polypeptide domains within
defense proteins such as lactoferrin and Limulus anti-LPS-factor
(LALF). This has successfully been done in previous studies [for
an overview, see Ref. (3)]. Using these domains as templates
and performing a rational design focused on optimizing their
lipid A-binding and neutralizing activity, we developed the
Aspidasept® family of compounds (also called SALP, synthetic
anti-LPS peptides). Within this family, Pep19-2.5 and Pep19-4LF
are undergoing preclinical testing. Although these polypeptides
exhibit a more modest antimicrobial activity against Gramnegative bacteria compared to polymyxin B, they are endowed
with a remarkable capacity to kill Gram-positive bacteria.
In addition, these AMPs have an increased ability to neutralize
toxins from both type of organisms, namely lipopolysaccharides
(LPS) and lipoproteins (LP) (4, 5). Interestingly, we demonstrated
that Pep19-2.5 efficiently counteracts the pro-inflammatory
activity of some antibiotics such as ciprofloxacin and ceftriaxone
[Figure 1 and Ref. (6), respectively] and cooperates in vivo with
several structurally unrelated antibiotics to neutralize serum
Frontiers in Immunology | www.frontiersin.org
FIGURE 1 | Antimicrobial and anti-inflammatory therapies. (A) Therapeutic
efficacy of Pep19-2.5 combined with antibiotics to control bacteremia and
endotoximia caused by intraperitoneally injected Salmonella enterica sv.
Minnesota (107 cfu/mouse). Bacteremia was treated either with various
antibiotics or antibiotics plus Pep19-2.5 and the TNFα serum levels were
measured 90 min after bacterial challenge and treatment (4). (B) Antibiotics
(red triangle) kill the bacteria, while Pep19-2.5 (blue semicircle) destabilizes
the bacterial membranes and neutralizes the toxins. As presented in the last
row, the peptide may bind to the toxins as constituents of the bacteria or in
isolated form, thus inhibiting the strong inflammation reaction.
levels of TNF-alpha induced by a bacterial infection (Figure 1).
Therefore, a combined medication based on antibiotics and toxinneutralizers offers great promise for the treatment of patients
with inflammatory diseases caused by bacterial infections, such
as sepsis.
In addition to peptides from Aspidasept, polymyxin, or
lactoferrin families, other interesting compounds are under
current investigation. Thus, for example, the well-known human
cathelidicin LL-37 is a peptide with multiple biological activities
including the potential to act as an anti-endotoxin, immunomodulatory, and wound-healing compound (7). This peptide
has the capacity to kill Gram-negative and Gram-positive bacteria, and it is able to neutralize endotoxin by sequestering soluble
LPS (7–9). It was found that its activity against bacteria is in close
relationship to its immunomodulatory function (4). The potency
of this peptide to kill bacteria was found to be lower than that of
polymyxin B or Pep19-4.LF, but it has similar LPS neutralizing
ability compared to polymyxin B and Pep19-2.5 derivatives.
Another interesting AMP is the cecropin d-like peptide (Gm1),
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Fundamentals of Peptide Therapies of Infections
a non-cationic AMP from Galleria mellonella. Gm1 was shown to
have a broad spectrum of antimicrobial activity and may represent a good template for the peptide-based drug development of
antisepsis compounds (10–12). The ability of this peptide to bind
and neutralize LPS demonstrates that a polycationic character
is not necessarily a prerequisite for an effective binding to LPS
(11). This conclusion opens the prospect of developing a whole
new peptide-based strategy to control sepsis.
spectrum of anti-inflammatory activity for multifaceted infections, as well as sufficient bactericidal activity. A peptide possessing this dual behavior against bacterial infections in several
sepsis models but also in other disease models is Pep19-2.5.
Notably, Pep19-2.5 and Pep19-4.LF inhibit inflammatory
responses triggered by LP and LPS and mediated by the pattern recognition receptors (PRRs), toll-like receptor 2 (TLR2)
and TLR4, respectively. This has also been confirmed in skin
cells including keratinocytes, dermal fibroblasts, and dendritic
cells (17). Interestingly, an additional mode of action has been
identified in keratinocytes. Both peptides accelerated already at
low concentrations artificial wound closure and increased cell
migration of keratinocytes via purinergic receptor activation.
These findings are particularly relevant to bacterial skin infections, which are often associated with impaired wound healing.
Besides transmembrane, PRRs, LPS, and LP are recognized
intracellularly by cytosolic PRRs, which sense intracellular
infections (Figure 2). The intracellular LPS sensor caspase-11
and its human orthologs caspase-4/5 cause activation of inflammasomes leading to production of IL-1β and cell death termed
pyroptosis (18). The intracellular effects of LPS are thought to be
crucial in the pro-inflammatory response during sepsis (19) and
may at least partially explain the failure of TLR4 inhibitors in
clinical trials. Given the neutralizing mode of action of SALPs,
it is likely that the peptides inhibit not only extracellular TLR2/4
signaling but also intracellular signaling cascades mediated by
inflammasomes. Indeed, Pep19.2-5 dampened intracellular
LPS-induced caspase-1 activation, IL-1β production, as well as
high mobility group box (HMGB)1 secretion and lactate dehydrogenase release in human cells (20). Although the underlying
mechanisms are not fully understood, Pep19.2-5 may bind
LPS extracellularly preventing its intracellular accumulation
or translocate across the cell membrane and neutralize LPS
LPS/LP-INDUCED INFLAMMATION
The basic mechanisms of LPS/LP-induced inflammation run
via stimulation of cell-surface receptors on immune cells,
toll-like receptor 4 (TLR4) for LPS and TLR-2 for LP, which
subsequently leads to an intracellular reaction by recruiting
transcription factors such as NF-κB in the nucleus followed by
the secretion of chemokines and cytokines. In septic patients, this
reaction gets out of control with the subsequent life-threatening
“cytokine storm.”
Research in the field of sepsis prevention, but also of other
serious infection-triggered inflammations, has been plagued
with many failed clinical trials (13, 14). A major reason for this
could be the overly specific—and thereby, narrow spectrum of
biological activities displayed by the drugs under development.
Prominent examples include the monoclonal anti-LPS antibodies E5 (15) and H1-A1 (16), which failed to improve survival
of septic patients in clinical Phase III trials. These compounds
were selected by their ability to bind to a collection of different
endotoxins. This apparently promising approach ignores the
fact that there is an innumerable diversity of Gram-negative as
well as Gram-positive pro-inflammatory PAMPs (i.e., pathogenassociated molecular patterns) inducing, often in parallel, sepsis.
Thus, for therapeutic efficiency, AMPs need to display a broad
FIGURE 2 | The synthetic peptide Pep19-2.5 inhibits signaling of LP and LPS mediated by transmembrane and cytosolic PRRs. The activated signaling cascades
lead to inflammation and a form of cell death termed pyroptosis. LP, lipopeptides; LPS, lipopolysaccharides; OMV, outer membrane vesicle.
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intracellularly. A similar mode of action can be assumed for
acetylated LP such as fibroblast-stimulating lipid 1 (FSL-1),
which activates the NLRP7 inflammasome in the cytosol (21).
Since most Gram-negative bacteria that activate caspase11/4/5 do not reach the cytosol, mechanisms are required that
mediate internalization of LPS. Recent studies suggest that
LPS is delivered into the cytosol via outer membrane vesicles
(OMVs) that are secreted by Gram-negative bacteria (22).
OMVs have been implicated in the pathogenesis of infectious
diseases such as sepsis, thus, it would be interesting to know
whether LPS-neutralizing peptides are able to interfere with
OMVs to prevent intracellular LPS-mediated IL-1β production
and pyroptosis.
assembly (27). These findings in the SAXS experiment were
independently confirmed by freeze-fracture electron microscopy data, indicating stacks of membraneous arrangements of
LPS with periodicities of the same size (9.0 nm) as found above.
Similar results were reported also for the aggregate structures of
wild-type LPS (from E. coli O55:B5 and Salmonella abortus equi)
(28). Here, the binding of Pep19-2.5 to the latter LPS leads to a
change from a mixed lamellar/non-lamellar aggregate structure
into a multilamellar one with a periodicity of 9.20 nm. This
value fits with that produced by the bioactive LPS Ra fraction
(the rough LPS form) within the heterogeneous wild-type LPS.
The observation that the endotoxically active unit within wildtype LPS corresponds to a Ra-LPS, was originally reported by
Jiao et al. (29). Also here, the findings regarding multilamellarization events are in agreement with those reported for
polymyxin B (30).
Data on the measurements of the electrophoretic mobility of
LPS aggregates (Zeta potential) in the presence of peptides with
either a potent (Pep19-2.5) or a weak (Pep19-8) LPS-neutralizing
activity showed a compensation of the LPS head group charges
by both peptides, with an even stronger action of the latter
peptide (24). This is a clear indication that the neutralization
of the negative charges within the LPS backbone is necessary
but not sufficient for an effective anti-inflammatory action.
These data could be convincingly confirmed by ITC measurements (4, 23), which showed binding of Pep19-2.5 to LPS as
an exothermic process, which was saturated at a much lower
molar ratio for Pep19-2.5 [(Pep19-2.5):(LPS) = 0.3], compared
to Pep19-8 [(Pep19-8):(LPS) = 1.2]. This binding process can be
explained as a two-step event consisting of a Coulomb interaction between the basic AA (R and K) with the negative charges
of LPS (phosphates, carboxylates), followed by the hydrophobic
interaction of the C-terminal region of the peptides (FWFWG)
with the lipid A moiety of LPS. This interpretation is backed by
the observation that a peptide variant (Pep19-2.5gek) lacking
the C-terminal region was nearly unable to neutralize LPS, most
likely because the second step of interaction could not take place.
Pep19-2.5 was also reported to efficiently bind and neutralize a
model toxin from a non-Gram-negative (mycoplasmic) bacteria,
called FSL-1. Binding of the peptide to FSL-1 was associated with
a strong exothermic reaction with saturation characteristic (31)
similar as found for LPS.
Consistent with this explanation, the interaction of the
inflammation-enhancing peptide Hbγ35 with LPS—although an
exothermic process—exhibited no saturation characteristics (26).
To study the interaction of peptides with phospholipid bilayers, FRET spectroscopy was applied (24). It was found that
selected peptides (i.e., Pep19-2.5, its scrambled version Pep192.5KO, and the compound with low LPS-neutralizing activity,
Pep19-8), all intercalated readily into phosphatidylcholine and
phosphatidylserine liposomes as well as into LPS aggregates.
There was no correlation between the different LPS antagonistic
activities of the peptides and their ability to interact with lipid
bilayers. Similarly, when peptides were added to aggregates
formed by amphiphilic molecules other than LPS, such as FSL-1
and the lipoprotein SitC from S. aureus, all SALPs incorporated
readily (31).
BIOPHYSICAL MECHANISMS OF THE
TOXIN-NEUTRALIZATION PROCESS
The interaction of LPS with Pep19-2.5 was investigated with a
variety of physical techniques: (I) Fourier-transform infrared
spectroscopy (FTIR) and differential scanning calorimetry to
analyze the gel to liquid crystalline phase transition of the acyl
chains of the toxins; (II) small-angle X-ray scattering (SAXS) with
synchrotron radiation and freeze-fracture electron microscopy
to determine their aggregate structure; (III) Zeta sizer analysis
for surface charge and electrophoretic mobility determination;
(IV) isothermal titration calorimetry for measuring the binding
constants and saturation of the toxin:peptide complexes; and
(V) Förster resonance energy transfer spectroscopy for intercalation experiments of the peptide into the toxin aggregates or
target cell membranes (4, 23, 24).
The data on the gel to liquid crystalline phase transition
showed a fluidization of the hydrocarbon chains of LPS from
Salmonella enterica rough mutant chemotype R60 due to
binding of Pep19-2.5. This phenomenon was detectable to
a lesser extent in the FTIR experiment, but led to a complete
disappearance of the phase transition in the calorimetric scan.
In the former method, the fluidization could be deduced from
the increase of the wave numbers of the symmetric stretching
vibration of the methylene groups at 2,850 cm−1 in the gel phase
below the phase transition temperature at around 36°C. Parallel
to this increase, the change of the heat capacity between the two
phases decreased considerably. Similar behavior was reported
for the interaction of LPS with polymyxin B (25). Interestingly,
the interaction of LPS with a peptidic portion of the human
hemoglobin gamma-chain, called Hbγ35, led to a decrease
in fluidization (i.e., a rigidification) of the LPS assembly (26).
In contrast to the Pep19-2.5 series, the hemoglobin-derived
peptide does not antagonize endotoxin activities upon binding
to LPS but even enhances the LPS-induced cytokine secretion.
Investigations into the change of the aggregate structures of
LPS due to peptide binding revealed a drastic reorientation of
the LPS aggregates from a bilayered conformation, possibly in
cubic symmetry, into a multilamellar arrangement (24). This
could be proven in the SAXS experiments by the occurrence of
reflections around 9.00 and 4.50 nm, which—as shown earlier—
corresponds to the main reflections of LPS R60 in a multilayered
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Finally, binding of LPS to either the inflammation-inhibiting
peptide Pep19-2.5 or to the inflammation-enhancing peptide
Hbγ35 led to antagonistic results. Thus, whereas the former
peptide caused an increase in aggregate size, connected with the
adoption of a multilamellar structure, the latter decreased that
parameter, probably coupled with the production of smaller
bilayered LPS-aggregates with cubic symmetry (26).
of endotoxin released by antibiotics in vivo and cooperates with
conventional antimicrobials to reduce inflammation caused by
bacterial infections. This is accompanied by a disturbance of
the bacterial membranes, which enhances the activity of conventional antibiotics. Thus, this compound in combination with
antibiotics could be a life-saving contribution for the treatment
of sepsis and other infectious diseases.
CONCLUSION
AUTHOR CONTRIBUTIONS
Peptides combine features that make them attractive candidates for the treatment of infectious and inflammatory diseases.
On the one hand, they can be produced in high amounts using
simple and affordable procedures. In addition, since AMPs
consist of natural amino acids, these compounds are rapidly
metabolized in the body without generating toxic by-products.
Pep19-2.5 neutralizes with high efficiency both extracellular and
intracellular bacterial cell wall-derived toxins, such as LPS and
LP. This property is connected with a conformational change
of the toxins (aggregate structure, fluidity of their acyl chains,
surface charges) converting them into a bioinactive conformation. The peptide also counteracts the pro-inflammatory activity
LH, GW, GM-d-T, and KB conceived and wrote the manuscript.
WC, ThG, PG, SS-G, SB-V, and ToG edited the manuscript.
All the authors read and approved the final manuscript.
FUNDING
LH is supported by the Deutsche Forschungsgemeinschaft
(DFG, project DR797/3-1 611672). GW acknowledges support by the DFG grant RA 895/16-1. GM-d-T was funded by a
grant (reference number PIUNA-P2011-17) from University of
Navarra (Spain). WC is a recipient of a grant by “Brandenburg
Antiinfektiva GmbH.”
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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Copyright © 2018 Heinbockel, Weindl, MartinezdeTejada, Correa, SanchezGomez,
BárcenaVarela, Goldmann, Garidel, Gutsmann and Brandenburg. This is an open
access article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
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July 2018 | Volume 9 | Article 1704