Therapeutic Potential of Cathelicidin Peptide LL-37, an Antimicrobial Agent, in a Murine Sepsis Model

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International Journal of Molecular Sciences

Review Therapeutic Potential of Cathelicidin Peptide LL-37, an Antimicrobial Agent, in a Murine Sepsis Model

Isao Nagaoka 1,2,* , Hiroshi Tamura 3 and Johannes Reich 4 1 Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 2 Department of Physical Therapy, Faculty of Health Science, Juntendo University, 3-2-12 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 3 Laboratory Program Support (LPS) Consulting Oﬃce, 4-7-13 Nishi-Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan; [email protected] 4 Microcoat Biotechnologie GmbH, Am Neuland 3, 82347 Bernried am Starnberger See, Germany; [email protected] * Correspondence: [email protected]; Tel.: +81-3-5802-0422

Received: 18 June 2020; Accepted: 10 August 2020; Published: 19 August 2020

Abstract: Among the mechanisms put-up by the host to defend against invading microorganisms, antimicrobial peptides represent the first line. In different species of mammals, the cathelicidin family of antimicrobial peptides AMPs has been identified, and in humans, LL-37 is the only type of cathelicidin identified. LL-37 has many different biological activities, such as regulation of responses to inflammation, besides its lipopolysaccharide (LPS)-neutralizing and antimicrobial and activities. Recently, employing a murine septic model that involves cecal ligation and puncture (CLP), we examined the effect of LL-37. The results indicated that LL-37 exhibits multiple protective actions on septic mice; firstly, the survival of CLP mice was found to be improved by LL-37 by the suppression of the macrophage pyroptosis that induces the release of pro-inflammatory cytokines (such as IL-1β) and augments inflammatory reactions in sepsis; secondly, the release of neutrophil extracellular traps (NETs), which have potent bactericidal activity, is enhanced by LL-37, and protects mice from CLP-induced sepsis; thirdly, LL-37 stimulates neutrophils to release antimicrobial microvesicles (ectosomes), which improve the pathological condition of sepsis. These findings indicate that LL-37 protects CLP septic mice through at least three mechanisms, i.e., the suppression of pro-inflammatory macrophage pyroptosis and the release of antimicrobial NETs (induction of NETosis) and ectosomes from neutrophils. Thus, LL-37 can be a potential therapeutic candidate for sepsis due to its multiple properties, including the modulation of cell death (pyroptosis and NETosis) and the release of antimicrobial NETs and ectosomes as well as its own bactericidal and LPS-neutralizing activities.

Keywords: antimicrobial peptide; cathelicidin; sepsis; pyroptosis; NETs; ectosome

1. Introduction Sepsis is a frequent cause of mortality in the noncoronary intensive care unit (ICU). Sepsis results from harmful or detrimental host response to infection and it is a systemic host response [1,2]. Multiple organ failure, which mostly results from the excessive production of pro-inflammatory cytokines, arises because of the dysregulated inflammatory/immune responses during sepsis. Recent statistics indicate a decrease in sepsis mortality in hospitals, probably due to advances in patient care. Unfortunately, clinical trials revealed that many of the therapeutic approaches with anti-inflammatory cytokines are not effective [2,3]. Therefore, it is necessary to develop an effective and novel approach for sepsis treatment [2,3]. Many recent studies addressed the host cell death mechanisms, which likely underlie the dysregulated inflammatory/immune responses in sepsis [4–6].

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Int.mechanisms, J. Mol. Sci. 2020 which, 21, 5973 likely underlie the dysregulated inflammatory/immune responses in sepsis2 of [4– 16 6]. Macrophages and dendritic cells, which are found in bacterial infection, undergo caspase-1- dependentMacrophages cell death, and dendritic called cells, pyroptosis which are [7]. found The in bacterialcells undergoing infection, undergo pyroptosis caspase-1-dependent release pro- cellinflammatory death, called cytokines, pyroptosis such [7]. Theas interleukin cells undergoing (IL)-1 pyroptosisβ and IL-18, release extracellularly pro-inflammatory [7,8]. IL-1 cytokines,β not suchonly β β asenhances interleukin both (IL)-1 systemicand and IL-18, local extracellularly inflammatory/imm [7,8]. IL-1une responsesnot only enhances[9] but also both leads systemic to tissue and injury local inflammatoryin sepsis, by synergistically/immune responses acting [ 9along] but alsowith leadsother tocytokines tissue injury [10]. in sepsis, by synergistically acting alongNeutrophils with other cytokines are the most [10]. abundant leukocytes and as an important part of the innate immune systemNeutrophils in humans, are protect the most the abundant host against leukocytes invading and microorganisms as an important [11,12]. part of theUpon innate stimulation, immune systemneutrophils in humans, go through protect NETosis the host (a againstkind of invadingprogrammed microorganisms cell death) [and11,12 trigger]. Upon the stimulation, release of neutrophilsneutrophil extracellular go through NETosis traps (NETs) (a kind [13]. of programmedNETs are capable cell death) of trapping and trigger microorganisms the release ofand neutrophil exerting extracellularanti-bacterial traps activity (NETs) through [13]. NETs the areNET-associat capable of trappinged components’ microorganisms action and(e.g., exerting DNA, anti-bacterialhistone and activitygranule-derived through proteins the NET-associated and peptides) components’ [14,15]. Furthermore, action (e.g., stimulated DNA, histone neutrophils and granule-derived extracellularly proteinsrelease microvesicles and peptides) (0.1-1.0 [14,15]. μ Furthermore,m in diameter), stimulated called neutrophilsectosomes, during extracellularly the inflammatory release microvesicles response µ (0.1-1.0[16,17]. Ectosomesm in diameter), contain called functional ectosomes, proteins during of theneutrophils inﬂammatory [17,18], response and exhibit [16,17 ].bacteriostatic Ectosomes containpotential functional [19]. Notably, proteins the ectosome of neutrophils level is [17 reported,18], and to exhibit be augmented bacteriostatic in surviving potential patients [19]. of Notably, sepsis the[20,21]. ectosome level is reported to be augmented in surviving patients of sepsis [20,21]. Antimicrobial peptides are evolutionarily conservedconserved among various species (vertebrates as well as invertebrates, the latter encompassing arthropods)arthropods) [[22,23].22,23]. These are also known as host defense peptides and take part in thethe innateinnate immuneimmune responseresponse byby exhibitingexhibiting antimicrobialantimicrobial activities against both Gram-negativeGram-negative and and -positive -positive bacteria, bacteria, and and also al virusesso viruses and fungi. and fungi. Antimicrobial Antimicrobial peptides peptides (AMPs) belong(AMPs) to belong two main to two families, main families, the defensins the defensins and cathelicidins. and cathelicidins. Besides Besides acting asacting antimicrobials, as antimicrobials, AMPs areAMPs also are capable also capable of enhancing of enhancin immunityg immunity by acting by onacting host on cells host as cells immunomodulators as immunomodulators to link to innate link andinnate adaptive and adaptive immunity immunity [22,23] [22,23] (Figure (Figure1). 1).

Figure 1. Roles of antimicrobial peptides in host defense. Antimicrobial peptides (AMPs) participate in theFigure innate 1. Roles immune of responseantimicrobial by showing peptides antimicrobial in host defense. actions Antimicrobial against Gram-negative peptides and (AMPs) -positive participate bacteria, virusesin the innate and fungi. immune Besides response having by direct showing antimicrobial antimicrobial properties, actions AMPs against also haveGram-negative the capability an tod -positive augment immunitybacteria, viruses and to linkand adaptive fungi. andBesides innate having immunity direct by antimicrobial acting on host cellsproperties, as immunomodulators. AMPs also have HNPs, the humancapability neutrophil to augment peptides; immunity hBDs, and human to linkβ-defensins. adaptive and innate immunity by acting on host cells as immunomodulators. HNPs, human neutrophil peptides; hBDs, human β-defensins. Nearly 30 different cathelicidin types have been described in various species of mammals. In humans,Nearly 30 however, different therecathelicidin is only types one have type been of cathelicidin, described in knownvarious asspecies the humanof mammals. cationic In antibacterialhumans, however, protein there of 18is only kDa on (hCAP18).e type of cathelicidin, The hCAP18 known has aas C-terminalthe human maturecationic antibacterialantibacterial peptideprotein of LL-37, 18 kDa which (hCAP18). consists The hCAP18 of 37 amino has a C-te acidrminal residues mature with antibacterial the first two peptide leucine LL-37, residues which (Lconsists1LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES of 37 amino acid residues with37 ) andthe hasfirst been foundtwo mostlyleucine in epithelialresidues cells(L1LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES and neutrophils [24–26]. Besides having antimicrobial37) and and lipopolysaccharidehas been found mostly (LPS)-neutralizing in epithelial activitiescells and [27neutrophils,28], LL-37 also[24–26]. shows Besides many biologicalhaving antimicrobial activities, such and as regulationlipopolysaccharide of responses (LPS)- to inflammationneutralizing activities [26,29]. [27,28], Importantly, LL-37 wealso demonstrated shows many before biological that activities, in neutrophils such theas regulation spontaneous of

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apoptosis is inhibited by LL-37 through purinergic receptor P2X7 as well as formyl-peptide receptor-like 1 (FPRL1) [30]. We also showed that LL-37, by neutralizing the action of LPS, decreases the apoptosis of endothelial cells induced by LPS [31]. These above results suggested that cell death is modulated by LL-37. Recently, we assessed the inﬂuence of LL-37 in a murine cecal ligation and puncture (CLP) sepsis model and revealed that the survival of CLP septic mice is improved by the administration of LL-37, which also caused the suppression of macrophage pyroptosis [32] and the release of NETs [33] and ectosomes [34] from neutrophils. Thus, in this article, based on our recent ﬁndings, the therapeutic potential of LL-37 on a murine sepsis model is reviewed.

2. Inhibition of the Macrophage Pyroptosis and Improvement of the CLP Mouse Survival by LL-37 Pyroptosis is a cell death pathway that is dependent on caspase-1 and occurs mostly in dendritic cells and macrophages, in association with the release of pro-inflammatory cytokines (IL-1β and IL-18) [7] (Figure2). In addition, membrane perforation leads to cytosolic content release, which augments the inflammatory reactions [7,35]. Two separate stimuli, microbial-pathogen-associated molecular patterns (PAMPs) (including bacterial lipoproteins and LPS) and endogenous damage-associated molecular patterns (DAMPs) (for example, ATP and uric acid) are required for inducing pyroptosis [8,36]. These two stimuli trigger the formation of an inflammasome, a multi-protein complex (typically including caspase-1, NALP3 (NACHT domain-, leucine-rich repeat-, and pyrin domain (PYD)-containing protein 3) and ASC (apoptosis-associated speck-like protein containing a CARD)), which facilitates the conversion of pro-caspase-1 to active caspase-1 [37]. Subsequently, the activated caspase-1 catalyzes the cleavage of pro-IL-1β and pro-IL-18 to release IL-1β and IL-18, respectively, and N-terminal fragments of Gasdermin D, generated by activated caspase-1, oligomerize and create pores in the plasma membrane, leading to cell death (pyroptosis) [8,37]. It is important to note that caspase-1 genetic deletion leads to suppressed IL-1β levels and confers a protective effect on murine sepsis models and improves the survival of mice [38,39]. Therefore, the activation of caspase-1 and the resultant pyroptosis plays a significant part in sepsis pathogenesis and mortality [7,35]. Recently, we described that the LPS/ATP-induced pyroptosis as well as production of IL-1β are suppressed by LL-37 in macrophages by both curtailing the effect of LPS on CD14/TLR4 (Toll-like receptor 4) and preventing the P2X7 response to ATP in vitro [40] (Figure3). Accordingly, we hypothesize that LL-37 exhibits protective effects in the murine septic model Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 16 by inhibiting the pyroptosis of macrophages in vivo [32].

Figure 2. Mechanism for the induction of pyroptosis. Pyroptosis occurs by two diﬀerent pathways. Figure 2. Mechanism for the induction of pyroptosis. Pyroptosis occurs by two different pathways. First, canonical pyroptosis is supported by the caspase (CASP)-1 activation by inﬂammasomes, which First, canonical pyroptosis is supported by the caspase (CASP)-1 activation by inflammasomes, which recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)(red arrows). Second, the noncanonical pyroptosis is conducted by the activation of caspase-1 and caspase-4/-5 (caspase 11 in mice), which can be directly triggered by lipopolysaccharide (LPS) independent of TLR4 (Toll-like receptor 4)(purple arrows). Gasdermin D N- terminal fragments, generated by activated caspases, oligomerize and create pores in the plasma membrane due to their binding to certain lipids in the plasma membrane inner leaflet, leading to the release of cellular contents and cell death (violet arrows). Caspase-4/-5/-11, on the other hand, activates the Pannexin-1 channel and opens the P2X7 pore to induce pyroptosis. Activated Pannexin- 1 can trigger the NLRP3 inflammasome through K+ efflux, and caspase-1 is activated by cleaved Gasdermin D through the combination of NLRP3 and ASC (dashed arrows). Caspase-1 activation results in the proteolytic cleavage of pro-IL-1β and pro-IL-18 and the formation of mature IL-1β and IL-18 [41].

Figure 3. Suppression of LPS/ATP-induced macrophage pyroptosis by LL-37. Gram-negative bacterial LPS and dead/dying cell-derived ATP induce macrophage pyroptosis via the action on

CD14/TLR4 and P2X7, respectively. LL-37 reduces the LPS/ATP-stimulated pyroptosis of macrophages and IL-1β production by both curtailing the action of LPS on CD14/TLR4 and blocking

the P2X7 response to ATP.

The results indicated that the administration of LL-37 (2 μg/mouse) intravenously, to the CLP septic mice, improves their survival (Figure 4), and the effect was dose-dependent, since LL-37 (1 μg/mouse and 2 μg/mouse) improved the survival rate to 14.3% and 36.4%, respectively [32]. Interestingly, the pyroptosis of peritoneal macrophages and the CLP-induced caspase-1 activation

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Figure 2. Mechanism for the induction of pyroptosis. Pyroptosis occurs by two different pathways. Int. J. Mol.First, Sci. canonical2020, 21, pyroptosis 5973 is supported by the caspase (CASP)-1 activation by inflammasomes, which4 of 16 recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)(red arrows). Second, the noncanonical pyroptosis is conducted by the activation recognizeof caspase-1 pathogen-associated and caspase-4/-5 molecular (caspase patterns 11 in (PAMPs) mice), andwhich damage-associated can be directly molecular triggered patterns by (DAMPs)(redlipopolysaccharide arrows). (LPS) Second, independent the noncanonical of TLR4 (Toll-like pyroptosis receptor is conducted 4)(purple by thearrows). activation Gasdermin of caspase-1 D N- andterminal caspase-4 fragments,/-5 (caspase generated 11 in by mice), activated which caspas can bees, directly oligomerize triggered and bycreate lipopolysaccharide pores in the plasma (LPS) independentmembrane due of to TLR4 their (Toll-likebinding to receptor certain lipids 4)(purple in the arrows). plasma membrane Gasdermin inner D N-terminal leaflet, leading fragments, to the generatedrelease of bycellular activated contents caspases, and oligomerizecell death (vio andlet create arrows). pores Caspase-4/-5/-11, in the plasma membrane on the other due to hand, their binding to certain lipids in the plasma membrane inner leaflet, leading to the release of cellular activates the Pannexin-1 channel and opens the P2X7 pore to induce pyroptosis. Activated Pannexin- contents and cell death (violet arrows). Caspase-4/-5/-11, on the other hand, activates the Pannexin-1 1 can trigger the NLRP3 inflammasome through K+ efflux, and caspase-1 is activated by cleaved channel and opens the P2X pore to induce pyroptosis. Activated Pannexin-1 can trigger the NLRP3 Gasdermin D through the 7combination of NLRP3 and ASC (dashed arrows). Caspase-1 activation inflammasome through K+ efflux, and caspase-1 is activated by cleaved Gasdermin D through results in the proteolytic cleavage of pro-IL-1β and pro-IL-18 and the formation of mature IL-1β and the combination of NLRP3 and ASC (dashed arrows). Caspase-1 activation results in the proteolytic IL-18 [41]. cleavage of pro-IL-1β and pro-IL-18 and the formation of mature IL-1β and IL-18 [41].

Figure 3. Suppression of LPS/ATP-induced macrophage pyroptosis by LL-37. Gram-negative bacterial Figure 3. Suppression of LPS/ATP-induced macrophage pyroptosis by LL-37. Gram-negative LPS and dead/dying cell-derived ATP induce macrophage pyroptosis via the action on CD14/TLR4 bacterial LPS and dead/dying cell-derived ATP induce macrophage pyroptosis via the action on and P2X7, respectively. LL-37 reduces the LPS/ATP-stimulated pyroptosis of macrophages and IL-1β CD14/TLR4 and P2X7, respectively. LL-37 reduces the LPS/ATP-stimulated pyroptosis of production by both curtailing the action of LPS on CD14/TLR4 and blocking the P2X response to ATP. macrophages and IL-1β production by both curtailing the action of LPS on CD14/TLR47 and blocking Thethe P2X results7 response indicated to ATP. that the administration of LL-37 (2 µg/mouse) intravenously, to the CLP septic mice, improves their survival (Figure4), and the e ffect was dose-dependent, since LL-37 (1 µg/mouse The results indicated that the administration of LL-37 (2 μg/mouse) intravenously, to the CLP and 2 µg/mouse) improved the survival rate to 14.3% and 36.4%, respectively [32]. Interestingly, Int.septic J. Mol. mice, Sci. 2020 improves, 21, x FOR their PEER survival REVIEW (Figure 4), and the effect was dose-dependent, since LL-375 of (116 the pyroptosis of peritoneal macrophages and the CLP-induced caspase-1 activation are inhibited by μg/mouse and 2 μg/mouse) improved the survival rate to 14.3% and 36.4%, respectively [32]. LL-37are inhibited (Figure by5). LL-37 In addition, (Figure the 5). levelsIn addition of inflammatory, the levels of cytokines inflammatory (IL-1 β cytokines, IL-6 and (IL-1 TNF-βα, )IL-6 inboth and Interestingly, the pyroptosis of peritoneal macrophages and the CLP-induced caspase-1 activation theTNF- peritonealα) in both fluids the andperitoneal sera were fluids reduced and bysera LL-37, were which reduced also blockedby LL-37, the which peritoneal also macrophagesblocked the activationperitoneal (asmacrophages assessed by activation the elevated (as assessed intracellular by the levels elevated of IL-1 intracellularβ, IL-6 and levels TNF- ofα). IL-1 Finally,β, IL-6 LL-37 and decreasedTNF-α). Finally, the bacterial LL-37 loaddecreased in both the the bacterial peritoneal load fluids in both as well the asperitoneal blood samples fluids as (Figure well 6as). Thus,blood oursamples work (Figure indicates 6). thatThus, LL-37 our work administration indicates that improves LL-37 administration the survival of CLPimproves septic the mice survival by inhibiting of CLP activationseptic mice and by pyroptosis inhibiting of activation macrophages, and productionpyroptosis ofof inflammatorymacrophages, cytokines, production and of bacterial inflammatory growth. cytokines, and bacterial growth.

Figure 4. EffectEﬀect of LL-37 on the survival of cecal ligation and puncture (CLP)(CLP) septicseptic mice.mice. Mice were divided into the Sham (without CLP; □),), CLP CLP ( (●)) and and LL-37 LL-37 ( (○) )groups. groups. In In the the LL-37 LL-37 group, group, mice mice were • intravenously administered with 2 μg per mouse LL-37 immedeiately# after CLP, and the survival rates of the mice were monitored for 7 days. Survival data were analyzed using the Kaplan-Meier method and survival curves were compared using the log-rank test and Gehan-Breslow-Wilcoxon test in univariate analysis. n = 8–15 per group. * p < 0.05 [32].

Figure 5. Effect of LL-37 administration on the pyroptosis of peritoneal macrophages in CLP mice. Peritoneal cells were collected from mice of Sham, CLP and LL-37 groups at 5 h after the surgery. Thereafter, peritoneal cells were evaluated for pyroptosis by detecting caspase-1 activation (FLICA positive) and cell death (7AAD positive) of the peritoneal macrophages (F4/80 positive) by flow cytometry. In panel (a), upper halves, right halves and upper-right quadrants show cell death, caspase-1 activation and pyroptosis (FLICA/7AAD-double positive), respectively, among peritoneal macrophages. Panels (b) and (c) show the percentage of caspase-1 activation and pyroptosis, respectively. Values are compared between the CLP and LL-37 groups. * p < 0.05, ** p < 0.01. FLICA, fluorescent-labeled inhibitor of caspases; 7AAD, 7-amino-actinomycin D [32].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 16 are inhibited by LL-37 (Figure 5). In addition, the levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in both the peritoneal fluids and sera were reduced by LL-37, which also blocked the peritoneal macrophages activation (as assessed by the elevated intracellular levels of IL-1β, IL-6 and TNF-α). Finally, LL-37 decreased the bacterial load in both the peritoneal fluids as well as blood samples (Figure 6). Thus, our work indicates that LL-37 administration improves the survival of CLP septic mice by inhibiting activation and pyroptosis of macrophages, production of inflammatory cytokines, and bacterial growth.

Figure 4. Effect of LL-37 on the survival of cecal ligation and puncture (CLP) septic mice. Mice were Int. J. Mol. Sci. 2020, 21, 5973 5 of 16 divided into the Sham (without CLP; □), CLP (●) and LL-37 (○) groups. In the LL-37 group, mice were intravenously administered with 2 μg per mouse LL-37 immedeiately after CLP, and the survival rateswere intravenouslyof the mice were administered monitored with for 27 µdays.g per Surv mouseival LL-37 data immedeiatelywere analyzed after using CLP, the and Kaplan-Meier the survival methodrates of theand micesurvival were curves monitored were forcompared 7 days. usin Survivalg the datalog-rank were test analyzed and Gehan-Breslow-Wilcoxon using the Kaplan-Meier testmethod in univariate and survival analysis. curves n were= 8–15 compared per group. using * p < the 0.05 log-rank [32]. test and Gehan-Breslow-Wilcoxon test in univariate analysis. n = 8–15 per group. * p < 0.05 [32].

Figure 5. Effect of LL-37 administration on the pyroptosis of peritoneal macrophages in CLP mice. FigurePeritoneal 5. Effect cells of were LL-37 collected administration from mice on of the Sham, pyroptos CLPis and of peritoneal LL-37 groups macrophages at 5 h after in the CLP surgery. mice. PeritonealThereafter, cells peritoneal were collected cells were from evaluated mice of for Sham pyroptosis, CLP and by LL-37 detecting groups caspase-1 at 5 h activationafter the surgery. (FLICA Thereafter,positive) and peritoneal cell death cells (7AAD were evaluated positive) offor the pyro peritonealptosis by macrophagesdetecting caspase-1 (F4/80 activation positive) by(FLICA flow positive)cytometry. and In panelcell death (a), upper (7AAD halves, positive) right halvesof the and peritoneal upper-right macrophages quadrants show(F4/80 cell positive) death, caspase-1by flow cytometry.activation and In pyroptosispanel (a), (FLICAupper /halves,7AAD-double right halves positive), and respectively, upper-right among quadrants peritoneal show macrophages. cell death, caspase-1Panels (b) activation and (c) show and thepyroptosis percentage (FLICA/7AAD-d of caspase-1 activationouble positive), and pyroptosis, respectively, respectively. among peritoneal Values Int. J. Mol.macrophages.are comparedSci. 2020, 21 between,Panels x FOR PEER( theb) and CLPREVIEW ( andc) show LL-37 the groups. percentage * p < 0.05, of **caspase-1p < 0.01. activation FLICA, fluorescent-labeled and pyroptosis,6 of 16 respectively.inhibitor of caspases; Values are 7AAD, compared 7-amino-actinomycin between the CLP D and [32]. LL-37 groups. * p < 0.05, ** p < 0.01. FLICA, fluorescent-labeled inhibitor of caspases; 7AAD, 7-amino-actinomycin D [32].

Figure 6. Effect of LL-37 administration on the bacterial burdens in peritoneal fluids and blood samples Figure 6. Effect of LL-37 administration on the bacterial burdens in peritoneal fluids and blood of CLP mice. Peritoneal fluids and blood samples were collected from mice of Sham, CLP and LL-37 samples of CLP mice. Peritoneal fluids and blood samples were collected from mice of Sham, CLP groups at 15 h after the surgery, and serially diluted in PBS. Then, diluted samples were plated on and LL-37 groups at 15 h after the surgery, and serially diluted in PBS. Then, diluted samples were Trypto-Soya agar plates, and the plates were incubated for 20 h at 37 ◦C. CFU was counted and corrected platedfor the on dilution Trypto-Soya factor. agar Panels plates (a) and, and ( bthe) show plates the were CFU incubated in the peritoneal for 20 h at fluids 37 °C. and CFU blood was samples,counted andrespectively. corrected Values for the are dilution compared factor. between Panels the (a CLP) and and (b) LL-37show groups.the CFU * pin< the0.05, peritoneal *** p < 0.001 fluids [32 and]. blood samples, respectively. Values are compared between the CLP and LL-37 groups. * p < 0.05, *** pHowever, < 0.001 [32]. the mechanism by which LL-37 prevents pyroptosis and the caspase-1 activation in the CLP model, in vivo, is not known. In addition, LL-37, due to its antimicrobial activity, may protect the CLPHowever, septic micethe mechanism by lowering by the which burden LL-37 of bacteria prevents in thepyroptosis blood and and by the decreasing caspase-1 the activation pyroptosis in theof macrophages. CLP model, in vivo, is not known. In addition, LL-37, due to its antimicrobial activity, may protect the CLPFurthermore, septic mice it by has lowering been reported the burden that LPS, of bact a majoreria in component the blood and of bacteria, by decreasing is elevated the pyroptosis in the sera ofand macrophages. peritoneal fluids of sepsis models [42]. Moreover, dead/dying cells release ATP extracellularly, and thusFurthermore, it is increased it has in been the plasmareported of that CLP LPS, mice a [43major] and component peritoneal of fluid bacteria, of E. coli is -inducedelevated in septic the sera and peritoneal fluids of sepsis models [42]. Moreover, dead/dying cells release ATP extracellularly, and thus it is increased in the plasma of CLP mice [43] and peritoneal fluid of E. coli- induced septic mice [44]. Importantly, we earlier described that LPS and ATP together induce macrophage pyroptosis, which is blocked by LL-37 through both LPS neutralization and inhibition of the P2X7 activation by ATP [40]. Thus, we propose that in the CLP septic mice, LPS and ATP are the primary inducers of caspase-1 activation and pyroptosis, as in the case of other sepsis models [45,46], and that LL-37 prevents the actions of LPS and ATP, both in vivo and in vitro, thereby inhibiting pyroptosis (caspase-1 activation) and improving the survival of CLP mice (Figure 3).

3. LL-37 induces NET Release from Neutrophils and Reduces the Inflammatory Response in CLP Mice Neutrophils function as the first line of host defense against invading microorganisms [11,12]; and they exert antimicrobial activity through phagocytosis and the subsequent killing of microorganisms by the actions of reactive oxygen species and antimicrobial granule proteins or peptides. Moreover, upon stimulation, neutrophils undergo NETosis and release NETs [13] (Figure 7). NETs are capable of trapping the microorganisms and by the action of NET-associated components (DNA, histone and granule proteins and peptides) show anti-bacterial activity [14,15].

Int. J. Mol. Sci. 2020, 21, 5973 6 of 16 mice [44]. Importantly, we earlier described that LPS and ATP together induce macrophage pyroptosis, which is blocked by LL-37 through both LPS neutralization and inhibition of the P2X7 activation by ATP [40]. Thus, we propose that in the CLP septic mice, LPS and ATP are the primary inducers of caspase-1 activation and pyroptosis, as in the case of other sepsis models [45,46], and that LL-37 prevents the actions of LPS and ATP, both in vivo and in vitro, thereby inhibiting pyroptosis (caspase-1 activation) and improving the survival of CLP mice (Figure3).

3. LL-37 induces NET Release from Neutrophils and Reduces the Inﬂammatory Response in CLP Mice Neutrophils function as the ﬁrst line of host defense against invading microorganisms [11,12]; and they exert antimicrobial activity through phagocytosis and the subsequent killing of microorganisms by the actions of reactive oxygen species and antimicrobial granule proteins or peptides. Moreover, upon stimulation, neutrophils undergo NETosis and release NETs [13] (Figure7). NETs are capable of trapping the microorganisms and by the action of NET-associated components (DNA, histone Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 16 and granule proteins and peptides) show anti-bacterial activity [14,15].

Figure 7. Mechanism for the release of neutrophil extracellular traps (NETs). Bacteria, protozoa, diFigurefferent 7. fungiMechanism (yeast andfor the hyphae release forms) of neutrophil or their productsextracellular cause traps the stimulation(NETs). Bacteria, of neutrophils, protozoa, leadingdifferent to: fungi (a) ultrastructural(yeast and hyphae changes forms) in theor shapetheir products of nuclei cause by the the decondensation stimulation of of neutrophils, chromatin, nuclearleading membraneto: (a) ultrastructural swelling and changes fragmentation, in the shape facilitate of nuclei the by chromatin the decondensation association withof chromatin, granules andnuclear cytoplasmic membrane proteins, swelling and and (b fragmentation,) release of extracellular facilitate the structures chromatin that association contain histones with granules decorated and withcytoplasmic DNA, granular proteins, and and cytoplasmic (b) release proteins of extracellular of neutrophils, structures which that are contain capable histones of trapping decorated and killing with theDNA, microorganisms granular and [cytoplasmic47]. proteins of neutrophils, which are capable of trapping and killing the microorganisms [47]. As described above, the intravenous administration of LL-37 enhanced the survival of murine CLP sepsisAs described model [above,32]. Moreover, the intravenous it is demonstrated administrati thaton LL-37 of LL-37 induces enhanced NET release the survival from neutrophils of murine inCLP vitro sepsis[48 ].model Thus, [32]. we Moreover, hypothesized it is demonstrated that LL-37 may that induce LL-37 theinduces release NET of release NETs fromfrom neutrophilsneutrophils inin vivovitro and[48]. protect Thus, we mice hypothesized from CLP-induced that LL-37 sepsis. may Toinduce test thisthe release hypothesis, of NETs we evaluatedfrom neutrophils the effect in ofvivo LL-37 and onprotect the levels mice offrom inflammatory CLP-induced cytokines sepsis. (TNF-To testα thisand hypothesis, IL-1β), triggering we evaluated receptor the expressed effect of onLL-37 myeloid on the cells levels (TREM)-1 of inflammatory and DAMPs cytokines (histone-DNA (TNF-α and complex IL-1β), and triggering high-mobility receptor group expressed protein on 1myeloid (HMGB1)) cells as (TREM)-1 well as NETsand DAMPs (determined (histone-DNA as myeloperoxidase complex and (MPO)-DNA high-mobility complex) group inprotein plasma 1 and(HMGB1)) peritoneal as well fluids as ofNETs CLP (determined septic mice [ 33as]. myeloperoxidase (MPO)-DNA complex) in plasma and peritonealThe findings fluids of suggested CLP septic that mice LL-37 [33]. administration, intravenously, prevented the upsurge in DAMPsThe as findings well as TNF-suggestedα, IL-1 βthatand LL-37 soluble administrati TREM-1 inon, peritoneal intravenously, fluids and prevented plasma (Figures the upsurge8 and9 ).in Interestingly,DAMPs as well LL-37 as TNF- significantlyα, IL-1β reducedand soluble the increaseTREM-1 in in the peritoneal number fluids of peritoneal and plasma polymorphonuclear (Figures 8 and cells9). Interestingly, (neutrophil) during LL-37 sepsis. significantly In addition, reduced LL-37 lowered the increase the microbial in burdenthe number in circulation of peritoneal and also inpolymorphonuclear peritoneal fluids. cells Importantly, (neutrophil) LL-37 during administration sepsis. In addition, significantly LL-37 lowered increased the the microbial level of burden NETs inin circulation plasma and and peritoneal also in peritoneal fluids of CLPfluids. mice Import (Figureantly, 10 LL-37). In addition,administration we established significantlyin vitroincreasedthat the level of NETs in plasma and peritoneal fluids of CLP mice (Figure 10). In addition, we established in vitro that LL-37 directly stimulates bone marrow-derived neutrophils to release NETs and the NETs possess the bactericidal activity (Figure 11).

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LL-37Int. J. Mol. directly Sci. 2020 stimulates, 21, x FOR PEER bone REVIEW marrow-derived neutrophils to release NETs and the NETs possess8 of 16 Int.the J. bactericidal Mol. Sci. 2020, activity21, x FOR(Figure PEER REVIEW 11). 8 of 16

Figure 8. Effect of LL-37 administration on the levels of TNF-α, IL-1β and soluble TREM-1 in plasma Figureand peritoneal 8. EffectEffect fluidsof LL-37 of CLPadministration mice. Plasma on theand levels peritoneal of TNF- fluidsα, IL-1 wereβ and collected soluble from TREM-1 mice in of plasma Sham, andCLP peritonealand LL-37 fluidsfluidsgroups of at CLP 20 h mice.mice after. Plasma Plasmathe surgery, and and peritoneal peritonealand assayed fluids fluids for were wereTNF- collectedα collected, IL-1β and from from soluble mice mice of ofTREM-1 Sham, CLPby ELISA. and LL-37 TNF- groups α levels at (a 20) and h after (b), IL-1 the surgery,β levels ( andandc) and assayedassayed (d), and for TREM-1 TNF-TNF-α,, IL-1levelsIL-1β and(e) and soluble (f) in TREM-1 plasma byand ELISA. peritoneal TNF- fluids,α levels respecti ( a) and vely. (b), Values IL-1β levelslevels are compared ( (c) and ( d amon), and g TREM-1 Sham, CLP levels and ( e )LL-37 and ( groups.*f) in plasma p < and0.05, peritonealperitoneal *** p < 0.001 fluids,fluids, [33]. respectively. respectively. Values Values are are compared compared among amon Sham,g Sham, CLP CLP and and LL-37 LL-37 groups.* groups.*p < 0.05, p < ***0.05,p <***0.001 p < 0.001 [33]. [33].

Figure 9. Effect of LL-37 administration on the levels of HMGB1 and histone-DNA complex in plasma andFigure peritoneal 9. Effect fluids of LL-37 of CLP administration mice. Plasma on and the peritoneallevels of HMGB1 fluids were and collectedhistone-DNA from complex mice of Sham, in plasma CLP Figureand peritoneal 9. Effect fluidsof LL-37 of administrationCLP mice. Plasma on the and levels peritoneal of HMGB1 fluids and were histone-DNA collected from complex mice in of plasma Sham, andCLP peritonealand LL-37 fluidsgroups of at CLP 20 h mice after. Plasmathe surgery, and peritonealand assayed fluids for HMGB1were collected and histone-DNA from mice ofcomplex Sham, CLPby ELISA. and LL-37 HMGB1 groups levels at 20 (a h) andafter ( theb), andsurgery, histone-DNA and assayed complex for HMGB1 levels and(c) andhistone-DNA (d) in plasma complex and byperitoneal ELISA. fluids,HMGB1 resp levelsectively. (a) and Values (b), are and compared histone-DNA among complex Sham, CLPlevels and (c) LL-37 and ( dgroups.) in plasma * p < 0.05,and peritoneal*** p < 0.001 fluids, [33]. respectively. Values are compared among Sham, CLP and LL-37 groups. * p < 0.05, *** p < 0.001 [33].

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Int. J. Mol.and LL-37Sci. 2020 groups, 21, x FOR at 20 PEER h after REVIEW the surgery, and assayed for HMGB1 and histone-DNA complex by ELISA.9 of 16

Int. J. HMGB1Mol. Sci. 2020 levels, 21 (,a x) FOR and (PEERb), and REVIEW histone-DNA complex levels (c) and (d) in plasma and peritoneal ﬂuids,9 of 16 respectively. Values are compared among Sham, CLP and LL-37 groups. * p < 0.05, *** p < 0.001 [33].

Figure 10. Effect of LL-37 administration on the levels of MPO-DNA complex in plasma and peritoneal fluids of CLP mice. Plasma and peritoneal fluids were collected from mice of Sham, CLP FigureFigure 10.10.E ffEffectect of of LL-37 LL-37 administration administration on the on levels the oflevels MPO-DNA of MPO-DNA complex complex in plasma in and plasma peritoneal and and LL-37 groups at 20 h after the surgery, and NETs were assayed as MPO-DNA complex by ELISA. fluidsperitoneal of CLP fluids mice. of PlasmaCLP mice. and Plasma peritoneal and fluidsperitoneal were fluids collected were from collected mice offrom Sham, mice CLP of Sham, and LL-37 CLP groupsLevels of at MPO-DNA 20 h after the complex surgery, (a and) and NETs (b) werein plasma assayed and as peritoneal MPO-DNA fluids, complex respectively. by ELISA. Values Levels are of and LL-37 groups at 20 h after the surgery, and NETs were assayed as MPO-DNA complex by ELISA. MPO-DNAcompared among complex Sham, (a) andCLP (andb) in LL-37 plasma groups. and peritoneal * p < 0.05, fluids,** p < 0.05, respectively. *** p < 0.001 Values [33]. are compared Levels of MPO-DNA complex (a) and (b) in plasma and peritoneal fluids, respectively. Values are among Sham, CLP and LL-37 groups. * p < 0.05, ** p < 0.05, *** p < 0.001 [33]. compared among Sham, CLP and LL-37 groups. * p < 0.05, ** p < 0.05, *** p < 0.001 [33].

Figure 11. Induction of NET release and bactericidal activity of NETs. Mouse bone marrow-derived Figure 11. Induction of NET release and bactericidal activity of NETs. Mouse bone marrow-derived neutrophils (106 cells) were incubated without (unstimulated) or with 100 nM PMA (phorbol myristate neutrophils (106 cells) were incubated without (unstimulated) or with 100 nM PMA (phorbol acetate)Figure 11. or Induction 5 µM LL-37 of NET for 4 release h at 37 and◦C. bactericidal Then, the supernatants activity of NETs. were Mouse recovered, bone andmarrow-derived assayed for myristate acetate) or 5 μM LL-37 for 4 h at 37 °C. Then, the supernatants were recovered, and assayed MPO-DNAneutrophils complex(106 cells) by ELISAwere incubated (a). Relative without NET release (unsti frommulated) PMA- or or with LL-37-stimulated 100 nM PMA neutrophils (phorbol for MPO-DNA complex by ELISA (a). Relative NET release from PMA- or LL-37-stimulated ismyristate expressed acetate) as a ratioor 5 μ toM thatLL-37 from for 4 unstimulatedh at 37 °C. Then, neutrophils. the supernatants Bactericidal were recovered, activity of and NETs assayed was neutrophils is expressed as a ratio to that from unstimulated neutrophils. Bactericidal activity of NETs assayedfor MPO-DNA by incubating complex the supernatantsby ELISA ( froma). Relative unstimulated NET orrelease PMA-stimulated from PMA- neutrophils or LL-37-stimulated with E. coli was assayed by incubating the supernatants from unstimulated or PMA-stimulated neutrophils with (10neutrophils7 cells) in is LB expressed broth at as room a ratio temperature to that from forunstimulated 30 min (b). neutrophils. Mixtures wereBactericidal appropriately activity diluted,of NETs E. coli (107 cells) in LB broth at room temperature for 30 min (b). Mixtures were appropriately diluted, andwas platedassayed on by Trypto-Soya incubating the agar supernatants plates. The from plates unstimulated were incubated or PMA-stimulated for 20 h at 37 ◦C, neutrophils and CFU were with and plated on Trypto-Soya agar plates. The plates were incubated for 20 h at 37 °C, and CFU were countedE. coli (10 and7 cells) corrected in LB broth for the at dilution room temperature factor. Values for are 30 comparedmin (b). Mixtures between were unstimulated appropriately and PMA-ordiluted, counted and corrected for the dilution factor. Values are compared between unstimulated and LL-37-stimulatedand plated on Trypto-Soya neutrophils. agar * p plates.< 0.05 [The33]. plates were incubated for 20 h at 37 °C, and CFU were PMA-or LL-37-stimulated neutrophils. * p < 0.05 [33]. counted and corrected for the dilution factor. Values are compared between unstimulated and TREM-1PMA-or LL-37-stimulated is expressed mainly neutrophils. on monocytes * p < 0.05 [33]./macrophages and neutrophils and is recognized as a novel receptor, which participates in the amplification of inflammatory responses in sepsis [49]. Moreover, membrane-anchored TREM-1 is shed by metalloproteinases as a soluble form of TREM-1 [50], and solubleTREM-1 TREM-1 is expressed can bemainly used on as monocytes/macrophages a potential marker for identifying and neutrophils clinically and illis recognized patients with as infectiona novelTREM-1 receptor, [51]. is expressed which participates mainly on inmonocytes/macrophages the amplification of inflammatory and neutrophils responses and is inrecognized sepsis [49]. as Moreover,a novelDAMPs receptor, membrane-anchored are host which cell-derived participates TREM-1 biomolecules in the is shed amplific thatby metalloproteinasesation function of inflammatory as potent as activators a responsessoluble of form innate in ofsepsis immuneTREM-1 [49]. system[50],Moreover, and initiating soluble membrane-anchored systemicTREM-1 inflammatorycan be TREM-1used as response a is potential shed syndromeby markermetalloproteinases (SIRS), for identifying multiple as a organclinically soluble failure formill patients and of deathTREM-1 with in sepsisinfection[50], and [52 ].[51].soluble DAMPs TREM-1 consist can of nuclearbe used or as cytosolic a potential molecules, marker for such identifying as DNA, histone,clinically HMGB1 ill patients and ATP,with andinfection areDAMPs released [51]. are outsidehost cell-derived the cells following biomolecules tissue that injury function or cell as death potent [8, 53activators]. of innate immune systemBasedDAMPs initiating on are these systemichost findings, cell-derived infla ourmmatory observationsbiomolecules response suggest that syndrome function that LL-37(SIRS), as potent stimulates multiple activators organ the release,of failure innate inand immune vivo death, of bactericidalinsystem sepsis initiating [52]. NETs, DAMPs systemic thereby consistsuppressing infla ofmmatory nuclear the responseor bacterial cytoso licsyndrome growth molecules, and (SIRS), improving such multiple as DNA, the inflammatoryorgan histone, failure HMGB1 and responses death and ofATP,in sepsis,sepsis and [52]. asare evidenced released DAMPs outsideconsist by the suppressionofthe nuclear cells following or of cytoso inflammatory tissuelic molecules, injury cytokines, or suchcell death as soluble DNA, [8,53]. TREM-1histone, andHMGB1 DAMPs and (hostATP,Based celland death) are on releasedthese and thefindings, outside change our the of inflammatoryobservationscells following suggest cell tissue number, thatinjury LL-37 and or protectscell stimulates death mice [8,53]. the from release, lethal sepsisin vivo [33, of]. bactericidalBased onNETs, these therebyfindings, suppressing our observations the bact suggesterial thatgrowth LL-37 and stimulates improving the release,the inflammatory in vivo, of responsesbactericidal of sepsis,NETs, asthereby evidenced suppressing by the suppression the bacterial of inflammatory growth and cytokines,improving soluble the inflammatory TREM-1 and DAMPsresponses (host of sepsis, cell death) as evidenced and the changeby the suppression of inflamma oftory inflammatory cell number, cytokines, and protects soluble mice TREM-1 from lethal and sepsisDAMPs [33]. (host cell death) and the change of inflammatory cell number, and protects mice from lethal sepsis [33].

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4. LL-37 Stimulates the Release of Antimicrobial Ectosomes from Neutrophils and Improves the the Septic Condition Septic Condition Various host cells release extracellular vesicles including ectosomes (100-1000 nm in diameter) Various host cells release extracellular vesicles including ectosomes (100-1000 nm in diameter) and exosomes (30–150 nm in diameter) that mediate intercellular communications [16,17] (Figure 12). and exosomes (30–150 nm in diameter) that mediate intercellular communications [16,17] (Figure 12). During the inflammatory response, neutrophils release microvesicles, named ectosomes, which During the inflammatory response, neutrophils release microvesicles, named ectosomes, which bud bud off from the cell membrane [16,17]. Ectosomes express the cell surface molecules originating off from the cell membrane [16,17]. Ectosomes express the cell surface molecules originating from from neutrophils such as Ly6G and phosphatidylserine, and contain functional proteins of neutrophils such as Ly6G and phosphatidylserine, and contain functional proteins of neutrophils neutrophils [17,18]. Interestingly, ectosomes released from bacteria-stimulated neutrophils exhibit [17,18]. Interestingly, ectosomes released from bacteria-stimulated neutrophils exhibit bacteriostatic bacteriostatic potential [19]. Moreover, the ectosome level is reported to be augmented in surviving potential [19]. Moreover, the ectosome level is reported to be augmented in surviving patients of patients of sepsis [20,21]; thus, ectosomes are speculated to play a protective role in sepsis. Since sepsis [20,21]; thus, ectosomes are speculated to play a protective role in sepsis. Since the survival of thea murine survival CLP of asepsis murine model CLP sepsisis improved model isby improved LL-37 [32,33], by LL-37 we [investigated32,33], we investigated the mechanisms the mechanisms of LL-37- ofmediated LL-37-mediated protective protective action against action againstsepsis, sepsis,by addressing by addressing the effect the eff ectof ofLL-37 LL-37 on on the the release release of ectosomes in the CLP model [[34].34].

Figure 12. Schematic representation of extracellular vesicles released from cells. Three subtypes of extracellularFigure 12. Schematic vesicles are representation secreted by cells, of extracellular and these are vesicl exosomes,es released ectosomes from (sheddingcells. Three microvesicles) subtypes of andextracellular apoptotic vesicles bodies. Exosomesare secreted arise by via exocytosis,cells, and whereasthese are plasma exosomes, membrane ectosomes outward (shedding budding generatesmicrovesicles) ectosomes. and apoptotic On the otherbodies. hand, Exosomes apoptotic arise bodies via exocytosis, are produced whereas by cells plasma undergoing membrane later stagesoutward of apoptosis.budding generates MVB: multivesicular ectosomes. On body the [ 17othe]. r hand, apoptotic bodies are produced by cells undergoing later stages of apoptosis. MVB: multivesicular body [17]. The ﬁndings showed that administration of LL-37 enhances the level of ectosomes in the peritoneal exudatesThe andfindings plasma showed of CLP-operated that administration mice as well of asLL-37 Sham enhances (the same the procedure level of but ectosomes without ligation in the andperitoneal puncture) exudates mice (Figureand plasma 13), suggesting of CLP-operated that the mice enhanced as well level as ofSham ectosomes (the same may procedure be associated but withwithout the ligation survival and of CLP puncture) mice. Inmice addition, (Figure the 13), bacterial suggesting load that was the decreased enhanced in LL-37-injectedlevel of ectosomes CLP micemay be compared associated with with PBS-injected the survival CLP of CLP mice; mice. thus, Init addition, could be the speculated bacterial thatloadthe was enhanced decreased level in LL- of ectosomes37-injected isCLP associated mice compared with the lowerwith PBS-injected bacterial load CLP in LL-37-injected mice; thus, it CLPcould mice. be speculated To further conﬁrmthat the theenhanced antibacterial level of activity ectosomes of ectosomes, is associated we with assessed the lower the antibacterial bacterial load activity in LL-37-injected of ectosome CLP fractions mice. isolatedTo further from confirm PBS- and the LL-37-injected antibacterial CLPactivity mice. of Importantly,ectosomes, we both assessed fractions the possessed antibacterial the antibacterial activity of potential,ectosome andfractions interestingly, isolated the from fraction PBS- from and LL-37-injectedLL-37-injected CLP CLP mice mice. displayed Importantly, higher both potential fractions than thatpossessed from PBS-injected the antibacterial CLP micepotential, (Figure and 14 a).interestin These observationsgly, the fraction suggest from that LL-37-injected LL-37 elevates CLP the levelmice ofdisplayed ectosomes higher with potential higher antibacterial than that from potential, PBS-injected thereby reducingCLP mice the (Figure bacterial 14a). load These in CLP observations mice. suggest that LL-37 elevates the level of ectosomes with higher antibacterial potential, thereby reducing the bacterial load in CLP mice.

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Figure 13. Effect of LL-37 administration on the ectosome level in peritoneal exudates and plasma of Figure 13. Effect of LL-37 administration on the ectosome level in peritoneal exudates and plasma of CLPFigure mice. 13. EffectPeritoneal of LL-37 exudates administration and plasma on were the ectosome collected levelfrom in mice peritoneal of Sham exudates and CLP and groups plasma with of CLP mice. Peritoneal exudates and plasma were collected from mice of Sham and CLP groups with PBS-CLP mice.or LL-37-injection Peritoneal exudates at 14–16 and h after plasma the surgery,were collected microvesicles from mice isolated of Sham from and peritoneal CLP groups exudates with PBS- or LL-37-injection at 14–16 h after the surgery, microvesicles isolated from peritoneal exudates andPBS- plasma or LL-37-injection were incubated at 14–16 with h afterPE-anti-Ly6G the surgery, IgG microvesicles and FITC-Annexin isolated V, fr omand peritoneal analyzed exudatesby flow and plasma were incubated with PE-anti-Ly6G IgG and FITC-Annexin V, and analyzed by flow cytometry.and plasma Ectosomes were incubated express with the cell PE-anti-Ly6G surface molecu IgGles and originated FITC-Annexin from neutrophils V, and analyzed such as Ly6Gby flow (a cytometry. Ectosomes express the cell surface molecules originated from neutrophils such as Ly6G neutrophilcytometry. surfaceEctosomes marker) express and the phosphatidylserine, cell surface molecu andles originateddefined as fromdouble-positive neutrophils (Ly6G such as+/Annexin Ly6G (a (a neutrophil surface marker) and phosphatidylserine, and defined as double-positive (Ly6G+/Annexin Vneutrophil+ particles) surface for both marker) anti-Ly6G and phosphatidylserine, antibody and Anne andxin definedV (a substance as double-positive with an ability (Ly6G to+/Annexin bind to V+ particles) for both anti-Ly6G antibody and Annexin V (a substance with an ability to bind to phosphatidylserine).V+ particles) for both ( aanti-Ly6G) Representative antibody cytograms and Anne of microvesicxin V (a substanceles from peritoneal with an ability exudates to bindof PBS- to phosphatidylserine). (a) Representative cytograms of microvesicles from peritoneal exudates of PBS- orphosphatidylserine). LL-37-injected Sham (a) orRepresentative CLP mice are cytograms shown. Th ofe countsmicrovesic of ectosomesles from peritoneal in peritoneal exudates exudates of PBS- (b) or LL-37-injected Sham or CLP mice are shown. The counts of ectosomes in peritoneal exudates andor LL-37-injected plasma (c) are Sham shown. or ValuesCLP mice were are compared shown. Th betweee countsn PBS-injected of ectosomes Sham in peritoneal and CLP mice,exudates LL-37- (b) (b) and plasma (c) are shown. Values were compared between PBS-injected Sham and CLP mice, injectedand plasma Sham (c )and are shown.CLP mice, Values PBS-injected were compared and LL- betwee37-injectedn PBS-injected Sham mice, Sham and andPBS-injected CLP mice, and LL-37- LL- LL-37-injected Sham and CLP mice, PBS-injected and LL-37-injected Sham mice, and PBS-injected 37-injectedinjected Sham CLP and mice. CLP * pmice, < 0.05, PBS-injected ** p < 0.01, and*** p LL-< 0.001.37-injected N.D. (not Sham detected); mice, an ectosomesd PBS-injected could and not LL- be and LL-37-injected CLP mice. * p < 0.05, ** p < 0.01, *** p < 0.001. N.D. (not detected); ectosomes could counted37-injected in theCLP plasma mice. *of p PBS-injected< 0.05, ** p < Sham0.01, *** mice p < [34].0.001. N.D. (not detected); ectosomes could not be not be counted in the plasma of PBS-injected Sham mice [34]. counted in the plasma of PBS-injected Sham mice [34].

Figure 14. AntibacterialAntibacterial activity activity and and detection detection of of antibact antibacterialerial molecules in ectosome fractions isolated from peritoneal exudates of CLP mice. (a) Ectosome fractions (1, 2.5 or 5 µg protein) isolated from fromFigure peritoneal 14. Antibacterial exudates activity of CLP and mice. detection (a) Ectosome of antibact fractionserial molecules (1, 2.5 or in 5 ectosome μg protein) fractions isolated isolated from peritoneal exudates of PBS-injected (PBS-injected CLP) or LL-37-injected CLP (LL-37-injected CLP) peritonealfrom peritoneal exudates exudates of PBS-injected of CLP mice. (PBS-injecte (a) Ectosomed CLP) fractions or LL-37-injected (1, 2.5 or 5 CLPμg protein) (LL-37-injected isolated CLP)from mice were incubated with E. coli for 20 min at 37 C. The relative bacteria viability is calculated miceperitoneal were incubatedexudates of with PBS-injected E. coli for 20(PBS-injecte min at 37d ° C.CLP)◦ The or relative LL-37-injected bacteria CLPviability (LL-37-injected is calculated CLP) and and expressed as the percentage by dividing the colony-forming unit (cfu) of bacteria incubated with expressedmice were asincubated the percentage with E. coliby dividingfor 20 min the at colo37 °ny-formingC. The relative unit bacteria (cfu) of viability bacteria is incubated calculated with and ectosome fractions by that with vehicle (PBS). Values were compared between ectosome fractions ectosomeexpressed fractionsas the percentage by that with by vehicledividing (PBS). the colo Valuesny-forming were comp unitared (cfu) between of bacteria ectosome incubated fractions with from PBS-injected CLP and LL-37-injected CLP mice, and among ectosome fractions (containing 1, 2.5 fromectosome PBS-injected fractions CLP by andthat LL-37-injectedwith vehicle (PBS). CLP mice Values, and were among comp ectosomeared between fractions ectosome (containing fractions 1, 2.5 and 5 µg protein) isolated from PBS-injected CLP or LL-37-injected CLP mice. * p < 0.05, ** p < 0.01, andfrom 5 PBS-injectedμg protein) isolated CLP and from LL-37-injected PBS-injected CLP CLP mice or LL-37-injected, and among ectosome CLP mice. fractions * p < 0.05, (containing ** p < 0.01, 1, ***2.5 *** p < 0.001. (b) Ectosome fractions (1 µg protein) isolated from peritoneal exudates of PBS-injected pand < 0.001. 5 μg protein)(b) Ectosome isolated fractions from PBS-injected (1 μg protein) CLP isolated or LL-37-injected from peritoneal CLP exudates mice. * p of< 0.05, PBS-injected ** p < 0.01, CLP *** CLP and LL-37-injected CLP mice were subjected to SDS-PAGE, followed by western blotting using andp < 0.001. LL-37-injected (b) Ectosome CLP fractions mice were (1 μsubjectedg protein) to isolated SDS-PAGE, from followed peritoneal by exudates western ofblotting PBS-injected using anti-CLP anti-lactoferrin, MPO, and CRAMP antibodies. Each lane shows an ectosome fraction isolated from lactoferrin,and LL-37-injected MPO, and CLP CRAMP mice were antibodies. subjected Each to SDS-PAGE, lane shows followedan ectosome by western fraction blottingisolated usingfrom eachanti- each of PBS-injected CLP and LL-37-injected CLP mouse [34]. oflactoferrin, PBS-injected MPO, CLP and and CRAMP LL-37-injected antibodies. CLP Each mouse lane [34]. shows an ectosome fraction isolated from each of PBS-injected CLP and LL-37-injected CLP mouse [34].

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 16 Since neutrophil granule molecules are contained in ectosomes [17,18], we examined Since neutrophil granule molecules are contained in ectosomes [17,18], we examined the the involvement of neutrophil granule molecules in the antibacterial activity of ectosomes. Western blot involvement of neutrophil granule molecules in the antibacterial activity of ectosomes. Western blot analysis indicated that the neutrophil granule-derived antimicrobial molecules, such as lactoferrin, MPO analysis indicated that the neutrophil granule-derived antimicrobial molecules, such as lactoferrin, and CRAMP (cathelicidin-related antimicrobial peptide, a mouse ortholog of human cathelicidin peptide) MPO and CRAMP (cathelicidin-related antimicrobial peptide, a mouse ortholog of human were detected in the ectosome fractions isolated from peritoneal exudates of both PBS- and LL-37-injected cathelicidin peptide) were detected in the ectosome fractions isolated from peritoneal exudates of CLP mice. In addition, the fraction from LL-37-injected CLP mice had increased amounts of these both PBS- and LL-37-injected CLP mice. In addition, the fraction from LL-37-injected CLP mice had antimicrobial molecules compared with that from PBS-injected CLP mice (Figure 14b). In addition, increased amounts of these antimicrobial molecules compared with that from PBS-injected CLP mice anti-lactoferrin or anti-CRAMP antibody partially but substantially abrogated the antibacterial activity (Figure 14b). In addition, anti-lactoferrin or anti-CRAMP antibody partially but substantially of ectosome fractions from PBS- and LL-37-injected CLP mice, suggesting the involvement of lactoferrin abrogated the antibacterial activity of ectosome fractions from PBS- and LL-37-injected CLP mice, and CRAMP in the antibacterial potential of ectosomes. Collectively, these findings indicate that LL-37 suggesting the involvement of lactoferrin and CRAMP in the antibacterial potential of ectosomes. stimulates the release of ectosomes containing higher amounts of antibacterial molecules (lactoferrin Collectively, these findings indicate that LL-37 stimulates the release of ectosomes containing higher and CRAMP) in CLP mice, which may exert the antibacterial action and reduce the bacterial load, amounts of antibacterial molecules (lactoferrin and CRAMP) in CLP mice, which may exert the thereby improving the survival of septic mice. antibacterial action and reduce the bacterial load, thereby improving the survival of septic mice. Moreover, we examined whether LL-37 is able to stimulate neutrophils ex vivo, to release Moreover, we examined whether LL-37 is able to stimulate neutrophils ex vivo, to release ectosomes and whether the administration of ectosomes ameliorates a murine septic CLP model ectosomes and whether the administration of ectosomes ameliorates a murine septic CLP model in in vivo. Importantly, LL-37 directly activated mouse bone marrow-derived neutrophils, ex vivo, to vivo. Importantly, LL-37 directly activated mouse bone marrow-derived neutrophils, ex vivo, to release ectosomes (Figure 15a). In addition, the LL-37-triggered ectosomes have the antibacterial release ectosomes (Figure 15a). In addition, the LL-37-triggered ectosomes have the antibacterial potential, and the administration of these ectosomes to CLP mice led to improved survival of mice potential, and the administration of these ectosomes to CLP mice led to improved survival of mice and reduced bacterial load (Figure 15b). and reduced bacterial load (Figure 15b).

Figure 15. EffectEffect of LL-37 on the release of ectosome ectosomess from from mouse mouse bone bone marrow-derived marrow-derived neutrophils, neutrophils, and eeffectffect of ectosomeectosome administration on the survival of CLPCLP mice.mice. (a) Mouse bone marrow-derived 6 neutrophils (2 × 106 cells) were stimulated with fMLF or LL-37 for 45 min at 37 °C, and the released neutrophils (2 × 10 cells) were stimulated with fMLF or LL-37 for 45 min at 37 ◦C, and the released ectosomes in in the the supernatant supernatant were were analyzed analyzed by by flow flow cytometry. cytometry. Values Values were were compared compared between between the theincubation incubation with with vehicle vehicle (PBS) (PBS) and and fM fMLFLF or orLL-37, LL-37, and and among among 1, 1, 3 3and and 10 10 μµg/mLg/mL of of LL-37. An inlet shows aa representative representative cytogram cytogram of ectosomes of ectosomes released released from LL-37 from (10 LL-37µg/mL)-stimulated (10 μg/mL)-stimulated neutrophils. (b) CLP mice were intraperitoneally injected with PBS (PBS-injected CLP) or ectosomes (3 105 neutrophils. (b) CLP mice were intraperitoneally injected with PBS (PBS-injected CLP) or ectosomes× ectosomes)(3 × 105 ectosomes) isolated isolated from LL-37-stimulated from LL-37-stimulated neutrophils neutrophils (LL-37-MV-injected (LL-37-MV-injected CLP) 2 h CLP) after 2 the h after surgery. the Shamsurgery. mice Sham were mice also injectedwere also with injected PBS (PBS-injected with PBS (PBS-injected Sham) or ectosomes Sham) or (LL-37-MV-injected ectosomes (LL-37-MV- Sham) 2injected h after Sham) the surgery. 2 h after The the survival surgery. was The monitored survival was for 10monitored days, and for the 10 survival days, and rate the was survival compared rate betweenwas compared PBS-injected between CLP PBS-injected and LL-37-MV-injected CLP and LL-37-MV-injected CLP mice by CLP Kaplan-Meier mice by Kaplan-Meier method for 10method days p p p andfor 10 by days chi squareand by analysis chi square for analysis each day. for * each< 0.05, day. ** * p< < 0.01,***0.05, ** p < 0.01,***0.001 [34 p ].< 0.001 [34]. Collectively, these findingsfindings indicate that LL-37 stimulates neutrophils to release antibacterial ectosomes, thereby eliminating bacteria, and protectingprotecting micemice fromfrom lethallethal sepsis.sepsis.

5. Perspective Cationic antimicrobial peptides target cell surfacesurface anionic lipids such as phosphatidyl glycerol and cardiolipin that that are are abundant abundant in in microorganisms; microorganisms; the the action action is is not not receptor-based receptor-based but but involves involves a aless less specific speciﬁc interaction interaction with with microbial microbial membrane membrane components components [54,55]. [54,55]. In In contrast, contrast, the mammalian cell membrane is mainly composed of electrically neutral phospholipids such as phosphatidylcholine and sphingomyelin, sphingomyelin, for for which which the the affinity aﬃnity of ofthe the antimicrobial antimicrobial peptides peptides is generally is generally low [53]. low [The53]. simple electrostatic interaction between cationic antimicrobial peptides and microbial membrane lipids provides selective toxicity (bacteria versus mammalian cells) as well as a broad spectrum of

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TheInt. J. simpleMol. Sci. electrostatic2020, 21, x FOR interaction PEER REVIEW between cationic antimicrobial peptides and microbial membrane 13 of 16 lipids provides selective toxicity (bacteria versus mammalian cells) as well as a broad spectrum antimicrobial activities. Moreover, the development of microbial resistance is assumed to be low, of antimicrobial activities. Moreover, the development of microbial resistance is assumed to be because the target molecules (anionic lipids) are important components conserved among low, because the target molecules (anionic lipids) are important components conserved among microorganisms, and the molecular recognition between cationic peptides and target molecules is microorganisms, and the molecular recognition between cationic peptides and target molecules is rather lenient [54,55]. In addition, the peptides are small and relatively easy to synthesize. From these rather lenient [54,55]. In addition, the peptides are small and relatively easy to synthesize. From these points of view, cationic antimicrobial peptides could be promising candidates for new antibiotics points of view, cationic antimicrobial peptides could be promising candidates for new antibiotics with with therapeutic value. Moreover, cationic host defense peptides are expected to protect the host therapeutic value. Moreover, cationic host defense peptides are expected to protect the host against against pathogens not only by being directly antimicrobial but also by modulating the immune pathogens not only by being directly antimicrobial but also by modulating the immune responses responses and boosting infection-resolving immunity while dampening potentially harmful pro- and boosting infection-resolving immunity while dampening potentially harmful pro-inflammatory inflammatory (septic) responses [56–59]. (septic) responses [56–59]. However, it should be noted that as cationic antimicrobial peptides act principally via However, it should be noted that as cationic antimicrobial peptides act principally via electrostatic electrostatic attraction, and hydrophobic partitioning into the membrane targets, they could also bind attraction, and hydrophobic partitioning into the membrane targets, they could also bind to various to various host components such as anionic constituents of host cell membranes, leading to host components such as anionic constituents of host cell membranes, leading to potentially harmful potentially harmful side effects on the host [55]. In this context, it has been demonstrated that high side effects on the host [55]. In this context, it has been demonstrated that high concentrations of concentrations of cationic antimicrobial peptides are occasionally toxic to host cells [26]. Thus, cationic antimicrobial peptides are occasionally toxic to host cells [26]. Thus, cationic antimicrobial cationic antimicrobial peptides should be cautiously administered in vivo, considering their toxic peptides should be cautiously administered in vivo, considering their toxic effects on host cells. effects on host cells. Our studies have revealed that LL-37 exhibits multiple functions in sepsis. Firstly, LL-37 enhances Our studies have revealed that LL-37 exhibits multiple functions in sepsis. Firstly, LL-37 the survival of CLP mice by reducing the macrophage pyroptosis that induces the secretion of enhances the survival of CLP mice by reducing the macrophage pyroptosis that induces the secretion pro-inflammatory cytokines (such as IL-1β) and augments inflammatory reactions in sepsis [32]. of pro-inflammatory cytokines (such as IL-1β) and augments inflammatory reactions in sepsis [32]. Secondly, LL-37 induces the release of NETs with potent bactericidal activity and protects mice from Secondly, LL-37 induces the release of NETs with potent bactericidal activity and protects mice from CLP-induced sepsis [33]. Thirdly, LL-37 stimulates neutrophils to release antimicrobial ectosomes, CLP-induced sepsis [33]. Thirdly, LL-37 stimulates neutrophils to release antimicrobial ectosomes, which improve murine sepsis [34]. These findings indicate that LL-37 protects CLP septic mice which improve murine sepsis [34]. These findings indicate that LL-37 protects CLP septic mice through at least three mechanisms, i.e., the suppression of pro-inflammatory macrophage pyroptosis through at least three mechanisms, i.e., the suppression of pro-inflammatory macrophage pyroptosis and the release of antimicrobial NETs (induction of NETosis) and ectosomes from neutrophils. Thus, and the release of antimicrobial NETs (induction of NETosis) and ectosomes from neutrophils. Thus, LL-37 can be a promising candidate as a therapeutic agent for sepsis because of its multiple functions, LL-37 can be a promising candidate as a therapeutic agent for sepsis because of its multiple functions, including modulation of cell death (pyroptosis and NETosis) and release of antimicrobial NETs including modulation of cell death (pyroptosis and NETosis) and release of antimicrobial NETs and and ectosomes as well as its own bactericidal and LPS-neutralizing activities (Figure 16). Furthermore, ectosomes as well as its own bactericidal and LPS-neutralizing activities (Figure 16). Furthermore, our findings likely open novel paths for designing immunomodulatory peptide drugs, using LL-37 our findings likely open novel paths for designing immunomodulatory peptide drugs, using LL-37 as the lead molecule with multiple actions on the pathogenesis of sepsis. as the lead molecule with multiple actions on the pathogenesis of sepsis.

Figure 16.16. Therapeutic action action of of antimicrobial antimicrobial cathelicidin cathelicid peptidein peptide LL-37 LL-37 on a murineon a murine sepsis sepsis model. model. LL-37 protectsLL-37 protects CLP septic CLP mice septic through mice at through least three at mechanisms, least three i.e.,mechanisms, the suppression i.e., the of pro-inflammatory suppression of pro- inflammatory macrophage pyroptosis (yellow arrows) and the release of antimicrobial NETs (blue arrows) and ectosomes (red arrows) from neutrophils, in addition to its own bactericidal and LPS- neutralizing activities.

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macrophage pyroptosis (yellow arrows) and the release of antimicrobial NETs (blue arrows) and ectosomes (red arrows) from neutrophils, in addition to its own bactericidal and LPS-neutralizing activities.

Author Contributions: I.N. prepared this manuscript by discussing with H.T. and J.R., based on the data already published [32–34]. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded in part by a Grant-in-Aid (grant numbers 23590519, 26460538, 17K08840 and 20K07486) for Scientific Research from Japan Society for the Promotion of Science, and a Grant-in-Aid (grant numbers S0991013 and S1201013) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) for the Foundation of Strategic Research Projects in Private Universities. Acknowledgments: The author is grateful to Zhongshuang Hu and Yumi Kumagai (Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine), and Hiroshi Hosoda (Department of Molecular Microbiology, Faculty of Life Sciences, Tokyo University of Agriculture) for performing excellent experiments and publishing their data in the journals, described in this review; Niyonsaba François (Atopy Research Center, Juntendo University, Graduate School of Medicine), Kaori Suzuki (Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine), Taisuke Murakami (Department of Microbiology and Immunology, Gifu University of Medical Science), and Toshiaki Iba (Department of Emergency and Disaster Medicine, Juntendo University, Graduate School of Medicine) the helpful suggestion and discussion. Conflicts of Interest: The authors declare no conflict of interest.

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