Tài liệu Báo cáo khoa học: Multifunctional host defense peptides: Antimicrobial peptides, the small yet big players in innate and adaptive immunity docx

Cationic peptides are divided into several subfamilies, of which the most extensively studied are the mammalian gene families of antimicrobial peptides, the cathelicidins and defensins [

Multifunctional host defense peptides: Antimicrobial

peptides, the small yet big players in innate and adaptive immunity

Constance Auvynet1,2,* and Yvonne Rosenstein1

1 Instituto de Biotecnologia, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Mor Mexico

2 FRE 2852, Peptidome de la peau des amphibiens, CNRS ⁄ Universite´ Pierre et Marie Curie, Paris, France

Introduction

Antimicrobial peptides constitute a heterogeneous

group of peptides with respect to their primary and

secondary structures, their antimicrobial potentials,

their effects on host cells, and the regulation of their

expression Most antimicrobial peptides are small (12–

50 amino acids), have a positive charge provided by

Arg and Lys residues, and an amphipathic structure

that enables them to interact with bacterial membranes Cationic peptides are divided into several subfamilies, of which the most extensively studied are the mammalian gene families of antimicrobial peptides, the cathelicidins and defensins [1–3] A comprehensive view of the field can be obtained through recent reviews that have covered this subject extensively [4–7]

Keywords

antimicrobial peptides; cathelicidins;

defensins; gene expression; immunity

Correspondence

Y Rosenstein, Instituto de Biotecnologia,

Universidad Nacional Auto´noma de Me´xico,

Av Universidad 2001, Col Chamilpa,

Cuernavaca, Mor 62210, Mexico

Fax: +52 777 317 2388

Tel: +52 777 329 1606

E-mail: yvonne@ibt.unam.mx

*Present address

INSERM UMR-S 945 Immunite´ et

Infection ⁄ Universite´ Pierre et Marie Curie,

Paris, France

(Received 31 May 2009, revised 3

September 2009, accepted 4 September

2009)

doi:10.1111/j.1742-4658.2009.07360.x

The term ‘antimicrobial peptides’ refers to a large number of peptides first characterized on the basis of their antibiotic and antifungal activities In addition to their role as endogenous antibiotics, antimicrobial peptides, also called host defense peptides, participate in multiple aspects of immunity (inflammation, wound repair, and regulation of the adaptive immune sys-tem) as well as in maintaining homeostasis The possibility of utilizing these multifunctional molecules to effectively combat the ever-growing group of antibiotic-resistant pathogens has intensified research aimed at improving their antibiotic activity and therapeutic potential, without the burden of an exacerbated inflammatory response, but conserving their immunomodula-tory potential In this minireview, we focus on the contribution of small cationic antimicrobial peptides – particularly human cathelicidins and defen-sins – to the immune response and disease, highlighting recent advances

in our understanding of the roles of these multifunctional molecules

Abbreviations

CRAMP, murine cathelin-related antimicrobial peptide; EGFR, epidermal growth factor receptor; ET, extracellular trap; GM-CSF, granulocyte– macrophage colony-stimulating factor; HD, human defensin; hBD, human b-defensin; HNP, human neutrophil peptide (a-defensins); IFN-c, interferon-c; IL, interleukin; LPS, lipopolysaccharide; NFjB, nuclear factor kappaB; NK, natural killer; SCCE, stratum corneum chymotryptic enzyme; SCTE, stratum corneum tryptic enzyme; TCF-4, transcription factor-4; TLR, Toll-like receptor; TNF-a, tumor necrosis factor-a; VDR, vitamin D receptor.

Herein, we have centered our attention on the most

recent findings regarding the transcriptional regulation

of cathelicidins and defensins, and the mechanisms

through which they modulate different facets of

immu-nity and disease

Defensins are cationic peptides containing six Cys

residues forming three intramolecular disulfide bonds

On the basis of the position of the six conserved Cys

residues and on sequence identity, members of this

family of peptides have been classified into a-defensins,

b-defensins, and h-defensins Defensins are widely

expressed [8], and in mammalian species more than

100 have been identified Depending on the cell, they

will exert their function either in the intracellular or

extracellular compartment They exhibit bactericidal,

fungicidal and antiviral activity [9–11] Defensins are

either stored in granules of neutrophils or Paneth cells

or secreted by monocytes, macrophages, mast cells,

natural killer (NK) cells, keratinocytes, and epithelial

cells When released into the extracellular milieu, they

exert their antimicrobial activity directly by attacking

the microbe membrane, and in the intracellular

com-partment, they contribute to the oxygen-independent

killing of phagocytosed microorganisms Furthermore,

defensins are mediators in the crosstalk between the

innate and adaptive immune systems [4]

In addition to a highly conserved cathelin domain,

cathelicidins have an N-terminal signal peptide and a

structurally variable antimicrobial peptide at the

C-ter-minus Humans and mice have only one cathelicidin

gene, whereas other mammals, such as pigs and cattle,

have several genes [12] In humans, the cathelicidin

antimicrobial peptide gene encodes an inactive

precur-sor protein (hCAP18) that is processed to release a 37

amino acid peptide (LL-37) from the C-terminus of

the precursor protein Several cell types produce

cath-elicidins: keratinocytes, macrophages, mast cells,

neutrophils, and eccrine glands [13] Cathelicidins kill

Gram-positive and Gram-negative bacteria and

Trypanosoma cruzi Similar to defensins, cathelicidins

participate actively in linking innate and adaptive

immunity and in modulating the amplitude of immune

responses [14]

Expression pattern and gene regulation

In general, mature, biologically active peptides require

proteolytic cleavage from a precursor peptide [15] The

expression pattern of antimicrobial peptides is not

uni-form across species, and within a species it is regulated

by the cellular lineage, the differentiation⁄ activation

state of the cell, and the tissue type [16] Some

antimi-crobial peptides are synthesized in the absence of

infec-tion or inflammainfec-tion, whereas others are upregulated

in response to endogenous or infectious ‘alarm’ signals, suggesting different functions for these peptides under different physiological settings Moreover, differential proteolytic processing can modulate their activity and,

by extension, their ability to modulate immunity [17] Consequently, the combination of defense peptides produced by different cell types in a given tissue can positively or negatively modify cell functions, ulti-mately promoting bacterial clearance, albeit not neces-sarily through direct killing, but through the establishment of immune cell circuits

Defensins Genes for antimicrobial peptides tend to cluster within

a chromosomal region In the human genome, the genes encoding most human defensins are grouped within the same chromosomal region (8p21–23) [18], suggesting evolution from a single precursor gene as well as the existence of a master switch to orchestrate the synthesis of these molecules However, the genes encoding the defensin family secreted in epididymis, testis, pancreas, kidney and skeletal muscle are located

in chromosome 20 These peptides seem to be unique

in the sense that they are only synthesized in those locations, and not in the skin or airways, the common sites for b-defensins, indicative of an as yet undiscov-ered biological function [19] Interestingly, the number

of defensin genes on chromosome 8 appears to fluc-tuate among individuals, partially explaining genetic susceptibility to infection [20]

Human a-defensins [human neutrophil peptides (HNPs) 1–4] are produced by leukocytes, Paneth cells

of the small intestine, and epithelial cells of the female urogenital tract [1] On stimulation through Toll-like receptor (TLR)-2, TLR-3, and TLR-5, neutrophils,

NK cells and Paneth cells will release stored a-defen-sins to the extracellular milieu, where they will exert their antimicrobial activity Interestingly, in addition

to its antimicrobial capacity, a-defensin HNP1 has antiviral activity, as it inhibits HIV and influenza virus replication, following viral entry into target cells It diminishes HIV replication by, on the one hand, block-ing steps subsequent to reverse transcription and inte-gration, and on the other by hindering a cellular protein kinase C-dependent mechanism that partici-pates in viral infection [21,22] Similarly, it can inacti-vate herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and adenovirus [23] Whether the molecular mechanisms that mediate these antiviral effects are common or virus-specific remains an open question

Human b-defensins (hBDs) 1–4 show unique as

well as overlapping expression patterns The hBD-1

b-defensin is constitutively synthesized by epithelia that

are in direct contact with the environment or microbial

flora, such as lung, salivary gland, mammary gland,

prostate, gut, as well as by leukocytes; it is upregulated

by lipopolysaccharide (LPS) and peptidoglycan [24]

Although the expression pattern of hBD2 overlaps

with that of hBD1, it is also present in skin, pancreas,

leukocytes, and bone marrow In addition to epithelia,

hBD3 has been detected in nonepithelial cells, in the

heart, liver, and placenta [4], and hBD4 mRNA has

been detected in the testis, epididymis, lung tumor

tis-sue [25], and gastric epithelial cells [26] hBD1 and

hBD2 have predominant antibacterial activity against

Gram-negative bacteria and some fungi, whereas

hBD3 has a broader spectrum and kills many

patho-genic Gram-positive and Gram-negative bacteria and

opportunistic yeasts such as Candida albicans [27]

b-Defensin expression is modulated in response to

bacterial-derived molecules and⁄ or to cytokines and

chemokines produced by the immune system or

dam-aged cells [16] In keratinocytes stimulated by bacteria,

interferon-c (IFN-c), tumor necrosis factor-a (TNF-a),

interleukin (IL)-b, IL-17, or IL-22, hBD2 and hBD4

gene expression is upregulated, like that of hBD1 and

hBD3 in airway, intestinal or uterine epithelial cells

[28,29], whereas it is inhibited by retinoic acid [30] and

heat shock [31] In immune cells, their production is

also upregulated following exposure to bacteria, LPS,

IFN-c, or IL-b [29]

Cathelicidins

The human cathelicidin gene is located on

chromo-some 3 (3p21.3), in close proximity to the genes

encod-ing TLR-9 and Myd88 (3p22) Cathelicidins are

constitutively synthesized in thymus, spleen, bone

mar-row, liver, skin, stomach, intestine, and testis Besides

epithelial cells, they are produced by neutrophils,

monocytes, T-lymphocytes, B-lymphocytes, and NK

cells Upon epidermal injury, the concentration of

human cathelicidin LL-37 is augmented significantly in

keratinocytes and epidermal mast cells [32], and it has

been detected in wound and blister fluid as well

[33,34] In keratinocytes, synthesis of LL-37 is induced

in response to insulin-like growth factor 1, TNF-a [35],

IL-1a, and IL-6 [36], and upon contact with

Staphylo-coccus aureus [37] Cathelicidin peptides have potent,

direct antimicrobial activity against Gram-positive and

Gram-negative bacteria and, importantly, against some

antibiotic-resistant bacteria [38] Conversely, virulence

proteins of pathogenic microorganisms can negatively

modulate the transcription of antimicrobial peptides, notably hBD-1 and LL-37 in intestinal epithelial cells [39], through a signaling pathway dependent on cAMP, protein kinase A, extracellular signal-related kinase, and Cox2 [40], counterbalancing the positive signals of alarmins

LL-37 was assumed to be the only active form of cathelicidin in the skin However, LL-37 is susceptible

to proteolytic processing, generating multiple cathelici-din-derived peptides that are present in normal human skin LL-37 actually represents < 20% of the cathelic-idin-derived peptides, smaller forms of the peptide being more abundant These smaller peptides result from proteolytic processing by two serine proteases belonging to the tissue kallicrein family: stratum

corne-um tryptic enzyme (SCTE) (kallicrein-5) and stratcorne-um corneum chymotryptic enzyme (SCCE) (kallicrein-7) Based on its specificity, each enzyme generates a differ-ent set of peptides SCTE generates three main pep-tides (KS30, KS22, and LL29), whereas the cleavage

of LL-37 by SCCE yields two peptides (RK31 and KR20) SCTE is considered to be the generator of the cathelicidin-derived antimicrobial activity (KS30, KS22 and LL29 are very potent antimicrobial compounds, but lack chemotactic activity), and SCCE may be considered as the inactivator of LL-37, rather than a generator of antimicrobial peptides [17] Ultimately, the relative proportions of these peptides may set the balance between antimicrobial activity and immuno-modulatory function

Expression of defensin-coding and cathelicidin-coding genes

The final combination of peptides at a specific location reflects the signaling of pattern⁄ pathogen-associated receptors as well as that of other molecules that sense the environmental conditions A proof of this was pro-vided by experiments showing that frogs do not syn-thesize and produce the same combinations and relative proportions of antimicrobial peptides in a ster-ile environment as they do in their natural one More-over, once they are pharmacologically depleted of antimicrobial peptides, frogs will not reaccumulate skin antimicrobial peptides until they are re-exposed to bacteria [41] In agreement with the different environ-mental cues that promote antimicrobial peptide syn-thesis, multiple signaling pathways are involved Upregulation of cathelicidin and defensin gene expres-sion in response to bacterial products and proinflam-matory molecules depends on the activation of the nuclear factor kappaB (NFjB), AP-1, JAK2 and STAT3 signaling pathways [16]

Transcription of the human defensin (HD)5 and

HD6 genes in Paneth cells is under the control of

tran-scription factor-4 (TCF-4) (also named TCF7L2), a

Wnt signaling pathway transcription factor, also

involved in Paneth cell differentiation [42] Reduced

amounts of HD5 and HD6 peptides have been

associ-ated with the development of Crohn’s disease [43,44]

Consistent with this, heterozygous TCF-4 knockout

mice show decreased production of Paneth cell

a-de-fensins and diminished bacterial killing capacity The

promoter region of neutrophil-derived defensins

con-tains recognition sequences for transcription factors

such as the hematopoietic-specific Ets family

transcrip-tion factor PU.1 and C⁄ EBP-a [16]), as well as an

NFAT binding site overlapping the Pu.1 site

Interest-ingly, NFAT was found to be associated with the

pro-moter in response to hepatitis C infection, thus

suggesting a correlation between a-defensin expression

and liver fibrosis [45] In human skin, during wound

healing, the synthesis of antimicrobial peptides by

incoming neutrophils, and notably that of hBD-3, is

induced through an LL-37-mediated mechanism of

transactivation of the epidermal growth factor receptor

[46]

The promoter regions of cathelicidin genes have

consensus binding sites for NFjB, IL-6, acute phase

response factor and IFN-c response element as well

[16] In mice, murine cathelin-related antimicrobial

peptide (CRAMP) is dependent on hypoxia-inducible

factor-1a, a factor now understood to play a key role

in the bactericidal capacity of phagocytic cells such as

macrophages and neutrophils [47] In different human

cell types (keratinocytes, monocytes, neutrophils, and

bone marrow-derived macrophages), cathelicidin gene

expression is under the control of vitamin

D-respon-sive elements [48] In turn, upregulation of the

vita-min D receptor (VDR) and Cyp27B1, the enzyme

that catalyzes the conversion of 25-hydroxyvitamin

D3 to the active 1,25-hydroxyvitamin D3, is

depen-dent on TLR-mediated signals Moreover,

1,25-hy-droxyvitamin D3 increases CD14 and TLR-2

synthesis All together, these data reveal a direct link

between 1,25-hydroxyvitamin D3, TLR activation, the

VDR and downstream targets such as cathelicidin,

ultimately regulating the antibacterial response [49]

Interestingly, many autoimmune patients are deficient

in vitamin D, and providing greater quantities of it

reduces the symptoms [50] Likewise, VDR-deficient

mice or vitamin D-deficient mice show increased

sen-sitivity to autoimmune diseases such as inflammatory

bowel disease and type I diabetes [51] Whether there

is a direct connection between low levels of

1,25-di-hydroxyvitamin D3, low levels of cathelicidin

produc-tion, poor clearance of bacterial pathogens and autoimmunity is certainly a challenging concept that needs to be further investigated

A recent computational analysis of the promoter region of 61 genes belonging to 29 families of mouse, rat and human antimicrobial peptide-encoding genes identified factors that regulate the transcription of anti-microbial peptides In addition to predicting most of the transcription factors already described individually for antimicrobial peptides, this study suggests that the influence of the VDR and new nuclear hormone recep-tors (glucocorticoid receptor, retinoic receptor, etc.) is not restricted to cathelicidins, and that it extends to other antimicrobial peptides, in particular a-defensins Furthermore, this in silico study identified a core set of transcription factors regulating the transcription of the majority of antimicrobial peptides considered The transcription factors were grouped in tissue specific-categories, of which the liver-specific, neuron-specific and nuclear hormone-specific factors occupied the first positions, underscoring new functions for antimicrobial peptides in energy metabolism and neuroendocrine regulation [52], in addition to their role in immunity

Immunomodulatory properties of antimicrobial peptides

By disrupting bacterial membranes, antimicrobial pep-tides participate as direct effectors of innate immunity Multiple antimicrobial peptides are simultaneously present at the same site, and they are thought to work in concert, to effectively fight infection It has frequently been argued that the minimal inhibitory con-centration of antimicrobial peptides needed to effec-tively combat microbial infection is rarely found in

in vivo conditions, despite the fact that antimicrobial peptide gene expression is mostly under the control of innate immunity-related transcription factors However,

in addition to the concentration of these natural antibi-otics, the resistance of the microbial membrane (i.e the target of the antimicrobial peptides) in a given ionic environment is the counterpart to effectiveness of anti-microbial activity In support of this, it has recently been shown that S aureus and Escherichia coli grown

in carbonate-containing solutions are more susceptible

to physiological concentrations of antimicrobial pep-tides, as a result of changes in bacterial gene expression that translate into changes in cell wall thickness and the expression of several genes related to virulence [53] Thus, the balance in the host’s ionic condition is an important element to consider when evaluating the antimicrobial activity of a given peptide Also, it should

be considered that the microbicidal activity of most

antimicrobial peptides is very potent in the intracellular

compartment, in phagocytic vacuoles, and on the

exter-nal surface of skin and mucosa, three low-salt

compart-ments Updating the molecular mechanisms involved in

this microbicidal effect is beyond the scope of this

minireview, but it is a field of extensive research

Innate immunity cells such as neutrophils, mast cells

and eosinophils can form extracellular traps (ETs) that

consist of a chromatin–DNA backbone, to which

anti-microbial peptides and enzymes are attached, ultimately

forming a net in which microbes are entrapped and

killed [54] An NAPDH oxidase-dependent mechanism

initiates a signaling cascade that leads to the

disintegra-tion of the nuclear and cellular membrane [55], leading

to cell death and the formation of ETs Besides

augmenting the local concentration of antimicrobial

peptides and effectively killing the microbes, it is

possi-ble to think that ETs limit the diffusion of

microbe-derived alarmins, minimizing tissue damage

Data from experiments with knockout and

trans-genic mice highlight the direct antimicrobial effect of

antimicrobial peptides [7,16] However, given the

cen-tral role that antimicrobial peptides seem to play in

the outcome of an infection⁄ injury, it is surprising to

see that all knockout mice lacking antimicrobial

pep-tides are quite healthy, with only modest alterations in

susceptibility to specific infectious agents For example,

mice lacking b-defensin-1 are inefficient at clearing

Haemophilus influenzae from their lungs [56], and

CRAMP-deficient mice are impaired in their ability to

clear skin infections caused by group A Streptococcus

[57] These results underline the fact that antimicrobial

peptides work in concert, and that their ranges of

activity frequently overlap

Apart from efficient antimicrobial activity,

antimi-crobial peptides modulate immunity They seem to

participate in every facet of it, by boosting the immune

response to prevent infection, and also by suppressing

other proinflammatory responses to avoid uncontrolled

inflammation Furthermore, some antimicrobial

pep-tides synergize with cytokines and modify their

immuno-modulatory activity

Chemotactic activity

In addition to their direct microbicidal activity,

antimi-crobial peptides are chemotactic for leukocytes and

other nonimmune cells at nanomolar concentrations

Despite a certain overlap, antimicrobial peptides work

in concert, as they complement each other to direct

effector cells to the site of inflammation, organizing the

order of appearance of the different players in different

scenarios, and modulating the local immune response

Phagocytic cells, neutrophils and monocytes that are recruited through a-defensins, HNP1–3 and b-defen-sins hBD3 and hBD4, and mast cells that are attracted through LL-37, HNP1–3 and hBD2 contribute to increase the local density of neutrophils [58] In addi-tion, hBD1 and hBD3 are chemotactic for immature dendritic cells and memory T-cells, whereas human a-defensins selectively induce the migration of human naı¨ve CD4+CD45+and CD8+cells [7] The combina-tion of these peptides and cytokines present at the site

of injury will contribute to the maturation of these immature dendritic cells, enabling them to process antigen and to migrate to proximal lymph nodes to present antigens to naı¨ve cells, thus setting in motion the adaptive immune response machinery, and shaping the outcome of the response Besides their intrinsic chemoattractant properties, which directly promote the locomotion and arrival of different cohorts of cells to the site of injury, antimicrobial peptides indirectly favor chemotaxis by inducing or increasing the secre-tion of chemokines For example, LL-37 has been shown to induce IL-8 release by lung epithelial cell lines [59,60], and human defensins HNP1–3 also favor the recruitment of neutrophils by inducing the activa-tion and degranulaactiva-tion of mast cells, augmenting neu-trophil influx and further stimulating the transcription and production of IL-8 by bronchial epithelial cells [61–64]

Antimicrobial peptide-induced chemotaxis is pre-sumably mediated through G-protein-coupled recep-tors, as pretreatment of the cells with pertussis toxin

or phospholipase C, phosphoinositide-3-kinase and Rho kinase inhibitors abolishes cell migration [65] According to the peptide and the cell, several receptors have been identified LL-37, like the frog peptides tem-porin A and probably Drs S9, attracts cells through formyl peptide receptor-like-1, whereas defensins hBD2 and hBD3 use CC-chemokine receptor-6, pres-ent on memory T-cells, immature dendritic cells, and human colonic epithelial cells [66–69] CC-chemokine receptor-6 is also the receptor for macrophage inflam-matory protein-3a, a chemokine involved in homeo-static lymphocyte homing as well as in epithelial cell migration, further suggesting a function for hBD2

in healing and protection of the intestinal epithelial barrier [70]

Proinflammatory and anti-inflammatory signals

of antimicrobial peptides Antimicrobial peptides have a dual identity: they pro-tect the host against potentially harmful pathogens through their antimicrobial activity and by stimulating

innate immune functions, yet, at the same time, they

protect the organism from the detrimental effects of an

excessive inflammatory response

In addition to their direct antimicrobial capacity, the

in vivo contribution of antimicrobial peptides to

anti-microbial defense depends on their capacity to induce

the production of proinflammatory cytokines, to

pro-mote the recruitment of dendritic cells and monocytes

to the site of injury, and to enhance phagocytosis and

the maturation of dendritic cells All of these effects

will augment the uptake, processing and presentation

of antigen, and stimulate the clonal expansion of

T-lymphoctes and B-lymphocytes B-lymphocytes will

produce antibodies that are highly specific for

patho-gen antipatho-gens, contributing to the clearance of microbes

through phagocytosis [16]

Some of the molecular mechanisms that control this

positive feedback loop have been described recently

Human a-defensins and b-defensins induce the release

of histamine and prostaglandin D2 in a G-protein–

phospholipase C-dependent manner [62] [71]; HPN1–3

bind C1q and activate the classic complement pathway

[72], increase the production of TNF-a and IL-1b, and

decrease the production of IL-10 by monocytes

[61,62,73] Furthermore, as an endogenous ligand for

TLR-4, b-defensin-2 activates immature dendritic cells

through TLR-4-dependent mechanisms, triggering a

robust Th1 response [74] Consistent with their role in

wounding, b-defensin-mediated signals positively

regu-late the expression of matrix metalloproteinase genes

and negatively regulate that of tissue inhibitor of

matrix metalloproteinase genes, thus modulating tissue

repair [75,76] LL-37 induces the release of 1b,

IL-8, TNF-a, IL-6 and granulocyte–macrophage

colony-stimulating factor (GM-CSF) by keratinocytes, and of

TNF-a and IL-6 by immature dendritic cells [58,77]

Moreover, LL-37 and GM-CSF synergize, as the

pres-ence of GM-CSF augments LL-37-mediated

mitogen-activated protein kinase activation and reduces the

amount of LL-37 required for this activation and for

cytokine production [78,79]

Cathelicidins function as anti-inflammatory

mole-cules as well In in vivo models, administration of

LL-37 protects mice and rats from LPS-mediated

lethality [60,80] Indeed, LL-37 binds and neutralizes

LPS, possibly by dissociation of LPS aggregates,

limit-ing the extent of inflammation [60,81–84]

Addition-ally, cathelicidin abrogates the expression of

proinflammatory molecules such as TNF-a and IL-6

and the nuclear translocation of NFjB p50⁄ p65

induced by TLR-2 and TLR-4 in response to lipoteic

acid and LPS, respectively, through a partially defined

mechanism involving mitogen-activated protein kinase

p38 inactivation [85] This immunomodulatory effect

of the TLR response is mediated through the binding

of the mid-region of LL-37, comprising amino acids 13–31, to TLR ligands through an LPS-binding mech-anism [86] Moreover, LL-37 was found to selectively permeabilize the membranes of apoptotic human leukocytes through a mechanism similar to the direct microbicidal effect, independently of known surface receptors or cell signaling, leaving viable cells un-affected This causes the cells to empty the cytoplasm

as well as intragranular molecules to the extracellular compartment, shifting the balance between proinflam-matory and anti-inflamproinflam-matory signals [87] Further-more, the fact that, as mentioned, LL-37 is shortened

by a serine protease-dependent mechanism, generating novel antimicrobial peptides with enhanced antimicro-bial action, but reduced proinflammatory activity, con-tributes to controlling the inflammatory response [88]

In addition, these data point to the role of hydropho-bicity in the immunomodulatory capacity of LL-37 [86] and potentially in new synthetic peptides designed

to downmodulate inflammatory responses Accord-ingly, IDR-1, a synthetic peptide derived from LL-37, although devoid of direct antimicrobial activity, is effective in limiting a broad range of Gram-positive and Gram-negative pathogens, through signaling path-ways that increase the level of monocyte cytokines while diminishing proinflammatory responses [89] Such peptides, capable of suppressing the host’s harm-ful proinflammatory responses without losing the beneficial infection-fighting components of host innate defenses, are desirable tools for antisepsis therapies Defensins play the same dual role as cathelicidins The activation of TLR-4, mediated through murine b-defensin-2, leads to atypical death of dendritic cells, through upregulation of membrane-bound TNF-a and tumor necrosis factor receptor 2 This suggests that b-defensins participate in the triggering of an immune response and in the natural process of elimination of activated antigen-presenting cells and termination of the immune response [90]

Healing Infection and injury provoke tissue damage Immedi-ately after injury, innate immune cells, mostly neu-trophils and macrophages, together with antimicrobial peptides, produced by immune cells or secreted by local cells, will take care of microbe clearance and removal of debris Other cells, such as T-lymphocytes, secrete cytokines and chemokines that will further activate macrophages and induce inflammation and vasodilatation, and enhance vessel permeability Tissue

regeneration requires multiple events Following

removal of bacteria and debris, the release of growth

factors will promote the migration and proliferation of

fibroblasts, which will deposit the extracellular matrix

over which epithelial cells will crawl and cover the

wound bed [91]

A recent report showed the secretion of hBD3 and

other antimicrobial peptides by human keratinocytes,

after the disappearance of neutrophils and before the

re-establishment of the physical barrier, in sterile

wounds as well as in microbe-induced wounds [35,46]

The expression of antimicrobial peptides at that time is

probably protective against subsequent infections

However, the fact that growth factors such as IGF-1,

transforming growth factor-a, and epidermal growth

factor, in combination with IL-1, induce the secretion

of LL-37, hBD3 and other antimicrobial peptides by

human keratinocytes [35,59] suggests that

antimicro-bial peptides participate in additional tasks LL-37 is

mostly present in the inflammatory infiltrate as well as

in the epithelium migrating over the wound, but not at

the wound edge Interestingly, its highest level in the

skin wounds is reached 48 h postinjury, whereas the

normal level is achieved only upon wound closure,

after infection resolution, suggesting direct

participa-tion of LL-37 in wounding [92] Indeed, through

trans-activation of epidermal growth factor receptor, LL-37

induces keratinocyte migration [93], and through

for-myl peptide receptor-like-1, it induces angiogenesis

[94] Consistent with this role in wound healing, and in

addition to increased bacterial colonization, mice

lack-ing the cathelicidin gene have longer periods of wound

healing than their wild-type counterparts [57,95]

Simi-larly, hBD-2 was recently described as also being a

potent promoter of human endothelial cell migration,

proliferation and, in the presence of angiogenic factors,

tube formation [96], accelerating wound closure LL-37

may also have antifibrotic activity during the wound

repair process, as it inhibits baseline and transforming

growth factor-b-induced collagen expression at

nanom-olar concentrations, through an extracellular

signal-related kinase-dependent and G-protein-dependent

pathway [97]

These data regarding the role of antimicrobial

pep-tides in wounding provide evidence for their dual role;

they serve as sentinels and they actively participate in

tissue regeneration Whether noninducible

antimicro-bial peptides function in a similar way during

infec-tion, under normal conditions or during development

is an attractive possibility In conclusion, the multiple,

yet sometimes opposite, functions of antimicrobial

peptides are complementary, and they control

homeo-stasis through complex regulatory loops that involve

different cells responding to multiple signaling path-ways

Antimicrobial peptides and disease Dysregulated production of antimicrobial peptides is associated with disease As we recognize that these molecules are multifunctional and that they modulate multiple events, the list of diseases in which anti-microbial peptides participate is growing Throughout previous sections of this minireview, we have pointed

to the participation of antimicrobial peptides in several diseases In this section, we will highlight recent data

on psoriasis, rosacea, atopic dermatitis and Crohn’s disease

It was long considered that skin passively obstructed the entrance of pathogens, thus constituting a natural barrier against potential microbial pathogens and other assaults from the external environment It is now clear that, through the antimicrobial activity and inmuno-modulatory functions of antimicrobial peptides, the skin plays a major and active role in the onset and development of an immune response to injury and microbial insult Recent publications have narrowed the role of cathelicidins and defensins in psoriasis, ros-acea and atopic dermatitis, providing evidence that the concentration, processing and signaling of antimicro-bial peptides are critical parameters for maintaining the delicate equilibrium between effective protection and autoimmunity

As mentioned, cathelicidin and hBD1–4 are present

in low amounts in healthy skin keratinocytes, but in response to injury or infection, their synthesis is signifi-cantly enhanced [98] In atopic dermatitis, the continu-ous bacterial and viral infections produce chronic inflammation It was recently shown that the cytokine milieu (IL-4 and IL-13) of this Th2-type inflammatory skin disease downregulates the gene expression of

LL-37, and thus contributes to a partially uncontrolled cutaneous innate immune response in those patients [99] A recent report showed that Bcl-3, a protein with close homology to IjB proteins and that interacts with p50 NFjB homodimers, is overexpressed in skin lesions of patients with atopic dermatitis, and that its silencing reverses the inhibitory effect of IL-4 on hBD3 gene expression Moreover, Bcl-3 silencing upregulates the 1,25-dihydroxyvitamin D3-dependent production of cathelicidin in keratinocytes, and 1,25-dihydroxyvita-min D3 suppresses Bcl-3 expression [100] In addition, Bcl-3 synthesis is upregulated in the presence of IL-4 [101], thus generating a negative feedback loop that will reduce the cathelicidin concentration, favoring skin infections and chronic inflammation

Unlike atopic dermatitis, psoriasis, a common

auto-immune disease of the skin, results partially

cathelici-din overproduction By binding to damaged or

apoptotic skin cells self-DNA, cathelicidin converts it

into aggregated and condensed structures That will be

delivered to plasmocytoid dendritic cells These, in

turn, will infiltrate the psoriatic skin, triggering

en-dosomal TLR-9 and subsequent IFN-c production,

thus driving autoimmune skin inflammation [102]

Patients with rosacea have abnormal inflammation

and vascular reactivity in facial skin These individuals

have high levels of cathelicidin and higher levels of the

enzyme that processes the propeptide into the LL-37

biologically active peptide and of other unusual

iso-forms of the peptide The current thinking is that, at

least partially, the chronic inflammation results from

the increased chemotactic and angiogenic activity of

the LL-37-derived peptides [103]

Crohn’s disease is an inflammatory disease of the

small intestine and⁄ or the colon As mentioned

already, in the small intestine, the pathogenesis is

asso-ciated with a reduced expression of the Wnt signaling

pathway TCF-4, involved in Paneth cell differentiation

and in a-defensin gene expression [42] Consequently,

a-defensin-2 and a-defensin-3 genes are deficiently

expressed, regardless of the inflammation Moreover,

single-nucleotide polymorphisms in TCF-4 are directly

related to ileal Crohn’s disease incidence, providing

evidence that low levels of HD5 and HD6 are directly

associated with the disease [43] In contrast, in the

colon, Crohn’s disease is associated with impaired

expression of the genes encoding hBD5 and hBD6

Patients affected with this form of disease tend to have

fewer gene copy numbers in the locus of b-defensin in

chromosome 8 As a result of this deficiency in

a-de-fensins and b-dea-de-fensins, luminal microbes invade the

mucosa and trigger inflammation [104]

Concluding remarks

Initially described as molecules with bactericidal

capac-ity, antimicrobial peptides are now considered to be

multifunctional molecules They stimulate the

produc-tion and release of proinflammatory and

anti-inflam-matory molecules, they recruit inflamanti-inflam-matory cells to

the site of injury, they function as antimicrobial

mole-cules directly and by promoting ingestion of microbes

by phagocytic cells, and they participate in damage

repair These pleiotropic effects reflect the diversity of

effector molecules and their targets, as well as the

sometimes overlapping, yet very specific, functions

Through elaborate feedback mechanisms, they control

immune cells as well as nonimmune cells, link innate

immunity to adaptive immunity, and maintain homeo-stasis Alterations in their physiological concentrations correlate with disease Their antimicrobial activity, immunomodulatory functions, adjuvant properties and low toxicity make antimicrobial peptides the object of intense investigation in order to develop new therapeu-tic agents with specific activities A deeper understand-ing of the signalunderstand-ing pathways underlinunderstand-ing these effects and of the physiological processes that are controlled

by antimicrobial peptides will help in the better exploi-tation of the potential use of these peptides

Acknowledgements

We thank Drs L Perez, G Pedraza, Claire Lacombe and G Corzo for their helpful discussions and com-ments, and S Ainsworth for her librarian support Work in the Y Rosenstein laboratory is supported by CONACyT and DGAPA⁄ UNAM, Mexico

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