Defensins: Antimicrobial Peptides of Innate Immunity

REVIEWS

DEFENSINS: ANTIMICROBIAL PEPTIDES OF INNATE IMMUNITY

Tomas Ganz The production of natural antibiotic peptides has emerged as an important mechanism of innate immunity in plants and animals. Defensins are diverse members of a large family of antimicrobial peptides, contributing to the antimicrobial action of granulocytes, mucosal host defence in the small intestine and epithelial host defence in the skin and elsewhere. This review, inspired by a spate of recent studies of defensins in human diseases and animal models, focuses on the biological function of defensins.

Antimicrobial peptides are polypeptides of fewer than cysteines (FIGS 1 and 2). During studies of the antimicro- 100 amino acids that are found in host defence settings, bial activity of rabbit and guinea-pig leukocyte lysates in and that have antimicrobial activity at physiological con- the 1960’s, the so-called arginine-rich cationic peptides centrations under conditions prevailing in the tissues of were defined by their high cathodal electrophoretic origin. In humans and other mammals, the two main mobility, and attracted attention because of their abun- antimicrobial peptide families are defensins and catheli- dance and broad spectrum of antimicrobial activity8. cidins. Individual members of these two families of Subsequent technical developments facilitated their iso- antimicrobial peptides have been implicated in antimi- lation and detailed chemical characterization9–11.The crobial activity of phagocytes, inflammatory body fluids discovery of structurally similar peptides in human and epithelial secretions. Defensins1,2 are widely distrib- leukocytes1,2 indicated that such peptides were widely uted in mammalian epithelial cells and phagocytes, and distributed in nature, and were named ‘defensins’ based are often present at high (up to millimolar) concentra- on their association with host defence settings. After their tions. Cathelicidins3,4 are structurally and evolutionarily isolation from leukocytes, defensins were also found to distinct antimicrobial peptides that are similar to be produced by various types of epithelial cell12,13. defensins in abundance and distribution. Other mam- The two main defensin subfamilies, α- and malian antimicrobial peptides, including histatins5, β-defensins, differ in the length of peptide segments dermcidin6 and ‘anionic peptides’7 are restricted to a few between the six cysteines and the pairing of the cys- animal species and tissues. The arsenal of antimicrobial teines that are connected by disulphide bonds (FIG. 1). peptides differs from one animal species to another. I Several structures that are representative of these two focus on defensins, indicating the anthropocentric per- families have been solved by two-dimensional NMR spective of a physician: among all the antimicrobial pep- and by X-ray crystallography14–21. Although crystal tides, defensins are particularly prominent in humans, as structures of some defensins are made up of dimers or evidenced by the large number of expressed human multimers, it is not yet clear whether these multimers genes, the various forms that are present in human tis- are the biologically relevant defensin forms. Both α β β Departments of Medicine sues, and the ubiquitous occurrence of defensins in - and -defensins consist of a triple-stranded -sheet and Pathology, David Geffen inflamed or infected human tissues. with a distinctive ‘defensin’ fold (FIG. 2). Recently, School of Medicine, UCLA, another structurally distinct subfamily of θ-defensins22 Los Angeles, California Definition and structure has been identified in rhesus macaque monkey leuko- 90095-1690, USA. Defensins1,2 are a family of evolutionarily related verte- cytes. The mature θ-defensin peptides arise by an e-mail: tganz@mednet. β ucla.edu brate antimicrobial peptides with a characteristic -sheet- as-yet-uncharacterized process that generates a cyclic doi:10.1038/nri1180 rich fold and a framework of six disulphide-linked peptide by splicing and cyclization from two of the

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secretory granules that are released into narrow intestinal HNP3: α-defensin CYCRIPACIAGERRYGTCIYQGRLWAFCC pits, known as crypts. The concentration of defensins in the crypts can also reach >10 mg ml–1 (REF.31).Various barrier and secretory epithelial cells produce defensins, in β HBD2: -defensin GGIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP some cases constitutively32 and in other cases in response to infection33. The average concentration of defensins in these epithelial cells reaches the 10–100 µg ml–1 range33,34, RTD1: θ-defensin R C L F C C R but because the peptides are not evenly distributed the G R local concentrations are higher. T G Patterns of tissue distribution (TABLE 1) vary even R V when closely related species are compared. Among C R C R C 35 36 Figure 1 | Sequences and the disulphide pairing of cysteines of α-, β- and θ-defensins. rodents, mice lack leukocyte defensins , rats have them , The corresponding cysteines in α- and β-defensins are indicated by dotted lines, disulphide bonds and both species have numerous Paneth cell defensins by solid lines. Whereas in α-defensins the six cysteines are linked in the 1–6, 2–4, 3–5 pattern130, and epithelial cell β-defensins. In pigs, there is expression in β-defensins the pattern is 1–5, 2–4, 3–6 (REF.131). Because cysteines 5 and 6 are adjacent in of a β-defensin in the tongue epithelium34, but defensins both types of defensins, this difference in cysteine connectivity does not substantially alter the have not been detected in pig granulocytes37. Instead, 19 structure . The structure of θ-defensins is circular without a free N- or C-terminus. The pig granulocytes have abundant cathelicidins including θ α β θ disulphide pairing of -defensins is also different from - and -defensins, as -defensins are protegrins37, prophenins, PR-39 and others. Ungulate formed by peptide splicing from two hemi-α-defensins, each of which contributes three cysteines22. The peptide bonds between the two hemi-defensins are shown as arrows. HBD, granulocytes, best studied in the cow, have abundant 38 human β-defensin; HNP, human neutrophil peptide (α-defensin); RTD, rhesus θ-defensin. β-defensins that are encoded by many genes , but also express other β-defensins in the trachea13 (tracheal antimicrobial peptide, TAP), tongue39 (lingual antimi- nine-amino-acid segments of α-defensin-like precursor crobial peptide, LAP) and the intestine40 (enteric peptides. The θ-defensins apparently evolved in primates, β-defensin, EBD). In some cases, defensin expression but are inactivated in humans owing to mutations that seems to be induced by a combination of a specific cell encode premature stop codons23. Based on their adja- type and tissue environment. Inflammatory macro- cent chromosomal location and similar peptide precur- phages are leukocytes that arise by differentiation from sor and gene structure, it is probable that all vertebrate circulating blood monocytes, under the influence of local defensins arose from a common gene precursor24. tissue signals. In the rabbit, alveolar (lung) macrophages Antimicrobial peptides from invertebrates and plants have abundant α-defensins in amounts that are compa- that contain six or eight cysteines in disulphide linkage rable to rabbit neutrophils, but defensins are absent from are also known as defensins (for example, insect and their peritoneal macrophages41. Although defensin plant defensins). Their evolutionary relationship to ver- expression by monocytes, macrophages and lymphocytes tebrate defensins is uncertain. Insect defensins contain of some mammals can be detected by highly sensitive an α-helix that is disulphide-linked to a β-sheet (FIG. 1) techniques42–44, high levels of defensins in macrophages with cysteine linkage (1–4, 2–5, 3–6) that differs from have only been shown in rabbits. It is probable that these vertebrate defensins. peculiarities in the pattern of expression of defensins in certain animal species are related to evolutionary Distribution pressures from species-specific pathogens. Typical defensin peptides have been found in all mammals that have been examined, as well as in chickens and Genes and biosynthetic pathways turkeys25–28. Defensin-like peptides (growth arresting In humans, at least eight genes encoding α- and peptide29 and crotamines) have also been isolated from β-defensins are located in a cluster on chromosome snake venom in which they might represent an adapta- 8p23 (REFS 24, 45–49). Despite the reported completion of tion of epithelial host defence peptides for efficacy the Human Genome Project, this defensin cluster is against larger predators. Notably, the specific tissue dis- incompletely mapped. This is probably due to its poly- tribution of defensins diverged rapidly during verte- morphic nature, with individuals and their chromo- brate evolution (TABLE 1). somes differing in the number of copies of specific Defensins are abundant in cells and tissues that are defensin genes (for example, neutrophil defensins)49, involved in host defence against microbial infections. In which makes it difficult to assemble a unique sequence many animals, the highest concentrations of defensins in this region. Four additional defensin clusters have (>10 mg ml–1) are found in granules, the storage been identified50 and contain transcribed genes, but organelles of leukocytes1,30. When leukocytes ingest their peptide products have not yet been characterized. microorganisms into phagocytic vacuoles, the granules All five human clusters correspond to syntenic mouse fuse to these vacuoles and deliver their contents onto the defensin clusters. target microorganism. As there is little free space in phagocytic vacuoles, the microorganism is exposed to Amino-acid sequence and composition. The amino-acid minimally diluted granule material (FIG. 3). Paneth cells sequences of mature defensins are highly variable except — specialized host defence cells of the small intestine for the conservation of the cysteine framework in each — are another site of high defensin concentration in defensin subfamily. Clusters of positively charged amino many animal species. Paneth cells contain defensin-rich acids are characteristic of most α- and β-defensins, but

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their specific distribution in the defensin molecule is cationic defensin (~30 amino acids)56,57. In many cases, variable. In leukocytes and Paneth cells of the small the charge of the propiece and the mature defensin intestine, defensins are stored in granules — subcellular approximately balance58, and this arrangement might be storage organelles that are rich in negatively charged important for folding and/or to prevent intracellular glycosaminoglycans51,52. With the exception of chicken interactions with membranes59,60. Neutrophil α-defensins gallinacins, these α- and β-defensins contain arginine as are synthesized in the bone marrow (FIG. 3 and TABLE 1), the predominant cationic amino acid. By contrast, in neutrophil precursor cells known as promyelo- β-defensins that are secreted from epithelial cells contain cytes61–63. Mature neutrophils that circulate in the blood similar amounts of arginine and lysine. The preferential or that are found in inflamed tissues contain large levels use of arginine in defensins that are stored in granules of defensins, but are no longer synthesizing the peptides might reflect the constraints imposed by packing cationic or their mRNAs. Only small amounts of partially peptides and proteins into the glycosaminoglycan matrix processed intermediates are detectable in mature neu- of granules53–55. trophils64. In the case of mouse Paneth cell defensins (cryptdins), the metalloproteinase matrilysin (MMP7) Defensin synthesis. α-defensins are generally encoded as is required for processing, as mice with homozygous tripartite prepropeptide sequences, in which a 90–100 disruption of the matrilysin gene do not process Paneth amino-acid precursor contains an amino (N)-terminal cell defensin past the removal of the signal sequence. signal sequence (~19 amino acids), an anionic propiece Matrilysin processes mouse prodefensins before their (~45 amino acids) and a carboxy (C)-terminal mature storage in granules (TABLE 2). However, some mouse

HNP3 dimer (α-defensin) HBD2 (β-defensin)

Insect defensin A θ-defensin

Figure 2 | Cartoon structures of representative mammalian defensins and an insect defensin. β-sheet structures are indicated by flat ribbons and arrows. Human neutrophil peptide 3 (HNP3, α-defensin) forms a β-sheet-rich dimer14. Human β-defensin 2 (HBD2) in solution is a monomer21 with the same general shape (defensin-fold) despite the change of the disulphide-bond pattern (FIG. 1). In addition, there is a short α-helical segment at the N-terminus. The conformation of insect defensins132 is distinct, with a prominent α-helical segment that is linked by two disulphide bonds to the C-terminal β-sheet. The θ-defensin structure is cyclic, forming a simple β-sheet.

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Table 1 | Diverse patterns of defensin expression in vertebrates intestinal crypts after exposure to bacteria or cholinergic stimuli31. The specific receptors and transduction path- Species Neutrophil Paneth cell Epithelial cell References defensins defensins defensins ways that regulate defensin release from Paneth cells are of interest, but have not yet been characterized. Human αααand β 1,33,74,134–141 Comparison of the promoter regions of pairs of defensin Rhesus α and θ N.D. β 22,142–144 monkey genes with the same site of expression, for example, human defensin neutrophil peptide 1 (HNP1) and Mouse none ααand β 12,35,141,145–153 HNP4, or HD5 and HD6 (Paneth cell specific) reveal Rat ααβ36,61,154,155 marked similarities, even when, as is the case for the Pig Not detected in N.D. β 34,37,156 Paneth cell genes HD5 and HD6, the non-promoter granule extracts regions of the genes have completely diverged69.The Cow β none β 13,39,40,157,158 promoter region of human neutrophil defensin genes Chicken β N.D. β 25–27,159 contains several characteristic myeloid transcription N.D., not determined. factor-binding sites that are essential for transcription by the HL-60 myeloid cell line70. The specific motifs that direct defensin expression to other tissues are not known. intestinal defensins undergo further processing that HBD1, expressed mainly by the urinary tract, but does not involve matrilysin. In humans, the key enzymes also by the skin and other epithelial cells, seems to be that are responsible for the processing of intestinal synthesized constitutively. HBD2 synthesis in the skin is prodefensin HD5 are the three forms of trypsin that are induced by bacteria through a pathway that requires expressed by the same Paneth cells that synthesize, store intermediate interleukin-1 (IL-1) synthesis71 by myeloid and secrete HD5. Unlike in mice, Paneth cell defensins cells (dendritic cells or macrophages). Dependence of are stored as proforms that undergo processing during HBD2 induction on the intermediate production of IL-1 or after release from Paneth cells. This step might be reg- was also seen in a model system consisting of a co-culture ulated in Paneth cell granules65, as indicated by the pres- of the A549 lung epithelial cell line and MM6 or U937 ence of the trypsin inhibitor α1-proteinase inhibitor monocyte-like cell lines exposed to lipopolysaccharide72. (α1-antitrypsin). The induction of HBD2 mRNA by IL-1 was dependent The structure of β-defensin precursors is simpler, con- on a specific nuclear factor-κB (NF-κB) site (–205 to sisting of a signal sequence, a short or absent propiece, –186) and its interaction with the p65–p50 NF-κB and the mature defensin peptide at the C-terminus. heterodimer. HBD3 (REF.73) and HBD4 (REF.74) are regu- The lack of an anionic propiece in β-defensin precur- lated by distinct NF-κB-independent mechanisms, but sors contrasts with the relatively large anionic propiece these remain to be characterized. The bovine epithelial in α-defensin precursors — a difference that has not cell β-defensins39,75,76 in the trachea, tongue and the been satisfactorily explained. In epidermal keratinocytes, intestine served as an important early model for human β-defensin 2 (HBD2) is secreted in lamellar defensins that were induced by infection and inflamma- bodies66 — lipid-containing vesicles that are secreted tion. The production of the bovine β-defensin tracheal into the intercellular space and that make the skin antimicrobial peptide is transcriptionally regulated impermeable to water. Apparently, these vesicles also through the NF-κB pathway75. generate the antimicrobial barrier in the epidermis. In some cases, the synthesis of defensins is also regulated developmentally, as illustrated by rabbit alveolar Regulation of defensin synthesis and release macrophages and mouse Paneth cells, both of which Defensin synthesis and release is regulated by microbial synthesize and accumulate defensins during the first signals, developmental signals, cytokines and in some postnatal month12,77. cases neuroendocrine signals in a tissue-specific manner (TABLE 3). Human neutrophil defensins are synthe- Activity sized constitutively by the bone-marrow precursors Most defensins show antimicrobial activity against bacte- of neutrophils during specific differentiation stages of ria and fungi, especially when tested under low ionic neutrophil development, in promyelocytes and early strength conditions11,78,79 and with low concentrations of myelocytes63. Defensins are packaged in primary divalent cations, plasma proteins or other interfering sub- (azurophil) granules — a population of granules that are stances. Under these optimal conditions, antimicrobial destined to fuse with phagocytic vacuoles67. By contrast, activity is observed at concentrations as low as 1–10 µg secondary granules contain low levels of defensins, but ml–1 (low µM). In general, metabolically active bacteria are rich in CAP18 — the precursor of cathelicidin LL-37. are more sensitive to defensins than are bacteria that have Having matured in the bone marrow and assembled been made inactive by nutrient deprivation or metabolic their arsenal of granules, neutrophils cease granule syn- inhibitors. Increasing the concentration of salts and thesis, are released into the blood and enter tissues. plasma proteins competitively inhibits the antimicrobial During phagocytosis, defensin-rich primary granules activity of defensins, in a manner that depends on both fuse with phagocytic vacuoles in which they generate the specific defensin and its microbial target. Certain high concentrations of defensins68 (FIG. 3). enveloped viruses are also inactivated by defensins80,81. The synthesis of human Paneth cell defensins is prob- Recently, human neutrophil defensins have been impli- ably also constitutive. They are released into the lumen of cated among the molecules that are responsible for the

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Bone-marrow promyelocyte Blood neutrophil Tissue neutrophil became permeable to small molecules (chromogenic (phagocytosis) enzyme substrates and trypan, respectively). In bacteria, NucleusER Golgi Maturing granule permeabilization coincided with the inhibition of RNA, DNA and protein synthesis and decreased bacterial viability as assessed by the colony forming assay. Conditions that interfered with permeabilization also prevented the loss of bacterial viability, indicating that permeabilization is essential for bacterial killing. The permeabilized K569 cells could be rescued for up to 1 hour by removing the defensin, raising the possibility that additional intracellular sites of action contribute to PreprodefensinProdefensin Defensin Pathogen cell death85. Figure 3 | Human neutrophil peptide α-defensin synthesis and release onto In experiments with artificial membranes comprised α microorganisms. For human neutrophil peptide -defensins, synthesis takes place in the bone of phosphatidylethanolamine, phosphatidylcholine and marrow, in neutrophil precursor cells known as promyelocytes61–63. The ribosomally-synthesized form is a 94 amino-acid preprodefensin (pink), which is converted by the removal of the N-terminal phosphatidylserine in 2:2:1 ratio, defensins NP1 (rabbit) 19 amino-acid signal sequence to 75 amino-acid prodefensin (purple) and further N-terminal and HNP1 (human) formed channels when a negative proteolytic shortening to a 29–30 amino-acid mature defensin (red)57. During defensin synthesis by potential was applied to the membrane side opposite to myeloid cell lines, the signal sequence is rapidly removed, but the subsequent proteolytic the defensin-containing solvent89 (FIG. 4). This is consis- processing to mature defensins takes many hours, and the final proteolytic cleavage occurs in tent with the idea that the insertion of defensin mole- maturing granules57. Defensins are packaged in primary (azurophil) granules — a population of 67,133 cules into the membrane depends on electrical forces granules that are destined to fuse with phagocytic vacuoles . Having matured in the bone that act on the positively charged defensin molecule. The marrow and assembled their arsenal of granules, neutrophils cease granule synthesis, are released into the blood and enter tissues. During phagocytosis (the target microorganism is represented as effect of these forces is evident even when no transmem- a black sphere), defensin-rich primary granules (red) fuse with phagocytic vacuoles in which they brane potential is applied externally. Unlike another generate high concentrations of defensins68. ER, endoplasmic reticulum. cationic peptide, melittin, which indiscriminately permeabilized vesicles composed of neutral or anionic phospholipids, defensins were more active against vesi- antiviral activity secreted by CD8+ T cells of HIV-non- cles that included negatively charged phospholipids90. progressors82 — HIV-infected individuals who, for many In general, the activity of defensins against vesicles was years after infection, do not develop the signs, symptoms diminished in the presence of increased salt concentra- and laboratory markers of AIDS. It is not yet clear tions, supporting the importance of electrostatic forces whether defensin secretion by CD8+ T cells occurs in vivo, between the anionic phospholipid headgroups and the and if so, whether it is the cause or the result of non-pro- cationic defensins. gression. The specific molecular signals that might induce In other experiments, large vesicles composed of the CD8+ T cells to secrete defensins are also not known. negatively charged phospholipid palmitoyloleoylphos- Retrocyclin — a θ-defensin that was artificially created by phatidylglycerol were permeabilized by human defensin correcting the stop mutation found in the human HNP2, but the addition of neutral phospholipids to the pseudogene — exerts substantial activity against HIV23 by lipid mix inhibited both defensin binding and permeabi- an as-yet-incompletely characterized mechanism. lization91. Using a different method, the importance of At higher concentrations, some defensins are cyto- anionic phospholipids for the membrane interactions toxic to mammalian cells83–85. As high concentrations with defensins was clearly shown by calorimetric mea- of defensins are present in inflamed tissues and cells surements of the effects of defensins on membrane phase exposed to defensins generate pro-inflammatory sig- transitions (collective reordering of the membrane mole- nals86, defensins could contribute to tissue injury, mainly cular pattern and fluidity)92. Taken together, we interpret in the lungs87. these studies as showing that defensin molecules enter into the membrane under the influence of cell-generated Mechanisms of antimicrobial activity. Permeabilization transmembrane potentials and local electrostatic fields. of target membranes is the crucial step in defensin- What happens once the defensin molecules are in the mediated antimicrobial activity and cytotoxicity. Model membrane is less certain. The observed leakage of dye bacteria (Escherichia coli ML-35)88 and the mammalian markers from liposomes implies that pores (we use this cell line K562 (REF. 85) that were treated by defensins term to refer to any ion or water-permeable structure in

Table 2 | Post-translational processing of defensins Defensin Storage form Site of processing Processing enzymes References Human neutrophil Mature peptides Golgi and maturing Not known 1,57 peptides 1–3 granules in promyelocytes Human defensin 5 Propeptide During or after release Paneth cell 65,160 from Paneth cells trypsins Mouse cryptdins Mature peptide Golgi and maturing granules? Matrilysin (MMP7) + ? 97 Human β-defensin 2 Mature peptide Endoplasmic reticulum? Signal peptidase only

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cecropin) or β-loop (for example, protegrin) peptides94–96. Although it is probable that the larger and more com- Defensin plex defensins act by similar mechanisms, a unitary hypothesis of how defensins permeabilize membranes is complicated by the marked differences in net charge, amino-acid sequence and quaternary structure (monomers versus dimers) among the defensins. It is possible that these differences have evolved so that various defensins can target different types of bacteria with differing structures of cell walls and membranes. Further complexity is introduced by the flexibility of the basic amino-acid side chains that allow various potential spatial interactions with phospholipid headgroups or water. Although the interactions of defensins 91 Electric with membranes have been modelled , there are only field rudimentary experimental data on the structure of the defensin complexes in the membrane. Finally, there is some evidence that having penetrated the cell wall, defensins can also interact with periplasmic and cytoplasmic macromolecules. These late interactions could also contribute to the lethal effect of defensins on microorganisms.

Role of defensins in host defence When initially proposed1, the name ‘defensins’ represented a conjecture, as it was largely based on in vitro antimicrobial activity and the peptide’s location in neutrophils — the prototypic host defence cells. Figure 4 | The carpet–wormhole model of action of defensins. Most defensins (shown as Experiments with transgenic mice are particularly large ovals) are amphipathic molecules that have clusters of positively charged amino-acid side informative in determining the non-redundant func- chains (pink) and hydrophobic amino-acid side chains (green). This allows them to interact with tions of many genes. Unfortunately, mice naturally lack microbial membranes, shown schematically with their negatively charged phospholipid granulocyte defensins, and the transgenic expression of headgroups (blue) and hydrophobic fatty acid chains (green). In the top panel, electrostatic human granulocyte defensins results in relatively low attraction and the transmembrane bioelectric field pull the peptide molecules towards and into concentrations of mature defensins in granulocytes the membrane. As peptide molecules accumulate in a ‘carpet’, the membrane is strained and the peptides transition into another arrangement (shown in the lower panel) that lowers the strain but (R. Linzmeier and T. G., unpublished observations). results in the formation of membrane ‘wormholes’ or pores92,94–96. For defensins, the specific Direct ablation of Paneth-cell-defensin expression is arrangement of peptide molecules in the pores is not yet known. made difficult by the many copies of similar defensin genes that encode these peptides. However, mice with homozygous disruption of the matrilysin gene failed the membrane) form either stably or transiently. For to activate intestinal prodefensins to defensins, and some defensins, the release of internal markers from were more susceptible to infection with Salmonella each vesicle occurred in an all-or-nothing manner91, typhimurium, requiring an eight-fold lower oral dose indicating that the pores that formed were stable. Using for 50% mortality97. After oral administration of E. coli, markers of various sizes, the pore diameter was esti- the counts of viable bacteria were similar in the proxi- mated at 25 Å. The authors proposed a model of a mal intestine of wild-type and matrilysin-knockout defensin pore (a hexamer of dimers) that generates an mice, but the wild-type mice had lower bacterial counts opening of the observed size. However, stable pore for- in the mid- and distal small intestine, where Paneth cells mation is not the only mechanism of defensin interac- are present at a higher density. In vitro, segments of the tion with membranes. The more cationic rabbit intestine from wild-type mice contained and secreted defensins induced a partial release of markers from indi- more antimicrobial activity than those of matrilysin- vidual vesicles, indicating that the pores that formed knockout mice31. Moreover, in wild-type mice the were not stable. It is possible that electrostatic repulsion antimicrobial activity could be largely neutralized by between the highly cationic rabbit defensin molecules defensin-specific antibodies, indicating that defensins destabilizes the pores. were responsible for most of the activity. Taken together, Some defensins also bind avidly to membrane glyco- these experiments provided important circumstantial proteins93 and this could be in part responsible for their evidence for the protective role of defensins in the early antiviral activity. stages of infection. More recently, a gain-of-function model was reported, Structure–function correlations. Most biophysical in transgenic mice expressing the human Paneth cell studies on the mode of action of antimicrobial peptides defensin gene HD5 (REF.98). HD5, compared with mouse have used simple α-helical (for example, magainin and Paneth cell defensins, has greater antibacterial potency

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Table 3 | Regulation of defensin synthesis and release Defensin Main cell Main regulators Cause of stored References or tissue of synthesis defensin release Human neutrophil Neutrophils Constitutive in promyelocytes Phagocytosis 30,63 peptides 1–3 Negligible in neutrophils Human defensin 5 Paneth cells Constitutive? Bacterial or cholinergic stimuli? Mouse cryptdins Paneth cells Constitutive? Bacterial or cholinergic stimuli 31,161 Human β-defensin 1 Distal renal tubular Constitutive N.D. epithelia, epidermis and other epithelia Human β-defensin 2 Epidermis and IL-1 N.D. 71,72 other epithelia Human β-defensin 3 Epidermis and Growth factors N.D. 73 other epithelia Bovine β-defensin tracheal Trachea Bacteria and N.D. 75,162 antimicrobial peptide lipopolysaccharide IL, interleukin; N.D., not determined.

against the mouse pathogen S. typhimurium. HD5- the expression of many genes involved in the resistance transgenic mice were fully protected against death from to cationic peptides, and also exerts some of its activity S. typhimurium infection at oral doses that killed all of by modulating a second two-component regulator the wild-type mice. Protection from infection was PmrA–PmrB. The function of the downstream genes seen as early as six hours, and correlated with lower includes the covalent modification of lipopolysaccha- S. typhimurium counts in the intestinal lumen, and rides that decreases their affinity for cationic peptides106 prevention of the spread of infection to other organs. and the expression of membrane proteases that The effect of transgenic defensin was local, as intraperi- degrade cationic peptides107.In Neisseria gonorrhoeae toneal inoculation that bypassed the intestine caused — a bacterium naturally resistant to defensins — the equal mortality in the transgenic and wild-type strains. energy-dependent efflux system mtr increases the resis- The intestinal lumen-specific effects of transgenic tance to protegrins — potent β-loop peptides of pig defensin early in the course of infection provide the neutrophils108.In Staphylococcus aureus, the disruption strongest evidence to date that defensins act as locally of either of the two genes, Dlt or MprF, increases the secreted antibiotics (FIG. 5). sensitivity of the bacteria to defensins109,110. The gene Mice deficient in mouse β-defensin 1 show only Dlt is required for covalent modification of cell wall tei- mild defects99,100 in host defence of the urinary and choic acid by alanine, and MprF is required for the respiratory tracts, probably due to the redundancy covalent modification of membrane phosphatidylglyc- among mouse defensin genes. Unlike the many defensin erol with L-lysine. These modifications probably act by genes that are present in the mouse genome, there is decreasing the negative charge of the cell wall and bacte- only one, or, at most, a few mouse cathelicidins (the num- rial membrane, respectively, and diminish their attrac- ber depends on how the family is defined). The mouse tion for the cationic defensins. Homologues of these cathelicidin (cathelin-related antimicrobial peptide, resistance genes have been identified in many bacterial CRAMP) is similar to its human orthologue, LL-37, species, indicating that these mechanisms might be and both are mainly expressed by neutrophils. Mice widespread. The existence of specific bacterial counter- with homozygous disruption of the Cramp gene measures argues that antimicrobial peptides or other showed diminished resistance to skin infection with functionally similar molecules have exerted significant group A Streptococcus101. Taken together, data from influence on microbial evolution. loss-of-function models support a host defence role for cathelicidins and defensins. Additional studies in Other activities of defensins these and related models would be desirable to docu- Various defensins have been reported to have chemotac- ment the main mechanism of action of defensins and tic activity for monocytes, T cells and dendritic cells111–114. cathelicidins in vivo. In the case of HBD1 and HBD2, which attract memory T cells and immature dendritic cells, the chemoattrac- Bacterial countermeasures tant activity might be due to defensin binding to the Specific mechanisms that confer increased bacterial chemokine receptor CCR6 (REF.113). Although the physi- resistance to defensins have been identified by inser- ological importance of this interaction has not yet been tional mutagenesis. Disruption of the two-component shown, the high concentrations of HBD2 in inflamed transcriptional regulator phoP–phoQ increases the skin make it probable that this defensin could compete sensitivity of S. typhimurium to defensins and other effectively with the natural CC chemokine ligand 20 cationic peptides102–105. PhoP–phoQ directly regulates (CCL20), despite the higher affinity of the latter for

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CCR6. Recent structural analysis of CCL20 indicated not yet been identified. Mouse β-defensin 2 acted as a marked similarities to HBD2 in the putative receptor- peptide adjuvant when it was linked to a non-immuno- binding region of CCL20. The role of this region in the genic tumour antigen115. This immunostimulatory chemotactic activity of HBD2 needs to be confirmed by activity was shown to depend on Toll-like receptor 4 mutating the amino-acid residues that are suspected (TLR4) and its ability to induce dendritic-cell matura- to be involved in its interaction with CCR6. Human tion. It is not yet certain how this receptor can bind neutrophil defensins HNP1–3 have been reported to mouse β-defensin 2 as well as the many other ligands be chemotactic for monocytes111, naive T cells and attributed to it, or whether some of these molecules immature dendritic cells114, but a specific receptor has function as efficient carriers for lipopolysaccharide — the main ligand of TLR4. Some defensins (known as corticostatins)116–118 oppose the action of adrenocorticotropic hormone Paneth 119 cells (ACTH) by binding to ACTH receptor without acti- vating it. Although such activity would inhibit the production of the immunosuppressive hormone cortisol, and could therefore be useful in responding to infections, the physiological role of this in vitro interaction has not yet been shown. Another reported activity of some defensins is their ability to activate nifedipine-sensitive calcium channels in mammalian cells120,121. This effect required only nanomolar concentrations of defensins. The structural basis of this effect is not understood. Certain mouse Paneth cell defensins activate chloride secretion, probably by forming channels in the apical membrane of epithelial cells122,123. This activity is limited to a subset of mouse Paneth cell defensins, and its structural basis is not yet known.

Salmonella Role of defensins in human diseases. Cystic fibrosis (CF) is a common autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. The CFTR gene encodes a regulated chloride channel that also functions as a regulator of other ion channels. The main cause of morbidity and mortality in CF is respiratory failure due to progressive destruction of the airways and lungs by Blood vessel recurrent infections and inflammation. Typically, in infancy or early childhood, the epithelial surfaces of the nose, sinuses and lower airways can be colonized by S. aureus and/or Hemophilus influenzae, but eventually Lymphatics these bacteria are succeeded by Pseudomonas aeruginosa and this infection heralds the onset of progressive lung disease. These events indicate a local defect in epithelial Crypt host defence, as the infection almost always remains localized in the lung and does not spread elsewhere or affect non-respiratory epithelia. Smith et al.124 proposed that the local host defence impairment is due to the inhibition of defensin activity by the abnormal ionic environment of CF airway fluid. The ‘high salt’ hypothesis was based on studies of ion transport in normal and CF sweat glands and in cultures of respiratory epithelia, as well as more limited analyses of human respiratory flu- Figure 5 | Killing of Salmonella by human defensins secreted by Paneth cells. The small ids. According to this theory, the thin layer of fluid that intestine is lined by finger-like absorptive villi interspersed with crypts — narrow pits containing coats respiratory epithelia normally has a low salt content a cluster of defensin-rich Paneth cells at the bottom. The granules of Paneth cells have high relative to blood plasma, and these conditions favour the concentrations of prodefensin 5, consisting of a propiece segment (blue circles) joined to the activity of defensins and other cationic antimicrobials N-terminus of mature human defensin 5 (red circles), together with Paneth cell trypsin (green that are attracted to their microbial targets by electrosta- triangles). After Paneth-cell degranulation, induced by the entry of bacteria into the intestinal lumen, trypsin activates defensin 5 by cleaving off its propiece. This process might function to tic forces. In CF,the ability to absorb salt from epithelial protect the absorptive epithelium, as well as the crypt, with its intestinal stem cells that generate cell fluid is compromised by the lack of functional chlo- the absorptive enterocytes98. Image courtesy of D. Schumick, Cleveland Clinic Foundation, USA. ride channels, the sweat (and by implication, respiratory

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fluid) becomes too salty and this diminishes the activity mammals, defensin gene ablation (or its functional of defensins and other cationic antimicrobials. The pro- equivalent) in non-mouse models might be required posal stimulated several alternative hypotheses125–127,but for the full understanding of the role of defensins in the nature of the connection between the defective neutrophils. With regard to β-defensins, the discovery CFTR and pulmonary infections has not yet been estab- of several previously unrecognized human and mouse lished with certainty, and the role of defensins in CF β-defensin genes further complicates the attempts to pathogenesis remains speculative. understand their biological roles. Exploration of the In another series of studies, polymorphisms in HBD1 function of the many epithelial cell β-defensins in genes have been linked with susceptibility to chronic animal models might be advanced by technologies for obstructive pulmonary disease with bronchitis128 and the disruption of entire gene clusters, the products high levels of oral Candida carriage in diabetics129.If of which have overlapping effects. In view of the these specific linkages are confirmed in larger groups of hypothesis that defensins are evolving rapidly under patients, they would further implicate β-defensins in the pressure from the microbial flora of each animal control of resident microbial flora on mucosal surfaces. species, the effects of experimental alterations in the arsenal of defensins on the composition of resident Concluding remarks microbial flora are of particular interest. We must Defensins are abundant and widely distributed pep- also learn how the variations in the amino-acid tides in human and animal tissues that are involved in sequences of defensins determine their specificity to host defence. Evidence from several models indicates various microbial targets, and conversely how struc- that defensins and other antimicrobial peptides func- tural changes in microorganisms modulate their sen- tion mainly as natural antibiotics in infected tissues. sitivity to specific defensins. Finally, the lack of fully However, alternative roles of defensins as immune or representative animal models increases the impor- inflammatory regulators have also been proposed and tance of analyses of the impact of defensin gene poly- need to be explored in ablative or additive animal morphisms on human health, as well as of other models, and in human subjects. As mice lack neutrophil studies of defensin biology in healthy volunteers and defensins that are so prominent in humans and other patients with various diseases.

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