Chapter 2.1

Innate immune defense system of the skin

Maryam Afshar and Richard L. Gallo

Division of Dermatology, Department of Medicine, University of California, San Diego, 8899 University Center Lane, San Diego, CA 92122, USA

Correspondence: Richard L. Gallo, Division of Dermatology, Department of Medicine, University of California, San Diego, CA 92126, USA. E-mail: rgallo@ucsd.edu

Background – Antimicrobial peptides (AMPs) have a pivotal role in cutaneous innate immunity. They are present in the skin of many animals, including mammals, and are both constitutively present and inducible by infection and injury.

Functions – Antimicrobial peptides exhibit antimicrobial activity against bacteria, viruses, fungi and parasites, with different potencies depending on their peptide structure. They also act as multifunctional effector molecules that influence diverse cellular processes, including cell migration, proliferation and differentiation, cytokine production, angiogenesis and wound healing. Suppressed AMP production has been associated with increased susceptibility to microbial insults and the pathogenesis of atopic dermatitis.

This review highlights recent observations on the expression and role of AMPs, particularly the AMPs cathelicidin and β-defensin, in healthy and diseased skin.

Introduction

Skin is the largest organ of the body and provides an effective immune barrier between the internal and external environment. The cutaneous immune system can be arbitrarily divided into innate and adaptive components.¹ The innate immune system of the skin is the first-line, fast, nonspecific and active mechanism against environmental toxins and invading microbes. The innate immune response is followed by acquired immune responses, particularly T- and B-cell activation and proliferation against specific antigens.^1–3

The innate immune system of the skin is comprised of both a physical and a chemical shield. The uppermost layer of the epidermis, the stratum corneum, consists of keratinocytes tightly linked by desmosomes in a hydrophobic cellular matrix, thereby creating an anatomical barrier against irritants, allergens and shear force.^4–6 In addition to an immediate physical barrier, this intrinsic resistance system functions through chemical mediators (cytokines), specialized signalling pathways, the complement cascade, leukocytes and host defense peptides [antimicrobial peptides (AMPs)]. In particular, AMP production is a critical mechanism for immune response of the skin to cutaneous infection and injury. This review discusses the seminal role of AMPs in skin innate immunity.

Antimicrobial peptides

Antimicrobial peptides are evolutionarily conserved major contributors to host innate immune defense against bacteria, viruses, fungi, parasites and tumour cells.^3,7,8 Over 1700 AMPs are currently known.⁹ They have been described in plants, insects, invertebrates and vertebrates;¹⁰ however, great variability among species exists in AMP tissue distribution, genetic make-up and regulation of expression.¹¹ These differences suggest that AMPs are of varying significance for each species’ antimicrobial or innate immune response.¹²

Skin expression of AMPs has been demonstrated in humans¹³ and many animals, including primates,¹⁴ pigs,¹⁵ cows,¹⁴ sheep,¹⁶ chickens,¹⁷ rats,¹⁸ horses¹⁹ and dogs.¹² Cutaneous AMPs are constitutively present and further induced by infection or injury or both (Figure 1).⁴

Figure 1. Antimicrobialpeptide (AMP) expression in normaland infected or injured skin. (a) Normalskin is composed of an epidermallayer (keratinocytes) and a dermis containing capillaries. Keratinocytes and sweat glands produce low levels of AMPs to minimize microbial colonization. (b) Upon infection or injury, keratinocytes increase AMP production, which initiates neutrophil and mast cell recruitment to the site of injury via chemotaxis. These infiltrating leukocytes also make AMPs, and the consequent robust increase of AMPs at the injured site stimulates angiogenesis and keratinocyte proliferation to regenerate the damaged tissue. Abbreviations: C, constitutive expression; and I, inducible.

(Adapted from Radek and Gallo,²⁶ with permission of Springer Science+Business Media.)

In normal human skin, the main source of AMPs is the keratinocyte.²⁰ Synthesis of AMPs primarily occurs in the stratum granulosum; AMPs are then packaged into lamellar bodies and transported to the stratum corneum.²¹ Sebocytes, mast cells and neutrophils are also important sources of AMPs in normal human skin.²⁰ Antimicrobial peptides are also present in skin secretions, such as saliva and sweat.⁴

Upon skin infection or injury or both, recruited neutrophils, mast cells and other leukocytes contribute to the majority of AMP production.²⁰ Production is triggered by activation of pattern recognition receptors, such as Toll-like receptors (TLRs), mannose receptors and helicases.⁴ These receptors are activated by lipopolysaccharides from Gram-negative bacteria, lipoteichoic acid and peptidoglycans from Gram-positive bacteria, mannans from yeast and fungi, and nucleic acids from pathogens and self.^4,22 Antimicrobial peptide upregulation is a secondary response, which limits the severity of infection or injury when the primary line of defense (constitutive AMP expression) fails.²⁰

A major role of AMPs in innate immunity is their direct antibiotic-like inhibition of microbial pathogens. Most AMPs carry an overall net positive charge; this ensures their interaction with the negatively charged phospholipids in the cell membranes of both Gram-positive and Gram-negative bacteria, as well as the anionic components of fungi and viruses.^23,24 The peptides attach to, align with, and then insert into the microbial phospholipid bilayer.²⁵ As the peptides associate with the lipid head groups of the phospholipid bilayer, transmembrane ‘pores’ form.^25,26 The ‘pores’ disrupt and destabilize the bacterial cell membrane, ultimately leading to bacterial lysis.^25,26 Antimicrobial peptides preferentially target replicating bacteria, especially at the site of cell division.²⁷

There are many AMPs expressed in human and animal skin. Together, they carry out a variety of functions in cutaneous host defense, ranging from influencing cell signalling to inhibiting the growth of a wide spectrum of pathogens. The expression, structures, processing, induction and antimicrobial and immunomodulatory properties vary between the peptides.¹⁹ Cathelicidins and β-defensins are the well-characterized AMPs.^11,28,29 Key features of human cathelicidin and β-defensins are summarized in Table 1.

Table 1. Key features of human antimicrobial peptides (adapted from Izadpanah and Gallo,⁴⁰ with permission of Elsevier)

Cathelicidins

Cutaneous expression

Cathelicidins were the first AMPs found in mammalian skin.³⁰ Since their discovery, cathelicidins have been identified in the skin of humans,³¹ marsupials,³² dogs³³ and cats.³⁴

In humans, cathelicidin predominantly resides in granules of the superficial epidermis and in the extracellular spaces in the stratum corneum.³⁵ Likewise, in dogs, it resides in the stratum granulosum and less so in the stratum corneum.^33,36

Structure

Only one cathelicidin is encoded by humans (LL-37), mice (CRAMP) and dogs (cCath), while multiple cathelicidins have been discovered in pigs, horses, cattle and chickens.^11,37

The human cathelicidin gene (CAMP) encodes an 18 kDa α-helical precursor protein [human cathelicidin antimicrobial protein (hCAP-18)]⁴ that consists of an N-terminal prodomain region, cathelin, the amino-acid sequence of which is highly conserved across species.^38–40 In contrast, the C-terminal domain of hCAP-18 varies between 20 and 40 amino acids among different species; this variability contributes significantly to the broad-spectrum antimicrobial activity of cathelicidins.²⁶ The hCAP-18 has a 68% mRNA sequence similarity and a 57% protein sequence similarity to cCath.³⁶

Peptide processing is essential for providing both the regulation and diverse biological activity of cathelicidin.²⁹ The full-length precursor, hCAP-18, is thought to be inactive in its immature form.²⁹ The hCAP-18 is commonly cleaved by serine proteases, such as kallikrein 5 and 7 in keratinocytes, and by neutrophil proteases, such as serine protease 3, to release the mature 37-amino-acid peptide that begins with two leucine residues, LL-37.^37,41-43 In sweat, LL-37 is further processed, generating KR-20, a 20-amino-acid derivative, KS-30, a 30-amino-acid derivative, and RK-31, a 31-amino-acid derivative.^44,45

Unlike hCAP-18, processed cathelicidins demonstrate fast, potent, broad-spectrum antimicrobial activity.²⁶ The expression of cathelicidin as a full-length precursor that requires proteolysis for its activation allows for the stable control of its antimicrobial and proinflammatory action in the presence of particular stimuli.²⁶

Induction

Infection, cutaneous injury and vitamin D₃ are all potent inducers of cathelicidin expression. LL-37 is induced by both bacterial components, such as lipopolysaccharide, and proinflammatory mediators, such as interleukin-6 and retinoic acid.^46,47 Cathelicidin expression levels are upregulated in the presence of Gram-positive bacteria.^48,49 LL-37 is induced within the epidermis during the development of verruca vulgaris.⁵⁰ Cutaneous injury induces the release of cathelicidin active against group A Streptococcus.³¹ Vitamin D₃ can also induce hCAP-18/LL-37 expression in keratinocytes.⁴²

Antimicrobial functions

Cathelicidins are potent antimicrobial agents. Cathelicidins possess an intrinsic ability to kill Gram-negative and Gram-positive bacteria, fungi and viruses.^44,46 Cathelicidin expression levels correlate with resistance to cutaneous infections.⁵¹ The CRAMP knockout mice show increased susceptibility to skin infections with group A Streptococcus and vaccinia.⁵² LL-37 has been shown to neutralize lipopolysaccharide endotoxin.⁵³

Immunomodulatory functions

Antimicrobial peptide LL-37 interacts with mammalian cells to induce a host response by triggering inflammatory cell recruitment and cytokine release (Figure 2). LL-37 acts as a chemoattractant through binding of formyl peptide receptor-like 1, which belongs to the G_i protein-coupled receptor family.⁵⁴ LL-37 promotes the recruitment of neutrophils, T cells, mast cells and monocytes to sites of injury and infection, and stimulates angiogenesis.^54–56 LL-37 also induces histamine release from mast cells and intracellular Ca²⁺ mobilization.⁵⁷ Cathelicidin may help repair skin wounds by inducing keratinocyte migration via transactivation of the epidermal growth factor receptor.⁵⁸ LL-37 significantly inhibits the lipopolysaccharide-induced release of proinflammatory cytokines by macrophages.⁵⁹ LL-37 influences TLR signalling in immune cells, specifically by inhibiting TLR4- but not TLR2-mediated induction of dendritic cell maturation and cytokine release.⁶⁰ LL-37 also binds extracellular self-DNA fragments, enabling them to enter plasmacytoid dendritic cells, which initiates TLR9 activation to produce type I interferons.²⁸ LL-37 changes the expression of phagocytic receptors, significantly enhancing the ability of dendritic cells to undergo phagocytosis.⁶¹ The immunomodulatory mechanisms of cathelicidin complements its antimicrobial properties and reinforce its role as an integral defense molecule in innate immune responses.

Figure 2. Several of the functions of AMPs in host defense. Antimicrobial peptides recruit leukocytes to the infection site, block or activate TLR signalling, protect against infection, control inflammation and promote wound healing. Abbreviations: AMP, antimicrobial peptide; LPS, lipopolysaccharide; pDC, plasmacytoid dendritic cell; and TLR, Toll-like receptor

(adapted from Lai and Gallo,²⁸ with permission of Elsevier).

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