Carbon Chapter 101 HBD1

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The human intestine also interacts with a large burden of commensal as well as potentially pathogenic microorganisms. Intestinal AMPs can fend off ingested pathogens, and they also play a crucial role in maintaining a healthy balanced relationship between the host and the extreme number of commensal inhabitants. Paneth cells are the major source of AMPs in the small intestine, producing α-defensins HD-5 and HD-6, lysozyme C, secretory phospholipase 2 (sPLA2), angiogenin 4 (ANG4) and lectins. HD-5 and HD-6 are the most abundant AMPs in Paneth cells and are constitutively expressed at high levels in these cells. In contrast to α-defensins, the expression of β-defensins is more variable throughout the gastrointestinal tract. Six human β-defensins and five mouse β-defensins (mBDs) have been identified and characterized. While hBD1 and its ortholog mBD1 are constitutively expressed in enterocytes, hBD2, hBD3 and hBD4 are induced during infection or inflammation. Cathelicidins constitute another major class of AMPs expressed by the intestinal epithelium at the upper crypts of the colon. Antimicrobial activity has also been observed in a number of epithelium-derived chemokines, including CCL20/MIP3α and an additional 17 chemokines. Other peptide molecules, including elafin (a peptidase inhibitor), secretory leukocyte protease inhibitor (SLPI), bactericidal/permeability-increasing protein (BPI) and REG3a are all anti-infective molecules present in the intestinal mucosa.

AMPs produced by the commensal microorganisms resident on epithelial surfaces of vertebrates also contribute to the antimicrobial shield of these structures. Staphylococcus epidermidis, the most abundant skin commensal bacterium, produces several AMPs, including epidermin (Staphylococcin 1580), Pep5 and epilancin K7, all of which belong to a unique group of AMPs known as lantibiotics because these peptides contain the thioether amino acids lanthionine and/or methyllanthionine. Our group has shown that S. epidermidis also produces the phenol-soluble modulins PSMγ and PSMδ, which are AMPs that selectively exhibit bactericidal activity against skin pathogens, such as S. aureus, group A Streptococcus and Escherichia coli, but are not active against S. epidermidis, their host organism. Emerging data have also shown that many other potent and selective AMPs are secreted on the epidermal surface by the commensal bacteria on the skin and may be both diagnostically and therapeutically useful.

In contrast to vertebrates, insects lack an adaptive immune system, but have a robust innate immune system that effectively kills invading microbes, mainly by humoral responses. This involves the rapid synthesis and release of different types of AMPs. Secretion of AMPs from the fat body (the insect equivalent of the vertebrate liver) into hemolymph (the fluid portion of insect blood) is the dominant mechanism for host defense in infected insects. Furthermore, as in vertebrates, insect gut epithelia also secretes tissue-specific AMPs, a response that is important in insect resistance to intestinal parasites (Figure 3). Insect defensins are structurally characterized by two or three β-sheets stabilized by disulfide bonds formed between cysteine residues. Three-dimensional structural analyses have revealed that there is a closer relationship between invertebrate defensins and vertebrate β-defensins than between vertebrate α- and β-defensins, suggesting that defensins are ancient molecules that are conserved across the eukaryotic kingdom. Cecropins are cysteine-free cationic peptides that adopt an α-helical structure in a hydrophobic environment. Cecropin was originally found in the pupae of the Hyalophora cecropia moth and the first identified genes encoding Drosophila AMPs were the cecropin genes. To date, eight distinct classes of AMPs have been identified in Drosophila melanogaster, and these can be further classified into three groups based on their main biological targets: Drosophila defensins are active against Gram-positive bacteria; cecropins, attacins, diptericins, and drosocin are active against Gram-negative bacteria; and drosomycin and metchnikowin have activity against filamentous fungi. Andropin, the only Drosophila AMP that is not induced during infection, is expressed specifically in male flies during mating to protect the reproductive tract. Extensive genetic analysis in Drosophila has shown that production of AMPs is controlled by the Toll and the Imd signaling pathways. The Toll pathway is activated primarily by Gram-positive bacteria or fungi, whereas the Imd pathway responds to Gram-negative bacterial infection. The common downstream feature of both pathways is activation of the fly homologues of the NF-κB signaling pathway, involving degradation of the IκB homolog Cactus followed by nuclear translocation of the NF-κB homologs Dorsal/Dif, leading to induction of the effector AMP genes.

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Figure 3. Local and systemic AMP response in Drosophila.

Drosophila produces AMPs in response to microbial infection, either locally by epithelial cells or systemically by the fat body which secretes AMPs into hemolymph. Activation of the Toll pathway by Gram-positive bacteria or fungi, or the Imd pathway by Gram-negative bacteria, triggers NFκB activation followed by induction of AMPs, the effector molecules that mediate microbial lysis. The table on the right lists the main AMPs and their antimicrobial targets in Drosophila.

Many AMPs are derived from larger precursor proteins with a signal sequence at the amino terminus and the antimicrobial peptide domain at the carboxyl terminus. The active peptide is generated by post-translational modification through proteolytic cleavage. The precursor protein holds the carboxy-terminal peptide in an inactive form before it is further processed and prepared for release when needed. Because of the potential detrimental effect of mature AMPs on mammalian cell membranes, the processing of many AMPs is also regulated by storage in secretory granules. α-defensins are stored in the secretory granule of the intestinal Paneth cell as inactive pro-peptides (∼10 kDa), which are cleaved by trypsin in humans or by matrix metalloproteinase 7 (MMP7) in mouse to generate the mature α-defensin peptides (∼4.5 kDa) with antimicrobial activity. The human cathelicidin hCAP18 precursor protein is cleaved by serine proteinases to its active 37 amino acid peptide (LL37). These proteases appear to be specific to the cell type expressing the AMP because proteinase 3 and elastase are active in the granules of neutrophils whereas kallikrein-related peptidase is active in the lamellar bodies of epidermal keratinocytes.

Immunoregulatory and non-antimicrobial functions of AMPs

One of the key advantages of AMPs over pharmaceutical antibiotics is the ability of some of these peptides to also modulate immune responses. For example, in addition to their direct antimicrobial activity, AMPs can protect the host by a range of mechanisms: chemotactic activity, attracting leukocytes; modulation of host-cell responsiveness to TLR ligands; stimulation of angiogenesis; enhancement of leukocyte/monocyte activation and differentiation; and modulation of the expression of proinflammatory cytokines/chemokines (Figure 1). For example, LL37 has been shown to attract neutrophils, monocytes, mast cells and T cells. Different groups of AMPs appear to have distinct chemotactic activities from each other. Human α-defensins selectively chemoattract CD4+ CD45RA+ naïve T cells but not CD4+ CD45RO+ memory T cells, whereas β-defensins are chemotactic for both CD4+ CD45RO+ resting memory T cells and immature dendritic cells. LL37 can suppress TLR signaling and bacterial-induced inflammation by binding to bacterial factors including lipoteichoic acid or lipopolysaccharide. On the other hand, LL37 can also promote inflammation by binding to nucleic acids to facilitate recognition of self-DNA by TLR9, self-RNA by TLR7 and TLR8, or double-stranded RNA by TLR3. A recent study from our lab has also found that LL37 enables dsRNA recognition by the MAVS/RIG-I pattern recognition signaling pathways in keratinocytes, thus further amplifiying the response to self-RNA, a known 'danger' signal. These interactions indicate that, during infection, AMPs can kill pathogens, initiate immunocyte recruitment/activation, and at the same time neutralize an excessive inflammatory response at the site of infection. However, sustained expression of AMPs could increase the host's responsiveness to self-nucleic acids, leading to the development of autoimmune disorders, such as psoriasis and rosacea.

AMPs and human disease

AMPs are pivotal for host defense and have several relevant associations with human disease. One indirect effect of the AMPs is their role in shaping the composition of the commensal microbiota and thereby contributing to the maintenance of skin and intestinal homeostasis. Disruption of the stability of the microbiome can lead to severe health consequences, including obesity, recurrent infections, and diseases such as inflammatory bowel syndrome and atopic dermatitis.

Numerous clinical studies have linked chronic intestinal inflammation to defective antibacterial mechanisms, possibly due to altered expression and secretion of AMPs. For example, Crohn's disease of the ileum has been associated with a deficiency in Paneth cell α-defensins. Crohn's disease of the colon is associated with a low-gene copy number polymorphism of the human β-defensin locus, resulting in a reduced epithelial induction of β-defensin AMPs in the colonic mucosa. Defensin deficiency weakens the antimicrobial defense properties of the intestinal mucosa, leading to alterations in the composition of intestinal flora that ultimately may promote bacterial invasion of the mucosa and predispose to the chronic inflammation of Crohn's disease. Similarly, abnormal expression of AMPs has also been linked to the development of atopic dermatitis, a common and chronic relapsing inflammatory skin disease. Atopic dermatitis patients usually bear abnormally high burdens of S. aureus on their skin, and this correlates with reduced expression of AMPs, such as LL37 and β-defensins (hBD2 and hBD3).

In contrast, overproduction of AMPs can directly initiate inflammatory diseases. For example, patients with psoriasis, a chronic auto-inflammatory skin disease characterized by high levels of cytokines produced by Th1 and Th17 T helper cells, express excessive amounts of AMPs, such as cathelicidin, hBD2, hBD3 and S100A8/9 in their skin. In rosacea, another auto-inflammatory skin disease mainly affecting the central portions of the face, proteolytic activation of cathelicidin protein is enhanced due to elevated expression of cutaneous proteinases such as KLK5 and KLK7. These observations implicating AMPs in skin disease pathogenesis have provided new opportunities for disease therapy.

Conclusions

All living organisms are constantly threatened by large numbers of microorganisms seeking to exploit the same environmental space. To cope with this substantial microbial threat, most cells produce natural antibiotic-like molecules that directly kill or inhibit the growth of foreign microorganisms. Since the discovery of lysozyme more than 90 years ago, more than 2,500 AMPs have been identified in single-celled organisms, plants, insects and animals. These molecules have selective antimicrobial activity against a wide range of organisms, largely due to their relatively strong electronic interaction with negatively charged membranes. In vertebrate species, some AMPs also promote host defense by modulating host cellular immunity. Furthermore, in mammals it has recently become clear that AMPs not only fend off pathogens but also shape the composition of the microbiome, and this is important for the maintenance of many aspects of health. The growing problem of resistance to and overuse of conventional antibiotics has stimulated interest in the development of AMPs as the next generation anti-infectives and as methods to more selectively combat pathogens. Although several limitations remain, control of endogenous AMP production may represent the next major paradigm for treatment of a broad range of human and animal diseases.

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