Cationic host defense peptides are a widely distributed family of immunomodulatory molecules with antimicrobial properties. The biological functions of these peptides include the ability to influence innate and adaptive immunity for efficient resolution of infections and simultaneous modulation of inflammatory responses. This unique dual bioactivity of controlling infections and inflammation has gained substantial attention in the last three decades and consequent interest in the development of these peptide mimics as immunomodulatory therapeutic candidates. In this review, we summarize the current literature on the wide range of functions of cationic host defense peptides in the context of the mammalian immune system.
Cationic host defense peptides (CHDPs) are immunomodulatory molecules that have evolved to provide broad range of protection against a variety of pathogenic microbes. Early research in the field focused primarily on the antimicrobial functions of CHDPs, consequently these molecules are also known as antimicrobial peptides. However, studies have shown that certain microbial pathogens can be resistant to the direct microbicidal activity of CHDPs at physiological concentrations. Moreover, it has been demonstrated that the direct antimicrobial property of certain CHDPs are often compromised in the presence of physiological salt concentrations and specific host factors, yet these peptides are essential for the protection against pathogens. This highlights the role of CHDPs in immunity, which is to enhance microbial clearance and maintain immune homeostasis. Therefore, the term CHDPs has now been adopted to encompass the antimicrobial, immunological and other biological functions of these molecules that have been defined over the last three decades. A wide range of species from microbes to plants and mammals express CHDPs. Cationic peptides with antimicrobial activity were originally identified from lysosomal fractions of leukocytes from guinea-pigs in the 1960s. Subsequently, Lehrer and Selsted purified cysteine-linked antimicrobial peptides from rabbit leukocytes in the 1980s. Subsequent research has led to the characterization of more than 2600 cationic peptides from a wide variety of organisms ranging from microbes to plants to humans.
The two most well characterized families of CHDPs in mammals are cathelicidins and defensins. We will focus on these two families of mammalian CHDPs in this review. These CHDPs are expressed by various cell types including macrophages, polymorphonuclear leukocytes such as neutrophils, mast cells, stromal bone marrow and mucosal epithelial cells. CHDPs are small amphipathic peptides typically 12–50 amino acids in length, with a net positive charge ranging from +2 to +9 due to an abundance of basic amino acids such as lysine and arginine. CHDPs are derived from immature precursor proteins that subsequently undergo proteolytic cleavage to render the biologically active mature peptides. CHDPs have diverse sequences and structures, and are broadly classified into two categories, α-helical peptides and β-sheet class peptides, based on their length, structure and the presence of disulfide linkages.
A prominent example of amphipathic α-helical CHDPs is the cathelicidin family of peptides. Cathelicidins are comprised of a highly conserved 14 kDa N-terminal cathelin-like pro-domain, followed by a signal peptide and a C-terminal ‘mature’ peptide region. The sole human cathelicidin is expressed as an 18 kDa precursor pro-protein called hCAP18, which is subsequently cleaved to the well characterized, biologically active, 37 amino acid cationic peptide LL-37. This can be subsequently cleaved to generate smaller peptides that have been identified from epithelial surfaces. LL-37 is expressed constitutively or induced by the presence of infections and/or inflammatory stimuli, in both immune and structural cells, e.g., secretory granules of neutrophils, mast cells, B-cells, T-cells, monocytes, natural killer cells, keratinocytes, squamous and airway epithelial cells. Additionally, epithelial cells lining the upper digestive tract, salivary glands, sweat glands, small intestine, epididymis, testis, vagina and cervix also express LL-37. A recent study has also demonstrated that LL-37 can be expressed by adipocytes. Structurally, LL-37 is devoid of disulphide bonds and acquires an α-helical conformation when interacting with lipid bilayers. Solid-state NMR studies and CD spectroscopy suggest that α-helical LL-37 can also form tetramers or higher oligomers in solution, rendering the peptide cytotoxic at higher concentrations. Extensive solid-state NMR studies have elucidated the conformation, orientation, dynamics and the oligomeric nature of LL-37 in membranes.
Amongst the β-sheet class of CHDPs, the mammalian defensins are the best characterized. These are 30–45 amino acid long cyclic peptides that are rich in cysteine, arginine and aromatic amino acid residues and contain cysteine disulphide bridges. Defensins are categorized further as α, β and θ-defensins. The α-defensins were identified from azurophilic neutrophil granules of primates, and are also known as human neutrophil peptides (HNP). There are four different types of HNPs (HNP1-4), of these HNP1-3 are highly homologous, varying by one amino acid at the amino terminus. Thus, it is not surprising that HNP1-3 exhibit an exceptionally high degree of sequence and functional similarity. A wide range of cells and tissues such as neutrophils, peripheral blood mononuclear cells, bone marrow, spleen and thymus express α-defensins. HNPs are endogenously expressed as inactive precursor pro-protein (proHNPs) that are proteolytically cleaved to generate the mature peptide. Intestinal proHNPs are processed by serine protease trypsin in humans and matrix metalloprotease in mice. However, a recent study has demonstrated that processing of neutrophil proHNPs may be independent of serine proteases, which indicates that the mechanisms underlying the posttranslational processing of HNPs are not completely elucidated and that the activity of other classes of proteases need to be examined in this context.
β-defensins are primarily secreted by epithelial cells, however, immune cells, such as monocytes, macrophages and dendritic cells, also express these peptides. β-defensins are expressed in mucosal secretions of gastrointestinal, urogenital and respiratory tracts. More than 20 potential β-defensin homologues have been described based on human genome analyses, of which hBD1-3 are most well characterized. Similar to HNPs, β-defensins are also expressed as a pro-peptide that is subjected to proteolytic cleavage to release the mature peptide. However, the proteases involved in the processing of β-defensins are not completely defined. A recent study using computational analyses has demonstrated that β-defensins differ from α-defensins in the pattern of cysteine pairing and spacing, as well as having a shorter pro-peptide. Despite the high degree of similarity in tertiary structures, β-defensins exhibit poor sequence similarities, suggesting that their bioactivity maybe independent of the primary amino acid sequences.
θ-defensins are macrocyclic peptides expressed in leukocytes and the bone marrow of nonhuman primates, such as macaques and baboons, and have not yet been conclusively identified either in humans or in other higher primates such as gorillas and chimpanzees. θ-defensins exist in humans as a pseudogene due to the presence of a premature stop codon. Therefore, these peptides are called rare defensins with cyclic structures. The most well characterized θ-defensins are from rhesus macaque, and the rhesus θ-defensin 1 is the most abundant isoform in macaque granulocytes. Furthermore, there are also CHDPs that lack typical secondary structure but are able to endorse novel folds, which include bovine indolicidin and porcine tritrpticin.
As mentioned above, research in the last three decades has demonstrated that CHDPs elicit a wide range of biological functions including an influence on the host’s immune responses for the resolution of infections and inflammation. CHDPs affect both innate and adaptive immunity, playing a critical role in immune homeostasis. CHDPs contribute to both immune activation and immune regulation, which is dependent on cellular composition, environmental stimuli and the kinetics of the inflammatory response. In this review, we will focus on the immune functions influenced by mammalian CHDPs.
Innate immune response is the first line of host defense in response to infections. Microbial components, such as lipopolysaccharides (LPS), interact with host pattern recognition receptors such as toll-like receptors (TLRs). This results in the activation of multiple intracellular signaling networks and the production of numerous effector molecules, including CHDPs, that orchestrate host immune responses. Accumulating evidence suggests that released CHDPs in response to an infectious challenge contribute to the enhancement of innate immune processes by inducing classical pro-inflammatory responses, such as recruitment of leukocytes to the site of infection, generation of reactive oxygen species and aiding phagocytosis, which collectively facilitate resolution of infections. In this context, one of the primary immune activating functions of CHDPs is to facilitate recruitment of leukocytes to the site of infections, either by direct chemoattractant properties or by mediating the induction of chemokines. CHDPs, e.g., LL-37, HNP-1, HNP-2, hBD-1 and hBD-2 can directly promote chemotaxis of neutrophils, monocytes, eosinophils, dendritic cells (DCs) and T-lymphocytes to the site of infection (reviewed in). In addition, CHDPs such as LL-37 and β-defensins (hBD-2 and hBD-3) at physiological concentrations can stimulate the production of various chemokines, e.g., Gro-α/CXCL1, IL-8/CXCL8, M
