Host-defense peptides (HDPs) are vital components of innate immunity in all vertebrates. While their antibacterial activity toward bacterial cells was the original focus for research, their ability to modulate immune and inflammatory processes has emerged as one of their major functions in the host and as a promising approach from which to develop novel therapeutics targeting inflammation and innate immunity. In this review, with particular emphasis on the cathelicidin family of peptides, the roles of natural HDPs are examined in managing immune activation, cellular recruitment, cytokine responses, and inflammation in response to infection, as well as their contribution(s) to various inflammatory disorders and autoimmune diseases. Furthermore, we discuss current efforts to develop synthetic HDPs as therapeutics aimed at restoring balance to immune responses that are dysregulated and contribute to disease pathologies.
Host defense peptides (HDPs) have evolved across all species of animals and are recognized as vital components of innate immune processes. HDPs are short, gene-encoded polypeptides (10–50 residues in length) that are broadly characterized by a net positive charge and a high proportion of hydrophobic amino acids. They can exhibit potent bactericidal activity in buffer, which is why they are often referred to as antimicrobial peptides (AMPs), although this activity is often abrogated by host physiological conditions including (especially divalent) cation concentrations and the presence of polyanions such as glycosaminoglycans. Conversely, under host-like physiological conditions and in animal models, many natural AMPs are able to modulate the host innate immune response. Indeed, the immunomodulatory activity of these molecules might be more representative of their natural functions and potential for development as therapeutic agents. Numerous studies have focused on unraveling the mechanisms that underlie the various immunomodulatory functions of HDPs in diverse scenarios. While no general mechanism has been described for all HDPs, several features of the immunomodulatory response to HDPs have been described for a variety of cell types and animal models, including cellular recruitment, anti-inflammatory activity, and wound healing, among others.
Current knowledge about the activities of HDPs has been largely derived from the study of naturally-occurring peptides from vertebrates. Some HDPs are expressed constitutively by immune cells, whereas the local concentration of others can be upregulated in response to a particular stimuli and/or secreted into the local environment or released from phagocytes by degranulation. Several HDPs are also expressed by epithelial, cells of the skin, gastrointestinal, genital, and respiratory tracts as well as a variety of other cell types. The most abundant and best characterized HDPs in mammals are those classified as cathelicidins and defensins. Numerous cathelicidins have been described in mammals as well as other phyla including birds, reptiles, amphibians, and fish.
Here we focus on the features of cathelicidins that contribute to their immunomodulatory properties and highlight the potential for developing synthetic HDP derivatives as novel therapies for various inflammatory conditions. An overview of the structure, function, and expression of naturally-occurring cathelicidins across vertebrates is provided with a particular emphasis on their ability to maintain homeostasis by influencing immune signaling and mitigating damaging inflammatory responses. In addition, we discuss disorders that are made more severe by cathelicidins acting as self-antigens, and describe various diseases associated with dysregulated expression of cathelicidins. Several examples of synthetic peptides that have been designed to harness the beneficial effects of natural peptides are highlighted, particularly for their capacity to modulate innate immune processes. In addition, we examine an emerging role for cathelicidins and synthetic HDP derivatives in the management of dysregulated immunity present in sepsis. Finally, we highlight several ongoing clinical trials aimed at exploiting the immunomodulatory functions of HDPs and discuss emerging peptide formulation strategies and studies in animal models that bridge the gap between pre-clinical and clinical development of novel peptide therapies.
The cathelicidin family of HDPs exhibits a broad diversity in structure and function across all vertebrates. The number of genes encoding cathelicidin analogs can vary by species. For instance, only a single cathelicidin gene is encoded in humans, mice, and dogs, while 2–11 cathelicidin-coding genes have been identified in certain species of fish, amphibians, reptiles, birds, and most other mammals. The organization of the coding sequence seems to be well conserved among vertebrates and is comprised of four exons that collectively encode the precursor peptide consisting of a signal peptide sequence, the cathelin pro-domain, and the mature cathelicidin sequence. Although there is high amino acid sequence identity for the cathelin domain between species, the mature form of the cathelicidin peptide is remarkably diverse in length, composition, net charge, and structure.
Mature cathelicidin peptides can be loosely grouped into four structural classes: α-helical or linear peptides that can adopt helical conformations under physiological conditions or in the presence of biological membranes; linear peptides that are disproportionately high in particular amino acids such as glycine, serine, proline or tryptophan; and two classes stabilized by disulfide bridges, namely β-structured and cyclic peptides. The α-helical peptides are the most widely distributed and present in all vertebrate groups, but other structural classes are observed across species.
It has been suggested that HDPs found in multicellular organisms arose as a protective mechanism against microbes, particularly against bacteria. In such a scenario, it is assumed that host-microbial interactions and direct antimicrobial activity drove the evolution of HDP sequences to optimize them collectively for anti-bacterial potency. However, as mentioned above, the antimicrobial potency of most HDPs remains rather modest in host-like environments. A recent study of mammalian homologs of LL-37 proposed that the driving force behind the evolution of cathelicidins might be their interaction with host receptors, which is consistent with the concept that immune response elements are one of the most highly evolving groups of proteins across mammalian species.
An earlier study suggested that the disordered C-terminus of LL-37 interacts with the N-formyl peptide receptor-like (FPR) family of proteins, as part of the process to mediate chemotaxis. Sequence analysis of the human FPR2 receptor indicated high variability in the ligand-binding extracellular loop domain, while the C-terminus of mammalian LL-37 homologs was disordered; thus statistical analysis revealed a possible co-evolution of this peptide as a cognate binding partner for FPR2 was proposed. Furthermore, the elimination of the disordered N- and C-terminal regions in the rabbit LL-37-homolog (CAP18-FV) or their replacement with disordered regions from the dolphin (ttLL-37) or human homologs had no impact on the anti-bacterial activity. Unfortunately, since the immunomodulatory properties of the resulting species-hybrid mutants of LL-37 were not evaluated, the direct influence of this proposed interaction was not confirmed. Regardless, several other host receptors with immune functions have been proposed to interact with cathelicidins, including purinergic receptors P2Y11 and P2 × 7, the CXC chemokine receptor type 2, Mas-related gene X2 (MrgX2), GAPDH, and others. This provides strong evidence that receptor binding directly impacts the biological functions of cathelicidins. Curiously, a similar evolutionary analysis did not identify highly variable residues in avian cathelicidins, suggesting that this putative co-evolutionary relationship might be specific to mammalian LL-37 homologs.
Since the repertoire and cell/tissue distribution of cathelicidins varies by species, we focus below on discussing the expression and activity of the human cathelicidin antimicrobial peptide (CAMP) gene found on chromosome 3p21. The CAMP gene encodes the 18 kDa precursor human cationic antimicrobial protein, hCAP18, which is cleaved by proteases to generate the active peptide known as LL-37. It is expressed in a variety of tissues and cell types, including epithelial cells and many cells of the immune system. Expression of hCAP18 is highest in the bone marrow in healthy individuals, although expression can be detected in many organs and tissues. Secretory glands enhance basal expression at mucosal surfaces, with hCAP18 secreted in the semen, saliva, and sweat. Most studies of the regulation of CAMP expression in various tissues reflect recognition of inflammatory stimuli by neutrophils and monocytes, since these cell types produce more hCAP18/LL-37 than other immune cells. In addition, neutrophils store the inactive hCAP18 precursor in specific (azurophilic) granules for rapid deployment during immune responses.
