Lung antimicrobial proteins and peptides: from host defense to therapeutic strategies

Abstract

Representing severe morbidity and mortality globally, respiratory infections associated with chronic respiratory diseases, including complicated pneumonia, asthma, interstitial lung disease, and chronic obstructive pulmonary disease, are a major public health concern. Lung health and the prevention of pulmonary disease rely on the mechanisms of airway surface fluid secretion, mucociliary clearance, and adequate immune response to eradicate inhaled pathogens and particulate matter from the environment. The antimicrobial proteins and peptides contribute to maintaining an antimicrobial milieu in human lungs to eliminate pathogens and prevent them from causing pulmonary diseases. The predominant antimicrobial molecules of the lung environment include human α- and β-defensins and cathelicidins, among numerous other host defense molecules with antimicrobial and antibiofilm activity such as PLUNC (palate, lung, and nasal epithelium clone) family proteins, elafin, collectins, lactoferrin, lysozymes, mucins, secretory leukocyte proteinase inhibitor, surfactant proteins SP-A and SP-D, and RNases. It has been demonstrated that changes in antimicrobial molecule expression levels are associated with regulating inflammation, potentiating exacerbations, pathological changes, and modifications in chronic lung disease severity. Antimicrobial molecules also display roles in both anticancer and tumorigenic effects. Lung antimicrobial proteins and peptides are promising alternative therapeutics for treating and preventing multidrug-resistant bacterial infections and anticancer therapies.

CLINICAL HIGHLIGHTS.

• 1) Antimicrobial proteins and peptides contribute to maintaining an antimicrobial milieu in human lungs to eliminate pathogens and inhalants and prevent them from causing pulmonary diseases.
• 2) Besides direct antimicrobial and antibiofilm activity against bacteria, viruses, and fungi, antimicrobial proteins and peptides also have roles in immunomodulatory function, regulating inflammatory and repair responses.
• 3) The major antimicrobial molecules of the lung environment include human α- and β-defensins, cathelicidins, PLUNC (palate, lung, and nasal epithelium clone) family proteins, lactoferrin, lysozymes, secretory leukocyte proteinase inhibitor, and surfactant proteins SP-A and SP-D.
• 4) The lung antimicrobial proteins and peptides are significant in helping the host combat upper and lower respiratory tract infections and chronic inflammatory airway diseases, including cystic fibrosis, chronic obstructive pulmonary disease, asthma, and lung cancer.
• 5) Lung antimicrobial proteins and peptides are promising alternative therapeutics for treating and preventing multidrug-resistant bacterial infections and anticancer therapies.

1. INTRODUCTION

Human respiratory diseases remain a serious clinical concern amplified by the global dissemination of microbial pathogens. 2021 United States mortality data provided by the Centers for Disease Control and Prevention determined that lung cancer, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and chronic lower respiratory diseases were the second, third, and sixth leading causes of death from 2020 to 2021 (1). The respiratory system’s main function is gas exchange by transporting oxygen and removing extra carbon dioxide. Our respiratory system, including the trachea, lungs, blood vessels, and chest wall diaphragm muscles, makes breathing and gas exchange possible with a coordinated effort with the brain and nerves. An average sedentary adult breathes ∼10,000 liters of air daily (2), with each individual’s respiratory system in constant contact with the external environment. Pulmonary diseases are pathological conditions that impair respiratory function and overall lung health. These diseases or disorders may develop through inhalation of pathogens, including bacteria, viruses, and fungi, or exposure to environmental chemicals and particles. The pathology of both infectious and noninfectious lung disease involves the activity of innate host defense molecules to enact direct killing mechanisms against respiratory pathogens and have roles in the immunomodulation of pulmonary disease (3, 4).

The airway provides a critical first-line defense against pathogenic microorganisms and polluting contaminants inhaled from the environment (5, 6). Maintenance of overall lung health and the prevention of lung disease relies on the mechanisms of airway surface liquid (ASL) secretion, mucociliary clearance (MCC), and adequate immune response to eradicate pathogens and particulate matter from the environment (5). Another critical mechanism of surface airway defense is the antimicrobial milieu provided by the production of antimicrobial molecules, including antimicrobial peptides (AMPs) and proteins. AMPs, or host defense peptides, are a group of evolutionarily conserved molecules in virtually all kingdoms’ innate host defense mechanisms. These peptides have integral roles in the host’s response to infection and inflammation. Sources of AMPs range from bacteriophages, bacteria (both Gram-positive and Gram-negative), fungi, plants, invertebrates, fish, amphibians, reptiles, birds, and mammals (7). Natural antimicrobial proteins and AMPs (AMPPs) in the lung are mainly produced by epithelial cells and neutrophils (6).

This review highlights concepts, mechanisms, and applications related to lung antimicrobial proteins and peptides. We discuss the relevance of innate antimicrobial molecules of the lung in association with upper and lower respiratory tract infections and chronic inflammatory airway diseases, including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), asthma, and lung cancer. First, we describe the discovery and structure of the antimicrobial proteins and peptides. Second, we delineate the antimicrobial activity and mechanisms of these antimicrobials. Third, we highlight the nonantimicrobial activity in the immunomodulatory host response. Fourth, we describe the antimicrobial molecules as having physiological consequences correlating with various pulmonary diseases. Finally, we discuss how natural and engineered antimicrobial molecules can be utilized as clinical therapeutics. Improved knowledge regarding lung antimicrobial proteins and peptides could lead to novel therapeutic options using antimicrobial molecules to treat pulmonary diseases.

2. NATURAL ANTIMICROBIAL ACTIVITIES AND HOST DEFENSE

Antimicrobial agents typically have antiviral, antibacterial, antifungal, and antiparasitic effects. For thousands of years, humans have employed natural substances from many different sources to treat various infections, as documented in ancient China, Egypt, and Rome, by using herbs, honey, and moldy bread (8). The discovery of penicillin by Alexander Fleming in 1928 originating from the fungus Penicillium notatum represented an unprecedented breakthrough in medicinal practice, pushing the search for compounds with similar efficacy primarily from microbial sources (9). In fact, under limited nutrient conditions, microbes produce toxic metabolites against other “competitive” microbial species to gain growth and survival advantage (10). The following period of rapid antibiotic discovery (defined as the “golden era”) contributed to these agents’ current essential role in medicine and surgery. During the golden era in the forties and early fifties of the twentieth century, almost all classes of antibiotics (aminoglycosides, tetracyclines, cephalosporins, macrolides) were identified, mainly from soil bacteria (11).

However, in the next period, between the seventies and nineties, specific resistances of bacterial strains to antibiotics emerged quickly, abolishing the benefits of antibiotics and leading to a gradual return to the preantibiotic era (12). Significantly, during these years, chemically modified penicillin compounds were developed, resistant to the cleavage by penicillinase. Nevertheless, once derivatives of existing antibiotics began to lose their effectiveness against multidrug-resistant (MDR) organisms, several pharmaceutical companies stopped working on antibiotic discovery for other applications (e.g., anticancer drugs), ensuring higher profits (13). This provoked a dramatic reduction in the number of approved antibiotics for commercial use, with only three new drugs developed between 2005 and 2009 (13). However, the entrance of 12 new antibiotics into the market between 2010 and 2018 indicates that although the capacity to eliminate MDR strains is limited, improvements can be made in the near future (14, 15).

Besides microbes, various microscopic algae, as well as higher green plants such as gymnosperms and angiosperms alike, produce structurally unique secondary metabolites (alkaloids, flavonoids, terpenoids, etc.) with antimicrobial features (16–18). Although extensive research has been carried out to identify antimicrobial substances from microbial and plant sources, the enormous variety of terrestrial and marine organisms has played an essential role. In their ecosystem, invertebrates, fishes, amphibians, reptiles, birds, and mammals, including humans, are regularly exposed to different stressful environmental conditions, which have prompted them to develop robust host defense mechanisms against infections, later found to be related to antimicrobial proteins and peptides.

3. THE ORIGINS OF ANTIMICROBIAL PROTEINS AND PEPTIDES

Several years after the clinical use of penicillin, multiple AMPs were discovered and characterized as molecules active against Gram-positive and Gram-negative bacteria. Gramicidin from the bacterium Bacillus brevis was the first natural peptide-based drug mainly active against Gram-positive bacteria and was launched in 1939 despite its cytotoxicity (19). However, the real explosion of AMPs can be traced back to the early 1980s, when Hans Boman isolated cecropin from the hemolymph of the silkworm moth Hyalophora cecropia (20). Later, in 1987, the relevance of AMPs was emphasized by the discovery of magainins by Michael Zasloff from the skin of Xenopus laevis (21). Zasloff demonstrated, for the first time, the existence of AMPs in vertebrates, as subsequently confirmed by Ganz (22) and Lehrer with the identification of defensins from neutrophils of higher vertebrates, including humans.

Historically, the immune system of human newborns was thought to be an immature version of that of adults. Unlike fetuses, which typically experience a sterile environment before delivery, newborns are challenged by various microorganisms soon after birth (23). Without adaptive immunity, infant defense relies primarily on innate mechanisms mediated by antimicrobial proteins and AMPs (AMPPs). AMPs are usually cationic molecules synthesized by ribosomes to counteract microbial infections (24). In humans and other mammals, many AMPs are stored within granules of circulating immune cells (neutrophils, monocytes, and macrophages), from where they are secreted locally at sites of infection and inflammation. AMPs are also found in the skin and in body fluids (e.g., tears, saliva, nasal secretion, sweat, breast milk, semen) as well as in the intestinal, respiratory, gastrointestinal, and urinary tracts (FIGURE 1) to help protect against the continuous exposure to external microorganisms while maintaining homeostasis of the host microbiota (25–27). The production of AMPs is tightly regulated, with many being expressed as inactive precursors and then activated through proteolytic cleavage (28). Their expression can be constitutive (as in neutrophils) or induced upon contact with microbial components or cytokines during infection or inflammation processes (29).

FIGURE 1.

Principal features of antimicrobial peptides (AMPs): expression, structural features, and production sites. TLR, toll-like receptor.

Among human AMPs, defensins, cathelicidins, and histatins can reach antimicrobial concentrations at sites of infection in the body or in leukocyte granules that are extremely harmful to microorganisms (FIGURE 1). However, in most anatomical sites, their antimicrobial activity is hindered by the physiological concentrations of salts and serum proteins (4, 30).

Human defensins include α- and β-defensins (see sects. 4 and 5 for more details). Six members belong to the α-defensin family, which are α-defensins 1–4 produced by leukocytes and natural killer (NK) cells (31) and α-defensin 5 (HD5) and HD6 expressed by the Paneth cells of the small intestine (22, 32–34). In comparison, human β-defensins (HBDs) are widely distributed throughout the body and synthesized mainly by epithelial cells of those regions accessible to pathogen invasion. HBD-1 is constitutively expressed, whereas HBD-2 is inducible upon nuclear factor-kappa B (NF-κB) activation following contact with bacteria (31).

Cathelicidins are synthesized as prepropeptides containing an amino-terminal signal peptide, a cathelin-like domain followed by a single copy of the mature peptide at the carboxy terminus. Once secreted, the procathelin domain is cleaved by serine proteases (35). LL-37 is the only human cathelicidin transcribed from the cathelicidin AMP (CAMP) gene (36). Although LL-37 is produced constitutively in many cell types, its synthesis in immune and epithelial cells is controlled by infection or tissue damage (36, 37). In addition, vitamin D₃ plays a relevant role in the upregulation of LL-37 (38), and studies based on vitamin D₃ intake are currently underway in Asia to treat tuberculosis by promoting LL-37 expression (38).

Histatins are produced by salivary glands and are rich in histidine amino acids (39). The first histatin was discovered in 1988 from human parotid salivary gland secretions (40). The natural variants of these peptides are histatin-1 (38 amino acids), histatin-3 (32 amino acids), and histatin-5 (24 amino acids) (41). Histatin-1 and histatin-3 derive from the Htn1 and Htn3 genes, respectively (39). Histatin-5 originates from the parent histatin-3 and contains a highly reactive NH₂ terminal that binds to metals. These peptides have gained popularity in therapeutic and dental medicine.

In addition to AMPs, AMPPs include bactericidal/permeability-increasing protein (BPI), S100 proteins (e.g., calprotectin), lactoferrin, lysozyme, cathepsin, and RNases (e.g., 4, 5, and 7). Inhaled microorganisms can colonize the human host through the airways. Still, the lung tissue is well protected from infection because of the presence of many AMPPs in the epithelial lining fluid, including LL-37, defensins, PLUNC, and lysozyme (42, 43), and their level significantly rises during the context of pneumonia (44). Histones and thrombin fragments, usually found in neutrophil extracellular traps (NETs), belong to AMPPs with a broad spectrum of antimicrobial activities (45, 46). Lactoferrin is another example of a protein with antimicrobial activity. It is not confined to milk but is produced by several tissues, and its proteolysis in the gastrointestinal tract gives rise to fragments that are more active than the native protein (47).

Remarkably, antimicrobial proteins also display additional functions to prevent pathogenic species’ invasion of host tissues by sequestration of metals that often serve as cofactors for essential enzymes in microbes (48) like Pseudomonas aeruginosa and Staphylococcus aureus (49). Indeed, calprotectin is one metal-sequestering protein that binds free multiple metal ions, limiting metal ion access to microbes as nutrients (49).

4. THE PROPERTIES OF ANTIMICROBIAL PROTEINS AND PEPTIDES

AMPs are usually short polypeptides with 12–50 amino acids with a cationic character (generally a net charge of ≥ +2) and ∼30–50% hydrophobic amino acids. These structural features are two fundamental parameters accounting for the antimicrobial activity of AMPs (50). The cationic AMPs bind electrostatically to the negatively charged components of the surface of the target microbial cell. At the same time, the hydrophobicity facilitates the interaction of AMPs with the phospholipid membrane, followed by its disintegration or AMPs’ translocation into the cytosol.

The common classification of AMPs is based on their secondary structure, as described below:

• 1) The major group is given by cationic and amphipathic α-helical AMPs when interacting with target cell membranes (51). Cecropins, magainins, and cathelicidins belong to this class. Generally, α-helical AMPs are unfolded in solution and form amphiphilic structures when in contact with biological membranes.
• 2) The second group consists of cationic β-sheet AMPs bearing cysteine residues that form intramolecular disulfide bonds (52), which are essential for their structural stabilization and biological functions. The mammalian defensins belong to this group and include α- and β-defensins according to the position of disulfide bonds (53). The family of α-defensins have a three-stranded chain and a cyclic structure stabilized by hydrogen and disulfide bonds, respectively (54). In the β-defensin family, some peptides contain both α-helix and β-sheet portions (55).
• 3) The θ-defensins (α-defensins produced after primate differentiation and isolated from leukocytes of rhesus monkeys and baboons) are uniformly cyclized peptides with antiparallel β-sheets (56). Studies revealed that the number and position of disulfide bonds influence the structure and stability of defensins. At the same time, the cyclic backbone affects their antibacterial and membrane-binding properties (57).
• 4) The third group is AMPs rich in arginine, proline, tryptophan, glycine, and histidine (58) and with an extended conformation. Even though they do not have a defined secondary structure, they adopt an amphiphilic conformation upon membrane interaction.
• 5) The fourth subgroup involves anionic AMPs after proteolysis, like dermicidin, which is processed in sweat glands, or cryptic peptides produced upon proteolytic digestion of proteins (59). The anionic AMPs need metal ions to form oligomeric complexes in the bacterial membrane, resulting in ion channel formation and membrane depolarization (59, 60).

5. LUNG ANTIMICROBIAL PROTEINS AND PEPTIDES

Many, but not all, antimicrobial molecules are expressed in the lungs. Except for PLUNC family proteins and pulmonary surfactant proteins specifically expressed in the lung and secreted by lung epithelial cells, most other AMPPs are also expressed in different organs. Thus, we describe the abundant major AMPPs that play significant roles in pulmonary pathophysiological conditions (FIGURES 2 AND 3).

FIGURE 2.

Antimicrobial proteins and peptides of the upper and lower airways. Antimicrobial molecules are critical mediators of first-line defense against pathogens, modulate the immune response, and regulate inflammation associated with respiratory disease. AMP, antimicrobial peptide; HBDs, human β-defensins; HNPs, human neutrophil peptides, Lf, lactoferrin; Lz, lysozyme; PLUNC, palate, lung, and nasal epithelium clone; PNEC, pulmonary neuroendocrine cell; SLPI, secretory leukocyte proteinase inhibitor; SP-A/D, surfactant proteins A/D.

FIGURE 3.

Antimicrobial proteins and peptides of the alveoli. AMP, antimicrobial peptide; SP-A/D, surfactant proteins A/D.

5.1. Human β-Defensins

Human β-defensins (HBDs) of the lung are primarily synthesized by mucosal epithelial cells and serve as mediators of host defense against infection by microorganisms and regulate the innate and adaptive immune response (61). HBDs 1–3 are most prevalent in the lung and are secreted and released to the epithelial surface mucus layer (61). In structure, HBDs are small (25–45 amino acids), cationic amphipathic peptides comprised of β-sheet folds formed by three intramolecular disulfide bond-cysteine linkages (22). In addition to direct broad-spectrum antimicrobial killing, HBDs are active mediators in the adaptive immune response through the chemotaxis of monocytes, macrophages, T cells, and dendritic cells (61). HBDs have also been shown to activate and promote the migration of mast cells (61).

5.2. Human Neutrophil Peptides

Human neutrophil peptides (HNPs), or α-defensins, are originally synthesized in the bone marrow by promyelocytes, where they are packaged in azurophil granules until they are released in masses upon neutrophil activation (22, 61). Neutrophils, some lymphocytes, and natural killer (NK) cells express HNP1–4 (62). Like HBDs, HNPs are small (25–45 amino acids), cationic amphipathic peptides with six cysteines linked by three intramolecular disulfide bonds, forming a structure of β-sheets and a defensin fold (22). The length of peptide segments and the characteristic pattern of cysteine linkages distinguish the structure of the defensin subfamilies from one another (22). Apart from broad-spectrum killing of invading microorganisms, HNPs exhibit chemotactic activity for T cells and dendritic cells and elevate dendritic cell activation as part of the adaptive immune response (61). HNPs have both pro- and anti-inflammatory effects in immunomodulation. For instance, HNPs upregulate the expression of interleukin-8 (IL-8) and interleukin-1beta (IL-1β) in airway epithelial cells. Also, they ameliorate inflammatory injury by reducing reactive oxygen species production from stimulated neutrophils and releasing proinflammatory cytokines by macrophages (61). Additionally, HNPs promote phagocytosis of both viruses and bacteria by neutrophils and macrophages (63).

5.3. Cathelicidin hCAP18/LL-37

The human cathelicidin, hCAP18/LL-37, is synthesized in immune cells and epithelia (61) (FIGURES 2 AND 3). Cathelicidins have a cationic COOH-terminal domain (hCAP18) that may be cleaved by proteases, leaving the NH₂-terminal domain (LL-37), which enacts antimicrobial activity (64). LL-37 has two leucine residues at the NH₂ terminus with a total length of 37 amino acids and a curved amphipathic helix-bend-helix tertiary structure (61, 64). LL-37 binds to and neutralizes lipopolysaccharide (LPS) and has activity against Gram-positive and Gram-negative bacteria, as well as viruses and fungi (64). In addition, LL-37 modulates the adaptive immune response by promoting the chemotaxis of neutrophils, monocytes, and T cells and enhancing the activation of dendritic cells (61). LL-37 has been shown to combat toll-like receptor (TLR)-mediated inflammation by inhibiting the release and expression of proinflammatory cytokines (65).

5.4. PLUNC Family Proteins

PLUNC antimicrobial proteins are abundantly secreted by airway epithelial cells of the upper respiratory tract and share structural similarity to bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) (66). The PLUNC genes are on human chromosome 20, along with phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), BPI, and LBP (67). This superfamily of proteins may bind to lipid A of LPS, enacting bactericidal activity against Gram-negative bacterial species. PLUNC proteins appear to have multiple roles at the air-liquid interphase, including regulation of surface tension of epithelial cell secretions as a surfactant and in vitro inhibition of biofilm formation (66). Short-palate lung nasal epithelium clone 1 (SPLUNC1), also known as bacterial permeability family member A1 (BPIFA1), is a 25-kDa secretory protein highly expressed in serous secretory cells of the pharynx, trachea, larynx, and bronchi regions of the airways (68). SPLUNC1 has important roles in host defense against pathogens and shows antibiofilm activity against Klebsiella pneumoniae and P. aeruginosa by reducing the surface tension of airway epithelial cell secretions (69, 70). In addition, transgenic mice expressing human SPLUNC1 exhibit enhanced protection against Mycoplasma pneumoniae (71) and P. aeruginosa (72). SPLUNC1 also modulates airway hydration by inhibiting the epithelial Na⁺ channel (ENaC), but its activity is hampered by the acidic cystic fibrosis (CF) environment, allowing for pathogenic invasion (73, 74).

5.5. Pulmonary Surfactant Proteins

Pulmonary surfactant plays a crucial role in lung function and gas exchange. It is essential in maintaining the integrity of the lung alveolar epithelium, reducing surface tension, facilitating breathing, and preventing alveolar collapse (75). Pulmonary surfactant is secreted into the alveoli and comprises a complex mixture of lipids and associated proteins. All four surfactant proteins SP-A, SP-B, SP-C, and SP-D are synthesized by alveolar type II epithelial (ATII) cells, with roles in downregulating inflammation in the lung (76) (FIGURE 3). SP-B and SP-C are low-molecular-weight, hydrophobic surfactant proteins cosecreted with the surfactant lipids and interact closely with them (77) by altering lipid packing and spreading to enhance the surface tension-lowering activity of the lipids (78). SP-A and SP-D are lipoproteins and relatively large macromolecules with four domains: an NH₂-terminal domain, a collagen-like domain, the α-helical coiled neck, and the COOH-terminal domain for carbohydrate binding (75). SP-A and SP-D are structurally related members of the collectin family, which is named based on the presence of collagen and lectin in their structure that interacts with carbohydrates in the presence of calcium (75). SP-A binds LPS and various microbial pathogens, enhancing their clearance from the lung. SP-D has antimicrobial activity against viral, fungal, and bacterial pathogens and is necessary to suppress pulmonary inflammation. SP-A and SP-D modulate antiviral activity by binding to regions of viruses, thereby neutralizing or inhibiting infection, promoting phagocytosis of viruses by macrophages, and downregulating proinflammatory cytokines associated with viral infection (75). SP-A and SP-D also have roles in immunomodulation, irrespective of pathogenic infection (75, 79). Approximately half of the alveolar surfactant pool is cleared through a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent alveolar macrophage pathway. Most of the remaining surfactant is taken up by ATII cells and recycled to the lamellar body through the multivesicular body/late endosome for resecretion, while a portion is degraded in lysosomes (for review, see Ref. 78). Changes in surfactant integrity and composition contribute significantly to chronic inflammatory lung diseases.

5.6. Secretory Leukocyte Protease Inhibitor

Secretory leukocyte protease inhibitor (SLPI) is synthesized by epithelial cells, neutrophils, macrophages, and mast cells, is expressed in the human lung epithelia, and is present at high levels in saliva and mucus secretions (80). SLPI is a low-molecular-weight (107 amino acids) evolutionarily conserved pleiotropic protein with two spiral polypeptide domains bound together by four disulfide bridges (81). Critical to host defense and immune response, SLPI mediates inflammation by inhibiting protease activity and transcription of central inflammatory mediator protein nuclear factor-kappa B (NF-κB) and also aids in immune defense against pathogenic infection (80). SLPI ameliorates damaging inflammation by reducing the production of proinflammatory cytokines, disrupts protease activity, and indirectly inhibits the TLR-dependent innate immune response by reducing binding with LPS (80). Furthermore, SLPI shows broad-spectrum antimicrobial activity through direct killing of both Gram-positive and Gram-negative bacterial species, inhibition of viral infection, and antifungal activity (80, 82).

5.7. Lactoferrin

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins, found in a variety of human secretory fluids and neutrophil granules, and is primarily secreted by neutrophils (83). Lactoferrin is ∼80 kDa in size, with two homologous domain lobes bound by a short peptide three-turn α-helix (84). Besides iron transport, lactoferrin has roles in immunomodulation and has activity against viruses, bacteria, fungi, and protozoa (85). Lactoferrin may bind to lipid A of Gram-negative LPS or Gram-positive teichoic acid, leading to increased membrane permeability and bacteria cell death (85). Lactoferrin has also been shown to inhibit biofilm formation through iron chelating, promoting surface twitching motility (86). Inhibition of bacterial invasion and viral infection has been demonstrated by lactoferrin activity (85). Through iron chelation, lactoferrin reduces oxidative stress released by damaged tissue. It binds immune cells to reduce the proinflammatory response, inhibit TLR-mediated cytokine production, and further alter immune cell recruitment and activation (87). Therefore, lactoferrin modulates the host inflammatory response and disease pathogenesis.

5.8. Lysozyme

Lysozyme is one of the primary antimicrobial proteins secreted by airway epithelial cells and neutrophils with antibacterial activity in airway secretions (88). Lysozyme is abundant in the secretions of the respiratory mucosa and in macrophages, neutrophils, and dendritic cells (89). Lysozyme is a 14-kDa polypeptide (129 amino acids), globular in structure, and possesses enzymatic activity against peptidoglycan, a major bacterial cell wall component (90). Lysozyme also facilitates bacteria killing through membrane pore formation (91). Furthermore, lysozyme exhibits an anti-inflammatory response by defending against pathogen invasion at the epithelial barrier and through clearance of cleaved peptidoglycan soluble fragments that may trigger the host immune response or macrophage activation (91). On the other hand, lysozyme can also elicit a proinflammatory immune response by recruiting phagocytes following bacteria killing and subsequent release of pathogen-associated molecular patterns (PAMPs) (91). The release of PAMPs triggers proinflammatory cytokines and stimulates inflammation. Therefore, the immunomodulatory activities of lysozyme are complex and important in the immune response to pathogens.

6. ANTIMICROBIAL ACTIVITY AND MECHANISMS BY ANTIMICROBIAL PROTEINS AND PEPTIDES

6.1. Direct Microbicidal Activity

By virtue of their cationicity and amphipathicity, many AMPs directly kill microbes through an initial electrostatic interaction with the anionic teichoic acids (92), peptidoglycan layer, or LPS that is in the outer membrane of Gram-negative bacteria. This is then followed by the formation of salt bridges with the bacterial cytoplasmic membranes that are rich in negatively charged phospholipids (phosphatidylglycerol, cardiolipin, and phosphatidylserine) (93–95) compared to the mammalian cell membranes that are mainly made of neutral phospholipids and cholesterol (96). This difference is one of the major reasons accounting for the preferential activity of AMPs toward bacterial rather than mammalian cells (97). In addition to ionic bonds, some AMPs, e.g., nisin and mesentericin, bind the bacterial membrane through receptor-mediated recognition (98–100).

The electrostatic interaction of AMPs with the bacterial membrane is imperative for their direct killing activity, occurring through physical destabilization of the membrane itself or inhibition of intracellular processes (e.g., DNA/RNA/cell wall synthesis, protein synthesis and folding, enzymatic activity, etc.) after the translocation of AMPs into the cytosol and subsequent interplay with specific intracellular targets (101), limiting the onset of resistance to AMPs due to this multitarget mechanism with complementary effects. The following sections explain the different mechanisms of direct antimicrobial activity.

6.1.1. Membrane-perturbing mode of action.

Before interacting with the anionic cytoplasmic membrane, AMPs have to cross the peptidoglycan cell wall rich in lipoteichoic acids (and mannans in fungi) or pass through the LPS-outer membrane of Gram-negative bacteria by a self-promoted mechanism (FIGURE 4A) (92, 102). Once they reach the cytoplasmic membrane, AMPs adopt an amphiphilic structure by orienting their cationic domain toward the anionic phospholipid head groups. Contextually, the hydrophobic region is directed toward the fatty acid chains of the lipid bilayer (103). When a threshold peptide concentration is reached, AMPs permeabilize the membrane (FIGURE 4B) by pore formation (according to the “barrel-stave” and “toroidal pore” models) or by a micellization process in a detergent-like manner (“carpet” model), as further described below. This ultimately results in the leakage of ions and metabolites, depolarizing the transmembrane potential and impairing membrane function to cause eventual cell lysis. Alternatively, as stated in the previous paragraph, peptides translocate into the cytoplasm, hindering biosynthetic processes upon interaction with specific targets (FIGURE 4B) (101).

FIGURE 4.

Schematic representation of the mode of action of membranolytic antimicrobial peptides (AMPs). A: self-promoted uptake to allow AMP translocation through the LPS-outer membrane in Gram-negative bacteria. B: different models of cytoplasmic membrane perturbation and pore formation (left), compared to the mechanism of AMPs, which inhibit different intracellular biosynthetic processes once they enter the bacterial cytosol.

6.1.1.1. barrel-stave model.

According to this model, peptides self-associate on the surface of the bacterial membrane and then insert into the phospholipid bilayer to form transmembrane bundles. The hydrophobic surfaces of the helices interact with the hydrocarbon chains of the membrane phospholipids, while the hydrophilic surfaces point inward to form a transmembrane pore. Progressive recruitment of additional peptide monomers leads to an increase in pore size. The barrel-stave model represents the main activity for the AMPs gramicidin and alamethicin and other cytolytic toxins (104).

6.1.1.2. toroidal pore model.

In the toroidal pore model, first described by Makovitzki et al. (105) and Ludtke et al. (106), peptide helices insert into the membrane and interact with lipids to form pore complexes. The first step is the binding of the helical peptides in an orientation parallel to the plane of the bilayer until a threshold concentration is reached. Above this threshold, which generally depends on the lipid composition of the bilayer, the peptide orients perpendicular to the plane of the bilayer to form a toroidal or a wormhole pore. This model differs from the barrel-stave model because the lipid bends back on itself to line the pore through the entire permeation process (104).

6.1.1.3. carpet model.

According to this model, AMPs first bind onto the surface of the target membrane via electrostatic interaction and cover it in a “carpet-like” manner. Subsequently, the peptides reorient their hydrophobic face toward the lipids and the hydrophilic face toward the polar phospholipid head groups. Practically, regardless of the type of permeation pathway, described as toroidal pores or channel aggregates, higher peptide concentrations can lead to membrane disintegration (membrane packing collapses into fragments with physical disruption of the microbial cell). The carpet-like mechanism represents the activity of most α-helical AMPs, which do not principally self-aggregate before insertion into the bacterial membrane (107, 108).

6.1.2. Intracellular mode(s) of action.

A model that combines the interaction of the peptide with microbial membranes followed by their destabilization and the peptide’s entry into bacterial cells to hamper different biological processes is called the Shai–Matsuzaki–Huang (SMH) model.

6.1.2.1. amps acting on dna/rna.

Among AMPs acting on intracellular targets, buforin II binds to DNA/RNA without altering membrane permeability (109). Likewise, indolicidin penetrates bacterial membranes and inhibits DNA synthesis without lysing bacteria (109). In addition to direct binding to DNA and induction of DNA damage, AMPs can also indirectly affect DNA transcription or translation. For example, Bac5 inhibits bacterial protein synthesis through binding to the ribosome tunnel. This prevents the transition from the initiation to the elongation stage of translation (110).

6.1.2.2. amps act on the activity of enzymes.

Some AMPs inhibit intracellular enzymes implicated in microbial metabolism and proliferation (4). Otvos and collaborators (111, 112) showed that the proline-rich AMP pyrrhocoricin binds the Escherichia coli heat shock protein DnaK and blocks its ATPase activity. In a published study (113), the AMP microcin J25 interacted with RNA polymerase and hampered its catalytic activity, preventing the entry of substrates to the active site (114). Yang and colleagues (115) discovered that the antibacterial effect of LL-37 on E. coli occurs through the inhibition of palmitoyl transferase PagP activity, increasing outer membrane permeability. In comparison, other AMPs, like defensins, interact with precursor molecules needed for cell wall synthesis, like the peptidoglycan precursor lipid II (116–119).

6.1.2.3. other mechanisms.

Other studies have shown that the coaggregation of amyloid peptides and amyloid proteins is another relevant mechanism of action of AMPs (120). Although AMPs and amyloid peptides are not similar in secondary structure or sequence, the formation of fibrils by AMPs and the discovery of antimicrobial activity by amyloid peptides suggest that their actions are similar (121). The formation of protein aggregates with functional proteins of the target pathogenic organism could provoke changes in bacterial growth rate and virulence (120, 122). Chu and colleagues (123) demonstrated that α-defensin-6 released by Paneth cells can self-assemble into nanonets and fibrils to capture bacteria and protect stem cells from bacterial damage.

6.2. Antibacterial Activity: Bacteriostatic vs. Bactericidal

AMPs exert activity against bacteria by a membrane- or non-membrane-perturbing mechanism. As mentioned above, the membrane permeation caused by AMPs induces the loss of cellular contents or favors intracellular uptake of AMPs followed by their diffusion into the cytosol, where AMPs can bind to specific intracellular targets. However, the mechanism of antimicrobial action of AMPs depends on their concentration. Previous studies performed with amphibian skin peptides, such as temporins, demonstrated that bacteria could still be alive even if AMPs affect the permeability of the bacterial membrane, making it accessible to small molecules (124), strengthening the notion that membrane perturbation is not per se a lethal event. Furthermore, subinhibitory concentrations of derivatives of the membrane-active frog skin AMP esculentin-1a were found to modify the protein expression profile of E. coli and provoke a significant reduction in the expression level of virulence genes of P. aeruginosa (125), without killing bacteria. It was also demonstrated that the in vitro activity of different AMPs depends on the density of bacterial cells used for bacterial growth inhibition assays. This phenomenon is called the inoculum effect and has relevant consequences for the therapeutic efficacy of drugs since bacterial loads change by several orders of magnitude in clinical infections (126). It was suggested that 1) bacterial killing by membrane-perturbing α-helical AMPs, such as PMA23, takes place only when bound peptides completely saturate bacterial membranes, supporting the carpet model for the membrane perturbation in actual bacteria; 2) the peptide/cell binding equilibrium dictates the active concentration of the peptide; and 3) the effective selectivity of AMPs hinges on the amounts of host and microbial cells; whereas 4) the antibacterial activity does not depend on the concurrent presence of a large surplus of host cells.

6.3. Antibiofilm Activity

Many pathogenic bacteria protect themselves by secreting an extracellular matrix in which they remain entrapped while entering a metabolically inactive dormant state. These sessile communities, named biofilm, are not attackable by traditional antibiotics and work like a shield. Therefore, preventing biofilm formation at its early stage is another advantageous strategy to ward off bacterial infections (FIGURE 5). The capability of AMPs to inhibit biofilm formation is not related to their efficacy in killing or inhibiting the growth of the planktonic form of microorganisms. As stated by Luo and Song (128), inhibition of biofilm formation can occur in different ways, including the following: 1) Alteration of the two main Las I and Rhl quorum-sensing intercellular communication systems controlling the architecture and maintenance of biofilms in P. aeruginosa. For example, LL-37 reduces the attachment of bacterial cells to surfaces and negatively affects Las and Rhl quorum-sensing systems with the downregulation of genes essential for biofilm development (129). 2) Suppression of alarmone production. Generally, the exposure of bacteria to nutrient-free or other stressful environmental conditions triggers the upregulation of the signal nucleotide guanosine tetra and pentaphosphate (pppGpp) (130, 131), which controls the expression of a variety of virulence genes promoting biofilm formation. For example, peptide 1018 prevents biofilm production by interacting with (p)ppGpp and degrading it, thus impeding its signaling effects (132). Derivatives of the frog skin AMP esculentin-1, namely Esc peptides, downregulate the expression of genes implicated in bacterial motility, likely upon binding to ppGpp, thus lowering the concentration of such available nucleotides (128, 133). This would hamper bacteria from reaching the surface on which to start biofilm formation. 3) Downregulation of the expression of other biofilm-associated genes (134). Human β-defensin 3 significantly reduces the expression of icaA and icaD genes (accountable for biofilm production in Staphylococcus epidermidis) while increasing the expression of the icaR gene (encoding for a transcriptional repressor of the ica operon expression), resulting in a significant attenuation of biofilm production (135). In comparison, another way to eradicate preformed biofilm can rely on the membrane perturbation of biofilm cells. For example, nisin A compromises the membrane potential of methicillin-resistant biofilm cells of S. aureus and elicits the formation of stable pores with ATP leakage (136). Esc(1–21) kills P. aeruginosa biofilm cells by destroying the cell membrane and breaking down the extracellular matrix (137).

FIGURE 5.

Antibiofilm activity of antimicrobial peptides (AMPs). AMPs prevent the formation of or destroy produced biofilm through suppression of alarmones, alteration of quorum-sensing communication systems, downregulation of biofilm formation-associated genes, and membrane perturbation of biofilm cells (127). EPS, extracellular polymeric substance; (p)ppGpp, guanosine tetra and pentaphosphate.

6.4. Antiviral Activity

Considering the involvement of AMPs in the host defense system, it is not surprising that some of them display antiviral activity. The first report dates back to 1986, when a member of the defensin family, HNP-1, was found to inactivate a large variety of enveloped viruses, including influenza and Herpes simplex (138). Viruses are noncellular pathogens that must be internalized into a host cell to replicate and survive. A viral particle contains a nucleocapsid with genetic material (single- or double-stranded RNA or DNA) surrounded by a protein capsid and an envelope membrane (139). Below are the three most common mechanisms AMPs use to inactivate viruses (FIGURE 6).

FIGURE 6.

Schematic representation of the principal mechanism of antiviral activity of antimicrobial peptides (AMPs).

6.4.1. Destabilization of the viral envelope to damage the virions.

For example, temporin isoforms destroy the enveloped membrane of herpes simplex virus type 1 (HSV-1), preventing the attachment of the virus to the host cell (140, 141). In addition, LL-37 is active against the influenza A virus (IAV), human immunodeficiency virus, dengue virus, and Zika virus by physical destruction of the viral membrane and inhibition of DNA replication (141–145).

6.4.2. Inhibition of viral attachment to the host cell.

For example, indolicidin and protegrin are active against HSV, targeting the viral membrane glycoprotein. This blocks the adhesion and entry of the virus (146, 147). HNP-1–4 and α-defensin 6 (HD-6) interfere with HSV binding to the host cell receptor by direct interaction with the HSV glycoprotein or by binding to the cell surface glycosaminoglycan heparan sulfate, thereby preventing viral entry (148).

Remarkably, AMPs not only directly affect the viral particle and its replication cycle but also indirectly inhibit the growth of viral particles by activating the host immune response through the attraction of immune cells to the site of infection and production of cytokines to potentiate viral clearance (149, 150).

6.4.3. Inhibition of viral spreading by acting intracellularly.

It has been reported that the frog skin AMP temporin G can interact with the viral hemagglutinin protein of the influenza virus, hampering the conformational rearrangements of the HA2 subunit, a fundamental step to enable the fusion of the viral envelope with the intracellular endocytic vesicles to release viral particles into the cytosol of the host cells (151). The same peptide was found to block the later stages of parainfluenza respiratory virus replication, preventing the extracellular diffusion of viral particles. Some other peptides, like melittin, have also been found to inhibit viral gene expression by interfering with cellular signal transduction (152).

6.5. Antifungal Activity

AMPs with antifungal activity include peptides that typically act by directly targeting the invading fungal pathogen or stimulating an inflammatory response. Various killing mechanisms concerning yeasts and fungi have been reported, ranging from effects on mitochondrial functions of Candida albicans by histatin 5 to membrane permeabilization by cathelicidins, Esc(1–18), and temporins (153–156). All three major types of histatins inhibit and kill Candida species, although their effectiveness can vary (157). The mechanism by which they act as fungistatic and fungicidal agents involves disruption of the plasma membrane, leading to the loss of intracellular components (40). Histatin-3 and Histatin-5 can bind to metals (158), particularly copper and nickel, generating reactive oxygen species that damage cell organelles and DNA, ultimately leading to fungal and bacterial cell death (159). Denture wearers with compromised immunity are more prone to opportunistic pathogens like C. albicans (160). Several studies have shown that histatins inhibit the growth of these microorganisms on polymethyl methacrylate, which is used in dentures, making histatins a powerful tool for the prevention and treatment of fungal infections (161).

Some AMPs, like echinocandin, inhibit the biosynthesis of the fungal cell wall component 1,3-β-glucan, thus increasing susceptibility to osmotic stress and cell death (162). Others interact with the membrane ceramide, causing membrane permeability and promoting the production of intracellular reactive oxygen species that are toxic to fungal cells (163).

7. IMMUNOMODULATORY ACTIVITY BY ANTIMICROBIAL PROTEINS AND PEPTIDES

The function of AMPs can go beyond direct antimicrobial activity by regulating a broad range of immunity-related functions that can enhance the host’s response to infection (FIGURE 7) (37, 164–166).

FIGURE 7.

Schematic representation of immunomodulatory properties of antimicrobial peptides (AMPs).

As depicted in FIGURE 7, most AMPs are able to 1) suppress proinflammatory cytokines and display an antiendotoxin activity to avoid exaggerated and harmful inflammatory conditions, 2) stimulate chemotaxis of inflammatory cells to the site of infection, NET formation, immune cell differentiation, and phagocytosis to facilitate bacterial clearance (167), and 3) display a wound healing activity to restore tissue integrity, preventing pathogen penetration. Several of these activities are further discussed in the subsections below. Because of these multifaced features, AMPs could be more appropriately referred to as host defense peptides. This definition is also supported by the observation that many AMPs lose antimicrobial activity under physiological conditions. In contrast, their immunomodulatory functions can still be detected both in vitro and in vivo (168).

The molecular mechanism underpinning the ability of AMPs to modulate immune responses is highly complex, involving intracellular uptake of the peptides, interaction with several intracellular/membrane protein partners or receptors, and alteration of signaling pathways (128).

7.1. Anti-inflammatory/Proinflammatory Function

Inflammation is the first response of the human body to harmful and foreign stimuli to maintain tissue and organ homeostasis (169). The anti-inflammatory mechanism of AMPs generally consists of neutralizing toxic effects produced by bacterial components, such as LPS, released from the cell walls of Gram-negative bacteria, especially upon antibiotic treatment. LPS contains three portions: lipid A, which is an acylated and phosphorylated disaccharide unit; the o-antigen, which is an oligosaccharide chain; and the polysaccharide core, which joins the two parts (170).

Once released, LPS stimulates immune cells, causing an increased secretion of proinflammatory cytokines, e.g., tumor necrosis factor-alpha (TNF-α), and excessive inflammation leading to sepsis (171). Indeed, LPS first binds to lipopolysaccharide-binding protein to form the LPS-LBP complex (172, 173), which then interacts with the CD14 pattern recognition receptor on macrophages. CD14 then transfers LPS to TLR4, triggering the intracellular pathway, which ends with the expression of inflammatory factors (172). As reported by Luo and Song (128), the anti-inflammatory activity of most AMPs is generally expressed in different ways.

7.1.1. LPS detoxification.

Cationic AMPs can neutralize LPS and prevent the secretion of inflammatory mediators by binding to the negatively charged phosphate groups of LPS (174). In comparison, the LPS acyl chains interact with the nonpolar hydrophobic portion of AMPs (175). Gutsmann et al. (176) showed that AMPs convert the active cubic aggregate structure of LPS to an inactive multilamellar structure.

7.1.2. Inhibition of LPS binding to LBP.

In most cases, the binding of peptides to LPS induces dissociation of LPS aggregates (177), hampering LPS binding to LBP. For example, temporins and esculentin-1a derivatives were found to display antiendotoxin activity by promoting the disassembly of LPS aggregates (178, 179).

7.1.3. Inhibition of LPS transport.

Some AMPs compete with LPS for binding to CD14, thus impeding LPS from interacting with TLR4 receptors (180). The anti-inflammatory function of AMPs is supported by studies highlighting that an increased inflammatory response accompanies a deficiency of these peptides. For example, SPLUNC1-deficient and cathelicidin-deficient mice display a more severe inflammatory phenotype than wild-type (WT) mice (70, 181, 182). Analogously, a decreased expression of β-defensins in human enterocytes has been found to be associated with Crohn’s disease (183).

7.2. Wound Healing

Wound healing is a physiological process to reestablish tissue entirety and function upon injury (184, 185). Many studies have evidenced the crucial role of AMPs in promoting this process by multiple mechanisms encompassing modulation of cytokine production, cell migration, and proliferation (186). As an example, HBD-2, which is induced in human skin wounds upon activation of the epidermal growth factor receptor (EGFR) (187), increases cytokine production and migration of keratinocytes, the most abundant cells in the epidermis (188). The production of LL-37 is also enhanced upon wound formation and in an ex vivo human skin wound healing model (141). Lately, Esc peptides have also been found to stimulate migration of human keratinocytes through a mechanism that implies EGFR activation and involvement of metalloproteinase MMP-9 (189). Remarkably, the beneficial effects of these AMPs on wound healing are not limited to the skin but extend to other tissues exposed to the external environment, such as the corneal epithelium, promoting healing at the ocular surface (139, 140). This makes these peptides excellent and valuable templates for producing new multifunctional drugs potentially useful for injured lung epithelium.

8. ANTIMICROBIAL PROTEINS AND PEPTIDES IN HUMAN LUNG DISEASES

Skin and mucosal surfaces, such as the respiratory tract (190), are usually considered natural barriers where a host encounters pathogens and microbial infections. In healthy individuals, these infections are primarily controlled by the innate defense responses that allow the host to maintain its homeostasis and integrity (191) (FIGURE 8). Studies using knockout mice have confirmed the crucial role of many AMPs in limiting microbial proliferation at various anatomical locations and the spread of infections.

FIGURE 8.

The innate defense mechanisms of the human lung. The airway surface liquid is comprised of the mucus and periciliary layers, which contain mucins and ciliated epithelial cells that allow for the self-clearance of foreign agents. The secretion of antimicrobial molecules is the first line of defense against respiratory pathogens. AMPPs, antimicrobial proteins and peptides.

8.1. Upper Respiratory Tract Infections

Acute upper respiratory tract infections (URTIs), including the common cold (rhinovirus), nasopharyngitis, pharyngitis, otitis media, and tonsillitis, are prevalent health care burdens, costing billions of dollars annually. Viral agents predominantly cause URTIs, but some bacterial species such as Haemophilus influenzae, Moraxella catarrhalis, S. aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are commonly associated with sinusitis, which is often observed along with the common cold (192). Human respiratory syncytial virus (RSV) is an RNA virus from the Paramyxoviridae family and impacts humans worldwide (193). RSV is one of the leading viruses that infect children but also affects adults and the immunocompromised. RSV manifests as a range of various symptoms and most commonly presents as a URTI but may progress to a lower respiratory tract infection (LRTI) with small airway obstruction (193). IAV is another RNA virus that may present as a mild URTI or cause infection of the lower respiratory tract severe enough to induce alveolar damage and lung injury (194). It is understood that human α- and β-defensins have antiviral activity against both enveloped and nonenveloped viruses, their glycoproteins, glycolipids, and capsids (195). For example, an increase in HBD-2 and HBD-3 expression correlated with stimulated release of IL-8 following rhinovirus-16 strain (RV16) infection of bronchial epithelial cells in vitro (196). In addition, human neutrophil peptides HNP1 and HNP2 have been shown to enhance the uptake of IAV by neutrophils and promote neutrophil-mediated IAV clearance (63).

In contrast, SP-D may enhance IAV uptake by neutrophils and stimulate viral aggregation (63). Antimicrobial surfactant proteins SP-A and SP-D show a range of human defense against viruses, including IAV, RSV, and SARS coronavirus (197). Both SP-A and SP-D may inhibit viral infection by independent mechanisms of binding to IAV or RSV, and viral titers and inflammation were shown to increase in both SP-A^−/− and SP-D^−/− mice (197). Also, single-nucleotide polymorphisms in Thr11 and Ala160 of SP-D are linked to less severe or reduced risk of RSV infection (197). SP-D inhibition of SARS coronavirus infectivity has been demonstrated in vitro, and elevated serum levels of SP-D are associated with patients with SARS-induced pneumonia, with a risk of alveolar damage (197).

The antimicrobial activity of SPLUNC1 has been demonstrated in several in vitro and in vivo experimental models. For instance, 2 h of incubation of M. pneumoniae with mouse SPLUNC1 at various concentrations led to a dose-dependent inhibition of Mp proliferation and a reduction in the release of IL-8 (198). Recombinant orthologs of human SPLUNC1 have also shown antimicrobial activity against P. aeruginosa and H. influenzae in vitro (199, 200). CCSP-SPLUNC1 transgenic mice characterized by overexpression of human SPLUNC1 driven by the Clara cell secretory protein (CCSP) promotor were generated to assess the in vivo antimicrobial activity of SPLUNC1, localized from airway epithelial secretory cells. After intratracheal administration of P. aeruginosa and K. pneumoniae in wild-type or transgenic mice, it was observed that CCSP-SPLUNC1 mice showed decreased susceptibility to bacterial infection, reduced concentration of inflammatory cells and neutrophils, and lessened lung inflammation and injury compared to WT mice (68). Pathogenic colonization in the upper airways may be deadly, especially to vulnerable populations, when the innate immune response is unsuccessful at clearing the infection, leading to progressive infection into the lower respiratory tracts with heightened mortality and morbidity.

8.2. Lower Respiratory Tract Infections

Acute lower respiratory tract infections, including bronchitis, bronchiolitis, bronchopneumonia, and tracheitis, occur in regions below the larynx (201). Chronic, frequent LRTIs may contribute to the development of pulmonary fibrosis, damaged and widening of the airways, defined as bronchiectasis, and overall impaired lung function (201). Globally, nearly 500 million absolute episodes of LRTIs occur, contributing to over 2 million deaths in 2019 (202). The most prevalent LRTIs in children include acute bronchiolitis and community-acquired pneumonia (201, 203). The predominant viral agents in LRTIs include adenovirus, coronavirus, influenza, human metapneumovirus, parainfluenza, rhinovirus, and RSV, whereas H. influenzae, Chlamydia pneumoniae, M. catarrhalis, M. pneumoniae, and S. pneumoniae are the most common bacterial pathogens associated with LRTIs (201). It is evident that several antimicrobial molecules play important roles in the innate immune response of the lower respiratory tract (TABLE 1). HBD-4 production is localized to the bronchial region by bronchial epithelium and has been demonstrated to have antimicrobial activity against P. aeruginosa (232). LPS-treated small airway epithelial cells resulted in increased secretion of HBD-4 compared to untreated control cells (232).

Table 1.

Antimicrobial molecule expression associated with pulmonary disease

Pulmonary Disease	AMP Expression	Antimicrobial Protein Expression	Reference(s)
Chronic obstructive pulmonary disease	↑ LL-37, HNPs; ↓ hBD-2	↑ LL-37, SP-A; ↓ SPLUNC1 by NE, lysozyme, SLPI	(204–208)
Cystic fibrosis	↑ LL-37, ↓ hBD-2	↑ Lactoferrin, lysozyme, SLPI; ↓ SPLUNC1, SP-A/SP-D	(73, 209–213)
Allergic asthma	↑ HNPs, LL-37	↑ SPLUNC1, lactoferrin,SP-A/SP-D; ↓ SLPI	(214–220)
Lung cancer	↑ LL-37, hBD-1, hBD-2	↑ SPLUNC1, SP-A, SLPI	(221–226)
SARS-CoV-2	↑ HNPs	↑ SP-D	(197, 227)
Bronchiectasis	↑ LL-37	↑ Lactoferrin	(228)
Influenza A virus	↑ HNP1, HNP2	↑ SP-D	(63, 229)
Tuberculosis	↑ LL-37, hBD-2, hBD-3	↑ SP-A	(230, 231)

AMP, antimicrobial peptide; HBD, human β-defensin; HNP, human neutrophil peptide; NE, neutrophil elastase; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SLPI, secretory leukocyte proteinase inhibitor; SPLUNC1, short-palate, lung, and nasal epithelial clone 1; SP-A, surfactant protein A; SP-D, surfactant protein D.

Increased levels of proinflammatory cytokines and neutrophils were associated with the severity of SARS-CoV-2 infection, with significantly higher levels of α-defensins (DEFA1) linked to patients with mild disease severity (227). Expression of neutrophil secretory proteins DEFA1, calprotectin, and myeloperoxidase (MPO) decreased to normal levels in healthy subjects for patients recovering from the infection (227). Based on the marked increase in DEFA1 levels within the early course of disease onset, it is possible that these AMPPs could be used to predict and monitor disease severity (227). The heightened expression in genes related to neutrophil activity and the primary defense mechanism of neutrophil degranulation in SARS-CoV-2 are likely reasons for the elevated levels of HNPs in patients with the disease (233). It has also been observed that the host antiviral defense response in the early stages of SARS-CoV-2 infection involves heightened expression of azurophilic granule proteins proteinase 3, MPO, elastase, and cathepsin G, leading to potential tissue damage stimulated by the release of antiviral enzymes through neutrophil degranulation as part of the mechanism of viral clearance (233).

Persistent LRTI can lead to the lung disease bronchiectasis, which involves thickening and dilating of the bronchi due to repeated infection, chronic inflammation, and frequent exacerbations (228). Lactoferrin showed high expression levels and was detected in the sputum and serum of patients with mild, moderate, or severe bronchiectasis. At the same time, elevation in LL-37 concentrations was associated with disease severity levels (228). In addition, levels of proinflammatory mediators LL-37, lactoferrin, and lysozyme in sputum appeared to be positively correlated with elastase activity (228). SP-D binds to carbohydrates of IAV proteins in a calcium-dependent manner, and levels are shown to increase upon viral infection (229). In patients with fatal influenza A subtype H5N1 infection, SP-D levels decrease, likely contributing to epithelial damage, viral pneumonia, and acute lung injury due to a deficiency in antiviral defense (229).

Adenovirus (AdV) is a DNA virus that most prevalently impacts the upper and lower respiratory tracts and is most common in young children or the immunocompromised (234). The hydrophobic residues on the structure of human α-defensin 5 (HD5) and HNP1 stabilize the structure of the peptides and aid in binding to human AdV viruses, thereby facilitating viral neutralization (235). RSV is the main viral agent in the development of bronchiolitis, which is a leading cause of infant hospitalization in the United States (236). LL-37 levels were lower in infants with severe bronchiolitis or those with high rates of intensive care and prolonged hospital stay (236). These observations further support the antiviral and immunomodulatory activity of LL-37 in the lung as an important host defense mediator.

8.3. Cystic Fibrosis

CF is an autosomal recessive genetic disease most prevalent in Caucasians caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to both acute and chronic lung infection by respiratory pathogens, resulting in airway inflammation and progressive decline in pulmonary function (237) (FIGURE 9). The leading pathogen in the colonization of chronic CF infection is P. aeruginosa, which has shown enhanced biofilm formation and antimicrobial resistance within the acidic CF airway environment (238). However, the acidification of the CF airway has been shown to inhibit AMP activity, thereby reducing the eradication of bacteria colonization (3). The acidic microenvironment also affects the antibiotic susceptibility and biofilm formation of P. aeruginosa (238). The expression of innate AMPs is highly influential in lung disease progression (TABLE 1). In CF, there is an increase in LL-37 and a decrease in HBD-2 levels, directly impacting host disease severity (209). Lung deterioration in patients with mild CF is associated with increased LL-37 and decreased HBD-2 bronchoalveolar lavage fluid (BALF) levels (209). Increased levels of LL-37 within the airways contribute to inflammatory cell recruitment, thereby contributing to the enhanced inflammation of CF airways and subsequent deterioration of pulmonary function (209). In contrast, a decrease in HBD-2 expression observed with advanced CF and other inflammatory lung diseases likely promotes and sustains microbial colonization (3, 209). For patients with severe CF, the expression of SPLUNC1 is decreased, especially during acute pulmonary exacerbations, resulting in increased morbidity (73). Furthermore, the acidic airway surface liquid of CF airways inhibits SPLUNC1 activity, promoting further colonization by Gram-negative bacteria and worsening disease severity (239).

FIGURE 9.

Physiology of chronic respiratory diseases. AMP, antimicrobial peptide; ASL, airway surface liquid; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; COPD, chronic obstructive pulmonary disease.

In addition, plasma and serum levels of the cationic glycoprotein lactoferrin are significantly elevated in CF patients (210). A CF murine infection model in which C57BL/6 CFTR-deficient and WT mice were infected with P. aeruginosa through intratracheal inoculation showed that aerosolized bovine lactoferrin treatment led to a significant decrease in lung bacteria load and leukocyte invasion in CF compared to WT mice (240). Levels of lysozyme in the serum of CF patients are also significantly increased compared to healthy individuals, without evidence of in vitro antibacterial activity against CF isolates of Staphylococcus aureus or P. aeruginosa (211).

Additionally, lower levels of pulmonary surfactant proteins SP-A and SP-D measured in the BALF of young CF patients are correlated with increased inflammation and high neutrophil infiltration in response to bacterial infection (212). It is possible that chronic inflammation decreased SP-A and SP-D synthesis, elevated neutrophil levels, and increased macrophage protease support the decline in collectin levels in CF airways (212). The balance between levels of proteases versus antiproteases is influential in modulating innate host defense. Higher levels of proteases such as neutrophil elastase (NE) and antiproteases, including SLPI, were detected in lower airways (sputum) versus upper airway (nasal lavage) in patients with CF, which may enhance tissue damage and decline in lung function (213).

8.4. COPD

Similar to CF, lower expression of HBD-2 is observed in smokers (a risk factor for lung disease), patients with COPD, and asthma (204) (FIGURE 9). It is suggested that smoking promotes pathogenic infection by inhibiting the innate immune system and suppressing the activity of AMPs (204). For instance, in patients with community-acquired pneumonia, levels of HBD-2 were significantly lower for smokers, with a decline in antimicrobial activity (204). In addition, smoking significantly impacts the expression or activation of TLRs and HBD-2 in the airways dependent on their localization, with an increase observed for both TLR4 and HBD-2 in the distal airway epithelium and peripheral lung tissues of smokers with COPD (241, 242). TLR4 is activated by host exposure to certain environmental stimuli and the endotoxin LPS found on the Gram-negative bacterial membrane, which triggers the secretion of HBD-2. However, the central airways showed reduced HBD-2 epithelial expression for smokers with COPD compared to smokers without COPD and former smokers with COPD (241). Reduced HBD-2 in the central airway epithelium of smokers may promote the development of COPD and chronic bacterial colonization, leading to chronic airway inflammation and eventual damage to lung tissue (241, 243, 244).

In addition, LL-37 localized from neutrophils and airway epithelial cells shows heightened expression in smokers and COPD patients, which promotes fibroblast collagen synthesis and the density of the airways (205). Therefore, elevated LL-37 expression with smoking is tightly linked to small airway remodeling in patients with COPD (205). Furthermore, levels of HNPs, NE, Interleukin-8 (IL-8), and Metalloproteinase-9 (MMP-9) measured from human sputum are biomarkers used to monitor airway inflammation and COPD progression (206). Higher levels of HNPs and NE have been observed in patients with severe airway obstruction and those with mild COPD. In contrast, levels of HNP, NE, IL-8, and MMP-9 were significantly increased for patients with COPD compared to those who smoked without airway obstruction (206). Prolonged smoking and repeated exposure to noxious chemicals induce chronic inflammation in the airways and lung parenchyma. This leads to tissue remodeling and airway obstruction similar to features observed in COPD, making it an important risk factor for many chronic respiratory diseases (245).

NE is elevated in the lungs of COPD subjects, which, along with other inflammatory mediators, promotes acute exacerbations of COPD (246). It has been shown that degradation of SPLUNC1 by NE increases airway susceptibility to pathogenic infection by nontypeable Haemophilus influenzae (NTHI), the leading bacterial agent in acute exacerbations of COPD (246). Furthermore, sputum samples from COPD patients collected over a 6-yr time period showed a significant decrease in lysozyme production during exacerbations due to NTHI compared to baseline levels (207). In addition, decreased SLPI and increased LL-37 levels were associated with exacerbation due to NTHI and Moraxella catarrhalis infection (207). These data provide evidence for heightened LL-37 expression following bacterial infection, whereas other lung antimicrobial proteins, including lysozyme and SLPI, are decreased. Most substantial fluctuations in the regulation of lung antimicrobial molecules are observed during periods of COPD exacerbations, compared with bacterial colonization (207). Another comparative yearlong study found that plasma SP-A levels showed significant increases in the healthy smoker and exacerbated subject populations compared to those with stable COPD (208). SP-A of lung surfactant lines the alveolar epithelium, which is a target for exposure to inhaled toxins, such as cigarette smoke (208). The pulmonary inflammation caused by smoking and COPD exacerbations leads to excessive concentrations of SP-D being released into the blood.

8.5. Allergic Asthma

The expression of HNPs also has roles in the airway inflammation and airflow limitation observed in various asthma phenotypes (FIGURE 9). The proinflammatory effect of HNPs suggests their role in allergic asthma exacerbation. It has been reported that with allergen stimulus patients with allergic asthma showed a significant increase in the secretion of HNPs in venous blood leukocyte culture compared to healthy individuals (214). In addition, after stimulus with phorbol myristate acetate, individuals with asthma and a respiratory tract infection showed significantly higher HNP levels than those with asthma without infection (214). Along with antimicrobial activity, HNPs are involved in epithelial wound healing and regulation of the inflammatory response by stimulating the release of chemokines and cytokines, induction of neutrophil chemoattractants from alveolar macrophages, and upregulating histamine synthesis and mucin expression by mast cells and the lung epithelium, respectively (165, 247–249). Hypersecretion of mucus in the airway epithelium is critical to airway remodeling observed in asthma patients relative to healthy individuals, and this feature has been a recent target for clinical management of the disease (250). Asthma exacerbations are commonly triggered by rhinovirus (RV) infection, which involves increased release of neutrophil chemokines such as CXCL8/IL-8 and enhanced secretion of HNP1–3 in the lower airways (215). Asthma exacerbations triggered by RV lead to chemoattraction of neutrophils in the airway and increased release of HNPs, which both act to defend against viral infection but also enhance neutrophilia.

LL-37 has a proinflammatory role in allergic asthma by recruiting immune and mast cells. Also, it triggers the release of cysteinyl leukotrienes from eosinophils, leading to further bronchus constriction (216). Eosinophilic LL-37 expression and secretion are significantly higher in asthma patients than in healthy individuals (216). Respiratory infections stimulate LL-37 expression, and more recently identified human rhinovirus strains more prevalently show an association with asthma onset and exacerbations (251). In addition, SPLUNC1 detected in the sputum of asthma patients is decreased compared with healthy individuals and contributes to two notable features of the disease: exaggerated smooth muscle contraction and airway hyperresponsiveness (217). It is understood that SPLUNC1 acts as an epithelium-derived smooth muscle relaxing factor modulating airway smooth muscle calcium influx, thereby altering the degree of contraction (217).

Furthermore, in stable asthma patients, lactoferrin levels in BALF significantly increase compared to healthy individuals. This is likely due to increased secretion by stimulated airway epithelial cells and potentially the degranulation of neutrophils and eosinophils (218). Lactoferrin demonstrates protective effects against airway hyperresponsiveness, reduces lung inflammation and damage, promotes dendritic cell maturation, and reduces Th2-related cytokine production associated with asthma in ovalbumin-sensitized BALB/c mice (252). Similarly, collectins SP-A and SP-D ameliorate allergic inflammation through downregulating eosinophil-driven inflammation and direct binding with inhaled allergens, including pollen grains, dust mites, and Aspergillus fumigatus, thereby preventing allergen-IgE interaction, uptake of allergens by dendritic cells, and prevention of asthmatic reaction (219). Several in vivo studies show SP-A and SP-D upregulation in the BALF of allergen-sensitized mice (219). In contrast, severe asthma patients show a decrease in SLPI and an increase in IFN-γ (Th1) immune response expression, and this inverse relationship has been shown to contribute to airway hyperresponsiveness (220). SLPI inhibition of proteases synthesized by mast cells, including tryptase, prevents activation of protease-activated receptor 2, which is known to promote a neurogenic mechanism of airway hyperresponsiveness (253).

8.6. Lung Cancers

AMPs have also shown potential roles in several human cancers as well as in both anticancer and tumorigenic effects. For instance, the roles of LL-37 in immune response and pro- or anticancer effects are highly tissue specific. LL-37 is upregulated in expression, suggesting protumorigenic effects in breast, lung, ovarian, pancreatic, and prostate cancers (221, 254–256) (FIGURE 10). However, LL-37 is significantly downregulated in hematologic, colon, and gastric cancers and oral squamous cell carcinoma (254, 255, 257–259). Myeloid cells such as macrophages synthesize LL-37, which is critical in regulating tumor cell proliferation through inflammatory cell recruitment and shows increased expression within lung cancer tissues (222, 260). It has been shown that activated secretion of LL-37 by the inflammatory cytokine interleukin-33 (IL-33) activates the ST2 signaling pathway in mouse monocyte macrophages and induces the expression of interleukins IL-6 and IL-1β, leading to destructive inflammation and proliferation of Lewis lung carcinoma cells of mice (221).

FIGURE 10.

Antimicrobial activity associated with lung cancers. HBDs, human β-defensins; NSCLC, non-small cell lung cancer; SLPI, secretory leukocyte proteinase inhibitor; SPLUNC1, short-palate, lung, and nasal epithelium clone; SP-A, surfactant protein A.

Similar to LL-37, HBD-1 shows a tissue-dependent role in human cancers. Serum HBD-1 and HBD-2 levels are heightened in patients with lung cancer compared to healthy individuals (FIGURE 10). Defensins are known to be induced by and stimulate cytokine activity, which is important in tumor tissue angiogenesis and growth. Levels of HBDs are elevated in early versus advanced-stage lung cancer and may serve as an effective tumor marker for monitoring cancer progression (223). SPLUNC1 was discovered as a novel tumor marker for lung cancer, with significant expression in adenocarcinomas, bronchioalveolar carcinoma, large cell carcinoma, and mucoepidermoid tumors (224) (FIGURE 10).

Furthermore, significantly elevated levels of SP-A are associated with malignant pleural effusions in non-small cell lung cancer (NSCLC) (225). SP-A is an indicator of cellular damage, which supports its heightened concentration in damaged lungs with NSCLC progression (225). In addition, SP-A expression is significantly elevated in the serum of patients with pulmonary tuberculosis compared to healthy control subjects, likely because of enhanced vascular permeability associated with lung injury, which similarly happens in tumorigenesis (231). Active Mycobacterium tuberculosis (Mtb) infection and lung cancer share similar disease pathology, and it is even suggested that Mtb may increase the potential for lung cancer development (261). Colonization of Mtb infection promotes the secretion and antimicrobial response of LL-37 and defensins HBD-2 and HBD-3 (230).

Upregulation of SLPI is also observed in NSCLC, with significantly higher expression in the serum of lung cancer patients respective to healthy control subjects, especially with later more progressive stages (III and IV) (226). The roles of SLPI in tissue regeneration by induction of tissue regenerating factor hepatocyte growth factor (HGF) and ameliorating inflammation through suppression of NF-κB support its upregulation during tumorigenesis (226).

9. INTERPLAY OF ANTIMICROBIAL PROTEINS AND PEPTIDES AND LUNG MICROBIOME IN HEALTH AND DISEASE

The upper respiratory tract, including the nasal cavity and oropharynx, is normally colonized by bacteria such as Moraxella, Staphylococcus, Corynebacterium, Haemophilus, Streptococcus, and Streptococcus species (262). In contrast, the lower respiratory tract, which consists of the bronchi, bronchioles, and lungs, has a relatively low biomass, playing a crucial role in exchanging oxygen and carbon dioxide. Therefore, it must maintain a low bacterial burden and homeostasis through actions of mucociliary clearance, coughing, and innate and adaptive immune responses (263). Once bacteria colonize the respiratory tract, they rapidly form sessile communities (264). In a healthy lung, a wide range of AMPs and proteins are released into the lumen of the airways to fight infections, along with the physical removal of inhaled microbes by mucociliary clearance and the production of chemokines and proinflammatory cytokines by immune cells. The expression and existence of the lung AMPs and proteins interact with commensal microbes and shape the lung microbiome.

Several studies have indicated that exposure to microbes begins early in life and has important implications for immune system development (265). As neonates grow, their lung microbiome becomes more diverse, consisting of a mixture of oral commensals. These changes in the airway and lung microbiome contribute to lower airway immune maturation and have been linked to the development of respiratory conditions like asthma (263, 266). Microbial dysbiosis, characterized by a reduction in bacterial diversity, is implicated in the pathogenesis of various lung diseases like CF, COPD, and idiopathic pulmonary fibrosis (267). Still, it is unclear whether the imbalance in the microbial community causes these diseases or is the result of the disease process itself (268–273). For example, in the case of CF, acidification of the lung environment inactivates lung AMPs like LL-37. At the same time, the higher production of host proteases gives rise to tissue injury, compromising the antimicrobial activity of AMPs. Vitamin D₃ has been shown to regulate the expression of LL-37 through specific elements in the CAMP gene promoter region (274) in various types of cells, e.g., monocytes, keratinocytes, neutrophils, and epithelial cells, and this expression is enhanced in the presence of bacterial infection (275). Mechanistically, LL-37 binds to and activates multiple receptors, leading to the transcription of proinflammatory genes. LL-37 is known to have protective effects against invading pathogens in the mucosa (276), but it is also implicated in COPD. Several studies have shown that the production of LL-37 is elevated in COPD patients compared to healthy individuals. On the contrary, COPD patients with vitamin D deficiency have significantly lower LL-37 levels (277–280). Changes in AMPs have been observed in dysbiosis, with increasing levels of LL-37 and decreasing amounts of lysozyme during bacterial infection of the airways.

In diseases like asthma, where airway inflammation is a key feature, dysregulated AMP expression may exacerbate symptoms. Individuals with sarcoidosis exhibit reduced levels of AMPs (SLPI and hBD-1) in their respiratory airways, in parallel with an imbalance in the microbial composition, dominated by fungi like Aspergillus (281). In the context of exposure to cigarette smoke, research studies have demonstrated that low levels of LL-37 are associated with a decrease in forced expiratory volume in 1 s (FEV₁), which is particularly significant in individuals with deficient vitamin D₃ production (282). However, it is still unclear whether enhanced susceptibility to bacterial infections leads to inflammation and subsequent tissue damage in individuals with low LL-37 levels or if the peptide directly impacts lung function. Recent studies support the former hypothesis, indicating that inhibition of LL-37 promotes bacterial infection, dysbiosis, and inflammation (283). On the other hand, earlier in vitro studies lean toward the latter hypothesis, showing that the mouse ortholog of cathelicidin, mCRAMP, reduces the severity of emphysema, likely because of its anti-inflammatory and healing properties (284).

Imbalances in the airway microbiome can lead to gastrointestinal issues such as irritable bowel disease (285). However, this is presumably due to antibiotics and other therapies for chronic respiratory diseases, which disrupt the gut microbiome, causing related conditions. Emerging literature suggests that using prebiotics and probiotics to restore the normal gastrointestinal microbiome can have therapeutic benefits in various respiratory diseases, including lung cancer, asthma, tuberculosis, and CF. This concept is known as the “gut-lung axis” (286). Furthermore, some papers have highlighted that exposure of neonatal mice to high levels of oxygen can reduce the expression of AMPs in their intestine and modify the gut microbiota composition. However, suppose that oral supplementation of the antibacterial lysozyme is given to neonatal mice exposed to high oxygen levels. In that case, their microbiota is less affected by oxygen exposure, and the animals experience less lung injury (287). The complexity and diversity of microbiomes have shown how they can be a valuable source of novel bioactive compounds for biotechnological/therapeutic use (288) and contribute to defense barriers against infectious agents, including SARS-CoV-2 (289).

The interplay between AMPs and airway and lung microbiome is critical in lung health and disease. Further research into this complex relationship may uncover novel therapeutic approaches for preventing and treating lung diseases.

10. OPTIMIZATION OF ANTIMICROBIAL PROTEINS AND PEPTIDES

Despite natural AMPs having appealing antibiotic properties, several disadvantages must be overcome for their development as new therapeutics. Among these are the susceptibility to proteases, potential cytotoxicity toward mammalian cells, and high costs of large-scale production. As explained by Bee Ha Gan and colleagues (290), to improve the biological properties of AMPs and resistance to proteolytic degradation, various chemical approaches can be followed to modify naturally occurring AMPs by substitution of one or more residues of the AMP template with natural or noncoded l- or d-amino acids; by COOH-terminal amidation, NH₂-terminal acetylation, cyclization, PEGylation, or lipidation; as well as by the synthesis of hybrids or dendrimeric molecules. The following sections describe the major known modifications to enhance and optimize AMPs.

10.1. Simple Amino Acid Substitutions

The most straightforward strategy to improve the antimicrobial activity and selectivity of AMPs consists of substituting one or more residues with other proteinogenic l-amino acids. This is the case of pexiganan, the chemically modified isoform of the amphibian AMP magainin II, where the replacement of neutral and anionic residues with cationic amino acids significantly improved the antimicrobial efficacy of the peptide (291).

10.2. l-to d-Amino Acid Replacement

Numerous studies have shown how l- to d-amino acid replacement can retain or even improve the antimicrobial activity of the original peptide while increasing its biostability. As an example, the substitution of Leu¹⁴ and Ser¹⁷ in the frog skin-derived Esc(1–21) with the corresponding d-enantiomers conferred the peptide’s higher antibiofilm activity and in vivo antipseudomonal efficacy than the parental all-l isoform, while being more resistant to human and bacterial elastases (178). Furthermore, Di and coworkers (292) designed a substitution of d-Val for l-Val of an engineered cationic AMP WLBU2 named D8, which significantly increased resistance to protease enzymatic degradation, higher activity against bacteria in biofilm, and drastically lowered toxicity to erythrocytes and white blood cells. The improved safety in mice and the demonstrated efficacy in attenuating P. aeruginosa infection in mouse lungs resulted in a considerably increased therapeutic index of >140 that warrants clinical exploration by aerosolized delivery to mitigate MDR pathogen-related respiratory infections (292).

10.3. Incorporation of Nonnatural Amino Acids

These chemical modifications can increase the stability of proteases and the antimicrobial efficacy of AMPs. For example, ornithine can replace Lys to change the number of side chain methylene groups, and experiments performed with temporins have indicated that the presence of the amine group of ornithine instead of a guanidinium group at position 11 reduced the hemolytic effect of the original peptide without significantly altering its antimicrobial activity (293). The helicogenic alpha aminoisobutyric acid (Aib) can also be used to increase the alpha-helical content of AMPs and to expand the antibacterial activity spectrum. Casciaro and colleagues (294) demonstrated how the insertion of three Aib into the sequence of Esc(1–21) made this latter more stable and efficient against Gram-positive bacteria.

10.4. COOH-Terminal Amidation and NH₂-Terminal Acetylation

These changes are other common methodologies used to ameliorate the stability of either naturally occurring or synthetic peptides by blocking the activity of carboxypeptidase and aminopeptidases, respectively. Ovchinnikova and colleagues (295) demonstrated that N-acetylation and C-amidation of tachyplesin I significantly improved its proteolytic stability. Note that COOH-terminal amidation is a common posttranslational change of most amphibian skin AMPs to enhance their cationicity by neutralizing the negative charge of the carboxyl group and, therefore, their resistance to skin carboxypeptidases.

10.5. COOH or NH₂ Terminus Peptide Lipidation

The peptide lipidation generally results in improved activity against fungal species. Shai and coworkers (105) designed a series of lipopeptides containing an NH₂-terminal fatty acid chain with 12, 14, or 16 carbon atoms and the amino acid sequence KXXK-NH2 (where X was equal to l-leucine, alanine, lysine, glutamic acid, or glycine), each peptide bearing a single d-amino acid. Biological analysis indicated that although these molecules contained a short sequence, they possessed potent antimicrobial activity and increased target cell selectivity toward fungi without being hemolytic. Also, in the case of temporins, it was demonstrated that the length of the aliphatic moiety is a crucial factor in controlling target cell specificity as well as the oligomeric state of peptides either in aqueous solution or in a membrane-mimicking environment (296).

10.6. Peptide Cyclization

Several naturally occurring cyclic AMPs have already been approved by the Food and Drug Administration and are used in the clinic. Cyclization can be obtained through side chain-side chain, head-to-tail, backbone-backbone, or side chain-backbone bonds. Cyclization generally confers the peptides’ more favorable antimicrobial properties due to better proteolytic stability and conformational rigidity compared to the linear AMPs. Hancock and colleagues (297) used the side chain-to-tail cyclization to make the innate defense regulator peptide (IDR)-1018 more efficient in reducing inflammation and treating skin infections.

10.7. Single Hybrid Peptide with AMP Fragment Combination

This approach is another promising alternative to ameliorate AMPs’ biological properties. Shan and coworkers (298) developed hybrids of the antibiofilm peptide FV7 with the AMPs LL-37, magainin II, and cecropin A with higher membranolytic activity against Gram-negative and Gram-positive bacteria compared to the parent AMPs. Cecropin-melittin hybrids have the same antibacterial potency as melittin but with reduced hemolytic activity (299). Cell penetrating peptides (CPPs) are a group of short, structurally different peptides (not longer than 35 amino acids) capable of translocating across cell membranes without provoking membrane damage. Matsuzaki and colleagues studied a disulfide-linked chimera between PGLa and magainin II (310). They discovered a synergistic antibacterial effect attributed to the formation of a heterodimer between the two peptides. Indeed, either the mixture of the two peptides or the hybrid had a stronger antibacterial activity against E. coli and Staphylococcus epidermidis compared to the single peptides alone, but the hybrid was more cytotoxic toward mammalian cells (300, 301).

10.8. Dendrimer Peptides

Dendrimers have attracted considerable attention as a novel class of synthetic AMPs, and the scaffold of peptide dendrimers provides several benefits compared to linear peptides, including higher stability and a reduced risk of peptide aggregation during the synthesis process. Pini’s and Bracci’s groups assembled four copies of the peptide KKIRVRLSA to a three-lysine core. This compound, M33, exhibited antimicrobial efficacy in either the free or pegylated form at the COOH terminus and the ability to bind LPS, preventing septic shock in vivo (302).

10.9. Peptidomimetics

Peptidomimetics are molecules that mimic the biological effect of AMPs while being stable to enzymatic degradation without resembling natural peptide structures (303).

10.10. Tertiary Structure Modifications

Part of the natural antimicrobial arsenal includes a number of small- and medium-sized proteins that act via an ordered tertiary structure. Cabalteja and coworkers (304) designed variants of lasiocepsin, a 27-residue disulfide-rich AMP found in bee venom that adopts a compact tertiary fold. The modified peptide variants were extensively examined for their antimicrobial activity against a large panel of many ESKAPE pathogens. These experiments showed effective functional mimicry by some artificial backbone analogs. In particular, tuning the type and density of modifications present in a loop connecting the two helices in the tertiary fold is beneficial, with Gly → d-Ala substitution more compatible with the native fold and function than α → β3 replacement. The results suggest backbone engineering as a way to modify peptide and protein functions by exerting site-specific control over the protein folded structure (304).

11. LUNG ANTIMICROBIAL PROTEINS AND PEPTIDES AS CLINICAL THERAPEUTICS

Despite the significant roles of natural lung antimicrobial proteins and AMPs in protecting the host from many lung illnesses, as described above, the therapeutic applications of utilizing these AMPPs for treating pulmonary diseases are surprisingly sparse. The optimization strategies described in sect. 10 could generate a new generation of AMPs to be used as novel therapies for infectious and noninfectious lung diseases, with improved efficacy and lower propensity for resistance and host toxicity. Selected examples of clinical trials targeting pulmonary diseases with AMPPs include investigators trying to determine the association of cathelicidin and vitamin D levels with COPD (NCT05431218) and others evaluating the links between trefoil family factor (TFF) peptides and COPD and asthma (NCT00839735). The safety and tolerability of hLF 1–11, a fragment of human lactoferrin, were also evaluated in neutropenia patients as autologous hematopoietic stem cell transplant recipients (NCT00430469). Currently, only AMPs such as polymyxins, nisin, gramicidin, daptomycin, and melittin are in clinical use as an alternative to antibiotics based on their antimicrobial potency. Although there has been a considerable rise in the number of clinical trials for synthetic AMPs, very few have been targeting lungs or approved as therapeutic candidates, such as polymyxin. Polymyxins are cationic cyclic polypeptides naturally produced by Gram-positive Paenibacillus polymyxa. Colistin, or polymyxin E, is a last-resort antibiotic reserved for treating complex MDR infections. Unfortunately, increasing use of last-line antibiotics such as colistin has shown an association with the emergence of resistant strains during therapy, highlighting the urgent need for novel antimicrobials.

Novel peptides Esc(1–21), or Esc peptides, have shown to be promising agents in combating CF pathogens, especially P. aeruginosa, without damaging the lung epithelia or inducing inflammatory cell recruitment in lung alveoli of treated mice, compared to vehicle control mice (305). Esc peptides have also been shown to activate with the CFTR protein, acting as potentiators of its mutated form and altering ion currents regulated by the protein, with a promising role in blocking the persistent lung infection predominantly established by P. aeruginosa (306). Another novel AMP, A4-short, derived from a functional motif “α4” of lung-specific host defense protein SPLUNC1, possesses potent antimicrobial activity, with the most common minimum inhibitory concentration (MIC) of 2 µM against a panel of 39 MDR ESKAPE pathogens (307). Red blood cell (RBC) lysis and peripheral blood mononuclear cell (PBMC) toxicity by A4-short were negligible, similar to the nontoxic properties of the parent peptide α4. A4-short showed favorable in vitro bactericidal and antibiofilm activity with very low toxicity. Importantly, A4-short was more efficacious in the murine pneumonia model than untreated control mice with decreased bacterial lung burden (>2 logs) after P. aeruginosa infection (307).

As novel synthetic AMPs continue to be designed, critical limitations regarding their use in preventing and treating pulmonary disease are mainly peptide stability, bioavailability, toxicity, and proteolytic degradation (123). One successful example is demonstrated by a newly developed AMP named D8 with a designed substitution of d-Val for l-Val of engineered cationic AMP WLBU2, which resulted in increased resistance to protease enzymatic degradation, higher activity against bacterial biofilm, noticeably lower cytotoxicity to erythrocytes and white blood cells, and increased safety in mice compared to the original WLBU2 (292). Significantly, direct airway delivery of D8 revealed a therapeutic index of >140 compared to <35 for WLBU2 in treating P. aeruginosa–induced respiratory infections (292).

Novel antimicrobial molecules are being designed as attractive alternatives or combined with chemoimmunotherapy models for lung cancer treatment because of their selective targeting of cancer cells apart from healthy cells (308). These AMPs are usually not human derived but are discovered from marine animals and skin secretions of amphibians or structurally modified for the enhanced killing of NSCLC cells (308). A recent push has been toward using engineered peptides with antimicrobial activity to target and destroy cancer cells. One example is the model AMP MAD1 (myco-membrane associated disruption 1), originally showing selective targeting activity against membranes of mycobacteria, which was subsequently engineered into two anticancer peptides, DAP1 and DAP2 (de novo designed anticancer peptide) and successfully shown to target and kill or impede the growth of human ovarian carcinoma cells (309). Therefore, the clinical importance of AMPs as dual-therapeutic agents targeting both pathogens and cancer cells should be further explored.

One of the major gaps in knowledge regarding antimicrobial peptides and proteins for clinical application is the lack of comprehensive studies of how and why these antimicrobial molecules work in addition to direct killing. Many AMP researchers who conduct in vitro testing lack the rigor to select AMPs likely to display in vivo efficacy. One consideration is to use physiologically relevant in vitro conditions such as blood and eukaryotic cell culture media, which contain many of the physiologically relevant cations challenging to classical AMPs. Another limitation is that the antimicrobial potency of AMPs often correlates with toxicity. Although AMPs are highly attractive as therapeutics, their potential remains unfulfilled because of potential toxicity issues, which curb most AMPs to topical rather than systemic applications currently. If possible, the coordination of an interactive structural-activity relationship framework of peptide designs and testings, uncoupling antimicrobial potency from toxicity would be a significant breakthrough and allow for the systemic administration of AMPs to fight various MDR bacterial infections. In addition, there are very few successful examples of preclinical and clinical development and applications of AMPs. The exact clearance mechanisms of AMPs from the body and tissues vary depending on their structures, size, and administered routes. Some AMPs are filtered by the kidneys and excreted in urine, whereas tissue enzymes may break others down. Research in AMP degradation and excretion is ongoing and likely to be peptide and context dependent. The crucial experiments to advance AMPs for clinical applications are those required for classic drug development, for which AMPs must go beyond the individual academic investigations, adapt to the pharmaceutical standard, and pass through vigorous and extensive testing, including but not limited to pharmacodynamics, pharmacokinetics, safety, and toxicity.

12. CONCLUSION

Lung antimicrobial proteins and AMPs play essential roles in both innate and adaptive immunity. These antimicrobial molecules protect against pathogenic microbial infection and impact respiratory disease progression by regulating inflammation and mediating lung injury. The primary roles of AMPPs are interconnected with antimicrobial activities, triggering the immune system, regulating the inflammatory response and immunomodulatory activities, and facilitating anticancer effects in the host. Peptide drugs have shown growing prominence in the pharmaceutical industry, and recently, a 31-amino acid linear peptide named “semaglutide” (aka Ozempic) has exhibited great success in treating type 2 diabetes and obesity. Peptide drugs such as AMPs could have significant therapeutic potential and emerging roles in addressing complex lung health challenges. Although the cost of synthesizing peptides is higher than that of small-molecule-based drugs, the high mortality rate resulting from severe pulmonary diseases, such as complicated pneumonia, COPD, and lung cancer, highlights the unmet medical need for a new class of therapeutics despite the higher drug costs. As respiratory diseases and lung cancer continue to be primary sources of all-cause mortality and burden, research in advancing their design and delivery is critical for enhanced lung health by developing enhanced therapeutic alternatives that overcome the deficiencies of traditional antibiotics and cancer immunotherapies.

GRANTS

The authors are funded by the National Institutes of Health (Grant Nos. R01 AI133351, R01 AI176537) and Fondazione Italiana per la Ricerca sulla Fibrosi Cistica (Project FFC#4/2022) Delegazione FFC Ricerca di Roma e della Franciacorta e Val Camonica.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Y.P.D. conceived and designed research; Y.P.D. analyzed data; Y.P.D. interpreted results of experiments; Y.P.D. and J.M.K. prepared figures; Y.P.D., J.M.K., and M.L.M. drafted manuscript; Y.P.D., J.M.K., and M.L.M. edited and revised manuscript; Y.P.D. and M.L.M. approved final version of manuscript.