Cathelicidins In Humans And Animals

These peptides in humans are represented by the cathelicidin LL-37, which is characterized by a linear structure with α-helix structure [2, 118]. It may exist in the form of a monomer, dimer or tetramer [74, 76], creating cationic, amphipathic structures composed of three parts [74, 76]. These are: the N-terminal and C-terminal part of the α-helix and the C-terminal region, where the α-helix at the N-terminus, participates in the oligomerization of the peptide and provides the molecule with resistance to proteases, since the C-terminal section is important for the formation of tetramers [101, 110]. Initially, cathelicidin LL-37 was called hCAP18, which referred to a peptide with size up to 18 kDa, which contained two disulphide bonds between the cysteine residue C85-C96 and C107-C124, produced by extracellular proteolysis of the C-terminal human CAP (Cationic Antimicrobial Protein) [110]. When the peptide was found to consist of 37 amino acids starting with two leucines, the name was changed from hCAP18 to LL-37. Currently, hCAP18 refers only to the propeptide, whereas LL-37 alone denotes a mature peptide having pleiotropic properties upon release from the C-terminus of hCAP18. Human cathelicidin – the LL-37 peptide, is encoded by the CAMP gene (Cathelicidin Antimicrobial Peptide), which is found in the locus 21 of chromosome 3 (3p21.3) [110]. The LL-37 peptide is synthesized in the human body in response to bacterial, viral and fungal infections or is a result of neutrophil elastase, which does not activate its peptides accumulated in granular granulocytes, and breaks them down into active components secreted from these cells [97]. Human LL-37 peptide appears at a very early stage of development, because it has already been detected in new-borns in the skin and trachea [61, 81]. In adults, it undergoes expression, among others, in the gastrointestinal epithelium, including the epithelium of the oral cavity and intestines [28] and airways [11, 27, 28, 30, 32, 23, 34], as well as in keratinocytes [33, 66, 118]. It is also synthesized in neutrophils, monocytes-macrophages, NK cells, mast cells, dendritic cells and T and B lymphocytes, as well as conjunctival epithelial cells, urogenital and biliary tracts and in the liver, cervix, vagina, epididymis, testicles and is also found in blood plasma, saliva, sweat, semen and secretion in the trachea [4, 6, 8, 16, 22, 26, 28, 33, 48, 65, 66, 4, 92, 107, 118]. The LL-37 peptide in neutrophils, in response to bacteria or their products, is produced constitutively, while in monocytes-macrophages, NK cells, mast cells, T and B lymphocytes, enterocytes and keratinocytes, it arises only through the action of proinflammatory cytokines (TNF, IL1α, IL-6, IL-17A, IFN-γ), growth factors (IGF-1) and due to the active form of vitamin D [2, 733, 92, 101, 118]. Studies have demonstrated that as a result of proteolytic activity of serine proteases, which represent the tissue kallikrein family, derivatives of the LL-37 peptide are formed, which indicates its heterogeneity [112]. It was found that as a result of SCTE (Stratum Corneum Tryptic Enzyme), three peptides are formed – that is KS30, KS22 and LL29, while under the influence of SCCE (Stratum Corneum Chymotryptic Enzyme) action, two peptides are formed – RK31 and KR20. Therefore, it is assumed that the LL-37 peptide is only 20% of the cathelicidins in humans, while the rest are its derivatives, which exhibit both antimicrobial and immune system-modifying properties, and affect epithelial cells and keratinocytes [18, 20, 33, 35, 54, 64, 96, 108, 112].

The antibacterial activity of the LL-37 peptide is associated with its high concentration and the presence of divalent ions [18, 64]. In women in the reproductive tract (vagina), among its derivatives, the ALL-38 peptide has been described, which is a very important element in the defence of this section [21, 96, 108]. The LL-37 peptide, in addition to the mentioned anti-carcinogenic effect and modulation of the immune system, is characterized by pro-inflammatory and anti-inflammatory activity, proangiogenic and antiapoptotic as well as anti-carcinogenic activity [2, 15, 18, 28, 33, 41, 59, 64, 70, 89, 101, 107, 118]. It exhibits a strong direct effect against bacteria and viruses possessing a cell envelope as well as fungi, [50, 69, 104, 108, 110]. Gram-positive bacteria are particularly sensitive to this peptide, like i.a. Staphylococcus spp., Enterococcus spp., Streptococcus spp., Bacillus spp., Lactobacillus acidophilus, Listeria monocytogenes, Propinibacterium acnes, as well as Gram-negative bacteria, i.a. Acinetobacter baumannii, Escherichia coli, Salmonella Typhimurium, Pseudomonas aureginosa, Stenotrophomonas maltophilia, Proteus vulgaris, Klebsiella pneumioniae and Neisseria gonorrhoeae [1, 11, 14, 26, 28, 51, 60, 69, 90, 98, 101]. The impact of this peptide on the germs – mainly bacteria, is associated with its effect on their cell membrane, which leads to its fragmentation and the formation of pores therein. The LL-37 peptide can also induce the death of a bacterial cell by inhibiting the synthesis of its bacterial components, including the cell wall. It also has a neutralizing effect on bacterial LPS, which is important during infection with Gramnegative bacteria [60]. In vitro LL-37 exhibits an inhibitory effect on fibroblasts isolated from clinically healthy gingiva in mammals which have been treated with LPS derived from E. coli [60], exerting an inhibitory influence on LPS. It is assumed that it can be used i.a. in the oral control of pathogens such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans [44]. The inhibitory effect of this peptide on the formation of bacterial biofilm Pseudomonas aeruginosa, Francisella novicida or Staphylococcus epidermidis was also recorded, as well as its direct destructive effects on the biofilm of these bacteria [5, 24, 379, 58]. It also has a destructive effect against mycobacteria, and in particular Mycobacterium (M.) smegmatis, M. bovis and M. tuberculosis, which is probably due to its exogenous action after penetrating into infected macrophages through endocytosis, which leads to the inactivation of mycobacteria [79, 91]. Another way of LL-37 activity against mycobacteria is the endogenous pathway through its synthesis in macrophages, as a result of the stimulation of these cells with vitamin D [79, 91]. Its antimicrobial action through the activation of immune cells takes place through TLR receptors [18, 44], except that it works via TLR5 in keratinocytes, via TLR2 and TLR3 monocytes, and in B-lymphocytes, dendritic cells and neutrophils through TLR9 [3, 43, 71]. It has now been demonstrated, on the example of cathelicidin – CATH2 (chicken), that this peptide supplying signal through TLRs 2 and 4, points to a new mechanism of “tuning” the immune response, resulting in the reduction of inflammation, which allows the immune system to distinguish between live and dead Gram-negative bacteria (E. coli), which can be very important, e.g. in sepsis [59]. It has been demonstrated that the LL-37 peptide can also activate other receptors than TLRs, because in neutrophils and lymphocytes it also stimulates the G Protein-Coupled Receptor (GPCR) e.g. FPRL-1 (Formyl Peptide Receptor-like 1), receptor of tyrosine kinases e.g. EGFR (Epidermal Growth Factor Receptor), as well as the receptor channel P₂X₇R, which leads to a strong activation of innate immunity elements and activation of the pro-inflammatory signal cascade [3, 110]. It has been shown that the LL-37 peptide, acting on peripheral blood T and B lymphocytes, enhances the synthesis of, among others IL-6, IL-10 and chemokines CCL2 (CC-chemokine Ligand 2) and CCL7 (CC-chemokine Ligand 7) and increase the secretion of IL-1β, which is a potent inflammation activator [33, 44, 108, 110]. In contrast, induction with LL-37 peptide of FPRl-1 receptors in endothelial cells leads to an increase in their number, and in neutrophils increases the activation of FPRL-1 markers ₂X₇R. This results in the suppression of apoptosis of these cells and, as a result, prolongs their lifespan, resulting in a relative increase in the duration of neutrophil during infection [12, 67, 108, 110]. This peptide, by activating intracellular factors, induces autolysis of phospholipase A2 and enhances natural immunity [83]. In the case of airway epithelial cells, it activates not only the EGFR receptor, but also affects many molecular elements associated with the membrane. An example is the activation of metalloproteinase and the MAPK / ERK pathway (Mitogen-Activated Protein Kinases / extracellular Signal-regulated Kinases) [110]. It has been shown that an increase in the concentration of sodium chloride in the airway epithelial cells, causes a four or fivefold decrease in the activity of the LL-37 peptide, resulting in a reduction in the activity of various factors in these cells, which leads to the defect of the transmembrane expression regulator CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) [74].

In the case of viral infection with the background of double-stranded viral RNA, the LL-37 peptide, increases the pro-inflammatory signalling in epithelial cells through TLR3 [20, 50]. However, when humans are infected with the HPV virus (Human Papillomavirus), this peptide seems to increase its activity in the epidermis, both in the development of normal and infectious warts, which proves that it is an important element of immunity during this infection [72]. This peptide is also active against HIV1 [104]. In the case of fungal infections with Candida albicans and Trichophyton (T) mentagrophytes and T. rubrum, increased expression of LL-37 has been recorded, which leads to inhibition of epidermal cell proliferation [28, 57, 110]. In non-infectious diseases, for example in the case of psoriasis, contact dermatitis and Lupus Erythematosus, the LL-37 peptide, as in the case of fungal infection, strongly activates keratinocytes [16]. It has been proven that the increased expression of this peptide in the case of psoriasis correlates with a low rate of secondary bacterial infections [16]. Although in people with atopic dermatitis, who are particularly susceptible to this type of bacterial and even viral infections, there is no secondary infection [22, 72]. In addition, the LL-37 peptide, acting as an inflammatory regulator, enhances wound healing and renewal of the superficial layer of the skin [28, 33], as it contributes to increased IL-8 and IL-6 synthesis in these cells cooperating in keratinocytes with IL-17 and IL-22, which leads to an increase in local immunity in the skin [20, 33]. The physiological concentration of LL-37 peptide in human plasma is 27.2 ng/ml, in saliva 30.5 ng/ml, in sweat 447 ng/ml, in sputum 3.0–5.0 ng/ml, in bronchial fluid – vesicles 4.8 ng/ml, while in the case of diseases such as tuberculosis and cystic fibrosis, it increases even 30–50 times [118].

These peptides in monkeys, farm animals, carnivores, laboratory animals, wild animals and birds, fish, reptiles, amphibians and insects, occur in the cells of lymphatic organs-bone marrow (mammals), bursa of Fabricius, (birds), in the cells of the immune system – neutrophils, although they also occur in the intestines, liver, showing large structural differences in relation to cathelicidins in humans, although in the field of biological activity, they are very similar to those occurring in humans [6, 10, 11, 19, 25, 46, 48, 52, 53, 56, 64, 69, 75, 86, 7, 88, 96, 97, 99, 100, 102, 108, 111, 114, 116, 118, 119].

In rhesus monkeys, cathelicidins rhLL-37 and rhCAP18 [10] or rhCAP18 – CAP18 [48] have been described, which occur in particular in gastrointestinal and respiratory epithelial cells, and have similar α-helix structure in humans and are similar in biological activity to human cathelicidin LL-37, exhibiting anti-germ activity, including LPS of Gram-negative bacteria [10, 48]. These animals (rhesus monkeys) also have a cathelicidin RL-37 having an α-helix structure, whereas in macaques and orangutans, an analogous structure of the ppp RL-37 has been recorded, which has also been described in gibbons, in which it was designated as a peptide hmd SL-37 [62.86].

However, in farm animals (cattle, sheep and goats), these peptides most often also have an α-helix structure, although in pigs these substances not only have an α-helical structure, e.g. cathelicidin PAMP (Porcine Antimicrobial Peptide), but also protegrins having a β-sheet structure. In sheep, cyclic cathelicidins, e.g. bactenecin-Bac and cathelicidins rich in proline and arginine residues have been recorded analogously to Bac in cattle, sheep and goats and prophenins (PG1 and 2) in pigs, as well as tryptophan-rich peptides, e.g. indolicidin in cattle or PR protegrins in pigs were have been recorded. [48, 52, 56, 96, 108, 114]. Genes encoding cathelicidins in cattle, sheep, goats and pigs, have the same organization and are 2 kbp in size. They are characterized by a high percentage of identical nucleotides, which indicates their origin from the same gene, and which in part confirms their location in the chromosome (in cattle, sheep and pigs, they are located close to each other) [114].

In cattle, the best known cathelicidins occurring in neutrophils is bactenecin 1 (Bac-1-cathelicidin 1), 5 (Bac5-cathelicidin 2) and 7 (Bac7-cathelicidin 3) – peptides rich in proline and arginine and indolicidin (cathelicidin 4) rich in tryptophan [48, 96] . These peptides have a strong antibacterial effect acting on the cell membrane and on their intracellular organelles, mainly against Gram-negative bacteria: E. coli, S. Typhimurium, Klebsiella pneumoniae and P. aeruginosa, as well as in Gram-positive bacteria, although for Enterobacter cloace, Leptospira (L) interrogans and L. biflexa, exhibit bacteriostatic activity [48, 96]. Furthermore, indolicidin, mentioned in cattle and occurring in neutrophilic granulomas, is characterized not only by antibacterial but also anti-fungal activity, e.g. against C. albicans, Cryptococcus neoformans and anti-protozoan activity against Leismania donovani and Giardia lamblia. In addition, it activates chemokine secretion, neutrophil autophagy, which is very important, in particular in the mammary gland in cattle during the drying period, where the neutrophil autophagy process is very intense [13, 85, 96]. In cattle a cathelicidins in a form of BMAP peptide have been described ( bovine myeloid antimicrobial peptides) 27 (cathelicidin 6), 28 (cathelicidin 5) and 34 (cathelicidin 7) [48, 52, 56, 108] that affect mesosomes in bacteria and mitochondria in fungi and affect the secretion of TNF-α in mammary epithelial cells, which is valuable for mastitis in these animals, although they also have anti-carcinogenic effects [48, 56, 95, 108].

In sheep, the important cathelicidins are: a cyclic dodecapeptide under the names Oa Bac 5, 6, 7.5 and 11, (bactenecin 5, 6, 7.5 and 11) and cathelicidin 1 (bactenecin 1), 2 (bactenecin 5) and 3 (bactenecin 7), and also the SMAP peptide (sheep myeloid antimicrobial peptide) (SC5-cathelin related peptide) and MAP (myeloid antimicrobial peptides) 29 and 34, rich in proline and arginine [48, 52]. All these peptides in these animals are characterized by antibacterial (Gram-negative and positive) and antifungal activity (C. albicans) [48, 52]. These compounds in sheep are synthesized by neutrophils and mammary epithelial cells, exhibiting inhibitory activity against Staphylococcus aureus, Streptococcus uberis and Mycoplasma agalactie – germs which are a very common cause of infection of this gland [23]. In addition, cathelicidin in sheep activates PMN cells in the scope of their antimicrobial activity and NET network formation. This peptide is a specific marker indicating udder inflammation [23].

In goats, cathelicidins Bac 7.5, 3, 4 and cathelicidin 2 (Bac5) are known – peptides rich in proline and arginine, which are 50% similar in structure to Bac5 in cattle [48, 52, 108]. These peptides in goats, even in low and high concentration of sodium chloride, exhibit antibacterial activity against Gram-negative bacteria, e.g. E. coli, P. aeruginosa) and some Gram-positive ones, e.g. Bacillus subtilis, S. aureus, while in low concentration they only eradicate C. albicans [48, 52].

In contrast, in pigs, cathelicidins are represented by Pell (Porcine antimicrobial peptide), 23.36 and 37 (α-helical structure), PR-39, prophenin 1 and 2 (PF 1 and 2) (rich in proline and arginine) and protegrin 1–5 (PG 1–5), which exhibit a β-sheet structure and have SS bridges. They are synthesized in neutrophils, bone marrow, and also occur in the bronchioles and tongue cells, small intestines, trachea and urogenital cells, demonstrating an activating effect on the elements and phenomena of the immune system, in particular the neutrophil phagocytosis [48, 52, 56, 75, 106, 108]. These peptides in pigs also show activity against Gramnegative bacteria such as E. coli, although for example: Prophenins 1 and 2 (PF 1 and 2) additionally combat Gram-positive bacteria, e.g. L. monocytogenes, and protegrins 1–5. (PG1-5) together with prophenins 1 and 2 (PF1 and 2), are particularly destructive towards Chlamydia trachomatis, C. albicans, N. gonorrhoeae and some viruses, as well as nematodes and flat worms [40, 48, 52, 56, 55, 105, 106]. In addition, protegrin 1 (PG1) in pigs impacts M. tuberculosis and bacteria which cause wound infections [77]. It has been reported that purified LPS of Gram-negative bacteria, increases the expression of PR-39 cathelicidin in bone marrow cells of these animals [109], and which peptide, affects wound healing and has an inhibitory effect on apoptosis [40, 77].

In horses, among the cathelicidins peptides called eCATH 1, 2 and 3 are known, which are synthesized in the bone marrow, are characterized by an α-helical structure and are rich in lysine. In terms of the impact on germs, they are characterized by potent destructive effect on Gram-negative and Gram-positive bacteria and fungi, such as: Cryptococcus neoformans and Rodotorula rubrum [25, 48, 56, 88].

However, in carnivorous animals – dogs, cathelicidin K9CATH has been described, whereas in cats FeCates – peptides which have an α-helical structure and occur in the bone marrow and neutrophils, and are characterized – mainly in dogs – by strong antibacterial properties, e.g. N. gonorrhoeae and Ureaplasma sp. This explains the low susceptibility of these animals to sexually transmitted diseases [48, 56, 62, 112].

In laboratory animals, cathelicidin was found in rabbits, rats, mice and guinea pigs [48, 56]. Cathelicidins in rabbits are represented by the CAP 18 peptide and PI5A and PI5B proteins present in their neutrophils and kidneys; in rats by the rCRAMP peptide, whose origin in available literature is not mentioned; in mice the Cramp peptide is indicated, which occurs in the testicles of males, spleen, liver and gastrointestinal tract; in guinea pigs cathelicidins are represented by CAP11 peptide, found in neutrophils and bone marrow [48, 56]. These peptides in these laboratory animals exhibit antibacterial activity, including inhibitory effect on LPS of Gram-negative bacteria [48, 56].

In wild animals, cathelicidins have been described in deer in the form of bactenecin, which occurs in neutrophils and kidneys, characterized by antimicrobial activity against Gram-negative and Gram-positive bacteria [97]. Cathelicidin has also been described in these animals in the form of the P-9 peptide , which is rich in proline and arginine and has a bactericidal effect on many germs [29]. These compounds have also been recorded in buffaloes of the Bovidae family (Bubalus bubalis) in the form of a fragment of myeloid cathelicidin and a fragment of cathelicidin 4, found in the bone marrow and genital tract, whose activity has been described as anti-germ [17, 97]. Cathelicidins called EACATH 1 have been described in donkeys, characterized by α-helical structure [37] and in platypus in the form of PA1 and PA2 peptide [29], as well as in pandas in the form of the AM peptide [113]. The cathelicidins in these animals have not been well characterized yet in respect of their structure and biological activity.

The cathelicidins were also recorded in birds, in particular in chickens in which they are represented by cathelicidin 1 (CATH 1), 2 (CATH 2), 3 (CATH 3), cathelicidin B-1 (CATH B1) and peptide CMAP 27 (Chicken Myeloid Antimicrobial Peptide-27). These peptides have been found, among others, in their bursa of Fabricius, in the bone marrow, gastrointestinal tract, liver, respiratory system, kidneys, spleen, brain and muscles [48, 53, 56, 100, 107, 111]. They exhibit strong activity against Gram-negative and Gram-positive bacteria, including those resistant to antibiotics. It is assumed, however, that in chickens it is primarily the CMAP 27 peptide, which determines their natural immunity. According to Linde et al. [56] in chickens as well as turkeys, these peptides are observed in the bone marrow in the form of protegrin. It has also been reported [101, 111] that the cathelin region of cathelicidin in birds displays low homology in comparison to this region in mammalian vertebrates.

Cathelicidin has also been described in fish (Atlantic salmon, rainbow trout, Atlantic cod) in the form of peptide 29 (HFIAP-3) and 37 (HFIAP 1.2) and cathepsin H and cathelicidin 2, which are found in the cells of the digestive tract, liver, kidneys, the skin and which are similar to some mammalian cathelicidins, because they have proline and cysteine in their structure and exhibit, in particular, antibacterial activity [19, 48, 52]. In addition, it is assumed [108] that in salmons there occurs a cathelicidin called rtCATH 1 – rich in glycine, while in the Atlantic cod there occurs the Cod Cath cathelicidin – rich in glycine and serine.

Cathelicidins have also been recorded in snakes in the form of the OHCATH peptide – king cobra, NACATH peptide – cobra and BF-CATH peptide and BF cathelicidin in the banded krait [102, 108].

Literature data [48, 64, 93, 96, 108] also indicate the existence of cathelicidins in amphibians and insects, but mainly in amphibians; details on the structure of these peptides and their biological activity is lacking. On the other hand, in 1980 it was demonstrated in insects that apart from the isolation of cecropins from the pupae of the Hyalophora cypropia moth, many peptides had been registered in various representatives of this cluster [64]. And thus in Drosophila melanogaster, 8 families of AMPs have been found, and in Galleria mellonella caterpillars 12 peptides which are synthesized in the fat body, haemocytes and epithelial cells have been described. These peptides affect mainly Gram-negative bacteria, but also Gram-positive bacteria, and also have viricidal activity. Among the bactericidal peptides, melittin, cecropins A, alloferon 1 and 2 and the myristoyalated peptide isolated from Heliothis virescens are distinguished. In contrast, viricidal peptides include representatives of four classes of AMPs and these are: linear, α-helical peptides, peptides with a structure stabilized by disulphide bridges, peptides containing a significant amount of amino acid of one type and peptides containing rare, modified amino acids [64].