CELL SOURCES AND TISSUE DISTRIBUTION
Human defensins are mainly produced by leukocytes and epithelial cells. HNPs 1-3 were first isolated from neutrophilic granulocytes (polymorphonucleated neutrophilic leukocytes; PMN), and account for 30-50% of total protein in azurophil granules of neutrophils [20].
They share high similarity in sequence with only a single amino acid difference [21]. No gene encoding HNP2 was found and thus it is proposed to be a proteolytic product of HNP1 or HNP3. HNP4 comprises less than 2% of defensins in neutrophils and has a relatively distinct sequence but similar structure to HNPs1-3 [2, 22]. While neutrophils produce the highest amount of HNPs, these peptides can be found in other immune cells including natural killer cells, B cells, γδ T cells, monocytes/macrophages and immature dendritic cells [23, 24]. In addition, cells can absorb and internalize HNPs intracellularly [25-27], underlining the complexity in defining true HNP producing cells and the questions regarding the function of the up-taken defensins. HNPs have been detected in placenta, spleen, thymus, intestinal mucosa, saliva, and cervical mucus plugs [24, 28-30]. Elevation of HNPs has been reported in the vaginal mucosa in women with N. gonorrhoeae (GC), T. vaginalis, or C. trachomatis (CT) [31-33], suggesting their role in mucosal immunity against infections in vivo [32, 34].Although leukocyte α defensins are conserved evolutionally and have been isolated from many species including human, rabbits, rats, guinea pigs and hamsters, mice lack α-defensin expression by neutrophils [2]. Mice express many enteric a-defensins known as cryptdins in intestinal Paneth cells [1, 2]. Similarly, HD5 and HD6 are produced predominantly by intestinal Paneth cells [2]. Interestingly, endogenous cryptdins do not protect mice against salmonellosis, whereas transgenic mice with an HD5 minigene that contains 2 exons and 1.4 kilobases of 5'-flanking sequence are markedly resistant to oral challenge with virulent Salmonella typhimurium [35].
In rhesus macaques, an animal model used for studying HIV pathogenesis, six Paneth cell defensins have been identified and their coding sequences are distinct from HD5 and HD6 [36]. HD5 is also found in other tissues such as the salivary glands, the female genital tract and the inflamed large bowel [29, 37-39]. In addition, increased levels of HD5 have been observed in urethral secretions of men with Neisseria gonorrhoeae and urethritis associated with Chlamydia trachomatis infection [10] and in cervicovaginal secretions from women with bacterial vaginosis [40].Six human β-defensins (HBD1, -2, -3, -4, -5,-6) have been identified and characterized [4, 41, 42], although an additional 28 human β-defensins [43] have been identified by gene-based searches. HBDs are expressed by epithelial cells and non-epithelial cells including monocytes, macrophages and monocyte-derived dendritic cells (DCs) [2, 4, 44]. While HBD1 is often constitutively expressed, expression of HBD2 and HBD3 can be induced by viruses, bacteria, microbial products and pro-inflammatory cytokines, such as tumour-necrosis factor (TNF) and interleukin-1 (IL-1) [2, 45-48]. HBD1, HBD2 and HBD3 have been detected in various epithelial tissues [29, 49, 50]. Both human α- and β-defensins have been found in breast milk [51, 52], suggesting a role for defensins in protecting infants from infection. Constitutive expression of HBD4 seems to be restricted to testis and gastric antrum, although HBD4 expression can be induced in human respiratory epithelial cells after exposure to phorbol 12-myristate 13-acetate (PMA) or bacteria infection in vitro [53]. HBD5 and HBD6 are specifically expressed in human epididymis [41].
Old World monkeys including rhesus macaques, orangutans and a lesser ape species express intact θ- defensins [54]. In contrast, primates including human, chimpanzees and gorillas contain pseudogenes of θ- defensin mRNAs with a conserved stop codon upstream of the signal sequence that prevents translation [54].
Three θ-defensins have been found in leukocytes of rhesus macaques: rhesus θ-defensin-1 (RTD1), RTD2 and RTD3 [11-13]. Retrocyclin, an artificially made circular peptide based on the sequence of the mature peptide that would be encoded by the human θ-defensin pseudogene, has been shown to display antiviral activity in vitro [55].Regulation
HNPs are primarily synthesized in promyelocytes and early myelocytes, the bone-marrow precursors of neutrophils [56]. HNP1 and HNP3 are transcriptionally regulated in promyelocytic cells by the binding of CCAAT/enhancer-binidng protein (C/EBP a) to C/EBP/c-Myb sites in the HNP promoter [57]. In response to bacterial infection, high concentrations of HNPs (mg/ml) are present in neutrophils phagosomes as the result of the fusion of granules and phagocytic vacuoles of neutrophils [2, 58]. HNPs can also be released by chemokines, FCg receptor cross-linking and PMA [25, 59-61]. Pathogen-associated molecular patterns (PAMPs) from the outer membrane protein A of Klebsiella pneumoniae and flagellin of Escherichia coli, which signal via toll-like receptors 2 and 5, respectively, trigger the release of HNPs 1-3 by CD3+CD56+ natural killer T cells [60]. Direct interaction of Mycobacterium bovis BCG with eosinophils induces the production and release of HNPs 1-3 in a TLR2 dependent manner [62].
HD5, the most abundant AMP in the small intestine, is constitutively expressed by Paneth cells but can be found in the colon of patients with inflammatory bowel disease [35, 63, 64]. A NOD2 mutation in patients with ileal Crohn’s disease (CD), a chronic mucosal inflammation, has been associated with a pronounced reduction in HD5 production [65]. A reduced expression Wnt signaling transcription factor Tcf-4 protein has been correlated with a decrease in HD5 and HD6 expression in the small intestine of patients with ileal CD, although this association is independent of the NOD2 genotype [66]. HD5 is induced in the genital mucosa in patients with bacterial vaginosis, Neisseria gonorrhoeae and Chlamydia trachomatis infections [10, 40], although the mechanism of induction remains to be defined.
The mechanisms of induction of HBD1, HBD2 and HBD3 have been shown to be distinct from each other [42]. HBD2 can be induced by TLR2, TLR3, TLR4, TLR7, NOD1 and NOD2 signaling in various epithelial cells and keratinocytes [67-71]. Stimulation of TLR3 has been shown to induce HBD1 and HBD2 expression in uterine epithelial cells [72]. Induction of HBD2 and HBD3 but not HBD1 in bronchial epithelial cells in response to human rhinovirus infection is mediated by nuclear factor-κB (NF-κB) activation but not of IL-1 [45]. As TLR3 activation also induces HBD2 and HBD3, it is possible that intracellular double-stranded RNA generated during replication of rhinovirus may be involved in the regulation of HBDs [45, 46]. Similarly, HBD2 and HBD3 are induced in normal human oral epithelial cells [73]. In oral epithelium, TLR2 and NOD1/2 ligands synergistically activate NF- κ B and induce HBD2 gene expression [74]. Cytokines such as IL-1 and IL-17 also play important roles in the regulation of HBD2 expression. Induction of HBD2 by IL- 17A is mediated by the PI3K and MAPK pathways to activate NF- κ B in airway epithelial cells, whereas regulation of HBD2 by the activation of NF- κ B is not dependent on PI3K pathway in bronchial epithelial cell, [75-77], indicating that specific pathways involved in regulation of HBDs are cell type dependent.
Functions
Defensins are originally thought to kill mammalian target cells and microorganisms though a common mechanism by permeabilization of target membranes, which involves electrostatic interactions between positively charged defensins and negatively charged membrane lipids (reviewed in [78]). Recent studies revealed that defensins have differential antibacterial activity and specificity. The varying degree of antibacterial activity of HBD3 against different bacteria is in part attributed to its lipid-specificity [79]. For example, HD6, distinct from other α-defensins including HNPs1-4 and HD5, does not exhibit anti-bacterial activity [80].
Additionally, HNPs and HBDs have differential effects on cytokine production in bronchial epithelial cells [81]. Thus, the functions of defensins appear to be specific to the defensin and target.Defensins have a wide range of functions in modulating innate and adaptive immunity [4] as well as a number of other defensin specific biological functions [82-87]. Many of these effects could directly or indirectly influence HIV. Both HNPs and HBDs exhibit chemotactic activity for T cells, monocytes and immature DCs and can induce production of cytokines and chemokines [4, 88, 89]. HNP1 also regulates the release of IL-1β and enhances phagocytosis [90, 91]. HBDs1-3 recruit memory T cells and immature DCs through binding to CCR6, the receptor for the CC-chemokine ligand 20 (CCL20; also known as MIP3α) [92, 93]. HBD2 has multiple activities on mast cells, including induction of cell migration, degranulation and prostaglandin D2 production [94]. Murine β-defensin-2 can recruit bone-marrow-derived immature DCs through CCR6 and can induce DC maturation through TLR4 [95]. HBD3 activates antigen-presenting cells such as monocytes and DCs through TLRs 1 and 2 [96]. HBD3 actives antigen presenting cells (DCs and monocytes) via TLR1/2 [96], suggesting a role for defensins in HIV transmission. Defensins are frequently induced by pro- inflammatory cytokines or TLR activation [2, 97]. Conversely, defensins can induce cytokines and chemokines. HNPs upregulate the expression of CC-chemokines and IL-8 in macrophages and epithelial cells, respectively [98, 99]. HBD2, known to be inducible in response to bacterial infection and pro-inflammatory cytokines [2, 4] can up-regulate IL-6, IL-8, IL-10, MCP-1, IL-1β, MIP-1β and RANTES in PBMCs [100]. HD5 can induce IL-8 [101] that enhances HIV infection in cervical tissues [102].
Defensins can bind to other host proteins to modulate immune or metabolic functions [82]. HNPs bind to low- density lipoprotein receptor-related proteins and interact with protein kinase Ca and β leading to decreased smooth muscle contraction in response to phenylephrine [103]. HNPs also interact with adrenocorticotrophic hormone (ACTH) receptors and heparan sulfate-containing proteoglycan (HSPGs) to modulate other biological activities [104, 105]. HNP1 has been shown to inhibit the activity of conventional PKC isoforms in a cell-free system [106]. This PKC inhibitory activity appears to be important for HNP1-mediated inhibition of HIV replication in primary CD4+ T cells [107]. As defensins display various biological functions, the roles of defensins in HIV-associated metabolic disorders or cancers in addition to HIV transmission and pathogenesis remain to be investigated.