Genes encoding AMPs in mammals are expressed throughout the body, both in circulating cells and epithelium (for reviews, see, for example, [4
]). The most abundant human AMPs, defensins and cathelicidins, are produced from genes that encode larger precursor proteins, each with a different precursor structure (). These structures lead to gene regulation both at the transcriptional and posttranscriptional levels, to provide controlled expression of these multifunctional molecules. The coordinated transcriptional regulation of AMP genes can lead to multiple AMP expression at a single site, providing a combination of host defense molecules [101
]. A recent computational study examined the upstream regions of numerous AMP genes in human, mouse and rat, including defensins and cathelicidins, and has characterized them according to similarities in their transcription factor binding sites [103
]. The results support the concept of coordinate transcriptional regulation of AMP genes, to provide the most comprehensive antimicrobial host defense.
Gene structure for human α-defensins (A), β-defensins (B) and cathelicidin (C)
In humans, the α-defensin family includes four peptides found in neutrophils (HNP 1-4) (reviewed in [104
]), and two peptides in Paneth cells within the small intestine (HD-5 and –6) (reviewed in [105
]), which are differentially expressed. Transcription of HNP1-4 is found in bone marrow and in immature myeolocytes, but stops with maturation of the neutrophil [106
]. Precursor α-defensins are produced in promyelocytes, which are then processed to the mature, active forms during granulogenesis [107
]. The active peptides are found in primary granules in the neutrophils, where they can then be used as part of the oxygen-independent antibacterial mechanism of these cells. In contrast, intestinal α-defensins are transcribed in the Paneth cells, where the pre-propeptides are translated. Upon removal of the signal peptide during the secretory process, the inactive propeptide is secreted into the lumen of the small intestine, where it is activated by a trypsin-mediated removal of the propiece in humans [108
], and by matrilysin in mice [109
In contrast, the β-defensins are found primarily in epithelial cells, at numerous sites throughout the body, including oral, airway and skin epithelium (reviewed in [2
]). As β-defensins lack an acidic propiece [110
], their expression is primarily at the level of transcription, producing active peptide. In epithelial cells, human β-defensin 1 (hBD1) is generally transcribed at a constitutive, low level. The mRNAs for other β-defensins, including hBD2, 3 and 4, are found at low levels, but transcription is induced by a variety of factors including microbes and cytokines (reviewed in [2
]). Specifically, this includes Toll-like receptor (TLR) agonists such as LPS, and inflammatory mediators such as TNF-α, Interleukin (IL)-1β and IL-17 [111
]. More recently, three cytokines, IL-12, 23 and 27 were shown to enhance the IL-1β-mediated induction of hBD2 [112
]. Together the multitude of factors that induce β-defensin transcription suggest a complex role for these peptides in innate immunity.
In the airway, which is a generally sterile tissue, the epithelial cells are highly responsive to the presence of microbes, including Gram-positive and –negative bacteria, which recognize the potential pathogens through a TLR pathway, and lead to an NF-κB-mediated induction of β-defensin gene expression [113
]. Similarly, induction by IL-17 proceeds through NF-κB as well [114
]. In contrast, the oral cavity, which is home to hundreds of bacterial species, expresses β-defensins at several sites, including the gingival epithelium (reviewed in [115
]). However, the genes are induced only by a subset of bacteria, and the induction utilizes different pathways, including p38, JNK [116
] and NF-κB [117
], depending on the species. Even within the NF-κB activation, there are different pathways leading to activation utilized by different microbes [118
] This control may be partially responsible for regulating the homoeostatic levels of bacteria at this site.
Some β-defensins have been observed in circulating blood cells, and their expression appears to be similarly regulated. In the viral defensive PDC, hBD1 can be found [54
], and appears to be induced by viral challenge (Ryan et al.
, manuscript in preparation). In macrophages and monocytes, hBD2 is induced by cytokines, including interferon-γ [119
]. Both α- and β-defensins have been observed in breast milk [120
], including the neutrophil peptides HNP 1-4 as well as the intestinal α-defensins HD-5 and –6. There is some evidence suggesting that these α-defensins may play a role in this tissue in the protection against transmission of HIV through this route [121
Alongside β-defensins, LL-37 is also expressed in the surface epithelia of conducting airways [122
], and in bronchoalveolar lavage fluid (BALF) [123
]. Its expression is induced by bacteria and cytokines, similar to β-defensins, confirming the computationally observed similarities in their promoter regions. To demonstrate this peptide's role in airway defense, a complex animal model was used. Specifically, human respiratory epithelial cells were seeded on denuded rat tracheas. These tracheas were implanted in the flanks of nude mice, creating a xenograft [124
]. These xenografted tracheas secrete hBD-1 and -2 and LL-37 into the airway surface fluid (ASF), which exhibits antibacterial activity. While airway cell cultures from patients with cystic fibrosis (CF) have reduced antibacterial activity, overexpression of LL-37 in xenografts developed from human cells exhibiting the CF defect results in normal antibacterial activity, compared with untransfected cells [124
], supporting the role of this peptide in antibacterial host defense.
Similar to the expression pattern in the airway, LL-37 is observed in both healthy gingival epithelium and in neutrophils, with an increase in expression observed in inflamed gingiva [127
By computational examination of putative promoter sequences, Wang et al.
] discovered the presence of a Vitamin D Response Element (VDRE) upstream from the cathelicidin antimicrobial peptide (CAMP) gene. This promoter element is recognized by a nuclear receptor (VDR) which heterodimerizes with the retinoid X receptor upon activation by the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3
) (reviewed in [129
]). The presence of such a sequence suggested that LL-37 mRNA might be inducible by vitamin D. Further studies demonstrated this to be the case in several cell types, including monocytes and primary keratinocytes, as well as established cell lines such as U937 (a monocyte line), HL-60 (a promyelocyte) and SCC25 (tongue carcinoma) [128
]. LL-37 mRNA, protein and antimicrobial activity was also induced in primary cultures of airway epithelium by 1,25(OH)2
]. Further studies demonstrated that the induction was in response to a VDRE-mediated increase in transcription, which led to the increase in LL-37 mRNA and peptide, as well as an increase in the antimicrobial activity of the cell culture medium [130
]. More recently, Liu et al.
demonstrated that the activation of TLR 2/1 (a receptor for patterns including those found on Mycobacterium tuberculosis
) increased transcription of the VDR and the Vitamin-D1-hydroxylase genes. This led to a VDRE-mediated increase in LL-37 levels in macrophages, and a subsequent increased killing of M. tuberculosis
]. Together, these studies support the potential use of this less toxic agent such as Vitamin D in the increase in antibacterial activity of tissues to prevent bacterial colonization.
Such a therapeutic modulation may be useful in the oral cavity as well. Preliminary data from our laboratory show that a similar induction of LL-37 by 1,25(OH)2D3 occurs in cultured gingival epithelial cells (Yim et al., manuscript in preparation).
The identification of single nucleotide polymorphisms in the 5′ untranslated region (UTR) of hBD1 that are associated with clinical phenotypes (see below), however, suggest that there may be some level of posttranscriptional regulation as well. And as with the transcriptional regulation, LL-37 peptide expression also appears to be controlled by elements in the 5′ UTR [133