Human and murine enteric alpha defensin genes consist of two exons, whereas alpha defensin genes expressed in human neutrophils consist of three exons, two of which (second and third) are homologous to enteric defensins [¹]. Search for orthologous sequences in promoters of these genes revealed presence of putative binding sites for transcription factors AP1, OCT1.4, and GCN4 [²⁰,²¹]. Upstream regions of human DEFA5 and murine Defa4 genes contain highly conserved cis-acting elements as evident from the expression of functionally active human peptide in small intestinal crypts of mice transgenic for human DEFA5 minigene construct [²²].

Analysis of postnatal changes in concentration of murine enteric defensins in the small intestine of conventional and germ-free mice revealed two patterns of gene expression: gradual increase in production of defensins 1, 3 and 6 and rapid raise in secretion of defensins 2, 4 and 5 in the jejunum (but not in the ileum), which is presumably induced by the presence of luminal bacteria [²³]. In adult mice alpha defensins are equally expressed along the small intestine except Defa4, which is more abundant in the ileum as compared to the duodenum [²⁴]. Recently, it has been shown, that in human tropical populations secretion of enteric alpha defensins may decrease up to ten times, as compared to Europeans, due to down-regulation of gene expression in response to infections, inflammatory conditions, and malnutrition [²⁵].

Human enteric alpha defensins are synthesized in vivo as precursor proteins, some of which have antimicrobial activity. For example human DEFA5 prepropeptide (aa 1-94), contains signal peptide (aa 1-19) and propeptide (aa 20-94), which is subsequently processed into major mature peptide (aa 63-94), and minor mature peptide (aa 56-94) [²⁶]. The prosequence flanking the mature peptide is necessary for correct intracellular sorting and trafficking of the propeptide into the secretory vesicles, where it is proteolytically processed by trypsin during secretion [²⁷,²⁸]. Conversely, murine defensins are stored in vesicles in a mature form after propeptide processing by matrilysin (also known as matrix metalloproteinase Mmp7) [²⁹]. It has been shown that abrogation of murine alpha defensin processing by targeted disruption of the matrilysin gene increases susceptibility of Mmp7 knockout mice to oral challenges with enteric bacteria, whereas transgenic mice overexpressing human DEFA5 are markedly resistant to orally administered virulent Salmonella typhimurium, due to proper processing of human propeptide by murine matrilysin [²²].

The mucosal surface of the small intestine consists of crypts of Lieberkühn and villi. Due to the constant shedding of gut epithelial cells in the lumen the whole epithelium renews once in four-five days, except for Paneth cells, which live approximately 70 days. Intestinal epithelium is derived from multipotent columnar stem cells located at the base of the crypt, which expess LGR5 receptor and other stem cell-specific protein markers [³⁰]. An alternative pool of stem cells is positioned higher in the crypt wall [^30-32]. Columnar stem cells give rise to actively proliferating transit amplifying cells differentiating into four major epithelial cell lineages: 1) enterocytes absorbing nutrients; 2) goblet cells producing mucus; 3) enteroendocrine cells secreting hormones in the capillaries of the underlying connective tissue (lamina propria); and 4) Paneth cells secreting enteric alpha defensins, as well as other antimicrobials in the mucous layer. Besides, stem cells generate less documented microfold cells (M-cells) responsible for the uptake of mucosal antigens [³³] and the recently described tuft cells secreting endogenous intestinal opioids [³⁴]. Paneth cells protect the adjacent stem cells and the whole gut epithelium from microbial infection and regulate bacterial colonization of the gut [³⁵].

Enteric alpha defensins are the most abundant products secreted by Paneth cells [³⁶]. The main inducers of their secretion are the products of degradation of Gram-positive and Gram-negative bacteria, inhabiting the gut including: muramyl dipeptide, bacterial lipopolysaccharide, flagellin, lipid A, and unmethylayed CpG sequences in bacterial DNA. Their presence in the mucous layer of the intestinal epithelium is constantly monitored by the three receptor types that are expressed in Paneth cells and enterocytes: 1) toll-like receptors (TLRs, including the most abundant cell surface receptors 2 and 4); 2) cytoplasmic nucleotide oligomerization domain-like receptors (NLRs, in particular NOD1 and 2); and 3) retinoic acid inducible gene 1-like receptors (RLRs, including the most abundant receptor RIG1) (Figure 1) [³⁷]. Receptor signals transmitted by the MAP kinase signaling pathway induce translocation to the nucleus of transcription factors, which initiate transcription of genes that are involved in functioning of the innate and adaptive immune systems and stimulation of inflammation, wound healing, and angiogenesis in the adjacent connective tissues [^38-40].

Detection of microbial presence in the mucous layer of the gut results in massive release of enteric alpha defensins by Paneth cells that leads to an enhanced killing of microbes in the lumen. Simultaneous secretion of alpha defensins in lamina propria [¹²] triggers two opposite defense mechanisms of pro- and anti-inflammatory response. According to the first mechanism, human enteric alpha defensins bind to as yet unidentified Gαi-protein coupled receptors located on the surface of macrophages and T lymphocytes of lamina propria (Figure 1) [^41-43], which leads to proliferation of T lymphocytes and their chemotaxis to the site of inflammation [⁴⁴]. Moreover, it was shown that murine defensins 2 and 3 (as well as beta defensins) may induce reversible formation of ion channels on apical membranes of undifferentiated Cl^-secreting cells of the crypt. This results in salt and water secretion, causing the crypt lumen to be flushed after Paneth cell discharge [⁴⁵].

At the same time enteric alpha defensins moderate intestinal anti-iflammatory response, as a consequence of suppression of IL1 beta release from bacterial lipopolysaccharide-activated monocytes [⁴⁶] and inhibition of interleukin 17-producing T helper cell proliferation in lamina propria (Figure 1) [³⁶]. In order to avoid the immune response, microbes inject effector proteins into intestinal epithelial cells that either block their immune and inflammatory response (mostly due to inactivation of transcription factors NF-κB or AP-1) or reprogram signaling pathways sensing and killing bacteria [⁴⁷,⁴⁸]. Besides, microbes are able to block expression of enteric alpha defensins and other antimicrobial substances by Paneth cells using as yet unknown mechanisms [⁴⁹].

Changes in secretion of enteric alpha defensins have been registered in the intestinal epithelium of patients with inflammatory bowel disease (IBD) - a large group of inflammatory illnesses of the gastrointestinal tract, which are caused by immune system over-activation due to a loss of tolerance to gut microflora [^50-52]. Two major types of IBD are Crohn's disease (major location - terminal ileum) and ulcerative colitis (predominant lesions in colon and rectum). Susceptibility to Crohn's ileitis in European and North American populations is most strongly associated with mutations in the gene encoding cytoplasmic protein NOD2 - bacterial muramyl dipeptide receptor from NLR family, which is essential for activation of enteric alpha defensins secretion by Paneth cells (Figure 1) [⁵³]. Accordingly, mutations in NOD2 encoding gene (most frequently a frame shift Leu1007fsX1008, caused by single nucleotide insertion 3020insC or "snp13") lead to a decreased level of enteric alpha defensins in the intestinal mucosal extracts [⁵⁴]. Analysis of Nod2-deficient mice confirmed decreased secretion of alpha defensins 1-6 by Paneth cells and demonstrated inability of such mice to develop intestinal inflammation, which results in their susceptibility to bacterial infection by oral administration [⁵⁵]. Part of the cases of Crohn's ileitis is independent of NOD2 genotype and is linked to changes in the WNT pathway, mostly due to a decreased activity of transcription factor TCF4 that binds to enteric defensins gene promoters [⁵⁶].

The second major type of IBD - ulcerative colitis is associated with the infection of gastrointestinal tract by bacterium Helicobacter pylori [⁵⁷]. Subsequent over-activation of the intestinal immune system results in elevated secretion of enteric alpha defensins (as well as TNF alpha and IL1 beta) suggesting that Helicobacter is able to avoid the innate and adaptive immune response in human intestine by as yet unknown mechanisms [⁵⁸].

Similar to ulcerative colitis, 15% of gastrointestinal malignancies arise as a consequence of chronic microbial infections, as demonstrated by the raised chance of hepatocellular carcinoma in patients with chronic hepatitis, association between Helycobacter pylori infection and higher gastric cancer risk, and increased chance of colon cancer in patients with inflammatory bowel disease [⁵⁹]. At initial stages of colon carcinogenesis, mutations in intestinal epithelial cells lead to constitutive activation of the Wnt pathway in early adenoma cells, which simultaneously follow differentiation programs of progenitor and Paneth cells [⁶⁰]. This results in 60 fold increase of DEFA5 and DEFA6 production by tumors, as compared to normal colonic epithelium [⁶¹,⁶²]. Besides, both early adenomas and adenocarcinomas acquire the ability to secrete these proteins into the bloodstream. Thus, enteric alpha defensins are promising markers for early diagnosis of colon cancer provided that test sensitivity is sufficient for their robust detection in sera.