Introduction

Host defense peptides (HDPs) are polypeptides assembled from fewer than 100 amino acids. These peptides tend to have a high proportion of positively charged and hydrophobic residues.1,2 Based on the Host Defense Peptides Database in 2022 (https://wangapd3.com/main.php), scientists have identified or predicted a total of 3257 HDPs. These HDPs are derived from various organisms, including 365 from bacteria, five from archaea, eight from protists, 30 from fungi, 371 from plants, and 2521 from animals. Among these, 348 are classified as defensins and have an average length of 41.26 residues, with an average net charge of 4.61.

Based on the amino acid composition, length and structural characteristics, mammalian HDPs are generally categorized into two prominent families: defensins and cathelicidins. The cathelicidins comprise a conserved gene family, initially thought to produce small proteins with cysteine protease inhibitor activity, as well as antimicrobial activity.3,4 Recently, however, the notion of protease inhibitor activity of cathelicidins has been refuted.5 Although pro-defensins are inactive, pro-cathelicidins and cathelicidins are equally bactericidal.5 Initially, direct activity against microorganisms was deemed to be the primary role of HDPs. For example, mouse cryptdins and human alpha defensin-5 (HD5) directly kill Salmonella, and human alpha defensin 6 (HD6) traps Salmonella in a high-ordered “nanonet” structure to prevent infection.6,7 However, many HDPs lose their antimicrobial potency in some localized microenvironments. Even so, it is becoming increasingly clear that HDPs act as immunomodulatory mediators that regulate the mammalian innate immune response and moderate the establishment of adaptive immunity.8,9 The structure, function and mechanism of action of cathelicidins4,10,11,12,13 and defensins14,15,16,17,18,19,20 have been reviewed over the past few years. Nonetheless, given the enormous number of defensins known, the diversity of their biological activities, the intricate ways in which they function, and the multitude of targets they interact with, publishing a comprehensive review on this topic is an arduous, if not impossible, feat.

Thus, this review is focused on defensins in host defense. It mainly summarizes and discusses their properties, biological function, related clinical diseases, and therapeutic potential, as well as their nutritional regulation. We will also cover the function of defensins in promoting the chemotaxis of immune cells, their influence on multiple signaling pathways involved in inflammation and immunity, how they maintain gut microbial homeostasis and their regulation of epithelial injury and the promotion of proper organ development and eukaryotic cell death, as well as their contribution to clinical diseases and their therapeutic potential. We will highlight the current knowledge base regarding mammalian defensins and their roles in regulating host health, thus providing a theoretical basis for clinical therapeutic strategies targeting defensins to treat disease.

History of defensins

In 1985, Dr. Robert Lehrer from the University of California, Los Angeles, was the first to discover and name defensins. He reported that rabbit defensins MCP-1 and MCP-2 had strong antibacterial and antiviral activities21,22 (Fig. 1a). That same year, he and his team discovered and characterized the structure of human neutrophil peptides (HNP1-3)23 (Fig. 1a). Over time, more defensins were found, such as HNP4 (ref. 24) in 1989, HD5 (ref. 25) and HD6 (ref. 26) in 1992 and 1993, respectively, and human β-defensins (hBD1-3)27,28,29 in 1995, 1997, and 2001, respectively (Fig. 1a). The first θ-defensin was found in 1999 (ref. 30) (Fig. 1a). Since then, with the widespread use of in silico analyses, researchers have been able to predict the sequence and structure of defensins31 (Fig. 1a). Meanwhile, in the late 20th and early 21th centuries, the scientific community widely studied the processing and storage mechanisms of defensins6,32,33,34,35,36 (Fig. 1a). In addition, from 1988 to 2010, the antibacterial mechanisms of defensins have been established, which involve a membrane penetration mechanism and targeting lipid II by inhibiting cell wall synthesis37,38 (Fig. 1a). During this period, the role of defensin dimers, disulfide bonds and other biochemical structures in their antibacterial function and mechanisms have also been analyzed39,40,41,42,43 (Fig. 1a).

Fig. 1
figure 1

Introduction to the history of defensin research. a Timeline of defensin characterization, processing and storage mechanisms and antibacterial mechanisms. b Timeline of regulation mechanism of defensin gene. c Timeline of studies on the role of defensin-mediated host immunity in various disease progression. SNP single-nucleotide polymorphism

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In 2007, chromosome 8 was fully sequenced and analyzed by the Human Genome Project, resulting in the description of the first human defensin gene family landscape44,45,46 (Fig. 1a). The first defensin database was established in the same year, incorporating 350 defensins47 (Fig. 1a). Then, in the early decade of the 21st century, researchers gradually analyzed the regulation pattern of defensin gene expression48,49,50,51,52,53 (Fig. 1b). These results provide essential data and technical support for the subsequent research into the genetic engineering and drug development of defensins.

Over the last two decades, defensins have been found to regulate immune cell chemotaxis and to be involved in regulating sperm activity, male infertility, thrombosis, melanin deposition, and other essential biological functions.54,55,56,57,58 Further, defensins have also been shown to induce the host’s innate immune response, enhance the host’s adaptive immune response and promote the activation of T cells, macrophages, and other immune cells59,60,61,62,63,64,19 β-defensin has a shorter pro-segment than α-defensin. It may be due to differences in the transcription patterns (α-defensins are usually constitutively produced, while most β-defensin expression occurs in response to stimuli15), leading to different processing and intracellular transport requirements for the mature peptides to rapidly react to immune responses. It is worth noting that crystal structure analyses of defensins show that defensins exist as dimers or multimers.90,91,92 Lu et al. have preliminarily studied the importance of dimerization for the biological roles of defensins. They found that these polymers have stronger antibacterial and membrane destruction activity and can enhance binding to multiple molecular targets compared to monomers.91,93,94,95

Based on the difference in the coding exons, α-defensins are classified into myeloid and enteric α-defensins (Fig. 2a). HNP1-4 are four of the six known myeloid α-defensins and are expressed primarily in the granules of neutrophils96 and certain lymphocytes,97 as well as natural killer (NK) cells.98 Notably, mouse neutrophils lack defensins.99 HNP1-4 are stored in the azurophilic granules as fully processed mature peptides.34,100 Upon fusing with phagasomes, α-defensins-laden azurophil granules then release large amounts of HDPs in the proximity of the pathogen surface, where they quickly penetrate the cell membrane due to their amphipathic nature.64,101 The other two α-defensins, HD5 and HD6, are enteric α-defensins that are mainly expressed by Paneth cells (PCs).102,103,104,105 Unlike pro-HNP1-4 processing and vesicle storage, HD5 and HD6 are stored in secretory vesicles as a pro-peptide and are processed by one or more isoforms of Paneth cell trypsin.32 However, whether the pro-HD5 peptide is converted into its mature form during secretion or within the lumen is unclear. In addition to PCs, the reproductive tract and oral cavity also express HD5 and HD6. Interestingly, these two peptides are functionally different. The antibacterial activity of HD5 is to kill bacteria directly,106 while HD6 does so indirectly by forming self-assembled nanonets in order to trap bacteria and prevent infection.7,107,108,109 Although mouse neutrophils lack defensins, mouse PCs express more than 20 α-defensins throughout the mouse small intestine,110,111,112 which are also called cryptdins. Seventeen cryptdins (Cryptdin1-17) have been identified at the protein level.113,114 All the peptides have potent in vitro bactericidal activity,41 with S. aureus appearing to be more susceptible to cryptdin-mediated killing than E. coli.41 Mouse cryptdins are processed into their active form by matrix metalloproteinase 7 (MMP7) during granulogenesis.6,33 Indeed, mice lacking MMP7 cannot process the precursors of pro-cryptdin, leading to a deficiency of mature cryptdins, thus impairing their ability to scavenge infections and regulate immune homeostasis.6 In mouse and human PCs, mature α-defensin is oxidized to prevent internal digestion.115

Compared with α-defensins, the localization of the cysteine residues along the amino acid sequence of β-defensins (BDs), the folding pattern of the peptide chain and the disulfide bond pattern are entirely different (Fig. 2b). The peptide chains of BDs fold to form three β-lamellae with four conserved glycine, proline, threonine and lysine residues. The synthesis and secretion of BDs are also different from α-defensins. BDs are directly secreted into the extracellular space in their mature form to exert immunomodulatory and antibacterial activities.116 BDs mainly display stimulated expression, but constitutive expression patterns also exist. For example, the promoter of DEFB1 does not contain response elements for NF-κB and AP-1, so DEFB1 gene expression is not upregulated in response to inflammatory factors but is physiologically expressed in epithelial cells.117 However, the expression of most BDs is limited to specific tissues or epithelial cells where they perform a particular function. For example, the production of macaque BD126 is confined to the epididymal epithelium, where it is attached to membranes of sperm cells as they traverse through the epididymis. This exclusive function safeguards macaque sperm from being attacked by the immune system within the female reproductive tract.118

Presently, the progress in studying θ-defensins is relatively slow compared with α-defensins and β-defensins. However, from an evolutionary perspective, it is clear that θ-defensin genes arose from mutated α-defensin genes.30,64 θ-defensins are the only cyclic peptides in animals (Fig. 2c) and have been isolated from rhesus macaques and baboons. Rhesus θ-defensins (RTDs) are primarily synthesized in the bone marrow and secreted by neutrophils, PCs and monocytes.119 Intriguingly, θ-defensins are chimeras of 18 residues formed by spliced heads and tails from two separate precursors, each of which contains nine amino acids.30,120 In humans, the θ-defensin gene has an early termination codon that hinders efficient translation of the desired precursor,121,122 indicating that θ-defensins are not exist in the human body and were most likely phased out by natural selection.

Antimicrobial mechanisms of defensins

Defensins possess wide-ranging antibacterial activity against both Gram-negative (G−) and Gram-positive (G+) bacteria in vivo and in vitro.123,124,125,126,127,128,129,130 For example, the anti-Staphylococcal and anti-E. coli activity of hBD3 is 1 mg/L and 4 mg/L, respectively.131 However, the cell membrane structure of G− and G+ bacteria differ (Fig. 3a) as the cell membrane of G− bacteria has three layers that include an outer membrane, a peptidoglycan layer and a plasma membrane, whereas G+ bacteria have only a peptidoglycan layer and a plasma membrane. G− surfaces contain many lipopolysaccharides (LPS) with a negative charge.132 By interacting with negatively charged components on the surface of G− bacteria, defensins destroy membrane barrier function. With the accumulation of defensins on the membrane (Fig. 3b), the electrostatic attraction and penetration of defensins bound to the membrane are enhanced, and the defensins freely diffuse and preassemble on the membrane surface,133,134,135 followed by hydrophobic interactions between the amphipathic peptide domain and the membrane phospholipids.136,137 There are three primary models for defensin-mediated transmembrane pore formation, which are barrel-stave, toroidal pore, and carpet models.135,138,139,140,141 The first proposed mechanism for permeabilization was the barrel-stave model, which serves as a prototype for defensin-mediated transmembrane pore formation. Defensins serve as staves that insert themselves vertically into the phospholipid bilayer, yielding barrel-like structures (Fig. 3c), such as HD5 for G− bacteria.128,142,143,144 The toroidal pore model depicts the insertion of defensins into the membrane, causing a consistent curvature of the phospholipid monolayer from the upper portion to the lower portion (Fig. 3d). In the carpet model, peptide-induced membrane disruption is similar to that of a detergent-like action (Fig. 3e). For example, the cell membrane adsorbs hBD3 through strong electrostatic interaction of Arg12 with POPG lipids in G+ bacteria.145

Fig. 3
figure 3

Antimicrobial mechanisms of defensin. a The cell membrane structure of G− and G+ bacteria. b Defensins accumulate on the cell membrane before destroying it. c–e Illustrations of the various modes of defensins-mediated cell killing, including the barrel-stave model, the toroidal pore model and the carpet model. f The structure Lipid II; g Cell wall biosynthesis begins in the cytoplasm where UDP-MurNAc-pentapeptide is formed. This soluble precursor is then linked to the membrane carrier bactoprenolphosphate (C55P) by MraY, yielding Lipid I (reaction I). MurG subsequently adds GlcNAc to form Lipid II (reaction II). After the formation of the interpeptide bridge (as seen in reaction III), the monomeric peptidoglycan unit undergoes translocation across the cytoplasmic membrane for incorporation into the cell wall (reaction III). It is noteworthy that this interpeptide bridge formation is limited to some Gram-positive bacteria, as highlighted by research.38 Note: To better demonstrate the crosstalk mechanism of defensins in regulating immune homeostasis, the intestine containing PCs and mucosal structures was used as the background of the regulatory network

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However, the membrane destruction model cannot fully explain the complete mechanism behind the defensin-mediated bacterial killing. Specifically, it is difficult for this model to account for how defensins can swiftly eradicate bacteria in the LPS-deficient outer membrane of G+ bacteria. Thus, it is likely that another mechanism exists for defensin-mediated bacterial killing. One possible alternative mechanism is that defensins disrupt cell wall synthesis (Fig. 3f, g) by targeting the membrane-anchored cell wall precursor, lipid II, which is crucial for the process.146,147,148,149 Plectasin, a fungal defensin secreted by Pseudoplectania nigrella, displays strong antibacterial activity against G+ bacteria, even against otherwise resistant clinical isolates.38 Tanja and colleagues found that plectasin does not cause any disruptions to membrane integrity as it had no influence on the typical features of the membrane penetration mechanism, such as membrane potential and intracellular K+ contents.38 Interestingly, plectasin treatment led to an accumulation of the cell wall precursor, UDP-MurNAc-pentapeptide.38 Plectasin effectively prevents the interaction between the lipid I and lipid II carriers and cell wall biosynthetic enzymes by bonding with them in a 1:1 molar ratio. The equilibrium-binding constants for lipid II and lipid I are 1.8 × 10−7 mol and 1.1 × 10−6 mol, respectively, indicating that the second sugar in lipid II, N-acetyl glucosamine (GlcNAc), plays a role in stabilizing this complex.38 In addition to plectasin, researchers have identified other defensins targeting lipid II, such as hBD3 and HNP1, Cg-Defh1-2 from crassostrea gigas, oryzeasin and eurocin from fungi, and lucifensin from maggots.146,147,150,151,152,153 For example, in S. aureus treated with hBD3, the UDP-MurNAc-pentapeptide, a cell wall precursor, was also found to accumulate,147 like in the case for plectasin. Further, hBD3 was shown to inhibit the activity of staphylococcal penicillin-binding protein 2 (PBD2) when the molar ratio of hBD3 to lipid II is 2:1. However, hBD3 treatment also resulted in a decreased membrane potential, and transcriptome data indicated that hBD3 treatment was partially like HDPs treatment exposed to membrane-active α-helices.147,150 Thus, hBD3 exhibits a pleiotropic antibacterial mechanism against S. aureus involving cell wall synthesis inhibition via targeting lipid II and effects on membrane permeabilization.

Recently, the Wehkamp lab found that in a reduced physiological environment, the disulfide bridges characteristic of defensins become disrupted, rendering them susceptible to protease degradation. This process liberates novel antimicrobial peptide fragments that enhance the antimicrobial repertoire and may thus be an evolutionary trait enabling the host to mount an effective broad-spectrum response towards invading pathogens with minimal resources. For example, duodenal fluid- and gastrointestinal-derived trypsin degrade full-length HD5, hBD1, and HNP4 into various bioactive fragments with different antibacterial properties. Other fragments showed different antibacterial activity. As an example, HNP41-11 exhibits superior antimicrobial potential in comparison to the intact peptide on mass and molar levels.154 Other fragments, including HD51-9, HD51-13, HD57-32, and HD5fl, substantially affected the growth of all tested bacterium, while others, like HD514-32 and HD510-27, were ineffective against the tested bacteria under the same experimental conditions.107 The minor differences in fragment sequences of HD51-9 and HD51-13 resulted in different antimicrobial activity.107 These results suggest that defensin fragmentation is a fine-tuning mechanism for host-microbe interactions.

The biological functions of defensins

As the number of studies of defensins increases, it has been found that these molecules act in numerous biological processes, including showing immunomodulatory and chemotactic activities, maintaining mucosal barrier function, balancing the gut microbiota and regulating organ development and cell death. Therefore, gradually defensins have been perceived to be innate immune factors. Here, we review the biological functions of defensins that have been discovered to date.

Immunomodulatory activity

Increasing evidence indicates that the direct bactericidal activity of defensins in regulating the antibacterial immune response is not the only essential role of defensins in regulating host immune homeostasis. Specifically, they also modulate both innate and adaptive immune responses as immune regulatory factors.1,14,101,155 Not surprisingly, dysregulation of defensins expression is associated with autoinflammatory and autoimmune diseases, including sepsis, irritable bowel syndrome (IBS), atherosclerosis, thrombosis, rheumatoid arthritis and type 1 diabetes.74,76,156,157,158,159,160,161,162 However, the involvement of defensins in immune regulation is very complicated, and their role goes far beyond simply acting as immunomodulators via a singular receptor or linear signaling within the immune system (Fig. 4). A case in point of the complex roles of defensins in the immune response is the protein–protein interaction network of hBD3. Notably, hBD3 interacts with no less than 46 proteins or receptors and 1779 genes show differential expression upon hBD3 stimulation of TLR4 agonist KDO2-lipid A-primed mouse macrophage cells.163 These varied responses suggest that defensins exert their effects mainly by interacting or trans-activating various extracellular and intracellular receptors.

Fig. 4
figure 4

Regulation role of defensins in immune homeostasis. a mBD14 promotes B cell proliferation via TLR2 and improves the M1/M2 macrophage balance and induces regulatory T cells. b Mature α-defensins prevent NLRP3 inflammasome activation and the release of IL-1β. c hBD3 is activated by EGFR-mediated MAP kinase and JAK/STAT signaling pathways after H. pylori infection. d By competitively inhibiting the LPS-induced activation of the NF-κB via TLR4, pBD2 can effectively restrict downstream inflammatory cytokine secretion. e HNP1 released by neutrophils enters macrophages to bind to mRNA, and then inhibits mRNA translation of various inflammatory factors. f, g hBD2, hBD3, and HNPs inhibit the secretion of inflammatory cytokine; h mBD2 promotes the maturation of DCs via TLR4 signal. i Defensins recruit various immune cell to clear out dead cells and pathogens. j hBD2 and hBD3 regulates the repair of barrier function via the CCR6-Rho-ROCK signaling pathway. k In a nutrition-deficient state, the continuously activated α-defensins promote the resistance to invasion by enteric pathogens through an mTOR-Hes1-Atoh1-MMP7-α-defensins axis

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Regulation of autoimmunity is one of the main functions of defensins. Miani et al. found that endocrine cell-expressed mBD14 promotes B cell proliferation and increases their secretion of IL-4 by acting on TLR2 (ref. 159). Subsequently, IL-4 further improves the M1/M2 macrophage balance and induces regulatory T-cell responses to prevent autoimmune diabetes159 (Fig. 4a). In addition, mBD2 functions as an endogenous TLR4 ligand that acts upon immature dendritic cells (iDCs), resulting in the enhanced expression of costimulatory molecules and the maturation of DCs50 (Fig. 4h). These findings indicate that defensins can regulate acquired immune responses. Further, in the nutritionally-deficient state, the continuously activated α-defensins promote resistance to enteric pathogen invasion via an mTOR-Hes1-Atoh1-MMP7-α-defensins axis164 (Fig. 4k). In addition, defensins also regulate the expression of inflammatory factors. Koeninger et al.70 found that hBD2 improves disease activity indices and prevents colitis-associated weight loss in three mouse models (dextran sodium sulfate (DSS), 2,4,6-Trinitrobenzenesulfonic acid (TNBS) and T-cell transfer into immunodeficient recipient mice). Furthermore, they found that hBD2 engages with CCR2 on DCs to inhibit NF-κB activity and to promote CREB phosphorylation, thus reducing the expression of inflammatory factors (Fig. 4f). Our previous studies showed that pBD2, a porcine β-defensin, competitively inhibits LPS- and DSS-induced activation of NF-κB signaling via TLR4, thus dampening the secretion of inflammatory cytokines69,165 (Fig. 4d). Similarly, Zhang et al. and Lian et al. observed that pBD2 decreases the adherence of E. coli to cells and alleviates inflammation via the TAK1-NF-κB pathway.166,167,314 In fact, in 2019 alone, they were responsible for over 13.7 million fatalities.315 Despite advances in medicine, our current antimicrobials have become less effective over the past few decades due to the increasing prevalence of drug resistance, as exemplified by multidrug-resistant tuberculosis.314,316 Notably, the immunomodulatory activity of defensins in clearing pathogenic infections is extensive and challenging for microorganisms to develop resistance to.

Numerous studies have highlighted the therapeutic potential of defensins as a form of treatment for various types of infections. One such example is the prevention of mycobacterium tuberculosis in mice through the subcutaneous injection of HNP1. Moreover, in vitro mechanistic experiments further demonstrated beneficial outcomes to verify using HNP1 as an anti-infective agent for tuberculosis.317 Exogenous supplementation of recombinant hBD1 or hBD2 effectively controlled Salmonella infection. Nearly 50% of infected mice that were inoculated with recombinant hBD1 or hBD2 were still alive 206 h post-inoculation compared to complete lethality within just 24 h for control mice, while in the liver and spleen, the abundance of live Salmonella was remarkably reduced in the treated mice.318,319 Deficiency of mBD2, an analog of hBD2, in a mouse model of local P. aeruginosa-mediated corneal infection showed a worse outcome than control mice, indicating that mBD2 promotes resistance to P. aeruginosa-induced keratitis.79 Likewise, synthetic nine-mer peptides, specifically ALYLAIRRR and ALYLAIRKR, developed based on the active fragment of insect defensins, have been observed to provide protection in mice infected with lethal Methicillin-resistant S. aureus (MRSA).320

Similarly, administering exogenous defensins has also achieved beneficial effects against viral pathogens. For example, HNP4 and HD6 can block herpes simplex virus (HSV) infection.321 In addition, studies have shown that recombinant mBD2, when given before or after exposure to human influenza A virus (IAV), can protect experimental mice from a lethal virus challenge by 70% and 30%, respectively.322 pBD2 inhibits the proliferation of pseudorabies virus in transgenic mice.75 It is worth noting that Zhou Rui’s laboratory constructed the first pBD2 transgenic pig and explored the role and mechanism of pBD2 transgenic pig in swine influenza virus (SIV) infection. Studies have shown that pBD2 transgenic pigs can effectively relieve SIV-related clinical symptoms. Mechanistically, pBD2 enters host cells, mediated by energy-dependent endocytosis, to bind SLC25A4, a pro-apoptotic molecule.80 This interaction inhibits SIV-induced cell apoptosis.80

These experimental data all confirm the excellent therapeutic potential of defensin in anti-infection. Despite these benefits, no clinical trials currently utilize human defensin molecules in infectious disease treatment. Still, several clinical trials have involved the use of two defensin analogs, which will be discussed later (6.2 Clinical Trials of Defensins).

Inflammatory bowel disease and defensins

IBD, including ulcerative colitis (UC) and CD, is a complex barrier disease marked by a loss of tolerance towards commensal microbes, altered microbial composition, barrier dysfunction and chronic inflammation of temporal intensity.323 In the intestine, defensins help strengthen host immunity and help maintain the correct balance between defending against harmful pathogens and tolerating beneficial microorganisms. However, when the expression of defensins decreases, it disrupts immune homeostasis and exacerbates intestinal inflammatory response. Therefore, the alteration of defensin expression is considered an indispensable factor in the pathogenesis of IBD.

β-defensins: focusing on hBD2

The most replicated finding in active IBD is an increase of hBD2. Patients with UC exhibit a ten-fold increase and patients with colonic CD have a 3–4-fold increase compared to controls, and thus both groups express hBD2 at relatively high levels, especially in the inflamed tissue vs the non-inflamed tissue; however, there was no obvious difference in patients with ileal CD.237,324,325,326,327,328,329,330 Notably, in UC, hBD2 levels increase with the degree of inflammation, whereas this is not observed in CD.330 Another study found that patients with colonic CD exhibit reduced functional antimicrobial activity against commensal gut microbiota compared to patients with UC,229 but it is unclear if this difference is hBD2-mediated. The differences in hBD2 abundance observed between UC, colonic CD and ileal CD have different mechanisms. The most pronounced genetic risk factor of CD, especially ileal CD, is a frameshift mutation in the Nod2 gene (around one-third of patients with CD carry this mutation), rendering them incapable of proper hBD2 expression.331,332,333,334 In contrast, patients with UC exhibit diminished colonic mucin production, which may prevent hBD2 (and other HDPs) from being chemostatically retained in the mucus layer.324,330,335 Thus, enhanced hBD2 expression in UC is likely a counter-response to protect against microbial encroachment caused by diminished barrier function, as well as defects in mucus production, whereas reduced or unaltered hBD2 expression in CD may instead relate to different disease pathology and etiology (such as frameshift mutations).

In addition, hBD2 is distributed differently among the colon cell population. Patients with UC exhibit notably higher hBD2 expression in the luminal/villous compartment (I/v-IEC) compared to the crypt compartment (c-IEC), suggesting that mature IECs facing the intestinal lumen are responsible for producing more hBD2 (ref. 328). The production of defensin by plasma cells is also thought to be clinically relevant in UC since these cells accumulate in large numbers between the distorted crypts and muscular mucosae.336 According to Rahman et al., there is a significant increase in plasma lineage cells observed in colonic samples of patients suffering from UC compared to those with CD and control patients, and hBD2 secreted by plasma cells was upregulated by two- to threefold.336 This highlights the potential mechanism by which plasma cells regulate UC through hBD2 at sites of intestinal inflammation. No independent studies have investigated the difference in hBD2 expression between plasma cells, I/v-IEC and c-IEC. We speculate that the potential mechanism of hBD2 to prevent microbial attack might be related to the distance between cells and the intestinal cavity. The closer the cell is to the intestinal cavity, the higher the expression is. A study involving systemic administration via subcutaneous administration of hBD2 in the scapular region in mice found that recombinant hBD2 reduced inflammation, improved disease activity indices and prevented colitis-associated weight loss.70 And another study demonstrated a potential improvement in DSS-induced changes in paracellular permeability and mucosal lesions through the intrarectal administration of pBD2, which may impact the activation of NF-κB signaling.69 However, to date, there have been no studies of hBD2 in clinical trials in IBD. Given the differences in the expression of hBD2 in cases of UC, ileum CD, and colonic CD, these three clinical phenotypes may respond differently after hBD2 treatment. We speculate from our previous description that a protective effect of hBD2 therapy might be observed more often in UC or colonic CD than in ileal CD. Nonetheless, it appears that no related studies have been conducted thus far.

The expression of hBD1 is constant in the intestinal epithelium, and its expression levels remain unchanged in patients with IBD.337 Despite this, the precise function and mechanism of hBD1 concerning IBD have not been fully elucidated. hBD3 and hBD4 are like hBD2 and are noticeably increased in expression levels within the colon of patients with UC and CD.326 This observation may be because hBD2, hBD3 and hBD4 are inducible rather than constitutively expressed. However, in patients with IBD, the concentration of hBD3 and hBD4 are much lower than hBD2, and there is no significant difference in serum hBD3 and hBD4 (ref. 337). This suggests that hBD3 and hBD4 may be able to regulate local immunity. In addition, Meisch et al. investigated the distribution of hBD3 in the terminal ileum of healthy individuals and patients with CD. According to their findings, in the healthy small intestine, hBD3 is primarily observed in the luminal surface of the intestinal epithelium, as well as inside PC granules. However, in cases of CD, hBD3 relocates to the basolateral surface of the villus epithelium and accumulates in the lamina propria of the terminal ileum.326 We speculate that in patients with CD, hBD3 may, on the one hand, resist the microbial attack on the surface of the intestinal cavity and, on the other hand, enter the lamina propria and perform chemotaxis to recruit immune cells. Like with hBD2, there are still no clinical trials of hBD3 and hBD4 to treat IBD.

α-defensins: focusing on HD5

HD5 and HD6 are secreted mainly by PCs located in the small intestine and ileum, with a small amount coming from IECs.338 PCs continuously express HD5 and HD6 to protect nearby epithelial stem cells situated at the base of the crypts, thereby maintaining barrier integrity.337 Nevertheless, in IBD, the microbes and their metabolites and inflammatory factors interact to destroy PCs and IECs, thus disrupting HD5 and HD6 expression.338,339 Multiple studies have demonstrated a significant reduction in ileal HD5 and HD6 levels in patients with CD.340,341 As a result, antibacterial activity mediated by HD5 and HD6 is disrupted, resulting in a massive microbiome’s severe invasion of the intestinal mucosa and destruction of the epithelial barrier. Interestingly, patients with UC and colonic CD exhibit a significant increase in HD5 levels in their colon.328,342,343,344,345 This is mainly due to the absence of PCs in the colon of healthy people. However, after the occurrence of IBD, PC translocation hyperplasia occurs in the colonic crypts of patients with UC and colonic CD.337,344,346 We speculate that the possible mechanism is the colonic defensive response to microorganisms after the occurrence of IBD. Multiple mouse and cell studies have consistently confirmed the therapeutic effect of HD5 on colitis. For example, Shukla et al. found that HD5 administration improved ethanol- and colitis-triggered dysbiosis, inflammation response and barrier defects in the small intestine and colon.347 In addition, Zeng et al. created the recombinant NZ9000SHD-5 strain by transfecting the DEFA5 gene vector of pN8148-SHD-5 into Lactococcus lactis (L. lactis), which continuously produces mature HD5 (ref. 348). They found that NZ9000SHD-5 ameliorates intestinal damage and inflammation in mouse with DSS-induced colitis compared to the L. lactis + DSS group. These direct HD5 supplementation trials suggest that increased defensin expression is a potential avenue to treat colitis. Indeed, in a randomized clinical trial of anti-TNF therapy in patients with UC, HD5 was significantly upregulated in those who responded to the therapy compared to those that did not, with a lower microflora imbalance index in the responders.349 This suggests that the rise of HD5 may play a vital role in successfully treating UC with anti-TNF therapy. However, this needs to be confirmed experimentally; for example, in HD5 transgenic and knockout mice in a colitis model.

Unfortunately, to date, no clinical trials for IBD utilizing HD5 have been reported. However, some clinical retrospective and correlation studies have revealed the mechanism of PC regulation of HD5 and HD6 expression. This is helpful as it would then allow the targeting of the pathway of HD5 secretion by PCs as an additional means to treat IBD, to develop related inhibitors or agonists and to provide a solid foundation for the clinical application of HD5. For example, NOD2, a significant risk factor for ileal CD, is highly expressed in PCs, as shown by genome-wide association studies (GWASs).104,350,351 Economou and colleagues performed a meta-analysis and found that the CD risk is significantly increased in individuals with two mutated Nod2 alleles (17.1-fold) and Nod2 heterozygotes (2.4-fold).352 The mRNA expression of DEF5A (the gene encoding HD5) in PCs is significantly reduced in patients with a Nod2 mutation compared to patients with CD expressing wild-type Nod2 (ref. 62). These data suggest that NOD2 directly regulates HD5 in PCs to prevent CD and enhance mucosal protection. However, the Nod2 mutation does not fully explain the downregulation of HD5. This is because healthy patients with Nod2 mutations have higher HD5 expression levels than patients with CD expressing wild-type Nod2 (ref. 62). In addition, the DEFA5 gene promoter in PCs lacks NF-κB binding sites, indicating NOD2 is not directly involved in DEFA5 gene transcription,62,337 suggesting that other factors also influence the regulation of HD5. Notably, Wnt signaling regulates the positioning, differentiation and maturation of PCs.353 Blocking the Wnt signaling pathway disrupts HD5 production in PCs and induces CD.104 This is because HD5 is a transcriptional target of TCF1 and TCF4, which act downstream in Wnt signaling, and thus is directly regulated by Wnt signaling in PCs.104,271,354 Both adult and child patients with CD exhibit a decrease in the expression of TCF1 and its active isoforms, confirming its role in CD pathology.244,355 Reduced expression of TCF4 is also associated with reduced expression of HD5 in PCs in patients with ileal CD irrespective of the degree of inflammation. Nevertheless, this association is not observed in patients with colonic CD or UC. Moreover, in Tcf-4 knockout mice the α-defensins expression and bacterial killing activity were lower compared to wild-type mice, and in both species the reduced defensins expression occurred independently of the NOD2 genotype.340

Similarly, HNP1-3 expression is also dysregulated in IBD. Multiple studies have repeatedly confirmed that patients with IBD highly express HNP1-3 and patients with UC have significantly higher expression than patients with CD.340,356 It is worth noting that experiments in mice have confirmed that HNP1 has dual effects. On the one hand, low doses of HNP1 (5 μg/day) can ameliorate DSS-induced colitis.78 On the other hand, high doses of HNP1 (100 μg/day) can promote a macrophage-driven inflammatory response and aggravate the progression of DSS-induced colitis.357 In addition, data from clinical samples showed that individuals with active UC have significantly higher expression of HNP1 compared to those with UC in remission. Kanmura et al. confirmed that an increased gene copy number of HNP1-3 and the severity of UC are positively correlated.358 These data suggest that HNP1-3 may be a risk gene for severe UC, and its high expression in patients with UC may induce a hyperinflammatory response. However, it is still challenging to know where the critical concentration of HNP1-3 is for the concentration-transition-dependent effect in patients with UC and whether to consider the concentration between HNP1-3 alone or the concentration of the three in total. These answers will require further studies in patients with UC in remission.

Defensins in diabetes and obesity

Type 2 diabetes is closely linked to obesity, which is expected to affect 1 billion people worldwide by 2030 (ref. 359). Evidence of dyshomeostasis of defensin in serum and tissues of patients with diabetes has been reported. For example, hBD1-3 is down-regulated, and HNP1-3 is upregulated, in the serum of individuals with type 1 diabetes (T1D).360,361,362,363 According to a prospective study examining cardiovascular risk factors, individuals belonging to the highest quartile for plasma HNP1-3 show a significant correlation with being leaner, more insulin sensitive and possessing lower levels of total and LDL-cholesterol.364 On the other hand, those belonging to the lowest quartile for circulating HNP1-3 lack these benefits.364 Moreover, even after considering the factors of age, BMI, insulin sensitivity and smoking, the links with serum lipids remain solid.364 Another investigation conducted by Liu et al. found that HNP1 inhibits hepatic gluconeogenesis via a c-Src-dependent pathway, resulting in lowering blood glucose concentration in normal mice and Zucker diabetic fatty rats.71 In addition, a low number of HNP1-3 gene copies may increase the risk for renal dysfunction,83 which is closely related to diabetes.365 These data suggest that HNP1-3 has a practical clinical significance in the control of blood lipid levels and treating diabetes-related diseases. Of note, various studies have pointed to the role of HD5 in both obesity and diabetes. For example, the levels of HD5 in the jejunum have been found to have an inverse correlation with obesity in humans.366 In addition, when mice are fed a high-fat diet and are deficient in vitamin D, there is a decrease in the expression of the murine analog, α-defensin-5. Functionally, mice with α-defensin-5 knockout experience more severe liver steatosis and metabolic disorders than the HFD-fed mice. However, when these mice were given exogenous HD5, observed symptoms improved, indicating that the protein is an essential regulator of metabolic balance.367 In addition, Larsen et al. fed mice a 60% HFD for 13 weeks and treated them with physiologically relevant levels of HD5 (0.001%) or vectors for 10 weeks. They found that HD5-treated mice show better glucoregulatory performance, as well as improved plasma and liver lipid levels in comparison to those treated with vectors.72 These findings demonstrate that the implementation of human defensins may hold promise in enhancing host metabolism, as well as mitigating the commonly related triad of dyslipidemia, obesity and diabetes. Moreover, clinical sample data and in vivo studies in mice and in vitro cell experiments also support the therapeutic benefits of defensins in treating obesity and diabetes. Nevertheless, no trials have been conducted in people with related diseases. The difficulty in producing defensins remains a significant challenge. However, the Wehkamp laboratory recently demonstrated that intestinal proteases digest HD5 to form peptide fragments with potential antimicrobial activity.107 This newly generated peptide fragment may replace full-length peptides, providing a solution for the clinical use of HD5 active fragments.

Chronic inflammatory lung disease and defensins

The lungs inspire numerous pathogens daily. As defensins play a vital role in the fight against pathogens and mediate immune response, the role of specific defensins in regulating inflammatory lung disease has been investigated. Multiple studies have confirmed that single-nucleotide polymorphisms and copy number variations of DEFB1 and DEFB2 are associated with chronic obstructive pulmonary disease (COPD) and asthma.368,369,370,371,372,373 The ile38 variant (untranslated regions) of hBD1 was detected in 15.0% of patients, while only 2.8% of healthy individuals carried this variant. Its presence has been found to be significantly associated with the disease.369 Furthermore, over 80% of patients with this hBD1 ile38 variant reported experiencing sputum production for more than three months during their follow-up period. This suggests that the ile38 variant of hBD1 exacerbates the disease state of COPD. In addition, Andresen et al. and Baines et al. reported that hBD1 expression is elevated in bronchia biopsies of patients suffering from asthma or COPD.372,374 This rise in hBD1 expression is associated with COPD’s pathological changes and disease severity.372,374 Similar studies were replicated with hBD2. For example, levels of hBD2 were observed to correlate with IL-8 level as well as COPD severity.375 This result implies that it is an effector in the innate immune response involved in COPD’s pathogenesis. However, studies have also reported that hBD2 is decreased in central airways of COPD individuals who smoke, but not in distal ones.376 In addition, the concentration of hBD2 in pharyngeal washing fluid and sputum of smokers or former smokers is markedly lower than individuals who never smoked.377 Upon co-infection with viruses and bacteria, individuals with COPD have shown a decrease in the production of hBD2. Administering recombinant hBD2 has proven to be effective in reducing lung neutrophilia caused by exposure to cigarette smoke, while still maintaining proper immune function and promoting an appropriate response to bacterial stimuli.378 In addition, oral treatment with hBD2 is beneficial in mitigating the effects of house dust mite challenge in a murine asthma model, whether administered prophylactically or therapeutically.373,379 We speculate that the upregulation of hBD2 in COPD will play a pro-inflammatory role in inducing lung cell death. Due to the impaired immune function in patients with COPD, when smoking or when there is a large challenge by bacteria and viruses, hBD2 already expressed in COPD will be neutralized. In such cases apoptotic epithelial cells will not be able to continue to express hBD2. Thus, the immunomodulatory, antibacterial and antiviral effects of hBD2 are inhibited, and the inflammatory response of COPD is further aggravated.

Periodontitis and defensins

Periodontitis, which is responsible for a large percentage of tooth loss among adults, affects approximately 47% of adults.380 Defensins are biomarkers for the early diagnosis of periodontitis and regulate the interaction between the subgingival microbiota and host tissues.381 Research has indicated that the concentration of both α-and β-defensins in the saliva of individuals with chronic periodontitis is higher than in healthy cases.382,383,384,385,386,387 In addition, a recent bioinformatics study predicted that hBD1 might be able to bind effectively to the virulence factors of red complex bacteria in periodontitis, potentially reducing the severity of the infection.388 In vivo, hBD3 inhibits the severity of periodontitis induced by Porphyromonas gingivalis in mice and decreases osteoclast formation, while less alveolar bone loss was also observed.387

Cancer and defensins

The role of defensins in cancer development and progression has been a topic of intensive research, with some noteworthy findings.389,390 Human tumor tissue clinical samples show remarkable changes in the expression of defensins, while in vivo studies in mice and in vitro studies of related cancer cells show that defensins have anticancer and tumor progression effects. For example, in one study 82% of prostate cancer clinical tissues showed complete loss or minimal expression of hBD1 protein, while adjacent benign epithelial cells expressed it normally.297 Similarly, 90% of clinical renal cell carcinoma tissues show cancer-specific deletion of hBD1 protein.297 In addition, clinical samples of renal and prostate cancer reveal the discovery of three novel hBD1 promoter mutations.298 Synthetic hBD1 and overexpression of hBD1 can promote the death of bladder cancer cell and the renal cancer cell.298 These data suggest that hBD1 could possibly function as a tumor suppressor in urological cancers. In addition, hBD1 inhibits tumor growth of oral squamous cell carcinoma (OSCC) and lung cancer in vitro and in vivo in mice. However, hBD1 production appears to be closely linked to cancer type. For example, hBD1 expression is reduced in prostate, kidney and skin basal cell carcinoma (SBCC) and skin squamous cell carcinoma (SSCC), colon, liver, and OSCC but upregulated in lung squamous cell carcinoma (LSCC) and adenocarcinoma (AC). This pattern is further supported by studies indicating that serum hBD1 levels are notably elevated in patients with lung cancer as opposed to healthy people and patients with pneumonia.297,298,391,392,393,394,395

It should be noted that further confirmation of the potential therapeutic benefits of hBD1 is still lacking in transgenic animal studies and in vivo studies in primates. Phan and colleagues found that the sequence of hBD3 possesses a homologous β2-β3 loop that binds phosphoinositides to promote cytolysis of tumor cells.396 Continuous infusion of hBD3 in mice shows a remarkable inhibition in tumor growth in Lewis lung carcinoma cells and inhibits migration of colon cancer cells.397,398

However, some paradoxical results of hBD3 promoting tumor progression have also been found. For example, hBD3 contributes to the carcinogenesis of cervical cancer, HNSCC and OSCC via the activation of NF-κB signaling.399,400,401,402,403 Notably, defensins may also be regulated by bacteria or viruses before indirectly influencing cancer development. For example, Porphyromonas gingivalis, associated with oral cancer progression, actively triggers the transcription of α-defensins in oral tumor cells, which in turn is thought to promote the proliferation of these cells.389 In contrast, HD5 and HD6 are protective against colon cancer.404,405,406 For example, HD5 expression is reduced in colon cancer tissues from patients, and prognostic results indicate that patients with high HD5 expression have significantly longer survival than patients with low HD5 expression.405 HD5 overexpression also inhibits tumor growth in nude mice. Similarly, HD5 also inhibits the growth of gastric cancer.407 We summarized the expression of defensins in different cancer types in Fig. 6b.

Overall, the immunomodulatory activity of defensins offers the potential for them to be an effective anticancer therapy. Nonetheless, the development of defensin-based cancer therapies is complicated by the conflicting roles of defensins in different cancers. Future research is required to identify unique active structures of defensins that can be used to develop derived peptides with the discriminatory ability to target specific cancers.

Clinical trials of defensins

Brilacidin

Brilacidin, a synthetic defensin mimetic obtained from plants, has undergone extensive clinical testing involving more than 500 human patients for the treatment of various conditions, such as acute bacterial skin and skin structure infection (ABSSSI), UC, COVID-19 and oral mucositis (OM). For example, in vitro testing has revealed that brilacidin exhibits broad-spectrum antiviral activity, particularly against multiple human coronaviruses, including SARS-CoV-2. However, it does not possess antiviral activity against influenza or enterovirus.408,409,410 According to previous research, brilacidin has a dual anti-SARS-CoV-2 mechanism of action that involves targeting host cell surface heparan sulfate proteoglycans to prevent viral attachment and to inactivate viral particles.408 In fact, the US FDA has granted Fast Track status for brilacidin for COVID-19 treatment and a Phase 2 clinical trial (NCT04784897) on hospitalized patients has been conducted.411 Although the study did not meet its primary endpoint, the recovery time was significantly reduced for patients who received study treatment less than seven days after showing symptoms of COVID-19. Regarding two secondary endpoints, a higher number of patients treated with brilacidin (5-dose group) experienced clinical improvement by ten days after treatment initiation, as assessed using the National Emergency Warning Score 2 (NEWS2) criteria. The mean change in NEWS2 baseline was more remarkable for the brilacidin-treatment groups at all evaluated time points.

Brilacidin also effectively prevents and controls OM in patients undergoing head and neck cancer (HNC) chemotherapy. A Phase II clinical trial of brilacidin for this circumstance (NCT02324335) showed that patients with HNC who self-administered brilacidin three times a day for 7 weeks significantly reduced the incidence of OM compared with placebo (from 60 to 36.8%).412 Two randomized, phase II trials (NCT02052388 and NCT01211470) indicate that a single dose of intravenous brilacidin is just as safe and effective as FDA-approved daptomycin for the treatment of ABSSSI, with an early clinical response (7-day) rate of 90% (refs. 413,414,415).

Aside from its use in COVID-19, ABSSSI and OM, brilacidin is currently being developed as a preventative measure for UC. Most patients with UC that were treated with brilacidin achieved induction of clinical remission.416 After administering brilacidin, there were no reports of Serious Adverse Events (SAEs) and it was generally well-tolerated.416 In animal models, brilacidin also demonstrated a potential therapeutic effect in treating keratitis (topical drops) and pulmonary infection (intraperitoneal injection) induced by Aspergillus fumigatus.417,418 These studies all show the clinical potential of brilacidin, although further investigation is needed.

Pezadeftide (HXP124)

HXP124 is a novel plant defensin being clinically developed by Hexima Ltd as a novel topical candidate for treating onychomycosis.419,420 To evaluate its efficacy, a Phase I/IIa clinical trial was conducted using pezadeftide (Australian Clinical Trials ID: ACTRN12618000131257) in a double-blinded, randomized study with multiple ascending doses.420,421 Patients who received daily topical application of pezadeftide for 6 weeks were found to have reduced infection area compared to those receiving current best-in-class therapies, with a shorter treatment time and excellent safety profile.420,421 The clinical data from this trial demonstrated a 69% Mycological Cure rate at 12 weeks, a vast improvement over the 29% rate achieved by the control group.420,421 These results show very promising clinical efficacy. Hexima has since raised $11 million and initiated Phase IIb testing of 2% pezadeftide in three active arms to determine the optimal dosing frequency and evaluate further its safety and efficacy.422

Hurdles to the development of defensins-based therapy

From the extensive evidence presented previously, the role of defensins and targeted therapies offer new hope, but most natural defensins may not be suitable as drugs for direct application. The main challenges lie in the following areas. (1) The effect of defensins in vivo requires a suitable local microenvironment. The direct antibacterial activity requires high local concentrations, and many defensins exhibit specific cytotoxicity and inflammatory responses at high concentrations,17 as described in the Regulation of Cell Death section. In addition, specific disease environments lead to significant changes in mucosal pH and salt ion concentrations that do not allow defensins to function.423,424,425,4).

Table 4 Regulation effects of nutrients on the expression of defensins
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Fatty acids

Fatty acids (FAs), the main components of lipids, undergo various metabolic processes after absorption in the gut. Proper lipid metabolism in the gut is essential to ensure adequate energy for the body’s organs. In humans, defects in the absorption of lipids can cause severe symptoms, including IBD and IBS.436,437 The length of the aliphatic hydrocarbon chain of free FAs has been found to have a negative correlation with the ability of FAs to induce defensin expression.438 Undigested dietary fiber in the large intestine undergoes bacterial fermentation, yielding short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate. They enhance the expression of hBD1 and hBD2 in IECs439 (Fig. 7). In pigs, caprylic acid and nonanoic acid, both medium-chain fatty acids, significantly increase pBD2-3 and pBD1-2 expression, respectively, in porcine intestinal enterocytes (IPEC-J2) and decrease bacterial translocation, with augmented antibacterial activity440,441 (Fig. 7).

Fig. 7
figure 7

Signaling pathways of fatty acids-regulated expression of defensins. SCFAs induce the expression of β-defensins via GPR43-STAT3 and GPR43-mTOR-4E-BP signals; sodium butyrate (NaB) induces the expression of β-defensins via TLR2-p38/ERK- NF-κB, HDAC inhibition and EGFR signals; sodium phenylbutyrate (PBA) induces the expression of β-defensins via TLR2/TLR4-p38/ERK-NF-κB and EGFR signals; Caprylic acid and nonanoic acid induce the expression of β-defensins via HDAC inhibition-acetylation H3K9

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The expression of defensins mediated by SCFAs involves multiple signaling pathways. For instance, GPR43 mediates SCFA-regulated β-defensins-1, 3, and 4 expressions in IECs via activating mTOR and STAT3 (ref. 442) (Fig. 7). Sodium phenylbutyrate, an aromatic SCFA, promotes the expression of pBD1-3 through the TLR2/4-mediated NF-κB pathway443,444 (Fig. 7). In addition to TLRs or GPCRs, histone modification is involved in FA-induced expression of defensins. For example, caprylic and nonanoic acid attenuate histone deacetylase (HDAC) activity, leading to an elevation in the acetylation level of H3K9 and an upregulation of pBD1 and pBD2 (ref. 441) (Fig. 7). Sodium phenylbutyrate and butyrate further amplify defensin secretion through histone deacetylation and STATs phosphorylation in IPEC-J2 cells and crypt organoids443,445 (Fig. 7). In addition, in our previous study we found that butyrate upregulates pBD2 and pBD3 to enhance disease resistance, including promoting the removal of harmful bacteria and improving inflammation caused by E. coli O157:H7 infection in piglets via HDAC inhibition473 Similarly, the probiotic E. coli Nissle 1917 boosts hBD2 expression via flagellin-mediated NF-κB and AP-1 pathways, enhancing the mucosal barrier against luminal bacteria.474,475 With beneficial outcomes observed in human clinical studies, E. coli Nissle 1917 appears to show promise.476,477,478

Selenium-enriched Bacillus subtilis yb-1114246 activates the TLR2-NF-κB signal to control intestinal β-defensins expression, thereby improving the immune status of the intestine.435 Our prior research indicates that C. butyricum binds to the adhesion sites of IECs, prompting the secretion of pBD1-3 by IECs.479 C. butyricum and pBD1-3 synergistically positively regulate the composition of intestinal microbiota and SCFA production, culminating in an improvement in intestinal immune function in weaned piglets.479 Over- or under-production of defensins can adversely impact intestinal integrity. However, the beneficial effects of probiotics in adjusting abnormal defensin levels, be it an increase or decrease, have been fairly consistent in aiding host recovery. A deeper understanding of the interactions between probiotics and defensins is necessary. This will facilitate the comprehensive analysis of dysregulation of defensin homeostasis and microbial crosstalk in various gastrointestinal diseases, which is vital for treating gastrointestinal diseases.

Although several nutrients have been shown to regulate the expression of defensins, this screening approach excessively relies on reproducible experiments. A high throughput screening method was recently developed, in which the capacity of up to 584 compounds to induce the expression of specific defensins, such as LL-37 and AvBD9, could be determined in one in vitro experiment.480,481 Such a screening approach will significantly accelerate the speed of discovery of nutrient-induced defensin expression.

Conclusions

As a common defense mechanism among mammals, host-derived defensins comprise a critical innate immune barrier to external insults. A better understanding of the expression site, chemotactic activity, inflammation regulation, damage regulation and secretion regulation of host-derived defensins is critical to comprehending host defense mechanisms and disease processes. Although there is still a lack of solid clinical trials that adequately utilize the immune effectors of defensins in various diseases, both clinical and preclinical data obtained using mouse models highlight the vital role that defensins play in regulating the immune response. Meanwhile, in the future, the field should focus on exploring defensin functions and mechanisms in ameliorating specific diseases by establishing defensin knockout animal models or utilizing clinical samples. Moreover, as multiple defensins are present in the host, better tools and proteomic methodologies must explore how the synergies between defensins improve innate immunity or enhance resistance to infection. But even so, the combined data generated to date in the field point to a bright future for a role of defensins or their derivatives in the treatment of various human diseases.