Antimicrobial peptides and the gut microbiome in inflammatory bowel disease

John Gubatan, Derek R Holman, Christopher J Puntasecca, Danielle Polevoi, Samuel JS Rubin, Stephan Rogalla

Abstract Antimicrobial peptides (AMP) are highly diverse and dynamic molecules that are expressed by specific intestinal epithelial cells, Paneth cells, as well as immune cells in the gastrointestinal (GI) tract. They play critical roles in maintaining tolerance to gut microbiota and protecting against enteric infections. Given that disruptions in tolerance to commensal microbiota and loss of barrier function play major roles in the pathogenesis of inflammatory bowel disease (IBD) and converge on the function of AMP, the significance of AMP as potential biomarkers and novel therapeutic targets in IBD have been increasingly recognized in recent years. In this frontier article, we discuss the function and mechanisms of AMP in the GI tract, examine the interaction of AMP with the gut microbiome, explore the role of AMP in the pathogenesis of IBD, and review translational applications of AMP in patients with IBD.

Key Words: Antimicrobial peptides; Inflammatory bowel disease; Ulcerative colitis;Crohn’s disease; Gut microbiome; Biomarkers

INTRODUCTION

The gastrointestinal (GI) tract is a highly complex and dynamic ecosystem consisting of a protective epithelial barrier in constant exposure to commensal microorganisms that are collectively known as the gut microbiome[1]. An intricate balance between tolerance to commensal microorganisms and protection against enteric pathogens is required to maintain intestinal homeostasis. A breakdown in this balance has been recognized to play a role in the pathogenesis of inflammatory disorders of the GI tract such as inflammatory bowel disease (IBD)[2]. Antimicrobial peptides (AMPs) are diverse and bioactive compounds that play critical roles in host defense and maintaining tolerance to commensal microorganisms[3,4]. Here we provide a comprehensive review of the significant AMP functions in the GI tract and the gut microbiome, potential roles of AMPs in the pathogenesis and treatment of IBD based on preclinical animal models, and translational applications of AMPs in patients with IBD.

ANTIMICROBIAL PEPTIDES IN THE GASTROINTESTINAL TRACT

Human defensins

Table 1 summarizes the major classes of AMPs in the GI tract. Defensins, which consist of small cationic peptides, protect against bacterial infections by directly disrupting bacterial membranes. The two major classes of defensins include α-defensins and βdefensins which differ structurally in their cysteine pairings[5]. Human α-defensins are also known as human neutrophil peptides (hNP). Human defensin 5 and 6 (HD5 and HD6) are the only α-defensins produced in the GI tract by Paneth cells, highly specialized secretory epithelial cells with antimicrobial function[6]. Known functions of HD5 include conferring resistance to oral challenge with enteric pathogens[7] and regulating the intestinal microbiota by reducing levels of segmented filamentous bacteria[8]. HD6 has been shown to restrict infection by limiting intestinal epithelial cell invasion[9]. β-defensins are expressed by enterocytes of the small and large intestine. The most relevant intestinal β-defensins include human β-defensins 1-4(hBD-1, hBD-2, hBD-3, and hBD-4). hBD-2 and hBD-3 expression increases in response to infectious stimuli, whereas hBD-1 is constitutively expressed by the GI tract[10]. βdefensins hBD-2-4 have antimicrobial activity againstEscherichia coli(E. coli),Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes, whereas hBD-1 only has activity against gram positive commensals[11-13].

Cathelicidin

Cathelicidin is another class of cationic peptides that mediates its bactericidal effects through direct disruption and lysis of bacterial membranes. Cathelicidin, also known as LL-37 or hCAP18, is an 18 kDa antimicrobial peptide involved in innate immune defenses and is encoded by the CAMP gene in humans[14]. Cathelicidin has a broad-spectrum activity against bacteria, enveloped viruses, and fungi[15]. It is expressed by differentiated colonic epithelial cells as well as resident immune cells in the GI tract including neutrophils, monocytes, and macrophages, and mast cells[16,17].Cathelicidin expression has been reported to be increased in inflamed and noninflamed mucosa in ulcerative colitis patients[18]. Butyrate[18] and vitamin D[19,20] are known inducers of cathelicidin expression on colonic epithelial cells and immune cells. Cathelicidin deficiency increases susceptibility to infection with enterohemorrhagicE. coli(EHEC)[21]. Vitamin D induction of cathelicidin in human colonic epithelial cells has been shown to inhibitin vitro E. coligrowth[21]. Likewise,cathelicidin protects against colonization with epithelial adherent bacterial pathogens[22].

Table 1 Antimicrobial peptides in the gastrointestinal tract

Regenerating protein

Another class of antimicrobial peptides expressed in the GI tract include the soluble lectins belonging to the regenerating (Reg) Protein family. RegIIIγ and its human counterpart RegIIIα, also known as Hepatocarcinoma-Intestine Pancreas/Pancreatitis-Associated Protein (HIP/PAP), are expressed by enterocytes and Paneth cells in response to microbial and inflammatory stimuli[23,24]. RegIIIα selectively binds to cell wall peptidoglycan in gram-positive bacteria to induce pore formation[25]. RegIIIβ interacts with surface Lipid A structures to target gram-negative bacteria[26]. In mice,RegIIIγ maintains physical separation between the gut microbiota and the intestinal epithelial surface and regulates bacterial colonization and intestinal immune responses by the microbiota[27]. In mice, RegIII is strongly induced in gut epithelial cells following bacterial reconstitution and colitis[28]. In human studies, Reg Iα, Reg Iβ, and Reg IV are overexpressed in colon mucosa with ulcerative colitis, whereas Reg IV is overexpressed in Crohn's disease[29].

Metal sequestering antimicrobial peptides

Some antimicrobial peptides function by sequestering metal micronutrients which are required as co-factors for microbial growth. Lactoferrin is a secreted iron binding protein that is expressed by intestinal epithelial cells. Lactoferrin mediates its antimicrobial activity by sequestering free iron required for bacteria growth[30]. Lipocalin-2(neutrophil gelatinase-associated lipocalin, GAL) is expressed by intestinal epithelial cells after stimulation by proinflammatory cytokines IL-17 and IL-22. Lipocalin-2 sequesters the siderophore enterobactin which then prevents bacteria cells from binding iron[31]. Calprotectin, a heterodimer consisting of S100A8 and S100A9, is produced by intestinal epithelial cells and neutrophils. Calprotectin inhibits bacterial growth by sequestering zinc and manganese during infection[32]. The cationic peptide hepcidin plays a key role in regulating iron homeostasis through its binding to the iron exporter ferroportin. During infection and inflammation, hepcidin is upregulated and subsequently limits iron availability to bacterial pathogens. Hepcidin has antimicrobial activity againstE. coli,Pseudomonas aeruginosa, and group AStreptococcus[33].

Antimicrobial peptides with different mechanisms of action

Other AMPs of various mechanisms of action have also been characterized. Galectins are β-galactoside-binding lectins that can bind to galactose-containing glycans on glycoproteins and glycolipids. They are highly expressed by intestinal epithelial cells and innate immune cells. Galectin-3, -4, and-8 recognize human blood group B antigen-like determinants on the surface ofE. coliO86 and have bactericidal activity.Galectin-3 can bind to lipopolysaccharide (LPS) on gram-negative bacteria. Galectin-8 targets damaged vesicles for autophagy during bacteria invasion[34,35]. Another mechanism involves enzymatic degradation of bacterial membranes. Lysozyme which is secreted by Paneth cells preferentially binds to gram-positive bacteria and degrades bacterial membranes by hydrolyzing peptidoglycan linkages[36]. AMPs also function as protease inhibitors such as elafin and secretory leukocyte protease inhibitor (SLPI).Elafin is produced by epithelial cells of mucosal surfaces including the GI tract. Elafin mediates its antimicrobial activity by binding to LPS from gram-negative bacteria and modulating macrophages[37]. SLPI is a major serine proteinase inhibitor that is expressed and apically secreted by human intestinal epithelium as well as Paneth cells,neutrophils, and macrophages. SLPI has antimicrobial activity against the enteric pathogenSalmonella typhimuriumas well as gram-positive and gram-negative bacteria and fungi[38,39].

ANTIMICROBIAL PEPTIDES AND THE GUT MICROBIOME

The appropriate maintenance of the gut microbiome is critical for health. In addition to offering competitive protection against pathogen growth, the microbiome regulates gut development[40] modulates digestion[41] and provides nutrients[42]. Thus, the microbiome must be carefully cultivated, without being permitted to proliferate excessively. However, the rapid renewal of epithelial layers, particularly in the gut where renewal rates are amongst the most rapid[43,44], poses a unique challenge for maintaining microbial composition and distribution. AMPs are a critical mechanism for regulating the microbiome, and act as part of a complex interplay between the gut microbiome, the innate immune system, and epithelium renewal. Reduced AMP production is associated with disorders such as IBD[45], which will be discussed in more depth in section III. In wounds or acute infections, multiple classes of AMPs are rapidly upregulated, frequently through PAMP-dependent induction. Above threshold doses, they achieve rapid bacterial killing by synergistically targeting diverse yet critical microbial functions[46]. In contrast, direct interactions between AMPs and the gut microbiome occur at sub-lethal doses[47], though AMPs also act indirectly on the gut microbiome through the local modulation of immune response[48].

Evolutionary analysis of AMPs offers insights into AMP function

Across a wide array of species, the regional control of which AMP classes are expressed acts in concert with local environmental conditions to fine-tune both the microbiome’s spatial heterogeneities as well as bacterial phenotype[49]. The requirements for broad-spectrum pathogen resistance, coupled with carefully tuned microbiome maintenance, lead to fascinating AMP evolutionary behavior. While genes associated with immune defense are associated with rapid evolution, AMP amino acid sequences evolve more slowly than the genome average. Indeed, they can be highly conserved across multiple species[50]. The relatively slow evolution rate of AMP amino acid sequences therefore suggests that pathogen control is likely a result of a complex AMP mixture, and that any individual AMP exerts minimal co-evolutionary pressure[51,52].

Given the importance of microbiome composition for health, and in light of the highly conserved AMP amino acid sequences, one might expect strict control over AMP copy number and regulation. Unexpectedly, this is not what has been observed.While AMP coding sequences are highly conserved within a species, there is substantial variability in both copy number and regulatory sequences, as reviewed in[53]. This is particularly intriguing given that there is a high evolutionary cost associated with AMPs; when model organisms are propagated in germ-free environments, AMPs are rapidly lost[54]. Together, these data strongly suggest that the regulatory variability that is observed within humans may be a function of geography, specifically long-term local diets, local pathogens, and/or candidate microbiome components.

Dynamics of AMP-microbiome interactions

AMP serve as key regulators of host-gut microbiota interactions in a bi-directional and highly dynamic process[55]. AMP can shape the composition of the gut microbiome.For example, sublethal doses of AMPs could primeE. colito develop tolerance and increase persistence by production of curli or colonic acid[56]. Prior studies have demonstrated that species-specific AMP profiles in animals maintains species-specific bacterial communities. Loss-of-function experiments have also shown that antimicrobial peptide composition is a predictor of bacterial colonization[57]. Furthermore,AMP resistance patterns maintains the resilience of prominent gut commensals during perturbations such as inflammation[58]. Conversely, the gut microbiome produces a complex array of metabolites[59] that directly regulate AMP production and function[60,61]. For example, the microbiota metabolite short chain fatty acid promoted the production of the AMP RegIIIγ and β-defensins by intestinal epithelial cells[62].

Manipulation of gut microbiome composition has been shown to control AMP production and function. Cazorlaet al[63] demonstrated that oral administration of probiotics in mice increased Paneth cell and intestinal antimicrobial activity. In addition, treatment of mice with VSL #3, a common probiotic used in patients with IBD, was associated with restoration ofAMPgene expression in the small intestine and increased abundance of bacterial commensals in the gut[64]. Some probiotic strains produce AMP and has been proposed as a strategy to improve immune responses in immunocompromised patients[65]. Finally, fecal microbial transplant also modulates AMP expression in the GI tract. Tenget al[66] demonstrated that fecal microbial transplant of piglets resulted in increased expression in porcine betadefensins in the jejunum and subsequent increased gutFirmicutesand decreasedBacteroides.

Gut microbiome effects of different antimicrobial peptides

Different locations and cellular origins of AMP production are superimposed along the GI tract. Defensins, the most abundant AMPs in the gut, are notable for their multiple disulfide bridges which confer substantial structural resistance to bacterialderived peptidases[67]. Defensins exert antimicrobial activity through forming pores in target bacterial membranes. Above sufficient thresholds, this results in cell death.Although the effect of sub-lethal concentrations is still undergoing characterization in humans, it is notable that a similar strategy is used by plants[50]. Here, pore-forming AMPs are used to facilitate the release of endosymbiotic microbe-derived nutrients.

Local immune cell populations such as macrophages, T cells, and B cells[68] secrete both classes of defensins. The highly spatially restricted secretion of α-defensins, in comparison to the ubiquitous secretion of β-defensins, strongly suggests that their role is likely to prevent bacterial overgrowth[61]. Indeed, Paneth cells are positioned just beneath the actively proliferating epithelial stem cells which are critical for epithelium renewal. Single-crypt studies show that Paneth cell degranulation of α-defensins is induced by both gram-negative and gram-positive bacteria, regardless of whether they are alive or dead, as well as bacterial components such as lipopolysaccharide,lipoteichoic acid, lipid A, and muramyl dipeptide[69]. Furthermore, the antimicrobial products of Paneth cells are protective againstin vitromicrobial challenges many orders of magnitude (> 106) higher than those encounteredin vivo. Notably, degranulation is not induced by eukaryotic pathogens, including live fungi and protozoa[69].While α-defensin deficiencies in mouse models do not affect total bacterial load, they do result in reducedBacteroidesabundance and increasedFirmicutesabundance[70].

β-defensins act in the gut as a two-layered, ubiquitous defense system. β-defensin-1 is constitutively expressed at low levels, even in the gut of germ-free models[71]. βdefensin-2 and β-defensin-3 can be further induced by the local microbiome, and additionally act as potent chemo-attractants for neutrophils and memory T cells[72]. In contrast to α-defensins, cell culture models suggest that gut β-defensin induction may rely on live bacteria; pre-incubation of Caco-2 epithelial cells withEnterococcus faeciumreducedSalmonella typhimuriumuptake, while pre-incubation with heat-killedE.faeciumdid not[72]. Unlike α-defensins, at least one (β-defensin-3) has anti-fungal activity[72].

Cathelicidins (in humans: LL-37) have broad anti-microbial and immunomodulatory function, and act to maintain epithelial barrier integrity[73,74]. Cathelicidins also have a two-tiered anti-microbial activity. While their primary mechanism of activity at high concentrations is to disrupt bacterial membranes, their immunomodulatory functions occur at substantially lower concentrations. Epithelial barrier integrity maintenance is accomplished primarily through increasing tight junction protein expression, as well as post-translational effects including the redistribution of tight junctions[75]. Together, this suggests that cathelicidins are primarily used when the epithelial barrier becomes compromised. Furthermore, LL-37 has also been shown to alter the composition of the gut microbiome in mice. Cathelicidin knockout mice had significantly more OTUs belonging to the phylumVerrucomicrobiaand had lower amount of OTUs belonging to phylumProteobacteriaand the genusLactobacillusthan the other genotypes[76].

Reg III AMPs, primarily secreted by Paneth cells and epithelial cells[28,61], are soluble lectins that appear to primarily govern spatial relationships between the microbiome host tissuesviathe mucosa. In mice, Reg IIIβ/γ are co-regulated; Reg IIIα is the human ortholog[27,77]. Thinning of the mucosa driven by dietary restrictions in microbiota-accessible carbohydrates resulted in increased Reg IIIβ[78], as did increased mucosal inflammation[28]. Reg IIIγ-/- mice exhibited increased mucosal bacterial burden and impaired spatial relationships between bacteria and their host tissues[27].

FUNCTION AND MECHANISMS OF ANTIMICROBIAL PEPTIDES IN THE PATHOGENESIS OF IBD

Alpha defensins: HNP-1

Several prior studies have linked defects or alterations in GI tract AMPs with the pathogenesis of IBD. Table 2 summarizes studies exploring the function and mechanisms of AMPs in IBD. HNPs and their role in IBD continues to be investigated.Maedaet al[79] found that mild transgenic overexpression of HNP-1 reduces the susceptibility to murine dextran sulfate sodium (DSS) induced colitis. Not only did the colon of HNP-1 transgenic mice show less tissue damage, but mice also had significantly lower disease activity index (DAI) scores when compared to wild type mice. Additionally, the authors found intraperitoneal injection of low dose HNP-1 mitigates DSS-induced colitis and results in reduced expression of pro-inflammatory cytokines in the colon of mice. This improvement of colitis from low-dose HNP-1 could be from its antimicrobial activity[79].

Furthermore, Hashimotoet al[80] found that intraperitoneal injection of high concentrations of HNP-1 exacerbate DSS-induced colitis in pathogen free (BALB/c)mice and severe combined immunodeficient (SCID) mice. Clinically, HNP-1 treated BALB/c mice had significantly decreased weight and colon length as well as significantly increased DAI score, histologic score and myeloperoxidase (MPO)activity when compared to control mice. Furthermore, inflammatory cytokines IL-1β and TNF-α were significantly higher in colon of HNP-1 treated mice. In both murine models, an increased recruitment of F4/80-positive macrophages in the inflamed colonic mucosa after HNP-1 injection has been observed. This enhanced disease activity is thought to be due in part to HNP-1 induced cytokine production in macrophages.

Table 2 Antimicrobial peptides in preclinical models of inflammatory bowel disease

Beta defensins: Porcine B-defensin and hBD-2

Beta defensins are epithelial cell derived AMPs that have immunomodulating properties. Koeningeret al[81] found that subcutaneous recombinant hBD-2 reduced intestinal inflammation in three distinct animal models of IBD: chemically induced mucosal injury (DSS), loss of mucosal tolerance (TNBS), and T cell transfer into immunodeficient recipient mice. Mice treated with hBD-2 had less weight loss, better stool score and improved DAI scores in comparison to the T cell colitis control group.Additionally, mice given hBD-2 had less mucosal damage and inflammation as they maintained crypt anatomy and had reduced colon weight.

In addition to the protective effects of hBD-2, Hanet al[82] found that intrarectal administration of porcine beta-defensin 2 (pBD2) ameliorated colonic inflammation in mice during the induction of DSS-induced colitis. Mice in the pBD2 plus DSS group had less symptoms, including less weight loss, firmer and less bloody stools compared to the DSS-treated group. Mice treated with pBD2 plus DSS also had less evidence of macroscopic and histological colitis in addition to reduced production of TNF-a, IL-6 and IL-8 when compared to the DSS-treated group. Through colon cell culture, the effects of pBD2 seemed to occurviaan upregulation of genes associated with tight junctions and mucins. This may explain how pBD2 can improve DSS-induced changes in the mucosa and paracellular permeability through possible activation of the NF-kB signaling.

Cathelicidin (LL-37)

Koonet al[73] demonstrated that genetic knockout of LL-37 in mice had more severe forms of DSS-induced colitis and that inflamed colon in wild type mice in DSS colitis models had increased cathelicidin expression. The authors suggested that this upregulation of cathelicidin involves activation of TLR9-ERK signaling from bacterial DNA,which may play a role in the development of colitis. In addition to its protection against the induction of colitis, Fabisiaket al[83] showed that intraperitoneal injection of LL-37, and its shortest active metabolite, KR-12, decreases ulcer and macroscopic scores in DSS-induced and TNBS-induced models of colitis. The study showed that intraperitoneal injection of KR-12 altered the microbiomes of TNBS-induced colitis mice by reducing total andE. coligroup bacteria.

In addition to the protective and antimicrobial properties of LL-37, Yooet al[84]found that intracolonic cathelicidin or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic collagen deposition TNBSinduced colitis mice when compared to TNBS-induced mice not receiving LL-37. These results suggest that cathelicidin reverses fibrosis in the intestinesviainhibition of collagen synthesis in colonic fibroblasts.

Another unique property of LL-37 was investigated by Taiet al[85], who describe that intrarectal administration of plasmids containing cathelicidin to DSS-induced colitis mice reestablished colonic mucus thicknessviaincreased expression of mucin genes and reduced severe symptoms compared to cathelicidin knockout mice with DSS-induced colitis. This increase in mucin genes protected against mucosal damage and was linked to the activation of MAP kinase.

Gubatanet al[21] found that cathelicidin is a key mediator of the protective role of vitamin D in ulcerative colitis (UC). The authors found higher levels of 25(OH)D correlate with increased levels of both serum and colonic LL-37 in UC patients, and these higher levels are associated with decreased histologic inflammation and probability of clinical relapse. Intrarectal LL-37 reduced the severity of DSS-induced colitis in mice, but did not alter the intestinal microbial imbalance, whereas 25(OH)Dinduced cathelicidin in human colonic epithelial cells suppressedE.coligrowth. The study demonstrated that 25(OH)D is an independent predictor of cathelicidin in UC patients in remission and may protect against microbial associated gut inflammation.

Arachidonic acid and its metabolism also play a role in the regulation of antimicrobial peptides in inflammatory bowel disease. Arachidonic metabolites such as leukotrienes and are elevated in both animal models of colitis and patients with IBD[86]. Leukotrienes have been shown to trigger release of human cathelicidin from neutrophils[87], whereas prostaglandins suppress cathelicidin in human macrophages[88]. In addition, cyclooxygenase-2 (COX-2), an enzyme that metabolizes arachidonic acid, is also induced in colonic epithelial cells in IBD[89]. Cox-2 selective inhibitors have been shown to inhibit production of human beta defensins but not cathelicidin[90].

Elafin

Mottaet al[91] showed that in TNBS or DSS-induced mouse models of colitis,transgenic expression of elafin or disruption of enzymes that elafin inhibits protected against development of colitis. Transgenic mice expressing elafin had reduced inflammation as measured by a reduction in macroscopic tissue damage and myeloperoxidase (MPO) activity when compared to TNBS or DSS-induced mice that were not expressing elafin. Authors showed that adenoviral delivered elafin inhibited inflammatory parameters. The authors demonstrated that elafin is involved in inflammatory mediators and its protective effect could in part be from a bolstering of epithelial and mucosal barriers.

SLPI

Reardonet al[92] reported that thymic stromal lymphopoietin-deficient (TSLP-/-) mice led to endogenous SLPI deficiency, which prevented recovery from DSS-induced colitis and resulted in death. The authors demonstrated that the mechanism by which the absence of SLPI prevents healing of the colon is from increased neutrophil elastase(NE) activity in TSLP-/- mice. When TSLP-/- mice were treated with oral recombinant SLPI (rSLPI) there was reduced DSS-induced mortality.

Reg III (HIP/PAP)

Ogawaet al[28] aimed to identify genes that were modulated by bacterial flora to better understand mucosal inflammation in IBD patients. The authors found that expression of Reg III (HIP/PAP) was increased in DSS-induced colitis. Furthermore,the upregulation of Reg III may be due to an increase in the acute phase reactant IL-6 that occurs during gut inflammation.

Donkey milk lysozyme

Donkey milk contains high lysozyme levels and was studied by Jianget al[93] due to its antimicrobial properties. Authors found that mice given donkey milk lysozyme(DML) orally in a DSS-induced colitis model had improved symptoms of colitis measured by a reduction in weight loss, loose stools, rectal bleeding and mucosal inflammation. The authors showed that 50% DML treatment brought cytokines, TNF-a and IL-13, a pleiotropic cytokine that has proinflammatory effects on intestinal epithelial cells resulting in apoptosis and epithelial barrier dysfunction in intestinal inflammation[94] back to basal levels similar to control mice. They hypothesized that DML improves the intestinal barrier by increasing expression of tight junction proteins in the colon. They also presume that DML increases gut microbiota diversity and reduces detrimental bacteria thereby restoring the gut microflora.

Lactoferrin

Lactoferrin, a known immunomodulator, was studied by Togawaet al[95] and was found to reduce DSS-induced colitis in a dose-dependent manner after oral administration to rats. The DAI, shortening of colon length, histological/macroscopic damage score, tissue levels of MPO activity, WBC, and reduction in hemoglobin were decreased when DSS-induced colitis rats were treated with lactoferrin. The authors postulate that the protective properties of lactoferrin were tied to its modulation of the immune system by reducing pro-inflammatory cytokines TNF-a, IL-1B and IL-6 as well as the augmented levels of anti-inflammatory cytokines IL-4 and IL-10 in colonic tissue of DSS-induced colitis rats given lactoferrin.

Hepcidin

Hepcidin is regulator of iron metabolism and is upregulated during the inflammation in IBD, often resulting in anemia. Shanmugamet al[96] investigated the mechanisms that control hepcidin during periods of inflammation. They showed that the proinflammatory cytokine TNF-a inhibits hepcidin in both a DSS-induced colitis and T cell transfer colitis model in mice with downregulation of Smad1 protein mediating this effect.

TRANSLATIONAL APPLICATIONS OF ANTIMICROBIAL PEPTIDES AS BIOMARKERS IN PATIENTS WITH IBD

The diagnosis and long-term monitoring of IBD commonly involve invasive and costly endoscopy combined with histologic screening. Consequently, a biomarker that reflects the ongoing severity of disease is attractive as a non-invasive, cost-effective,and convenient alternative for diagnosing new IBD cases and identifying flares of disease. Given their involvement in disease pathophysiology, AMPs represent such potential markers, and several have been studied to determine their utility in differentiating CD and UC from other conditions, such as celiac disease and IBS, as well as active from quiescent disease states. In addition to reflecting ongoing severity of inflammation, several AMPs have shown promise as predictors of relapse,complication risk, and treatment response in the setting of IBD. Table 3 summarizes the application of AMPs as biomarkers in IBD.

Calprotectin

Among all known AMPs, calprotectin is the one most frequently used in the clinical diagnosis and monitoring of IBD. It has been known for decades that fecal calprotectin(FC) concentrations are markedly increased in the setting of both CD and UC[97-100].Elevated FC is a highly sensitive marker and is thus a particularly useful tool in the initial diagnosis and discrimination of IBD from non-inflammatory causes of abdominal discomfort and bowel dysfunction like IBS[97-103]. Based on this diagnostic utility, current practice guidelines from the World Gastroenterology Organization support measuring FC in the initial work-up of suspected IBD in both adult and pediatric patients[101,102]. Recent research has supported using FC measurements for the early diagnosis of IBD in at-risk populations, such as patients with ankylosing spondylitis[104].

FC is also particularly useful in the evaluation of IBD severity and the early identification of disease flares[104-106]. Data suggest that FC concentrations positively correlate with histologic inflammation in IBD, and assays can be used to accurately classify inactive, mild, moderate, and severe disease[102,103]. Cut-off values of fecalcalprotectin to differentiate active diseasevsremission in patients with IBD have been previously evaluated[107]: A cutoff value of 50 mg/g had a pooled sensitivity of 0.92 and specificity of 0.60 (0.52-0.67), a cutoff value of 100 mg/g had a pooled sensitivity of 0.84 and specificity of 0.66, a cutoff value of 250 mg/g had a pooled sensitivity of 0.80 (0.76-0.84) and specificity of 0.82 (0.77-0.86). Decreased levels of FC after therapy are associated with clinical, endoscopic and histological improvement with a normal ization of FC (< 50 mg/g) signifying deeper remission[108].

Table 3 Biomarker applications of antimicrobial peptides in patients with inflammatory bowel disease

IBD: Inflammatory bowel disease: UC: Ulcerative colitis; CD: Crohn's disease.

Notably, FC has been found to correlate more strongly with IBD activity than other markers of inflammation, including C-reactive protein and blood leukocytes[104,105].FC elevations are more pronounced in patients with pan-colonic CD than in those with isolated small bowel disease, indicating that concentrations may reflect disease location[105]. Rapid bedside and at-home FC assays are currently available as tools for monitoring IBD activity, with elevated concentrations detectable early in disease flares[104,109]. FC can be used to predict the risk of relapse for patients with quiescent CD and UC[105]. FC monitoring also plays a role in the treatment of IBD, as levels decrease following effective medical and diet-based management of disease[107,110].

Despite its clear clinical utility, FC remains an imperfect biomarker for the diagnosis and monitoring of IBD. Like many other inflammatory biomarkers, FC is not 100%specific for IBD. Other factors, including the use of NSAIDs, can also result in elevated FC, thereby introducing potential inaccuracy when using the biomarker to evaluate IBD[104,105].

Defensins

Previous studies have revealed increased defensin concentration at the intestinal surface epithelium in the setting of IBD, and dysregulation of defensin gene expression has been proposed as one pathogenic mechanism of disease[110,111]. Thus, defensins have been explored as potential biomarkers of IBD[112,113]. Among the 10 known human defensins, the alpha defensins HNP-1, HNP-2, and HNP-3 have been found to be significantly elevated in the sera of both UC and CD patients[114,115]. In CD, serum HNP-1-3 Levels have been shown to correlate with disease severity, as measured by Crohn’s disease Activity Index (CDAI)[114]. In UC, these levels are significantly greater in active disease than in inactive disease, and serum HNP-1-3 Levels decrease following successful treatment with corticosteroids[113]. Notably, serum HNP-1-3 Levels do not decrease following corticosteroid administration in non-responders,signifying the potential use of defensins in the monitoring of treatment efficacy[114].Fecal HNP-1-3 Levels are also significantly elevated in both CD and UC as well, with greater elevations measured during UC flares than in remission[113]. In the same study, fecal HNP-1-3 Levels correlated more closely with endoscopic severity than calprotectin. Results involving the ability to differentiate between UC and CD using defensin levels remain mixed[110-113].

Cathelicidin

Significantly elevated levels of serum LL-37 have been detected in both adult and pediatric IBD cohorts[115,116]. Multiple studies have indicated that cathelicidin can be used to reliably differentiate both CD and UC from healthy controls, reflecting the AMP’s potential diagnostic utility[115,116]. While cathelicidin levels are increased in both active and remission-stage IBD patients relative to controls, these levels seem to inversely correlate with disease activity, histologic inflammation, and risk of clinical relapse[21,116,117]. In moderate to severe IBD, higher serum cathelicidin prior to treatment is associated with better prognosis and may therefore serve as a predictor of treatment response[21]. Cathelicidin may also be a useful indicator of complication risk, as reduced serum levels correlate with significantly increased risk of intestinal stricture in CD[117]. Serum levels positively correlate with 25(OH)D levels, and the apparent protective effect of elevated cathelicidin is likely at least partially dependent on this increase in vitamin D[21].

Lactoferrin

Lactoferrin is among the most thoroughly explored AMPs in the diagnosis and clinical evaluation of IBD. Fecal concentrations of lactoferrin are consistently elevated among both children and adults with IBD relative to healthy controls[118-123]. While estimates of fecal lactoferrin sensitivity in identifying CD and UC vary, several studies have confirmed the AMP’s utility as a highly specific marker of IBD-related inflammation[120-122]. This specificity makes lactoferrin a particularly valuable biomarker for differentiating IBD from IBS, with studies indicating that lactoferrin levels can discriminate between the two conditions with a specificity at or near 100%[119-121].Lactoferrin levels positively correlate with disease activity, with significantly higher fecal concentrations found in those with moderate to severe IBD relative to those with mild or inactive disease[122]. Unlike some of the other AMPs, lactoferrin has not been shown to predict responsiveness to corticosteroid treatment, and only insignificant concentration changes have been detected following both effective and ineffective treatment regimens[123].

Galectin

Many members of the galectin family of proteins have been studied as potential biomarkers of IBD. Though several galectins are known to be expressed by intestinal epithelial cells, only galectin-1 and -3 have been shown to be significantly elevated in the serum of IBD patients[124,125]. Unlike those of galectin-1 and -3, serum levels of galectins-2, -4, -7, and -8 have not been shown to differentiate IBD patients from healthy controls[125]. Of note, galectin-1 and -3 Levels cannot reliably distinguish active from remission-stage CD or UC, nor can they distinguish CD and UC from each other[125,126]. Evidence also suggests that galectin-1 is a slightly more sensitive marker of IBD than galectin-3[125]. Nevertheless, galectins-1 and -3 may have use as biomarkers either alone or when combined with other molecules, and their upregulation in the intestinal cells of IBD patients may indicate their potential as therapeutic targets[124,125].

Hepcidin

Data regarding the utility of hepcidin as a diagnostic biomarker remain mixed[126-129]. However, given hepcidin’s crucial role in regulating iron absorption, the AMP may be useful in the monitoring of iron deficiency and related anemia, which are two common comorbidities seen in IBD patients[126,127]. These comorbidities are most frequently seen in pediatric IBD patients[126,127]. Consequently, multiple studies have aimed to elucidate the relationship between hepcidin expression and these comorbidities in pediatric IBD cohorts. In pediatric patients with IBD, elevated hepcidin levels negatively correlate with iron absorption and serum iron levels[125,126]. Elevated hepcidin corresponds with decreased response to iron supplementation in these patients, suggesting that the biomarker may serve a role in predicting response to oral iron supplementation in the setting of IBD[129].

Elafin

Elafin is known to be markedly upregulated in the intestinal mucosa of UC patients[130,131]. Intestinal expression seems to correlate closely with disease progression, as elevated concentrations are detectable in the right colon of patients with pan-colonic disease, but not those with exclusively left-sided disease[130]. This finding is further supported by enhanced colonic mRNA immunostaining in inflamed relative to noninflamed UC samples[131]. While serum elafin levels are increased in UC patients relative to healthy controls, some evidence suggests an inverse correlation between serum elafin and disease severity within UC cohorts[131,132]. Among UC patients,significantly elevated serum elafin tends to correlate with decreased disease activity scores, with the highest elafin levels measured during disease remission[133,134]. Data involving elafin as a biomarker in CD remain mixed, with most results indicating only weak correlations between elafin and CD activity[132-134]. However, serum elafin measurements may play a role in the evaluation of complication risk in CD, as elevations are significantly associated with increased risk of intestinal stricture[132].

CONCLUSION

AMPs produced by innate immune cells of the GI tract and cells that support barrier function such intestinal epithelial cells and Paneth cells play critical roles in protecting against enteric pathogens while maintaining tolerance to support a complex ecosystem of commensal gut microbiota. These highly dynamic molecules have broad spectrum antimicrobial activity against bacteria, fungi, and enveloped viruses and mediate their protective effects through diverse mechanisms of action from disrupting cell membranes, binding microbial components such as LPS, and sequestering metal cofactors to limit microbial growth. AMPs also play major roles in regulating gut microbiome composition and spatial relationships between the microbiota and intestinal barrier.

Defects in endogenous AMP expression and function have been linked with intestinal inflammation in mice. Conversely, exogenous delivery of AMPs such as defensins, cathelicidin, and elafin have been shown to attenuate intestinal inflammation in murine models of IBD. AMPs such as calprotectin and lactoferrin have found clinical applications as biomarkers of intestinal inflammation in patients with IBD. Other AMPs including alpha- and beta-defensins, cathelicidin, and elafin may be useful biomarkers for disease activity and predicting clinical outcomes in patients with IBD. Although the protective effects of AMPs have been demonstrated in murine models of IBD, there are currently no AMP-based therapies approved or in clinical trials for IBD. Future studies should focus on translation of AMPs as potential therapies in patients with IBD. Several challenges with AMPs including limited stability due to enzymatic degradation by endogenous proteases[135 ,136] and crossreactivity of AMPs with host cells leading to cytotoxicity[137] pose major barriers to their application as therapies. Biochemical modifications to enhance AMP stability,selectivity, and delivery are being explored[46 ,137].