Antimicrobial peptides and the gut microbiome in inflammatory bowel disease

Table 1 Antimicrobial peptides in the gastrointestinal tract

Antimicrobial peptide class	Gene	Specific antimicrobial peptides	Tissue expression	Biologic function
α-defensins (human neutrophil peptides)[5-9]	DEFA	Human defensin 5 and 6 (HD5 and HD6)	Paneth cells	Confers resistance to oral challenge with enteric pathogens, regulates the intestinal microbiota by reducing levels of segmented filamentous bacteria, restricts infection by limiting intestinal epithelial cell invasion
β-defensins[5,10-13]	DEFB	Human β-defensins 1–4 (hBD-1, hBD-2, hBD-3, and hBD-4)	Intestinal epithelial cells	Antimicrobial activity (hBD-2-4) against bacterial pathogens including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes, antimicrobial activity (hBD-1) against gram-positive commensals
Cathelicidin[14-22]	CAMP	Cathelicidin (LL-37/hCAP18)	Colonic epithelial cells, neutrophils, monocytes, macrophages, mast cells	Cationic peptide that directly disrupts bacterial cell membranes, deficiency increases susceptibility to infection with enterohemorrhagic E. coli (EHEC)
Regenerating (Reg) protein[23-29]	REG	RegIII; Hepatocarcinoma-intestine pancreas (HIP)/pancreatitis-associated protein (PAP)	Paneth cells, intestinal epithelial cells	Regulates intestinal homeostasis by maintaining a physical separation between epithelial cells and the microbiota, selective for gram-positive bacteria through interaction with cell wall peptidoglycan
Lactoferrin[30]	LTF	Lactoferrin	Epithelial cells	Secreted iron binding protein, sequesters free iron required for bacterial growth
Lipocalin[31,129]	LCN2	Lipocalin-2 (neutrophil gelatinase-associated lipocalin, GAL)	Neutrophils, granulocytes, macrophages, epithelial cells	Binds to bacterial siderophore enterobactin and inhibits bacterial growth by sequestering iron
Calprotectin[32]	S100A8, S100A9	Calprotectin	Intestinal epithelial cells, neutrophils	Chelates and sequesters metal co-factors (manganese, zinc, iron) during infection and inhibits bacterial growth
Hepcidin[33]	HAMP, LEAP1	Hepcidin antimicrobial peptide	Intestinal epithelial cells	Regulates iron absorption and homeostasis, inhibits bacterial growth by limiting iron availability
Galectin[34,35]	LGALS	Galectin-3, Galectin-4, Galectin-8	Intestinal epithelial cells	Galectins has bactericidal activity against bacteria expressing blood group antigen, Gal-8 targets damaged vesicles for autophagy during bacteria invasion
Lysozyme[36]	LYZ	Lysozyme	Paneth cells	Enzymatic degradation of bacterial membranes, preference towards Gram-positive pathogens
Elafin[37]	PI3	Elafin (peptidase inhibitor 3)	Intestinal epithelial cells	Binds to bacterial lipopolysaccharide (LPS) and modulates innate immunity
Secretory Leukocyte Protease Inhibitor (SLPI)[38,39]	SLPI	SLPI	Intestinal epithelial cells, paneth cells, neutrophils, macrophages	Protease inhibitor binds to bacterial mRNA and DNA, dose-dependent bactericidal properties of SLPI against both Gram-positive and Gram-negative bacteria, has fungicidal properties

Table 2 Antimicrobial peptides in preclinical models of inflammatory bowel disease

Ref.	Antimicrobial peptides (expression location)	Antimicrobial peptide delivery	Preclinical models (animal, human cell culture)	Key findings
Maeda et al[79]	Alpha defensins: Human neutrophil peptide-1 (HNP-1) murine colon	Genetic overexpression, intraperitoneal	Murine dextran sulfate sodium (DSS) colitis	Mild transgenic overexpression of HNP-1 reduces the susceptibility to DSS-induced colitis; Intraperitoneal injection of low-dose HNP-1 ameliorates DSS-induced colitis; The amelioration of colitis by low-dose HNP-1 may be explained by its indirect antimicrobial activity
Hashimoto et al[80]	Alpha defensins: Human neutrophil peptide-1 (HNP-1): Murine colon, human colon cells	Intraperitoneal	Murine dextran sulfate sodium (DSS) colitis, SCID mice, human colon cell cultures	Body weight and colon length significantly decreased, and the disease activity index score, histologic score, and myeloperoxidase activity significantly increased in HNP-1-treated mice compared with PBS-treated mice. High concentrations of HNP-1 aggravate DSS-induced colitis, including upregulated expression of such macrophage-derived cytokines as IL-1β
Han et al[82]	Porcine β-defensin (pBD)2: Murine colon	Intrarectal	Murine dextran sulfate sodium (DSS) colitis, human colon cell cultures	Administration of pBD2 effectively attenuated colonic inflammation in mice with DSS induced colitis. pBD2 reduced the increased serum and colon levels of TNF-a, IL-6 and IL-8 all caused by DSS. The effects of pBD2 appeared to be through upregulation of the expression of genes associated with tight junctions and mucins
Koeninger et al[81]	Beta defensins: human beta defensin 2 (HBD-2): Murine colon	Subcutaneous	Murine dextran sulfate sodium (DSS) colitis, 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis, T cell transfer colitis model	Treatment improved disease activity index and hindered colitis-induced body weight loss on par with anti-TNF-α and steroids. Mechanistically, hBD2 engaged with CCR2 on its DC target cell to decrease NF-κB, and increase CREB phosphorylation, hence curbing inflammation
Koon et al[73]	Cathelicidin (LL-37): Murine colon	Genetic knockouts	Murine dextran sulfate sodium (DSS) colitis	Increased expression of cathelicidin in the colon of DSS-exposed mice; Compared with WT mice, cathelicidin KO mice developed a more severe form of DSS-induced colitis; Cathelicidin protects against induction of colitis in mice; Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9-ERK signaling by bacterial DNA
Fabisiak et al[83]	Cathelicidin (LL-37) KR-12 (active fragment of LL-37): Murine colon	Intraperitoneal	Murine dextran sulfate sodium (DSS) colitis, 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis, T cell transfer colitis model	LL-37 and KR-12 (1 mg/kg, ip, twice daily) showed a decrease in macroscopic and ulcer scores in the acute TNBS-induced model of colitis. KR-12 (5 mg/kg, ip, twice daily) reduced microscopic and ulcer scores in the semi-chronic and chronic TNBS-induced models of colitis compared with inflamed mice
Yoo et al[84]	Cathelicidin (LL-37): Murine colon	Intracolonic, intravenous	2,4,6-trinitrobenzenesulfonic acid (TNBS) Colitis,	Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice compared with vehicle administration. Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts
Tai et al[85]	Cathelicidin (LL-37): Murine colon	Genetic knockouts, intrarectal	Murine dextran sulfate sodium (DSS) colitis	Cathelicidin knockout mice had more severe symptoms and mucosal disruption than the wild-type mice in response to DSS colitis. Intrarectal administration of plasmids encoding cathelicidin reversed colitis in cathelicidin knockout mice
Gubatan et al[21]	Cathelicidin (LL-37): Murine colon, human colon cells	Intrarectal	Murine dextran sulfate sodium (DSS) colitis, human colon cell cultures	Vitamin D-induced cathelicidin in human colonic epithelial cells suppressed Escherichia coli growth. Intrarectal cathelicidin reduced the severity of DSS colitis but did not mitigate the impact of colitis on microbial composition
Motta et al[91]	Elafin: Murine colon	Transgenic expression, adenoviral delivery	Murine dextran sulfate sodium (DSS) colitis, 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis	In mice given TNBS or DSS, transgenic expression of elafin protected against the development of colitis. Similarly, adenoviral delivery of Elafin significantly inhibited inflammatory parameters. Elafin modulated a variety of inflammatory mediators in vitro and in vivo and strengthened intestinal epithelial barrier
Ogawa et al[28]	RegIII (HIP/PAP): Murine colon	Endogenous expression	Murine dextran sulfate sodium (DSS) colitis	Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon
Jiang et al[93]	Donkey milk lysozyme (DML): Murine colon	Oral	Murine dextran sulfate sodium (DSS) colitis	DML ameliorated weight loss, colon damage and mucosal inflammation in DSS colitis mice. DML improved mechanical barrier function and increased gut microbiota composition diversity, promoting growth of probiotics and inhibiting pernicious bacteria
Reardon et al[92]	Secretory leukocyte peptidase inhibitor (SLPI): Murine colon	Genetic SLPI deficiency, oral	Murine dextran sulfate sodium (DSS) colitis, T cell transfer colitis model	Tslp−/− mice lead to endogenous SLPI deficiency which exacerbated DSS colitis. Treatment with recombinant SLPI (rSLPI) reduced DSS-induced mortality in Tslp−/− mice
Togawa et al[95]	Lactoferrin: Rat colon	Oral	Rat dextran sulfate sodium (DSS) colitis	DSS-induced colitis was attenuated by oral administration of lactoferrin in a dose-dependent manner. Reduced inflammation in response to lactoferrin was correlated with the significant induction of the anti-inflammatory cytokines and with significant reductions in the pro-inflammatory cytokines
Shanmugam et al[96]	Hepcidin: Murine colon	Endogenous expression	Murine dextran sulfate sodium (DSS) colitis, T cell transfer Colitis model	TNFα inhibits hepcidin expression in two distinct types of innate colitis, with down-regulation of Smad1 protein playing an important role in this process

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

Ref.	Antimicrobial peptides	Type of IBD	Biomarker application	Key findings
Holgersen et al[110]	Alpha defensins 5 and 6 (DEFA5/DEFA6)	UC	IBD diagnosis	Marked upregulation of DEFA5 and DEFA6 in terminal ileal biopsies of inflamed ulcerative colitis relative to normal controls
Wehkamp et al[111]	Alpha defensin (HD -5/6)	UC/CD	IBD diagnosis	HD-5/6 both decreased in ileal Crohn's, and this correlated with a decrease in transcription factor Tcf-4, a known regulator of Paneth cell differentiation. Normal levels were observed in UC and colonic Crohn's
Yamaguchi et al[112]	Alpha defensin (HNP1-3), beta-defensin (HBD-2)	UC/CD	Disease activity	HNP-1-3 all elevated in IBD patients, while HBD-2 levels normal; serum HNP1-3 levels correlated with disease severity for Crohn's
Kanmura et al[113]	Alpha defensin (HNP)	UC/CD	Disease activity	Fecal-HNP levels were markedly elevated in both UC and Crohn's, but slightly more so in Crohn's; F-HNP was significantly higher during flares of UC than remission. For UC, HNP levels correlated with Mayo endoscopic score
Cunliffe et al[114]	Alpha defensin (HNP 1-3)	UC/CD	Disease activity	Surface epithelial cells strongly immunoreactive for neutrophil defensins and lysozyme were seen in active ulcerative colitis and Crohn's disease (but not normal or inactive IBD) mucosal samples. Many of these cells coexpressed both antimicrobial proteins.
Tran et al[116]	Cathelicidin	UC/CD	Disease activity	Cathelicidin levels were significantly increased in IBD patients and were inversely correlated with CD activity. In moderate to severe IBD, higher cathelicidin levels before treatment correlated with better prognosis.
Krawiec et al[115]	Cathelicidin	UC/CD	IBD diagnosis	Cathelicidin was significantly increased in patients with ulcerative colitis (1073.39 ± 214.52 ng/mL) and Crohn’s disease (1057.63 ± 176.03 ng/mL) patients compared to controls (890.56 ± 129.37 ng/mL) (P = 0.0003)
Gubatan et al[21]	Cathelicidin	UC	Disease activity, clinical relapse	In ulcerative colitis patients, serum 25(OH)D positively correlated with serum and colonic cathelicidin. Higher serum cathelicidin is associated with decreased risk of histologic inflammation and clinical relapse but not independent of 25(OH)D or baseline inflammation
Borkowska et al[118]	Lactoferrin	UC/CD	IBD diagnosis, disease activity	Fecal concentration of lactoferrin in children with IBD was significantly higher than in the controls. The sensitivity and specificity were 80.7% and 92.7%, respectively, and its positive and negative prognostic values were 96.8% and 63.3%, respectively
Sugi et al[119]	Lactoferrin, lysozyme	UC/CD	Disease activity	Lactoferrin and lysozyme were significantly increased in the active phases of CD and UC relative to inactive. They both correlated with fecal Hb concentration in UC, and with alpha 1-AT concentration in CD
Sidhu et al[120]	Lactoferrin	UC/CD	IBD diagnosis, disease activity	Lactoferrin levels were significantly higher in IBD patients compared with IBS/healthy controls (P < 0.001). The sensitivity, specificity, positive and negative predictive values of lactoferrin in distinguishing active IBD from IBS/healthy controls were 67% and 96%, 87% and 86.8% respectively
Wang et al[121]	Lactoferrin	UC/CD	IBD diagnosis	FL test has a high sensitivity (82%) and specificity (95%) for the discrimination of patients with IBD against non-IBD patients
Kane et al[122]	Lactoferrin	UC/CD	Disease activity	Fecal lactoferrin was 90% specific for identifying inflammation in patients with active IBD. Elevated fecal lactoferrin was 100% specific in ruling out IBS
Turner et al[123]	Lactoferrin	UC	IBD diagnosis	Lactoferrin levels significantly were elevated in pediatric UC patients, but were not responsive to change or predictive of response to corticosteroids
Wang et al[132]	Elafin	CD	Disease activity, intestinal strictures	High serum elafin levels were associated with a significantly elevated risk of intestinal stricture in CD patients. Serum elafin levels had weak positive correlations with clinical disease activity but not endoscopic disease activity
Zhang et al[133]	Elafin	UC/CD	Disease activity	The expression of elafin mRNA in peripheral blood in active IBD patients is decreased, which may be correlated with the activity of IBD, and negatively correlated with corresponding disease activity score
Motta et al[130]	Elafin	UC	Disease activity	Study identified a previously unrevealed production of elastase 2A (ELA2A) by colonic epithelial cells, which was enhanced in IBD patients. Study demonstrated that ELA2A hyperactivity is sufficient to lead to a leaky epithelial barrier and modified the cytokine gene expression profile with an increase of pro-inflammatory cytokine transcript
Schmid et al[134]	Elafin and SLPI	UC/CD	Disease activity	Levels of mRNA and immunostaining of the antiproteases elafin and SLPI were enhanced strongly in inflamed versus noninflamed UC
Frol'ová et al[124]	Galectin-3	UC/CD	Disease activity	Serum concentrations were significantly increased in specimen of patients with active and remission-stage ulcerative colitis and Crohn's disease (relative to healthy controls)
Yu et al[125]	Galectin-1, -3	UC/CD	IBD diagnosis	Serum level of galectin-1 and -3, but not galectins-2, -4, -7 and -8, were significantly higher in IBD patients than in healthy people. None of the galectins however were able to distinguish active disease from remission in UC or CD
Tibble et al[97]	Calprotectin	CD	IBD diagnosis	The cross-sectional study showed a sensitivity of 96% for calprotectin in discriminating between normal subjects and those with Crohn's disease. With a cutoff point of 30 mg/L fecal calprotectin has 100% sensitivity and 97% specificity in discriminating between active CD and irritable bowel syndrome
Moniuszko et al[100]	Calprotectin	UC/CD	Disease activity, progression	Rapid bedside FC test reliably detected disease flares in patients with both UC and CD. FC levels increased even with early signs of inflammations; values were lower in isolated small bowel disease for CD patients
Pous-Serrano et al[101]	Calprotectin	CD	Disease activity	FC was the only inflammatory marker significantly associated with the degree of histologic inflammation in surgical specimens
Scheopfer et al[102]	Calprotectin	CD	Disease activity	FC correlates more closely with endoscopic disease activity that CRP, blood leukocytes, and CDAI. It was the only marker that reliably discriminated inactive from mild, moderate, and highly active disease, underscoring its value in disease monitoring
Ferreiro-Iglesias et al[103]	Calprotectin	UC/CD	Relapse	In IBD patients under Infliximab maintenance therapy, high FC levels allow predicting relapse within the following 2 mo. Long-term remission is associated with low calprotectin levels
Klingberg et al[104]	Calprotectin	CD	IBD diagnosis, treatment monitoring	FC was a useful predictor of the development of CD in patients with ankylosing spondylitis; NSAIDs increase FC levels; FC levels drop following TNF-blocker treatments
Godny et al[109]	Calprotectin	CD	Treatment monitoring	FC decreases following successful diet-based treatment of active CD
Karaskova et al[126]	Hepcidin	UC/CD	IBD diagnosis	Serum hepcidin concentration was significantly decreased in IBD children compared with controls; levels did not differ significantly between patients with CD and UC
Martinelli et al[128]	Hepcidin	UC/CD	IBD diagnosis, iron deficiency Monitoring	Serum hepcidin was significantly higher in IBD patients with active disease versus healthy and celiac patients. Hepcidin levels corresponded with iron malabsorption and other inflammatory biomarkers like ESR
Aksan et al[129]	Hepcidin	UC/CD	Response to iron supplementation	Higher hepcidin and other inflammatory markers correlated with decreased iron absorption follow supplementation
Zollner et al[127]	Lipocalin	CD	Clinical and endoscopic activity	Fecal lipocalin-2 levels of 78.4 and 0.56 μg/g in Crohn’s disease patients for clinical and endoscopic activity, respectively, corresponded well with fecal calprotectin levels in UC patients (R = 0.87, P < 0.001)

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