Nutritional Modulation of Host Defense Peptide Synthesis: A Novel Host-Directed Antimicrobial Therapeutic Strategy?

Abstract

The escalating threat of antimicrobial resistance underscores the imperative for innovative therapeutic strategies. Host defense peptides (HDPs), integral components of innate immunity, exhibit profound antimicrobial and immunomodulatory properties. Various dietary compounds, such as short-chain fatty acids, vitamins, minerals, sugars, amino acids, phytochemicals, bile acids, probiotics, and prebiotics have been identified to enhance the synthesis of endogenous HDPs without provoking inflammatory response or compromising barrier integrity. Additionally, different classes of these compounds synergize in augmenting HDP synthesis and disease resistance. Moreover, dietary supplementation of several HDP-inducing compounds or their combinations have demonstrated robust protection in rodents, rabbits, pigs, cattle, and chickens from experimental infections. However, the efficacy of these compounds in inducing HDP synthesis varies considerably among distinct compounds. Additionally, the regulation of HDP genes occurs in a gene-specific, cell type–specific, and species-specific manner. In this comprehensive review, we systematically summarized the modulation of HDP synthesis and the mechanism of action attributed to each major class of dietary compounds, including their synergistic combinations, across a spectrum of animal species including humans. We argue that the ability to enhance innate immunity and barrier function without triggering inflammation or microbial resistance positions the nutritional modulation of endogenous HDP synthesis as a promising host-directed approach for mitigating infectious diseases and antimicrobial resistance. These HDP-inducing compounds, particularly in combinations, harbor substantial clinical potential for further exploration in antimicrobial therapies for both human and other animals.

Statement of Significance.

This review systematically categorizes various dietary compounds capable of inducing host defense peptides by synthesizing a large body of evidence and further summarizes the mechanistic underpinnings of each compound class. This review provides a foundation for future work aimed at developing these compounds as innovative antibiotic alternatives for disease control and prevention, addressing the escalating challenge posed by antimicrobial resistance.

Introduction

The pervasive and indiscriminate usage of antibiotics in human health care and agriculture has led to the emergence of antibiotic-resistant bacteria that are associated with ∼700,000 annual deaths globally [1,2]. Antibiotic resistance has become a major public health concern, arising from the ability of bacteria to survive and proliferate in the presence of antibiotics. Evolutionarily, bacteria exposed to antibiotics experience selective pressure, allowing resistant strains to thrive and multiply through mutation and horizontal gene transfer [3,4]. Biochemically, bacteria gain resistance through mechanisms such as producing enzymes that degrade antibiotics, altering targets for antibiotics, reducing the influx of antibiotics, and increasing the expression of efflux pumps to expel antibiotics [3,4].

In human medicine, antibiotic resistance complicates the treatment of infections, leading to longer hospital stays, higher medical costs, and increased mortality [5]. In veterinary science, the widespread use of antibiotics in livestock for growth promotion and disease prevention contributes to the emergence of resistant bacteria, which can transfer to humans through direct contact or the food chain [6]. A coordinated One Health approach across human health and veterinary medicine sectors is required to preserve the efficacy of current antimicrobials while novel approaches are developed to control infectious diseases [7]. A pressing need exists for novel approaches in the face of rapid resistance development. Modulation of the synthesis of host defense peptides (HDPs), also known as antimicrobial peptides, has emerged as a promising host-directed antimicrobial strategy [8,9].

HDPs, characterized by their short, positively charged, and amphipathic features, constitute an integral component of innate immunity across a wide spectrum of life forms, ranging from mammals, amphibians, and birds to plants, fungi, and bacteria [10,11]. Two major families of HDPs, namely cathelicidins and defensins (DEFs), are produced in vertebrate animals [[10], [11], [12]]. Cathelicidins are distinguished by the presence of a highly conserved cathelin precursor sequence that undergoes enzymatic cleavage to attain biological activity, while DEFs are defined by the conservation of multiple cysteines in defined positions forming intramolecular disulfide bonds [12]. The synthesis of HDPs is predominantly orchestrated by cell types in direct contact with invading pathogens, such as mucosal epithelial cells, neutrophils, and macrophages [12]. HDPs are either constitutively produced for constant host surveillance and protection or inducibly expressed in response to infection or inflammation [12].

As critical components of innate immunity, HDPs exhibit not only a direct antimicrobial activity against a broad spectrum of pathogens but also multifaceted immunomodulatory properties [[10], [11], [12]]. Their primary antimicrobial mechanism involves membrane disruption through electrostatic interactions with negatively charged microbial membranes, followed by membrane disruption and cell lysis owing to the amphiphilic nature of HDPs [[10], [11], [12]]. Additionally, HDPs play pivotal roles in orchestrating immune responses to pathogen invasion by recruiting different types of immune cells to the sites of infection through direct chemoattraction or stimulation of chemokine secretion [12]. Specific HDPs further contribute to host immune responses to pathogens by promoting phagocytosis or the formation of neutrophil extracellular traps [12]. Moreover, certain HDPs have demonstrated ability to neutralize bacterial endotoxins, modulate host signaling pathways, or promote wound healing to reduce inflammation and facilitate tissue repair [12].

Despite the clinical approval of a few HDPs, such as nisin and daptomycin, challenges persist in the application of the synthetic or recombinant form of HDPs due to high production costs and suboptimal pharmacokinetics [[10], [11], [12]]. Recent years have witnessed a burgeoning interest in identifying nutritional compounds capable of inducing endogenous HDP synthesis in humans and animals. These HDP-inducing nutritional compounds, such as fatty acids, vitamins, animal acids, sugars, minerals, bile acids, phytochemicals, epigenetic modulators, probiotics, and prebiotics, have potential to offer a cost effective alternative approach to disease mitigation [8,9,13] (Figure 1). Importantly, such HDP-inducing compounds pose minimum risk of triggering antimicrobial resistance as they target the host rather than the pathogen. This review aims to systematically summarize current findings regarding the regulation of HDP synthesis by diverse classes of nutritional compounds and the underlying mechanisms. Additionally, the review highlights the potential for synergistic induction of HDPs and improved disease mitigation through a combination of compounds, thereby providing a promising avenue for optimizing host-directed therapeutic strategies.

FIGURE 1.

Classification of dietary compounds with the ability to induce host defense peptides. Key examples are listed in each category.

Methods

Articles referenced in this systematic review were obtained by searching PubMed for relevant, peer-reviewed, full-text articles that were published in English since the year 1980. The search terms used included (“host defense peptide∗” OR “host defence peptide∗” OR “antimicrobial peptide∗” OR defensin∗ OR cathelicidin∗ OR LL-37) AND (butyrate OR “fatty acid∗” OR vitamin∗ OR “amino acid∗” OR sugar∗ OR mineral∗ OR “bile acid∗” OR polyphenol∗ OR phytochemical∗ OR epigenetic OR probiotic∗ OR prebiotic∗ OR nutrient∗) AND (regulate OR regulation OR modulate OR modulation). References from selected articles were further scanned for relevance and inclusion.

Results

Enhancing HDP expression by different classes of dietary compounds

Fatty acids

Butyrate, propionate, and acetate are major forms of short-chain fatty acids (SCFAs) that are produced by bacterial fermentation of undigested fiber in the intestine [14]. SCFAs and butyrate in particular are capable of upregulating cathelicidin (CAMP) and β-defensin (DEFB) expressions in various cell types including human intestinal epithelia [[15], [16], [17]], lung epithelial cells [18,19], and monocytes [20,21] (Table 1). Besides humans, butyrate induces HDP expression in rodents [[22], [23], [24]], chickens [25,26], cattle [27,28], and pigs [[29], [30], [31], [32], [33]], suggesting the conservation of such an innate defense mechanism. Several butyrate analogs, such as 4-phenylbutyrate (PBA), 4-hydroxybutyrate, isobutyrate, benzylbutyrate, trans-cinnamyl butyrate, and glyceryl tributyrate, had a similar or even superior potency than butyrate in HDP induction in humans [20], rabbits [37], rats [39], and pigs [29,38]. Because of rapid absorption and metabolism of butyrate in the gastrointestinal tract, some of these butyrate analogs may have better potential for in vivo applications.

TABLE 1.

Induction of HDP synthesis by different classes of dietary compounds

Compound	Cells or tissues	HDPs	References
Fatty acids
Butyrate	Human HT-29, SW620	CAMP	[15,16]
	Human HT-29, Caco-2	DEFB4	[17]
	Human A549	DEFB1	[18]
	Human EBC-1, THP-1	CAMP	[[19], [20], [21]]
	C57BL/6 mouse spleen	Camp	[22]
	Mouse Paneth cells	Defa1, Defb1, Reg3g	[23,24]
	Chicken HD11, HTC, primary monocytes, bone marrow cells, crop, jejunum, and cecum explants	AvBD3, AvBD4, AvBD5, AvBD8, AvBD9, AvBD10, AvBD14, CATHB1	[25,26]
	Bovine MEC	DEFB5, DEFB10, TAP, LAP	[27]
	Bovine MAC-T	DEFB5	[28]
	Porcine IPEC-J2, 3D4/31, primary monocytes	PBD2, PBD3, PG1-5, PEP2C	[29]
	Porcine ileum and colon, 3D4/2	PBD2, PBD3, PG1-5, PMAP37, PR-39	[30]
	Porcine PK-15	PBD3, PBD115, PBD123, PBD128, PEP2C	[31]
	Porcine IPEC-J2, jejunum, cecum	PBD3, PEP2C	[32,33]
Free fatty acids and their derivatives	Human HT-29, U937	CAMP	[20]
	Human SZ95 sebocytes	DEFB4	[34]
	Porcine IPEC-J2	PBD2, PBD3, PEP2C	[29]
	Chicken HD11, primary monocytes	AvBD9	[35]
4-Phenylbutyrate	Human VA10, U937, A498, HT-29	CAMP	[36]
	Human VA10	DEFB1	[36]
	Rabbit lung and rectum epithelium	CAMP	[37]
	Porcine IPEC-J2	PBD1, PBD3, PEP2C	[38]
4-Hydroxybutyrate	Rat bone marrow–derived macrophages	Camp, Defb4	[39]
Isobutyrate, propionate	Human SW620 colonocytes	CAMP	[15]
Valproic acid	Human A549	DEFB1	[18]
Caprylic acid, nonanoic acid	Porcine IPEC-J2	PBD1, PBD2	[40]
Oleic acid	Mouse hair follicle sebaceous glands	Defb1	[34]
Propionate, hexanoate	Bovine MEC	TAP	[41]
Vitamins
Vitamin D-3	Human keratinocytes, monocytes, neutrophils, neutrophil progenitors, SCC25, Calu-3, U937, HL60, bone marrow cells, HaCat, HT-29, airway epithelial organoids	CAMP	[[42], [43], [44], [45], [46], [47]]
	Human SCC25, Calu-3, primary keratinocytes	DEFB4	[42]
	Murine intestinal organoids	Defa5, Reg3g	[48]
	Chicken embryonic intestinal epithelial cells and PBMCs	Multiple β-defensins	[49]
	Bovine MEC	DEFB1, LAP, S100A7	[50]
	Bovine peripheral blood leukocytes	DEFB3, DEFB7, DEFB8, DEFB10, LAP, S100A8	[51]
Calcipotriol	Human skin biopsies	CAMP	[52]
Retinoic acid	Human Bronchial Epithelial Cells	DEFB3, DEFB4, CAMP	[53]
	Human U937	CAMP	[54]
	Murine intestinal organoids	Defa5, Reg3g	[48]
	Mouse Preadipocytes 3T3-L1	Camp	[55]
Retinol, retinaldehyde	Mouse skin	DEFB3	[56]
Nicotinamide	Human neutrophils	CAMP	[57]
	Mouse bone marrow cells	Camp	[57]
Niacin	Piglet ileum, jejunum, colon	PBD2, PG1-5, PR39	[58,59]
Vitamin C	Human keratinocytes	DEFB1	[60]
	Digestive gland of Haliotis discus hannai	DEFB	[61]
Amino acids
Isoleucine	Human HCT-116 colon cells	DEFB1	[62]
	Human A549 lung epithelial cells	DEFB4	[63]
	BALB/c mouse lung	DEFB3, DEFB4	[63]
	Bovine MDBK kidney cells	Bovine β-defensins	[64]
	Porcine IPEC-J2	PBD1, PBD2, PBD3	[65]
	Porcine IPEC-J2, jejunum and ileum	PBD1, PBD2, PBD114, PBD129	[66]
	Intestine of P. vachelli × L. longirostris	DEFB, HAMP	[67]
Leucine	Piglet jejunum and ileum, IPEC-J2	PBD1, PBD2, PBD114, PBD129	[66]
	Intestine of P. vachelli × L. longirostris	DEFB	[68]
Valine	Piglet jejunum and ileum, IPEC-J2	PBD1, PBD2, PBD114, PBD129	[66]
Tryptophan	Mouse ileal mucosa	Reg3g, Reg3b	[69]
	Rat jejunal and ileal mucosa	Defb2	[70]
	Porcine IPEC-J2	PBD1, PBD2, PBD3, PBD115, PG1-5, PR-39	[70]
	Piglet jejunal, ileal, colon mucosa	PBD2	[71]
Threonine	Porcine IPEC-J2	PBD1, PBD2, PBD3	[72]
	Head kidney of Ctenopharyngodon idella	DEFB1, HAMP, LEAP-2B	[73]
	Spleen of Ctenopharyngodon idella	DEFB1, HAMP, LEAP-2A, LEAP-2B	[73]
Arginine	Human HCT-116 colon cells	DEFB1	[62]
	Mouse Paneth cells	Defa29	[74]
Glutamine	Mouse Paneth cells	Reg3γ	[74]
	Mouse ileum	Defa4	[75]
Sugars
Lactose	Human HT-29, Caco-2, T84, THP-1	CAMP	[76]
	Chicken HD11 macrophages	AvBD9, AvBD14	[77]
	Piglet jejunal mucosa	PBD1, PBD2, PBD3	[78]
Glucose	Human NHK keratinocytes	DEFB1, CAMP	[60]
	Human HEK-293, HCT-116	DEFB1	[79]
	Rat kidney	DEFB1	[80]
	Chicken HD11 macrophages	AvBD9, AvBD14	[77]
Maltose, trehalose	Human HT-29 colon epithelia	CAMP	[76]
Galactose	Chicken HD11 macrophages	AvBD9, CATHB1, AvBD14	[77]
Trehalose	Chicken HD11 macrophages	AvBD9, CATHB1	[77]
Maltose, sucrose, fructose	Chicken HD11 macrophages	AvBD9	[77]
Minerals
Zinc	Human Caco-2	CAMP	[81]
	Porcine IPEC-J2	PBD1, PBD2, PBD3	[65]
Zinc gluconate	Human skin explants	DEFB4, Psoriasin	[82]
Calcium	Primary human keratinocytes	DEFB3, DEFB4, DEFB104	[83]
	Human HaCaT, PHK16-0b	DEFB1, DEFB3	[84]
Bile acids
LCA	Human HT-29, keratinocytes	CAMP	[16,85]
CDCA, UDCA	Human biliary epithelial cells	CAMP	[86]
TMCA, THDCA	Mouse 3T3-L1 adipocytes	Camp	[87]
Phytochemicals
EGCG	Human B11 gingival epithelial	DEFB1, DEFB4	[88]
	Human BEAS-2B	DEFB3	[89]
	Porcine IPEC-J2	PBD1, PBD2	[90]
	Chicken PBMCs	AvBD9	[91]
Quercetin	Human HepG2 hepatocytes, rat liver	HAMP	[92]
	Chicken HTC, PBMCs	AvBD4-7, AvBD9, AvBD14	[91]
	Chicken ileum	Multiple β-defensins	[93]
	Zebrafish liver	Defensin, LEAP2	[94]
Saponarin	Human HaCaT keratinocytes	CAMP	[95]
Apigenin	Human HaCaT keratinocytes	CAMP, DEFB1, DEFB3, DEFB4	[96]
Genistein	Human keratinocytes	CAMP	[97]
Xanthohumol	Porcine 3D4/31 macrophages	PBD3, PEP2C, PG1-5	[98]
	Porcine jejunal explants	PBD3, PG1-5	[98]
Calycosin	Porcine IPEC-J2	PBD2, PBD3, PEP2C	[98]
Datiscetin	Chicken HTC, jejunal explants	AvBD9	[99]
Isoloquiritigenin	Human Caco-2 colonic epithelial	DEFB3	[100]
	Porcine IPEC-J2	PBD3, PEP2C, PG1-5	[98]
Resveratrol	Human HepG2 hepatocytes, rat liver	HAMP	[92]
	Human periodontal ligament	DEFB4, DEFB3	[101]
	Human HaCaT keratinocytes, U937	CAMP	[102,103]
	Mouse ileum	Defa3, Defa5, Defa20	[104]
	Rat liver	Hamp	[92]
	Chicken PBMCs	AvBD9	[91]
Polydatin	Human HaCat keratinocytes	DEFB4	[105]
Pterostilbene	Human U937 monocytes	CAMP	[103]
	Porcine 3D4/31 macrophages	PBD3, PG1-5	[98]
Isorhapontigenin	Porcine IPEC-J2 intestinal epithelial	PBD3, PEP2C, PG1-5	[98]
	Porcine jejunal explants	PBD3, PG1-5	[98]
Ellagic acid	Human gingival epithelial	DEFB4	[106]
Caffeic acid	Mouse tongue	Defb3	[107]
Anacardic acid	Chicken PBMCs	AvBD9	[91]
Curcumin	Human U937 monocytes, HT-29	CAMP	[108]
	Grass carp liver, PBMCs	HAMP, LEAP-2, DEFB	[109]
Forskolin	Human HT-29, Caco-2, HaCaT, INT407, A549	CAMP	[110]
	Chicken HD11, HTC macrophages	AvBD9	[26]
Andrographolide	Human Caco-2 colonic epithelial	DEFB3	[100]
Oridonin	Human Caco-2 colonic epithelial	DEFB3	[100]
Tetrandrine	Chicken HTC cells, jejunal explants	AvBD9	[99]
Sanguinarine	Chicken HTC cells, jejunal explants	AvBD9	[99]
Deoxyshikonin	Porcine IPEC-J2, 3D4/31 cells	PBD3, PEP2C, PG1-5	[98]
	Porcine jejunal explants	PBD3, PG1-5	[98]
Epigenetic modulators
Trichostatin A	Human SW620 colon epithelial	CAMP	[15]
	Human A549 lung epithelial, human NCI-H727 lung epithelial	DEFB1	[18]
	Human primary gingival epithelial	DEFB4	[111]
	Human Caco-2 colonic epithelial, primary colonic	CAMP, DEFB3, DEFB4	[112]
	Human VK2/E6E7 vaginal keratinocytes	DEFB1	[113]
	Chicken HTC macrophages	AvBD9	[99]
	Porcine IPEC-J2 intestinal epithelial	PBD3	[114]
	Primary bovine mammary epithelial	DEFB3, DEFB4, DEFB7, DEFB10	[115]
	Rat testis, cauda	Defb1, Defb2	[116]
Vorinostat	Human THP-1 monocytes	DEFA1, DEFA5, DEFA6, DEFB4	[117]
	Human Caco-2/TC7 cells	DEFB4	[118]
	Human Caco-2 and primary colonic epithelial cells	CAMP, DEFB3, DEFB4	[112]
	Human Huh7 hepatocytes	LEAP-1	[119]
	Porcine IPEC-J2 intestinal epithelial, porcine 3D4/31 macrophages	PBD2, PBD3	[114]
	Chicken HTC macrophages	AvBD4, AvBD8, AvBD9, AvBD10, AvBD14	[120]
Entinostat	Human HT-29 colonic epithelial	CAMP	[121,122]
	Rabbit ileal and rectal epithelium	CAMP	[121,123]
	Chicken crop and jejunum	AvBD9, AvBD10, AvBD14, CATHB1	[124]
Aroylated phenylenediamines	Human HT-29 colonic epithelial	CAMP	[121]
ADP HO53	Human BCi and VA10 bronchial epithelial cells	CAMP, DEFB1	[125]
ADP HO56	Human BCi and VA10 bronchial epithelial cells	CAMP, DEFB1	[125]
Mocetinostat	Porcine IPEC-J2 intestinal epithelial	PBD3	[114]
	Chicken HTC macrophages	Multiple β-defensins	[126]
	Chicken jejunal explants	AvBD9	[126]
	Chicken HTC and HD11 macrophages	AvBD9	[120]
RGFP966	Human Huh7 hepatocytes	LEAP-1	[119]
Panobinostat	Porcine IPEC-J2 intestinal epithelial	PBD2, PBD3, PG1-5	[114]
LAQ824	Porcine IPEC-J2 intestinal epithelial	PBD2, PBD3, PG1-5	[114]
SB939	Porcine IPEC-J2 intestinal epithelial	PBD2, PBD3, PG1-5	[114]
Apicidin	Human A549 lung epithelial	DEFB1	[18]
	Chicken HTC macrophages	AvBD9	[99]
	Porcine IPEC-J2 intestinal epithelial	PBD2, PBD3	[114]
Depudecin	Human A549 lung epithelial	DEFB1	[18]
	Chicken HTC macrophages	AvBD9	[99]
	Porcine IPEC-J2 intestinal epithelial	PBD3	[114]
SGI-1027	Chicken HTC macrophages	AvBD9	[120]
BIX01294	Chicken HTC macrophages	AvBD9	[120]
UNC1999	Chicken HTC macrophages	AvBD9	[120]
5-Azacytidine	Human gingival epithelial cells	DEFB4	[111]
	Human OSC-19, BSC-OF, SAS, HSC-2, HSC-4, HSY oral squamous	DEFB4	[127]
	Human HSC-3 and HSC-4 oral squamous, HaCaT keratinocytes, TR146 buccal epithelial	LL-37	[128]
	Human VK2/E6E7 keratinocytes	DEFB1	[113]
	Human HC-OA chondrocytes	CAMP	[129]
	Bovine mammary epithelial	DEFB3, DEFB5, DEFB10, EBD, LAP, TAP	[115]
	A. pernyi larvae	Attacin, cercropin, and lebocin	[130]
	Rat cauda	Defb1, Defb2, Defb27	[116]
	Rat testis	Defb1, Defb2, Defb30	[116]
	Rat caput	Defb30, Defb36	[116]
Probiotics
Lactobacillus gasseri	Human VK2/E6E7 keratinocytes	DEFB1	[113]
Lactobacillus fermentum K11-Lb3 and K2-Lb6	Human Caco-2 colonic epithelial	DEFB4	[131]
Ligiactobacillus salivarius SMXD51	Human Caco-2/TC7 cells	DEFB4	[132]
Lacticaseibacillus paracasei CBA L74	Human Caco-2 colonic epithelial	CAMP, DEFB4	[133]
Lactobacillus helveticus SBT2171	Human Caco-2 colonic epithelial, HSC-4 tongue epithelial	DEFB4	[134]
	Human Ca9-22 gingival cells	DEFB3, DEFB4	[135]
	Mouse gingival tissue	Defb4, Defb14	[135]
Lactobacillus rhamnosus GG	Human SW480 colonic epithelial	DEFB4	[136]
Bifidobacterium longum	Human SW480 colonic epithelial	DEFB4	[136]
L. paracasei SD1, L. rhamnosus SD4, L. fermentum SD7, L. rhamnosus SD11, L. rhamnosus GG	Primary human gingival epithelial cells	DEFB4, DEFB104	[137]
L. rhamnosus Lcr35	Human VK2/E6E7 vaginal epithelia	DEFB4	[138]
Lactobacillus crispatus	Human HeLa cells	DEFB3, DEFB4	[139]
Lactobacillus johnsonii NCC 533	Human epidermis	CAMP, DEFB1, DEFB4, DEFB103B	[140]
Lactobacillus delbrueckii subsp. bulgaricus 8481	Human serum	DEFB4	[141]
BB12	Human infant stool samples	CAMP, DEFB4	[142]
Escherichia coli Nissle 1917, Pediococcus pentosaceus, Lactobacillus acidophilus, L. fermentum	Human Caco-2 colonic epithelial	DEFB4	[143]
Lactobacillus reuteri 5454, B. animalis ssp.	Mouse ileum	Reg3b, Reg3g	[144]
L. paracasei subsp. paracasei CNCM I-1518	Rat ileum	Defb1	[145]
Lactobacillus casei CRF28, UW1	C57BL/6 mouse ileum	Defa-rs1	[146]
L. casei 32G, CRF28, BL23, M36	C57BL/6 mouse ileum	Reg3β	[146]
Lactobacillus plantarum ZLP001	Piglet jejunum, ileum, IPEC-J2, 3D4/31	PBD2, PBD3, PBD114, PBD129, PG 1–5, PEP2C	[147,148]
Lactobacillus amylovorus SLZX20-1	Porcine IPEC-J2	PBD1, PEP2C, PG1-5	[149]
Bacillus subtilis CP9	Porcine IPEC-J2	PG1	[150]
B. subtilis and L. salivarius	Piglet duodenum	PBD2	[151]
L. rhamnosus GG	Piglet jejunum	PBD1, PMAP-37	[152]
L. reuteri Clostridium butyricum	Chicken cecum	AvBD4 AvBD1, CATH3	[153]
L. casei BL23	Bovine MEC	TAP	[154]
Saccharomyces cerevisiae CNCM I-1077	Bovine rumen and colon epithelium	DEFB1	[155]
Prebiotics
Inulin	Mouse Paneth cells	Defb1, Defa1, Defa4, Defa29, Defa5, Reg3g	[24]
Long-chain inulin-type fructans	Mouse colon	Defb1, Camp	[156]
Dendrobium huoshanense polysaccharides	Mouse jejunum and ileum	Total β-defensin protein	[157]

Abbreviations: AvBD, avian β-defensin; CAMP, cathelicidin; CDCA, chenodeoxycholic acid; DEFA, α-defensin; DEFB,β-defensin; EGCG, epigallocatechin gallate; HAMP, hepcidin antimicrobial peptide; HDP, host defense peptide; LAP, lingual antimicrobial peptide; LCA, lithocholic acid; LEAP, liver-enriched antimicrobial peptide; PBD, porcine β-defensin; Reg3, regenerating islet-derived protein 3; TAP, tracheal antimicrobial peptide; TDCA, taurodeoxycholic acid; THDCA, taurohyodeoxycholic acid; TMCA, α-tauromuricholic acid; UDCA, ursodeoxycholic acid.

Consistent with their HDP-inducing activity in cells, oral supplementation of sodium butyrate or PBA improved the clinical outcomes of shigellosis in both humans and rabbits by counteracting Shigella-induced downregulation of CAMP expression in the intestinal tract [37,158]. In chickens, dietary supplementation of butyrate significantly reduced Salmonella sp. colonization in the cecum of experimentally infected chickens [25]. Additionally, oral supplementation with sodium butyrate reduced the Corynebacterium pseudotuberculosis load, enhanced Camp expression, and alleviated lesions in the spleens of infected mice [22]. Similarly, butyrate, caprylic acid, and nonanoic acid reduced the bacterial load, mitigated intestinal inflammation, and upregulated HDP expressions in piglets challenged with Escherichia coli 0157:H7 [30,40]. A recent study also showed the ability of butyrate to counteract deoxynivalenol-mediated suppression of intestinal HDPs in weaned piglets [33].

Comparison of the HDP-inducing activity among free saturated fatty acids of 1–18 hydrocarbons in humans, pigs, and chickens revealed that SCFAs are the most potent, whereas fatty acids with longer aliphatic chains quickly lose HDP-inducing potency [20,29,39]. However, relative HDP-inducing potency of individual fatty acids varies among animal species. Butyrate appears to be the most efficacious HDP inducer in chicken and porcine epithelial and monocytic cells [29,35], whereas valerate, hexanoate, and heptanoate are capable of triggering stronger HDP expression than butyrate when used at higher concentrations in humans [20]. It will be interesting to evaluate the in vivo efficacy of valerate, hexanoate, and heptanoate and their analogs in HDP induction and bacterial clearance in humans.

It is abundantly clear that different HDPs are regulated differently by fatty acids. For example, PBA stimulated human CAMP and DEFB1 expression but not DEFB3, DEFB4, and DEFB104 in human lung epithelial cells [36]. Lauric acid, palmitic acid, and oleic acid upregulated DEFB4 but not CAMP or DEFB1 in human sebocytes [34]. In mice, oleic acid preferentially promoted the induction of mouse Defb2 in the hair follicle sebaceous glands of mouse ear skin [34]. Propionate and hexanoate potentiated bovine tracheal antimicrobial peptide expression but not DEFB5 [41].

A growing body of evidence has suggested that SCFAs and butyrate in particular induce HDP expression primarily by acting as histone deacetylase (HDAC) inhibitors [159]. HDAC1, HDAC2, and HDAC3 were downregulated by butyrate in human THP-1 monocytes [21], associated with an increase in histone acetylation along the promoters of human, bovine, and porcine HDPs, which is associated with active gene transcription [19,28,30]. Free fatty acid receptors, such as GPR41/FFAR3, GPR43/FFAR2, and GPR109a/HCAR2, are also involved in SCFA-mediated HDP induction, leading to downstream activation of the mammalian target of rapamycin and signal transducers and activator of transcription (STAT) 3 [23,160] (Figure 2).

FIGURE 2.

Molecular mechanisms of HDP induction by butyrate, vitamin D-3, bile acids, and lactose. An arrowhead indicates stimulation of a pathway, while a T-bar indicates inhibition of a pathway. See text for details. AC, acetyl group; AP1, activator protein-1; BRG1, Brahma-related gene 1; CDCA, chenodeoxycholic acid; C/EBPα, CCAAT enhancer binding protein α; CRE, cAMP-response element; CREB, cAMP-response element-binding protein; FXR, farnesoid X receptor; GPR, G protein-coupled receptors; HDAC, histone deacetylase; HDP, host defense peptide; LCA, lithocholic acid; MAPK, mitogen-activated protein kinases; mTOR, mammalian target of rapamycin; p50/p65, NF-κB proteins p50 and p65 heterodimer; PU.1, an ETS-family transcription factor; RXR, retinoid X receptor; SRC, steroid receptor coactivator; STAT, signal transducer and activator of transcription; TLR, toll-like receptor; UDCA, ursodeoxycholic acid; VDR; vitamin D receptor.

Additionally, Toll-like receptor (TLR) 2–mediated NF-κB activation is required for butyrate-induced expression of porcine β-defensins (PBDs) in porcine kidney and intestinal cells, while HDAC inhibition is insufficient [31,32,38]. NF-κB signaling is also required for butyrate-induced DEFB2 production by human colonic epithelial cells [118]. In fact, TLR2 expression is enhanced in bovine epithelial cells treated with butyrate, while concurrent p38 phosphorylation implicates the involvement of the mitogen-activated protein kinase (MAPK) pathway [27,28]. Canonical MAPK family members including MEK, extracellular signal-regulated kinase (ERK), and JNK regulate butyrate-induced CAMP expression in human colon, gastric, and bronchial cell lines but not in heptocarcinoma cell lines [15,36]. Furthermore, butyrate-enhanced CAMP expression in human colonic epithelial cells is mediated by PU.1, a transcription factor activated by MAPK [16]. Additional transcription factors, such as activator protein (AP) 1 and cAMP (cyclic adenosine monophosphate)-response element-binding protein (CREB), are key regulators in butyrate-induced human CAMP and DEFB4 expression [19,110,161].

Vitamins

Vitamin D-3 is a potent HDP inducer in several human cell types including epithelial cells, keratinocytes, monocytes, and neutrophils, leading to an increase in the antimicrobial activity of the host cells [[42], [43], [44], [45]]. For example, the growth of Mycobacterium tuberculosis was significantly suppressed in macrophages exposed to 1,25-dihydroxyvitamin D-3, also known as calcitriol [46,162] (Table 1 [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154], [155], [156], [157]]). The positive influence of treatment with vitamin D-3 or its analogs on HDP production was clearly demonstrated in vivo as well. Topical treatment with vitamin D-3 resulted in the upregulation of human CAMP in acute skin injuries [52]. Similarly, psoriatic skin biopsies treated with vitamin D-3 had elevated concentrations of LL-37 and reduced proinflammatory cytokines IL-12/23 p40, IL-1α, IL-1β, and TNF-α [163].

Conversely, a deficiency in vitamin D-3 is often associated with an increased susceptibility to infections, likely due to decreased synthesis of HDPs [164]. For instance, patients who experience severe congenital neutropenia, because of a lack of vitamin D-3, experience recurrent infections, whereas patients given vitamin D-3 show restored LL-37 concentrations and reduced infections [47]. In patients with atopic dermatitis, downregulation of serum 25-hydroxyvitamin D-3 and LL-37 concentrations was observed, with a reciprocal increase in the expression of proinflammatory cytokines [165]. In patients with cystic fibrosis, serum vitamin D concentrations are often deficient, perhaps contributing to the frequency of lung infections [166]. Although serum vitamin D concentrations are positively correlated with tuberculosis disease risk [167], a recent clinical trial with 8851 children with latent tuberculosis infection found no significant improvement in disease risk when a weekly dose of 14,000 IU of vitamin D-3 was given for 3 y [168].

In addition to humans, vitamin D-3 enhances the expression of HDPs in pigs [169], chickens [49], and cattle [50,51]. Administration of vitamin D-3 to cattle reduced internalization of Staphylococcus aureus in bovine mammary epithelial cells by inducing the expression of lingual antimicrobial peptide, DEFB10, and psoriasin [50]. Similarly, an increase in expression of HDPs after vitamin D-3 supplementation was accompanied by enhanced clearance of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine in bovine peripheral blood leukocytes [51]. It is noted that there is clearly a species-specific difference in the HDP-inducing ability of vitamin D-3. Although it is a strong inducer of cathelicidins and several DEFBs in humans and cattle, vitamin D-3 fails to do so in mice, dogs, and sheep, which is likely due to an absence of the vitamin D response (VDR) elements in the promoter of HDP genes in those species [43,170]. However, intestinal α-defensins (DEFAs) appear to be regulated by vitamin D-3 in mice [48].

Vitamin D-3 induces CAMP and DEFB4 gene expression by binding to VDR to activate respective gene promoters containing VDR elements in different human cell types [42,43] (Figure 2). More recently, transcription factors PU.1 and C/EBPα were found to participate in vitamin D-3 regulation of CAMP and DEFB4 gene expression [171]. Binding of PU.1 and C/EBPα recruits Brahma-related gene 1, a component of the SWI/SNF chromatin remodeling complex, to the CAMP gene promoter, leading to an increase in H4 acetylation [171]. Additionally, steroid receptor activator 3, which has intrinsic histone acetyltransferase activity, is critical for vitamin D-3–mediated induction of CAMP, as steroid receptor activator 3 knockdown prevented the upregulation of the CAMP gene [45].

Retinoic acid, a metabolite of vitamin A, has conflicting effects on HDP induction. Retinoic acid induces CAMP, DEFB3, and DEFB4 gene expression in human bronchial epithelial cells [53] but inhibits DEFB3, DEFB4, and DEFB104 in primary keratinocytes in response to proinflammatory cytokines, bacteria, phorbol myristate acetate, or calcium [83]. Although retinoic acid induces CAMP in human U937 monocytes, no change is seen in K562 lymphoblasts [54]. In mice, retinoic acid upregulated Defa1, Defa5, Defa21, and regenerating islet-derived protein 3-γ (Reg3g) in intestinal organoids [48], Camp in primary preadipocytes [55], along with Reg3b and Reg3g in lymphocytes [172]. Retinol and retinaldehyde significantly increased Defb3 expression in mouse skin, suggesting that topical retinols may be a potential treatment of cutaneous infections via increased expression of HDPs [56]. The expression of porcine cathelicidin PR-39 was enhanced in bone marrow cells by retinoic acid [173]. However, retinoic acid failed to induce HDP expression in ovine respiratory epithelial cells [174].

Niacin, also known as vitamin B-3, was found to elevate HDP gene expression in porcine IPEC-J2 cells as well as throughout the intestinal tract of piglets [58,59]. Notably, supplementation of niacin alleviated the disease severity of porcine deltacoronavirus [58] as well as enterotoxigenic E. coli K88 in weaned piglets [59]. Niacin appeared to induce porcine HDP gene expression through epigenetic modifications. Improved HDP mRNA concentrations coincided with increased expression concentrations of sirtuin (SIRT) 1 and reduced HDAC7 in the intestine of piglets in response to niacin supplementation [59]. Niacin enhanced phosphorylation of histone H3 at Ser10 (H3S10p) in the ileum as well as acetylation of lysine 9 on histone 3 (H3K9ac) and H3K27ac in the colon of piglets challenged with ETEC K88 [59].

Nicotinamide, an amide form of niacin, improved the expression of CAMP in human neutrophils but not in monocytes [57]. Increased Camp protein concentrations were also detected in bone marrow mononuclear cells of mice supplemented with nicotinamide, while the treatment enhanced S. aureus killing by ≤1000-fold in a systemic murine infection model [57]. Vitamin C, also known as ascorbic acid, increased DEFB1 but not CAMP gene expression in human keratinocytes, [60]. Vitamin C was similarly found to increase DEFB mRNA concentrations in the digestive gland of abalone [61].

Amino acids

Essential, branched-chain amino acids, including isoleucine, leucine, and valine, are each capable of upregulating HDP expression. Isoleucine increased DEFBs across multiple animal species, including humans [62,63], mice [63], cattle [64], pigs [65,175], and catfish [67] (Table 1 [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154], [155], [156], [157]]). For example, isoleucine supplementation increased Defb3 and Defb4 expression, which led to a decrease in intestinal bacillary loads and tissue damage after mice were inoculated with a multidrug resistant M. tuberculosis H37Rv [63]. Leucine increased Defa1 in mouse Paneth cells [23], 4 DEFBs in the jejunum and ileum of piglets [66], as well as DEFB transcription in the intestine of hybrid catfish [68]. Valine supplementation improved intestinal DEFB mRNA concentrations in piglets [66] and upregulated DEFB1 and CATH7 in cultured mammary epithelial cells of goats [176].

Tryptophan and threonine are essential amino acids to mammals that have also been shown to upregulate HDP production. Tryptophan improved both mRNA and protein concentrations of Defb2 in the jejunal and ileal mucosa of rats [177], as well as Reg3g and Reg3b mRNA concentrations in mouse ileal mucosa [69]. Tryptophan also increased the expression of multiple HDP genes in porcine IPEC-J2 cells [70]. Furthermore, diets with adequate tryptophan (0.21%, 0.28%, or 0.35%) elevated PBD2 protein concentration across the small intestine of piglets compared with a 0.14% tryptophan diet [71]. L-threonine similarly induced the transcription of DEFBs in porcine IPEC-J2 cells [72]. Supplementation with threonine compared with a threonine-deficient diet enhanced mRNA concentrations of hepcidin antimicrobial peptide (HAMP), liver-enriched antimicrobial peptide (LEAP) 2, and DEFB1 in the head kidney and spleen of grass carp [73].

Furthermore, arginine upregulated DEFB1 in human HCT-116 colonic epithelial cells [62,74]. Dietary arginine enhanced Defa29 and Reg3g expression in mouse Paneth cells and decreased E. coli colonization in the jejunum of challenged mice [74]. In porcine IPEC-J2 cells, L-arginine triggered DEFB transcription and could ameliorate LPS-induced inflammation [178]. Glutamine is a non-essential amino acid capable of HDP induction in mice. Defa4 mRNA was increased in the small intestine of mice supplemented with glutamine [75]. Similarly, glutamine augmented Defa29 and Reg3g mRNA expression and decreased E. coli colonization in mice [74].

Isoleucine, tryptophan, and threonine involve SIRT1 to initiate a signaling cascade that enhances HDP gene expression; however, whether SIRT1 is activated or inhibited varies among individual amino acids. In porcine intestinal epithelial cells and the intestine of hybrid catfish, isoleucine activated a SIRT1/ERK/90RSK signaling pathway to upregulate several HDP genes [66,67]. However, threonine suppressed SIRT1 expression in porcine intestinal epithelial cells, which enhanced acetylation of p65, promoted translocation of p65 into the nucleus, and activated NF-κB [72]. Tryptophan, on the contrary, had no direct effect on SIRT1 expression but suppressed LPS-induced SIRT1 expression in mouse ileal mucosa and porcine intestinal epithelial cells [69]. The mammalian target of rapamycin pathway is involved in induction of DEFBs by tryptophan [177] and arginine [178].

Sugars

Lactose, a disaccharide sugar, was shown to induce CAMP transcription in a dose-dependent and time-dependent manner in human HT-29 and T84 colonic epithelial cells and THP-1 monocytes/macrophages [76] (Table 1 [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154], [155], [156], [157]]). In fact, several other monosaccharide and disaccharide sugars such as glucose, galactose, trehalose, and maltose also showed varied potency in inducing CAMP gene expression [76]. Glucose supplementation upregulated DEFB1 and CAMP gene expression in human keratinocytes, leading to greater antimicrobial activity against both Listeria monocytogenes and S. aureus [60]. Interestingly, high glucose induced DEFB1 expression in human kidney and colon cells [79]. Similarly, Defb1 was also upregulated in the kidneys of hyperglycemic rats [80]. However, the expression of human CAMP, DEFB3, and DEFB4 were downregulated by glucose [179,180]. Multiple sugars, such as lactose, glucose, galactose, trehalose, maltose, sucrose, and fructose, increased HDP gene expression in chicken cells [77]. In pigs, lactose induced several DEFB genes in the jejunal mucosa and mitigated the negative effect of Rotavirus on animal growth [78]. Lactose-mediated HDP induction involves the MAPK pathway and histone acetylation. In HT-29 colonic epithelial cells, an inhibition of p38 MAPK and JNK reduced CAMP induction by lactose [76], and histone H4 acetylation was increased in chicken HD11 macrophage cells in response to lactose [77]. Additional studies are needed to clarify the mechanisms by which sugars upregulate HDP.

Minerals

Zinc exerts a plethora of benefits for mounting an effective immune response to infectious agents [181]. Among them is zinc’s ability to induce HDP synthesis. Zinc was found to induce CAMP in human Caco-2 colonic epithelial cells [81] and DEFB4 in LPS-induced inflammatory skin explants [82] (Table 1 [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154], [155], [156], [157]]). Zinc also upregulated DEFB concentrations in porcine intestinal epithelial cells [65]. On the contrary, zinc deficiency was accompanied by a decrease in Paneth cell DEFA synthesis in both humans [182] and mice [183]. Calcium is capable of inducing multiple DEFB gene transcription in human keratinocytes [83,84], while DEFB4 expression was reduced when calcium was chelated [184]. Chelation of calcium prevented cathelicidin-mediated killing of human Jurkat T leukemia cells [185] and inhibited DEFB-mediated fungicidal activity against Candida albicans [186].

Bile acids

Several bile acids have been reported to upregulate HDP gene expression. A primary bile acid, chenodeoxycholic acid, and a secondary bile acid, ursodeoxycholic acid, induced CAMP transcription in human biliary epithelial cells [86] (Table 1 [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154], [155], [156], [157]]). Another secondary bile acid, lithocholic acid (LCA), upregulated CAMP in human HT-29 colonic epithelial cells [16] and primary keratinocytes [85] but not in human Caco-2 colonic epithelial cells [85]. In mouse 3T3-L1 adipocytes, α-tauromuricholic acid and taurohyodeoxycholic acid, increased Camp mRNA expression, but cholic acid, deoxycholic acid, and taurodeoxycholic acid had no effect [87]. Two nuclear receptors, farnesoid X receptor and VDR, are involved in the upregulation of CAMP by bile acids (Figure 2). Although chenodeoxycholic acid induced CAMP through farnesoid X receptor, ursodeoxycholic acid activated VDR [187]. LCA likewise signaled through VDR to improve CAMP gene expression [16,85]. Moreover, MEK-ERK signaling was involved in CAMP gene regulation by LCA in primary human keratinocytes [85], while VDR recruited PU.1 to the CAMP gene promoter in HT-29 colon epithelial cells [16].

Phytochemicals

Polyphenols are a diverse group of naturally occurring compounds found in plants with anti-inflammatory, antioxidant, and anticancer properties [188]. Several classes of polyphenols, such as flavonoids, stilbenes, and phenolic acids, are capable of inducing HDP synthesis [189] (Table 1). For example, epigallocatechin gallate (EGCG), a flavonoid found in green tea, enhanced DEFB synthesis in humans [88,89], pigs [90], and chickens [91]. Quercetin is another flavonoid with an ability to improve HDP expression in rats [92], chickens [91,93], and zebrafish [94]. Saponarin (a flavone), genistein (an isoflavone), and apigenin (a trihydroxyflavone) enhanced CAMP transcription in human keratinocytes [[95], [96], [97]]. Additionally, xanthohumol (a prenylated flavonoid), calycosin (an isoflavone), datiscetin (a tetrahydroxyflavone), and isoliquiritigenin (a chalcone flavonoid) have been found to induce multiple DEFB genes in pigs [98], chickens [99], and humans [95,100], respectively. Resveratrol, a stilbene in grapes, stimulated HDP gene expression in humans, mice, rats, and chickens [91,92,[101], [102], [103], [104]]. Similarly, polydatin and pterostilbene, 2 resveratrol derivatives, were shown to increase HDP production in human keratinocytes [105] and monocytes [103] as well as in porcine macrophages [98]. Isorhapontigenin, a stilbenoid, augmented the expression concentrations of several DEFBs in porcine intestinal epithelial cells [98]. Phenolic acids, such as ellagic acid, caffeic acid, and anacardic acid, increased the DEFB expression in humans [106], mice [107], and chickens [91]. Curcumin, belonging to the curcuminoid group of polyphenols, stimulated CAMP expression in human monocytes, colonic epithelial cells, and keratinocytes [108] and enhanced HAMP, LEAP2, and DEFB mRNA concentrations in grass carp [109].

A few nonpolyphenol phytochemicals have also been found to upregulate HDP synthesis. For instance, forskolin (FSK), a diterpenoid produced by the Indian Coleus plant, was shown to enhance the expression of CAMP in human HT-29 epithelial cells [110], but suppress CAMP and DEFB1 concentrations in butyrate-differentiated HT-29 cells [161]. Chicken macrophage cells treated with FSK increased avian β-defensin 9 (AvBD9) transcription in chicken macrophage cells as well as in the crop of orally supplemented chickens [26]. Two diterpenoids, andrographolide and oridonin, were found to induce DEFB3 expression in human colonic epithelial cells [100]. The supernatant of cells treated with andrographolide and isoliquiritigenin limited the growth of 4 pathogenic bacteria [100]. A number of natural products and phytochemicals in particular were recently identified with the HDP-inducing activity in chicken and porcine cells through high throughput screening [98,99]. For instance, 1 plant alkaloids, tetrandrine and sanguinarine, were each capable of inducing chicken HDP gene expression [99], while deoxyshikonin, a plant naphthoquinone, increased PBD expression [98].

Many phytochemicals upregulate HDP synthesis through MAPK signaling pathways. For example, EGCG induced DEFB1, DEFB3, and DEFB4 via MAPK p38/ERK/JNK pathways in human bronchial or gingival epithelial cells [88,89] and required p38 activation for PBD2 induction in porcine intestinal epithelium [90]. Andrographolide, oridonin, and isoliquiritigenin activated MAPK pathways downstream of the epidermal growth factor (EGF) receptor and recruited c-Fos, c-Jun, and Elk1 or cMyc to the DEFB3 promoter in human colon epithelial cells [100]. Although andrographolide upregulated DEFB3 by EGFR/ERK/JNK, oridonin and isoliquiritigenin instead worked through an EGFR/ERK/p38 signaling pathway [100]. Sulforaphane required VDR and ERK1/2 but not p38 to increase DEFB4 transcription in human intestinal epithelium [17]. In chicken macrophage cells, inhibitors of p38 or JNK nearly abolished FSK-induced AvBD9 [26]. MAP kinases are required for the upregulation of HDP by many phytochemicals, albeit the specific signaling cascade varies by treatment.

A NF-κB pathway was activated by resveratrol, genistein, or sulforaphane for HDP upregulation. Resveratrol-induced CAMP in human keratinocytes required the spingosine-1-phosphate (S1P) signaling, followed by transactivation of NF-κB and transcription factor C/EPBα [102]. A similar ER-β→S1P→NF-κB →C/EPBα mechanism was found for CAMP induction in keratinocytes treated with genistein [97]. Sulforaphane incubated with a NF-κB inhibitor reduced DEFB4 transcription in human intestinal epithelium [17]. NF-κB was also required for quercetin-induced AvBD9 induction in chicken monocytes [91]. However, dietary quercetin supplementation downregulated NF-κB mRNA expression in the chicken ileum although multiple AvBD genes were enhanced [93]. The involvement of NF-κB in HDP regulation by phytochemicals should be confirmed in vivo for additional species.

Interestingly, resveratrol, EGCG, quercetin, anacardic acid, and garcinol are natural cyclooxygenase (COX)-2 inhibitors that are able to upregulate HDPs in chicken monocytes [91]. Soponarin and apigenin were also found to inhibit COX-2 in human keratinocytes stimulated with inflammatory cytokines [95], mouse macrophages, and rat basophils [96]. Although inhibition of COX-2 signaling has previously been linked to HDP expression [190], its involvement in HDP regulation after treatment with these polyphenols remains to be studied.

Nuclear factor erythroid 2-related factor 2 (Nrf2) facilitates HDP expression in the liver in response to phytochemical treatment. Quercetin treatment increased binding of Nrf2 to an antioxidant response element at the HAMP promoter in parallel to enhanced HAMP transcription in human hepatocytes [92]. Curcumin likewise upregulated Nrf2 mRNA concentrations while increasing HAMP, LEAP2, and DEFB in grass carp liver [109].

FSK elevates intracellular cAMP concentrations, and binding of CREB to the human cathelicidin promoter promoted active gene transcription [110]. However, cAMP inhibited by 2',3'-dideoxyadenosine improved quercetin-induced AvBD9 in chicken monocytes [91]. The role of cAMP in HDP regulation needs to be clarified for different species and HDP genes.

Epigenetic modulators

Epigenetic modifications, such as acetylation of histones and methylation of histones and DNA, play an essential role in regulating chromatin accessibility and gene expression [191]. Histone acetylation is facilitated by histone acetyltransferases to favor a more relaxed chromatin structure, whereas the removal of acetyl groups by HDACs favors a more condensed chromatin configuration [191]. Histones and DNA can also be methylated by DNA methyltransferases (DNMT) and histone methyltransferases (HMT), respectively, to affect gene transcription [191]. HDAC inhibitors have been demonstrated to be among the most potent inducers of HDPs in several high throughput screenings [99,114,121,124,126,192]. Members of all 4 major classes of HDAC inhibitors, including benzamides, hydroxamates, cyclic peptides, and SCFAs, have been shown to be HDP inducers. For example, trichostatin A, a hydroxamate HDCA inhibitor, induced mRNA expression of HDPs in humans [15,18], pigs [114], chickens [99], and cattle [115] (Table 1). Other hydroxamte HDAC inhibitors, such as vorinostat also known as suberoylanilide hydroxamic acid, induced the mRNA expression of DEFA1, DEFA5, DEFA6, and DEFB4 in human monocytic cells infected with Leishmania donovani, contributing to increased antiparasitic activity [117]. Vorinostat also enhanced the expression of several HDP genes in human intestinal epithelium [112,118] and hepatocytes [119], chicken macrophages, [120,126] and porcine intestinal epithelial cells and lung alveolar macrophages [114].

However, among all HDAC inhibitors, benzamides appear to be the most potent HDP inducers in humans, pigs, and chickens as revealed in several recent high throughput assays [114,121,124,126,192]. For example, entinostat, a benzamide HDAC inhibitor, potently enhanced DEFB1 and CAMP mRNA expression in intestinal epithelial cells [121,122] and protected rabbits from experimental cholera [123]. Its analogous compounds, known as aroylated phenylenediamines, had similar effects in human bronchial epithelial cells, associated with a significant reduction in the intracellular invasion of Pseudomonas aeruginosa [125]. Entinostat also similarly induced HDP gene expression in chicken jejunal explants, and an oral inoculation of entinostat led to an increased expression of multiple HDP genes in the crop and jejunum of broiler chicks [124]. Mocetinostat, an entinostat analog, potently increased AvBD9 mRNA concentrations in chicken jejunal explants [126]. Polyphenols, such as sulforaphane, curcumin, and EGCG, are known natural HDAC inhibitors [193] with an HDP-inducing activity [17,89,108].

In addition to HDAC inhibitors, inhibitors of DNMT and HMT are HDP inducers. For instance, 5-azacytidine, a well-known DNMT inhibitor, increased CAMP and DEFB concentrations in gingival epithelial cells [111], oral carcinoma cells [127,128], keratinocytes [113], and chondrocytes [129]. Additionally, 5-azacytidine stimulated HDP expression in bovine mammary epithelial cells [115], chicken macrophage cells [120], the larvae of silkworm [130], as well as in the caput, cauda, and testis of the male rats [116]. Polyphenols such as EGCG, quercetin, and genistein, known for their DNMT inhibitory properties [194], also possess the ability to induce HDP synthesis as discussed in greater detail in an earlier section. Similarly, inhibitors of HMT, such as BIX01294 and UNC1999, have been shown to promote the expression of multiple HDPs in chicken macrophages [120].

Inhibition of HDACs, DNMTs, and HMTs may facilitate chromatin relaxation and promote gene transcription. Several HDAC inhibitors have demonstrated the capacity to promote histone acetylation at HDP promoter regions, accompanied by increased HDP transcription [18,112,117,195]. HDAC inhibition may also activate transcription factors such as NF-κB to turn on the transcription of HDPs such as DEFB4 owing to the presence of multiple NF-κB binding sites in the promoter region [118] (Figure 3). Consistently, trichostatin A increased activation and translocation of NF-κB through acetylation of p65 and phosphorylation of the IKK complex, ultimately leading to an increased binding of NF-κB to the DEFB4 promoter in human colonic epithelial cells [112]. Entinostat and its structural analog, HO53, promoted STAT3 activation leading to increased HIF1α expression and binding of HIF-1α to the CAMP promoter [122,125]. Similarly, RGFP966, an HDAC3 inhibitor, increased acetylation of STAT3, C/EBPα, and HIF-1α in human liver cells, enhancing the affinity of these transcription factors to the LEAP1 promoter [119].

FIGURE 3.

Epigenetic mechanisms of HDP gene induction. See text for details. Ac, acetyl group; C/EBPα, CCAAT enhancer binding protein α; DNMTi, DNA methyltransferase inhibitor; HDACi, histone deacetylase inhibitor; HIF1α, hypoxia-inducible factor-1α; HMTi, histone methyltransferase inhibitor; IKK, Iκ B kinase; P, phosphate group; Me, methyl group; TSA, trichostatin A; p50/p65, NF-κB proteins p50 and p65 heterodimer; STAT, signal transducer and activator of transcription.

DNMT inhibitors, such as AZA, reduced DNA methylation at DEFB1 [113] and CAMP [128,129] promoter regions, resulting in an increase in their transcription. Although the mechanism to support HMT inhibitors, BIX01294 and UNC1999, has not been well studied, a strong correlation has been found between histone demethylase, JMJD3, and expression of DEFB3, S100A7, S100A8, and CAMP in human keratinocytes [196]. Moreover, JMJD3 knockdown led to significantly increased histone methylation concentrations and reduced mRNA expression of these HDPs, highlighting the importance of histone methylation in shaping HDP gene expression patterns [196].

Probiotics

Probiotics are beneficial microbes that promote intestinal health through a myriad of mechanisms [197]. A variety of lactic acid bacteria have been found to induce HDP expression. For example, several Lactobacillus species induced HDP in human intestinal epithelia [[131], [132], [133], [134],136], gingival epithelia [135,137], vaginal epithelia [113,138], cervical epithelia [139], and epidermal cells [140] (Table 1). Oral supplementation of Lactobacillus delbruckii subsp. bulgaricus 8481 decreased IL-8 and increased DEFB4 in elderly patients [141]. In infants, Bifidobacterium animalis subsp. lactis BB-12 elevated both CAMP and DEFB4 protein concentrations [142]. E. coli Nissle 1917 were found to enhance DEFB4 expression in human intestinal epithelial cells [143,198].

Probiotics also induced HDP expression in rodents, pigs, chickens, cattle, and sheep. For instance, lactic acid bacteria were found to enhance HDP production and alleviate colitis [144], vancomycin-resistant enterococcus infection [199], and cirrhosis in mice [145]. In pigs, Lactobacillus reuteri I5007, Lactobacillus amylovorus SLZX20, and Lactobacillus plantarum ZLP001 increased the mRNA expression of multiple DEFBs in porcine intestinal epithelial cell lines [147,149,200], while L. plantarum counteracted intestinal barrier dysfunction induced by enterotoxigenic E. coli [148]. Treatment of intestinal cells with Bacillus subtilis CP9 enhanced PG1 transcription, but not PBD3, while having anti-ETEC properties [150]. Co-administration of Lactobacillus salivarius B1 and B. subtilis RJGP16 significantly induced the expression of PBD2 in the duodenum of neonatal piglets [151]. Likewise, both L. reuteri D8 and Lactobacillus rhamnosus Gorbach-Goldin (GG) increased body weight gain and decreased the incidence of diarrhea by upregulating HDPs in the jejunum of piglets [152,201].

In chickens, modulation of intestinal HDPs by L. reuteri is gene specific: AvBD1 and CATH3 mRNA expressions were increased, while CATH2 and AvBD10 were decreased, with no change observed with AvBD2 or AvBD12 [153,202]. In bovine mammary epithelial cells, Lactobacillus casei BL23 sustained the expression of DEFs during infection with S. aureus [154]. Furthermore, DEFB1 was increased in colon mucosa from dairy cows supplemented with live Saccharomyces cerevisiae CNCM I-1077 [155]. S. cerevisiae similarly upregulated SBD1 in ovine ruminal epithelial cells [203]. However, not all probiotics have the capacity to induce HDP synthesis. For instance, Bifidobacterium breve M16V did not affect the expression of DEFB1, DEFB4, or CAMP in preterm human infants [204], and 3 of 7 L. casei strains tested failed to upregulated Reg3b or a DEFA in the ileum of mice [146].

Probiotics such as Lactobacillus [133,147] or S. cerevisiae [203] triggered TLR2 along with MAPK and/or NF-κB signaling pathways to induce HDP gene expression in human, porcine, and ovine cells (Figure 4). L. rhamnosus GG and L. plantarum increased VDR protein expression in both mouse and human intestinal epithelial cells and induce CAMP gene expression through VDR [205]. On the contrary, L. gasseri, but not L. reuteri, induced DEFB1 gene expression associated with increased concentrations of histone H3 acetylation, H3K4me3, and H2A.Z in the proximal promoter region of the DEFB1 gene [113]. E. coli Nissle 1917, Pediococcus pentosaceus, Lactobacillus fermentum, and Lactobacillus acidophilus required NF-κB signaling in addition to AP-1 activation to induce DEFB4 [143,198]. An increase in HDP was paralleled by expression of nucleotide-binding oligomerization domain (NOD1/2) in cells treated with L. rhamnosus GG., B. longum spp. Infantis S12, or BI 5764 [136,144]. However, L. reuteri I5007 enhanced HDPs though upregulation of peroxisome proliferator-activated receptor-γ and GPR41 in porcine epithelial cells [200], while L. casei BL23 had no effect on NOD2 nor NF-κB [154]. These discrepancies may be due to differences in probiotic-host interaction or variation in signaling mechanisms between host species. Regardless, the escalating interest in the use of probiotics to promote host health warrants continued research into the mechanisms behind probiotic-induced HDP expression.

FIGURE 4.

Molecular mechanisms of HDP gene expression by probiotics. See text for details. Ac, acetyl group; AP1, activator protein-1; HDP, host defense peptide; MAPK, mitogen-activated protein kinases; NF-κB, nuclear factor-kappa B; TLR, toll-like receptor, VDR, vitamin D receptor.

Prebiotics

Supplementation of inulin to a Western-style diet increased the expression of multiple Paneth cell DEFAs and DEFB1 as well as tight junction proteins in the intestinal tissues of mice, resulting in improved intestinal barrier integrity and reduced endotoxemia [24] (Table 1). Similarly, long-chain, but not short-chain, inulin-type fructans delayed the onset of type 1 diabetes by promoting gut health, including a significant increase in Defb1 and Camp in the colon of mice [156]. Additionally, oral administration of polysaccharides extracted from Dendrobium huoshanense, a medicinal plant, enhanced DEFB production and barrier function of the intestinal tract of mice [157]. The HDP-inducing activity of other prebiotics remains to be studied.

Synergistic induction of HDP synthesis by different classes of dietary compounds

Several different classes of dietary compounds have been found to synergize with each other to potentiate HDP synthesis (Table 2) [16,26,35,36,76,77,91,99,100,103,105,[120], [121], [122],206,207]. For example, vitamin D-3 is synergistic with butyrate or PBA in CAMP synthesis in bronchial epithelial cells and macrophages, showing a bacteriostatic effect against M. tuberculosis [36,206]. The combination of vitamin D-3 and PBA was also shown to induce HDPs in human dendritic cells, leading to effective killing of S. aureus [207]. Vitamin D-3 further synergized with entinostat, resveratrol, or pterostilbene to induce CAMP synthesis in human colon cells, monocytes, or keratinocytes [103,121,122]. Synergy in augmenting CAMP expression was also observed when vitamin D-3 was combined with calcium in normal and cystic fibrosis bronchial epithelial cells [208].

TABLE 2.

Synergistic induction of HDP synthesis by combinations of different classes of dietary compounds

Compounds	Cells or tissues	HDPs	References
Butyrate + lithocholic acid	Human HT-29	CAMP	[16]
Butyrate + forskolin	Chicken HD11, HTC, jejunal explants	AvBD9	[26]
Butyrate + propionate + acetate	Chicken HD11 macrophages	AvBD9	[35]
Butyrate/PBA + lactose	Human HT-29	CAMP	[76]
Butyrate + galactose/trehalose/lactose	Chicken HD11 macrophages	AvBD9, CATHB1	[77]
Butyrate + COX-2 inhibitors	Chicken HTC, PBMCs	AvBD9	[91]
Butyrate + wortmannin/tetrandrine/datiscetin	Chicken HTC, chicken monocytes	AvBD9	[99]
Vitamin D-3 + PBA	Human VA10, MDM, PBMCs	CAMP	[36,206,207]
Vitamin D-3 + resveratrol/pterostilbene	Human U937, HaCaT	CAMP	[103]
Vitamin D-3 + entinostat	Human HT-29	CAMP	[121,122]
Vitamin D-3 + calcium	Human NHBE	CAMP	[208]
Andrographolide + isoliquiritigenin	Human Caco-2	DEFB3	[100]
Polydatin + resveratrol	Human HaCaT	DEFB4	[105]
HDACi + HMTi/DNMTi	Chicken HTC, HD11 macrophages	AvBD9	[120]

Abbreviations: AvBD, avian β-defensin; CAMP, cathelicidin; CATH, chicken cathelicidin; COX, cyclooxygenase; DEF, defensin; DNMTi, DNA methyltransferase inhibitor; HDACi, histone deacetylase inhibitor; HDP, host defense peptide; HMTi, histone methyltransferase inhibitor; PBA, phenylbutyrate.

In addition to vitamin D-3, butyrate synergizes with several other classes of dietary compounds to enhance HDP synthesis. When combined with lactose, butyrate or 4-PBA showed a synergistic effect on human CAMP expression [76]. Similarly, butyrate synergized with other sugars, such as galactose or trehalose, to upregulate HDP expression in chicken macrophages [77]. Administration of butyrate and FSK showed a synergistic increase in AvBD9 in the crop and jejunum of chickens [26]. An even higher magnitude of HDP gene induction and protection against necrotic enteritis was observed with the combination of butyrate, lactose, and FSK over any of the 2-compound combinations in chickens [209]. Butyrate in combination with wortmannin, a naturally occurring fungal metabolite and a specific phosphoinositide 3-kinase inhibitor, synergistically increased HDP expression in chickens [99]. Additionally, butyrate synergized with tetrandrine or datiscetin in chicken HDP gene induction [99]. Several polyphenols and COX-2 inhibitors, namely quercetin, resveratrol, anacardic acid, EGCG, and garcinol had a strong synergy with butyrate in HDP transcription in chicken cells [91]. Additionally, butyrate synergized with other SCFAs such as acetate and propionate in enhancing chicken HDP expression both in vitro and in vivo [35].

Furthermore, polydatin, a natural precursor to resveratrol, synergistically increased DEFB4 production in human keratinocytes when combined with resveratrol [105]. Andrographolide and isoliquiritigenin cooperated to enhance DEFB3 expression and antibacterial activity of human colonic epithelial cells [100]. Different classes of epigenetic compounds also appear to act in a corporative manner in increasing HDP gene expression. For instance, an HDAC inhibitor paired with an inhibitor of either an HMT or a DNMT led to a drastic synergy in the transcription of multiple HDP genes in chicken macrophages [120]. Additionally, probiotics such as E. coli Nissle 1917 synergizes with HDCA inhibitors to enhance DEFB4 expression in human intestinal cell lines, but not in human colonic biopsies [118].

The mechanism for synergy between HDP inducers remains elusive. HDAC inhibitors including butyrate increase histone acetylation in favor of active gene transcription [30], so a proposed mechanism for synergy with butyrate involved additional enrichment of histone acetylation. Butyrate and lactose treatment led to the hyperacetylation of H4 preceding a synergistic response in chicken HDP gene induction [77]. However, no additional histone acetylation was observed for butyrate and quercetin in relation to their synergy in chicken HDP transcription [91]. Changes in histone acetylation are insufficient to describe the synergy observed between butyrate and a secondary HDP inducer.

Other studies looked at the signaling pathways activated by individual compounds compared with compound combinations. A similar involvement of p38 MAPK, JNK, NF-κB, and cAMP signaling pathways was observed in response to butyrate and lactose as for butyrate alone [77]. Likewise, the signaling pathways activated by lactose and PBA for synergistic induction of CAMP coincided with those found for the individual treatments [210].

The mechanism appears to be due to the cooperation between epigenetic modulation and regulation of signaling pathways and/or specific transcription factors as opposed to synergy in boosting any individual pathway. For example, induction of CAMP transcription involved cooperation among VDR, C/EBPα, PU.1, and chromatin remodeling [171]. On the contrary, inhibition of ERK1/2 and MAPK-p38 by resveratrol was found to contribute to the synergy with vitamin D-3 in augmenting CAMP gene expression [103]. The synergistic effect of andrographolide and isoliquiritigenin on DEFB3 expression was supported by higher phosphorylation of H3S10 and recruitment of transcription factors Fos and ELK1 than use of either molecule alone [100]. A plausible mechanism for synergy between HDP inducers could be due to increased transcription factor binding when 1 or both modulators can also induce epigenetic changes in favor of an accessible HDP gene promoter.

Conclusions and Future Prospects

Dietary modulation of endogenous HDP synthesis has potential to be developed as an alternative approach to antimicrobial therapy. Several HDP inducers, such as butyrate, 4-PBA, and vitamin D-3, have demonstrated protective efficacy against infectious diseases across multiple animal species. It is noted that nutritional modulation of endogenous HDP synthesis is unlikely to drive antimicrobial resistance, as these HDP-inducing dietary factors act on the host without exerting direct antimicrobial activity. Additionally, unlike other immune boosters that often nonspecifically trigger inflammation, many of the HDP-inducing compounds stimulate the synthesis of HDPs without eliciting a proinflammatory response while some even have anti-inflammatory effects [31,69,100]. This is beneficial, as inflammation can lead to detrimental effects on the host, such as tissue damage. However, additional human and animal trials are warranted to realize the clinical potential of HDP inducers as novel host-directed antimicrobials.

It is noted that different classes of dietary compounds have a strong capacity to induce HDPs in multiple cell types and animal species, although cell-specific and species-specific HDP induction is evident. Importantly, many compounds show a synergy in promoting HDP synthesis when combined. Mechanisms of dietary compound-mediated HDP induction are being investigated and a detailed understanding of the molecular mechanisms may allow further HDP-inducing efficiency. Current evidence indicates the involvement of histone acetylation as well as MAPK, NF-κB, VDR, cAMP, and COX-2 signaling pathways. Transcription factors such as AP-1, CREB, STAT3, and sphingosine-1-phosphate have also been implicated in nutritional regulation of HDPs. Further investigation may yield dietary compounds or their combinations as promising candidates as effective alternatives to antibiotics for both human and animal applications.

Author contributions

The authors’ responsibilities were as follows – GZ: conceived the study; MW, IT, AB, GZ: drafted the manuscript; IT, MW: contributed to visualization; GZ: edited the manuscript and has primary responsibility for final content; and all authors: read and approved the final manuscript.

Conflict of interest

The authors report no conflicts of interest.

Funding

This research was supported by the USDA National Institute of Food and Agriculture grants (2020-67016-31619 and 2023-67015-39095), the Ralph F. and Leila W. Boulware Endowment Fund, and Oklahoma Agricultural Experiment Station Project H-3112. MW was supported by a USDA National Institute of Food and Agriculture Predoctoral Fellowship grant (2021-67034-35184). However, the funders had no role in the design of the study, the collection or interpretation of data, the writing of the manuscript, or the decision to publish the results.