Mechanisms and regulation of defensins in host defense.

As a family of cationic host defense peptides, defensins are mainly synthesized by Paneth cells, neutrophils, and epithelial cells, contributing to host defense. Their biological functions in innate immunity, as well as their structure and activity relationships, along with their mechanisms of action and therapeutic potential, have been of great interest in recent years. To highlight the key research into the role of defensins in human and animal health, we first describe their research history, structural features, evolution, and antimicrobial mechanisms. Next, we cover the role of defensins in immune homeostasis, chemotaxis, mucosal barrier function, gut microbiota regulation, intestinal development and regulation of cell death. Further, we discuss their clinical relevance and therapeutic potential in various diseases, including infectious disease, inflammatory bowel disease, diabetes and obesity, chronic inflammatory lung disease, periodontitis and cancer. Finally, we summarize the current knowledge regarding the nutrient-dependent regulation of defensins, including fatty acids, amino acids, microelements, plant extracts, and probiotics, while considering the clinical application of such regulation. Together, the review summarizes the various biological functions, mechanism of actions and potential clinical significance of defensins, along with the challenges in developing defensins-based therapy, thus providing crucial insights into their biology and potential clinical utility.

Acceleration of Small Intestine Development and Remodeling of the Microbiome Following Hyaluronan 35 kDa Treatment in Neonatal Mice.

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.

The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model.

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect.

Associations between inflammatory bowel diseases and vitamin D.

Inflammatory Bowel Diseases (IBD) are increasing sharply, and the common medications are not effective for most patients. Vitamin D (VD) has been considerate to reduce inflammatory processes and may be helpful in IBD. The aim of this review was to perform an update on the potential role of VD in the IBD. We performed a search for articles associating VD and IBD published in MEDLINE-PubMed and EMBASE. The focused question used for the search was "What is the association between Inflammatory Bowel Disease and Vitamin D?" The exclusion criteria for this search were studies not in English, editorials, case reports, or poster presentations. VD prevents the inflammatory process such as negatively interfering with the release of Interleukin (IL)-1, IL-6, and Tumour Necrosis Factor-α; enhancing the function of the intestinal epithelial barrier; decreasing the occurrence of apoptosis; stimulating Toll-Like Receptor-4; inducing the production of an antimicrobial peptide in Paneth cells. Furthermore, deficiency of VD is related to the severity of the symptoms and increased the risk of cancer and surgery. In conclusion, VD shows a potential role in the management of IBD, the supplementation is inexpensive, safe, and leads to improvement of the quality of life.

ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides.

BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. METHODS: We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

N-3 polyunsaturated fatty acid-enriched lipid emulsion improves Paneth cell function via the IL-22/Stat3 pathway in a mouse model of total parenteral nutrition.

Total parenteral nutrition (TPN) is a life-saving therapy for patients with gastrointestinal dysfunction or failure. Long-term TPN impairs gut barrier function and contributes to infections and poor clinical outcomes. However, the underlying mechanisms of TPN-related gut barrier damage have not been fully elucidated, and effective measures are still rare. Here, we compared the effects of a predominantly n-6 polyunsaturated fatty acids emulsion (PUFAs; Intralipid) and a lipid emulsion containing n-3 PUFAs (Intralipid plus Omegaven) on antimicrobial peptides produced by Paneth cells. Our results show for the first time that n-3 PUFAs markedly ameliorated intestine atrophy, and increased protein levels of lysozyme, RegIIIγ, and α-cryptdin 5, and their mRNA expression, compared to the n-6 PUFAs emulsion. Importantly, our study reveals that downregulation of IL-22 and phosphorylated Stat3 (p-Stat3) is associated with Paneth cell dysfunction, which may mediate TPN-related gut barrier damage. Lastly, n-3 PUFAs upregulated levels of IL-22 and increased the p-Stat3/Stat3 ratio in ileal tissue, suggesting that n-3 PUFAs improve Paneth cell function through activation of the IL-22/Stat3 pathway. Therefore, our study provides a cogent explanation for the beneficial effects of n-3 PUFAs, and indicates the IL-22/Stat3 pathway as a promising target in the treatment of TPN-related gut barrier damage.

Functional relevance of intestinal epithelial cells in inflammatory bowel disease.

The intestinal epithelium constitutes a physical barrier between inner and outer side of our body. It also functions as a "hub" which connects factors that determine the development of inflammatory bowel disease, such as microbiota, susceptibility genes, and host immune response. Accordingly, recent studies have implicated and further featured the role of intestinal epithelial cell dysfunction in the pathophysiology of inflammatory bowel disease. For example, mucin producing goblet cells are usually "depleted" in ulcerative colitis patients. Studies have shown that those goblet cells exhibit various immune-regulatory functions in addition to mucin production, such as antigen presentation or cytokine production. Paneth cells are another key cell lineage that has been deeply implicated in the pathophysiology of Crohn's disease. Several susceptibility genes for Crohn's disease may lead to impairment of anti-bacterial peptide production and secretion by Paneth cells. Also, other susceptibility genes may determine the survival of Paneth cells, which leads to reduced Paneth cell function in the patient small intestinal mucosa. Further studies may reveal other unexpected roles of the intestinal epithelium in the pathophysiology of inflammatory bowel disease, and may help to develop alternative therapies targeted to intestinal epithelial cell functions.

Dietary L-glutamine supplementation modulates microbial community and activates innate immunity in the mouse intestine.

This study was conducted to determine effects of dietary supplementation with 1 % L-glutamine for 14 days on the abundance of intestinal bacteria and the activation of intestinal innate immunity in mice. The measured variables included (1) the abundance of Bacteroidetes, Firmicutes, Lactobacillus, Streptococcus and Bifidobacterium in the lumen of the small intestine; (2) the expression of toll-like receptors (TLRs), pro-inflammatory cytokines, and antibacterial substances secreted by Paneth cells and goblet cells in the jejunum, ileum and colon; and (3) the activation of TLR4-nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt) signaling pathways in the jejunum and ileum. In the jejunum, glutamine supplementation decreased the abundance of Firmicutes, while increased mRNA levels for antibacterial substances in association with the activation of NF-κB and PI3K-Akt pathways. In the ileum, glutamine supplementation induced a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes, and enhanced mRNA levels for Tlr4, pro-inflammatory cytokines, and antibacterial substances participating in NF-κB and JNK signaling pathways. These results indicate that the effects of glutamine on the intestine vary with its segments and compartments. Collectively, dietary glutamine supplementation of mice beneficially alters intestinal bacterial community and activates the innate immunity in the small intestine through NF-κB, MAPK and PI3K-Akt signaling pathways.

Selenoprotein S is a marker but not a regulator of endoplasmic reticulum stress in intestinal epithelial cells.

Selenoproteins are candidate mediators of selenium-dependent protection against tumorigenesis and inflammation in the gut. Expression and roles of only a limited number of intestinal selenoproteins have been described so far. Selenoprotein S (SelS) has been linked to various inflammatory diseases and is suggested to be involved in endoplasmic reticulum (ER) homeostasis regulation and antioxidative protection in a cell-type-dependent manner, but its protein expression, regulation, and function in the gut are not known. We here analyzed the expression and localization of SelS in the healthy and inflamed gut and studied its regulation and function in intestinal epithelial cell lines. SelS was expressed in the intestinal epithelium of the small and large intestine and colocalized with markers of Paneth cells and macrophages. It was upregulated in inflamed ileal tissue from Crohn's disease patients and in two models of experimental colitis in mice. We detected SelS in colorectal cell lines, where it colocalized with the ER marker calnexin. SelS protein expression was unaffected by enterocytic differentiation but increased in response to selenium supplementation and after treatment with the ER stress inducer tunicamycin. On the other hand, depletion of SelS in LS174T, HT29, and Caco-2 cells by RNA interference did not cause or modulate ER stress and had no effect on hydrogen peroxide-induced cell death. In summary, we introduce SelS as a novel marker of Paneth cells and intestinal ER stress. Although it is upregulated in Crohn's disease, its role in disease etiology remains to be established.

Intravenous administration of tetramethylpyrazine reduces intestinal ischemia-reperfusion injury in rats.

Intestinal ischemia-reperfusion injury (IIRI) is a life-threatening condition requiring prompt medical intervention. Tetramethylpyrazine (TMP) is a biologically active alkaloid isolated from Ligusticum wallichii. Previously, it was shown that TMP causes vasodilatation and inhibition of platelet aggregation as well as exhibits significant antioxidant effects. Therefore, the aim of the present study was to evaluate possible therapeutic effects of TMP in the prevention of IIRI. Wistar rats (n = 80) were randomly divided into eight experimental groups and subjected to a 1 h occlusion of cranial mesenteric artery followed by 0, 1, 12, and 24 h period of reperfusion. Thirty minutes before the IIRI animals received either TMP (30 mg/kg, i.v.) or identical volume of saline. In addition, a control group of 10 animals was not exposed to IIRI. Intestine morphology was evaluated by using histopathological injury index examination (HII), goblet and Paneth cells quantification as well as by applying immunofluorescent methods such as InSitu TUNEL and caspase-3 positivity assessment. Here we showed that preconditioning with TMP prior IIRI decreases the grade of injury. Significant reduction of HII was detected in TMP pretreated groups after 0, 1, and 12 h of reperfusion where injury reduction up to 75% was found. Lower histopathological damage in preconditioned groups was accompanied with increased number of secretory epithelial cells and decreased number of apoptotic cells. These results demonstrate the protective effect of TMP on the small intestine mucosa, suggesting administration of TMP as a molecule for pharmacological intervention against IIRI.

Growth promoting effect of thioredoxin on intestinal epithelial cells.

Paneth cells are located at the bases of intestinal crypts, and their cytoplasmic granules contain large amounts of zinc. We previously showed that administration of diphenylthiocarbazone (dithizone), a zinc chelater, to rats induced the selective death of Paneth cells. This was followed by a transient wave of epithelial cell proliferation in the entire crypts. In the study described here, we again applied this experimental model in an attempt to identify novel growth-promoting factors in the small intestine. Male Wistar rats were injected with dithizone and killed 6 hr later. Messenger RNAs (mRNAs) were extracted from the terminal ileum for the construction of a cDNA library. This library was then transfected into the human intestinal cell line Caco-2, and the cells that continued to grow in the medium containing only 1% FBS were cloned. One of the cDNA sequences identified from those transfection experiments was the full-length rat thioredoxin (TRX) gene. To confirm the growth-promoting effect of this cDNA, we transfected it into Caco-2 cells again. These clones proliferated in the medium containing only 1% FBS, while the control clones failed to show any growth. Transient oxidative stress exerted by the addition of oxidative reagents diamide and hydrogen peroxide partially suppressed the growth of TRX-transfected cells. Northern hybridization analysis revealed that TRX expression in rat ileum after dithizone treatment was altered in accordance with intestinal epithelial regeneration in the crypts. Single-cell RT-PCR also showed TRX mRNA expression in Paneth cells. These studies identify rat thioredoxin as a growth-promoting factor for intestinal epithelial cells.

Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells.

The adult mouse intestine contains an intricate vascular network. The factors that control development of this network are poorly understood. Quantitative three-dimensional imaging studies revealed that a plexus of branched interconnected vessels developed in small intestinal villi during the period of postnatal development that coincides with assembly of a complex society of indigenous gut microorganisms (microbiota). To investigate the impact of this environmental transition on vascular development, we compared the capillary networks of germ-free mice with those of ex-germ-free animals colonized during or after completion of postnatal gut development. Adult germ-free mice had arrested capillary network formation. The developmental program can be restarted and completed within 10 days after colonization with a complete microbiota harvested from conventionally raised mice, or with Bacteroides thetaiotaomicron, a prominent inhabitant of the normal mouse/human gut. Paneth cells in the intestinal epithelium secrete antibacterial peptides that affect luminal microbial ecology. Comparisons of germ-free and B. thetaiotaomicron-colonized transgenic mice lacking Paneth cells established that microbial regulation of angiogenesis depends on this lineage. These findings reveal a previously unappreciated mechanism of postnatal animal development, where microbes colonizing a mucosal surface are assigned responsibility for regulating elaboration of the underlying microvasculature by signaling through a bacteria-sensing epithelial cell.

Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel.

Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.