Expanding the insect defensin landscape.

In this issue of Structure, Walker et al.1 determined the NMR structure of a recently discovered defensin, Pp19, from the venom of an assassin bug. This peptide adopts an α-defensin-like structure, which had not been observed in insects before. Unlike mammalian α-defensins, which are generally antimicrobial, Pp19 has insecticidal activity.

Withania somnifera osmotin (WsOsm) confers stress tolerance in tobacco and establishes novel interactions with the defensin protein (WsDF).

Pathogenesis-related proteins (PR), including osmotins, play a vital role in plant defense, being activated in response to diverse biotic and abiotic stresses. Despite their significance, the mechanistic insights into the role of osmotins in plant defense have not been extensively explored. The present study explores the cloning and characterization of the osmotin gene (WsOsm) from Withania somnifera, aiming to illuminate its role in plant defense mechanisms. Quantitative real-time PCR analysis revealed significant induction of WsOsm in response to various phytohormones e.g. abscisic acid, salicylic acid, methyl jasmonate, brassinosteroids, and ethrel, as well as biotic and abiotic stresses like heat, cold, salt, and drought. To further elucidate WsOsm's functional role, we overexpressed the gene in Nicotiana tabacum, resulting in heightened resistance against the Alternaria solani pathogen. Additionally, we observed enhancements in shoot length, root length, and root biomass in the transgenic tobacco plants compared to wild plants. Notably, the WsOsm- overexpressing seedlings demonstrated improved salt and drought stress tolerance, particularly at the seedling stage. Confocal histological analysis of H2O2 and biochemical studies of antioxidant enzyme activities revealed higher levels in the WsOsm overexpressing lines, indicating enhanced antioxidant defense. Furthermore, a pull-down assay and mass spectrometry analysis revealed a potential interaction between WsOsm and defensin, a known antifungal PR protein (WsDF). This suggests a novel role of WsOsm in mediating plant defense responses by interacting with other PR proteins. Overall, these findings pave the way for potential future applications of WsOsm in developing stress-tolerant crops and improving plant defense strategies against pathogens.

Tick-Borne pathogens and defensin genes expression: A closer look at Ixodes ricinus and Dermacentor reticulatus.

The immune system of ticks, along with that of other invertebrates, is comparatively simpler than that of vertebrates, relying solely on innate immune responses. Direct antimicrobial defence is provided by the synthesis of antimicrobial peptides (AMPs), including defensins. The aim of this study was to investigate the differences in defensin genes expression between questing and engorged Ixodes ricinus (def1 and def2) and Dermacentor reticulatus (defDr) ticks, in the presence of selected pathogens: Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis in the natural environment. After pathogen screening by PCR/qPCR, the expression of defensin genes in pathogen positive ticks and ticks without any of the tested pathogens, was analysed by reverse transcription qPCR. The results showed an increased expression of defensin genes in I. ricinus ticks after blood feeding and I. ricinus and D. reticulatus ticks during in cases of co-infection. In particular, the expression of defensins genes was higher in questing D. reticulatus than in questing and engorged I. ricinus ticks, when borreliae were detected. This study contributes to uncovering the expression patterns of defensin genes in the presence of several known tick pathogens, the occurrence of these pathogens and possible regulatory mechanisms of defensins in tick vector competence.

Supplementation of Vitamin D3 and Fructooligosaccharides Downregulates Intestinal Defensins and Reduces the Species Abundance of Romboutsia ilealis in C57BL/6J Mice.

The activation of the vitamin D receptor (VDR) in the ileum has been shown to regulate Paneth cell-specific defensins, a large family of antimicrobial peptides; hence, this may serve as a potential mechanism to maintain intestinal homeostasis. Previously, we have demonstrated that a combination of vitamin D3 (VD) and fructooligosaccharides (FOSs) upregulates colonic Vdr in mice. Here, we aim to examine the effect of VD, alone or in combination with FOSs, on intestinal barrier integrity and the secretion of antimicrobial peptides, as well as the gut microbial community. Male and female C57BL/6J mice at 6 weeks old were randomized into three groups to receive the following dietary regimens (n = 10/sex/group) for 8 weeks: (1) standard AIN-93G control diet (CTR), (2) CTR + 5000 IU vitamin D3 (VD), and (3) VD + 5% fructooligosaccharides (VF). VD and VF differentially regulated the mRNA expressions of tight junction proteins in the colon and ileum. VF suppressed the upregulation of colonic ZO-1 and occludin, which was induced by VD supplementation alone. In the ileum, occludin but not ZO-1 was upregulated 20-fold in the VF-treated mice. While VD did not alter the mRNA expressions of Vdr and defensins in the ileum, these targets were downregulated by VF. Microbial analysis further reveals a shift of microbial beta diversity and a reduction in Romboutsia ilealis, a pathobiont, in VF-treated mice. Though the implications of these phenotypical and microbial changes remain to be determined, the administration of FOSs in the presence of VD may serve as an effective dietary intervention for maintaining intestinal homeostasis.

The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H+-ATPases.

Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and ß-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

Endoplasmic reticulum stress response modulator OsbZIP39 regulates cadmium accumulation via activating the expression of defensin-like gene OsCAL2 in rice.

Accumulation of cadmium (Cd) in rice is not only harmful to the growth of plants but also poses a threat to human health. Exposure to Cd triggers unfolded protein response (UPR) within cells, a process that is still not completely understood. The study demonstrated that the lack of OsbZIP39, an essential endoplasmic reticulum (ER)-resident regulator of the UPR, resulted in decreased Cd intake and reduced Cd levels in the roots, stems, and grains of rice. Upon exposure to Cd stress, GFP-OsbZIP39 translocated from ER to nucleus, initiating UPR. Further investigation revealed that Cd treatment caused changes in sphingolipid levels in the membrane, influencing the localization and activation of OsbZIP39. Yeast one-hybrid and dual-LUC assays were conducted to validate the interaction between activated OsbZIP39 and the promoter of the defensin-like gene OsCAL2, resulting in an increase in its expression. Different variations were identified in the coding region of OsbZIP39, which may explain the varying levels of Cd accumulation observed in the indica and japonica subspecies. Under Cd treatment, OsbZIP39ind exhibited a more significant enhancement in the transcription of OsCAL2 compared to OsbZIP39jap. Our data suggest that OsbZIP39 positively regulates Cd uptake in rice, offering an encouraging objective for the cultivation of low-Cd rice.

Reduced expression of central innate defense molecules in pancreatic biopsies from subjects with Type 1 diabetes.

AIMS/HYPOTHESIS: Defensins play a crucial role in the innate immune system's first defense against microbial threats. However, little is known about the defensin system in the pancreas, especially in relation to Type 1 diabetes. We explore the expression of defensins in different disease stages of Type 1 diabetes and correlated obtained findings to the degree of inflammation, providing new insights into the disease and the innate immune system. MATERIAL AND METHODS: Pancreases from non-diabetic human organ donors of different age groups and donors with Type 1 diabetes with different disease duration were examined. Sections from head, body and tail of the pancreas were stained for eight different defensins and for immune cells; CD3+, CD45+, CD68+ and NES+ (granulocytes). RESULTS: In non-diabetic adult controls the level of expression for defensins Beta-1,Alpha-1, Cathelicidin and REG3A correlated with the level of inflammation. In contrast, individuals with Type  1 diabetes exhibit a reduction or absence of several central defensins regardless of the level of inflammation in their pancreas. The expression of Cathelicidin is present in neutrophils and macrophages but not in T-cells in subjects with Type 1 diabetes. CONCLUSIONS: Obtained findings suggest a pancreatic dysfunction in the innate immune system and the bridging to the adaptive system in Type 1 diabetes. Further studies on the role of the local innate immune system in Type 1 diabetes is needed.

Characterisation of defensins across the marsupial family tree.

Defensins are antimicrobial peptides involved in innate immunity, and gene number differs amongst eutherian mammals. Few studies have investigated defensins in marsupials, despite their potential involvement in immunological protection of altricial young. Here we use recently sequenced marsupial genomes and transcriptomes to annotate defensins in nine species across the marsupial family tree. We characterised 35 alpha and 286 beta defensins; gene number differed between species, although Dasyuromorphs had the largest repertoire. Defensins were encoded in three gene clusters within the genome, syntenic to eutherians, and were expressed in the pouch and mammary gland. Marsupial beta defensins were closely related to eutherians, however marsupial alpha defensins were more divergent. We identified marsupial orthologs of human DEFB3 and 6, and several marsupial-specific beta defensin lineages which may have novel functions. Marsupial predicted mature peptides were highly variable in length and sequence composition. We propose candidate peptides for future testing to elucidate the function of marsupial defensins.

Enhancing plant defensins in a desert shrub: Exploring a regulatory pathway of AnWRKY29.

A distinct family of plant-specific WRKY transcription factors plays a crucial role in modulating responses to biotic and abiotic stresses. In this investigation, we unveiled a signaling pathway activated in the desert shrub Ammopiptanthus nanus during feeding by the moth Spodoptera exigua. The process involves a Ca2+ flux that facilitates interaction between the protein kinase AnCIPK12 and AnWRKY29. AnWRKY29 directly interacts with the promoters of two key genes encoding AnPDF1 and AnHsfB1, involved in the biosynthesis of plant defensins. Consequently, AnWRKY29 exerts its transcriptional regulatory function, influencing plant defensins biosynthesis. This discovery implies that A. nanus can bolster resistance against herbivorous insects like S. exigua by utilizing this signaling pathway, providing an effective natural defense mechanism that supports its survival and reproductive success.

Defensins regulate cell cycle: Insights of defensins on cellular proliferation and division.

Defensins are a class of small antimicrobial peptides that play a crucial role against pathogens. However, recent research has highlighted defensins exhibit the ability to influence cell cycle checkpoints, promoting or inhibiting specific phases such as G1 arrest or S/M transition. By regulating the cell cycle, defensins impact the proliferation of normal and cancerous cells, with implications for cancer development and progression. Dysregulation of defensin expression can disrupt the delicate balance of cell cycle regulation, leading to uncontrolled cell growth and an increased risk of tumor formation. Defensins contribute to the resolution of inflammation, stimulate angiogenesis, and enhance the migration and proliferation of cells involved in tissue repair. Furthermore, The ability of defensins to respond to microenvironmental changes further demonstrates the significance of these peptides in host defense mechanisms and immune function. By adjusting their expression, defensins continue to combat pathogens effectively and maintain homeostasis within the body. This review highlights the multifaceted role of defensins in regulating the cell cycle and their broader implications in cancer progression, tissue repair, and microenvironmental response.

Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to ß-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide.

A trace amine associated receptor mediates antimicrobial immune response in the oyster Crassostrea gigas.

Trace amine-associated receptors (TAARs) are a class of G protein-coupled receptors, playing an immunomodulatory function in the neuroinflammatory responses. In the present study, a TAAR homologue with a 7tm_classA_rhodopsin-like domain (designated as CgTAAR1L) was identified in oyster Crassostrea gigas. The abundant CgTAAR1L transcripts were detected in visceral ganglia and haemocytes compared to other tissues, which were 55.35-fold and 32.95-fold (p < 0.01) of those in adductor muscle, respectively. The mRNA expression level of CgTAAR1L in haemocytes significantly increased and reached the peak level at 3 h after LPS or Poly (I:C) stimulation, which was 4.55-fold and 12.35-fold of that in control group, respectively (p < 0.01). After the expression of CgTAAR1L was inhibited by the injection of its targeted siRNA, the mRNA expression levels of interleukin17s (CgIL17-1, CgIL17-5 and CgIL17-6), and defensin (Cgdefh1) significantly decreased at 3 h after LPS stimulation, which was 0.51-fold (p < 0.001), 0.39-fold (p < 0.01), 0.48-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in the control group, respectively. The nuclear translocation of Cgp65 protein was suppressed in the CgTAAR1L-RNAi oysters. Furthermore, the number of Vibrio splendidus in the haemolymph of CgTAAR1L-RNAi oysters significantly increased (4.11-fold, p < 0.001) compared with that in the control group. In contrast, there was no significant difference in phagocytic rate of haemocytes to V. splendidus in the CgTAAR1L-RNAi oysters. These results indicated that CgTAAR1L played an important role in the immune defense against bacterial infection by inducing the expressions of interleukin and defensin.

An endophytic fungus interacts with the defensin-like protein OsCAL1 to regulate cadmium allocation in rice.

Defensin-like proteins are conserved in multicellular organisms and contribute to innate immune responses against fungal pathogens. In rice, defensins play a novel role in regulating cadmium (Cd) efflux from the cytosol. However, whether the antifungal activity of defensins correlates with Cd-efflux function remains unknown. In this study, we isolated an endophytic Fusarium, designed Fo10, by a comparative microbiome analysis of rice plants grown in a paddy contaminated with Cd. Fo10 is tolerant to high levels of Cd, but is sensitive to the defensin-like protein OsCAL1, which mediates Cd efflux to the apoplast. We found that Fo10 symbiosis in rice is regulated by OsCAL1 dynamics, and Fo10 coordinates multiple plant processes, including Cd uptake, vacuolar sequestration, efflux to the environment, and formation of Fe plaques in the rhizosphere. These processes are dependent on the salicylic acid signaling pathway to keep Cd levels low in the cytosol of rice cells and to decrease Cd levels in rice grains without any yield penalty. Fo10 also plays a role in Cd tolerance in the poaceous crop maize and wheat, but has no observed effects in the eudicot plants Arabidopsis and tomato. Taken together, these findings provide insights into the mechanistic basis underlying how a fungal endophyte and host plant interact to control Cd accumulation in host plants by adapting defense responses to promote the establishment of a symbiosis that permits adaptation to high-Cd environments.

Effects of histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors on proliferative, differentiative, and regenerative functions of Toll-like receptor 2 (TLR-2)-stimulated human dental pulp cells (hDPCs).

OBJECTIVES: This in vitro study aimed to modify TLR-2-mediated effects on the paracrine, proliferative, and differentiation potentials of human dental pulp-derived cells using histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. MATERIALS AND METHODS: Cell viability was assessed using the XTT assay. Cells were either treated with 10 µg/ml Pam3CSK4 only, or pre-treated with valproic acid (VPA) (3 mM), trichostatin A (TSA) (3 µM), and MG-149 (3 µM) for a total of 4 h and 24 h. Control groups included unstimulated cells and cells incubated with inhibitors solvents only. Transcript levels for NANOG, OCT3-4, FGF-1 and 2, NGF, VEGF, COL-1A1, TLR-2, hßD-2 and 3, BMP-2, DSPP, and ALP were assessed through qPCR. RESULTS: After 24 h, TSA pre-treatment significantly upregulated the defensins and maintained the elevated pro-inflammatory cytokines, but significantly reduced healing and differentiation genes. VPA significantly upregulated the pro-inflammatory cytokine levels, while MG-149 significantly downregulated them. Pluripotency genes were not significantly affected by any regimen. CONCLUSIONS: At the attempted concentrations, TSA upregulated the defensins gene expression levels, and MG-149 exerted a remarkable anti-inflammatory effect; therefore, they could favorably impact the immunological profile of hDPCs. CLINICAL RELEVANCE: Targeting hDPC nuclear function could be a promising option in the scope of the biological management of inflammatory pulp diseases.

Increasing expression of STING by ERα antagonizes LCN2 downregulation during chronic endometritis.

Chronic endometritis has a high incidence in infertile women, which is caused by endometrial microbiome infection. In response to microbial infection, the role of defensins during chronic endometritis need explored. Besides, the expression of estrogen and its receptors vary in different menstrual cycles, but their roles in chronic endometritis are still unclear. In this study, we used the human endometrial tissues to examine the expression of antimicrobial peptides (AMPs) α-defensin hNP-1 and ß-defensins hBD-1, hBD-2, hBD-3, hBD-4 and LCN2. We found the expression of hBD-1 and LCN2 were downregulated in endometritis tissues, while the expressions of hBD-2, hBD-3, hBD-4, hNP-1, and estrogen and ERα were upregulated in chronic endometritis tissues compared to normal tissues. The expression and phosphorylation of STING, which is a crucial mediator of mammalian innate immunity in response to pathogens, was regulated with the treatment of ERα inhibitor raloxifene (Rx). Furthermore, using with the estrogen receptor inhibitor Rx and STING inhibitor H-151 significantly decreases the LCN2 expression. Taken together, these results suggested ERα was upregulated to modulate STING expression inducing LCN2 antimicrobial peptide expression to modulate the mucosal immunity during chronic endometritis.

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.

Reduction in PLANT DEFENSIN 1 expression in Arabidopsis thaliana results in increased resistance to pathogens and zinc toxicity.

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions.

Zinc deficiency-induced defensin-like proteins are involved in the inhibition of root growth in Arabidopsis.

The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions. DEFLs are small cysteine-rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn-deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202-DEFL208) were categorized into one subgroup. Among the seven DEFLs, the transcripts of five (not DEFL204 and DEFL205) were upregulated by Zn deficiency, consistent with the presence of cis-elements for basic-region leucine-zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP-tagged DEFL203 showed that DEFL203-sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn-deficient conditions through a reduction in root meristem length and cell number.

Plant Defensin-Dissimilar Thionin OsThi9 Alleviates Cadmium Toxicity in Rice Plants and Reduces Cadmium Accumulation in Rice Grains.

Thionins are important antibacterial peptides in plants. However, the roles of plant thionins, especially the defensin-dissimilar thionins, in alleviating heavy-metal toxicity and accumulation remain unclear. Here, cadmium (Cd)-related functions and mechanisms of the defensin-dissimilar rice thionin OsThi9 were investigated. OsThi9 was significantly upregulated in response to Cd exposure. OsThi9 was localized to the cell wall and was shown to bind Cd; these characters help to increase Cd tolerance. In Cd-exposed rice plants, OsThi9 overexpression significantly increased cell wall Cd binding, decreasing upward Cd translocation and subsequent Cd accumulation in shoots and straw, while OsThi9 knockout had inverse effects. Importantly, in rice plants grown in Cd-contaminated soils, OsThi9 overexpression significantly reduced Cd accumulation in brown rice (decrease of ≥ 51.8%) without negatively impairing the crop yield and essential elements. Thus, OsThi9 plays an important role in the alleviation of Cd toxicity and accumulation and has significant potential for developing low-Cd rice.

Paneth cell proteins DEFA6 and GUCA2A as tissue markers in necrotizing enterocolitis.

Previous studies suggest that Paneth cells are involved in NEC development. Defensin alpha 6 (DEFA6) and guanylate cyclase activator 2A (GUCA2A) are selective protein markers of Paneth cells. The objective was to explore DEFA6 and GUCA2A expression in intestinal tissue samples from newborn infants with and without NEC. Tissue samples from histologically intact intestine were analyzed from 70 infants: 43 underwent bowel resection due to NEC and 27 controls were operated due to conditions such as intestinal atresia, dysmotility, aganglionosis, pseudo-obstruction or volvulus. Each tissue sample was immunohistochemically stained for DEFA6 and GUCA2A. Semi-automated digital image analysis was performed to determine protein expression. Clinical data and protein expressions were compared between the groups. DEFA6 expression was lower in the NEC group (p = 0.006). Low DEFA6 correlated with risk of developing NEC in a logistic regression analysis, independently of gestational age and birth weight (OR 0.843 [CI 0.732-0.971]; p = 0.018). GUCA2A expression did not differ between the two groups. CONCLUSION: Lower expression of DEFA6 together with intact GUCA2A expression indicates that NEC patients have well-defined Paneth cells but diminished defensin activity. Our results suggest that DEFA6 could be used as a biomarker for NEC. WHAT IS KNOWN: • Previous studies of defensin activity in NEC have been inconsistent, showing that defensin levels may be increased or diminished in NEC. GUCA2A has to our knowledge never been studied in NEC. WHAT IS NEW: • This study benchmarks two specific Paneth cell markers (DEFA6 and GUCA2A) and their activity in individuals with and without NEC. • The key finding is that the NEC group had a lower DEFA6 expression compared to the Controls, while the expression of GUCA2A did not differ between the groups.