DEFA1A3 DNA gene-dosage regulates the kidney innate immune response during upper urinary tract infection.

Antimicrobial peptides (AMPs) are host defense effectors with potent neutralizing and immunomodulatory functions against invasive pathogens. The AMPs α-Defensin 1-3/DEFA1A3 participate in innate immune responses and influence patient outcomes in various diseases. DNA copy-number variations in DEFA1A3 have been associated with severity and outcomes in infectious diseases including urinary tract infections (UTIs). Specifically, children with lower DNA copy numbers were more susceptible to UTIs. The mechanism of action by which α-Defensin 1-3/DEFA1A3 copy-number variations lead to UTI susceptibility remains to be explored. In this study, we use a previously characterized transgenic knock-in of the human DEFA1A3 gene mouse to dissect α-Defensin 1-3 gene dose-dependent antimicrobial and immunomodulatory roles during uropathogenic Escherichia coli (UPEC) UTI. We elucidate the relationship between kidney neutrophil- and collecting duct intercalated cell-derived α-Defensin 1-3/DEFA1A3 expression and UTI. We further describe cooperative effects between α-Defensin 1-3 and other AMPs that potentiate the neutralizing activity against UPEC. Cumulatively, we demonstrate that DEFA1A3 directly protects against UPEC meanwhile impacting pro-inflammatory innate immune responses in a gene dosage-dependent manner.

Enteric α-Defensin Contributes to Recovery of Radiation-Induced Intestinal Injury by Modulating Gut Microbiota and Fecal Metabolites.

The effect of ionizing radiation on the gastrointestinal tract is a common complication of abdominal and pelvic radiotherapy. However, the pathological features of radiation enteropathy and its effective medical intervention regimen is still a global challenge. Here, we explored the role and mechanism of enteric alpha-defensins (EαDs) in protecting against radiation enteropathy. To address this, we utilized EαDs-deficiency mice, in which the matrix metallopeptidase 7 to activate Paneth cell α-defensins was knockout (KO) mice, and the complementary wild-type (WT) control mice for this study. Remarkably, the KO mice were more susceptible to 5.0 Gy total-body irradiation, resulting in worse clinic scores and lower survival rate, compared with the wild-type mice. Histological examination indicated that the KO mice were subjected to slow recovery of intestinal villus and mucosa function, characterized by the reduced expression of TFF3, Glut1 and Muc2. In addition, compared with the wild-type controls, the KO mice experienced serious inflammation response in intestinal tissue, indicated by the remarkably increased expression level of IL-1ß, IL-6 and IL-12. Using high-throughput sequencing analysis, we found that the intestinal bacterial community of the KO mice was more prone to dysbiosis than that of the WT mice, with significantly increased abundance of opportunistic pathogenic bacteria, such as Streptococcus sp. and Escherichia-Shigella sp., whereas remarkably decreased probiotics harboring Lactobacillus sp., Desulfovibrio sp. etc. Fecal metabolomics analysis indicated that the relative abundance of 31 metabolites arose significantly different between WT and KO mice on day 10 after radiation exposure. A subset of differential metabolites to regulate host metabolism and immunity, such as acetic acid, acetate, butanoic acid, was negatively correlated with the alteration of gut microbiota in the irradiated KO mice. This study provides new insight into EαDs contribution to the recovery of radiation-induced intestinal damage, and suggests a potential novel target to prevent the adverse effects of radiotherapy.

FOXO inhibition rescues α-defensin expression in human intestinal organoids.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.

Association between α-defensin 5 and the expression and function of P-glycoprotein in differentiated intestinal Caco-2 cells.

α-Defensin 5 is known to be secreted by Paneth cells in the small intestine and plays an important role in eliminating pathogenic microorganisms. It has been reported that a decrease in α-defensin 5 level in the human small intestine is a risk of inflammatory bowel disease (IBD). Furthermore, P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter superfamily, encoded by the ABCB1/MDR1 gene, plays an important role in the front line of host defense by protecting the gastrointestinal barrier from xenobiotic accumulation and may contribute to the development and persistence of IBD. Therefore, we examined the relationship between α-defensin 5 and the expression and function of P-gp using a human gastrointestinal model cell line (Caco-2). We found that MDR1 mRNA and P-gp protein level were increased in Caco-2 cells as well as α-defensin 5 secretion corresponded with the duration of cell culture. Exposure to α-defensin 5 peptide and recombinant tumor necrosis factor-α (TNF-α) significantly increased the expression and function P-gp. The mRNA levels of interleukin (IL)-8, IL-6, TNF-α, IL-1ß, and IL-2 were also increased following exposure to TNF-α, similar to α-defensin 5 treatment. These results suggest that α-defensin 5 regulates P-gp expression and function by increasing TNF-α expression in Caco-2 cells.

Multi-omic and comparative analyses revealed monocyte-derived alpha-defensin-1 correlated with COVID-19 severity and inhibited SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological pathogen of coronavirus disease 2019 (COVID-19), a highly contagious disease, spreading quickly and threatening global public health. The symptoms of COVID-19 vary from mild reactions to severe respiratory distress or even fatal outcomes probably due to the different status of host immunity against the virus. Here in the study, we unveiled plasma proteomic signatures and transcriptional patterns of peripheral blood mononuclear cells (PBMCs) using blood samples of 10 COVID-19 patients with different severity. Through systemic analysis, α-defensin-1 (DEFA1) was identified to be elevated in both plasma and PBMCs, and correlated with disease severity and stages. In vitro study demonstrated that DEFA1 was secreted from immunocytes and suppressed SARS-CoV-2 infection of both original and mutated strains with dose dependency. By using sequencing data, we discovered that DEFA1 was activated in monocytes through NF-κB signaling pathway after infection, and secreted into circulation to perturb SARS-CoV-2 infection by interfering protein kinase C expression. It worked mainly during virus replication instead of entry in host cells. Together, the anti-SARS-CoV-2 mechanism of DEFA1 has unveiled a corner of how innate immunity is against SARS-CoV-2 and explored its clinical potential in disease prognosis and therapeutic intervention.

Intestinal α-Defensins Play a Minor Role in Modulating the Small Intestinal Microbiota Composition as Compared to Diet.

The intestinal microbiota is at the interface between the host and its environment and thus under constant exposure to host-derived and external modulators. While diet is considered to be an important external factor modulating microbiota composition, intestinal defensins, one of the major classes of antimicrobial peptides, have been described as key host effectors that shape the gut microbial community. However, since dietary compounds can affect defensin expression, thereby indirectly modulating the intestinal microbiota, their individual contribution to shaping gut microbiota composition remains to be defined. To disentangle the complex interaction among diet, defensins, and small-intestinal microbiota, we fed wild-type (WT) mice and mice lacking functionally active α-defensins (Mmp7-/- mice) either a control diet or a Western-style diet (WSD) that is rich in saturated fat and simple carbohydrates but low in dietary fiber. 16S rDNA sequencing and robust statistical analyses identified that bacterial composition was strongly affected by diet while defensins had only a minor impact. These findings were independent of sample location, with consistent results between the lumen and mucosa of the jejunum and ileum, in both mouse genotypes. However, distinct microbial taxa were also modulated by α-defensins, which was supported by differential antimicrobial activity of ileal protein extracts. As the combination of WSD and defensin deficiency exacerbated glucose metabolism, we conclude that defensins only have a fine-tuning role in shaping the small-intestinal bacterial composition and might instead be important in protecting the host against the development of diet-induced metabolic dysfunction. IMPORTANCE Alterations in the gut microbial community composition are associated with many diseases, and therefore identifying factors that shape the microbial community under homeostatic and diseased conditions may contribute to the development of strategies to correct a dysbiotic microbiota. Here, we demonstrate that a Western-style diet, as an extrinsic parameter, had a stronger impact on shaping the small intestinal bacterial composition than intestinal defensins, as an intrinsic parameter. While defensins have been previously shown to modulate bacterial composition in young mice, our study supplements these findings by showing that defensins may be less important in adult mice that harbor a mature microbial community. Nevertheless, we observed that defensins did affect the abundance of distinct bacterial taxa in adult mice and protected the host from aggravated diet-induced glucose impairments. Consequently, our study uncovers a new angle on the role of intestinal defensins in the development of metabolic diseases in adult mice.

Segregation analysis identifies specific alpha-defensin (DEFA1A3) SNP-CNV haplotypes in predisposition to IgA nephropathy.

BACKGROUND: Immunoglobulin A (IgA) nephropathy is a disorder of the immune system affecting kidney function, and genome-wide association studies (GWAS) have defined numerous loci with associated variation, all implicating components of innate or adaptive immunity. Among these, single nucleotide polymorphisms (SNPs) in a region including the multiallelic copy number variation (CNV) of DEFA1A3 are associated with IgA nephropathy in both European and Asian populations. At present, the precise factors underlying the observed associations at DEFA1A3 have not been defined, although the key alleles differ between Asian and European populations, and multiple independent factors may be involved even within a single population. METHODS: In this study, we measured DEFA1A3 copy number in UK family trios with an offspring affected by IgA nephropathy, used the population distributions of joint SNP-CNV haplotypes to infer the likely segregation in trios, and applied transmission disequilibrium tests (TDT) to examine joint SNP-CNV haplotypes for over- or undertransmission into affected offspring from heterozygous parents. RESULTS AND CONCLUSIONS: We observed overtransmission of 3-copy class 2 haplotypes (raw p = 0.029) and some evidence for under-transmission of 3-copy class 1 haplotypes (raw p = 0.051), although these apparent effects were not statistically significant after correction for testing of multiple haplotypes.

Changes in gut microbiome correlate with intestinal barrier dysfunction and inflammation following a 3-day ethanol exposure in aged mice.

Alcohol use among older adults is on the rise. This increase is clinically relevant as older adults are at risk for increased morbidity and mortality from many alcohol-related chronic diseases compared to younger patients. However, little is known regarding the synergistic effects of alcohol and age. There are intriguing data suggesting that aging may lead to impaired intestinal barrier integrity and dysbiosis of the intestinal microbiome, which could increase susceptibility to alcohol's negative effects. To study the effects of alcohol in age we exposed aged and young mice to 3 days of moderate ethanol and evaluated changes in gut parameters. We found that these levels of drinking do not have obvious effects in young mice but cause significant alcohol-induced gut barrier dysfunction and expression of the pro-inflammatory cytokine TNFα in aged mice. Ethanol-induced downregulation of expression of the gut-protective antimicrobial peptides Defa-rs1, Reg3b, and Reg3g was observed in aged, but not young mice. Analysis of the fecal microbiome revealed age-associated shifts in microbial taxa, which correlated with intestinal and hepatic inflammatory gene expression. Taken together, these data demonstrate that age drives microbiome dysbiosis, while ethanol exposure in aged mice induces changes in the expression of antimicrobial genes important for separating these potentially damaging microbes from the intestinal lumen. These changes highlight potential mechanistic targets for prevention of the age-related exacerbation of effects of ethanol on the gut.

A Temporal Comparative RNA Transcriptome Profile of the Annexin Gene Family in the Salivary versus Lacrimal Glands of the Sjögren's Syndrome-Susceptible C57BL/6.NOD-Aec1Aec2 Mouse.

A generally accepted hypothesis for the initial activation of an immune or autoimmune response argues that alarmins are released from injured, dying and/or activated immune cells, and these products complex with receptors that activate signal transduction pathways and recruit immune cells to the site of injury where the recruited cells are stimulated to initiate immune and/or cellular repair responses. While there are multiple diverse families of alarmins such as interleukins (IL), heat-shock proteins (HSP), Toll-like receptors (TLR), plus individual molecular entities such as Galectin-3, Calreticulin, Thymosin, alpha-Defensin-1, RAGE, and Interferon-1, one phylogenetically conserved family are the Annexin proteins known to promote an extensive range of biomolecular and cellular products that can directly and indirectly regulate inflammation and immune activities. For the present report, we examined the temporal expression profiles of the 12 mammalian annexin genes (Anxa1-11 and Anxa13), applying our temporal genome-wide transcriptome analyses of ex vivo salivary and lacrimal glands from our C57BL/6.NOD-Aec1Aec2 mouse model of Sjögren's Syndrome (SS), a human autoimmune disease characterized primarily by severe dry mouth and dry eye symptoms. Results indicate that annexin genes Anax1-7 and -11 exhibited upregulated expressions and the initial timing for these upregulations occurred as early as 8 weeks of age and prior to any covert signs of a SS-like disease. While the profiles of the two glands were similar, they were not identical, suggesting the possibility that the SS-like disease may not be uniform in the two glands. Nevertheless, this early pre-clinical and concomitant upregulated expression of this specific set of alarmins within the immune-targeted organs represents a potential target for identifying the pre-clinical stage in human SS as well, a fact that would clearly impact future interventions and therapeutic strategies.

Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge.

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.

Human Neutrophil Defensins Disrupt Liver Interendothelial Junctions and Aggravate Sepsis.

Human neutrophil peptides 1-3 (HNP1-3), also known as human α-defensins, are the most abundant neutrophil granule proteins. The genes that encode HNP1-3, DEFA1/DEFA3, exhibit extensive copy number variations, which correlate well with their protein levels. Human and mouse studies have shown that increased copy numbers of DEFA1/DEFA3 worsen sepsis outcomes. Additionally, high concentrations of HNP1-3 in body fluids have been reported in patients with sepsis. However, direct evidence for the pathogenic role of HNP1-3 proteins during sepsis progression is lacking. In current study, sepsis was induced by means of cecal puncture and ligation. Various doses of HNP-1 (low dose with 0.5 mg/kg body weight and high dose with 10 mg/kg body weight) or phosphate buffer saline were intraperitoneally administered to mice at six hours after sepsis onset. Survival rate was monitored, and vascular permeability, endothelial cell pyroptosis, and immunofluorescence of endothelial adherens junction protein vascular endothelial-cadherin were evaluated. The administration of a high dose of HNP-1 after sepsis onset led to increased mortality, more severe liver injury, and increased vascular permeability in the liver and mesentery. The injection of high dose of HNP-1 did not directly induce liver endothelial cell death but destroyed interendothelial junctions in the liver. Moreover, genetic deficiency of nucleotide-binding oligomerization domain-like receptor protein-3 or caspase-1 abrogated the high mortality and disrupted liver interendothelial junctions caused by high dose of HNP-1 during sepsis. This study directly demonstrates that neutrophil defensins play a key role in regulating endothelial stability during sepsis development.

Vibrio cholerae senses human enteric α-defensin 5 through a CarSR two-component system to promote bacterial pathogenicity.

Vibrio cholerae (V. cholerae) is an aquatic bacterium responsible for acute and fatal cholera outbreaks worldwide. When V. cholerae is ingested, the bacteria colonize the epithelium of the small intestine and stimulate the Paneth cells to produce large amounts of cationic antimicrobial peptides (CAMPs). Human defensin 5 (HD-5) is the most abundant CAMPs in the small intestine. However, the role of the V. cholerae response to HD-5 remains unclear. Here we show that HD-5 significantly upregulates virulence gene expression. Moreover, a two-component system, CarSR (or RstAB), is essential for V. cholerae virulence gene expression in the presence of HD-5. Finally, phosphorylated CarR can directly bind to the promoter region of TcpP, activating transcription of tcpP, which in turn activates downstream virulence genes to promote V. cholerae colonization. In conclusion, this study reveals a virulence-regulating pathway, in which the CarSR two-component regulatory system senses HD-5 to activate virulence genes expression in V. cholerae.

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain.

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a ß-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.

VP4 Is a Determinant of Alpha-Defensin Modulation of Rotaviral Infection.

Fecal-oral pathogens encounter constitutively expressed enteric alpha-defensins in the intestine during replication and transmission. Alpha-defensins can be potently antiviral and antibacterial; however, their primary sequences, the number of isoforms, and their activity against specific microorganisms often vary greatly between species, reflecting adaptation to species-specific pathogens. Therefore, alpha-defensins might influence not only microbial evolution and tissue tropism within a host but also species tropism and zoonotic potential. To investigate these concepts, we generated a panel of enteric and myeloid alpha-defensins from humans, rhesus macaques, and mice and tested their activity against group A rotaviruses, an important enteric viral pathogen of humans and animals. Rotaviral adaptation to the rhesus macaque correlated with resistance to rhesus enteric, but not myeloid, alpha-defensins and sensitivity to human alpha-defensins. While mouse rotaviral infection was increased in the presence of mouse enteric alpha-defensins, two prominent genotypes of human rotaviruses were differentially sensitive to human enteric alpha-defensins. Furthermore, the effects of cross-species alpha-defensins on human and mouse rotaviruses did not follow an obvious pattern. Thus, exposure to alpha-defensins may have shaped the evolution of some, but not all, rotaviruses. We then used a genetic approach to identify the viral attachment and penetration protein, VP4, as a determinant of alpha-defensin sensitivity. Our results provide a foundation for future studies of the VP4-dependent mechanism of defensin neutralization, highlight the species-specific activities of alpha-defensins, and focus future efforts on a broader range of rotaviruses that differ in VP4 to uncover the potential for enteric alpha-defensins to influence species tropism. IMPORTANCE Rotavirus is a leading cause of severe diarrhea in young children. Like other fecal-oral pathogens, rotaviruses encounter abundant, constitutively expressed defensins in the small intestine. These peptides are a vital part of the vertebrate innate immune system. By investigating the impact that defensins from multiple species have on the infectivity of different strains of rotavirus, we show that some rotaviral infections can be inhibited by defensins. We also found that some, but not all, rotaviruses may have evolved resistance to defensins in the intestine of their host species, and some even appropriate defensins to increase their infectivity. Because rotaviruses infect a broad range of animals and rotaviral infections are highly prevalent in children, identifying immune defenses against infection and how they vary across species and among viral genotypes is important for our understanding of the evolution, transmission, and zoonotic potential of these viruses as well as the improvement of vaccines.

Human α-defensin 6 (HD6) suppresses CRC proliferation and metastasis through abolished EGF/EGFR signaling pathway.

The incidence of colorectal cancer (CRC) has increased significantly in the past decade. Early diagnosis and new therapeutics are still urgently needed for CRC in clinical practice. Human α-defensin 6 (HD6) plays a defense role against microbes in the gastrointestinal tract. However, the role and mechanism of HD6 in CRC is still unresolved. Specimens from CRC patients with higher HD6 showed better outcomes. Overexpressed HD6 in CRC cells caused a reduction of cell proliferative, migratory, and invasive ability in vitro and in vivo. HD6-overexpressed caused S phase arrest through changes in cyclin-A and B and CDK2 levels. In addition, serpine-1 may be negatively regulated by HD6 altering the translocation of c-Jun N-terminal kinases (JNK), extracellular regulated protein kinases (ERK), and p38. Higher HD6 and lower serpine-1 levels in CRC patients reflected better outcomes. Finally, we found that HD6 interacts directly with epidermal growth factor receptor (EGFR) by co-immunoprecipitated assay. EGF treatment caused an increase of the level of serpine-1 and pEGFR levels and then increased growth activity in HD6 overexpressing cells. Together, our study shows that HD6 may compete with EGF to bind to EGFR and interrupt cancer progression in CRC. We believe these findings may give new insights for HD6 in CRC therapy.

Recombinant HNP-1 Produced by Escherichia coli Triggers Bacterial Apoptosis and Exhibits Antibacterial Activity against Drug-Resistant Bacteria.

Human neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical applications have been hampered by challenges during mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that Escherichia coli expressing full-length preproHNP-1 secretes a soluble form of HNP-1, which can be recovered from the total cell lysate after isopropyl thio-ß-d-galactoside (IPTG) induction and ultrafiltration. Label-free quantitative proteomics and co-immunoprecipitation experiments revealed that HNP-1 induces cell apoptosis in bacteria by causing DNA and membrane damage. Notably, we found that HNP-1 disrupts the DNA damage response pathway by interfering with the binding of RecA to single-stranded DNA (ssDNA). Further experiments demonstrated that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results indicated that recombinant protein expression may be a simple and cost-effective solution to produce HNP-1 and that RecA inhibition via HNP-1 may serve as an alternative strategy to counteract antibiotic resistance. IMPORTANCE Human neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical application has been hampered by the difficulty of mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that recombinant protein expression combined with ultrafiltration may be a simple and cost-effective solution to HNP-1 production. We further found that HNP-1 induces bacterial apoptosis and prevents its SOS repair pathway from binding to the RecA protein, which may be a new antibacterial mechanism. In addition, we showed that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results provide new insights into the production and antibacterial mechanism of HNP-1, both of which may promote its clinical application.

RNA Sequencing in COVID-19 patients identifies neutrophil activation biomarkers as a promising diagnostic platform for infections.

Infection with the SARS-CoV2 virus can vary from asymptomatic, or flu-like with moderate disease, up to critically severe. Severe disease, termed COVID-19, involves acute respiratory deterioration that is frequently fatal. To understand the highly variable presentation, and identify biomarkers for disease severity, blood RNA from COVID-19 patient in an intensive care unit was analyzed by whole transcriptome RNA sequencing. Both SARS-CoV2 infection and the severity of COVID-19 syndrome were associated with up to 25-fold increased expression of neutrophil-related transcripts, such as neutrophil defensin 1 (DEFA1), and 3-5-fold reductions in T cell related transcripts such as the T cell receptor (TCR). The DEFA1 RNA level detected SARS-CoV2 viremia with 95.5% sensitivity, when viremia was measured by ddPCR of whole blood RNA. Purified CD15+ neutrophils from COVID-19 patients were increased in abundance and showed striking increases in nuclear DNA staining by DAPI. Concurrently, they showed >10-fold higher elastase activity than normal controls, and correcting for their increased abundance, still showed 5-fold higher elastase activity per cell. Despite higher CD15+ neutrophil elastase activity, elastase activity was extremely low in plasma from the same patients. Collectively, the data supports the model that increased neutrophil and decreased T cell activity is associated with increased COVID-19 severity, and suggests that blood DEFA1 RNA levels and neutrophil elastase activity, both involved in neutrophil extracellular traps (NETs), may be informative biomarkers of host immune activity after viral infection.

Protective Role of Spermidine in Colitis and Colon Carcinogenesis.

BACKGROUND & AIMS: Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis. METHODS: SMOX was quantified in human colitis and associated dysplasia using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. We used wild-type (WT) and Smox-/- C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed. RESULTS: SMOX messenger RNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox-/- vs WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of α-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox-/- mice and was reversed with Spd. CONCLUSIONS: Loss of SMOX is associated with exacerbated colitis and CAC, increased α-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.

Human α-defensin 5 suppressed colon cancer growth by targeting PI3K pathway.

Defensins are highly conserved antimicrobial peptides, which ubiquitously expressed in different species. In addition to the functions in host defense, their aberrant expression have also been documented in cancerous tissue including breast cancer, lung caner and renal carcinoma etc. Whereas, roles of Defensin Alpha 5 (DEFA5) in colon cancer has not been explored. Bioinformatic analysis was used to study the expression of DEFA5 and its correlation with clinical outcomes; Western blot, qPCR, Co-immunoprecipitation, xenograft models were used to the study the molecular mechanism. Decreased expression of DEFA5 at protein level was observed in colon tissues. Colon cancer cell lines proliferation and colony formation capacity were significantly suppressed by DEFA5 overexpression. Moreover, in vivo tumor growth in nude mice was also suppressed by DEFA5 overexpression, suggesting a tumor suppressor role of DEFA5 in colon cancer. Mechanistically, DEFA5 directly binds to the subunits of PI3K complex, thus attenuates the downstream signaling transduction, leads to delayed cell growth and metastasis. Collectively, we concluded that DEFA5 showed an inhibitory effect in colon cancer cell growth and may serve as a potential tumor suppressor in colon cancer.

Arsenic exposure in drinking water reduces Lgr5 and secretory cell marker gene expression in mouse intestines.

Arsenic is a global health concern that causes toxicity through ingestion of contaminated water and food. In vitro studies suggest that arsenic reduces stem and progenitor cell differentiation. Thus, this study determined if arsenic disrupted intestinal stem cell (ISC) differentiation, thereby altering the number, location, and/or function of intestinal epithelial cells. Adult male C57BL/6 mice were exposed to 0 or 100 ppb sodium arsenite (AsIII) through drinking water for 5 weeks. Duodenal sections were collected to assess changes in morphology, proliferation, and cell types. qPCR analysis revealed a 40% reduction in Lgr5 transcripts, an ISC marker, in the arsenic-exposed mice, although there were no changes in the protein expression of Olfm4. Secretory cell-specific transcript markers of Paneth (Defa1), Goblet (Tff3), and secretory transit amplifying (Math1) cells were reduced by 51%, 44%, and 30% respectively, in the arsenic-exposed mice, indicating significant impacts on the Wnt-dependent differentiation pathway. Further, protein levels of phosphorylated ß-catenin were reduced in the arsenic-exposed mice, which increased the expression of Wnt-dependent transcripts CD44 and c-myc. PCA analysis, followed by MANOVA and regression analyses, revealed significant changes and correlations between Lgr5 and the transit amplifying (TA) cell markers Math1 and Hes1, which are in the secretory cell pathway. Similar comparisons between Math1 and Defa1 show that terminal differentiation into Paneth cells is also reduced in the arsenic-exposed mice. The data suggests that ISCs are not lost following arsenic exposure, but rather, specific Wnt-dependent progenitor cell formation and terminal differentiation in the small intestine is reduced.