Paneth Cell Antimicrobial Peptides Research Articles

CD226 deficiency exacerbated intestinal immune dysregulation in mice with dinitrochlorobenzene-induced atopic dermatitis.

Intestinal mucosal immunity plays a pivotal role in host defence. In this study, we found that cluster of differentiation 226 (CD226) gene knockout (KO) led to more severe atopic dermatitis (AD)-related skin pathologies and bowel abnormalities in a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model. Following DNCB administration, the expression of CD226 was elevated in intestinal mucosal tissues, including group 3 innate lymphoid cells (ILC3s) and CD4+ T cells of Peyer's patches (PPs). CD226 deficiency led to an overactive intestinal immune response in the AD-like mice, as evidenced by increased inflammation and Th1/Th2-related cytokine levels as well as increased Paneth cell numbers and antimicrobial peptide (AMP) expression, which was likely due to the higher interleukin (IL)-22 production in the lamina propria. Additionally, CD226 deficiency increased the production of IL-4 and IL-17 in mesenteric lymph nodes as well as the number of PPs and expression of immunoglobulin (Ig) A in B cells. Moreover, insufficient expression of CD226 affected the characterization of intraepithelial and lamina propria lymphocytes in the intestinal mucosa. Finally, the number of PPs was increased in CD4+ T cell-specific CD226 KO and regulatory T (Treg) cell-specific CD226 KO mice; thus, loss of CD226 in Treg cells resulted in impaired Treg cell-suppressive function. Therefore, our findings indicate that CD226 deficiency alters intestinal immune functionality in inflammatory diseases.

INTERLEUKIN-17 RECEPTOR SIGNALING TO PANETH CELLS HELPS MAINTAIN INTESTINAL INTEGRITY AFTER GAMMA RADIATION INDUCED INJURY

Abstract BACKGROUND IL-17A/F signaling through the IL-17RA/RC receptor complex plays a vital role in immunity and inflammation. The expression of IL-17RA/RC by diverse intestinal epithelial cell types including Paneth cells and the observation that therapies neutralizing IL-17A or IL-17RA in Crohn’s disease (CD) patients induce adverse events suggest essential homeostatic functions of this cytokine. Paneth cells and their antimicrobial products (a-defensins, lysozyme etc.) play a critical role in mediating small intestinal host defense under homeostatic conditions as well as after injury or infection, and their dysregulation constitutes a pathogenic factor for CD. The specific role of intestinal IL-17A signaling in Paneth cells to regulate host defense at steady state or after intestinal inflammation remains poorly understood. Thus, we hypothesize that intestinal IL-17A signaling, specifically in Paneth cells, plays an important role in regulating microbiota colonization as well as maintaining intestinal integrity after gamma radiation induced injury. METHODS To examine the importance of IL-17RA signaling to Paneth cells, we utilized total, entire gut epithelium (Il17rafl/fl;villin-cre) and Paneth cell-specific IL-17RA knockout mice (Il17rafl/fl;Defa6-cre+). We subjected these mice with gamma irradiation (1200 cGy) to study intestinal injury and regenerative responses. Mice were sacrificed 3/5 days post irradiation. Terminal ilium was collected from naïve or irradiated mice for immunofluorescence imaging, RT-PCR, RNA sequencing, and enteroid cultures. RESULTS Our preliminary data from irradiated Il17ra-/- and Il17rafl/fl;villin-cre mice suggest that IL-17A signaling is important in maintaining intestinal barrier integrity. Specifically, these mice revealed increased microbial dissemination to liver and spleen compared to their respective control mice. Reduced expression of Lyz1 3 days post radiation in the terminal ileum of Il17ra-/- mice suggest impaired Paneth cell function. Cessation of Paneth cell specific IL-17RA signaling, also resulted in systemic bacterial dissemination 5 days post radiation. We also observed increased level of fecal Lcn2 in irradiated Il17rafl/fl;Defa6-cre+ mice. To understand Paneth cell-specific IL-17RA functions, we performed 16S microbial and RNA sequencing from ileum luminal contents and terminal ilea of naïve Il17rafl/fl;Defa6-cre+/- mice, respectively. While microbial 16s sequencing revealed no change to the overall microbial community, specific changes were observed at a select family level. Specifically, the level of Streptococcaceae was increased. RNA sequencing data showed no difference in Paneth cell antimicrobial peptides but did show reduced Caspase 4 expression in Il17rafl/fl;Defa6-cre+ mice. Collectively, our data revealed an important role of IL-17RA signaling in Paneth cell function at steady state and after injury.

T‐cell Protein Tyrosine Phosphatase is a Key Regulator of Paneth Cell Function

Loss‐of‐function single nucleotide polymorphisms (SNPs) in the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene, are associated with increased susceptibility to Inflammatory Bowel Disease (IBD). PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) which is a negative regulator of intracellular signaling pathways including JAK‐STAT. We previously presented that Paneth and goblet cell numbers are reduced in Ptpn2 knockout (KO) mice, consistent with altered differentiation of these intestinal epithelial cell (IEC) types in IBD. Alterations in IEC populations are detrimental for intestinal homeostasis and increase susceptibility to microbial infection.GoalTo identify whether Ptpn2‐KO mice display altered IEC differentiation and Paneth cell antimicrobial peptide (AMP) production.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old constitutive Ptpn2‐KO mice and control littermates, and from adult tamoxifen‐inducible intestinal epithelial‐specific Ptpn2ΔIEC mice (Vil‐Cre/Ptpn2), were harvested and processed for imaging, western blot, flow cytometry and qPCR.ResultsConstitutive Ptpn2‐KO mice display reduced numbers of Paneth cells and loss of the AMP lysozyme (p=0.0158; n=5). This was confirmed by Western blots of isolated IECs (~67% reduction; n=4). Expression of Paneth cell markers and differentiation factors (Mmp7, Defa5 and Sox9) were not altered in KO mice (n=10). Interestingly, Reg3‐γ, which like lysozyme also targets gram‐(+) bacteria and is secreted by Paneth cells and enterocytes, was increased in KO mice vs. control littermates (p=0.0066; n=10). Next, we identified by immunofluorescence that loss of Ptpn2 only in intestinal epithelium (Ptpn2ΔIEC) dramatically reduced colocalization of lysozyme and another Paneth cell marker, UEA‐1 (n=6). Decreased lysozyme expression in Ptpn2ΔIEC ileum was confirmed by western blot (p=0.0315; n=6). Since Paneth cells reside in the stem cell compartment, we investigated whether Ptpn2 deficiency affects the intestinal stem cells. Gene expression of Lgr5, an intestinal stem cell marker, Ephb3, a marker of the stem cell compartment, and Ascl2, a gene that controls stem cell renewal in the crypts, was unchanged in Ptpn2‐KO mice (n=10). In addition, expression of early IEC differentiation factors, Hes1 and Math1, were not altered (n=10). Il‐22 and IFN‐γ are known to stimulate secretion of AMPs by Paneth cells. Interestingly, flow cytometry showed increased abundance of Il‐22+ T cells (p=0.0187; n=5) and IFN‐γ+ T cells (p=0.0277; n=5) in the Ileum of constitutive KO mice, indicating that the defect in AMP production is likely not due to a lack of immune cell‐derived stimuli.ConclusionPtpn2‐deficiency decreases Paneth cell number and has opposing effects on their production of specific AMPs. These effects on Paneth cell number and function may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2‐deficient mice.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM) and the Crohn’s and Colitis Foundation (DFM)

Paneth Cell Dysfunction Mediates Alcohol-related Steatohepatitis Through Promoting Bacterial Translocation in Mice: Role of Zinc Deficiency.

Microbial dysbiosis is associated with alcohol-related hepatitis (AH), with the mechanisms yet to be elucidated. The present study aimed to determine the effects of alcohol and zinc deficiency on Paneth cell (PC) antimicrobial peptides, α-defensins, and to define the link between PC dysfunction and AH. Translocation of pathogen-associated molecular patterns (PAMPs) was determined in patients with severe AH and in a mouse model of alcoholic steatohepatitis. Microbial composition and PC function were examined in mice. The link between α-defensin dysfunction and AH was investigated in α-defensin-deficient mice. Synthetic human α-defensin 5 (HD5) was orally given to alcohol-fed mice to test the therapeutic potential. The role of zinc deficiency in α-defensin was evaluated in acute and chronic mouse models of zinc deprivation. Hepatic inflammation was associated with PAMP translocation and lipocalin-2 (LCN2) and chemokine (C-X-C motif) ligand 1 (CXCL1) elevation in patients with AH. Antibiotic treatment, lipopolysaccharide injection to mice, and in vitro experiments showed that PAMPs, but not alcohol, directly induced LCN2 and CXCL1. Chronic alcohol feeding caused systemic dysbiosis and PC α-defensin reduction in mice. Knockout of functional α-defensins synergistically affected alcohol-perturbed bacterial composition and the gut barrier and exaggerated PAMP translocation and liver damage. Administration of HD5 effectively altered cecal microbial composition, especially increased Akkermansia muciniphila, and reversed the alcohol-induced deleterious effects. Zinc-regulated PC homeostasis and α-defensins function at multiple levels, and dietary zinc deficiency exaggerated the deleterious effect of alcohol on PC bactericidal activity. Taken together, the study suggests that alcohol-induced PC α-defensin dysfunction is mediated by zinc deficiency and involved in the pathogenesis of AH. HD5 administration may represent a promising therapeutic approach for treating AH.

Impact of Maternal Malnutrition on Gut Barrier Defense: Implications for Pregnancy Health and Fetal Development.

Small intestinal Paneth cells, enteric glial cells (EGC), and goblet cells maintain gut mucosal integrity, homeostasis, and influence host physiology locally and through the gut-brain axis. Little is known about their roles during pregnancy, or how maternal malnutrition impacts these cells and their development. Pregnant mice were fed a control diet (CON), undernourished by 30% vs. control (UN), or fed a high fat diet (HF). At day 18.5 (term = 19), gut integrity and function were assessed by immunohistochemistry and qPCR. UN mothers displayed reduced mRNA expression of Paneth cell antimicrobial peptides (AMP; Lyz2, Reg3g) and an accumulation of villi goblet cells, while HF had reduced Reg3g and mucin (Muc2) mRNA and increased lysozyme protein. UN fetuses had increased mRNA expression of gut transcription factor Sox9, associated with reduced expression of maturation markers (Cdx2, Muc2), and increased expression of tight junctions (TJ; Cldn-7). HF fetuses had increased mRNA expression of EGC markers (S100b, Bfabp, Plp1), AMP (Lyz1, Defa1, Reg3g), and TJ (Cldn-3, Cldn-7), and reduced expression of an AMP-activator (Tlr4). Maternal malnutrition altered expression of genes that maintain maternal gut homeostasis, and altered fetal gut permeability, function, and development. This may have long-term implications for host-microbe interactions, immunity, and offspring gut-brain axis function.

Constitutive Ptpn2‐Loss in Mice Decreases Number and Compromises Function of Intestinal Goblet and Paneth cells

Among the ~200 susceptibility loci associated with Inflammatory Bowel Disease (IBD), are loss‐of‐function single nucleotide polymorphisms (SNPs) in the locus (18p11) containing the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene. PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) that functions as a negative regulator of diverse intracellular pathways repressing inflammatory signals. In Ptpn2‐deficient mice, we observed altered intestinal homeostasis in the form of intestinal epithelial barrier defects and altered intestinal microbiome composition. Intestinal epithelial homeostasis is maintained by active‐cycling and slow cycling stem cells confined within the crypt‐based niche and subsequent differentiation into secretory and absorptive epithelial lineages as cells migrate up the crypt‐villus axis. The goal of this study was to determine whether Ptpn2‐loss affects intestinal epithelial cell populations and function that could contribute to altered host‐bacterial interactions.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old wild type (WT), heterozygous (HET) and constitutive Ptpn2 knockout (KO) Balb/C mice were harvested and processed for imaging to detect proliferation (Ki‐67), apoptosis (TUNEL), Paneth cell (lysozyme), and goblet cell (periodic‐acid Shiff) numbers via staining of specific markers. cDNA was generated by reverse transcriptase, and mRNA expression of key genes associated with Paneth and goblet cells function were measured by PCR.ResultsProximal and distal colon of Ptpn2‐KO mice showed clusters of apoptotic cells at the crypt base in comparison to wildtype. In addition, the number of proliferative cells was higher in the crypts of proximal and distal colon of Ptpn2‐KO mice compared to WT (p=0.0032 and p=0.0005 respectively; n=3). Lysozyme‐positive Paneth cells were significantly reduced in HET mice (p=0.0452 vs. WT; n=2–5) and further reduced in KO mice (p=0.0040; n=4). Moreover, KO mice were almost completely devoid of both lysozyme positive and negative Paneth cells, the latter characterized by large cytosolic granules at the crypt base. mRNA expression of the Paneth cell antimicrobial peptide, Lyz1, was reduced in Ptpn2‐KO mice compared to WT (p=0.017; n=2), whereas expression of antimicrobial peptides Defa5 and Defa6 were not altered. Goblet cell number was slightly reduced in the ileum and distal colon of KO mice (n=2). There was also a preliminary trend towards reduced expression of goblet cell secreted mucin proteins, Muc2, a major mucus layer constituent, and Muc3, a mucin protein homologous to MUC17 in humans shown to be protective against enteroinvasive E. coli, in the cecum of Ptpn2‐KO mice compared to wildtype (n=2).ConclusionPtpn2‐deficiency compromises intestinal epithelial cell differentiation leading to altered numbers and function of Paneth and goblet cells consistent with increased susceptibility to bacterial infection.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM), the Crohn's and Colitis Foundation (DFM), and Science Without Borders Fellowship (VC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recipient single nucleotide polymorphisms in Paneth cell antimicrobial peptide genes and acute graft-versus-host disease: analysis of BMT CTN-0201 and -0901 samples.

Host genetics shape the gut microbiota, and gut dysbiosis increases the risk of acute graft-versus-host disease (aGVHD). Paneth cells and microbiota have interactions that contribute to immune regulation. α-defensin-5 (HD5) and regenerating islet-derived protein 3 alpha (Reg3A) are the most abundant Paneth cell antimicrobial peptides (AMPs). We hypothesized that single nucleotide polymorphisms (SNPs) in the genes for HD5 (DEFA5) and Reg3A (REG3A) predict aGVHD risk. We analysed pre-transplant recipient peripheral blood mononuclear cell samples from randomized Blood and Marrow Transplant Clinical Trials Network (BMT CTN) studies 0201 (94 patients with bone marrow and 93 with peripheral blood grafts) and 0901 (86 patients with myeloablative and 77 with reduced-intensity conditioning; all using peripheral blood grafts). In multivariable analysis (with a SNP×graft source interaction term in CTN-0201 and a SNP×conditioning intensity term in CTN-0901), DEFA5 rs4415345 and rs4610776 were associated with altered incidence of aGVHD grade II-IV [rs4415345 G vs. C: hazard ratio (HR) 0·58, 95% confidence interval (95% CI) 0·37-0·92, P=0·02; rs4610776 T vs. A: HR 1·53, 95% CI 1·01-2·32, P=0·05] in CTN-0201, but not CTN-0901, suggesting a stronger effect in bone marrow allografts. REG3A SNP was not associated with aGVHD. Host genetics may influence aGVHD risk by modulating Paneth cell function.

The association between acute graft-versus-host disease and antimicrobial peptide expression in the gastrointestinal tract after allogeneic stem cell transplantation.

Intestinal microbiota disruption is associated with acute gastrointestinal (GI) Graft-versus-Host Disease (GvHD) and poor outcome after allogeneic stem cell transplantation (ASCT). Here, in a retrospective analysis of 200 patients undergoing ASCT at the Regensburg University Medical Center, we assessed the relative expression of Paneth cell antimicrobial peptides (AMPs), Human Defensins (HD) 5 and 6 and regenerating islet-derived 3α (Reg3α), in 292 human intestinal biopsies as well as Reg3α serum levels in relation to acute GI GvHD. In the absence of GI GvHD, the relative expression of Paneth cell AMPs was significantly higher in the small intestine (duodenum to ileum) than in the stomach and large intestine (cecum to rectum) for Reg3α (p≤0.001), HD5 (p≤0.002) and HD6 (p≤0.02). Acute stage 2–4 GI GvHD was associated with reduced expression of AMPs in the small intestine (p≤0.01) in comparison to stage 0–1 disease, accompanied by a decrease in Paneth cell count in case of severe acute GI GvHD (p<0.001). The opposite held true for the large intestine as we found stage 2–4 GI GvHD correlated with significantly higher expression of HD5, HD6, and Reg3α compared to mild or no acute GI GvHD (p≤0.002). Severe GI GvHD in both the lower and the upper GI tract also correlated with higher serum concentrations of Reg3α (p = 0.002). As indirect markers of intestinal microbiome diversity low levels of urinary 3-indoxyl sulfate levels were associated with severe stages of acute GI GvHD compared to mild stage or no acute GI GvHD (p = 0.05). In conclusion, acute GI GvHD correlates with intestinal expression of HD5, HD6 and Reg3α as well as Reg3α serum levels and is associated with intestinal dysbiosis.

Dysbiosis of Intestinal Microbiota and Decreased Antimicrobial Peptide Level in Paneth Cells during Hypertriglyceridemia-Related Acute Necrotizing Pancreatitis in Rats.

Hypertriglyceridemia (HTG) aggravates the course of acute pancreatitis (AP). Intestinal barrier dysfunction is implicated in the pathogenesis of AP during which dysbiosis of intestinal microbiota contributes to the dysfunction in intestinal barrier. However, few studies focus on the changes in intestine during HTG-related acute necrotizing pancreatitis (ANP). Here, we investigated the changes in intestinal microbiota and Paneth cell antimicrobial peptides (AMPs) in HTG-related ANP (HANP) in rats. Rats fed a high-fat diet to induce HTG and ANP was induced by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct. Rats were sacrificed at 24 and 48 h, respectively. Pancreatic and ileal injuries were evaluated by histological scores. Intestinal barrier function was assessed by plasma diamine oxidase activity and D-lactate level. Systemic and intestinal inflammation was evaluated by tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-17A expression. 16S rRNA high throughput sequencing was used to investigate changes in intestinal microbiota diversity and structure. AMPs (α-defensin5 and lysozyme) expression was measured by real-time polymerase chain reaction (PCR) and immunofluorescence. The results showed that compared with those of normal-lipid ANP (NANP) groups, the HANP groups had more severe histopathological injuries in pancreas and distal ileum, aggravated intestinal barrier dysfunction and increased TNFα, IL-1β, and IL-17A expression in plasma and distal ileum. Principal component analysis showed structural segregation between the HANP and NANP group. α-Diversity estimators in the HANP group revealed decreased microbiota diversity compared with that in NANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. In the HANP group, at phyla level, Candidatus_Saccharibacteria and Tenericutes decreased significantly, whereas Actinobacteria increased. At genus level, Allobaculum, Bifidobacterium, and Parasutterella increased significantly, while Alloprevotella, Anaerotruncus, Candidatus_Saccharimonas, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, Ruminiclostridium_5, Ruminococcaceae_UCG-005, and Ruminococcaceae_UCG-014 decreased. Compared with those in the NANP rats, mRNA expression of lysozyme and α-defensin5 and protein expression of lysozyme decreased significantly in the HANP rats. Moreover, in the NANP rats and the HANP rats, Allobaculum abundance was inversely correlated with lysozyme expression, while Anaerotruncus abundance was positively correlated with it by Spearman test. In conclusion, intestinal microbiota dysbiosis and decreased AMPs of Paneth cells might participate in the pathogenesis of intestinal barrier dysfunction in HANP.

Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.

ObjectivesIntestinal barrier dysfunction plays an important role in acute necrotizing pancreatitis (ANP) and intestinal microbiota dysbiosis was involved in intestinal barrier failure. Paneth cells protect intestinal barrier and are associated with intestinal microbiota. Here, we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP.MethodsRats with ANP were established by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct and sacrificed at 24h and 48h, respectively. Injuries of pancreas and distal ileum were evaluated by histopathological score. Intestinal barrier function was assessed by plasma diamine oxidase activity (DAO) and D-lactate. Systemic and intestinal inflammation was evaluated by TNFα, IL-1β and IL-17A concentration by ELISA, respectively. 16S rRNA high throughput sequencing on fecal samples was used to investigate the changes in intestinal microbiota in the ANP group at 48h. Lysozyme and α-defensin5 were measured by real-time PCR, western blot and immunofluoresence.ResultsANP rats had more severe histopathological injuries in pancreas and distal ileum, injured intestinal barrier and increased expression of TNFα, IL-1β and IL-17A in plasma and distal ileum compared with those of the sham-operated (SO) group. Principal component analysis (PCA) showed structural segregation between the SO and ANP groups. Operational taxonomic unit (OTU) number and ACE index revealed decreased microbiota diversity in the ANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. At phyla level, Saccharibacteria and Tenericutes decreased significantly. At genus level, Escherichia-Shigella and Phascolarctobacterium increased significantly, while Candidatus_Saccharimonas, Prevotellaceae_UCG-001, Lachnospiraceae_UCG-001, Ruminiclostridium_5 and Ruminococcaceae_UCG-008 decreased significantly. Lysozyme and α-defensin5 mRNA expression levels decreased significantly in ANP group at 48h. Protein expression of lysozyme decreased in ANP groups at 24h and 48h. Meanwhile, the relative abundance of Escherichia-Shigella correlated inversely with the decrease in lysozyme.ConclusionThe disorder in intestinal microbiota and decreases of Paneth cell antimicrobial peptides might participate in the pathogenesis of intestinal barrier dysfunction during ANP.

P-136 ATG16L1 Mitigates Fasting-Induced Reduction of Paneth Cell Antimicrobial Peptide Expression

Background:ATG16L1 and IRGM are Crohn's disease (CD) risk allele-associated genes that encode factors important for cellular autophagy. Under certain conditions, mice deficient in orthologues of these genes display Paneth cell (PC) abnormalities (Cadwell et al., 2008; Liu et al., 2013). Furthermore,

Improved oral therapeutic potential of nanoencapsulated cryptdin formulation against Salmonella infection

An encapsulated system for cryptdin-2 (a Paneth cell antimicrobial peptide) was developed, with a view to help it sustain adverse gut conditions and to ensure its bioavailability on oral administration. The formulation was characterized on the basis of particle size, zeta potential and polydispersity index. Cryptdin-2 loaded nanoparticles of size 105±7nm, formulated by ionotropic gelation method using chitosan: tripolyphosphate (5:2), revealed 60% drug entrapment efficiency with 65% in vitro release in 4.5h. Developed system was evaluated for its therapeutic application against Salmonella Typhimurium infection in mice, on the basis of survivability of animals, bacterial load in tissues, histo-architecture and oxidative damage markers. Infected mice when treated with the encapsulated peptide showed 83% survivability and approximately 2 log unit reductions in the bacterial load in the tissues versus 100% mortality observed with the free peptide. The encapsulated cryptdin-2 also achieved a decrease in the level of oxidants, particularly nitrite by 3.25 folds and increased the level of antioxidant catalase by 2 folds when compared to the levels exhibited by the free peptide. The bacteriological and biochemical alterations illustrated by encapsulated peptide co-related well with the histo-architectural studies. The study is a first pre-clinical report on the oral effectiveness of cryptdin-2 by its suitable encapsulation and has potential for future clinical applications.

P-207 Mouse Paneth Cell Antimicrobial Peptide Expression Is Independent Of ATG16L1 Expression in the Absence of Murine Norovirus Infection

P-207 Mouse Paneth Cell Antimicrobial Peptide Expression Is Independent Of ATG16L1 Expression in the Absence of Murine Norovirus Infection

Mouse Paneth cell antimicrobial function is independent of Nod2

ObjectiveAlthough polymorphisms of the NOD2 gene predispose to the development of ileal Crohn's disease, the precise mechanisms of this increased susceptibility remain unclear. Previous work has shown that transcript expression...

In VitroandIn VivoSynergistic Effects of Cryptdin 2 and Ampicillin against Salmonella

In view of the emergence of multidrug-resistant Salmonella strains, there is a need for therapeutic alternatives. To reduce the dose of antibiotic required in order to decrease the associated side effects, the present study was aimed at evaluating the synergism between cryptdin 2 (a Paneth cell antimicrobial peptide) and ampicillin (Amp) against Salmonella enterica serovar Typhimurium. The synergy was evaluated in terms of the fractional bactericidal concentration (FBC) index, time-kill assay results (in vitro), macrophage functions, i.e., intracellular killing, lipid peroxidation, superoxide dismutase activity, and generation of nitrite (ex vivo), and decreases in CFU of salmonellae in livers, spleens, and small intestines of infected mice treated with cryptdin 2 and/or Amp (in vivo). In vitro synergism between the two agents was observed on the basis of the FBC index and time-kill assays. When the agents were used in combination, ex vivo studies revealed an enhanced effect on macrophage functions, particularly exhibiting a synergetic effect in terms of SOD levels. In vivo synergy was indicated by larger log unit decreases in all target organs of mice treated with the combination than those for the drugs used alone. These results point toward the possible use of cryptdin 2 as an adjunct to ampicillin and may help in developing alternate strategies to combat Salmonella infections.

Bacterial Translocation is Associated With Downregulation of Paneth Cell Antimicrobial Peptides in Ascitic Cirrhotic but Not in Pre-Hepatic Portal Hypertensive Rats

Bacterial Translocation is Associated With Downregulation of Paneth Cell Antimicrobial Peptides in Ascitic Cirrhotic but Not in Pre-Hepatic Portal Hypertensive Rats

Bacterial translocation is associated with downregulation of Paneth cell antimicrobial peptides in ascitic cirrhotic but not in pre-hepatic portal hypertensive rats

Bacterial translocation is associated with downregulation of Paneth cell antimicrobial peptides in ascitic cirrhotic but not in pre-hepatic portal hypertensive rats

Over-expression of paneth cell-derived anti-microbial peptides in the gut of patients with ankylosing spondylitis and subclinical intestinal inflammation

Subclinical gut inflammation has been demonstrated in patients with AS. Altered expression of paneth cell (PC) anti-microbial peptides have been reported in the inflamed ileum of patients with Crohn's disease (CD). Here, we investigated the expression of PC-derived peptides in subclinical gut inflammation in AS. Multiple adjacent mucosal biopsies from terminal ileum were obtained from 25 patients with AS, 30 CD and 15 healthy controls (HCs). Expression of human α-defensin 5 (HD-5), phospholipase A2 (PLA2), lysozyme and SOX-9 molecules was assessed by quantitative Taqman RT-PCR on mucosal samples. Immunohistochemistry with anti-human HD-5 antibody and genotyping of relevant NOD2 mutations was also performed. HD-5, PLA2 and lysozyme transcript levels were strongly increased in AS and CD with similar degrees of intestinal inflammation when compared with normal controls. Immunohistochemical evaluation showed a normal number of PCs in both AS patients with chronic gut inflammation and CD patients with less-inflamed ileal samples. Conversely, CD patients with higher degree of gut inflammation had a reduced number of PCs and low expression levels of HD-5. In this study, we provide evidence that over-expression of PC-derived anti-microbial peptides occurs in the ileum of AS patients with subclinical gut inflammation, likely representing an important early alteration of the mucosal innate immune component and intestinal host defence in AS.

Decreased expression of Paneth cell antimicrobial peptides coincide with bacterial translocation after starvation

The lack of enteral feeding, with or without parenteral nutritional support has been reported to result in increased translocation of bacteria, which is considered to play an important role in the high morbidity and mortality rates of critical illnesses such as acute pancreatitis. Recently, Paneth cells, important constituents of the innate intestinal immunity, were shown to be crucial in host protection against invasion of both commensal and pathogenic bacteria.This study investigates the influence of food deprivation on Paneth cell function and small intestinal integrity. Significant decreases in RNA expression of lysozyme (p&lt; 0.01), cryptdin (p&lt; 0.05) and RegIIIγ (p&lt;0.05) were seen after 48 hours of food deprivation in C57BL/6 mice (n=15/group). Accordingly, protein expression of lysozyme and the precursor form of RegIIIγ were also shown to decrease significantly (p&lt;0.01, p&lt;0.05 respectively). Furthermore, increased bacterial translocation to mesenteric lymph nodes occurred (p&lt;0.01), while HE staining showed normal intestinal architecture, distribution and expression of respectively tight junction proteins zonula occludens‐1 and claudin‐3 were unchanged.In conclusion, the current study demonstrates that enteral starvation causes Paneth cell dysfunction paralleled by increased bacterial translocation without disturbances of the small intestinal physical barrier.

Cryptdin-2: a novel therapeutic agent for experimental Salmonella Typhimurium infection

Salmonella infections represent a major health hazard and have been responsible for a number of epidemics. In view of the emergence of multidrug-resistant Salmonella strains, there is a need for therapeutic alternatives. The purpose of this study was to evaluate the therapeutic potential of cryptdin-2 (a Paneth cell antimicrobial peptide) against Salmonella infection. The bactericidal activity of cryptdin-2 against Salmonella enterica serovar Typhimurium NCTC74 was evaluated in vitro, ex vivo and in vivo on the basis of cfu enumeration. The MBC of cryptdin-2 for Salmonella Typhimurium was found to be 19 mg/L. The ex vivo study demonstrated significantly higher intracellular killing of the bacteria by macrophages treated with cryptdin-2 as compared with untreated macrophages. Treatment of infected mice with cryptdin-2 resulted in significant clearance of Salmonella from livers, spleens and intestines. The therapeutic efficacy of cryptdin-2 suggests that it may be a promising option to combat Salmonella infections or at least may act as an adjunct to conventional antibiotics.