Paneth Cells Of Small Intestine Research Articles

Hydrophobic Determinants of α-Defensin Bactericidal Activity

Mammalian α-defensins are approximately 4- to 5-kDa broad-spectrum antimicrobial peptides and abundant granule constituents of neutrophils and small intestinal Paneth cells. The bactericidal activities of amphipathic α-defensins depend in part on electropositive charge and on hydrophobic amino acids that enable membrane disruption by interactions with phospholipid acyl chains. Alignment of α-defensin primary structures identified conserved hydrophobic residues in the loop formed by the Cys(III)-Cys(V) disulfide bond, and we have studied their role by testing the effects of mutagenesis on bactericidal activities. Mouse α-defensin 4 (Crp-4) and rhesus myeloid α-defensin 4 (RMAD-4) were selected for these studies, because they are highly bactericidal in vitro and have the same overall electropositive charge. Elimination of hydrophobicity by site-directed mutagenesis at those positions in Crp-4 attenuated bactericidal activity markedly. In contrast to native Crp-4, the (I23/F25/L26/G)-Crp-4 variant lacked bactericidal activity against Salmonella enterica serovar Typhimurium and did not permeabilize Escherichia coli ML35 cells as a result of removing aliphatic side chains by Gly substitutions. Ala replacements in (I23/F25/L26/A)-Crp-4 restored activity, evidence that hydrophobicity contributed by Ala methyl R-groups was sufficient for activity. In macaques, neutrophil α-defensin RMAD-6 is identical to RMAD-4, except for a F28S difference, and (F28S)-RMAD-4 mutagenesis attenuated RMAD-4 bactericidal activity and E. coli permeabilization. Interestingly, (R31/32D)-Crp-4 lacks activity in these assays despite the presence of the Ile23, Phe25, and Leu26 hydrophobic patch. We infer that electrostatic interactions between cationic α-defensin residues and negative charge on bacteria precede interactions between critical hydrophobic residue positions that mediate membrane disruption and bacterial cell killing.

An optimized Fmoc synthesis of human defensin 5

Human α-defensin 5 (DEF5), expressed by the Paneth cells of human small intestine, plays an important role in host defense against microbial infections. DEF5, a 32-residue peptide adopting a three-stranded β-sheet fold stabilized by three internal disulfide bonds, is not efficiently produced by recombinant expression techniques and is, therefore, an interesting goal for chemical synthesis. While DEF5 production by Boc-based solid-phase synthesis has been described, to date no synthetic account by the more convenient Fmoc method has been published. Herein, we report an optimized solid-phase synthesis of DEF5 using the Fmoc strategy. Starting from a rather problematic initial synthesis using standard Wang resin and coupling protocols, the sequence elongation process has been monitored by mini-cleavage and MS analysis at strategic points, to identify problematic spots and act accordingly. For expediency, some of the optimization rounds have been run on defensin 5 amide. Main modifications have included the ChemMatrix(®) resin, known to decrease chain aggregation, and the use of pseudoproline dipeptide units at selected positions. Combination of some of these improvements results in a significantly purer product, to the extent that it can undergo in situ anaerobic oxidative folding to the native form without the need of an intermediate purification step. A typical synthesis run yielded about 15 mg of >95 % pure material. This approach should facilitate production of DEF5 and of selected analogs for structure-activity studies and other applications.

Immunohistochemical expressions of adropin and ınducible nitric oxide synthase in renal tissues of rats with streptozotocin-ınduced experimental diabetes

Diabetes is characterized by high blood glucose levels; it occurs in 30–35% of the population. Elevated glucose levels can damage a number of organs, including the kidneys. Several peptide hormones participate in maintaining glucose homeostasis including the recently discovered “adropin,” a 42 amino acid peptide hormone. Adropin also alters inducible nitric oxide synthase (iNOS) expression. Therefore, we studied how adropin and iNOS expression is altered in the renal tissues of streptozotocin (STZ) induced diabetic rats. Seven sham, seven control and seven Wistar albino male rats were fed standard rat pellets and water ad libitum for 10 weeks. The rats in the diabetic group were injected i.p. with a single dose of 60 mg/kg STZ dissolved in 0.1 M phosphate-citrate buffer, pH 4.5. After the 10-week experimental period, the rats in both groups were anesthetized and decapitated. Kidney tissues were excised and placed in 10% formaldehyde solution, taken through routine histological procedures, and embedded in paraffin. Sections 5–6 μm thick were stained immunohistochemically using the avidin-biotin complex (ABC) method. Adropin and iNOS immunoreactivity were co-localized in the glomeruli, peritubular interstitial cells and peritubular capillary endothelium of the cortex; the thin limb of the loop of Henle in the medulla; and medullary peritubular interstitial cells and endothelium of the peritubular capillaries in both the control and diabetic groups. The intensities of adropin and iNOS immunoreactivity increased with the severity of the diabetes. Intense adropin immunoreactivity was detected in both the smooth muscle and human small intestine Paneth cells that were used as positive controls. The elevated levels of adropin and iNOS in the kidney indicates that these substances are involved in the pathophysiology of diabetes; this constitutes a compensatory mechanism against the damage inflicted by the disease.

Reduction of Human Defensin 5 Affords a High-Affinity Zinc-Chelating Peptide

Human defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits three disulfide bonds in the oxidized form (HD5ox). It is abundant in small intestinal Paneth cells, which release HD5 into the intestinal lumen and house a labile Zn(II) store of unknown function. Here, we consider the redox properties of HD5 and report that the reduced form, HD5red, is a metal-ion chelator. HD5 has a midpoint potential of -257 mV at pH 7.0. HD5red utilizes its cysteine residues to coordinate one equivalent of Zn(II) with an apparent Kd1 value in the midpicomolar range. Zn(II) or Cd(II) binding perturbs the oxidative folding pathway of HD5red to HD5ox. Whereas HD5red is highly susceptible to proteolytic degradation, the Zn(II)-bound form displays resistance to hydrolytic breakdown by trypsin and other proteases. The ability of a reduced defensin peptide to coordinate Zn(II) provides a putative mechanism for how these peptides persist in vivo.

Rattusin, an Intestinal α-Defensin-Related Peptide in Rats with a Unique Cysteine Spacing Pattern and Salt-Insensitive Antibacterial Activities

Cationic antimicrobial peptides are essential components of the innate immune system. As a major family of mammalian antimicrobial peptides, defensins are expressed mainly by mucosal epithelial cells and promyelocytes. Despite the capacity to kill a broad spectrum of bacteria through physical disruption of membranes, most defensins show substantially reduced antibacterial activities in the presence of monovalent and divalent cations, thereby limiting their therapeutic potential, particularly for the treatment of systemic infections. Genome-wide computational screening of the rat genome led to the identification of the gene for a novel α-defensin-related peptide that we termed rattusin. Rattusin shares a highly conserved signal and prosequence with mammalian α-defensins, but instead of the canonical α-defensin six-cysteine motif, rattusin consists of five cysteines with a distinctive spacing pattern. Furthermore, rattusin is preferentially expressed in Paneth cells of the distal small intestine with potent antibacterial activity against a broad range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains. The MICs were mostly in the range of 2 to 4 μM, with no appreciable toxicity to mammalian cells at up to 100 μM. In contrast to classical α- and β-defensins, rattusin retained its activity in the presence of physiological concentrations of NaCl and Mg(2+), making it an attractive antimicrobial candidate for both topical and systemic applications.

The CSF-1 receptor fashions the intestinal stem cell niche

Gastrointestinal (GI) homeostasis requires the action of multiple pathways. There is some controversy regarding whether small intestine (SI) Paneth cells (PCs) play a central role in orchestrating crypt architecture and their relationship with Lgr5+ve stem cells. Nevertheless, we previously showed that germline CSF-1 receptor (Csf1r) knock out (KO) or Csf1 mutation is associated with an absence of mature PC, reduced crypt proliferation and lowered stem cell gene, Lgr5 expression. Here we show the additional loss of CD24, Bmi1 and Olfm4 expression in the KO crypts and a high resolution 3D localization of CSF-1R mainly to PC. The induction of GI-specific Csf1r deletion in young adult mice also led to PC loss over a period of weeks, in accord with the anticipated long life span of PC, changed distribution of proliferating cells and this was with a commensurate loss of Lgr5 and other stem cell marker gene expression. By culturing SI organoids, we further show that the Csf1r−/− defect in PC production is intrinsic to epithelial cells as well as definitively affecting stem cell activity. These results show that CSF-1R directly supports PC maturation and that in turn PCs fashion the intestinal stem cell niche.

Paneth Cell–Mediated Multiorgan Dysfunction after Acute Kidney Injury

Acute kidney injury (AKI) is frequently complicated by extrarenal multiorgan injury, including intestinal and hepatic dysfunction. In this study, we hypothesized that a discrete intestinal source of proinflammatory mediators drives multiorgan injury in response to AKI. After induction of AKI in mice by renal ischemia-reperfusion or bilateral nephrectomy, small intestinal Paneth cells increased the synthesis and release of IL-17A in conjunction with severe intestinal apoptosis and inflammation. We also detected significantly increased IL-17A in portal and systemic circulation after AKI. Intestinal macrophages appear to transport released Paneth cell granule constituents induced by AKI, away from the base of the crypts into the liver. Genetic or pharmacologic depletion of Paneth cells decreased small intestinal IL-17A secretion and plasma IL-17A levels significantly and attenuated intestinal, hepatic, and renal injury after AKI. Similarly, portal delivery of IL-17A in macrophage-depleted mice decreased markedly. In addition, intestinal, hepatic, and renal injury following AKI was attenuated without affecting intestinal IL-17A generation. In conclusion, AKI induces IL-17A synthesis and secretion by Paneth cells to initiate intestinal and hepatic injury by hepatic and systemic delivery of IL-17A by macrophages. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from AKI.

Critical role of interleukin-17A in murine intestinal ischemia-reperfusion injury

Intestinal ischemia-reperfusion (I/R) injury causes severe illness frequently complicated by remote multiorgan dysfunction and sepsis. Recent studies implicated interleukin-17A (IL-17A) in regulating inflammation, autoimmunity, and I/R injury. Here, we determined whether IL-17A is critical for generation of intestinal I/R injury and subsequent liver and kidney injury. Mice subjected to 30 min of superior mesenteric artery ischemia not only developed severe small intestinal injury (necrosis, apoptosis, and neutrophil infiltration) but also developed significant renal and hepatic injury. We detected large increases in IL-17A in the small intestine, liver, and plasma. IL-17A is critical for generating these injuries, since genetic deletion of IL-17A- or IL-17A-neutralizing antibody treatment markedly protected against intestinal I/R injury and subsequent liver and kidney dysfunction. Intestinal I/R caused greater increases in portal plasma and small intestine IL-17A, suggesting an intestinal source for IL-17A generation. We also observed that intestinal I/R caused rapid small intestinal Paneth cell degranulation and induced murine α-defensin cryptdin-1 expression. Furthermore, genetic or pharmacological depletion of Paneth cells significantly attenuated the intestinal I/R injury as well as hepatic and renal dysfunction. Finally, Paneth cell depletion significantly decreased small intestinal, hepatic, and plasma IL-17A levels after intestinal I/R. Taken together, we propose that Paneth cell-derived IL-17A may play a critical role in intestinal I/R injury as well as extraintestinal organ dysfunction.

Heterogeneity in histone 2B-green fluorescent protein-retaining putative small intestinal stem cells at cell position 4 and their absence in the colon

Stem cells have been identified in two locations in small intestinal crypts; those intercalated between Paneth cells and another population (which retains DNA label) are located above the Paneth cell zone, at cell position 4. Because of disadvantages associated with the use of DNA label, doxycycline-induced transient transgenic expression of histone 2B (H2B)-green fluorescent protein (GFP) was investigated. H2B-GFP-retaining putative stem cells were consistently seen, with a peak at cell position 4, over chase periods of up to 112 days. After a 28-day chase, a subpopulation of the H2B-GFP-retaining cells was cycling, but the slow cycling status of the majority was illustrated by lack of expression of pHistone H3 and Ki67. Although some H2B-GFP-retaining cells were sensitive to low-dose radiation, the majority was resistant to low- and high-dose radiation-induced cell death, and a proportion of the surviving cells proliferated during subsequent epithelial regeneration. Long-term retention of H2B-GFP in a subpopulation of small intestinal Paneth cells was also seen, implying that they are long lived. In contrast to the small intestine, H2B-GFP-retaining epithelial cells were not seen in the colon from 28-day chase onward. This implies important differences in stem cell function between these two regions of the gastrointestinal tract, which may have implications for region-specific susceptibility to diseases (such as cancer and ulcerative colitis), in which epithelial stem cells and their progeny are involved.

PWE-132 Enhanced expression of secretory phospholipase A2 and cryptdins in small intestinal paneth cells following Trichinella spiralis infection

IntroductionT spiralis infection leads to a T cell-dependent enteropathy characterised by villus atrophy, crypt hyperplasia and an increase in Paneth and goblet cells. Paneth cells express a number of antimicrobial...

Identification of a cKit+ Colonic Crypt Base Secretory Cell That Supports Lgr5+ Stem Cells in Mice

Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells. We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased. cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

Biosynthesis and Antimicrobial Evaluation of Backbone-Cyclized α-Defensins

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel β-sheet but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other α-defensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in the proximity of each other (d ≈ 3 Å) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when the protein was produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either native Crp4 or its cyclized variants. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone-cyclized defensins in the development of novel peptide-based antimicrobial compounds.

Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus

Human α-defensin 5 (HD5, HD5(ox) to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys(3)-Cys(31), Cys(5)-Cys(20), Cys(10)-Cys(30)) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser(hexa)], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S-S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5(ox) against this Gram-positive bacterial strain. This observation supports the notion that the HD5(ox) mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem. 284, 29180-29192] and that the native disulfide array is a requirement for its activity against S. aureus.

Paneth cell-derived interleukin-17A causes multiorgan dysfunction after hepatic ischemia and reperfusion injury

Hepatic ischemia and reperfusion (IR) injury is a major clinical problem that leads to frequent extrahepatic complications including intestinal dysfunction and acute kidney injury (AKI). In this study we aimed to determine the mechanisms of hepatic IR-induced extrahepatic organ dysfunction. Mice subjected to 60 minutes of hepatic IR not only developed severe hepatic injury but also developed significant AKI and small intestinal injury. Hepatic IR induced small intestinal Paneth cell degranulation and increased interleukin-17A (IL-17A) levels in portal vein plasma and small intestine. We also detected increased levels of IL-17A messenger RNA (mRNA) and protein in Paneth cells after hepatic IR with laser capture dissection. IL-17A-neutralizing antibody treatment or genetic deletion of either IL-17A or IL-17A receptors significantly protected against hepatic IR-induced acute liver, kidney, and intestinal injury. Leukocyte IL-17A does not contribute to organ injury, as infusion of wildtype splenocytes failed to exacerbate liver and kidney injury in IL-17A-deficient mice after hepatic IR. Depletion of Paneth cell numbers by pharmacological (with dithizone) or genetic intervention (SOX9 flox/flox Villin cre+/- mice) significantly attenuated intestinal, hepatic, and renal injury following liver IR. Finally, depletion of Paneth cell numbers significantly decreased small intestinal IL-17A release and plasma IL-17A levels after liver IR. Taken together, the results show that Paneth cell-derived IL-17A plays a critical role in hepatic IR injury and extrahepatic organ dysfunction. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from hepatic IR.

Critical role of Paneth cell‐derived IL‐17A in hepatic ischemia and reperfusion injury

Hepatic ischemia and reperfusion (IR) injury is a major clinical problem with frequent extra‐hepatic complications including intestinal dysfunction and acute kidney injury (AKI). We aimed to determine the mechanisms of hepatic IR‐induced systemic complications. Mice subjected to 60 min of hepatic IR not only developed severe hepatic injury but also developed significant AKI and small intestinal injury. Hepatic IR induced small intestinal Paneth cell degranulation and increased IL‐17A levels in portal plasma and small intestine. IL‐17A neutralizing antibody treatment or genetic deletion of either IL‐17A or IL‐17A receptors significantly protected against hepatic IR‐induced acute liver, kidney and intestinal injury. Leukocyte IL‐17A does not contribute to organ injury as wild type splenocytes infused into IL‐17A deficient failed to exacerbate liver and kidney injury after hepatic IR. Laser capture micro‐dissection of Paneth cells showed significant IL‐17A mRNA induction after hepatic IR. Finally, wild type mice treated with dithizone to deplete Paneth cells or Paneth cell deficient (Sox9flox/flox;VilCre) mice were protected against hepatic, renal and intestinal injury following liver IR. Taken together, the results show that Paneth cell derived IL‐17A plays a critical role in hepatic IR injury and extra‐hepatic organ dysfunction. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from hepatic IR.Supported by Department of Anesthesiology, Columbia University.

Heterogeneity in expression of antimicrobial proteins in normal and crohn's disease small intestinal paneth cells

IntroductionPaneth cells are normally located at the base of small intestinal crypts and express a number of antimicrobial factors that are believed to be important in host protection against luminal...

Paneth cell function - Implications in pediatric Crohn disease

Defects in the intestinal barrier play a central role in disease pathogenesis. Recently we have demonstrated that children with ileal Crohn´s disease show a reduced expression of small intestinal HD-5 at the age of onset suggesting that a compromised mucosal barrier function might be a key factor in the early disease pathogenesis. We also identified a disturbance of the Wnt signaling transcription factor TCF-4 as a major mechanism for this deficiency in children which might result in a compromised innate immune function of small intestinal Paneth cells via defensin secretion. Here we provide a summary on our recent findings and discuss the data in more detail especially focusing on the role of Paneth cell differentiation and function in the pathogenesis of pediatric ileal Crohn’s disease.

Response of Crypt Paneth Cells in the Small Intestine following Total-Body γ-Irradiation

Ionizing irradiation causes damage and functional failure of irradiation-sensitive systems and tissues such as small intestine. The molecular mechanisms underlying inflammatory and adaptive responses to acute irradiation damage are poorly understood. Using a mouse model of total-body γ-irradiation, we assessed the irradiation response of crypt host-defense Paneth cells by measuring alpha-defensin 4 (AD4) expression and correlated the gathered data with activation of the caspase-1/IL-1β inflammatory signaling cascade. The irradiation injury was produced in CD2F1 mice exposed to 9.25 Gy γ-radiation. This dose resulted in 85-100 percent mortality at the 15(th) day post-irradiation. Small intestine tissue samples were collected at the 7th day post-irradiation. Assessment of irradiation-associated pro-inflammatory alterations in small intestine tissue and expression of AD4 in Paneth cells was conducted using confocal immunofluorescence imaging, transmission electron microscopy (TEM), light microscopy, and immunoblotting techniques. The small intestine analysis revealed an increase in the precursor form of IL-1β, the activated form of IL-1β, and the activated form of caspase-1 (p10 CASP-1) at the 7(th) day post-irradiation. Immunoprecipitation analysis showed increased interaction between IL-1β and p10 CASP-1 after irradiation. This effect was observed in the irradiated small intestine and CD15-positive Paneth cells using confocal imaging techniques. The pro-inflammatory alterations in Paneth cells were accompanied by increases in AD4 mRNA and its 8 kD peptide product. Paneth cell secretory activity was observed at the sites of bacterial translocation in the crypt lumens. These data suggest that Paneth cells can contribute to small intestine inflammatory remodeling during the post-irradiation period.

Innate antimicrobial host defense in small intestinal Crohn's disease

Paneth cells (PCs) are specialized epithelial cells predominantly found in the small intestinal crypts of Lieberkuehn. They produce different broad spectrum antimicrobial peptides most abundantly the α-defensins HD-5 and -6 (DEFA5 und DEFA6). Both these PC products show a specific reduction in small intestinal Crohn′s disease (CD) – a form of inflammatory bowel disease (IBD). Their decrease is independent of current inflammation and an association with a NOD2 frameshift mutation has been demonstrated. More recently, another independent and even more frequent mechanism has been found which is linked to diminished levels of the Wnt pathway transcription factor TCF7L2 (also known as TCF4). Besides regulating the expression of HD-5 and HD-6 as TCF4 target genes, the Wnt pathway also orchestrates Paneth cell differentiation and maturation and controls stem cell maintenance in the small intestine. Besides NOD2 (which is predominantly expressed in PC) and ATG16L1 (inter alia important in the exocytosis of PC products), TCF4 is the third gene which is associated with small intestinal CD and Paneth cell antimicrobial function. Thus, Paneth cells seem to be key player emphazising a paramount importance of antimicrobial host defense in small intestinal CD pathogenesis.

Up‐regulation of autophagy in small intestine Paneth cells in response to total‐body γ‐irradiation

Macroautophagy (mAG) is a lysosomal mechanism of degradation of cell self-constituents damaged due to variety of stress factors, including ionizing irradiation. Activation of mAG requires expression of mAG protein Atg8 (LC3) and conversion of its form I (LC3-I) to form II (LC3-II), mediated by redox-sensitive Atg4 protease. We have demonstrated upregulation of this pathway in the innate host defense Paneth cells of the small intestine (SI) due to ionizing irradiation and correlation of this effect with induction of pro-oxidant inducible nitric oxide synthase (iNOS). CD2F1 mice were exposed to 9.25 Gy gamma-ionizing irradiation. Small intestinal specimens were collected during 7 days after ionizing irradiation. Assessment of ionizing irradiation-associated alterations in small intestinal crypt and villus cells and activation of the mAG pathway was conducted using microscopical and biochemical techniques. Analysis of iNOS protein and the associated formation of nitrites and lipid peroxidation products was performed using immunoblotting and biochemical analysis, and revealed increases in iNOS protein, nitrate levels and oxidative stress at day 1 following ionizing irradiation. Increase in immunoreactivity of LC3 protein in the crypt cells was observed at day 7 following ionizing irradiation. This effect predominantly occurred in the CD15-positive Paneth cells and was associated with accumulation of LC3-II isoform. The formation of autophagosomes in Paneth cells was confirmed by transmission electron microscopy (TEM). Up-regulation of LC3 pathway in the irradiated SI was accompanied by a decreased protein-protein interaction between LC3 and chaperone heat shock protein 70. A high-level of LC3-immunoreactivity in vacuole-shaped structures was spatially co-localized with immunoreactivity of 3-nitro-tyrosine. The observed effects were diminished in iNOS knockout B6.129P2-NOS2(tm1Lau)/J mice subjected to the same treatments. We postulate that the observed up-regulation of mAG in the irradiated small intestine is at least in part mediated by the iNOS signalling mechanism.