Effects And Mechanisms Of NOD2 On Regulating Expression Of Antimicrobial Peptides And Interleukin-23p19 In The Paneth Cell Lineage

Background and ObjectiveCrohn’s disease (CD), the main clinical phenotype of inflammatory bowel disease (IBD), is a chronic granulomatous inflammatory bowel disorder. Although CD can occur anywhere in the gastrointestinal tract, it primarily affects the terminal ileum where as many as 75% of CD patients have inflammation. The terminal ileum is characterized by two relevant features:the greatest number of Paneth cells and the highest bacteria concentration in human terminal ileum. And there are more and more Paneth cells and bacteria concentration from the proximal to the distal small intestine. These features indicate that Paneth cells and intestinal flora have an intimate relationship with the pathogenesis of CD.Human Paneth cells serve as a key arm of innate mucosal immunity to maintain the intestinal homeostasis between a host and its colonizing microbes by secreting antimicrobial peptides and by offering a physical barrier which is composed of absorptive enterocytes, goblet cells, Paneth cells and intestinal endocrine cells. Furthermore, a strongly advocated view on the pathogenesis of CD is that the ineffective bacterial clearance that results from the reduced expression of human enteric antimicrobial peptides induces and sustains the abnormal adaptive immune responses observed in CD patients.Human enteric antimicrobial peptides are composed predominantly of human enteric a-defensins as well as lysozyme and secretory phospholipase A2 (sPLA2), to a lesser extent. These peptides not only have a strong antibacterial function against Gram-positive and Gram-negative bacteria, but they also have activity against viruses, fungi and protozoa. In humans, only two enteric a-defensins have been found, namely human a-defensin 5 and 6 (HD5 and HD6). Their important function is illustrated by many experiments in transgenic mice. For example, matrilysin-deficient transgenic mice that do not product mature enteric a-defensins are more susceptible to orally administered enteric pathogen Salmonella typhimurium and have significant changes in microbiota composition, while HD5 transgenic mice enhance resistance to enteric infection with virulent Salmonella typhimurium and have strikingly loss of Segmented Filamentous Bacteria. In addition, HD6 transgenic mice protect against invasion by diverse enteric pathogens.Interleukin (IL)-23, a heterodimeric cytokine, is composed of an IL-23-specific subunit p19 and an IL-12-common subunit p40. Although IL-12 was predominantly investigated before the discovery of IL-23, many recent studies, using IL-23 deficient mice, have identified that IL-23 but not IL-12 has a key role in orchestrating an inflammatory cytokine cascade involving enhanced expressed levels of IL-17, TNFa, IFNy and IL-6 in the intestine. In addition, a recent study, using mucosal specimens from IBD patients, has further confirmed that IL-23 can orchestrate chronic intestinal inflammation via multiple pro-inflammatory pathways, especially important two of which are the IL-23/Thl pathway in CD and the IL-23/Th17 pathway in UC. Moreover, a recent study, using transgenic mice, has shown that IL-23p19 over-expression can lead to multiple organ inflammation, including intestinal inflammation. Furthermore, some recent studies, using blocking antibodies, has shown that t spontaneous enteritis in IL-10 mice and colonic inflammation in the bacteria-reactive Thl7-cell-mediated colitis mice can be significantly ameliorated through treatment with blocking anti-IL-23p19 monoclonal antibodies. These studies indicate that IL-23p19 plays a crucial role in the inflammatory response.NOD2 is the first identified susceptibility gene for CD and its variants exist in over 50% of CD patients in Western populations. Recently, it has been demonstrated that Paneth cells constitutively express the IL-23p19 gene under physiologic conditions and over-express the IL-23p19 gene in CD. Similarly, these cells have been shown to constitutively express the NOD2 gene under physiologic conditions and over-express the NOD2 gene in CD. Nevertheless, it remains unclear whether dysregulated IL-23p19 expression might be due to abnormalities in NOD2 in Paneth cell. In addition, some studies, using ileal biopsies from CD patients, have identified that the mRNA expression of human enteric antimicrobial peptides, especially human enteric a-defensins, was decreased in CD, while the mRNA expression of human enteric a-defensins was more pronouncedly decreased in the presence of NOD2 mutations. Although these studies have supported a link between NOD2 function and human enteric antimicrobial peptides, the specific function of NOD2 expressed in regulating the expression of human enteric antimicrobial peptides remains unknown at present.Because primary Paneth cells do not survive in vitro, it is necessary to find a suitable cell line for in vitro study to explore the NOD2 function in the Paneth cells. Some studies have shown that Caco2 cells can display characteristics of small intestinal epithelial differentiation in vitro, suggesting that they maintain intestinal stem cell functions. In addition, these cells express abundant FGFR-3, which is a critical regulator of Paneth cell differentiation during gut development. Furthermore, they constitutively express the NOD2 gene. Thus, Caco2 cells are suitable for in vitro study to investigate the role of the NOD2 protein in the Paneth cell lineage.Objective:1. To determine whether the activation of FGFR-3-mediated signaling can induce in vitro differentiation of Caco2 cells along the Paneth cell lineage.2. To determine whether NOD2 can regulate FGF9-induced expression of human enteric antimicrobial peptides during differentiation of the Paneth cell lineage.3. To determine whether NOD2 can regulate the expression of human enteric a-defensins and the potential regulatory mechanism.4. To determine whether NOD2 can regulate the expression of IL-23p19 and the potential regulatory mechanism.Methods1. Caco2 cells were used between passages 15 and 30. For all of the experiments, to better mimic the steric conditions existing in the intestine in vivo, Caco2 cells were plated at a subconfluent cell density onto 6-well Millicell hanging filter inserts (3 μm pore size, Polyethylene Terephthalate) that allow free access of media to their apical and basolateral sides but not allow cells to pass.2. To induce Caco2 cells differentiation along the Paneth cell lineage,10 ng/ml fibroblast growth factor 9 (FGF9), a high affinity ligand for the fibroblast growth factor receptor-3 (FGFR-3), was added to both sides of the inserts daily starting at 24 h postplating and ending at 72 h postplating. Medium was changed every 24 h. After a consecutive 3-day treatment with FGF9, the mRNA expression of SI and APOA1, which encode two enterocyte differentiation markers, were determined by real-time PCR, and the mRNA expression of HD5, HD6, lysozyme and sPLA2, which encode four Paneth cell differentiation markers, were determined by real-time PCR.3. To determine the role of NOD2 in regulating the expression of human enteric antimicrobial peptides, cells were treated with 10 μg/ml MDP,10 ng/ml FGF9 or 10 μg/ml MDP+10 ng/ml FGF9 daily for 3 days. Medium was changed every 24 h. Total RNA was isolated and analyzed for HD5, HD6, Lysozyme and sPLA2 mRNA by real-time PCR. In order to further confirm the role of NOD2 in regulating the expression of human enteric antimicrobial peptides, Caco2 cells were first transfected with NOD2-siRNA, then were treated with 10 ng/ml FGF9 daily for 3 days. Medium was changed every 24 h and last total RNA was isolated and analyzed for HD5, HD6, Lysozyme and sPLA2 mRNA by real-time PCR and whole-cell extracts were analyzed for the protein expression of HD5 and HD6 by western blot.4. To determine whether stimulation with FGF9 could affect the expression of NOD2 during the differentiation of the Paneth cell lineage, Caco2 cells were treated with 10 ng/ml FGF9 for 24h、48 h and 72 h, then whole-cell extracts were prepared and analyzed for the protein expression of NOD2 by western blot.5. To determine which signaling pathways might be involved in inducing the expression of human enteric a-defensins, Caco2 Cells were treated with 10μg/ml MDP or 10 ng/ml FGF9 daily beginning at 24 h postplating and ending at 72 h postplating. For the last 24 h before harvest at 96 h postplating, signaling inhibitors BAY117082 (5,10 and 50μM; NF-κB), LY294002 (5,10 and 50μM; PI3K), SB203580 (5,10 and 50μM; p38 MAPK), SP600125 (5,10 and 50μM; JNK), and U0126 (5,10 and 50μM; ERK1/2) were added 30 min prior to MDP or FGF9 addition. Last total RNA was isolated and the mRNA expression of HD5 and HD6 was determined by real-time PCR.6. To assess the activation of the MAPK pathway, Caco2 cells were stimulated with 10 ng/ml FGF9 for 10 min,30min,60min, and 120min, then whole-cell extracts were prepared and were analyzed for the protein expression of p-p38, p38, p-JNK, JNK, p-ERK1/2 and ERK1/2 by western blot.7. To determine which NF-κB subunits are activated and transduce MDP-NOD2 signaling to the nucleus, Caco2 Cells were treated with 10μg/ml MDP for 2 h,4 h and 6 h, then nuclear extracts were prepared and translocation of NF-κB subunits in nuclear extracts was determined by TransAM assay.8. To induce Paneth cell (PC)-like cells, Caco2 cells were treated with 10 ng/ml FGF9 daily for a consecutive 3-day. Medium was changed every 24 h. This induced cells serves as PC-like cells to determine whether NOD2 can regulate the expression of IL-23p19 and the potential regulatory mechanism.9. To determine which bacterial compounds can induce expression of IL-23p19, PC-like cells were stimulated with 10μg/ml PGN,1 μg/ml Pam3CSK4,10 μg/ml Poly (I:C),10μg/ml LPS,1μg/ml Flagellin,1 μg/ml FSL-1,1μg/ml Imiquimod,1 μg/ml ssRNA40 and lμM ODN2006 for 4 h,8h and 16 h, then total RNA was isolated and IL-23pl9 mRNA expression was determined by real-time PCR.10. To determine whether NOD2 can regulate the expression of IL-23p19, PC-like cells were stimulated with 10 ug/ml MDP,10 μg/ml PGN,10 μg/ml PGN+10 μg/ml MDP,1 μg/ml Pam3CSK4 andlμg/ml Pam3CSK410 μg/ml MDP for 4 h, then total RNA was isolated and IL-23pl9 mRNA expression was determined by real-time PCR. In order to further confirm the role of N0D2 in regulating the expression of IL-23pl9, PC-like cells were first transfected with NOD2-siRNA, then were stimulated w1 ug/ml Pam3CSK4+ 10 jig/ml MDP for 4 h, and last total RNA was isolated and IL-23pl9 mRNA expression was determined ith 10 μg/ml PGN,1μg/ml Pam3CSK4 and by real-time PCR and whole-cell extracts from PGN-stimulated cells were analyzed for the protein expression of IL-23p19 by western blot.11.To determine which signaling pathways might be in involved in the TLR2-mediated induction of IL-23pl9 expression, PC-like cells were pretreated for 30 min with 50 uM BAY117082 (NF-kB),50 μM LY294002 (PI3K),50 μM SB203580 (P38),50 μM U0126 (ERK1/2), and 50 μM SP600125 (JNK) and subsequently were stimulated for 4 h with 10 μg/ml PGN, and last total RNA was isolated and IL-23p19 mRNA expression was determined by real-time PCR.12. To assess the activation of the NF-κB pathway, PC-like cells were stimulated with 10μ/ml PGN for 5 min,10 min,30 min and 60 min, then whole-cell extracts were prepared and were analyzed for the protein expression of IκB<x and p-IicBa by western blot.13. To determine which NF-kB subunits are activated and transduce TLR2-mediated signaling to the nucleus, PC-like cells were stimulated with 1 ug/ml Pam3CSK4 and 10 ug/ml PGN for 2 h, and then nuclear extracts were prepared and translocation of NF-kB subunits in nuclear extracts was determined by TransAM assay.14. To determine the mechanism by which activation of TLR2 by PGN and Pam3CSK4 can induce differential expression of IL-23pl9. PC-like cells were first transfected with NOD2-siRNA, then were stimulated with 10 ug/ml PGN for 2 h, and last total nuclear extracts were prepared and translocation of NF-κB subunits in nuclear extracts was determined by TransAM assay.15. Data are shown as the mean ± standard deviation. SPSS 18.0 statistical software was used to analyze results. Statistical significance of comparison between two groups was determined by Student’s ｔ-test; Statistical significance of multiple comparisons was determined by one-way analysis of variance with Tukey’s multiple comparisons under equal variances or with Dunnett T3’s multiple comparisons under unequal variances. A value of P<0.05 was considered statistically significant.Results1. After a consecutive 3-day treatment with FGF9, the mRNA expression of SI and APOA1, which encode two absorptive enterocyte differentiation markers, was greatly decreased compared with untreated control cells and these significant decreases were sustained for at least 72 h. In contrast, the mRNA expression of HD5, HD6, lysozyme and sPLA2, which encode four Paneth cell differentiation markers, was greatly increased and these significant increases were sustained for at least 24 h.2. After a consecutive 3-day treatment with FGF9 or FGF9+MDP, the mRNA expression of HD5, HD6, lysozyme and sPLA2 was decreased approximately 10.6-, 9.6-,2.7-and 2.3-fold, respectively, in Caco2 cells treated with MDP plus FGF9 compared with FGF9 only. However, the mRNA expression of HD5, HD6, lysozyme and sPLA2 was significantly higher in FGF9-stimulated NOD2-siRNA-transfected cells than in untransfected cells. Consistent with this result, the protein expression of HD5 and HD6 was significantly increased in FGF9-stimulated NOD2-siRNA-transfected cells compared with untransfected cells.3. The mRNA expression of HD5 and HD6 was increased approximately 2.8-and 1.5-fold, respectively, in Caco2 cells after a consecutive 3-day treatment with MDP compared with untreated control cells, but the mRNA expression of lysozyme and sPLA2 was not significantly different between MDP-treated cells and untreated control cells. However, the mRNA expression of HD5 and HD6 was significantly decreased in MDP-stimulated NOD2-siRNA-transfected cells compared with untransfected cells.4. The expression of NOD2 protein was not significantly different between FGF9-treated cells and untreated control cells.5. The MDP-induced mRNA expression of HD5 and HD6 in Caco2 cells treated with BAY 117082 was the lowest in treated cells with inhibitors. However, the FGF9-induced mRNA expression of HD5 and HD6 was significantly decreased in SP600125-and SB203580-treated cells, respectively. In addition, the p38, JNK, and ERK1/2 kinases of the MAPK pathway were all phosphorylated in response to the concentration of FGF9 that induced expression of human enteric a-defensins in Caco2 cells.6. The DNA-binding activities of NF-κB subunit p52, p50, RelB and c-Relwere significantly increased after 4 h and 6 h of MDP stimulation,especially the NF-κB signaling molecules p50 and p52.7. The mRNA expression of IL-23p19 was greatly enhanced in Paneth cell (PC)-like cells stimulation by PGN and, to a lesser extent, by the synthetic TLR2 agonist Pam3CSK4, peaking at 4 h after stimulation. At the peaking time, its expression was 4-fold higher in PC-like cells stimulation by PGN than by Pam3CSK4. In addition, activation of PC-like cells by other non-TLR2 agonists did not evidently enhance the mRNA expression of IL-23p19.8. The mRNA expression of IL-23p19 was significantly increased in PC-like cells stimulated by the TLR2 ligands PGN and Pam3CSK4 in the presence of MDP as compared with that in the absence of MDP, but not by MDP stimulation alone. The mRNA expression of IL-23p19 induced by PGN or Pam3CSK4 plus MDP was significantly lower in PC-like cells transfected with NOD2-siRNA than in untransfected PC-like cells. However, the Pam3CSK4-induced mRNA expression of IL-23p19 did not significantly change in PC-like cells transfected with NOD2-siRNA compared with untransfected cells. In addition, we found no significantly difference in the IL-23p19 mRNA levels between Pam3CSK4-stimulated PC-like cells and PGN-stimulated NOD2-siRNA transfectant. Moreover, the protein expression of IL- 23p19 was decreased in PGN-stimulated NOD2-siRNA transfectant compared with untransfected cells.9. The PGN-induced mRNA expression of IL-23p19 was significantly decreased in PC-like cells treated by BAY117082, SB203580, SP600125, U0126 or LY294002, especially in BAY117082-treated cells. And IκBα was phosphorylated in response to the concentration of PGN that induced IL-23p19 expression in PC-like cells.10. The DNA-binding activities of NF-κB subunit c-Rel were significantly increased in nuclear extracts from Pam3CSK4-and PGN-stimulated PC-like cells compared with extracts from untreated (medium) cells. However, there were no differences in DNA-binding activities of other NF-κB subunits between extracts from Pam3CSK4-and PGN-stimulated PC-like cells and untreated cells. In addition, the c-Rel DNA-binding activity in extracts from PGN-stimulated cells was significantly higher than that in extracts from Pam3CSK4-stimulated cells, but the c-Rel DNA-binding activity was significantly decreased in extracts from PGN-stimulated transfectant compared with untransfected cells. However, the c-Rel DNA-binding activity was not significantly difference between extracts from PGN-stimulated transfectant and Pam3CSK4-stimulated cells.Conclusions1. Activation of FGFR-3-mediated signaling induces in vitro differentiation of Caco2 cells along the Paneth cell lineage.2. NOD2 signaling down-regulates the expression of human enteric antimicrobial peptides during differentiation of the Paneth cell lineage.3. Although NOD2 by itself can slightly up-regulate expression of human enteric a-defensins, it can strongly down-regulate FGFR-3-mediated expression ofhuman enteric a-defensins. NOD2 up-regulate and down-regulate expression of human enteric a-defensins through the NF-κB pathway and the MAPK pathway, respectively.4. NOD2 up-regulates TLR2-mediated IL-23p19 expression via NF-κ/ subunit c-Rel.