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. Author manuscript; available in PMC: 2012 Dec 17.
Published in final edited form as: Immunity. 2012 Aug 16;37(2):326–338. doi: 10.1016/j.immuni.2012.05.024

Sensing of commensal organisms by the intracellular sensor NOD1 mediates experimental pancreatitis

Yoshihisa Tsuji 1, Tomohiro Watanabe 1,2,3, Masatoshi Kudo 4, Hidenori Arai 5, Warren Strober 3, Tsutomu Chiba 1
PMCID: PMC3523885  NIHMSID: NIHMS424626  PMID: 22902233

SUMMARY

The intracellular sensor NOD1 has important host defense functions relating to a variety of pathogens. Here we showed that this molecule also participated in the induction of a non-infectious pancreatitis via its response to commensal organisms. Pancreatitis induced by high-dose cerulein (a cholecystokinin receptor agonist) administration depends on NOD1 stimulation by gut microflora. To analyze this NOD1 activity we induced pancreatitis by simultaneous administration of low-dose of cerulein (that does not itself induce pancreatitis) and FK156, an activator of NOD1 that mimics the effect of gut bacteria that have breached the mucosal barrier. The pancreatitis was dependent on acinar cell production of the chemokine MCP-1 and the intra-pancreatic influx of CCR2+ inflammatory cells. Moreover, MCP-1 production involved activation of the transcription factors NF-κB and STAT3, each requiring complementary NOD1 and cerulein signaling. These studies indicate that gut commensals enable non-infectious pancreatic inflammation via NOD1 signaling in pancreatic acinar cells.

INTRODUCTION

Although most episodes of acute pancreatitis are mild, a sub-population of patients with this condition develops a severe disease with local and extra-pancreatic complications (Frossard et al., 2008). Bacterial colonization of the inflamed pancreas is involved in the latter cases and, in fact, infection of necrotic pancreatic tissue is one of the most important causes of mortality in acute pancreatitis (Frossard et al., 2008). It is now generally accepted that such colonization and associated inflammation result from failure of intestinal barrier function and translocation of intestinal microflora into the splanchnic vascular bed (Frossard et al., 2008; Rychter et al., 2009).

Microbe-associated molecular patterns (MAMPs) derived from the intestinal microflora activate the host innate immune system via pattern recognition receptors such as Toll-like receptors (TLRs) and nucleotide-binding domain and leucine-rich repeat containing molecules (NLRs) (Akira and Takeda, 2004; Chen et al., 2009; Strober et al., 2006; Werts et al., 2011). Thus, it is likely that activation of TLRs and NLRs is involved in the mechanisms by which bacterial translocation accounts for the development of severe acute pancreatitis. Consistent with this idea, the severity of acute pancreatitis is ameliorated in mice lacking TLR4 (Sharif et al., 2009) and polymorphism in the TLRs genes is associated with susceptibility to acute pancreatitis (Gao et al., 2007; Takagi et al., 2009). In addition, NF-κB, a down-stream transcription factor of the TLR and NLR signaling pathways (Akira and Takeda, 2004; Strober et al., 2006), plays a critical role in the development of acute pancreatitis (Baumann et al., 2007; Rakonczay et al., 2008; Tando et al., 1999).

Studies have highlighted the role of the NLR family of proteins in the microbial-recognition system that functions in the intestinal milieu (Strober et al., 2006) (Chen et al., 2009; Werts et al., 2011). NOD1, which belongs to this family, is of particular interest because it has been shown to play a protective role in infection of the mucosal surface (Strober et al., 2006). NOD1 contains a leucine-rich repeat region that serves as an intracellular sensor of small peptide components derived from bacterial peptidoglycan (PGN). Such recognition leads to NOD1 activation and the production of proinflammatory mediators either through nuclear translocation of NF-κB or interferon regulatory factors (IRFs) and type I interferon signaling (Chamaillard et al., 2003; Fritz et al., 2007; Watanabe et al., 2010).

The above properties of NOD1 suggest the possibility that this NLR family member could contribute to the development of non-infectious inflammatory states particularly if it can be shown that NOD1 responds to gut commensal organisms as well as to pathogenic organisms(Girardin et al., 2003; Kim et al., 2004). Here, we addressed this possibility by defining the role of NOD1 in the development of cholecystokinin receptor (CCKR) agonist-induced acute pancreatitis (cerulein pancreatitis). In a key initial finding we showed that administration of high doses of cerulein, a well-established inducer of pancreatitis, requires the presence of gut commensal organisms acting through NOD1 for the development of pancreatic inflammation. This observation led us to develop a model of pancreatitis that would allow us to define the role of NOD1 signaling in pancreatitis. This consisted of the administration of low doses of cerulein that does not in itself cause pancreatitis and of the administration of NOD1 ligand, which in this case could be shown to mimic the activity of gut commensal bacteria that enter the circulation during pancreatitis. Using this model we showed that NOD1 stimulation facilitates the migration of CCR2+ myeloid cells to the pancreas in response to the robust acinar cell production of monocyte chemotactic protein-1 (MCP-1); the latter in turn results from cooperative NOD1-cerulein activation of NF-κB and STAT3. Overall, these findings reveal that NOD1 signaling plays a major role in the pathogenesis of cerulein-induced acute pancreatitis via its capacity to respond to commensal gut bacteria.

RESULTS

Bowel sterilization by broad-spectrum antibiotics inhibits high-dose cerulein pancreatitis

Repeated administration of high doses of cerulein (50–100 μg/kg), a CCKR agonist, is a well-established inducer of acute murine pancreatitis (Baumann et al., 2007; Sharif et al., 2009). To address the role of commensal organisms in the intestinal microflora in the development of high-dose cerulein pancreatitis, C57BL6 mice were administered a combination of antibiotics (ampicillin, vancomycin, neomycin and metronidazole) in the drinking water for three weeks, a regimen previously used to achieve bowel sterilization (Fagarasan et al., 2002) and then challenged with repeated systemic (IP) injections of high doses of cerulein (50 μg/kg). We found that mice administered normal drinking water and given seven hourly injections of cerulein developed severe pancreatitis associated with a marked increase in the serum concentrations of pancreatic enzymes, amylase and lipase; in contrast, mice pre-administered antibiotics-containing drinking water developed barely detectable pancreatic inflammation and only marginal elevations in the serum concentrations of pancreatic enzymes (Figure 1A, B). In complementary studies addressing the effects of a single commensal organism, mice were administered ampicillin (AMP) and kanamycin (KM) in the drinking water for three weeks and then challenged with high doses of cerulein as before. We observed that even with this less stringent bowel sterilization regimen the mice exhibited only a mild elevation in the serum concentrations of amylase associated with normal pancreatic architecture (Figure S1A, B). However, high serum amylase concentrations and severe pancreatic inflammation were again observed in mice administered AMP+KM-containing drinking water together with periodic oral administration of AMP+KM-resistant E.coli expressing LacZ (ECLACZ) (Yoshida et al., 2001) (Figure S1A, B). In contrast, periodic oral administration of ECLACZ alone did not induce a significant elevation of serum amylase levels (Figure S1C) and, in addition, did not enhance elevated serum amylase levels caused by administration of cerulein in the absence of AMP+KM in the drinking water (Figure S1D). Thus, ECLACZ used in this study behaved like a commensal organism with no capacity to induce pancreatitis on its own. Taken together, these data provide strong evidence that commensal organisms in the intestinal microflora are essential for the development of acute pancreatitis caused by high-dose cerulein.

Figure 1. Bowel sterilization by a broad range of antibiotics inhibits development of cerulein-induced pancreatitis.

Figure 1

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(A) Changes in serum levels of amylase and lipase in C57BL6 mice treated with or without antibiotics. Mice administered with ampicillin (AMP, 1 g/l), neomycin (NEO, 1 g/l), vancomycin (VCM, 0.5 g/l) and metronidazole (MNZ, 1 g/l) for three weeks in the drinking water were challenged with a high dose of cerulein (50 μg/kg IP) for a total of seven times; results are expressed as means ± SD. ** p<0.01, as compared with group not administered antibiotics. (B) Histopathology of mice treated with a broad range of antibiotics followed by systemic injection of a high doses of cerulein (50 μg/kg IP) for a total of seven times. Pancreatic tissue was obtained from mice at 8 hours after the first injection of cerulein. Lack of pancreatitis in mice treated with antibiotics (bottom panel). Magnification ×200. (C) Changes in serum levels of amylase in Tlr2−/−, Tlr4−/−, Tlr9−/− or Nod1−/− mice without antibiotics. Mice were challenged with a high dose of cerulein (50 μg/kg IP) for a total of seven times. Results are expressed as means ± SD. * p<0.05, **p<0.01, as compared with C57BL6 wild type mice.

NOD1 activation is necessary for the development of high-dose cerulein acute pancreatitis

The above results suggested that an intestinal microbial component recognized by a pattern recognition receptor is important in the development of high-dose cerulein pancreatitis. To investigate this possibility, we compared serumconcentrations of amylase after high-dose cerulein injections as described above in mice deficient in TLR2, TLR4, TLR9, or NOD1. Wild-type (WT) mice, TLR2-deficient (Tlr2−/−) mice, and Tlr9−/− mice all developed similar increases in the serum concentration of amylase, suggesting that TLR2 and TLR9 are not required for pancreatitis induction (Figure 1C). In contrast, Tlr4−/− mice exhibited a moderate and significant decrease in serum amylase as compared to wild WT mice. This is consistent with a report that TLR4 activation is involved in pancreatitis induction (Sharif et al., 2009). More surprisingly, however, cerulein-induced elevations in serum amylase concentrations were almost completely abrogated in Nod1−/− mice (Figure 1C). These data suggest a critical role for NOD1, which senses a peptide derived from PGN of the intestinal bacteria in the induction of high-dose cerulein pancreatitis.

Low-dose cerulein together with NOD1 ligand induces pancreatitis

We sought an experimental model in which NOD1 ligand administration could be used as a substitute for MAMPs derived from gut bacterial flora so that the effects of NOD1 signaling could be easily isolated from those of cerulein. In searching for such a model we became aware that whereas repeated administration of high doses of cerulein (50–100 μg/kg) induces acute pancreatitis as indicated above, administration of low doses of cerulein does not have this effect, although such doses are still capable of acinar cell signaling (Baumann et al., 2007; Sharif et al., 2009). This suggested that low-dose cerulein does not induce pancreatitis because it does not by itself cause an influx of gut micro-organisms sufficient to result in robust acinar cell NOD1 signaling and thus low-dose cerulein would cause pancreatitis if administered with NOD1 ligand. To test this possibility, we administered FK156 (NOD1 ligand) or muramyl dipeptide (MDP, NOD2 ligand) (each at a dose of 200 μg IP) to C57BL6 mice, two hours prior to initiation of a low-dose cerulein regimen consisting of three hourly injections of a dose of cerulein (20 μg/kg IP) that does not induce pancreatitis when administered alone (Figure 2, A and B). We found that serum concentrations of amylase and lipase were markedly elevated in mice treated with FK156 plus low-dose cerulein, but not in mice treated with MDP plus low-dose cerulein or in mice treated with either ligand or cerulein alone (Figure 2B). In addition, pancreatic edema, necrosis, and infiltration of immune cells were observed in mice treated with FK156 plus cerulein but not under the other conditions (Figure 2, C and D). Finally, we showed that comparable amounts of serum amylase were obtained when mice were administered FK156 before, at the same time or after cerulein administration (Figure S2A). Thus, activation of NOD1 causes severe pancreatitis in mice treated with a low sub-inflammatory dose of cerulein and thus combined low-dose cerulein and NOD1 ligand could be used as a model to test the role of NOD1 in cerulein-induced pancreatitis.

Figure 2. Induction of acute pancreatitis in mice treated with NOD1 ligand or ECLACZ in combination with cerulein.

Figure 2

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(A–D) Induction of acute pancreatitis in mice treated with NOD1 ligand and cerulein. (A) Experimental protocol: C57BL6 mice were administered FK156 (NOD1 ligand; 200 μg/mice IP) or MDP (NOD2 ligand; 200μg/mice) followed by a low dose of cerulein (CCKR agonist; 20 μg/kg IP) for a total of three times. (B) Changes in serum levels of amylase and lipase in mice treated with NOD ligands and/or cerulein. Each color corresponds to group shown in Fig. 2A. Results are expressed as means ± SD. * p<0.01, as compared with other groups. (C, D) H&E staining and histological scores of pancreatic tissue harvested at 24 hours after the start of experiments; magnification ×200; each color corresponds to group shown in Fig .2A; scores are expressed as means ± SD. * p<0.01, as compared with other groups. (E, F) Induction of acute pancreatitis in mice treated with ECLACZ and cerulein. (E) Experimental protocol. C57BL6 mice and Tlr4−/− or Nod1−/− mice exposed to AMP and KM in the drinking water were administered ECLACZ (1×106 CFU, IP) followed by three hourly injections of low doses of cerulein (CER). Changes in serum levels of amylase in C57BL6 mice treated with ECLACZ and/or cerulein (E, left panel). Changes in serum levels of amylase in C57BL6 mice and Tlr4−/− or Nod1−/− mice treated with ECLACZ and cerulein (E, right panel). Results are expressed as means ± SD. * p<0.01, as compared with other groups. Serum levels of amylase were determined 8 hours after the treatment with ECLACZ. (F) Lack of pancreatitis in Nod1−/− mice treated with ECLACZ and cerulein. Magnification ×200.

We sought to show that NOD1 ligand administration in the above described low-dose cerulein model is acting in place of commensal bacteria. In these studies, C57BL6 mice administered drinking water containing AMP and KM were given three hourly injections of low doses of cerulein alone, IP injection of ECLACZ alone or an IP injection of ECLACZ injections of low doses of cerulein (Figure 2E). Treatment with IP injection of ECLACZ and low-dose cerulein resulted in acute pancreatitis as well as increased serum amounts of amylase whereas treatment with ECLACZ or cerulein alone did not cause significant changes in these parameters. In further studies Nod1−/− mice were more resistant to pancreatitis induced by ECLACZ and low dose cerulein than WT mice and Tlr4−/− mice (Figure 2E, F). Thus, intestinal bacteria evoke a NOD1 response similar to that obtained with NOD1 ligand and therefore the latter is acting as a mimic of intestine bacteria in this setting.

NOD1 ligand and low dose cerulein are synergistic inducers of MCP-1

Prior work has shown that MCP-1 and IL-6 are associated with the local inflammation of the pancreas (Grady et al., 1997). We therefore determined the involvement of these pro-inflammatory mediators in the development of pancreatitis induced by FK156 in combination with low-dose cerulein. Consistent with previous reports (Fritz et al., 2007; Watanabe et al., 2010), systemic injection of FK156 but not MDP, induced increased serum amounts of the chemokines MCP-1 and IP-10. Moreover, MCP-1, but not IP-10 induction was greatly enhanced by co-administration of cerulein, even though low-dose cerulein treatment alone did not induce MCP-1 (Figure 3A).

Figure 3. Concomitant administration of NOD1 ligand and low dose cerulein induces synergistic production of MCP-1.

Figure 3

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(A) Changes in serum levels of pro-inflammatory cytokines and chemokines. C57BL6 mice received systemic administration of FK156 or MDP followed by intraperitoneal injection of cerulein for a total of three times. Results are expressed as means ± SD. * p<0.01, as compared with other groups. The results shown are representative one of three experiments. (B) Serum levels of MCP-1 in mice treated with ECLACZ and cerulein. Changes in serum levels of MCP-1 in C57BL6 mice treated with ECLACZ and/or cerulein (B, left panel). Changes in serum levels of MCP-1 in C57BL6 mice and Tlr4−/− or Nod1−/− mice treated with ECLACZ and cerulein (B, right panel). Results are expressed as means ± SD. * p<0.01, as compared with other groups. C57BL6 mice and Tlr4−/− or Nod1−/− mice treated with AMP and KM in the drinking water were challenged with IP injection of ECLACZ followed by three hourly injections of low doses of cerulein. Serum levels of MCP-1 were determined 8 hours after the treatment with ECLACZ.

FK156 treatment also led to increased serum IL-6 amounts that were again enhanced by low-dose cerulein, particularly at later time points. In contrast, induction of IL-12p40 was observed either with FK156 or MDP administration and neither response was enhanced by low-dose cerulein. Comparable increases in serum amounts of MCP-1 were observed when FK156 was administered at the time of or after cerulein administration (Figure S2A). These data indicate that the severe pancreatitis induced by treatment with NOD1 ligand and low dose cerulein is associated with the production of MCP-1 and IL-6. Finally, similar effects on MCP-1 could be elicited by administration of ECLACZ in that IP administration of the latter followed by treatment with low doses of cerulein also led to a marked increase of the serum level of MCP-1 (Figure 3B). In contrast, Nod1−/− mice mice administered ECLACZ and low doses of cerulein exhibited little if any increase in the serum level of MCP-1 (Figure 3B).

Previous studies have shown that interaction between MCP-1 and its receptor, CCR2, plays a role in macrophage recruitment and the macrophage-dependent inflammatory response in the development of pancreatitis (Grady et al., 1997) (Bhatia et al., 2005; Zhao et al., 2005). One might therefore predict that low-dose cerulein pancreatitis is mediated by cells susceptible to MCP-1 chemoattraction. In tissue staining studies (Figure 4A), combined FK-156+ow-dose cerulein administration was associated with massive infiltration of CD11b+ myeloid cells and cells expressing CCR2, the receptor of MCP-1. In contrast, these cells did not express CXCR3, the receptor of IP-10, indicating the induction of IP-10 by NOD1 probably has little or no role in this type of inflammation.

Figure 4. Migration of CCR2+ myeloid cells is necessary for the development of pancreatitis.

Figure 4

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(A) At 6 hours after the injection of MDP or FK156, pancreatic tissue from C57BL/6 mice was removed and subjected to immunohistochemical analysis for visualization of cells positive for CD11b, CCR2, and CXCR3. (B, C) CCR2-intact (Ccr2+/+) or CCR2-deficient (Ccr2−/−) mice were administered FK156 followed by repeated administration of cerulein according to the protocol described in Figure 2A. (B) Changes in serum levels of amylase and MCP-1 at the indicated time points and pathology scores of the pancreatic tissue at 24 hours; results are expressed as means ± SD. *p < 0.01, as compared with CCR2-intact mice; results shown are representative one of three experiments. (C) Pancreatic tissue was removed at 6 hours after the start of the experiments, and subjected to immunohistochemical analysis for visualization of cells positive for CD11b and CCR2; magnification ×200.

The relation between MCP-1 induction and the infiltration of the pancreas by CCR2+ cells shown was further explored by studies of induction of pancreatitis with FK156 and low-dose cerulein in CCR2-deficient (Ccr2−/−) mice. Systemic administration of FK156 and low-dose cerulein to Ccr2−/− mice was associated with a marked reduction in the serum amylase level and pathology score as compared with WT mice, even though CCR2-deficient mice were still capable of producing MCP-1 (Figure 4B). In addition, migration of both CD11b+ cells and CCR2+ cells into the pancreas was absent in Ccr2−/− mice (Figure 4C). These studies thus showed that induction of MCP-1 by FK-156 and low-dose cerulein was associated with the infiltration of inflammatory CCR2+ cells into the inflamed pancreas.

Low-dose cerulein pancreatitis does not depend on T cells or B cells

NOD1 is a component of the innate immune system likely to involve mainly non-T cells and non-B cells. To determine if this was in fact the case we administered FK156 and low-dose cerulein to Prkdcscid mice with severe combined immunodeficiency (SCID) that lack both T cells and B cells. This treatment induced equally increased amounts of serum amylase and MCP-1 in WT and SCID mice (Figure S2B), suggesting that the acute pancreatitis induced by systemic injection of NOD1 ligand and low-dose cerulein does not depend on on T cells or B cells.

Acute pancreatitis requires activation of NOD1 in non-hematopoietic cells

We next turned our attention to the cellular location of the NOD1 involved in the acute pancreatitis model studied above. Our initial approach to this question was to conduct bone marrow (BM) chimera studies to determine if NOD1 was acting in hematopoietic cells or non-hematopoietic cells for the development of pancreatitis, but first we had to define the responses of Nod1−/− cells to be used in such studies. To this end we subjected Nod1−/− mice and Nod1+/+ mice to treatment with FK156 or MDP and low-dose cerulein. Nod1+/+ but not Nod1−/− mice developed pancreatitis after such FK156+cerulein administration, as evidenced by serum amylase and MCP-1 elevations and histology scores (Figure 5A). These studies indicated that NOD1 stimulation is essential for the induction of FK156+low-dose cerulein pancreatitis.

Figure 5. The importance of NOD1-signaling in non-hematopoietic cells for the development of severe acute pancreatitis.

Figure 5

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(A) Nod1+/+ orNod1−/− mice were treated with NOD ligands (FK156 or MDP) and/or cerulein (CER) as described in Fig. 2A. Changes in serum levels of amylase and MCP-1 at the indicated time points and pancreatitis histology score at 24 hours. * p<0.01, as compared with the other groups. (B) Bone marrow cells from GFP transgenic mice or Nod1−/− mice were transplanted into irradiated GFP transgenic mice or NOD1−/− mice. These chimeric mice were treated with FK156 followed by repeated administration of cerulein (CER). Changes in serum levels of amylase and MCP-1 at the indicated time points and pancreatitis histology score at 24 hours. Results are expressed as means ± SD. * p<0.01, as compared with the other groups. * p<0.01 (pathology score). The results shown are representative one of three experiments.

We then performed the BM chimera studies mentioned above and showed that administration of FK156+low-dose cerulein led to pancreatitis in Nod1+/+ GFP-transgenic mice reconstituted with Nod1−/− -BM cells (Figure 5B) but not in Nod1−/− mice reconstituted with Nod1+/+ BM cells. Thus, the development of FK156+cerulein acute pancreatitis involves NOD1 signaling in non-hematopoietic cells.

In a second approach to the identification of the site of NOD1 activity in this model we conducted in vitro studies of isolated pancreatic acinar cells from untreated mice to determine if these cells exhibited NOD1 activity following stimulation with FK156 and/or low-dose cerulein (Figure S3A). While stimulation of acinar cells with FK156 alone induced a substantial amount of MCP-1, synergistic production of MCP-1 was seen upon stimulation with FK156 plus low-dose cerulein in an FK156 dose-dependent fashion. In contrast, FK156 alone was sufficient for the induction of IP-10 production. Finally, the induction of MCP-1 under these conditions required the presence of NOD1 (Figure S3B). These studies established first that pancreatic acinar cells are the non-hematopoietic cellular site of NOD1 activation in the FK156+low-dose cerulein model of acute pancreatitis. Second, they showed that MCP-1 induction requires dual NOD1 and low-dose cerulein signaling via NOD1 and CCKR, respectively.

As mentioned above, administration of FK156 and low-dose cerulein elicited elevations in circulating amounts of IL-6 as well as MCP-1 (Figure 3A). This finding correlates with the fact that acinar cells from Nod1+/+ mice but not cells from Nod1−/− mice produced IL-6 upon stimulation with FK156 and low-dose cerulein and suggests that as in the case of MCP-1, increased circulatingamounts of IL-6 originate from acinar cells (Figure S3B). It seemed likely that the MCP-1 and IL-6 secretion might be inter-related since IL-6 participates in MCP-1 production via STAT3 activation (Deshmane et al., 2009). To address this possibility, we measured acinar cell production of MCP-1 in cells isolated from Nod1+/+ mice and then treated with FK156 and low-dose cerulein in the presence or absence of neutralizing IL-6R Ab. Such blockade of IL-6 signaling inhibited the production of MCP-1, but only partially (Figure S3C); thus IL-6 is not the only inducer of MCP-1 in this model.

NOD1+low-dose cerulein signaling induces transactivation of STAT3 and NF-κB in vivo

Because acinar cell MCP-1 production is a key factor in the development of FK156-low-dose cerulein pancreatitis we next turned our attention to the signaling pathways controlling such production. Previous studies have shown that MCP-1 production depends on STAT3 and NF-κB activation (Deshmane et al., 2009), and, indeed, we verified that these factors were necessary for the induction of FK156-low-dose cerulein pancreatitis by measuring activation of both STAT3 and NF-κB in pancreatic tissue of mice with pancreatitis. Nuclear expression of phospho-STAT3 (pSTAT3) and phospho-IκBα (pIκBα) was seen in the pancreatic acinar cells expressing amylase of Nod1+/+ mice treated with low-dose cerulein and FK156, whereas little or no pSTAT3 or pIκBα was seen in acinar cells of NOD1-deficient mice (Figure S3D). Similar findings were obtained using conventional immunohistochemical analysis (Figure S3E).

To confirm the above findings we performed staining studies of tissue obtained from BM-chimeric mice consisting of irradiated Nod1+/+ mice bearing a GFP-expressing transgene and irradiated Nod1−/− mice, reconstituted with Nod1−/− GFP-transgene negative BM cells and Nod1+/+ GFP-transgene positive BM cells, respectively. Replacement of BM cells was confirmed by flow cytometric analysis of GFP expression at 8 weeks after the transplantation (Figure S4A). We found that amylase-expressing acinar cells were GFP-positive in irradiated GFP-transgene-positive mice reconstituted with GFP-transgene-negative Nod1−/− BM cells whereas they were GFP-negative in irradiated GFP-transgene-negative NOD1-deficient mice reconstituted with GFP-transgene-positive Nod1+/+ BM cells (Figure S4B). Thus the GFP status of the acinar cells reflected the GFP status of the recipient, not the donor. In further studies we showed that expression of pIκBα and pSTAT3 was seen in acinar cell nuclei of irradiated GFP-transgene-positive Nod1+/+ mice reconstituted with Nod1−/− BM cells (Figure S4B). In contrast, expression of these factors was barely seen in acinar cell nuclei of irradiated GFP-transgene-negative Nod1−/− mice reconstituted with GFP-transgene positive Nod1+/+ BM cells (Figure S4B). Similar findings were obtained using conventional immunohistochemical analysis (Figure S4C). These data thus provide additional evidence that activation of STAT3 and NF-κB in response to FK156 and low-dose cerulein administration is occurring in pancreatic acinar cells.

Administration of JSI-124, a specific STAT3 inhibitor, inhibited nuclear translocation of Stat3 and administration of IMD-0354, a NF-κB inhibitor, inhibited nuclear translocation of NF-κB subunit, p65 in the pancreas of mice administered FK156-low-dose cerulein as assayed in the Transfactor Binding Assay, establishing that these agents do in fact exert the expected inhibition of activation of Stat3 and NF-κB under these conditions (Figure S5A). In addition, administration of JSI-124 or IMD-0354 alone led to a partial reduction of serum levels of amylase and MCP-1 whereas administration of both agents led to complete reduction of serum levels of amylase and MCP-1 (Figure S5B). A similar result was obtained in respect to inflammation score of the pancreas (Figure S5C). These studies using pharmacological inhibitors for STAT3 and NF-κB thus provide evidence that activation of both STAT3 and NF-κB in acinar cells act in an additive fashion to bring about the development of pancreatitis induced by FK156/low-dose cerulein.

Molecular mechanisms underlying the activation of STAT3 and NF-κB in FK156-low-dose cerulein pancreatitis

Having established that Stat3 and NF-κB are critical signaling components in the induction of FK156-low-dose cerulein pancreatitis we were interested in establishing how FK156 and low-dose cerulein act together to induce these components whereas neither stimulant can do so alone. In initial studies we focused on the signaling pathways leading to STAT3 activation and showed that treatment with FK156 alone induced expression of pSTAT3 in the pancreas of Nod1+/+ mice but not Nod1−/− mice (Figure 6A). This is consistent with our previous report that NOD1 ligand induces type I IFN production (Watanabe et al., 2010) as well as a study showing the type I IFN activates various STATs including STAT3 (Yang et al., 1998). Similarly, treatment with cerulein alone induced pSTAT3 expression (in both Nod1+/+ and Nod1−/− mice), presumably as a result of direct CCKR activation of JAK2 (Ferrand et al., 2005). However, treatment of Nod1+/+ mice (but not Nod1−/− mice) with both FK156 and low-dose cerulein induced higher pSTAT3 expression as compared with either stimulus alone and this was associated with similarly increased nuclear translocation (Figure 6B). Thus, FK156 and low-dose cerulein reinforce one another in Stat3 activation.

Figure 6. NOD1 activation followed by cerulein challenge induces transactivation of STAT3 and NF-κB in vivo.

Figure 6

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Nod1+/+ or Nod1−/− mice were treated with FK156 and/or cerulein (CER) as described in Fig. 2A. Whole or nuclear extracts were prepared from the pancreas of mice at 5 hours after systemic challenge with FK156 and subjected to immuno-blotting. (A) The expression of phospho-STAT3, STAT3, phospho-STAT1, STAT1, phospho-PKC, PKCδ, phospho-IκBα, IκBα, and actin was shown by using whole pancreatic extract. (B) Activation of STAT1, STAT3, p65 and p50 by using nuclear extracts as assessed by Transfactor assay. Results are expressed as means ± SD. *p < 0.01, as compared with no treatment group. The results shown are representative one of three experiments. (C) Physical interaction between RICK and TAK1 or between PKC-δ and TAK1 in whole pancreatic extracts. Pancreatic extracts were immuno-precipitated with anti-TAK1 antibody followed by immuno-blotting with anti- PKC-δ or RICK antibody.

We also noted that expression of phospho-STAT1 (pSTAT1) was induced in the pancreas of Nod1+/+ mice treated with FK156, but in this case such expression was not enhanced by combined treatment with FK156 and cerulein (Figure 6A). Moreover, expression of pSTAT1 was not induced in the pancreas of Nod1−/− mice whether mice were treated with FK156 or cerulein. These results were corroborated by measurement of nuclear translocation (Figure 6B). Given the fact that STAT1 is a critical down-stream signaling molecule for type I IFN, these data suggest that FK156 also activates a type I IFN pathway as reported previously (Watanabe et al., 2010).

We next examined the signaling pathways leading to NF-κB activation. Although previous studies utilizing human embryonic kidney cells show rapid activation of NF-κB upon stimulation with NOD1 ligands (Chamaillard et al., 2003; Fritz et al., 2007), the roles of NF-κB in NOD1-mediated signaling pathways have been poorly defined in primary pancreatic aciinar cells. In initial studies we found that neither NOD1 ligand nor low-dose cerulein administration alone resulted in expression of pIκBα or degradation of IκBα (Figure 6A). This is consistent with our previous report that NOD1 stimulation alone is a poor inducer of NF-κB in intestinal epithelial cells (Watanabe et al., 2010) as well as previous reports by other investigators that cerulein is also a poor inducer of NF-κB at low concentrations (Yu et al., 2005). Nevertheless, combined stimulation did result in such induction as shown by expression of pIκBα and nuclear translocation of p65 and p50 (Figure, 6A and 6B). In explanation of this finding, we focused first on CCK signaling and noted that previous studies had shown that CCKR signaling induces activation of protein kinase C (PKC) activation (Rakonczay et al., 2008; Tando et al., 1999). To verify that this pathway is in play_in the low-dose cerulein model we treated mice with low-dose cerulein (in the presence or absence of FK156) and showed that such mice exhibited enhanced pancreatic cell expression of phospho-PKC (Figure 6A). Since both activated PKC generated by cerulein signaling and RICK generated by NOD1 signaling is known to activate NF-κB via phosphorylation and/or ubiquitination of TGF-β-activated kinase 1 (TAK1) (Hasegawa et al., 2008; Shinohara et al., 2005), we next considered the possibility that TAK1 is a signaling molecule that serves as a nodal point in the low-dose cerulein and FK156 signaling pathways that facilitates NF-κB activation. In initial studies, we performed immuno-precipitation studies of cell lysates isolated from the pancreas of Nod1+/+ and Nod1−/− mice. We found that treatment with cerulein, but not FK156 induced physical interaction between TAK1 and PKC-δ in pancreatic cells of Nod1+/+ and Nod1−/− mice (Figure 6C), suggesting induction of TAK1 activation through cerulein-mediated PKC activation. More importantly, as shown by immuno-precipitation with anti-TAK1 Ab followed by immuno-blotting with anti-RICK Ab, interaction between RICK and TAK1 was markedly increased in the pancreas of mice treated with both FK156 and low-dose cerulein as compared to the marginal interaction observed with FK156 alone (Figure 6C). Taken together, these data are consistent with the hypothesis that synergistic NF-κB activation by low-dose cerulein and NOD1 is due to cerulein-induced PKC-TAK1 activation followed by opportunistic RICK utilization of the activated TAK1 to induce NF-κB.

NOD1-induced activation of STAT3 via type I IFN

Finally, to more fully assess the role of NOD1 signaling in STAT3 activation via type I IFN we investigated FK156-low-dose cerulein induction of pancreatitis in IFN-αβreceptor (αβR)-deficient (Ifnar1−/−) mice. In initial studies we showed that Ifnar1−/− mice exhibited an attenuated pancreatitis along with a significant reduction of serum levels of amylase and MCP-1 as compared with those in Ifnar1+/+ mice (Figure 7, A and B). In addition, upon challenge with FK156 and low-dose cerulein, pancreatic expression of pSTAT3 in Ifnar1−/− mice was significantly reduced as compared to that in Ifnar1+/+ mice, suggesting that type I IFN signaling is necessary for maximal Stat3 activation in this model (Figure 7C).

Figure 7. Involvement of type I IFN signaling in the development of pancreatitis.

Figure 7

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(A, B, C) IFNαβ receptor-intact (Ifnar1+/+) or -deficient (Ifnar1−/−) mice were subjected to induction of FK156/low-dose cerulein pancreatitis as indicated in Figure 2. (A, B) Serum levels of amylase, MCP-1, IL-6, IFN-β, and IP-10 at 6 hours after systemic challenge with FK156 and pathology scores of the pancreatic tissue at 24 hours after systemic challenge with FK156. Results are expressed as means ±SD. *p < 0.01, as compared with Ifnar1+/+ mice. (C) Whole cell extracts were prepared from pancreatic tissue of mice at 5 hours after systemic challenge with FK156 and subjected to immunoblotting to determine expression of phospho-STAT3, STAT3, phospho-STAT1, STAT1, phospho-IκBα, IκBα, andactin. The results shown are representative one of two experiments. (D, E) C57BL6 mice were subjected to induction of FK156-low-dose cerulein pancreatitis; blood and peritoneal lavage fluid were collected 8 hours after the injection. (D) Percentage and number (parentheses) of mice that were positive for bacterial culture in the blood and peritoneal lavage fluids. (E) Bacterial loads in the blood determined by counting colonies in columbia agar, trypticase soy agar, and chocolate agar plates. Results are expressed as means ±SD and summary of three independent experiments.

As expected, pancreatic expression of pSTAT1 was completely absent in Ifnar1−/− mice upon challenge with FK156 and low-dose cerulein, suggesting that pancreatic STAT1 activation is dependent upon NOD1-mediated type I IFN signaling in this model. In addition, consistent with the results of the signaling studies presented above, no significant difference in NF-κB activation was seen between Ifnar1−/− and Ifnar1+/+ mice, as assessed by pancreatic expression of p-IκBα and IκBα. Serum levels of IFN-β and IP-10, whose production requires activation of type I IFN signaling, were markedly reduced in Ifnar1−/− mice treated with FK156 and low-dose cerulein as compared with those in Ifnar1+/+ mice. In contrast, no significant difference was seen in production of IL-6, whose production depends mainly upon activation of NF-κB. Taken together, these results indicate that type I IFN signaling induced by NOD1 activation is involved in the development of pancreatitis in this model.

Bacterial translocation in low-dose cerulein induced pancreatitis

Intestinal barrier dysfunction and translocation of intestinal microflora into the systemic circulation play a critical role in the development of severe acute pancreatitis (Frossard et al., 2008; Rychter et al., 2009). IWe therefore addressed the role of bacterial translocation with studies of bacteremia in mice treated with low-dose cerulein alone or in combination with FK156. We found that 5 of 7 mice treated with low-dose cerulein alone exhibited bacteremia but that the level of bacteremia was relatively low in each case (Figure 7D, E). On the other hand, 6 of 7 mice subjected to combined treatment with FK156 and low-dose cerulein exhibited bacteremia and in this case the level of bacteria was mich higher than treatment with low-dose cerulein alone.

To more clearly identify the role of bacterial translocation from the gut into the circulation in low-dose cerulein pancreatitis, we treated mice with drinking water containing AMP+KM and administered oral ECLACZ prior to FK156 and low-dose cerulein administration. We again observed bacteremia, in this case at a level that increased in parallel with the oral ECLACZ dose (Figure S6). As expected, the peak level of bacteremia was not as high as obtained with normal “unperturbed” microflora in the initial study, probably due to the relative paucity of organisms present in the gut under these circumstances. However, it should be noted that serum amylase levels increased in proportion to the oral ECLACZ dose, indicating that even these relatively low levels of bacteremia supported the development of pancreatitis in mice administered FK156 and low-dose cerulein (Figure S6). These data are consistent with the view that low-dose cerulein administration alone does not induce pancreatitis in the absence of NOD1 ligand and, in addition, does not induce sufficient bacterial translocation to facilitate de novo NOD1 activation due to exposure to low levels of translocated organisms. In contrast, FK156 and low-dose cerulein administration not only initiated pancreatitis but also caused bacterial translocation that can now sustain the pancreatitis.

DISCUSSION

We explored the mechanism by which commensal intestinal bacteria contribute to the development of pancreatitis induced by cerulein, a CCKR agonist. Our initial studies established that high-dose cerulein administration, capable of causing pancreatitis by itself, required the presence of a normal intestinal bacterial flora and quite surprisingly was acting via stimulation of NOD1, an intracellular sensor of a peptide derived from the bacterial wall of both gram-positive and negative bacteria. To further study this phenomenon we developed a model of pancreatitis in which mice were admininstered a dose of cerulein that itself was insufficient to cause pancreatitis but was nevertheless able to cause pancreatitis when co-administered with NOD1 ligand. This model thus allowed us to identify the specific roles of cerulein and NOD1 signaling in the induction of pancreatic inflammation.

Two key immunopathologic features of the pancreatitis in the low-dose cerulein model are that the inflammation is associated with acinar cells that produce large amounts of MCP-1, a chemoattractant of CCR2+ cells, and that Ccr2−/− mice are almost completely resistant to the development of pancreatitis in this model. These findings strongly suggest that the pancreatitis is driven by MCP-1-mediated migration of CCR2+ inflammatory cells into the pancreas. This conclusion is consistent with previous studies showing that abrogation of MCP-1-CCR2 interactions by administration of a plasmid expressing mutated MCP-1 or a blocker of MCP-1 synthesis protects animals from experimental pancreatitis (Bhatia et al., 2005; Zhao et al., 2005). In addition, in a clinical study, Regner et al. showed that serum levels of MCP-1 at the time of a patient's admission to hospital are strongly associated with development of severe acute pancreatitis (Regner et al., 2008). Thus, it appears that acinar cell production of MCP-1 and MCP-1-dependent cell migration is essential to the development of acute pancreatitis, not only in this model, but also in other models and in human pancreatitis as well. The importance of MCP-1 to pancreatitis accounts for the need for NOD1 signaling in the induction and maintenance of this inflammation.

Previous studies have shown that activation of both NF-κB and STAT3 is required for maximal production of MCP-1 (Deshmane et al., 2009; Ray et al., 2008). In the present studies we corroborated this finding in the context of pancreatitis by showing that both the NF-κB and STAT3 signaling pathways are activated during MCP-1 induction and, indeed, pharmacological inhibition of both pathways is required for the suppression of pancreatitis. In addition, we found that while FK156 or cerulein could induce STAT3 alone, both were necessary for optimal induction and, perhaps more importantly, while neither stimulant could induce activation of the NF-κB signaling pathway alone they could induce this pathway when acting in concert. It follows that NOD1 signaling is essential to pancreatitis induction because it is necessary for robust activation of the signaling pathways that lead to the production of MCP-1.

The poor NF-κB activation by NOD1 signaling alone in the present study is consistent with our previous studies of this signaling pathway. We showed in these studies that NOD1 activation of RICK (the adaptor protein immediately down-stream of NOD1) leads to RICK binding to TRAF3 and thus to activation of TBK1 and IRF7; this, in turn leads to induction of IFN-β and signaling via IFNαβR to induce IFN-stimulated gene factor 3, a complex composed of STAT1, STAT2, and IRF9 that acts as a transcription factor for chemokines including IP-10 (Watanabe et al., 2010). Thus, in effect, NOD1 signaling by-passes NF-κB to induce chemokines via a type I IFN-dominated pathway. How then does NOD1 signaling cause NF-κB activation in association with low-dose cerulein? We believe the answer lies in the fact that even low doses of cerulein induce activation of TAK1 via PKC (Li et al., 2009; Shinohara et al., 2005) and that in the presence of such activation, RICK activated by NOD1 forms a complex with TAK1 to activate NF-κB. This idea is supported by an enhanced physical interaction between RICK and TAK1 in the mice treated with both FK156 and cerulein.

With respect to STAT3 activation in this model, previous studies have shown that cerulein can induce STAT3 at least at sub-optimal levels (Yu et al., 2005). However, as shown in these studies, NOD1 signaling greatly augments such induction by at least two mechanisms. On the one hand, by facilitating NF-κB activation, NOD1 promotes the synthesis of IL-6, a cytokine capable of STAT3 activation. On the other hand, NOD1 signaling leads to production of type I IFN, a cytokine also capable of STAT3 activation (Yang et al., 1998).

Analysis of the level of bacteremia occurring after administration of a low dose of cerulein alone or in combination with NOD1 ligand provided insight into the role of bacterial entry into the circulation in the induction low-dose cerulein pancreatitis. Low level bacteremia was observed in mice treated with low doses of cerulein alone indicating that the level of activation of CCKR with this dose leads to some degree of translocation of bacteria into the systemic circulation. Evidently, however, this level of translocation does not provide a sufficient NOD1 signal in acinar cells to initiate and sustain pancreatitis. In contrast, higher levels of bacteremia accompanied administration of NOD1 ligand and low-dose cerulein. It appears that such bacteremia, by providing a continued source of NOD1 signaling, is necessary to sustain the pancreatitis initiated by exogenous NOD1 ligand. Thus, low-dose cerulein induced pancreatitis is equivalent to high-dose cerulein pancreatitis, except for the fact that the latter initiates a level of pancreatic inflammation that causes sufficient bacterial translocation and NOD1 signaling on its own.

It has been generally assumed that NOD1 activation is induced exclusively by pathogenic organisms and that such activation elicits NOD1-mediated host defense responses (Girardin et al., 2003; Kim et al., 2004). However, it was recently shown that NOD1 signaling can also be initiated by commensal organisms in the neonatal gut, where it plays a role in lymphoid tissue organogenesis via the induction of defensin and a chemotactic factor production (Bouskra et al., 2008). The present study extends this observation by showing that NOD1 activation by commensal organisms also occurs during an inflammatory response. These observations raise the question as to why NOD1 activation by commensal organisms does not cause inflammation in the normal gut. We believe the answer to this question is that NOD1 does respond to commensal organisms in the normal un-inflamed gut via type I IFN signaling rather than NF-κB signaling, and type I IFN signaling does not result in inflammation (Abe et al., 2007).

In conclusion, in the present study we provide evidence that activation of NOD1 by commensal organisms can facilitate a severe form of acute pancreatitis by acting in synergy with low doses of a CCKR agonist, cerulein. In addition, we show that such synergy is necessary for the production of MCP-1, a chemokine that mediates migration and infiltration of CCR2+ pathogenic myeloid cells into the pancreas. Finally, we show that a critical feature of this model of pancreatitis, as well as high dose cerulein pancreatitis, that does not require priming by NOD1 agonist, is that pancreatitis is driven by commensal organisms that stimulate NOD1 in acinar cells. Thus, while autodigestion of acinar cells mediated by activation of pancreatic enzymes such as trypsinogen is one necessary component of acute pancreatitis, bacterial translocation and the induction of acinar cell expression of inflammatory mediators are also necessary components (Dawra et al., 2011; Ji and Logsdon, 2011). It seems likely that therapeutic measures attacking these latter components, such as those directed at the inhibition of NOD1 signaling offer a new approach to the treatment of acute pancreatitis.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6 mice, GFP transgenic mice (Okabe et al., 1997) and Prkdcscid mice were purchased from CLEA Inc. Tlr2−/−, Tlr4−/−, or Tlr9−/− mice were purchased from Oriental Bioservice Inc. In some experiments, Nod1−/− (Watanabe et al., 2010) and Ccr2−/− mice (Boring et al., 1998) were used. Mice were reared under specific pathogen free condition. Animal use adhered to Kyoto-university animal care guideline and protocols of animal experiments were approved by the review boards of Kyoto University.

Induction of pancreatitis

Mice received IP injection of FK156 (200 μg) or MDP (200 μg) in combination with IP injection of cerulein (20 μg/kg) for a total of three times. Mice treated with ampicillin (1 g/l) and kanamycin (1 g/l) for two weeks in the drinking water were challenged with IP injection of ECLACZ (Yoshida et al., 2001)(1×106 CFU) followed by IP injection cerulein (20 μg/kg) for a total of three times. Mice treated with ampicillin (1 g/l), neomycin (1 g/l), kanamycin (0.5 g/l), or metronidazole (1 g/l) for three weeks in the drinking water were challenged with IP injection of cerulein (50 μg/kg) for a total of seven times. Sera and pancreas tissue were obtained at the indicated time points.

Bone marrow transplantation

For the generation of BM chimeric mice, recipient mice were irradiated with 10 Gy and were reconstituted with BM cells from the donor mice as previously described (Watanabe et al., 2010)

NF-κB and STAT3 activation assay

Whole extracts isolated from the pancreas were subjected to immuno-blotting as previously described (Watanabe et al., 2010). Anti-phospho-STAT1, anti-STAT3, anti-phospho-Stat3, anti-IκBα, anti-phospho-IκBα, anti-phospho-PKC and PKCδ Abs were obtained from Cell signaling. Anti-actin, anti-TAK1, and anti-STAT1 Abs were obtained from Santa Cruz (Santa Cruz). Binding activity of nuclear extract to NF-κB subunit (p50, p65), STAT1, and STAT3 was measured using a Transam kit, obtained from Active Motif, as previously described (Watanabe et al., 2010). 7 or 15 μg of nuclear extracts were subjected to assay. Physical interaction between RICK and TAK1 or between PKC-δ and TAK1 was analyzed by immuno-precipitation using whole pancreatic extract. Pancreatic extracts were immuno-precipitated with anti-TAK1 antibody followed by immuno-blotting with anti- PKC-δor RICK antibody (Cayman) as described previously (Watanabe et al., 2010).

Statistical analysis

Student's t test was used to evaluate the significance of the differences. A value of P < 0.05 was regarded as statistically significant.

Supplementary Material

HIGHLIGHTS

  • 1)

    NOD1 activation enhances a cholecystokinin receptor agonist-induced pancreatitis.

  • 2)

    The development of pancreatitis depends on the interaction between CCR2 and MCP-1.

  • 3)

    Activation of Stat3 and NF-κB is responsible for the development of pancreatitis.

  • 4)

    Commensal organisms facilitate pancreatic inflammation via NOD1 signaling.

ACKNOWLEDGEMENTS

This work is supported in part by grants from the Ministry of Education, Science and Culture, Japan, and the Japan Society for the Promotion of Science (21590532), Takeda Science Foundation, Astellas Foundation for Research on Metabolic Disorders, Yakult Bioscience Foundation, Cell Science Research Foundation, Kato Memorial Trust for Nambyo Research, and Uehara Memorial Foundation (to T. W.). We would like to thank Dr. Naoki Asano for critical reading of this manuscript.

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NIHMS424626-supplement.pdf (8.7MB, pdf)

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