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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2012 Jun;91(6):957–966. doi: 10.1189/jlb.1211627

Role of IL-6 in the resolution of pancreatitis in obese mice

Maria Pini *, Davina H Rhodes *, Karla J Castellanos *, Andrew R Hall †, Robert J Cabay †, Rohini Chennuri †, Eileen F Grady ‡, Giamila Fantuzzi *,1
PMCID: PMC3360474  PMID: 22427681

IL-6 contributes to the delayed resolution of inflammation and promotes a tumorgenic microenvironment in the pancreas of obese mice.

Keywords: STAT-3, MMP-7, chemokines, cytokines, inflammation

Abstract

Obesity increases severity of acute pancreatitis and risk of pancreatic cancer. Pancreatitis and obesity are associated with elevated IL-6, a cytokine involved in inflammation and tumorigenesis. We studied the role of IL-6 in the response of lean and obese mice to pancreatitis induced by IL-12 + IL-18. Lean and diet-induced obese (DIO) WT and IL-6 KO mice and ob/ob mice pretreated with anti-IL-6 antibodies were evaluated at Days 1, 7, and 15 after induction of pancreatitis. Prolonged elevation of IL-6 in serum and visceral adipose tissue was observed in DIO versus lean WT mice, whereas circulating sIL-6R declined in DIO but not lean mice with pancreatitis. The severe inflammation and lethality of DIO mice were also observed in IL-6 KO mice. However, the delayed resolution of neutrophil infiltration; sustained production of CXCL1, CXCL2, and CCL2; prolonged activation of STAT-3; and induction of MMP-7 in the pancreas, as well as heightened induction of serum amylase A of DIO mice, were blunted significantly in DIO IL-6 KO mice. In DIO mice, production of OPN and TIMP-1 was increased for a prolonged period, and this was mediated by IL-6 in the liver but not the pancreas. Results obtained in IL-6 KO mice were confirmed in ob/ob mice pretreated with anti-IL-6 antibodies. In conclusion, IL-6 does not contribute to the increased severity of pancreatitis of obese mice but participates in delayed recovery from acute inflammation and may favor development of a protumorigenic environment through prolonged activation of STAT-3, induction of MMP-7, and sustained production of chemokines.

Introduction

Obesity is associated with increased severity of acute pancreatitis (AP) [1]. However, the mechanisms linking obesity to increased severity of AP are not known: some suggested factors include augmented necrosis of peripancreatic fat, fatty pancreas, alterations in the inflammatory network, and reduced respiratory excursion [2]. Obesity also increases the risk of carcinogenesis, including pancreatic cancer (PC), which has the highest case-fatality rate of any major cancer [3–5]. The exact mechanisms linking obesity to increased PC risk have not been elucidated, although insulin resistance and underlying chronic inflammation are likely involved [6].

IL-6 and its main signaling pathway, STAT-3, have been implicated in AP and PC [7–9]. IL-6 plays a causative role in models of PC through classical and trans-signaling events, which lead to activation of STAT-3 in infiltrating and parenchymal cells [10, 11]. One of the mechanisms linking STAT-3 to PC is induction of MMP-7 [12]. In patients with PC, serum levels of MMP-7 correlate with metastatic disease and survival [12, 13]. In mouse models of PC, MMP-7 is up-regulated in the pancreas, and its deficiency prevents or delays disease, perhaps as a result of resistance to apoptosis in metaplastic ducts [12, 13]. IL-6 may also participate in up-regulation of other mediators that are elevated in PC patients [14], such as SAA, OPN, TIMP-1 [15–17], and chemokines [18], which regulate tumor-stromal interactions in experimental models of PC [19].

At variance with knowledge about the causative involvement of IL-6 in PC, the mechanistic role of this cytokine in AP remains to be clarified. In humans, IL-6 is one of the most accurate predictors of AP severity, and constitutively activated STAT-3 is present in lymphocytes of AP patients [7, 9, 20]. Production of IL-6 is increased in human and experimental AP, with obesity further increasing IL-6 levels [20–23]. Despite its involvement in the pathogenesis of several chronic inflammatory disorders and cancer, IL-6 is also a critical anti-inflammatory cytokine that promotes resolution of inflammation [18]. Whether IL-6 exerts beneficial or detrimental roles in AP remains unclear. Evidence obtained in the cerulein model of AP is controversial, with beneficial and deleterious effects reported with blocking of IL-6 [24, 25]. Moreover, it is unknown whether elevated production of IL-6 mediates the enhanced severity of AP observed in obesity [1, 22, 26–28]. Furthermore, production of IL-6 by adipose tissue in obesity and by muscle during exercise has apparently opposite effects on modulation of insulin sensitivity [29, 30]. Therefore, the specific role of this cytokine in regulating inflammation and metabolism is likely to be highly context-dependent.

In the present report, we used the IL-12 + IL-18 model of pancreatitis to elucidate the role of IL-6 in AP severity in lean and obese mice [22, 23, 31]. We also determined whether obesity promotes signaling pathways that have been implicated in pancreatic adenocarcinoma induction and progression through sustained production of IL-6. Our results demonstrate that IL-6 does not mediate the acute severity of pancreatic inflammation in the IL-12 + IL-18 model. However, heightened production of IL-6 in obese mice mediates prolonged induction of STAT-3, MMP-7, and chemokines, which may favor development of a protumorigenic environment when chronically up-regulated.

MATERIALS AND METHODS

Animals

Animal protocols were approved by the Animal Care and Use Committee of the University of Illinois at Chicago. For induction of DIO, male WT and IL-6 KO C57BL6 mice were fed a high-fat diet (60 Kcal% fat; Research Diets, New Brunswick, NJ, USA) ad libitum for 13 weeks, beginning at 4 weeks of age, whereas groups of WT and IL-6 KO control groups received standard chow diet for the same period. Leptin-deficient, obese ob/ob mice and their lean littermates fed a standard chow diet were examined at 7–8 weeks of age. All mice were from The Jackson Laboratory (Bar Harbor, ME, USA).

Induction of AP

Murine rIL-12 and rIL-18 (R&D Systems, Minneapolis, MN, USA) were administered i.p. at 150 ng/mouse and 750 ng/mouse, respectively, at 24 h intervals, for a total of two injections, as described previously [22]. For neutralization experiments, WT and ob/ob mice received a neutralizing anti-IL-6 antibody (1 mg/mouse; BioLegend, San Diego, CA, USA), previously shown to neutralize IL-6 activity [32, 33], at the same time of the first injection of IL-12 + IL-18. Mice were euthanized at 1, 7, or 15 days after the second injection of IL-12 + IL-18. Control groups received vehicle and were euthanized 1 day after the second injection. Severity of VAT necrosis was quantified macroscopically as 0 (absent), 1 (few pinhead-sized necrotic areas without retropancreatic necrosis), 2 (moderately extended necrotic areas with moderate/extensive retropancreatic necrosis), and 3 (extensive areas of necrosis with extensive retropancreatic necrosis). Blood was collected at time of euthanasia and serum prepared. Pancreas, liver, and VAT obtained from non-necrotic and necrotic areas were frozen immediately in liquid nitrogen and stored at −70°C for subsequent processing or fixed in formalin for histological evaluation.

Miscellaneous measurements

Levels of IL-6, sIL-6R, leptin, OPN, SAA, and TIMP-1 were measured using ELISA kits from R&D Systems, eBioscience (San Diego, CA, USA), and Invitrogen (Carlsbad, CA, USA). Serum amylase, ALT, and calcium levels were measured using kits from Teco Diagnostics (Anaheim, CA, USA). Pancreas homogenates were prepared by homogenizing tissue in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), followed by sonication. Protein concentration was adjusted to 1 mg/ml for measurement of CXCL1, CXCL2, CCL2, OPN, and TIMP-1 using ELISA kits from R&D Systems and to 0.1 mg/ml for evaluation of Tyr705-pSTAT-3 levels using a PathScan assay (Cell Signaling Technology).

Histological analysis and immunohistochemistry

For histological assessment, the pancreas was fixed in 10% buffered formalin and sections stained with H&E for scoring by two pathologists (R.J.C. and R.C.), blinded to the experimental groups, using a scoring system described previously [23]. For immunohistochemistry, antibodies directed against rat anti-mouse Ly6G, rat anti-mouse F4/80 (BD Biosciences, San Diego, CA, USA), rabbit anti-Tyr705-pSTAT-3, or rabbit anti-mouse MMP-7 (Cell Signaling Technology) were used.

RNA expression analysis

Total RNA was isolated from VAT, obtained from areas containing necrotic and saponified tissue (necrotic VAT) and from VAT collected at sites distant from necrotis areas (non-necrotic VAT), as well as from liver using Trizol, and reverse transcribed. Gene expression of IL-6 and SAA-1 was assessed by real-time RT-PCR using the TaqMan system and primers from Applied Biosystems (Foster City, CA, USA). Relative expression was calculated using the ΔΔcomparative threshold method after normalizing for expression of GAPDH.

Statistical analysis

Data are expressed as mean ± sem. Statistical significance of differences was determined by ANOVA as appropriate. The Kaplan-Meyer method was used for analysis of survival data. Statistical analyses were performed using MedCalc software (Belgium).

RESULTS

Increased IL-6 levels in DIO mice with AP

We demonstrated previously that administration of IL-12 + IL-18 induces AP in mice, with more severe and prolonged disease in DIO and ob/ob mice compared with their lean controls [22, 23]. In this model of AP, circulating levels of IFN-γ are comparable in lean and DIO mice, whereas induction of the STAT-3-activating cytokines IL-6, IL-22, and IL-10 is significantly higher in obese compared with lean mice [22, 23]. In agreement with these previous data, we found a significantly higher and prolonged elevation of serum IL-6 in DIO mice compared with lean mice (Fig. 1A). Whereas lean mice had near-baseline levels of IL-6 at 15 days after the injection of IL-12 + IL-18, DIO mice had ∼200 pg/ml serum IL-6. Several deleterious activities of IL-6 are mediated through its ability to trans-signal by binding to its soluble receptor (sIL-6R) and activate cells, such as the endothelium, which do not express membrane-bound IL-6R [18]. Circulating levels of sIL-6R were comparable in lean and DIO control mice. Administration of IL-12 + IL-18 did not significantly alter serum sIL-6R levels in lean mice, whereas a significant reduction was observed in DIO mice at Days 1 and 7, with return to baseline by Day 15 (Fig. 1B).

Figure 1. IL-6 and sIL-6R levels in DIO mice with AP.

Figure 1.

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Lean and DIO WT mice received two injections of vehicle or IL-12 + IL-18. Blood and VAT were obtained at the indicated time-points. (A and B) Circulating IL-6 (A) and sIL-6R (B) levels in lean (black bars) and DIO (white bars) WT mice. C, Control. (C) Expression of IL-6 mRNA in VAT from lean WT mice (black bars) and non-necrotic (white bars) and necrotic (hatched bars) VAT from DIO WT mice. Data in C are expressed as fold increase over lean control mice. (D) VAT necrosis score in lean (black bars) and DIO (white bars) mice. Data are mean ± sem of four to seven mice/group. a, P < 0.01 versus respective control; b, P < 0.01 versus lean at the same time-point; c, P < 0.05 versus non-necrotic VAT at the same time-point.

We demonstrated previously that the pancreas significantly contributes to production of IL-6 in response to administration of IL-12 + IL-18 [23]. As indicated in Fig. 1B, VAT also contributed to production of IL-6 during AP in lean and DIO mice. A 20-fold increase in IL-6 mRNA expression was observed in VAT of lean mice at Day 1 post-AP, with levels returning to baseline by Day 7 (Fig. 1C). Compared with lean mice, non-necrotic VAT of DIO mice had a significantly higher and prolonged increase in IL-6 expression, with an almost 30-fold increase at Day 1 post-AP and an ∼10-fold increase on Day 7 post-AP. Necrotic VAT was only present in DIO mice with AP (Fig. 1D and Supplemental Fig. 1; see refs. [23, 31] for examples of macro- and microscopic VAT necrosis in ob/ob mice). Areas of VAT, which included necrotic tissue, as evaluated by the presence of macroscopic saponification, expressed the highest IL-6 mRNA levels, with sustained high levels even at Day 15 post-AP (Fig. 1C).

IL-6 does not mediate the increased AP severity of obese mice

To evaluate whether elevated production of IL-6 in obese mice causes the increased disease severity observed in DIO and ob/ob mice [22, 23], we used lean and DIO WT and IL-6 KO mice, as well as genetically obese ob/ob mice pretreated with anti-IL-6 antibodies. Administration of IL-12 + IL-18 caused no lethality in lean mice (Fig. 2A). We found comparable lethality (30–40%) in DIO WT and DIO IL-6 KO mice (Fig. 2A). In lean WT and IL-6 KO mice, AP did not alter body weight or serum leptin levels during the induction or the resolution phase. In marked contrast, AP induced significant and comparable body weight loss, accompanied by reduced leptin levels, in DIO WT and DIO IL-6 KO mice (Fig. 2B and C). In ob/ob mice pretreated with anti-IL-6 antibodies, neutralization of IL-6 did not significantly alter lethality rates or body weight loss induced by AP (Supplemental Fig. 2A and B). We verified that the antibody effectively neutralized IL-6 activity in vivo by measuring its ability to suppress induction of the acute-phase protein SAA (Supplemental Table 1).

Figure 2. Survival, body weight, and serum leptin in lean and DIO WT and IL-6 KO mice.

Figure 2.

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Lean and DIO WT and IL-6 KO mice received two injections of vehicle or IL-12 + IL-18. (A) Kaplan-Meyer curves for survival. (B) Body weight (BW). (C) Serum leptin levels. Data are mean ± sem of 10 mice/group. a, P < 0.01 versus respective time 0.

Injection of IL-12 + IL-18 induced edema, acinar cell necrosis, and fat necrosis in all mice (Fig. 3A–C and J). In lean mice, these parameters of histological damage were diminished at Days 7 and 15 after induction of AP. In sharp contrast, in DIO WT and DIO IL-6 KO mice, we found prolonged pancreatic damage with increased edema, acinar cell necrosis, and fat necrosis (Fig. 3A–C and J). Thus, prolonged histopathological damage in DIO mice occurred in an IL-6-independent manner.

Figure 3. Pancreatic tissue damage and induction of SAA in lean and DIO WT and IL-6 KO mice with AP.

Figure 3.

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Lean and DIO WT and IL-6 KO mice received two injections of vehicle or IL-12 + IL-18. Pancreas, liver, and blood were obtained at the indicated time-points. Quantification of edema (A), acinar necrosis (B), and fat necrosis (C) in the pancreas of lean WT (black bars), lean IL-6 KO (gray bars), DIO WT (white bars), and DIO IL-6 KO (hatched bars) mice. Levels of amylase (D), ALT (E), calcium (F), IL-22 (G), and SAA (H) were measured in serum. mRNA expression of SAA was measured in liver (I). (J) Representative samples of H&E-stained sections of the pancreas. Original magnification, 10×/0.3. Data are mean ± sem of four to seven mice/group. a, P < 0.01 versus respective control group; b, P < 0.01 versus lean groups at the same time-point; c, P < 0.05 versus DIO WT at the same time point; d, P < 0.05 versus lean WT at the same time-point.

As seen previously with administration of IL-12 + IL-18 [22, 23], at Day 1, serum amylase and ALT increased in each group, whereas serum calcium decreased significantly only in DIO mice, irrespective of genotype (Fig. 3D–F). Similarly, administration of anti-IL-6 antibodies did not significantly modify the effect of IL-12 + IL-18 on serum amylase and calcium levels in ob/ob mice (Supplemental Table 1). In contrast, induction of SAA was reduced significantly at the mRNA and protein level in lean and DIO IL-6 KO compared with WT mice (Fig. 3H and I). Induction of SAA in IL-6 KO mice with AP, although of lower magnitude than that seen in WT mice, was still significant compared with healthy controls, possibly as a result of production of IL-22 (Fig. 3G), a cytokine that is increased during AP induced by IL-12 + IL-18 and that is known to induce SAA expression [22, 34]. In summary, IL-6 did not mediate the increased lethality, body weight loss, or tissue damage observed in DIO mice with AP but was a critical mediator for induction of SAA.

Delayed resolution of the neutrophil infiltrate in the pancreas of obese mice with AP is mediated by IL-6

A comparable inflammatory infiltrate was present in the pancreas of lean WT, DIO WT, lean IL-6 KO, and DIO IL-6 KO at Day 1 (Fig. 4A). Thus, the initial recruitment of inflammatory cells was unaffected by DIO or IL-6. However, in DIO WT mice, the degree of infiltration continued to increase at Days 7 and 15, whereas it stabilized in each other group, including DIO IL-6 KO mice. At Days 7 and 15, the neutrophil infiltrate of DIO WT mice was significantly higher compared with the other groups, whereas the mononuclear infiltrate was not affected significantly by IL-6 deficiency (Fig. 4B–D). Immunohistochemistry for Ly6G, which identifies neutrophils, confirmed the presence of a significant neutrophil infiltrate in DIO WT mice but not DIO IL-6 KO mice at Day 7, whereas no significant differences between the two groups were observed at the same time-point for F4/80+ macrophages (Supplemental Fig. 3A). Thus, in contrast to initial recruitment, a prolonged retention/recruitment of neutrophils was dependent on DIO and IL-6.

Figure 4. Inflammatory infiltrate in the pancreas of lean and DIO WT and IL-6 KO mice with AP.

Figure 4.

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Lean and DIO WT and IL-6 KO mice received two injections of vehicle or IL-12 + IL-18. The pancreas was obtained at the indicated time-points. Quantification of the total inflammatory infiltrate (A), neutrophils (NE; B), macrophages (MΦ; C), and lymphocytes (LY; D) by histological analysis. Levels of the chemokines CXCL1 (E), CXCL2 (F), and CCL2 (G) in pancreatic homogenates of lean WT (black bars), lean IL-6 KO (gray bars), DIO WT (white bars), and DIO IL-6 KO (hatched bars) mice. Data are mean ± sem of four to seven mice/group. a, P < 0.01 versus respective control group; b, P < 0.01 versus lean groups at the same time-point; c, P < 0.05 versus DIO WT at the same time-point.

Chemokine levels in pancreatic homogenates were increased in lean mice at Day 1 after AP, but CXCL1 was increased more dramatically in DIO WT and DIO IL-6 KO mice (Fig. 4E–G). The kinetics of chemokine levels paralleled the delayed resolution of the inflammatory infiltrate of DIO WT mice. In fact, levels of CXCL1 and CXCL2 remained significantly elevated in the pancreas of DIO WT but not DIO IL-6 KO mice, up to 7 and 15 days post-AP, respectively (Fig. 4E and F). Pancreatic levels of CCL2 were comparable in each group at Day 1 and remained elevated only in DIO WT mice at Days 7 and 15 (Fig. 4G). In ob/ob mice, neutralization of IL-6 significantly reduced CXCL1, CXCL2, and CCL2 levels at Day 1, but the effect was lost at Day 7 (Supplemental Fig. 3B), possibly as a result of antibody clearance.

Prolonged STAT-3 activation in the pancreas of obese mice with AP is mediated by IL-6

To verify whether the increased and prolonged production of IL-6 in obese mice with AP was associated with activation of STAT-3 in the pancreas, we quantified levels of Tyr705-pSTAT-3. A significant increase in pSTAT-3 levels was observed at Day 1 in homogenates of pancreas obtained from each group of mice injected with IL-12 + IL-18 compared with that seen in mice without AP (controls), with the highest levels observed in DIO WT mice and significantly lower levels in IL-6 KO mice compared with WT mice (Fig. 5A). Residual activation of STAT-3 in IL-6 KO mice could be the consequence of induction of IL-22 (Fig. 3G), a STAT-3-activating cytokine whose receptor is present on acinar cells [35]. Activated STAT-3 returned to baseline by Day 7 in lean WT, lean IL-6 KO, and DIO IL-6 KO mice. In DIO WT mice, activated STAT-3 remained elevated up to Day 15 after AP.

Figure 5. Activation of STAT-3 and induction of MMP-7 in the pancreas of lean and DIO WT and IL-6 KO mice with AP.

Figure 5.

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Lean and DIO WT and IL-6 KO mice received two injections of vehicle or IL-12 + IL-18. The pancreas was obtained at the indicated time-points. (A) Quantification of Tyr705-pSTAT-3 in pancreatic homogenates of lean WT (black bars), lean IL-6 KO (gray bars), DIO WT (white bars), and DIO IL-6 KO (hatched bars) mice. Data are mean ± sem of four to seven mice/group. a, P < 0.01 versus respective control group; b, P < 0.01 versus lean groups at the same time-point; c, P < 0.05 versus DIO WT at the same time-point; d, P < 0.05 versus lean WT at the same time-point. (B) Representative samples of immunohistochemistry for Tyr705-pSTAT-3. (C) Representative samples of immunohistochemistry for MMP-7. Original magnification, 20×/0.5; original bars, 100 μm.

To confirm the finding that the AP-induced increase in activated STAT-3 was dependent on IL-6, we used ob/ob mice. Significantly higher levels of activated STAT-3 were observed in the pancreas of ob/ob compared with WT mice at Days 1 and 7 postinduction of AP (Supplemental Fig. 4A). Neutralization of IL-6 reduced levels of activated STAT-3 in ob/ob mice at Day 1 but not Day 7.

Immunohistochemistry for pSTAT-3 confirmed results of the PathScan assay. Nuclear staining for pSTAT-3 occurred in acinar cells and infiltrating inflammatory cells at Day 1 after induction of AP in all groups (Fig. 5B). However, more pronounced staining was present in WT versus IL-6 KO mice. At Day 1, some ductal cells were also pSTAT-3-positive in lean and DIO WT mice (Supplemental Fig. 4B), whereas endocrine cells (Supplemental Fig. 4C) and the ileum (Supplemental Fig. 4D) remained pSTAT-3-negative. At Day 7, a subset of infiltrating cells was still positive for pSTAT-3 in the pancreas of lean WT mice (Supplemental Fig. 4E), whereas acinar cells were no longer positive in this group (Fig. 5B). In contrast, in DIO WT mice, a strong immunoreactivity for pSTAT-3 was still present at Day 7 in acinar and inflammatory cells (Fig. 5B and Supplemental Fig. 4F). By Day 15, pSTAT-3 immunoreactivity subsided in each group except DIO WT mice. Thus, the prolonged increase in immunoreactive pSTAT-3 in the pancreas of DIO mice injected with IL-12 + IL-18 depends on IL-6.

Induction of MMP-7, OPN, and TIMP-1

Up-regulation of MMP-7 in pancreatic ducts has been causally linked to development of PC [12, 13]. A significant induction of MMP-7 was observed in pancreatic ducts of lean and DIO WT mice at Day 1, with lower induction in IL-6 KO mice (Fig. 5C). At Day 7, the number of immunoreactive MMP-7 cells sharply increased in DIO WT mice, whereas tissue from each other group was negative or only faintly positive. At Day 15, MMP-7-positive ductal cells were still present in the pancreas of DIO WT mice and were absent in each other group.

Elevated levels of OPN and TIMP-1 are present in patients with PC and chronic pancreatitis, a condition that favors development of PC [14, 36–39]. Administration of IL-12 + IL-18 significantly increased serum OPN levels in lean and DIO WT mice at Day 1 in an IL-6-dependent manner and with a more pronounced increase in obese animals (Fig. 6A). By Day 7, DIO WT mice were the only group in which serum OPN levels were still elevated significantly. Hepatic OPN expression paralleled the results of circulating levels and thus, was increased in an IL-6-dependent manner (Fig. 6B). In contrast, a comparable induction of OPN was observed in pancreatic homogenates from each group at Day 1 (Fig. 6C), with levels remaining significantly elevated in DIO mice, irrespective of genotype, until Day 7.

Figure 6. Levels of OPN and TIMP-1 in lean and DIO WT and IL-6 KO mice with AP.

Figure 6.

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Lean and DIO WT and IL-6 KO mice received two injections of vehicle or IL-12 + IL-18. Pancreas, liver, and blood were obtained at the indicated time-points. Levels of OPN and TIMP-1 in serum (A and D), mRNA expression in liver (B and E), and protein levels in homogenates of pancreas (C and F) of lean WT (black bars), lean IL-6 KO (gray bars), DIO WT (white bars), and DIO IL-6 KO (hatched bars) mice. Data are mean ± sem of four to seven mice/group. a, P < 0.01 versus respective control group; b, P < 0.01 versus lean groups at the same time-point; c, P < 0.05 versus DIO WT at the same time-point; d, P < 0.05 versus lean WT at the same time-point.

Similar to OPN, hepatic TIMP-1 expression was dependent on IL-6 and enhanced by obesity (Fig. 6E), whereas induction of TIMP-1 in the pancreas was IL-6-independent, with a comparable induction in each group at Day 1 and both DIO groups having significantly higher TIMP-1 levels at Day 7 relative to lean mice (Fig. 6F). At variance with OPN, circulating levels of TIMP-1 may have resulted from a combination of hepatic and pancreatic sources, with significantly higher serum TIMP-1 levels in DIO groups at Days 1 and 7 but no significant differences between DIO WT and IL-6 KO mice until Day 15 (Fig. 6D).

DISCUSSION

As we demonstrated previously, the injection of IL-12 + IL-18 induces a typical robust pancreatitis with elevated amylase levels and histological damage in lean and obese animals [22, 23]. In lean mice, AP resolves rapidly, with return to normal histology by Day 7, whereas more severe and prolonged disease, accompanied by elevated levels of IL-6 and acute-phase proteins, is observed in obese mice [22, 23], thus providing an excellent model to study the effect of obesity in AP. The novel finding of the current report is defining the role of the elevated production of the pleiotropic cytokine IL-6 in obese mice with AP. We demonstrate that high IL-6 in obesity prolongs inflammation and alters the resolution of pancreatic damage observed in lean mice through sustained activation of STAT-3 in the pancreas, delayed clearance of the neutrophil infiltrate, and up-regulation of CXC and CC chemokines, MMP-7, SAA, OPN, and TIMP-1. However, IL-6 does not mediate the heightened acute disease severity and lethality of obese mice with AP induced by IL-12 + IL-18. Comparison of DIO WT and IL-6 KO mice also points to a dissociation between resolution of tissue damage and kinetics of neutrophil clearance and STAT-3 activation, which requires further investigation.

Moreover, our data confirm that obesity, rather than leptin deficiency, mediates increased disease severity in this model of AP, as increased lethality and delayed resolution were observed in DIO and ob/ob mice [22, 23, 31]. Furthermore, age does not appear to play a major modulatory role, as comparable disease severity was observed in 17-week-old DIO mice and 7-week-old ob/ob mice. However, evaluation of aged mice is necessary to identify the contribution of senescense in determining AP outcomes, an issue of potential clinical relevance [40].

Evidence indicates that IL-6 is among the most accurate predictors of disease severity in AP [7]. The present report demonstrates that adipose tissue is an important contributor to the sustained production of IL-6 in obese animals with AP. In particular, areas of VAT containing necrotic and saponified tissue, which are only observed in obese animals, express high levels of IL-6, up to 15 days postinduction of AP. These data are in agreement with results obtained in the rat model of taurocholate-induced AP, in which necrotic VAT produces high levels of TNF-α and other mediators [41]. Although we did not directly address the specific source of IL-6 in VAT, it is likely that adipose tissue at the border between necrotic and non-necrotic areas has the highest inflammatory potential, as demonstrated in the taurocholate model [41]. It is also worth noting that VAT necrosis and sustained inflammation persisted up to 15 days in DIO mice with AP, thus mirroring the clinical situation and underlying the relevance of IL-12 + IL-18-induced AP to model human disease.

Thus, the presence of an expanded VAT not only contributes to the baseline chronic inflammation of obesity [42] but also actively participates in the systemic inflammatory response to pancreatic damage. However, whether sustained production of IL-6 in severe AP contributes to disease pathogenesis or represents a frustrated attempt at blunting inflammation remained unclear. Experimental results obtained using the cerulein model in lean mice are controversial [24, 25], whereas the interaction of obesity and IL-6 in AP severity had not been explored. The present results indicate that IL-6 does not mediate the increased disease severity and lethality of obese mice in the model of IL-12 + IL-18-induced AP, although it modulates induction of the acute-phase protein SAA.

Obesity is associated with delayed resolution of the neutrophil infiltrate in response to peritoneal inflammation [43, 44], although less clear-cut effects have been observed in models of pulmonary inflammation [45, 46]. Our data demonstrate that obesity leads to delayed resolution of the pancreatic inflammatory infiltrate in response to IL-12 + IL-18. Elevated intrapancreatic levels of CXCL1, CXCL2, and CCL2 likely contribute to the prolonged influx of inflammatory cells in the pancreas of DIO mice, but factors such as reduced clearance of apoptotic cells by macrophages of obese animals might also participate [47]. Our results also demonstrate that IL-6 mediates the sustained neutrophil infiltrate and chemokine production in the pancreas of DIO mice with AP and that acute neutralization of IL-6 with antibodies suppresses intrapancreatic chemokine levels. This is in agreement with reports demonstrating reduced neutrophil infiltrate and suppressed production of CXCL1 and CXCL2 in IL-6 KO mice under various experimental conditions [48–50]. Stimulation with IL-6 leads to induction of CCL2 through activation of STAT-3 [51]. Although classically considered a mononuclear cell-attracting chemokine, CCL2 is involved in neutrophil recruitment and delayed apoptosis [15, 51, 52]. Thus, up-regulation of CCL2 may also contribute to the prolonged neutrophil infiltrate observed in DIO mice. In addition to mediating recruitment of inflammatory cells, chemokines, particularly the CXC family, are involved in the pathogenesis of experimental PC by modulating tumor-stromal interactions [19]. Therefore, sustained production of CXC chemokines in the pancreas of obese mice may favor development of a tumorigenic environment, at least in part through IL-6. Trans-signaling events mediated by sIL-6R have been implicated in several of the effects of chronically elevated IL-6 levels, including modulation of the neutrophil-monocyte transition [18]. Our data demonstrate that obesity did not significantly affect serum sIL-6R levels, whereas a significant reduction in circulating sIL-6R was observed in DIO mice with AP, perhaps as an attempt to limit excessive IL-6 trans-signaling. These data are in agreement with reports showing lack of effect of obesity but reduced sIL-6R levels in women with polycystic ovary syndrome, a condition associated with elevated serum IL-6 [53].

Chronic activation of STAT-3 has been causally linked to tumorigenesis in several organs, including the pancreas [8]. Here, we report that administration of IL-12 + IL-18 leads to activation of STAT-3 in acinar and infiltrating cells in an IL-6-dependent manner and that obesity leads to prolonged activation of this signaling pathway. Pancreatic activation of STAT-3 plays a critical role at each stage of tumorigenesis in the Kras model of PC [10–12]. However, our data indicate that pancreatic inflammation superimposed on obesity leads to prolonged activation of STAT-3 even in the absence of Kras mutations, thus possibly generating an environment conducive to pancreatic tumorigenesis if sustained over long periods. This concept is in agreement with the role of obesity as a tumor promoter in hepatocellular carcinoma [54]. Activation of STAT-3 is accompanied by induction of MMP-7 in ductal cells, again in an IL-6-dependent manner [12]. Up-regulation of MMP-7 is a STAT-3-mediated response, which regulates acinar-to-ductal metaplasia, tumor size, and metastasis in mouse models and correlates with metastatic disease and survival in humans with PC [12, 13].

In addition to up-regulation of MMP-7, we demonstrate the presence of elevated levels of SAA, OPN, and TIMP-1 in DIO mice with AP. Circulating levels of these factors are elevated in PC, although specificity remains a challenge [14, 36]. Despite the unclear involvement of these mediators in the pathogenesis of PC, their concomitant upregulation, together with pancreatic activation of STAT-3 and induction of MMP-7, supports the argument that obesity favors development of a tumorigenic environment after a pancreatic insult. Induction of SAA by IL-12 + IL-18 is almost exclusively IL-6-dependent, in agreement with results obtained in mouse models of obesity and sterile inflammation but at variance with induction by microbial stimuli [17, 54, 55], suggesting a closer relationship of the IL-12 + IL-18 model with models of tissue damage rather than models of sepsis or infection. Regulation of hepatic expression of OPN and TIMP-1 parallels that of SAA, in that it is enhanced by obesity in an IL-6-dependent manner. In contrast, local production of OPN and TIMP-1 in the pancreas is IL-6-independent, suggesting differential regulation that deserves further investigation.

In conclusion, obesity exacerbates disease severity in response to administration of IL-12 + IL-18 in an IL-6-independent manner, despite elevated and sustained production of IL-6 in DIO mice from pancreas and VAT. However, in obese mice IL-6 is a major factor in prolonging inflammation and altering the resolution from pancreatic damage, possibly favoring development of a microenvironment conducive to tumorigenesis. However, caution should be used when intepreting these results, as a limitation of our study is the use of a model of acute rather than chronic pancreatitis, which would have had more direct relevance to the pathogenesis of PC.

Supplementary Material

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ACKNOWLEDGMENTS

This study was supported by NIH grants DK083328 to G.F. and DK080787-03S3 to E.F.G.

Footnotes

ALT
alanine aminotransferase
AP
acute pancreatitis
DIO
diet-induced obesity
KO
knockout
MMP-7
matrix metalloproteinase-7
OPN
osteopontin
p
phospho
PC
pancreatic cancer
s
soluble
SAA
serum amyloid A
VAT
visceral adipose tissue

AUTHORSHIP

M.P., D.H.R., K.J.C., and A.R.H. performed and intepreted experiments and helped draft the manuscript. R.J.C. and R.C. evaluated histological samples and helped draft the manuscript. E.F.G. helped draft the manuscript and intepret results. G.F. planned and directed the studies performed and intepreted experiments, wrote the manuscript, and provided funding through NIH grants.

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