Sentinel acute pancreatitis event (SAPE) increases severity of subsequent episodes in mice

INTRODUCTION

Pancreatitis is the inflammatory disorder of the pancreas, which often forms a disease continuum starting with an acute pancreatitis (AP) attack followed by recurrent acute pancreatitis (RAP) episodes and eventual progression to chronic pancreatitis (CP) [1]. Progression to RAP and ultimately to CP is associated with environmental and/or genetic risk factors. The Sentinel Acute Pancreatitis Event (SAPE) hypothesis has been proposed to explain how a single episode of AP could lead to CP [2-5]. This model posits that in the setting of susceptibility factors, an initial “sentinel” episode of AP sensitizes the pancreas to recurring/continual inflammation and fibrosis through activation of the immune system. The initial AP attack is called the sentinel event because it signals the beginning of CP and alerts the physician to initiate therapy for the prevention of progression. To test the SAPE concept experimentally, we set out to investigate whether an episode of experimental AP in mice would change the severity of later attacks. We also examined whether sensitization of the pancreas by the sentinel AP event is mediated by retention of inflammatory cells or by altered protease activation.

METHODS

Experimental procedures are described in the Supplementary Material.

RESULTS

Modeling RAP in mice.

To investigate whether a sentinel AP attack impacts severity of subsequent episodes, we induced 1, 2 or 3 attacks of AP in C57BL/6N mice with 10 hourly injections of cerulein. After each episode, mice were allowed to recover for 7 days. Mice were sacrificed either 1 h after the last cerulein injection or after the 7 day recovery period. Overall, 5 groups of mice were analyzed: mice with 1 episode of pancreatitis, mice that recovered after the first episode, mice with 2 episodes of pancreatitis, mice that recovered after the second episode and mice with 3 episodes of pancreatitis (Figure 1A).

Figure 1.

Recurrent acute pancreatitis in C57BL/6N mice. A, Experimental protocol of modeling recurrent acute pancreatitis. Solid triangles indicate an episode of acute pancreatitis elicited by 10 hourly injections of cerulein. Vertical lines indicate time of euthanasia. B, Hematoxylin-eosin stained histological sections of the pancreas from mice during the 1st, 2nd and 3rd pancreatitis episodes and from mice that recovered from the 1st and 2nd episodes. Scale bars correspond to 100 μm. C, Pancreas weight, D, plasma amylase activity and E, pancreas myeloperoxidase (MPO) content in mice during the 1st, 2nd and 3rd pancreatitis episodes and from mice that recovered from the 1st and 2nd episodes. For clarity, data from the saline-treated control groups of mice were pooled and graphed as a single group. F, Immunohistochemistry staining for the F4/80 macrophage marker in pancreas sections from mice with 1 or 2 episodes of acute pancreatitis and mice that recovered from an acute episode. Scale bars correspond to 50 μm. G, Quantitative analysis of F4/80 positive cells per visual field. Individual values with means ± SD are shown. The difference of means between two groups was analyzed by two-tailed unpaired t-test.

Pancreas histology.

Repeated injections of cerulein induced edematous AP with inflammatory cell infiltration and scattered necrosis (Figure 1B). Induction of a second episode of AP resulted in significantly increased edema (Supplementary Figure 1A) and inflammatory cells (Supplementary Figure 1B) relative to the initial attack, while acinar cell necrosis remained scarce (Supplementary Figure 1C). Importantly, complete histological recovery was achieved in 7 days after the first and second episodes, although the number of infiltrating inflammatory cells remained slightly elevated. No difference in severity was apparent between the second and third episodes.

Pancreas edema.

We assessed pancreas edema by measuring the pancreas weight and pancreatic water content. Cerulein injections induced a significant increase in pancreas weight (Figure 1C, Supplementary Figure 1D) and pancreatic water content (Supplementary Figure 1E), relative to saline treatment. In agreement with the histology data, the increase in pancreas weight and pancreatic water content during the second and third episodes of cerulein-induced pancreatitis were higher than those in the first episode. No significant difference in these parameters was evident when the second and third episodes were compared. Following the first and second AP episodes, the pancreas weight returned to normal levels within 7 days.

Plasma amylase activity.

Repeated cerulein injections induced a significant increase in plasma amylase activity relative to saline-treated mice (Figure 1D). Amylase activities were significantly higher in mice during the second and third attacks of AP relative to the first episode. Amylase increases were comparable during the second and third attacks. Within the 7-day recovery periods after the first and second attacks, amylase activities receded to normal levels.

Inflammatory cells.

To evaluate neutrophil granulocyte infiltration, we measured MPO levels in the pancreas (Figure 1E). Relative to mice given saline injections, cerulein-treated mice exhibited a marked increase in pancreatic MPO content during their first episode of pancreatitis, which was further elevated during the second and third attacks. MPO content returned to baseline levels within 7 days after the first and second episodes, indicating complete cessation of the acute inflammatory response. No change was observed between the second and third episodes.

We noted that after recovering from an AP episode, the pancreas still contained slightly elevated number of inflammatory cells (Supplementary Figure 1B). Since MPO measurement indicated resolution of neutrophils, we hypothesized that the lingering cells are macrophages. Indeed, pancreas sections from cerulein-treated mice showed positive staining for the F4/80 macrophage marker mostly in the interlobular space but also inside the lobules and around the acini during the second AP episode (Figure 1F). Importantly, mice that recovered from the first attack of pancreatitis also showed positivity for F4/80, indicating that macrophages remained resident in the pancreas. Quantitative analysis indicated an average of 65 macrophages per visual field during their first attack, 48 after recovery, and 200 during the second episode (Figure 1G).

Intrapancreatic trypsin and chymotrypsin activity.

A sentinel episode of AP might sensitize the pancreas to intrapancreatic protease activation and result in more severe subsequent episodes. In support of this theory, we found significant increases in the levels of the protease zymogens trypsinogen (1.2-fold, Supplementary Figure 1F) and chymotrypsinogen (1.4-fold, Supplementary Figure 1G) in the pancreas of mice that recovered from pancreatitis. To test whether the increased zymogen content would cause higher intrapancreatic protease activation, we compared cerulein-induced trypsin (Supplementary Figure 1H) and chymotrypsin (Supplementary Figure 1I) activities in the pancreas of mice with no previous AP to those that recovered from an AP episode. Interestingly, despite the elevated protease zymogen content, intrapancreatic trypsin and chymotrypsin activation was not increased significantly in mice that recovered from an episode of AP relative to mice with no previous pancreatitis.

DISCUSSION

Here, we provided direct experimental support for the SAPE hypothesis by showing that the initial episode of AP increases the severity of subsequent attacks. In our approach, we were inspired by prior rodent studies on gene expression during recovery after pancreatitis [6] and disease severity in recurrent episodes [7,8]. In addition to demonstrating proof of concept, we also identified persistently infiltrating macrophages as a plausible mechanism for the enhanced injury and more severe inflammatory response during successive episodes. The findings argue that novel strategies to arrest pancreatitis progression should target prevention and/or clearing of macrophage infiltration.