Transgenic Expression of PRSS1R122H Sensitizes Mice to Pancreatitis

Abstract

Background & aims: Mutations in the trypsinogen gene (PRSS1) cause human hereditary pancreatitis. However, it is not clear how mutant forms of PRSS1 contribute to disease development. We studied the effects of expressing mutant forms of human PRSS1 in mice.

Methods: We expressed forms of PRSS1 with and without the mutation encoding R122H (PRSS1^R122H) specifically in pancreatic acinar cells under control of a full-length pancreatic elastase gene promoter. Mice that did not express these transgenes were used as controls. Mice were given injections of caerulein to induce acute pancreatitis or injections of lipopolysaccharide to induce chronic pancreatitis. Other groups of mice were fed ethanol or placed on a high-fat diet to induce pancreatitis. Pancreata were collected and analyzed by histology, immunoblots, real-time polymerase chain reaction, and immunohistochemistry. Trypsin enzymatic activity and chymotrypsin enzymatic activity were measured in pancreatic homogenates. Blood was collected and serum amylase activity was measured.

Results: Pancreata from mice expressing transgenes encoding PRSS1 or PRSS1^R122H had focal areas of inflammation; these lesions were more prominent in mice that express PRSS1^R122H. Pancreata from mice that express PRSS1 or PRSS1^R122H had increased levels of heat shock protein 70 and nuclear factor (erythroid-derived 2)-like 2, and reduced levels of chymotrypsin C compared with control mice. Increased expression of PRSS1 or PRSS1^R122H increased focal damage in pancreatic tissues and increased the severity of acute pancreatitis after caerulein injection. Administration of lipopolysaccharide exacerbated inflammation in mice that express PRSS1^R122H compared to mice that express PRSS1 or control mice. Mice that express PRSS1^R122H developed more severe pancreatitis after ethanol feeding or a high-fat diet than mice that express PRSS1 or control mice. Pancreata from mice that express PRSS1^R122H had more DNA damage, apoptosis, and collagen deposition and increased trypsin activity and infiltration by inflammatory cells than mice that express PRSS1 or control mice.

Conclusions: Expression of a transgene encoding PRSS1^R122H in mice promoted inflammation and increased the severity of pancreatitis compared with mice that express PRSS1 or control mice. These mice might be used as a model for human hereditary pancreatitis and can be studied to determine mechanisms of induction of pancreatitis by lipopolysaccharide, ethanol, or a high-fat diet.

Keywords: Animal Model; Digestive Enzyme; Endotoxin; Immune Cells.

Fig. 1.. Human cationic trypsinogen was conditionally expressed in transgenic mice.

A. Human cationic trypsinogen (PRSS1) and its R122H mutant form were tagged with HA and cloned behind a loxp-GFP loxp cassette for conditional expression upon Cre-mediated recombination. B. This cassette was targeted between the translation stop and 3-UTR of the mouse elastase gene in a bacterial artificial chromosome by recombineering and transgenic mice were developed by pronuclear injection. C. The floxed trypsinogen transgenic mice were crossed with BAC Elastase-CreERT(BAC) mice and induced with tamoxifen (100mg/kg body weight) for 5 days. Expression of PRSS1 trypsinogen was detected by Western blot using an anti-PRSS1 antibody. A lower band, which may be a cross-reacting mouse PRSS, was also noted. D. Immunohistochemical images showed efficient expression of the HA-tagged human trypsinogens.

Fig. 2.. Focal inflammation and stress responses occurred in mice expressing mutant R122H trypsinogen.

A. Despite no obvious spontaneous pancreatitis, small focal areas of immune cell infiltrations were more often found in the pancreas of mice expressing mutant R122H trypsinogen than in wild-type trypsinogen (PRSS1) and control mice (BAC). Representative immunohistochemical images for CD45 positive cells in control mice (BAC) and R122H and PRSS1 mice. B. Quantification under light microscope of CD45 positive cells for control (BAC), R122H and PRSS1 mice. C-E. Relative mRNA expression of nuclear factor (erythroid-derived 2)-like 2 (NRF2) (C), heat shock protein 70 (HSP70) (D) and CTRC (E) in R122H, PRSS1 and control mice (BAC) was measured by real-time RT-PCR. (*, p<0.05; **, p<0.01; ns, p>0.05).

Fig. 3.. High-dose of caerulein increased the severity of acute pancreatitis in transgenic trypsinogen expressing mice.

A. Representative H&E images showed more severity damages in trypsinogen transgenic (R122H and PRSS1) mice compared to control (BAC) mice after 12 repeated caerulein injections. Magnification 10x, scale bar 200μm. Immunohistochemistry indicated there were more cleaved caspase 3 positive cells in trypsinogens transgenic mice. B. ER stress-related molecules were detected by Western blot. C. Serum amylase levels were higher in trypsinogens transgenic mice after treatment with caerulein (*, p<0.05; **, p<0.01). D. Semiquantitative histological score evaluation including edema, inflammatory cell infiltration, and cell damage indicated elevated damage in trypsinogen transgenic mice (*, p<0.05). E. After caerulein treatment for 1 hour, the level of trypsin activity in PRSS1 and R122H mice demonstrated a significant increase compared to control (BAC) mice. Trypsin activity in R122H mice was significantly higher than that of PRSS1 mice (*, p<0.05).

Fig. 4.. Human mutant PRSS1 ^R122H transgenic expression caused extensive chronic inflammatory changes after LPS stimulation.

A. The representative histological images of pancreas of R122H, PRSS1, and BAC mice examined by H&E staining. The treatment with LPS led to development of prominent chronic inflammatory changes in R122H mice. In contrast, smaller chronic lesions were observed in PRSS1 mice and only small focal changes in BAC controls. The representative immunohistochemical images of pancreas indicating an increased infiltration of leukocytes (CD45 positive inflammatory cells), macrophages (F4/80 positive cells), and phosphorylated form of histone 2A (γH2AX) (reflecting DNA damage) that were prominent in R122H mice treated with LPS. B-C. Quantification under light microscope (40x) for control (BAC), R122H and PRSS1 mice treated with LPS of CD45 positive cells, F4/80 positive cells(B), and γH2AX positive cells(C) (**, p<0.01; ***, p<0.001; ns, p>0.05). Western blot of Bip, which is induced by ER stress, indicated that LPS treatment increased ER stress in R122H compared to PRSS1 mice (D).

Fig. 5.. Feeding with ethanol or high fat diet induced chronic inflammatory changes in pancreas of transgenic trypsinogens expressing mice.

A. The scheme of treatment. The trypsinogen expressing and control mice were fed with a Liber – De-Carli Diet for 4 weeks. B. The representative H&E images of pancreas of control (BAC) and trypsinogens (R122H and PRSS1) expressing mice fed with ethanol diet for 4 weeks. Corresponding representative immunohistochemical images showing focally localized DNA damage (nuclear staining for γH2AX) (magnification: 40 x, error bar: 100μm). C. Quantification of γH2AX positive cells under light microscope (40x) for control (BAC), R122H and PRSS1 mice fed with ethanol diet. Trypsin activity in control (BAC), R122H and PRSS1 mice (D) (*, p<0.05; **, p<0.01; ns, p>0.05).

Fig. 6.

A. The scheme of treatment. The trypsinogen expressing and control mice were fed with a high fat diet (60% fat) for 4 weeks. B. The representative H&E images of pancreas of control (BAC) and trypsinogens (R122H and PRSS1) expressing mice fed with high fat diet for 4 weeks. Corresponding representative immunohistochemical images showing focally localized DNA damage (nuclear staining for γH2AX). C-D. Quantification of yH2AX positive cells (C) and cleaved caspase 3 (D) under light microscope (40x) for control (BAC), R122H and PRSS1 mice fed with high fat diet (*, p<0.05; **, p<0.01; ns, p>0.05).