Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer

Highlights

• We found some bacteria in pancreatic juice and tissue of pancreatic cancer.

• We identified E. faecalis infection in pancreas with chronic pancreatitis.

• Serum antibody titers against E. faecalis are increased in pancreatic diseases.

• Addition of E. faecalis antigens increased expression of inflammatory cytokine.

• E. faecalis might be a novel target for preventing chronic pancreatic diseases.

Abstract

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue–associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the last malignancies still resistant to treatment with available cancer therapeutics. This disease reportedly causes approximately 227,000 deaths per year worldwide [1], and the prognosis of PDAC is extremely poor compared with other carcinomas [2], likely due to impediments to early diagnosis and the lack of available methods for screening groups at high risk for developing PDAC. Family history is a major risk factor for developing PDAC, suggesting that genetic factors are involved in the pathogenesis of this disease. No clinicopathologic features that distinguish young patients (<40 years old) from older patients with PDAC have been identified [3], and abnormalities in PDAC-associated genes reportedly occur long before the disease can be diagnosed [4]. These data suggest that genetic disorders conferring a risk for PDAC might be common in the germline and that acquired mutations leading to PDAC develop slowly from a very young age. Unless detected while the tumor is < 1 cm in size, PDAC is difficult to cure completely [5].

We recently reported that most PDAC patients have subclinical chronic pancreatitis (CP) in the pancreatic tissue surrounding tumors [6]. In addition, many people with no overt pancreatic disease also have subclinical CP, as determined from post-mortem pathologic analyses. Although whether subclinical CP is associated with PDAC remains unclear, a mouse model of pancreatic cancer (PC) showed a high incidence of PDAC in animals harboring mutations that promote induction of CP [7]. The question remains, however, as to what factors lead to development of subclinical CP in humans. Although aging is likely important in development of subclinical CP, as this is a known risk factor for tissue fibrosis, we hypothesized that multiple etiologic factors are associated with subclinical CP development.

Many cancers have been linked with chronic inflammation arising from viral/bacterial infection [[8], [9], [10], [11], [12]]. It was previously thought that bacteria were incapable of surviving in the stomach due to its low pH. However, this assumption was disproven, and a clear causal link between Helicobacter pylori infection and resulting inflammation leading to stomach cancer was established [10]. Similarly, although a variety of bacteria survive in bile and the duodenum, it is thought that most bacteria cannot survive in pancreatic juice, which is highly alkaline and contains numerous proteases. However, at least one report has suggested that bacterial lipoteichoic acid (LTA) and/or lipopolysaccharide (LPS) are involved in development of CP [13].

In this study, we investigated the presence of bacteria in pancreatic juice and bile obtained from patients with PDAC. We found that Enterobacter and Enterococcus spp. were the major organisms present in bile samples. Antibodies against Enterococcus faecalis capsular polysaccharide (CPS) were increased in serum from patients with CP and PC, as compared with normal subjects and colon cancer patients. The antibodies were also elevated in serum of mice with caerulein-induced CP. Enterococcus faecalis was also detected at a greater frequency in pancreatic tissue from CP and PC patients and in CP mice, as compared with controls, and both Enterococcus LTA and heat-killed E. faecalis induced expression of pro-fibrotic cytokines by BxPc3 PC cells. These results indicate that E. faecalis infection could be involved in the progression of CP and subsequent development of PC.