Pancreatitis pathogenesis and its connection to gut microbiome and diabetes

Pancreatitis pathogenesis and its connection to gut microbiome and diabetes

In India, Pancreatitis, which refers to inflammation of the pancreas, may primarily be acute and chronic, while autoimmune (a form of chronic pancreatitis) constitutes a very small proportion. Globally, the most common risk factors ascribed to acute pancreatitis are alcohol, smoking and gallstones. Acute pancreatitis is characterised by inflammation of the pancreas that could be associated with pancreatic injury (necrosis), systemic inflammatory response and multiorgan failure resulting in death. A substantial proportion of patients with pancreatic necrosis can develop infection of necrotic pancreas, especially from the second week of disease onwards. Fortunately, up to 75% patients with acute pancreatitis develop mild disease that resolves without the need for much intervention. However, there are no specific curative modalities for the remaining 25% who develop moderate to severe disease; and treatment is restricted predominantly to organ support and treatment of complications. The absence of specific treatment stems from a relative lack of understanding of the early pathogenesis of the disease in humans.

Challenging the role of proteases in the development of pancreatic injury and inflammation

Several elaborate mechanistic studies had been conducted previously in experimental models based on which treatment modalities such as the use of protease inhibitors (gabaxate, nafamostat) have been shown to be beneficial in experimental models, but results in clinical trials were heterogeneous. This led to an important question- is protease activation really required for acute pancreatitis? The concept of pancreatic intra-acinar activation of trypsinogen to trypsin had been held as the central dogma in the pathogenesis of acute pancreatitis. We challenged this century old question couple of years back, and conducted experiments where we induced acute pancreatitis in wild-type and trypsin knock-out mice. We observed that there was pancreatic injury even in the absence of trypsin, and there was parallel activation of the inflammatory cytokine, NF-kB (Gastroenterology, 2011). These data clearly implied that the protease, trypsin, though important, is not mandatory for development of pancreatic injury and inflammation.

In order to delve further into the role of trypsin in the pathogenesis, we conducted experiments in Prof Ashok Saluja’s lab, University of Minnesota, USA, where we could demonstrate a novel paradigm of pancreatic acinar cell apoptosis or necrosis. Our data clearly showed that it is the concentration of the hydrolytic enzyme, cathepsin B in the cytosol of the acinar cells that determines whether the acinar cell will undergo apoptosis or necrosis. An important early event that has been consistently shown to occur in acute pancreatitis is co-localization of lysosome and zymogen containing organelles, which now has been proven to be impaired autophagy. In our studies, we observed that the role of trypsin is to make the membranes of autophagic vacuole permeable, through which cathepsin B leaks out into the cytosol and lead to acinar cell apoptosis or necrosis (Gastroenterology 2016).

However, these events occur very early (in a matter of minutes) in the pathogenesis of acute pancreatitis. In the clinical context, by the time a patient visits the hospital with severe abdominal pain, these events have already passed; and what the patient manifests is the result of the early events, i.e. a systemic inflammatory response. Therefore, in order to treat human pancreatic inflammation one needs to study human pancreatitis. This notion led our group to embark into experimental pancreatitis using human pancreatic tissue with bile acid and alcohol. Our studies revealed that autophagy occur in human biliary acute pancreatitis and the acinar cell is the earliest source of cytokine release. These cytokines then activate the peripheral blood mononuclear cells (PBMCs) that flow through the pancreatic circulation. The cytokines liberated by the activated PBMCs lead to a systemic inflammatory response syndrome and also cause a second wave of injury to the pancreatic acinar cells. We believe it is the second wave of cytokines (from the PMBCs) that determine the severity of the disease in patients (Submitted and under revision).

Identifying inflammatory biomarkers to predict infection of necrotic tissue in patients with acute pancreatitis

As patients with acute pancreatitis go into the second week, a proportion with necrosis develops infection in the necrotic tissue. The question here is why only a proportion of patients with necrosis develop infection? In sepsis literature there is an entity called Compensatory Anti-Inflammatory Response Syndrome (CARS), in which there is down regulation of important cell surface receptor, HLA-DR, involved in immune response, and up regulation of the anti-inflammatory cytokine IL-10, among other mediators. Earlier, studies had indicated that HLA-DR down regulation could be associated with infected pancreatic necrosis in humans. We tested this in patients with acute pancreatitis admitted within 72 hours of disease onset, where we divided patients with and without infected pancreatic necrosis and looked for IL-10 and HLA-DR expression during the first and second week of illness. We observed that irrespective of eventual development of infected pancreatic necrosis, the HLA-DR is down regulated in nearly 90% of patients during the first week. However, HLA-DR down regulation persisted till second week only in those patients who developed infected necrosis. The relative risk (95% CI) for developing infected necrosis in the patients who continued to have HLA DR down-regulation into the second week of the disease was 2.8 (0.9-8.8). Along with this, IL-10 increased during second week only in the patients who developed infected necrosis (manuscript in preparation). These data show that dynamic changes in these markers could be potential biomarkers that could predict development of infected necrosis. This needs further validation though. Currently we are evaluating in an experimental setting and also in patients with acute pancreatitis who present early in the disease if gut microbial dysbiosis could increase the susceptibility to infections and severity of the disease.

Diabetes in chronic pancreatitis is associated with infiltration of pancreatic islets with immune response regulating cells

Recurrent acute pancreatitis, especially those related to alcohol intake and those that are idiopathic (most of these are associated with genetic polymorphisms), progress to chronic pancreatitis. It was earlier shown that 50% of alcohol-related recurrent acute pancreatitis develops features of chronic pancreatitis in two years.  We had recently shown that recurrent acute pancreatitis in the presence of Claudin2 rs7057398 CC genotype was associated with seven fold higher risk of progressing to chronic pancreatitis (Journal of Gastroenterology and Hepatology, 2015). The pathological hallmark of chronic pancreatitis is pancreatic fibrosis, while clinically it is characterized by recurrent intractable pain, malnutrition resulting from Pancreatic Exocrine Insufficiency (PEI), and Type 3c diabetes that is distinct from Type 1 and 2 diabetes. In India, chronic pancreatitis and the related manifestations, including diabetes, mostly occur early in life, and we have recently shown that diabetes in chronic pancreatitis is associated with infiltration of immune response regulating cells (Th1 and Th17) into the pancreatic islets that is associated with secretion of the cytokine, interferon-g (Pancreas 2016).

Alteration of gut microbiota in chronic pancreatitis

Since there is fat maldigestion due to PEI in chronic pancreatitis, it is likely that there will be an alteration in the gut microbiota. We tested for this by metagenomic 16S rDNA V3-V4 region sequencing and observed that there was a significant reduction in the relative abundance of the species Fecalibacterium prausnitzii in patients with chronic pancreatitis who had diabetes compared to those who did not. This bacterium is known to maintain the gut barrier integrity, implying that its reduction could result in disrupted gut barrier. We also observed an increase in LPS synthetic pathways among the intestinal bacteria and a parallel increase in plasma endotoxin. This correlated positively with blood glucose and negatively with relative abundance of Fecalibacterium prasunitzii. Taken together, these findings led to the hypothesis that pancreatic exocrine insufficiency and fat malabsorption results in gut microbial imbalance (dysbiosis) in chronic pancreatitis, which results in gut barrier alteration and translocation of endotoxin into the circulation. The circulating endotoxin could then contribute to islet dysfunction, along with other possible mechanisms. It is known from earlier experimental studies that endotoxins could result in islet injury via TLR4. We also observed correlation of the relative abundance with several bacteria genera with nutritional parameters (Scientific Reports, 2017). The current attempt in the lab has been to elucidate the mechanistic insights of the above findings that would provide better understanding of the connection between pancreatitis and gut microbiome.