Premature trypsinogen activation

The central role of trypsin activation in the pathogenesis of AP has long been demonstrated, but the mechanisms by which trypsinogen gets activated are not entirely understood. The most substantial evidence supporting the importance of trypsin activation comes from the hereditary forms of pancreatitis. Mutations in the genes encoding cationic trypsinogen (PRSS1) determine an increased susceptibility to acute and chronic pancreatitis by making molecules more susceptible to activation. Similarly, the mutations in the gene encoding the serine protease inhibitor (SPINK1) cause an autosomal recessive susceptibility by secreting an inactive molecule (Refs Reference Dixit13, Reference Watanabe, Kudo and Strober14). Additional support for this hypothesis comes from studies with experimental models of pancreatitis. In a study that used knockout mice lacking trypsinogen isoform-7, there was no intra-acinar trypsinogen activation, but there was evidence of NF-κB activation, and systemic inflammation was not altered (Refs Reference Dixit13, Reference Dawra15). These findings concluded that trypsinogen activation could be an important factor in initiating acinar injury and inflammation cascade from AP but it is not mandatory for the appearance of the disease; other mechanisms determine cellular damage and inflammatory response (Refs Reference Dixit13, Reference Watanabe, Kudo and Strober14).

Colocalization of zymogens with lysosomes

Various pancreatic injuries like trauma, ductal obstruction or alcohol can determine the fusion between the lysosome and zymogen granules inside the acinar cell. This process is called colocalization, and in these vacuoles, cathepsin B, a lysosomal enzyme, determines the activation of trypsinogen to trypsin. After the enzymes are released from the vacuoles, trypsin causes autodigestion, but cathepsin B also plays an important role in cellular destruction. Experimental studies demonstrated that small amounts of cytosol cathepsin B initiate cell death by apoptosis. In contrast, large amounts shift the pathway towards necrosis, thus being an important molecule in the initial phases of AP (Refs Reference Dixit13, Reference Lee and Papachristou16–Reference Talukdar18).

Calcium signalling

Usually, intracellular calcium handling is an important mechanism for the function of the acinar cell. Calcium release from endoplasmic reticulum (ER) is associated with zymogen exocytosis in the apical pole and stimulates ATP production in the mitochondria. This increase in cytosolic levels of calcium is transient and, in physiological conditions, the levels rapidly decreased by two ATP-dependent calcium channels: the smooth-ER calcium channel, which moves back the calcium into the ER (SERCA- ssarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase), and the membrane calcium channel, which removes calcium from the cell (PMCA - plasma membrane Ca²⁺ ATPase) (Refs Reference Lee and Papachristou16, Reference Wen19). Alcohol and bile acids have been demonstrated to disrupt intracellular calcium homeostasis by increasing calcium levels from the ER store and, afterwards, entering extracellular calcium (Refs Reference Weber and Adler12, Reference Lee and Papachristou16, Reference Voronina20). Furthermore, other aetiologies have been associated with the calcium signalling mechanism, like medication (asparaginase) and post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis, making it one of the most important pathways for cellular injury (Refs Reference Pallagi21, Reference Peng22). The build-up of calcium levels in the acinar cell triggers several important mechanisms that eventually lead to cellular death: trypsinogen auto-activation, vacuole formation, cytoskeletal damage, NF-κB activation and maybe, most importantly, mitochondrial dysfunction. The calcium overload results in a loss of membrane potential to produce ATP, accentuating calcium accumulation by decreasing the function of ATP-dependent channels SERCA and PMCA (Refs Reference Weber and Adler12, Reference Lee and Papachristou16, Reference Raraty23–Reference Raraty25). All this evidence suggests that increased intracellular calcium has a central role in the initiating phases of AP. This observation led to the search for potential therapeutic targets that could prevent calcium overload, some of which had encouraged initial results (Ref. Reference Pallagi21).

Autophagy and endoplasmic reticulum stress

Autophagy is an essential cytoprotective process through which cytoplasmatic proteins and organelles are degraded and recycled. Targeted molecules are entrapped into autophagic vacuoles, which merge with lysosomes and are digested by proteolytic enzymes. In AP, autophagy is impaired, resulting in ER stress, premature trypsinogen activation and mitochondrial dysfunction (Refs Reference Watanabe, Kudo and Strober14, Reference Lee and Papachristou16).

ER stress means the accumulation of misfolded proteins inside the ER that happens when the normal function of the ER is overwhelmed and protein elimination is impaired. In AP, ER stress is determined by an increased demand for protein production and defects in recycling proteins (Refs Reference Lee and Papachristou16, Reference Biczo24, Reference Wu26).

Ductal cell dysfunction

The most investigated association between ductal cell involvement in AP is with the cystic fibrosis transmembrane regulator mutations that cause increased susceptibility to developing the disease by reduced secretion (Ref. Reference Lee and Papachristou16). Secretion of bicarbonate from the ductal cells is an essential mechanism for the neutralization of acidic content produced by acinar cells. It is regulated by pH-sensing mechanisms and membrane transporters (Refs Reference Grapin-Botton27, Reference Hegyi28). Recent studies regarding ductal cell function in AP revealed an increased secretion in the initial phase, followed by a reduction afterwards, which seems to be a protective mechanism intended to wash out the toxins (Ref. Reference Venglovecz, Rakonczay and Hegyi29). Another mechanism of ductal damage is increased pressure caused by gallstone obstruction or contrast injection during ERCP, which can activate the pathological calcium signalling pathway in the acinar cells. Other modifications are fluid stasis, which promotes premature enzyme activation, intraductal acidification and ductal cell exposure to bile acids (Ref. Reference Lee and Papachristou16).

NF-κB activation

NF-κB is a transcription factor that regulates several genes involved in immune and inflammatory response by increasing the production of cytokines and chemokines and the activation of innate immune cells and inflammatory lymphocyte T cells (Refs Reference Liu30, Reference Pai and Thomas31). The involvement of NF-κB in acute pancreatitis comes from experimental studies that demonstrated reduced severity of pancreatitis in a model with NF-κB deletion. The exact activation mechanisms are not fully understood but evidence suggests calcium signalling and the production of reactive oxygen species are the most important ones. NF-κB activation in AP determines cytokine synthesis and initiation of local inflammatory response through transcriptional induction of proinflammatory cytokines, chemokines and other inflammatory mediators like inflammasomes (Refs Reference Liu30, Reference Jakkampudi32).

MicroRNAs

MicroRNAs (miRNAs) are single-stranded nucleotides that belong to the non-coding RNA class. These nucleotides play an important role in regulating gene expression and, thus, are involved in cell growth, development and death (Refs Reference O’Brien33, Reference Yang34). The biogenesis of miRNAs is a complex process. Typically, they bind to a complementary sequence in the 3′-untranslated region of messenger RNA (mRNA), causing mRNA silencing or degradation. A single miRNA can regulate the expression of hundreds of genes (Ref. Reference Bartel35). Recently, there has been increasing evidence of the functional role of miRNAs in acinar cell injury, inflammatory response and distal organ involvement (Ref. Reference Yang34). The first evidence to support miRNAs in AP came from studies on animal models that revealed significant upregulation of miR-135a and miR-22 in the pancreatic tissue (Ref. Reference Qin36). Afterwards, many miRNAs, like miR-29a and miR-21-5p, seem to be upregulated in pancreatic acinar cells during AP and induce cellular death through apoptosis, necrosis or pyroptosis (Refs Reference Ma37, Reference Gao38). Furthermore, miRNAs participate in the immune cell regulation in the progression of AP by interacting and upregulating macrophages, mononuclear cells and even T lymphocytes (Refs Reference Yu, Odenthal and Fries39–Reference Wang41). miRNAs seem to be involved in AP-associated lung injury, miR-21 being upregulated in lung tissue during severe AP (Ref. Reference Li42). Due to this evidence and the good stability of these markers, miRNAs could be considered as possible predicting markers for severity stratification (Refs Reference Gao38, Reference Yang43).

Apoptosis and necrosis

The final result of all the above-described changes in the acinar cell is cellular death, which is produced either by apoptosis, necrosis or pyroptosis. While apoptosis is a form of programmed cellular death mediated by caspases, necrosis is a process of self-destruction caused by internal or external factors (Ref. Reference Kang44). In AP, necrosis seems to be the main type of pancreatic acinar cell death and the release of damage-associated molecular pattern molecules (DAMPs) from necrosis is implicated in the inflammatory response. The mechanism leading to one or another form of cellular death is still unknown but may be a crucial factor for disease progression (Refs Reference Raraty23, Reference Kang44).

Pyroptosis

Pyroptosis is a form of cellular death that involves the death of immune cells mediated by inflammasomes secondary to an immune stimulus (Ref. Reference Kang44). This type of cellular death has some features similar to apoptosis and necrosis, like DNA fragmentation and cytoplasmic swelling. Unlike apoptosis, pyroptosis has a proinflammatory effect due to the rapid plasma membrane permeabilization and early membrane rupture, as well as the secretion of proinflammatory cytokines (Refs Reference Kang44, Reference Lamkanfi and Dixit45). Pyroptosis progression is a caspase-dependent process. Both the canonical pathway, which involves caspase-1 activation, as well as the noncanonical pathway, involving caspase-4, -5 and -11, can initiate pyroptosis (Ref. Reference Li46). There is increasing evidence that shows the importance of the inflammasomes and, subsequently, pyroptosis in the progression of inflammation in AP. Therefore, pyroptosis-related molecules could be an independent prognostic marker for AP severity but pyroptosis could also provide a therapeutic target for severe forms in the future (Ref. Reference Al Mamun47).

The pathogenetic modifications are the same after initiating the inflammatory process, regardless of the aetiology. Nevertheless, the trigger may differ between the different aetiologies. The main aetiologies and the triggering mechanisms are summarized in Table 1.

Table 1.

The main mechanisms for the initiation of AP associated with the most important causes

Note: AP, acute pancreatitis; CFTR, cystic fibrosis transmembrane regulator; ERCP, endoscopic retrograde cholangiopancreatography; NF-κB, nuclear factor-κB.

Neutrophils

Neutrophils are the predominant leukocyte subpopulation in the blood, derived from bone marrow haematopoietic stem cells (Ref. Reference Castanheira and Kubes56). It has been demonstrated that neutrophils are an important factor involved not only in infections but also in sterile inflammation (Ref. Reference Peng, Li and Yu57). Thus, neutrophils are one of the first cells recruited in the pancreas by DAMPs or by the chemokines and leukotriene released by the surrounding tissue to respond to DAMPs production. DAMPs include histone, DNA, IL-1α and many others. From the chemokine family, IL-8 (CXCL8) has the most important role in neutrophil chemotaxis and has been demonstrated to be an essential predictive marker for severity (Refs Reference Peng, Li and Yu57–Reference Wang59).

The first step of the recruitment is the interaction with the endothelium, which is enhanced by adhesion molecules from the selectin and integrin family, like intercellular adhesion molecule 1 (ICAM1). Another essential molecule for neutrophil migration is metalloproteinase, especially matrix metalloproteinase-9 (MMP-9), which helps by degrading some components of the basement membrane so the neutrophils can reach the pancreatic tissue (Refs. Reference Wang59–Reference Wan61). Once infiltrated in the pancreas, neutrophils perform their functions by phagocytosis, degranulation and neutrophil extracellular trap (NET) production. The most important molecules released by neutrophils are elastase, reactive oxygen species (ROS) and myeloperoxidase, which are involved in the activation of trypsinogen, cytokine release and inflammation enhancement (Refs Reference Peng, Li and Yu57, Reference Wang59).

NETs are another way for neutrophils to fight bacterial infections, but they have also been involved in sterile inflammation. NETs contain DNA, histones and cytosolic proteins and they are usually formed once neutrophils are dead (Refs Reference Castanheira and Kubes56, Reference Wang59).

Neutrophils are also involved in the systemic complications that are associated with severe AP, especially with acute lung injury (ALI). The first phase of ALI is the exudative phase, which involves microvascular damage and endothelial barrier dysfunction induced by inflammatory mediators. These changes determine neutrophil recruitment in the pulmonary interstitium and alveolar space that become activated and produce an increased inflammatory response, similar to the effect on the pancreas (Ref. Reference Zhou62).

Although neutrophils play a significant role in exacerbating inflammation, they also contribute to tissue repair. This is achieved by the phagocytosis of cellular debris, but most importantly, by the apoptosis of neutrophils cleared by macrophages. The clearance process activates the secretion of anti-inflammatory cytokines like tissue-repairing cytokines transforming growth factor-β (TGF-β) and IL-10 (Refs Reference Wang59, Reference Soehnlein and Lindbom63).

Macrophages

Macrophages are one of the most important cells involved in the evolution of AP. Pancreatic macrophages include tissue-resident macrophages and migrating macrophages (circulating monocytes) (Ref. Reference Hu64). DAMPs, proinflammatory mediators released by pancreatic injured tissue, necrotic cells like monocyte chemoattractant protein-1 (MCP-1) and TNF-α induce resident macrophage activation and monocyte migration (Ref. Reference Castanheira and Kubes56). After macrophage recruitment, the predominant cytokine environment determines the orientation of macrophage polarization, meaning that there are two distinct subsets: M1, classically activated, and M2, alternatively activated. M1 macrophages induce a Th1-type response by releasing cytokines and chemokines, accentuating the proinflammatory response (Refs Reference Hu64, Reference Mantovani65). Mosser proposed an alternative classification of macrophages based on three homeostatic activities, which are host defence, wound healing and immune regulation. Furthermore, tumour-associated macrophages have also been identified as a separate group extensively studied in the last few years (Refs Reference Mosser and Edwards66, Reference Mamilos67). The process of macrophage differentiation is called plasticity and describes the ability of cells to respond to different microenvironmental influences by displaying diverse functional phenotypes. Unlike other cells, which lose their heterogeneity during maturation, macrophages retain their plasticity and transform according to environmental signals (Refs Reference Mamilos67, Reference Katkar and Ghosh68).

The severity of the pancreatitis episode is correlated with the degree of macrophage activation and the M1 to M2 ratio imbalance; M1 predominance is associated with more severe forms (Refs Reference Peng, Li and Yu57, Reference Hu64, Reference Perides69). The central role of pancreatic macrophages in the evolution of AP is due to the involvement, besides the cytokine secretion, in trypsinogen activation, NF-κB activation and adaptive immune response (Ref. Reference Hu64).

In addition to pancreatic macrophages, peritoneal, liver and alveolar macrophages play an important role in the evolution of AP. The proinflammatory cytokines and enzymes are released in the bloodstream and the peripancreatic area, inducing the activation of peritoneal macrophages to the M1 subtype. The activated peritoneal macrophage can synthesize chemokines and cytokines like IL-1β, TNF-α and IL-8, aggravating the inflammatory response (Refs Reference Hu64, Reference Samanta70).

Kupffer cells are the resident macrophages of the liver and become activated in the evolution of pancreatitis by the cytokines and enzymes that reach the liver through the portal vein. They influence the magnitude of inflammatory systemic response and pulmonary involvement (Refs Reference Hu64, Reference Liu71).

The alveolar macrophages are activated by the proinflammatory cytokines released by the pancreas and the Kupffer cells, which reach the lung through the blood. The resident pulmonary macrophages determine the severity of pulmonary involvement by cytokine and ROS release (Refs Reference Hu64, Reference Gea-Sorlí72, Reference Gea-Sorlí and Closa73).

DCs

DCs link innate and adaptative immune systems by functioning as phagocytes and antigen-presenting cells. In AP, DCs infiltrate the pancreas and increase inflammation by secreting IL-6, TNF-α and MCP-1 (Ref. Reference Wan61). Also, it has been described that acinar cells can transition to DCs and activate CD4+ T cells, thereby accentuating the local inflammation (Refs Reference Peng, Li and Yu57, Reference Xu74). On the other hand, some studies have demonstrated the protective effect of DCs on the pancreas by inducing severe forms in mice depleted of DCs (Refs Reference Peng, Li and Yu57, Reference Bedrosian75).

NK cells

NK cells are a subtype of circulating lymphocyte that has a cytotoxic effect on tumoural and virus-infected cells. The cytotoxic effect is accomplished by inducing cell apoptosis by releasing perforins and lytic proteins from cytoplasmatic granules (Refs Reference Marshall54, Reference Vivier76). Besides this function, NKs are involved in inflammation through cytokine secretion (interferon-γ (IFN-γ), TNF, IL-5, IL-10 and IL-13) and chemokines (CCL3, CCL4 and IL-8) (Refs Reference Peng, Li and Yu57, Reference Zitti and Bryceson77).

NK cells regulate immune response by activating macrophages, DCs and T cells through the cytokine secreted and surface receptors. On the other hand, NK can kill these cells (DCs and macrophages) by cytotoxic effect (Ref. Reference Vivier76).

Regarding the involvement of NK in AP, evidence suggests that the number of NKs in the peripheral blood is decreased compared to the healthy individual. Moreover, in severe forms, there is an even more considerable decrease and this depletion is correlated to the infectious complications in AP (Ref. Reference Peng, Li and Yu57).

Inflammasomes

Inflammasomes are a cluster of multiprotein complexes found in the cytosol involved in the inflammatory response as part of innate immunity (Refs Reference Ferrero-Andrés78, Reference Martinon, Burns and Tschopp79). These complexes have two important components: one is the upstream sensor protein that belongs to the NLR (NOD like receptor) or ALR (AIM2 like receptor) family, and the other one is the adaptor protein, containing a caspase recruitment domain (ASC - apoptosis-associated speck-like protein containing a caspase activating and recruitment domain) (Ref. Reference Ferrero-Andrés78). The most investigated inflammasome from the NLR family is the NLR pyrin domain-containing protein 3 (NLRP3), which is expressed in monocytes, neutrophils, lymphocytes, DCs and epithelial cells (Ref. Reference Ferrero-Andrés78). NLRP3 is activated by both PAMPS and DAMPs. However, in AP, the recognition of DAMPs by the NLRP3 is the first step in its activation and afterwards in the induction of sterile inflammatory response (Ref. Reference Ferrero-Andrés78). Inflammasomes need two different signals for activation. The first signal requires the upregulation of inflammasome mRNA by NF-κB and the second signal begins the activation of pro-caspase-1 to its active form, caspase-1 (Refs Reference Tsomidis, Voumvouraki and Kouroumalis80, Reference Guo, Callaway and Ting81). There are also two pathways for NLRP3 inflammasome action. The canonical pathway activates caspase-1 and subsequently induces the maturation of IL-1β and IL-18, as well as pyroptosis, a form of programmed cell death discussed earlier. In contrast, noncanonical activation of caspase-1 requires the presence of caspase-11 in addition to the NLRP3 inflammasome (Ref. Reference Lamkanfi and Dixit45). NLRP3 inflammasome is involved in AP inflammation by activating IL-1β and IL-18. These two cytokines are secreted by activated macrophages but require activation from the precursor form, pro-IL-1β and pro-IL-18, to the mature activated form (IL-1β and IL-18). There are two steps in the activation process: the first one is the increased expression of inflammasome components and pro-IL-1β by the NF-κB transcription factor and the second one is the proteolytic maturation of the pro-molecules in the presence of NLRP3 inflammasome and caspase-1 (Ref. Reference Glaubitz82). The involvement of NLRP3 in AP pathogenesis is sustained by experimental studies, which showed that the absence of caspase-1, ASC or NLRP3 substantially reduced oedema and inflammation in a pancreatitis mice model (Refs Reference Ferrero-Andrés78, Reference Hoque83). Furthermore, there is also clinical evidence of the involvement of inflammasomes in AP. In a study conducted by Algaba-Chueca et al. (Ref. Reference Algaba-Chueca84), the levels of NLRP3 and derived IL-1β and IL-18 were elevated in the first stages of AP. Another evidence of the importance of these molecules in AP comes from experimental studies that showed decreased severity of AP when NLRP3 inhibitor was used (Ref. Reference Sendler85).

T cells

In the initial phases of pancreas injury, there is a reduction in the number of T cells (CD4 and CD8) in the circulation due to an excessive expression of Fas and FasL ligands on the cell surface. Fas (also known as CD25 or Apo-1) is a member of the TNF receptor family, which promotes lymphocyte apoptosis (Refs Reference Sun87, Reference Zhang88). FasL (named CD95L or CD178) is a type-II transmembrane protein expressed on cytotoxic T lymphocytes and NK cells, which also induces programmed cell death (Ref. Reference Pardalis89). This overexpression of Fas ligands results in a significant loss of peripheral T lymphocytes, immunosuppression and bacterial superinfection. The number of peripheral T cells is also reduced due to their migration to the inflamed pancreas (especially CD4 cells infiltrating the inflamed acini). Usually, the reduction is transient in moderate AP (MAP), with the number of cells normalizing after the fifth day of evolution. In the severe forms of AP (SAP), the severity of cellular destruction is correlated with persistent lymphopenia, especially with the reduction of Th cells, which amplifies the complication risk (Ref. Reference Stojanovic86).

Cytokines released secondary to the pancreas injury (IL-2, IL-4 and IL-12) will determine the differentiation of T cells in T helper (Th1, Th2, Th9, Th17, Th22 and follicular T helper) and regulatory T cells (Treg cells).

B cells

B cells play an important immunomodulatory role. They secrete anti-inflammatory cytokines (IL-10, IL-35 and TGF-β) that inhibit the activation and proliferation of proinflammatory immune cells (Ref. Reference Rosser and Mauri114). By producing IL-10, B cells block the immune response of Th1 and Th-17 cells, reducing their proinflammatory activity (Ref. Reference Flores-Borja115). IL-35 is a recently discovered anti-inflammatory cytokine from the IL-12 family produced by a wide range of regulatory lymphocytes. It has an important role in blocking the development of Th1 and Th17 cells (Ref. Reference Behzadi, Behzadi and Ranjbar116). In a study conducted by Aksoy et al. (Ref. Reference Aksoy117), the level of IL-35 was significantly lower in people with AP compared with healthy people, this marker being considered in the diagnosis and follow-up of patients with AP.

In the first stages of AP, there is an increased level of B cells, correlated with mild forms of disease. Reversely, in patients with SAP, a reduced number of circulating B cells, especially of regulatory B cells that produce IL-10, and a decreased level of immunoglobulins M and G (IgM and IgG) are often found. IgG levels seem to be an important prognostic marker, being very low in patients who developed infectious complications or those who died (Ref. Reference Ueda118). Furthermore, increased B-cell-activating factor (BAFF) levels were detected in patients with SAP. BAFF is a major cytokine that regulates B-cell survival, maturation and differentiation. BAFF acts as an acute-phase reactant and, like IL-6 or procalcitonin, can predict the severity of AP; these parameters were found to be significantly higher in patients who developed necrosis or who required intensive care hospitalization for more than 7 days (Ref. Reference Pongratz119).

Studies conducted on mice proved that the value of amylase and the histological score of AP increases inversely proportional to the reduction in B lymphocytes. It is now established that B lymphocytes contribute to the severity of AP but the exact role of B subsets in PA etiopathogenesis still needs to be elucidated.