Organ Failure due to Systemic Injury in Acute Pancreatitis

Abstract

Acute pancreatitis may be associated with both local and systemic complications. Systemic injury manifests in the form of organ failure which is seen in approximately 20% of all cases of acute pancreatitis and defines ‘severe acute pancreatitis’. Organ failure typically develops early in the course of acute pancreatitis, but may also develop later due to infected pancreatic necrosis induced sepsis. Organ failure is the most important determinant of outcome in acute pancreatitis. We review here the current understanding of the risk factors, pathophysiology, timing, impact on outcome and therapy of organ failure in acute pancreatitis. As we discuss the pathophysiology of severe systemic injury, the distinctions between markers and mediators of severity are highlighted based on evidence supporting their causality in organ failure. Emphasis is placed on clinically relevant end points of organ failure and the mechanisms underlying the pathophysiological perturbations, which offer insight into potential therapeutic targets to treat.

Acute pancreatitis (AP) causes major morbidity and mortality. According to global estimates, the incidence of AP was shown to be 33·74 cases (95% CI 23·33-48·81) per 100 000 person-years and a mortality of 1·60 (95% CI 0·85-1·58) per 100 000 person-years due to AP.¹ The severity of AP can either be mild, moderate or severe, which depends on the extent of local injury in and around the pancreas, and more importantly systemic injury to remote organs.² Mild AP has no major local or systemic complications. More severe form of the disease seen in around 20% of all patients with AP is associated with significant local complications in the form of necrosis and often systemic injury due to systemic inflammation.³

Systemic inflammation presents initially as systemic inflammatory response syndrome (SIRS). Patients with persistent SIRS are prone to develop systemic organ dysfunction and later organ failure (OF)^{4, 5}. OF can develop either due to involvement of a particular organ system by a primary disease process or due to systemic effects of injury/inflammation at another site. Acute respiratory failure due to severe pneumonia is an example of the former and OF due to AP is an example of the latter. The most common cause of OF in clinical practice is sepsis. However, OF can develop due to non-infectious etiologies as well: AP and trauma are prime examples. OF is a conditio sine quo non of severe acute pancreatitis (SAP). SAP is defined by the presence of persistent OF as per the revised Atlanta classification of severity of AP.² OF largely governs the outcome and mortality in patients with AP, and therefore it is important to understand its epidemiology, risk factors, pathophysiology, potential mediators, impact on outcome and management. This review focuses on relevant pathophysiological and clinical aspects of OF in patients with AP and identifies unmet needs.

Definition of Organ Failure:

OF, as a generic term, can be defined as significant functional impairment of an organ system that is critical to sustenance of life. The severity of organ dysfunction can be quantified based on the parameter best defining the primary function of that particular organ e.g. partial pressure of arterial oxygen (PaO2) for pulmonary function or serum creatinine for renal function. In the case of AP, 3 organ systems are considered most important i.e. respiratory, renal and cardiovascular which are most commonly involved.² The severity of organ dysfunction is graded by the modified Marshall grading in AP ² which is preferred over the sequential organ failure assessment (SOFA) score used in sepsis. Any organ dysfunction of ≥grade 2 severity persisting for >48 hours is considered as persistent OF and defines SAP. Transient organ failure of <48 hours is considered a criterion for moderate AP.

How common is OF in AP?

The proportion of patients with AP who develop OF varies in different studies and primarily depends on the setting. Data from population-based cohort studies show a lower proportion of patients with OF while tertiary care hospital based studies have shown a much higher frequency of OF. Population based studies have shown the proportion of OF (severe AP) between 8% and 20%.^6-8 On the other hand, the proportion of patients with OF in large series of patients from tertiary care hospitals may reach up to 40%.^{9, 10}

Risk Factors for Organ Failure:

Why some patients develop OF and others do not is a matter of great importance. Host factors such as age, co-morbid conditions, obesity, triglyceride levels, etiology, extent of local pancreatic injury, and genetic predisposition have been reported to predict the development of OF in patients with AP. Older age is a risk factor for OF and worse outcome which could also be due to co-morbidities.^{11, 12} Co-morbidities, as measured by Charlson comorbidity index, may become worse due to AP and contribute to poor outcome but have not been directly related to another organ dysfunction.¹³ Obesity is an established bad prognostic marker of outcome in patients with AP.¹⁴ Visceral obesity predisposes to the development of OF.¹⁵ Lipotoxicity causes multi-organ failure and exacerbates AP in obesity.¹⁶ Peripancreatic visceral fat necrosis has been shown to worsen AP independent of pancreatic necrosis via unsaturated fatty acids resulting in OF.¹⁷ Triglyceride levels at admission have been correlated with the severity of AP and even mild to moderate hypertriglyceridemia was found to be associated with the development of persistent OF.¹⁸ Etiology has not been found to be an independent risk factor for OF although patients with alcohol induced AP may have a higher risk of early onset OF.¹⁹ Association between the extent of local pancreatic injury as measured by the extent of necrosis and development of OF has been reported but the causality is not established.^{3, 20-22} The mechanisms of local tissue injury leading to pancreatic necrosis and systemic injury manifesting as OF are intimately linked.²³ The issue from the pathophysiology point of view is if the extent of necrosis is causally related to the development of OF or do they both signify the end result of a severe response to acute pancreatic injury. The association between extent of necrosis and OF could be bidirectional. A complex inflammatory network in which the extent of (peri)pancreatic necrosis influences the severity of OF and OF exacerbates the development of pancreatic necrosis might exist.^24,25

Given the apparent similarities in the etiology and phenotype of patients with varying grades of severity of AP, differences in inter-individual inflammatory responses might explain the variability in the severity of AP. It is conceivable that the highly variable inflammatory response might be related to an underlying genetic predisposition. However, the data regarding the role of genetic polymorphisms in determining the severity of AP are scant and equivocal. TNF-α gene polymorphism was associated with severity but this finding has not been validated in other studies.^26-28 MCP-1 gene polymorphism was associated with increased risk of severe AP with the G allele acting as the risk factor but the data were inconsistent.^{29, 30} One study has shown genetic polymorphisms of IL-6 gene to alter the level of IL-6 but did not find any association with the severity of AP.²⁶

Clinical determinants/Characteristics of Organ Failure:

There are various characteristics of OF that affect the clinical course and outcome. The important determinants are – (i) grade of OF as per the Modified Marshall score, (ii) specific type of OF e.g. respiratory/renal, (iii) number of organs affected i.e. single or multi-organ failure, and (iv) the timing of OF from the onset of AP. A higher grade of OF naturally has a greater impact on outcome. Patients with grade 3 or 4 OF requiring organ assistance such as mechanical ventilation have a worse outcome.³¹ Respiratory failure is the commonest OF.^{10, 32, 33} Respiratory and renal failures are quite similar in their impact on the outcome but cardiovascular failure leads to the worst outcome.¹⁰ Multi-organ failure has a worse prognosis than single OF.³² The timing of onset of OF has important connotation regarding the likely cause of OF and possibly its impact on survival. Inflammation is the key pathological response both at the local and systemic levels in AP. Organ failure may develop early within a few days of onset of AP, which is termed as early severe acute pancreatitis and carries a high mortality.^{19, 31} This is primarily due to a sterile inflammatory response. Organ failure may also develop late during the course of AP due to sepsis as we discuss next.

Primary (Early) Sterile and Secondary (Late) Septic Organ Failure in Acute Pancreatitis:

Although organ failure and its consequences are well recognized in AP, there is limited understanding about primary OF that develops early due to pancreatitis per se (sterile inflammation) and may precede necrosis, and late secondary OF due to infected pancreatic necrosis (IPN) induced sepsis (Figure 1). Infection of the necrotic pancreatic tissue is an ominous development during the course of AP. IPN is the cause of most of the late mortality during the course of AP. Although many studies have shown development of early and late OF in patients with AP, the relative contributions of primary OF and secondary OF to mortality have not been well studied. One recent study of 805 patients with AP has provided the concept of primary and secondary OF and shown several differences between the two (Table 1).⁹ The window of opportunity is small in case of primary OF because it leads to early mortality while temporally there is a larger window of opportunity to intervene in those with sepsis and secondary OF. The treatment is largely supportive for primary OF while control of sepsis is the goal in secondary OF. Prognosis is poorer in primary OF and somewhat better in secondary OF.

Figure 1:

A conceptual model of early sterile injury and inflammation due to damage associated molecular patterns (DAMPs)/ Unsaturated fatty acids (UFAs), and late secondary septic inflammation due to pathogen associated molecular patterns (PAMPs) that may lead to organ failure in acute pancreatitis.

Table 1:

Differences between primary and secondary organ failure

Characteristic	Primary Organ Failure	Secondary Organ Failure
Cause	Sterile inflammation	Sepsis
Timing	Early	Late
Therapeutic window of opportunity	Small	Large
Treatment	Supportive	Control of sepsis
Prognosis	Poor	Relatively better

Table 1 is adapted from reference 9

Clinical correlates of OF: Can we predict Organ Failure?

By definition, persistent OF should last >48 hours. Therefore, it will take at least 3 days to document persistent OF even if a patient develops OF within a day of onset of pain. From triage and prognostic points of view, it is important to predict development of persistent OF in patients who present early to the hospital. Prediction of severe AP is largely based on several clinical scores, such as APACHE II, which are multifactorial and somewhat tedious to use. BISAP score has been developed and validated to predict development of severe AP and outcome.^{34, 35} Single prognostic markers are also used; CRP and IL-6 being sensitive markers.^{36, 37} Persistent SIRS is a reliable clinical marker to predict development of OF. However, despite a reasonably good sensitivity of 50-95%, SIRS has a lower specificity of 75% and suboptimal positive predictive value (PPV) of 16%-56%.^{38, 39} Although almost all patients with persistent OF have persistent SIRS, a significant proportion of patients with non-severe AP may also have persistent SIRS. PPV is an important attribute for a test variable as a low PPV may lead to unnecessary and avoidable referrals from primary/secondary care centers, increase monitoring and the overall cost of hospitalization. Recently, a study has shown that a combination of serum IL6 >160 pg/ml and SIRS at admission had a much higher PPV of 85% and a higher specificity of 95% for the development of severe AP.⁴⁰

Clinical trajectory of Organ Failure - Impact on outcome:

OF accounts for almost all the mortality in patients with AP. Patients who develop OF early on are at risk of having a severe course of the disease. In a population based study of 1024 deaths due to AP, the median interval between the onset of AP and death was 6 days and from the onset of OF to death was 3 days.³³ The concept of transient and persistent OF was introduced in 2004.⁴ Transient OF lasting <48 hours also has a negative impact on outcome.⁴ The mortality in patients with transient OF was 1.4% to 10% although the mortality was most likely due to other contributing factors such as IPN.^{4, 10, 13} The overall mortality in patients with persistent OF is >40% (Table 2). Patients with persistent OF have a high risk of early mortality within the first 2 weeks.^{9, 10, 33} particularly those with very early onset high-grade single OF or multi-organ failure termed as fulminant pancreatitis.^{19, 31}

Table 2:

Summary of Results of Recent Studies highlighting Mortality in Patients with Persistent Organ Failure

Study*	Number of patients with AP	Number of patients with POF	Mortality in patients with POF	Mortality in patients with early onset POF$	Mortality in patients with late onset POF
Padhan et al 2018 (ref 9)	805	365	156 (42.7%)	104/225 (46%)	52/140 (37%)
Schepers et al 2018 (ref 10)	639	219	83 (38%)	47/112 (42%)	36/107 (33.6%)
Sternby et al 2018 (ref 13)	1655	113	59 (52.2%)	47/89 (52.8%)	12/24 (50%)

POF = persistent organ failure

^*These are recent large studies, which had categorized patients according to revised Atlanta classification and the patients were treated as per current standard of care.

^$Early OF defined by development of OF within the first week of onset of AP

The mortality due to OF is high even after the first 2 weeks. Patients with persistent OF who survive the first 2 weeks are prone to develop infected necrosis, which accounts for the late mortality. In a French study of 148 patients, 40 of 53 (75%) patients with persistent OF developed infected pancreatic necrosis (IPN).⁴¹ In another study, 76% of patients with persistent OF developed IPN after they survived the first 2 weeks.⁹ Hypotension in the first week of AP was an independent risk factor for IPN.⁴² There is not much difference between mortality due to early onset OF and late onset OF (Table 2) Two recent studies have focused attention on the issue of timing of onset of OF and outcome in AP. In a study of 614 patients, early onset primary OF resulted in early mortality in 15.8% of patients and a further 42.8% late mortality due to development of infected necrosis.⁹ In a Dutch study of 639 patients, 219 patients with persistent OF had a mortality of 38%. Mortality was not related to timing of onset of persistent OF. Mortality due to persistent OF developing within the first week, 1-2 weeks, 2-3 weeks and >3 weeks from the onset was 42%, 46%, 36% and 29% respectively in that study.¹⁰ Patients with OF and IPN have a high mortality termed ‘critical AP’ according to the Determinant based classification.⁴³ But the data are inconsistent as to whether they have a higher mortality than those with early persistent OF without IPN. In the Dutch study, similar mortality rates were observed in patients with OF with and without IPN (28% vs. 34%, p=0.33) after excluding patients with mortality within 10 days of admission.¹⁰

In summary, the progression of an early systemic inflammatory response to organ failure defines severe AP and is associated with a high risk of mortality. Development of infected necrosis later in the clinical course exacerbates the initial injury and worsens the outcome.

Pathophysiology of systemic injury in acute pancreatitis

The pathophysiology of systemic injury in AP has remained enigmatic so far. The biggest hurdle has been the identification of mediators that are released locally in the pancreas and cause systemic injury. More so, as mentioned above, the systemic severity can precede local severity and the extent of pancreatic necrosis may not correlate with systemic injury, unless it is complicated by concurrent infection. It is to be noted that neither systemic involvement, nor its severity are related to the etiology of AP. Figure 2 summarizes the potential pathophysiology of systemic injury. This is based on literature in pancreatitis, as well as where individual agents have been studied in their ability to cause systemic injury irrespective of pancreatitis. The end points relevant to human disease are summarized in lower panel of Figure 2 and more than one positive study showing that the agent can incite the endpoint is taken as positive.

Figure 2: Pathophysiology of systemic injury in AP:

The upper part of the figure describes the initiation of acinar injury by an AP etiology like high dose CCK (↑CCK) during caerulein pancreatitis. The intra-acinar signaling events include the increase in cytosolic calcium (Ca²⁺), which has a role in mitochondrial depolarization (Memb. Depol.) and cytochrome C leakage, along with activating NF-kB via dissociation and proteasomal degradation of IkB, nuclear translocation of p65. This upregulates inflammatory mediator synthesis, which include cytokines and chemokines, and thus leads to neutrophil infiltration into the pancreas. The trypsin generated due to impaired autophagy involving lysosomes (L) and zymogen granules (ZG) and increased oxidized glutathione (GSSG) [from its reduced form (GSH), due to reactive oxygen species (ROS)], along with concurrent deleterious mechanisms, cause acinar injury. These other mechanisms include the loss of apical microvilli, inhibition of apical secretion, the reorganization of F-actin, basolateral blebbing, release of DAMPs that can activate the inflammasome, and leakage of exocrine enzymes such as lipase, trypsin. The DAMPS can worsen local injury, and may also contribute to systemic injury. Similarly, cytokines can cause the leukocytosis associated with SIRS, which can enter the systemic circulation and are a part of systemic injury. The lower part of the figure describes the types of systemic injury that may occur due to these, along with the underlying mechanisms. The mechanisms include unregulated hydrolysis of adipocyte triglyceride (Adipo. TG) by pancreatic lipase, resulting in fat necrosis, which generates UFAs, that inhibit mitochondrial complex I and V, which decrease ATP and worsen local injury. The effects of UFAs, trypsin, cytokine entry into the systemic circulation, and leukocytosis (from left to right) on end points of systemic injury are mentioned in the table below, with a + indicating 2 or more reports citing the agent in causing the end point. Unclear or weaker evidence is shown as ± or a – respectively.

It is to be noted that there are numerous and parallel steps involved in acinar cell injury induced by a single agent like caerulein, which itself does not cause systemic injury. Therefore, the role of other important factors in amplifying the signaling to become deleterious on a systemic level is essential. A clue to such factors that worsen pancreatitis lies in several reports showing hypertriglyceridemic AP to have a higher grade of severity than is usually reported. ^44-46 This may be related to the fatty acids that compose the triglyceride, and is discussed in detail under the section unsaturated fatty acids.

Distinction between markers, mediators and end-points of systemic injury:

It is important to appreciate the difference between markers vs. mediators vs. end points of systemic injury. Markers of systemic injury such as SIRS, or serum cytokines are distinct from the endpoints that determine the severity of systemic injury during pancreatitis. The clinical or biochemical clues do not themselves depict end organ injury, but may be markers or mediators of it. A marker in contrast to a mediator, when administered to an organism or when inhibited would not affect the endpoint of systemic injury. However, a mediator when administered would elicit an endpoint of systemic injury or when it is neutralized or inhibited the systemic injury would be ameliorated. As discussed previously, the end points of systemic injury during AP are renal, respiratory and cardiovascular failure.

Animal models and systemic injury in AP:

Although there are several AP models, it is important to realize their limitations in measuring systemic injury. Most AP models have a strong emphasis on local pancreatic injury in the context of specific initiators or etiologies like caerulein. Recently, clinically relevant risk factors such as obesity have been shown to result in systemic injury by modifying the course of AP in animal models.^47-49 These studies have used clinically relevant end points such as renal failure. Thus, it is important to analyze whether the end points used in basic/ animal models equate to human disease. For example, a common end point of lung injury used in animal models is the accumulation of myeloid inflammatory cells in the lung, shown as increase in lung myeloperoxidase (MPO) activity. Since pulmonary inflammation can be protective (please see section on neutrophils), it is important to realize the limitations of lung MPO as a parameter of lung injury. More relevant end points may be pulmonary microvascular permeability studied as the leakage into the alveolar space of an intravenously administered fluorescently tagged macromolecule such as albumin^50-52, the oxygen saturation as determined by pulse oximetry⁴⁷, or dead cells in the alveolar space^{47-49, 53} which are relevant to pulmonary edema or adult respiratory distress syndrome (ARDS) in humans.⁵⁴ Similarly, clinically relevant end points for renal failure in animal models include a sustained increase in serum blood urea nitrogen (BUN)⁴⁸ like in sustained renal failure in humans.^{48, 55}

While basic models typically cannot distinguish between primary and secondary organ failure due to their short course, we cover markers and potential mediators (Table 3) increased in early human AP independent of pancreatic necrosis, and where relevant distinguish these from those associated with infection in the following discussion.

Table 3:

Markers and mediators of systemic injury in acute pancreatit

Markers and mediators of systemic injury

Released from acinar cells

Trypsin

Inflammatory cells and their products

Neutrophils

NET

Inflammasome

Cytokines

IL-6

IL-1β

TNF-α

IL-12

IL-18

Adipokines

Resistin

Visfatin

Lipid mediators:

Unsaturated fatty acids

Platelet activating factor

DAMPs

HMGB1

sRAGE

ds-DNA

Histones/ nucleosomes

S100 proteins

ATP

Extracellular matric (e.g. hyaluronan)

Potential mediators of systemic injury in AP:

Trypsin:

Trypsin generation is ubiquitous in AP ^56-59 in both rodents and humans^{56, 59}. Being associated with the auto-digestive hypothesis of pancreatitis for over a hundred years⁶⁰, trypsin has been an attractive target to reduce pancreatitis severity. The most direct proof of trypsin’s role as a mediator of systemic injury early on came from its intravenous (IV) infusion resulting in hypotension, shock^{61, 62} and coagulopathy, which is consistent with the coagulation cascade being a series of proteolytic steps. In support of this observation, elevated D-dimer; a fibrin degradation products at admission has been shown to predict development of OF with a sensitivity, specificity, positive and negative predictive values of 90%, 89%, 75% and 96% respectively⁶³. Whether this coagulation cascade plays a role in splanchnic venous thrombosis, which is rare in the absence of necrosis, but occurs in about half of the patients with pancreatic necrosis⁶⁴ remains unknown. Trypsin infusion also causes lung injury⁶⁵ which is dependent on neutrophils (please see next section). More recent studies have identified the protease activated receptor-2 (PAR-2)⁶⁶ to be regulated by trypsin during pancreatitis. The most direct evidence comes from hypotension resulting from IV infusion of PAR-2 agonists, possibly via PAR-2 receptors on endothelial cells ⁶⁷. Whether trypsin actually plays a major role in systemic injury during clinical pancreatitis remains debated, since small molecule trypsin inhibitors have not shown conclusive benefit^68-77 in improving systemic injury during AP and patients with hereditary pancreatitis due to trypsinogen gene mutations that result in its activation rarely develop systemic injury compared to other AP etiologies⁷⁸. The interpretation of trypsin’s role is further complicated by circulating anti-proteases such as alpha-2 macroglobulin ^{79, 80} which can inactivate it, and trypsin’s inherent tendency to auto-inactivate. Whether trypsin is indeed an initiator/mediator of systemic severity during pancreatitis, therefore still remains inconclusive.

Neutrophils:

Leukocytosis (>12000/mm³) is part of the SIRS criteria⁸¹, and is an early predictor of severity of AP². The leukocytosis in early pancreatitis is predominantly neutrophilic, and several studies have shown that depletion of neutrophils reduces inflammatory cell infiltration (e.g. reduced MPO) into the lungs and improves microvascular permeability^{52, 65, 82, 83}. Neutrophil infiltration into an organ is dependent on their adhesion to the endothelium, mediated by P- and E-selectin on the surface of endothelial cells. These bind adhesion molecules like L-selectins and integrins on the surface of neutrophils. Blood levels of P- and E-selectin were elevated in rodent⁸⁴ and in human⁸⁵ AP. These respectively correlated with severity of AP and lung injury. Interestingly, trypsin generation in the late phase of experimental pancreatitis has been shown to be neutrophil dependent⁶⁵, and trypsin can also stimulate neutrophils to secrete matrix metalloproteinase-9. Based on these observations, trypsin mediated lung injury has been hypothesized to be neutrophil dependent. Other studies have shown that neutrophil infiltration into the lungs is due to the chemokines CXCL2 and CXCL4, and that their neutralization reduces lung inflammation⁸⁶. However, neutrophils also have physiologic roles and their accumulation in the lungs, such as by increasing KC/CXCL1^87-89 expression does not cause damage, but conversely protects from fungal and bacterial infections. Moreover, neutropenia predisposes to infections^90-92. Therefore, it remains to be seen whether interference in neutrophil recruitment or their depletion during AP will improve systemic injury in human AP.

Neutrophil extracellular traps (NETs):

NETs are web like structures containing neutrophil granule proteins (e.g. myeloperoxidase, elastase) and chromatin. These are released by neutrophils and are increased in the sera of patients with severe pancreatitis⁹³. In addition, they occlude pancreatic ducts in human AP and may perpetuate pancreatitis⁹⁴. NET formation in pancreatitis is catalyzed by the enzyme Protein Arginine Deiminase 4 (PAD4)⁹⁵, which causes histone modification of arginine residues to citrulline⁹⁶. This modification weakens DNA- histone interactions and allows the neutrophils to expel the de-condensed chromatin. PAD4 inhibition reduces NET formation in humans⁹⁷ and rodents. Whether inhibition of NET formation will prove to be beneficial in reducing systemic injury in pancreatitis remains to be seen, more so since recent studies also show NET formation to wall off pancreatic necrosis from viable tissue in humans, and thus may play a role as a protective barrier to the progression of pancreatitis⁹⁸.

Damage associated Molecular patterns (DAMPs):

DAMPs, which are released from dying cells during necrosis correlate with human AP severity ^99-102, and are potential mediators of severity based on animal studies^103,104 DAMPs include small molecules such ATP, proteins- including S100 proteins, the soluble receptor for advanced glycation end products (sRAGE), and high-mobility group box 1 (HMGB1), nuclear components (e.g. histones, DNA, nucleosomes), and molecules released from the extracellular matrix such as hyaluronic acid. Serum HMGB1 significantly correlated with AP severity in humans in a meta-analysis¹⁰⁵. DAMPs can worsen inflammation by causing activation of the inflammasome^{103, 106}, as shown for sRAGE and HMGB1, and also directly via inducing a sterile inflammatory response¹⁰⁷ by disrupting the plasmalemma¹⁰⁸, and further increasing DAMP release (e.g. HMGB1¹⁰⁴). However, it remains to be confirmed if DAMPs alone can induce the clinically relevant end points of OF or their inhibition during pancreatitis models, which induce OF, averts these end points

Inflammasome:

The inflammasome¹⁰⁹ is expressed in myeloid cells and has been implicated in systemic inflammation during pancreatitis ^{103, 110} Inflammasome activation results in production of IL-1β¹¹¹, IL-18^112-114 and HMGB-1^{104, 115}, all of which are associated with systemic injury during pancreatitis. Activation of the inflammasome is thought to be downstream of various receptors, some of which may be relevant to pancreatitis. These include nucleosomes (i.e. DNA-histone complexes), dsDNA and RAGE activating the AIM2 (absent in melanoma 2) inflammasome^{110, 116} and extracellular ATP or NAD released from injured acinar cells activating the P2X7 receptor¹⁰³. Additionally, cell surface pattern recognition receptors including TLR 4 and TLR9 may be activated by these DAMPs¹⁰³. Interfering with nucleosomes activating the inflammasome or inhibiting RAGE signaling has been shown to reduce the severity of L-arginine and caerulein pancreatitis^{104, 116} including lung inflammation. Interestingly, antagonism of TLR9 reduced lung inflammation, but not edema¹⁰³. Similarly while IL-1β increased lung inflammation, it did not induce lung injury⁴⁷. Additionally while IL-18 (in combination with IL-12) induces pancreatitis¹¹⁷, the systemic severity of pancreatitis these agents induce and the associated mortality are largely dependent on an increase in visceral fat¹¹⁸ and its lipolysis to fatty acids⁵³. In the absence of known genetic polymorphisms that govern inflammasome activation in pancreatitis, it is unclear how inflammasome activation variably affects the severity of pancreatitis in different individuals. This is further highlighted by early OF in pancreatitis occurring in the absence of extensive pancreatic necrosis and thus having a smaller source of inflammasome activators such as dsDNA or nucleosomes than later in AP, when necrosis is progressing. Future studies are needed to clarify the role of the inflammasome in systemic injury during pancreatitis.

Adipokines and Cytokines:

Previous clinical studies have shown that severe AP is associated with elevated serum levels of adipokines including resistin and visfatin ^{119, 120}, and cytokines including IL-6 ^121-124, IL-β¹¹¹, IL-8^{121, 124, 125}, MCP-1¹²⁶, TNF-α,¹²². Serum cytokines are part of the criteria for severity stratification ^{111, 121-124, 126-128}. In a prospective study including 108 patients, IL-6 was one of the best discriminators between mild and severe AP¹²⁹. Another study showed that blood levels of IL-6 correlated with OF and mortality. At a cut-off value of 122 pg/mL on day 3, IL-6 predicted OF and severe pancreatitis with a sensitivity and specificity of 81.8% and 77.7%, respectively³⁷. While the increase in their levels is associated with worse local and systemic complications, the evidence to support them as potential mediators of clinically relevant end points of OF in pancreatitis is so far lacking. For example, while IL-1β induces fever and myeloid infiltration into the lungs, it does not induce respiratory failure⁴⁷. Neutrophil infiltration mediated by cytokines such as IL-8, CXCL1 has been shown to protect from lung infections ^{88, 89,130} Similarly, IL-6 infusion in humans inhibited endotoxin induced TNF-α increase¹³¹ and its long term infusion led to hypoferremia and anemia^{132, 133}, but not systemic injury. IL-6¹³⁴ and TNF-α¹³⁵ have also been shown to have a protective role in AP, and neither these or other cytokines have been shown to induce OF^136-140. Therefore, targeting these cytokine alone may not improve outcomes in severe AP.

Coagulation pathway:

Vascular injury is an integral part of pancreatic inflammation and systemic injury. Endothelial activation, injury, increased vascular permeability, activation of coagulation, and increased leukocyte rolling, sticking and transmigration to pancreatic tissue have been demonstrated in AP. The inflammatory process and proteases like trypsin may activate the coagulation system leading to microvascular thrombosis. Whether this coagulation cascade contributes to systemic injury is unknown. As mentioned before, portal or splenic vein thrombosis can occur in about half of the patients with pancreatic necrosis⁶⁴. Similarly while both anti-thrombin III and heparin have been shown to reduce the severity of AP in animal models, the clinical implications of this on systemic severity are unknown¹⁴¹.

Lipid mediators:

Platelet activating factor (PAF):

Apart from trypsin, PAF has been extensively targeted in pancreatitis. PAF is a phospholipid (acetyl-glyceryl-ether-phosphorylcholine) produced by myeloid cells, platelets and endothelial cells. Intra-arterial delivery of PAFs into the pancreas causes AP¹⁴². PAF production in inflammation is mediated by phospholipase A2, and it is degraded by PAF acetylhydrolase ¹⁴³. Its receptor is a G-protein receptor¹⁴⁴ . PAF has a broad range of effects including increasing vascular permeability, worsening inflammation and initiating cell death¹⁴⁵. Its levels are increased in severe biliary pancreatitis¹⁴⁶ in which it was thought to mediate shock and acute lung injury¹⁴⁷, and the protective effect of antagonizing it was shown in multiple models including biliary, choline deficient ethionine supplemented diet (CDE diet)¹⁴⁸, caerulein model in rats, and severe biliary pancreatitis in opossums⁵¹. While initial clinical trials using lexipafant, a PAF antagonist were promising¹⁴⁹, the large definitive clinical trial did not show benefit in OF or mortality, even though local complications and sepsis were reduced¹⁵⁰. Whether this was due to a limitation of targeting PAF or the high prevalence of early OF remains to be studied (please see below).

Unsaturated fatty acids (UFAs):

The pancreas by its location is proximal to visceral fat in humans. Several studies report an increased risk of severe pancreatitis to be associated with an increase in visceral fat ^151-154 which ranges from 1-10% of body weight ¹⁵⁵. This fat is composed of adipocytes, the mass of which is predominantly (>80%) triglyceride^156-158. This triglyceride is predominantly composed of UFAs^{159, 160}, covalently linked to a glycerol backbone, which when released by unregulated lipolysis affect severity of AP. Interestingly, adipocyte triglyceride has become enriched in UFAs like linoleic acid over the last few decades¹⁶¹, which mirrors the 15-25% linoleic acid composition of necrosectomy samples from severe AP patients^{47, 53}. Previous studies have shown pancreatic lipases to be present in the adipocytes, damaged during AP¹⁶². This results in a morphology known as fat necrosis, which can worsen pancreatic parenchymal necrosis^{49, 53}, but can also occur independently ^{163, 164}. This lipolytic fatty acid generation can increase systemic injury during pancreatitis, in parallel with an increase in serum UFAs such as linoleic and arachidonic acid^{165, 166}, which like visceral fat^{159,47, 48, 53, 160}are unsaturated. These UFAs when liberated in excess, inhibit mitochondrial complexes I and V⁵³, and increase apoptotic cells in the lungs^{17, 48, 49, 53} (similar to patients with acute respiratory distress syndrome^{54, 167, 168}), elevate serum BUN^{47, 49, 53, 169} due to renal tubular injury , and result in mortality. Interestingly, elevated serum levels of TNF-α, IL-1β, MCP-1 and IL-18 can all be induced during this fatty acid toxicity, perhaps due to the widespread release of DAMPs. Importantly, inhibition of this excessive lipolysis can result in prevention of systemic injury, hypercytokinemia and mortality^{47-49, 53}. While it remains to be seen if lipase inhibition will reduce systemic injury during human AP, it is encouraging to note that the cyclooxygenase inhibitor indomethacin, which is known to affect the metabolism of UFA products, may reduce progression of moderate-severe AP.¹⁷⁰.

Role of Intestine in systemic injury:

The small intestine and colon may contribute to bacterial translocation and infected necrosis due to its close proximity to the pancreas, and thus to the pathophysiology of systemic inflammation. Another role may be played by mesenteric lymph from the GI tract. In an experimental study, intravenous administration of mesenteric lymph from rats with intestinal ischemia exacerbated pancreatic microcirculatory disturbances and worsened the severity of pancreatitis in the recipient rats.¹⁷¹ Similarly, profound vascular and coagulation changes can lead to ischemia of the pancreas^172-174 and bowel^{175, 176}. A lower gastric pH which correlated with a higher mortality in AP also supported the hypothesis of splanchnic ischemia¹⁷⁷. Additionally, Ischemia-reperfusion can cause oxidative stress¹⁷⁸. The major effect of such perturbations is gut barrier dysfunction with increased intestinal permeability¹⁷⁹, as supported by human studies¹⁸⁰ including a meta-analysis of 18 studies, which showed a pooled prevalence of gut barrier dysfunction of 59%¹⁸¹. However, parallel clinical observations question the gut as being the sole player. These include patients with severe ulcerative colitis having a very low prevalence of sepsis¹⁸² despite severe colonic ulcers being exposed to fecal matter for long periods. Similarly, while patients with fistulizing Crohn’s disease do develop abdominal abscesses, the prevalence of sepsis remains low¹⁸². The translocation hypothesis as being the sole reason for developing infected necrosis is further challenged by the fact that bacterial translocation is common after dental¹⁸³ and endoscopic¹⁸⁴ procedures but is transient. Thus, whether translocation of bacteria is the sole reason for infections of (peri) pancreatic necrotic tissue and collections, and the sepsis that ensues remains to be determined.

Mechanisms of Organ Dysfunction due to systemic perturbations – Lessons from Sepsis induced Organ Failure:

Mitochondria are at the center of cellular perturbations from tissue hypo-perfusion, which may result from hypovolemia, hypotension, and microvascular thrombi causing cellular hypoxia. Since mitochondrial energy production is oxygen dependent, their ability to generate ATP is severely compromised in such states, and can result in cellular dysfunction. OF may primarily be due to such mitochondrial dysfunction rather than cell death.¹⁸⁵ The following observations support this hypothesis: (i) Cellular injury and death is minimal in post-mortem examination of failed organs,¹⁸⁶ (ii) functional recovery of the organ is swift once the underlying pathophysiological perturbations reverse, and (iii) mitochondrial structural and functional changes have been documented in such patients. These observations are important from the point of prognosis and identifying targets for therapy.

Treatment of Organ Failure:

Treatment for OF is largely supportive. Patients with predicted severe AP should be referred to a tertiary care center with intensive care unit (ICU) facility. Patients with OF must be managed in an ICU and often require organ support such as dialysis, mechanical ventilation, and vasopressors.

Fluid and electrolyte balance is critical in the beginning of the illness. Ringer’s lactate has been shown to be better than normal saline in reducing systemic inflammation.^{187, 188} Optimal amount of fluid administration remains a challenge.¹⁸⁹ Both under-and aggressive hydration can be detrimental. A randomized trial showed worsening of OF in patients given aggressive fluid therapy¹⁹⁰. On the contrary, relative hypovolemia due to under correction of fluid deficit can lead to increased risk of necrosis. A recent trial showed benefit of aggressive fluid administration in mild AP.¹⁹⁰ But the results cannot be extrapolated to patients with severe AP because systemic events leading to OF develop rapidly and overzealous fluid therapy may exacerbate the clinical condition in those with impending respiratory and renal failure. One of the important points to consider is that normal homeostatic mechanisms are disturbed in patients with systemic injury due to abnormalities such as increased vascular permeability and thus the capability to deal with extra fluid being infused is compromised unlike in patients with mild AP.

Enteral nutrition should be instituted as soon as possible and has been shown to reduce the length of hospital stay and possibly the risk of infected necrosis.¹⁹¹ A multidisciplinary team comprising of a critical care expert, gastroenterologist, intervention radiologist and surgeon should look after the patient.

Specific therapy for Organ Failure:

Since the systemic injury is a result of dysregulated and out of proportion systemic inflammation in response to the local injury, specific treatment aimed at putative critical pathways has been tried. As mentioned above, PAF antagonist, Lexipafant, failed in a randomized controlled trial (RCT) involving 290 patients with predicted severe AP having an APACHE II score of >6. However, the treatment failed to provide any therapeutic benefit mainly because the primary hypothesis was invalidated by the unexpected finding that 44% of patients had OF on entry and only 14% patients developed new OF.¹⁵⁰ Intravenous antioxidants too failed in a RCT of 43 patients, the primary endpoint i.e. OF developed in 32% and 17% in antioxidant and placebo groups respectively.¹⁹² Probiotics were tried with an aim to prevent gut derived infection, but unexpectedly increased mortality leading to premature termination of the trial.¹⁹³ TNF-α has been implicated as an important cytokine mediating systemic inflammation. In a proof of concept small study involving 28 patients with predicted severe AP, Pentoxifyllin, an oral TNF-α antagonist, resulted in significantly fewer ICU admissions and shorter hospital stay.¹⁹⁴ A larger trial is currently in progress. Infliximab, a TNF-α antagonist, is being tested in a RCT involving patients with AP of all grades of severity (www.isrctn.com/ISRCTN16935761).

Unmet needs and potential areas for future research:

Organ failure remains the proverbial Achilles heel of managing patients with severe AP. There is no specific therapy available either to treat or prevent the development of OF. The success of anticytokine therapy in chronic diseases such as inflammatory bowel disease, psoriasis and rheumatoid arthritis^{195, 196} may not translate to AP. Future approaches include targeting intermediary signaling such as the increase in cytosolic calcium using ORAI1 inhibitors¹⁹⁷. The benefits of this approach remain to be seen in human AP, since ORAI1 is involved in innate immunity as well¹⁹⁸. Similarly, NF-kB has been shown to play a critical role in AP ^199-201. Pancreas-specific truncation of its trans-activating unit (RelA/p65) worsens pancreatic injury and lung inflammation. It would, therefore be important to know whether clinically approved agents such as Bortezomib ²⁰² that interfere with NF-kB signaling affect the course of human AP. Some other exciting potential targets include UFAs, lipase, DAMPs, inflammasome, and kynurenine²⁰³, which remain to be tested in human studies.

Biography