Inflection points in sepsis biology: from local defense to systemic organ injury

Sepsis and septic shock lead to considerable morbidity and mortality in developed and developing countries. Despite advances in understanding the innate immune events that lead to septic shock, molecular therapies based on these advances have failed to improve sepsis mortality. The clinical failure of laboratory-derived therapies may be, in part, due to the pleiotropic consequences of the acute inflammatory response, which is the focus of this review. A brisk response to infecting organism is essential for pathogen containment and eradication. However, systemic spread of inflammation beyond a single focus leads to organ injury and higher mortality. The primary goal of this article is to discuss recent animal- and human-based scientific advances in understanding the host response to infection and to highlight how these defense mechanisms can be locally beneficial but systemically detrimental. There are other factors that determine the severity of sepsis that are beyond the scope of this review, including the virulence of the pathogen and regulation by Toll-like receptors. Specifically, this review focuses on how the effector mechanisms of platelets, mast cells, neutrophil extracellular traps (NETs), and the endothelium participate in combating local infections yet can induce organ injury during systemic infection.     

Caspase-1 is hepatoprotective during trauma and hemorrhagic shock by reducing liver injury and inflammation

Adaptive immune responses are induced in liver after major stresses such as hemorrhagic shock (HS) and trauma. There is emerging evidence that the inflammasome, the multiprotein platform that induces caspase-1 activation and promotes interleukin (IL)-1β and IL-18 processing, is activated in response to cellular oxidative stress, such as after hypoxia, ischemia and HS. Additionally, damage-associated molecular patterns, such as those released after injury, have been shown to activate the inflammasome and caspase-1 through the NOD-like receptor (NLR) NLRP3. However, the role of the inflammasome in organ injury after HS and trauma is unknown. We therefore investigated inflammatory responses and end-organ injury in wild-type (WT) and caspase-1(-/-)mice in our model of HS with bilateral femur fracture (HS/BFF). We found that caspase-1(-/-) mice had higher levels of systemic inflammatory cytokines than WT mice. This result corresponded to higher levels of liver damage, cell death and neutrophil influx in caspase-1(-/-) liver compared with WT, although there was no difference in lung damage between experimental groups. To determine if hepatoprotection also depended on NLRP3, we subjected NLRP3(-/-) mice to HS/BFF, but found inflammatory responses and liver damage in these mice was similar to WT. Hepatoprotection was also not due to caspase-1-dependent cytokines, IL-1β and IL-18. Altogether, these data suggest that caspase-1 is hepatoprotective, in part through regulation of cell death pathways in the liver after major trauma, and that caspase-1 activation after HS/BFF does not depend on NLRP3. These findings may have implications for the treatment of trauma patients and may lead to progress in prevention or treatment of multiple organ failure (MOF).     

Multiple organ dysfunction syndrome.

The multiple organ dysfunction syndrome (MODS), though newly described, has manifested itself in intensive care unit (ICU) patients for several decades. As the name implies, it is a syndrome in which more than one organ system fails. Failure of these multiple organ systems may or may not be related to the initial injury or disease process for which the patient was admitted to the ICU. MODS is the leading cause of morbidity and mortality in current ICU practice. While the pathophysiology of MODS is not completely known, much evidence indicates that, during the initial injury which precipitates ICU admission, a chain of events is initiated which results in activation of several endogenous metabolic pathways. These pathways release compounds which, in and of themselves, are usually cytoprotective. However, an over exuberant activation of these endogenous systems results in an inflammatory response which can lead to development of failure in distant organs. As these organs fail, they activate and propagate the systemic inflammatory response. No therapy has proven entirely efficacious at modulating this inflammatory response and the incidence and severity of MODS. In current ICU practice, treatment is focused on prevention and treating individual organ dysfunction as it develops. With increased understanding of the pathophysiology of MODS therapy will come newer modalities which inhibit or interfere with the propagation of the endogenous systemic inflammatory response. These newer therapies hold great promise and already some are undergoing clinical investigation.     

Elevated urinary leukotriene E4 excretion in patients with ARDS and severe burns.

Increased synthesis of peptidoleukotrienes may occur in a variety of inflammatory diseases. To test this theory, hospitalized patients with a variety of diseases were studied and urine LTE4 quantitated as an index of total body peptidoleukotriene synthesis. 10 patients with ARDS, 7 of which had additional organ involvement, and 5 patients suffering from severe burn injuries were studied. Patients with uncomplicated ARDS excreted approximately 6-fold higher amounts of LTE4 in urine compared to healthy subjects. When ARDS was complicated by multiple organ failure (MOF), urine LTE4 levels were 2- to 150-fold higher than in healthy volunteers. Patients with severe burn injuries had peak urine LTE4 levels which were approximately 20-fold higher than in healthy volunteers. As additional controls, patients with cardiac arrhythmias (absence of inflammatory disease) and patients with uncomplicated pneumonia (localized inflammation) showed normal or mildly elevated urinary LTE4 levels. The urinary LTE4 levels in ARDS patients did not correlate with serum creatinine, bilirubin, or LDH levels, or with the WBC, nor did renal or liver failure by itself predict extremely elevated urinary LTE4 levels. In conclusion, patients with ARDS or ARDS/MOF and patients with severe injuries and sepsis syndrome excrete higher levels of urinary LTE4 than patients healthy volunteers or patients with limited inflammatory disease. In certain situations, urinary LTE4 levels may be useful as a marker of the degree of inflammation.     

The regulation of organ size in Drosophila: physiology, plasticity, patterning and physical force

The correct regulation of organ size is a fundamental developmental process, the failure of which can compromise organ function and organismal integrity. Consequently, the mechanisms that regulate organ size have been subject to intense research. This research has highlighted four classes of mechanism that are involved in organ size regulation: physiology, plasticity, patterning and physical force. Nevertheless, how these mechanisms are integrated and converge on the cellular process that regulate organ growth is unknown. One group of animals where this integration is beginning to be achieved is in the insects. Here, I review the different mechanisms that regulate organ size in insects, and describe our current understanding of how these mechanisms interact. The genes and hormones involved are remarkably conserved in all animals, so these studies in insects provide a precedent for future research on organ size regulation in mammals.     

In vivo stress preconditioning

The heat shock or stress protein response is a highly conserved defense mechanism. Activation of the stress protein response by mild hyperthermia or by pharmacological agents allows cells to withstand a subsequent metabolic insult that would otherwise be lethal, a phenomenon referred as "thermotolerance" or "preconditioning." Heat shock response is characterized by increased expression of stress proteins that provide cellular protection, e.g., via increased chaperoning activity in all organisms, from bacteria to animals and humans. Indeed, there is experimental evidence that overexpression of specific heat shock proteins or heat shock factors produce protective effects similar to those observed after stress preconditioning. The purpose of this review is first to discuss the methods used to induce in vivo thermotolerance with mild hyperthermia or pharmacological agents. Then, as an example of the organ protection provided by in vivo stress preconditioning, the second part of this paper will examine how the induction of thermotolerance modulates the lung inflammatory response associated with acute lung injury, thus providing broad organ and tissue protection against oxidative stress associated this syndrome.     

Intestinal preconditioning prevents inflammatory response by modulating heme oxygenase-1 expression in endotoxic shock model

Gut mucosal injury observed during ischemia-reperfusion is believed to trigger a systemic inflammatory response leading to multiple organ failure. It should be interesting to demonstrate this relationship between gut and multiple organ failure in a sepsis model. Intestinal preconditioning (PC) can be used as a tool to assess the effect of intestinal ischemia in inflammatory response after LPS challenge. The aim of this study was to investigate the protective effect of PC against LPS-induced systemic inflammatory and intestinal heme oxygenase-1 (HO-1) expression. ES was performed with LPS (10 mg/kg iv) with or without PC, which was done before LPS. Rats were first subjected to sham surgery or PC with four cycles of 1 min ischemia and 4 min of reperfusion 24 h before LPS challenge or saline administration. PC significantly reduced fluid requirements, lung edema, intestinal lactate production, and intestinal injury. Inflammatory mRNA expressions for intestine and lung ICAM and TNF were significantly reduced after PC, and these effects were significantly abolished by zinc-protoporphyrin (a specific HO-1 activity inhibitor) and mimicked by bilirubin administration. Intestinal PC selectively increased HO-1 mRNA expression in intestine, but we have observed no expression in lungs. These findings demonstrate that intestinal injury is a important event for inflammatory response and multiple organ injury after LPS challenge. Intestinal HO-1 expression attenuates LPS-induced multiple organ failure by modulating intestine injury and its consequences on inflammatory response. Identification of the exact mechanisms responsible for intestine HO-1 induction may lead to the development of new pharmacological interventions.     

Adding insult to injury - Inflammation at the heart of cardiac fibrosis

The fibrotic response has evolutionary worked in tandem with the inflammatory response to facilitate healing following injury or tissue destruction as a result of pathogen clearance. However, excessive inflammation and fibrosis are key pathological drivers of organ tissue damage. Moreover, fibrosis can occur in several conditions associated with chronic inflammation that are not directly caused by overt tissue injury or infection. In the heart, in particular, fibrotic adverse cardiac remodeling is a key pathological driver of cardiac dysfunction in heart failure. Cardiac fibroblast activation and immune cell activation are two mechanistic domains necessary for fibrotic remodeling in the heart, and, independently, their contributions to cardiac fibrosis and cardiac inflammation have been studied and reviewed thoroughly. The interdependence of these two processes, and how their cellular components modulate each other's actions in response to different cardiac insults, is only recently emerging. Here, we review recent literature in cardiac fibrosis and inflammation and discuss the mechanisms involved in the fibrosis-inflammation axis in the context of specific cardiac stresses, such as myocardial ischemia, and in nonischemic heart conditions. We discuss how the search for anti-inflammatory and anti-fibrotic therapies, so far unsuccessful to date, needs to be based on our understanding of the interdependence of immune cell and fibroblast activities. We highlight that in addition to the extensively reviewed role of immune cells modulating fibroblast function, cardiac fibroblasts are central participants in inflammation that may acquire immune like cell functions. Lastly, we review the gut-heart axis as an example of a novel perspective that may contribute to our understanding of how immune and fibrotic modulation may be indirectly modulated as a potential area for therapeutic research.     

Loss of adaptation to oxidative stress as a mechanism for aortic damage in aging rats

Cells are armed with a vast repertoire of antioxidant defence mechanisms to prevent the accumulation of oxidative damage. The cellular adaptive response is an important antioxidant mechanism against physiological and pathophysiological oxidative alterations in a cell's microenvironment. The aim of this paper was to study, in the rat aorta, whether this adaptive response and the inflammation associated with oxidative stress were expressed throughout the aging process. We examined the rat aorta, as it is a very sensitive tissue to oxidative stress. Male Wistar rats of 1.5, 3, 12, 18 and 24 months of age were used. Superoxide anion (O2(-)) generation; levels of two antioxidant enzymes, superoxide dismutase (SOD) and catalase; and the levels of prostaglandin E2 (PGE2), an inflammatory marker, were measured. The results for rats at different ages were compared with those for 3 months of age. A balance between production of O2(-) and SOD activity was found in the aorta of rats from 1.5 to 12 months old. Oxidative stress was present in the aorta of old animals (18-24 months), due to a failure in the mechanisms of adaptation to oxidative stress. The observed increase in PGE2 levels in these rats reflected an inflammatory response. All together suggest that vascular oxidative stress and the inflammatory process observed in the old groups of rats could be closely related to vascular aging. Our results also remark the importance of the adaptative response to oxidative stress.     

Cardiac fibroblasts: friend or foe?

Cardiac function is determined by the dynamic interaction of various cell types and the extracellular matrix that composes the heart. This interaction varies with the stage of development and the degree and duration of mechanical, chemical, and electrical signals between the various cell types and the ECM. Understanding how these complex signals interact at the molecular, cellular, and organ levels is critical to understanding the function of the heart under a variety of physiological and pathophysiological conditions. Quantitative approaches, both in vivo and in vitro, are essential to understand the dynamic interaction of mechanical, chemical, and electrical stimuli that govern cardiac function. The fibroblast can thus be a friend in normal function or a foe in pathophysiological conditions.     

Neurological complications of acute liver failure: pathophysiological basis of current management and emerging therapies

One of the major causes of mortality in patients with acute liver failure (ALF) is the development of hepatic encephalopathy (HE) which is associated with increased intracranial pressure (ICP). High ammonia levels, increased cerebral blood flow and increased inflammatory response have been identified as major contributors to the development of HE and the related brain swelling. The general principles of the management of patients with ALF are straightforward. They include identifying the insult causing hepatic injury, providing organ systems support to optimize the patient's physical condition, anticipation and prevention of development of complications. Increasing insights into the pathophysiological mechanisms of ALF are contributing to better therapies. For instance, the evident role of cerebral hyperemia in the pathogenesis of increased ICP has led to a re-evaluation of established therapies such as hyperventilation, N-acetylcysteine, thiopentone sodium and propofol. The role of systemic inflammatory response in the pathogenesis of increased ICP has also gained importance supporting the concept that antibiotics given prophylactically reduce the risk of developing sepsis during the course of illness. Moderate hypothermia has also been established as a therapy able to reduce ICP in patients with uncontrolled intracranial hypertension and to prevent increases in ICP during orthopic liver transplantation. Ornithine phenylacetate, a new drug in the treatment of liver failure, and liver replacement therapies are still being investigated both experimentally and clinically. Despite many advances in the understanding of the pathophysiological basis and the management of intracranial hypertension in ALF, more clinical trials should be conducted to determine the best therapeutic management for this difficult clinical event.     

Nutrition and inflammatory events: highly unsaturated fatty acids (omega-3 vs omega-6) in surgical injury.

Given the poor prognosis and high cost of care for patients with acute inflammatory responses (often leading to organ failure and/or allograft rejection), immunomodulation of this hyperresponse represents an important priority for research in nutritional medicine. The primary goal of nutritional support in inflammatory disease is to provide adequate energy, particularly through use of novel lipids (to alter eicosanoid pathway toward a more regulated inflammatory state), and protein to meet endogenous requirements for tissue repair IL-1 production, and restored cellular function, thus preventing secondary infection (52). Manipulation of macrophage eicosanoid production by use of omega-3 PUFA may reduce the cellular immune response (by competing with arachidonic acid, which produces inflammatory eicosanoids of the 2- and 4-series), whereas inclusion of MCT found in coconut oil may lower the arachidonic acid content of membrane phospholipids. As more data are obtained on the use of such tailored therapies in critically ill patients, a new generation of parenteral and enteral diets will be developed to reduce inflammation and immune dysfunction.     

Fat tissue,aging, and cellular senescence

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age‐related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro‐inflammatory, tissue‐remodeling, senescent‐like state. Other mesenchymal progenitors also can acquire a pro‐inflammatory, adipocyte‐like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro‐inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress‐responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent‐like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro‐inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.     

Integrated gut/liver microphysiological systems elucidates inflammatory inter‐tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut‐liver tissue interactions under normal and inflammatory contexts, via an integrative multi‐organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long‐term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut‐liver crosstalk. Moreover, significant non‐linear modulation of cytokine responses was observed under inflammatory gut‐liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA‐seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut‐liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut‐liver interaction also negatively affected tissue‐specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi‐tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648–2659. © 2017 Wiley Periodicals, Inc.     

Pathophysiology, staging and therapy of severe sepsis in baboon models

We review our baboon models of Escherichia coli sepsis that mimic, respectively, the shock/disseminated intravascular coagulation (DIC) and organ failure variants of severe sepsis, and analyse the pathophysiologic processes that are unique to each. The multi-stage, multi-factorial characteristics of severe sepsis develop as a result of the initial insult, which - depending on its intensity - activates components of the intravascular compartment leading to overwhelming shock/DIC; or initiates a sequence of events involving both the intra- and extravascular (tissues) compartments that lead to organ failure. In the latter case, the disorder passes through two stages: an initial inflammatory/coagulopathic intravascular first stage triggered by E. coli, followed by an extravascular second stage, involving components unique to each organ and triggered by ischemia/reperfusion (oxidative stress and histone release). Although a myriad of overlapping cellular and molecular components are involved, it is the context in which these components are brought into play that determine whether shock/DIC or organ failure predominate. For example, inflammatory and thrombotic responses amplified by thrombin in the first case whereas similar responses are amplified by complement activation products in the second. Rather than blocking specific mediators, we found that attenuation of the thrombin and complement amplification pathways can effectively reverse the shock/DIC and organ failure exhibited by the LD(100) and LD(50) E. coli models of severe sepsis, respectively. Translation of these concepts to successful intervention in the respective baboon models of E. coli sepsis and the application to their clinical counterparts is described.     

Extracellular matrix degradation in liver fibrosis: Biochemistry and regulation

Fibrosis is a highly conserved wound healing response and represents the final common pathway of virtually all chronic inflammatory injuries. Over the past 3 decades detailed analysis of hepatic extracellular matrix synthesis and degradation using approaches incorporating human disease, experimental animal models and cell culture have highlighted the extraordinarily dynamic nature of tissue repair and remodelling in this solid organ. Furthermore emerging studies of fibrosis in other organs demonstrate that basic common mechanisms exist, suggesting that bidirectionality of the fibrotic process may not solely be the preserve of the liver. In this review we will examine the cellular and molecular mechanisms that govern extracellular matrix degradation and fibrosis resolution, and highlight how manipulation of these processes may result in the development of effective anti-fibrotic therapies. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Flow cytometry analysis of atherosclerotic plaque cells from human carotids: a validation study

BACKGROUND: Atherosclerotic plaques are heterogeneous vascular lesions. Changes in cell plaque composition are fundamental events inside the plaque microenvironment that are strictly related to the clinical outcome of these lesions (organ damage). The knowledge of these modifications may help to better understand the pathophysiological mechanisms of atherosclerosis. METHODS: We report on a flow cytometry method to characterize and quantify the cell subpopulations in human atherosclerotic plaques. Cells were obtained from endarterectomy specimens after collagenase digestion. Both surface and intracytoplasmic antigens were labeled. RESULTS: Our data demonstrated that the method we described allowed the characterization of cell populations that compose the atherosclerotic plaque, avoiding contamination by tunica media smooth muscle cells and the noise of cellular debris. Moreover this validation study showed that about 50% of cells in the atherosclerotic plaques are inflammatory mononuclear cells (T lymphocytes and monocytes/macrophages). CONCLUSIONS: Reproducible quantitative methods for cell population characterization may increase the understanding of pathophysiological mechanisms responsible for plaque progression. The methodology herein described gave us the possibility of quickly calculating the relative amount of each cell population and studying both surface and intracellular markers to analyze the functional stage of the cells. The clinical correlation was not assessed in the present study, because we used a small patient group to validate the method, but should be the subject of further analyses in a larger patient population.     

The role of IRES trans-acting factors in regulating translation initiation

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.