Sepsis-Associated Encephalopathy and Blood-Brain Barrier Dysfunction

Abstract

Sepsis is a life-threatening clinical condition caused by a dysregulated host response to infection. Sepsis-associated encephalopathy (SAE) is a common but poorly understood neurological complication of sepsis, which is associated with increased morbidity and mortality. SAE clinical presentation may range from mild confusion and delirium to severe cognitive impairment and deep coma. Important mechanisms associated with SAE include excessive microglial activation, impaired endothelial barrier function, and blood-brain barrier (BBB) dysfunction. Endotoxemia and pro-inflammatory cytokines produced systemically during sepsis lead to microglial and brain endothelial cell activation, tight junction downregulation, and increased leukocyte recruitment. The resulting neuroinflammation and BBB dysfunction exacerbate SAE pathology and aggravate sepsis-induced brain dysfunction. In this mini-review, recent literature surrounding some of the mediators of BBB dysfunction during sepsis is summarized. Modulation of microglial activation, endothelial cell dysfunction, and the consequent prevention of BBB permeability represent relevant therapeutic targets that may significantly impact SAE outcomes.

BACKGROUND

Sepsis is a life-threatening critical condition in which an overactive systemic immune response to a microbial infection leads to organ dysfunction (45). This overwhelming inflammatory response of the host immune system prominently represents an important challenge for the current healthcare system (45). The multi-faceted pathophysiology of sepsis makes it a difficult condition to treat. Infection triggers both a sustained exacerbated inflammatory response (5) and apoptosis-related immunosuppression (2, 22) within the host to amplify the physiological immune response. Initial onset of sepsis causes a hyperinflammatory immune response. As sepsis is prolonged, apoptosis-induced decreases in lymphocytes are observed (22). Sepsis-induced immunosuppression has been also reported to increase the susceptibility to opportunistic infections that further aggravate the severity of sepsis (2).

With over 19.4 million cases and 5.3 million deaths across the globe annually (41), sepsis is a major pathological syndrome that currently lacks a targeted therapeutic strategy. The complexity of sepsis pathology poses a unique challenge in diagnosing and treating the condition. Clinical presentation of sepsis is often heterogeneous because the sepsis-induced host response heavily depends on several external factors, including age, source of infection, virulence of the pathogen, and time point of diagnosis (10, 28). Other risk factors that contribute to the heterogeneity of sepsis include comorbidities such as cardiovascular disease and diabetes (43) or undergoing a recent surgical procedure (26). This heterogeneity is portrayed by common non-specific patient symptoms that encompass fever, increased respiratory and heart rate (23), and neurological symptoms (13), increasing the difficulty of timely sepsis diagnosis. Additionally, the presentation of sepsis is highly dependent on the organ systems affected which might include the heart (38), lungs (40), and central nervous system (CNS) (39), and several others as seen in sepsis-induced multiple organ dysfunction syndrome (MODS). This mini-review discusses recent advances in our knowledge on the specific effects of sepsis on the CNS and its role in triggering sepsis-associated encephalopathy (SAE) and blood-brain barrier (BBB) disruption.

COMPROMISE OF THE CNS BY SEPSIS

SAE is a poorly understood acute cerebral dysfunction that appears in the setting of sepsis, affecting as many as 71% of patients diagnosed with the condition (7). As an indicator of septic infection (42), diagnosis of SAE occurs primarily through the detection of abnormalities in electroencephalogram recordings (31) and abnormal mental status (13), along with clinical history, physical examination, laboratory tests, and neuroimaging evaluation (13). Delirium, in the form of confusion and emotional dysfunction, often occurs at the onset of SAE (13). This initial mental disturbance is quickly followed by rapidly increasing degradation of the mental state characterized by deficits in both memory and verbal abilities, and possible onset of coma (44). The implications of sepsis on cerebral function are profound and the damage generated by SAE on the CNS appears to be permanent as determined by an autopsy study; signs of ischemia can be observed in several brain areas including the hippocampus, amygdala, frontal cortex, and hypothalamus (20).

BLOOD-BRAIN BARRIER DYSFUNCTION IN SEPSIS

The blood-brain barrier (BBB) is a highly selective and dynamic interface between the brain parenchyma and cerebral circulation formed by brain endothelial cells, pericytes, astrocytes, and surrounding microglial cells. Under normal conditions, the BBB serves as a physical barrier because tight junctions (TJ) between adjacent endothelial cells restrict molecules from diffusing through endothelial cells, forcing most molecular traffic to take a controlled transcellular route across the BBB (4). The BBB helps to maintain homeostasis of the brain microenvironment by restricting ionic and fluid movements between the blood and the brain, supplying the brain with essential nutrients and mediating efflux of several waste products (1). The barrier also plays an important role in brain immunity by restricting the infiltration of leukocytes from the periphery into the CNS (4).

Increasing evidence indicates that BBB integrity is compromised during sepsis. A recent preclinical study demonstrated that BBB permeability is increased as early as 24 h in the cerebral cortex, perirhinal cortex, hippocampus, and thalamus of rats after a single intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) (10 mg/kg in 100 μL saline), the major component of the outer membrane of Gram-negative bacteria, when compared to saline-treated animals (49). In humans, brain tissue samples obtained from deceased sepsis patients revealed a significant down-regulation of the TJ proteins occludin, claudin-5, and zonula occludens-1(ZO-1) in microvascular endothelial cells (9), suggesting impaired BBB function.

Dysfunction of the BBB contributes greatly to the pathophysiology of sepsis and SAE, as the CNS becomes highly vulnerable to neurotoxic factors such as free radicals, inflammatory mediators, intravascular proteins, plasma, and circulating leukocytes (17, 50). Consequently, barrier deficiencies lead to brain edema formation and reduced microvascular perfusion, contributing to and exacerbating neuronal loss (50) in SAE. Several mechanisms and multiple cell types are likely to be involved in the BBB dysfunction induced by SAE (Fig. 1).

Fig. 1.

Systemic inflammation contributes to BBB dysfunction during sepsis. A Schematic figure depicting how sepsis triggers multiple pro-inflammatory signaling pathways and induces the systemic upregulation of TNFα, IL-6, IL-1β, and MCP1 cytokine production. Inflammatory cytokines produced systemically also contribute to brain endothelial cell activation, initiation of the NF-κB pathway in brain endothelial cells, and downregulation of tight junction proteins ZO-1 and claudin-5, a hallmark of BBB dysfunction. B Intravital microscopy of pial venules using epifluorescence was performed and leukocyte-endothelial interactions are seen in mouse representative images from wild-type C57BL/6 mice 2 h after intraperitoneal injection of saline or LPS (10 μg/mouse). Saline image depicts baseline pial venule without inflammation and LPS image depicts adherent fluorescently-labeled leukocytes (green) to venule during inflammation (images adapted from Gavins et al., (12) and permissions were provided by John Wiley and Sons and Copyright Clearance Center).

MEDIATORS OF BBB DYSFUNCTION DURING SEPSIS

Microglial Cells

Microglial cells are primary initiators of brain immune responses and active surveyors of the brain parenchyma. These cells are part of the neurovascular unit that constitutes the BBB and are in close association with cerebral blood vessels (19). Recent experimental evidence has shown that microglial cells migrate to cerebral vessels during systemic inflammation and that their activation represents one of the earliest changes observed in SAE (19).

Pro-inflammatory cytokines that are produced systemically during sepsis have been shown to enter the CNS by several mechanisms that include transcellular diffusion, solute carrier proteins, receptor-mediated transcytosis, and adsorptive transcytosis (8). Once these inflammatory mediators reach the brain tissue, microglial cells are activated (51) and, as a consequence, an inflammatory signaling cascade that upregulates the transcription of several inflammatory mediators such as tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), and monocyte chemoattractant protein 1 (MCP1) (46) is initiated (Fig. 2). While intended to be a defense response against sepsis, microglial activation generates a cytotoxic environment further inducing the release of reactive oxygen species (ROS), nitric oxide (NO), and glutamate (46). Persistent microglial activation and the excessive release of inflammatory mediators and free radicals perpetuate a vicious cycle leading to aberrant neuronal function and cell death, contributing to the progression of SAE. A recent study has shown that microglial cells have a critical dual role in regulating BBB permeability following systemic LPS administration (1 mg/kg, daily for up to 7 days). The authors have shown that during the early stages (day 3) of LPS-induced inflammation, surrounding microglial cells migrate to cerebral microvessels in response to the release of the chemokine CCL5 from brain microvascular endothelial cells. This triggers microglial cells to infiltrate through the neurovascular unit, to make close contact with brain microvascular endothelial cells, and to express the junctional protein Claudin-5. These observations strongly suggest that areas of contact between microglial cells and brain endothelial cells are formed to strengthen the integrity of the BBB during the early phase of systemic inflammation. Conversely, sustained inflammation induced by daily injections of LPS (1.0 mg/kg i.p.) for 7 days induces a microglial phagocytic phenotype associated with morphological changes, engulfment of astrocytic end-feet fragments, and increased BBB permeability (19).

Fig. 2.

Microglial activation exacerbates the inflammatory response of the CNS during sepsis. A Schematic representation of how cytokines produced systemically during sepsis are inducted into the CNS by transcytosis and carrier proteins. Induction of cytokines causes microglial cells to become activated, leading to increases in TNFα, IL-1β, and MCP1 transcription within the CNS. Microglial activation also leads to the release of inflammatory mediators, ROS, NO, and glutamate, causing cerebral inflammation to become amplified. B Series of typical images from a mouse motor cortex following single daily injections of LPS (1 mg/kg, i.p.) for up to 7 days showed microglial (green) migration to a brain vessel (red) and the associated changes in microglia morphology (18) (images adapted from Haruwaka et al., 2019 and permissions provided by a Creative Commons 4.0 license ).

In line with a detrimental role for sustained microglial activation in BBB dysfunction, several studies suggest that attenuation of microglial activation prevents increases in BBB permeability and reduces the extent of SAE, but mechanisms are incompletely understood. A critical role for the microglial CD40-CD40 ligand (L), a membrane protein found in inflammatory cells and its ligand found expressed in the surface of immune cells, signaling pathway was demonstrated in a cecal ligation and puncture (CLP) model of sepsis (32). Both CD40-CD40L protein levels were found upregulated in the hippocampus 48 h after CLP induction, which was significantly abrogated when rats were treated with minocycline, a microglial inhibitor. In a series of in vivo and in vitro studies, the authors further demonstrated that the treatment of animals with an anti-CD40 prevented CLP-induced TNFα, IL-β, and IL-6 increased protein levels in the hippocampus, as well as LPS-induced TNFα, IL-β, and IL-6 in cultured primary microglial cells (32). In addition, rats who received the anti-CD40 treatment exhibited decreased hippocampal BBB permeability and long-term cognitive dysfunction following CLP-induced SAE (32). These findings indicate that glial activation plays a key role in exacerbating SAE pathology and BBB dysfunction.

Brain Endothelial Cells

Brain endothelial cells are critical components of the BBB. The presence of specialized TJ between adjacent endothelial cells seals the interendothelial cleft leading to high electrical resistance and very limited molecular flux through these cells. The junctional complex is formed by proteins including occludin, claudins, and junctional adhesion molecules (JAM)-A, JAM-B, and JAM-C (1). These transmembrane junctional complex proteins interact with cytoskeletal scaffolding proteins such as ZO-1, ZO-2, and ZO-3, the Ca²⁺-dependent serine protein kinase (CASK), and MAGI-1, MAGI-2, and MAGI-3 (membrane-associated guanylate kinase with inverted orientation of protein-protein interaction domains) that anchor the junctional complex to the actin cytoskeleton (1).

Endotoxemia and pro-inflammatory cytokines produced systemically during sepsis lead to activation of brain endothelial cells, increases in brain endothelial permeability, and altered BBB function. In vitro studies have demonstrated that exposure of mouse brain endothelial cells to plasma obtained from polymicrobial septic mice (single 1-mL i.p. injection of murine fecal material [20% wt/vol in saline]) resulted in downregulation of occludin and increased monolayer permeability (17). In addition, LPS (single i.p. injection, 18 mg/kg) has been shown to increase BBB permeability by leading to the downregulation of the TJ proteins ZO-1 and occludin in rat brain microvascular endothelial cells (27).

Other consequences of endothelial activation by LPS include the activation of the nuclear factor kappa beta (NFκB pro-inflammatory signaling pathway in brain micro-vascular endothelial cells (18). This signaling process promotes transcription of the inflammatory mediator cyclooxygenase-2 leading to increased BBB permeability via prostaglandin E2 synthesis, as demonstrated in murine brain endothelial cells (29). Additionally, activation of the brain endothelium leads to leukocyte recruitment and infiltration, enhanced activity of the coagulation cascade, and microthrombus formation (53, 57). During endothelial damage, thrombin, which is the major regulator in the coagulation pathway, is activated from the cleavage of prothrombin by Factor X. Thrombin then converts soluble fibrinogen to fibrin and activates platelets, forming micro-occlusions (11, 24, 30). The continuous formation of microthrombus exacerbates focal ischemia by occluding the vasculature beyond the initial occlusion sites, and further contribute to increases in BBB permeability (3, 33).

A cytosolic protein, the inflammatory regulator protein kinase C-delta (PKCδ), is also involved in mediating BBB damage during SAE. PKCδ depletion in isolated cardiomyocytes was found to reduce ROS production and prevent changes in inner mitochondrial membrane potential after LPS treatment (20 ng/mL for 1 h) (25). In rats, CLP-induced PKCδ activation and BBB permeability, as evaluated by Evans Blue extravasation into the brain tissue, were attenuated by the treatment with a PKCδ peptide inhibitor, PKCδ-TAT (48). In the same study, in vitro data further revealed that inhibition of PKCδ in human brain microvascular endothelial cells suppressed TNFα-induced neutrophil-endothelial cell adhesion and TJ protein degradation and prevented barrier dysfunction as measured by transendothelial electrical resistance (48).

MAJOR SIGNALING PATHWAYS

Sphingolipid Metabolism

Sphingolipids are 18-carbon fatty acid chains that are constituents of the plasma membrane particularly abundant in the brain tissue (36). Sphingolipids are not merely structural elements but are also known to be involved in a variety of signaling pathways and physiological functions, including angiogenesis, vascular permeability, cell proliferation, cytokine/chemokine generation, and apoptosis (15). The signaling sphingolipid sphingosine-1-phosphate (S1P), in particular, has been shown to be essential for the maintenance of the functional integrity of the CNS for its role in mediating neuroinflammation and BBB dysfunction (52). S1P is generated by the phosphorylation of sphingosine by sphingosine kinase (SphK) (34). Published observations indicate that knockdown of SphK1 in mice resulted in elevated microglial activation after neuroinflammation induced by intracerebral LPS injection (1 mg/kg), suggesting that SphK1 activation is involved in the regulation of LPS-induced neuroinflammation (15).

S1P is considered to be important for promoting endothelial barrier maintenance and S1P plasma and brain concentrations seem to be inversely correlated with sepsis severity and BBB permeability, as demonstrated in LPS (4 mg/kg, i.p.,) and polymicrobial (human stool suspension, i.p.) sepsis models (52, 54, 56). In vitro treatment with S1P increased the baseline tightness of monolayers of brain endothelial cells in a dose-dependent manner, as measured by transendothelial electrical resistance (55). In an LPS-induced SAE mice model (4 mg/kg, single i.p. injection), a significant downregulation of S1P levels was observed in blood serum, cerebral vasculature, and brain 4 h after the treatment (52). However, LPS induced a selective upregulation of S1P receptor type 1 in the cerebral vasculature, while claudin-5 was found downregulated, suggesting a role for sphingolipid metabolism in LPS-induced BBB disruption (52).

Mitochondrial Dysfunction

As sepsis reaches CNS, deleterious changes in endothelial mitochondrial function have also been reported. Mitochondrial dysfunction in the form of cytochrome C release (6) and excessive mitochondrial fission (37) has been implicated in mediating BBB dysfunction in stroke, in Alzheimer’s disease, and in other neuroinflammatory conditions (6, 14, 47).

Recent studies have been also exploring the contribution of altered mitochondrial dynamics in the context of BBB dysfunction in SAE. A study investigating dynamin-related protein 1 (Drp1), a key protein involved in mitochondrial fission and dysfunction, found that the Drp1 inhibitor P110 reduced mitochondrial fragmentation and reactive oxygen species (ROS) production, subsequently improving mitochondrial membrane potential and mitochondrial integrity (37). P110 is a peptide inhibitor that prevents Drp1 from binding to the mitochondrial membrane protein Fis1, leading to inhibition of the mitochondrial fission signaling cascade (37). In a different study, the treatment of mice with P110 prevented ZO-1 and occludin downregulation as well as TNFα, ILI-β, and IL-6 pro-inflammatory cytokine secretion in the brain tissue following LPS-induced SAE (8 mg/kg, i.p.) (16). In addition, P110 reduced the levels of released mitochondrial cytochrome C, a potent apoptotic trigger, and decreased LPS-induced sepsis symptoms (16). The effects of P110 treatment were further investigated in an in vitro BBB model in which brain microvascular endothelial cells were co-cultured with astrocytes. Treatment with P110 decreased mitochondrial ROS levels, improved mitochondrial membrane potential, and prevented paracellular permeability to FITC-dextran induced by LPS (0.1 μg/mL for 24 h). This data suggest that sepsis-induced impairment of BBB appears to be dependent on Drp1-mediated mitochondrial dysfunction (16).

Another regulator of mitochondrial function and many other important fundamental cellular responses that have been associated in mediating BBB dysfunction during LPS-induced SAE is polymerasδ-interacting protein 2 (Poldip2) (27, 35). Studies have shown that Poldip2 mediates mitochondrial respiration (35) and alterations of mitochondrial morphology (21), while also loss of Poldip2 protects against in vitro LPS-induced (1 μg/mL)

endothelial permeability (27). Heterozygous depletion of Poldip2 prevented Evans Blue dye extravasation in mouse brains after LPS treatment (18 mg/kg, i.p.) through the cyclooxygenase-2 signaling pathway (27). As multiple mitochondrial-associated proteins seemingly also engage in instigating BBB dysfunction during sepsis, it is likely that mitochondrial dysfunction is a significant factor in cerebral inflammation associated with SAE.

CONCLUDING REMARKS

This mini-review highlights how BBB dysfunction is a prominent clinical manifestation of sepsis and SAE. Endotoxemia and pro-inflammatory cytokines produced systemically during sepsis reduce the integrity of endothelial junctional proteins and activate microglial cells further triggering a pro-inflammatory cytokine signaling cascade within the CNS, which has been shown to exacerbate SAE pathology. Major mediators of SAE-induced endothelial barrier dysfunction that have been discussed in recent literature include glial cell activation, endothelial barrier dysfunction, sphingolipid metabolism, and mitochondrial dysfunction, all four of which can serve as promising relevant therapeutic targets that may significantly impact SAE outcomes reducing susceptibility to cognitive disorders. However, many aspects of sepsis physiopathology remain to be understood, particularly how sepsis-associated BBB injury leads to the characteristic excessive BBB dysfunction seen in SAE.