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Traumatic brain injury (TBI) affects not only the brain but also peripheral organs like the heart and the lungs, which influences long-term outcomes. A heightened systemic inflammatory response is often induced after TBI, but the underlying pathomechanisms that contribute to co-morbidities remain poorly understood. Here, we investigated whether extracellular vehicles (EVs) containing inflammasome proteins are released after severe controlled cortical impact (CCI) in C57BL/6 mice and cause activation of inflammasomes in the heart that result in tissue damage. The atrium of injured mice at 3 days after TBI showed a significant increase in the levels of the inflammasome proteins AIM2, ASC, caspases-1, -8 and -11, whereas IL-1β was increased in the ventricles. Additionally, the injured cortex showed a significant increase in IL-1β, ASC, caspases-1, -8 and -11 and pyrin at 3 days after injury when compared to the sham. Serum-derived extracellular vesicles (EVs) from injured patients were characterized with nanoparticle tracking analysis and Ella Simple Plex and showed elevated levels of the inflammasome proteins caspase-1, ASC and IL-18. Mass spectrometry of serum-derived EVs from mice after TBI revealed a variety of complement- and cardiovascular-related signaling proteins. Moreover, adoptive transfer of serum-derived EVs from TBI patients resulted in inflammasome activation in cardiac cells in culture. Thus, TBI elicits inflammasome activation, primarily in the atrium, that is mediated, in part, by EVs that contain inflammasome- and complement-related signaling proteins that are released into serum and contribute to peripheral organ systemic inflammation, which increases inflammasome activation in the heart. [ABSTRACT FROM AUTHOR]

Traumatic Brain Injury (TBI) is a major cause of death and disability in the US and a recognized risk factor for the development of Alzheimer's disease (AD). The relationship between these conditions is not completely understood, but the conditions may share additive or synergistic pathological hallmarks that may serve as novel therapeutic targets. Heightened inflammasome signaling plays a critical role in the pathogenesis of central nervous system injury (CNS) and the release of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck from neurons and activated microglia contribute significantly to TBI and AD pathology. This study investigated whether inflammasome signaling after TBI was augmented in AD and whether this signaling pathway impacted biochemical and neuropathological outcomes and overall cognitive function. Five-month-old, 3xTg mice and respective wild type controls were randomized and underwent moderate controlled cortical impact (CCI) injury or served as sham/uninjured controls. Animals were sacrificed at 1 hour, 1 day, or 1 week after TBI to assess acute pathology or at 12 weeks after assessing cognitive function. The ipsilateral cerebral cortex was processed for inflammasome protein expression by immunoblotting. Mice were evaluated for behavior by open field (3 days), novel object recognition (2 weeks), and Morris water maze (6 weeks) testing after TBI. There was a statistically significant increase in the expression of inflammasome signaling proteins Caspase-1, Caspase-8, ASC, and interleukin (IL)-1β after TBI in both wild type and 3xTg animals. At 1-day post injury, significant increases in ASC and IL-1β protein expression were measured in AD TBI mice compared to WT TBI. Behavioral testing showed that injured AD mice had altered cognitive function when compared to injured WT mice. Elevated Aβ was seen in the ipsilateral cortex and hippocampus of sham and injured AD when compared to respective groups at 12 weeks post injury. Moreover, treatment of injured AD mice with IC100, an anti-ASC monoclonal antibody, inhibited the inflammasome, as evidenced by IL-1β reduction in the injured cortex at 1-week post injury. These findings show that the inflammasome response is heightened in mice genetically predisposed to AD and suggests that AD may exacerbate TBI pathology. Thus, dampening inflammasome signaling may offer a novel approach for the treatment of AD and TBI. [ABSTRACT FROM AUTHOR]

Background: The inflammasome plays an important role in the inflammatory innate immune response after central nervous system (CNS) injury. Inhibition of the inflammasome after traumatic brain injury (TBI) results in improved outcomes by lowering the levels of caspase-1 and interleukin (IL)-1b. We have previously shown that inflammasome proteins are elevated in the cerebrospinal fluid (CSF) of patients with TBI and that higher levels of these proteins were consistent with poorer outcomes after TBI when compared to patients that presented these inflammasome proteins at lower levels. Methods and findings: Here we extend our work by analyzing serum from 21 TBI patients and CSF from 18 TBI patients compared to 120 serum samples and 30 CSF samples from no-TBI donor controls for the expression of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), interleukin(IL)-1b and IL-18. Analysis was carried out using the Ella Simple Plex system (Protein Simple) to determine the sensitivity and specificity of inflammasome proteins as biomarkers of TBI. Receiver operator characteristic (ROC) curves, confidence intervals and likelihood ratios for each biomarker was determined. ROC curves, confidence intervals, sensitivity and specificity for each biomarker examined revealed that caspase-1 (0.93 area under the curve (AUC)) and ASC (0.90 AUC) in serum and ASC (1.0 AUC) and IL-18 (0.84 AUC) in CSF are promising biomarkers of TBI pathology. Importantly, higher protein levels (above 547.6 pg/ml) of ASC (0.91 AUC) were consistent with poorer outcomes after TBI as determined by the Glasgow Outcome Scale-Extended (GOSE). Conclusion: These findings indicate that inflammasome proteins are excellent diagnostic and predictive biomarkers of TBI. [ABSTRACT FROM AUTHOR]

Traumatic brain injury elicits acute inflammation that in turn exacerbates primary brain damage. A crucial part of innate immunity in the immune privileged central nervous system involves production of proinflammatory cytokines mediated by inflammasome signaling. Here, we show that the nucleotide-binding, leucine-rich repeat pyrin domain containing protein 1 (NLRP1) inflammasome consisting of NLRP1, caspase-1, caspase-11, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), the X-linked inhibitor of apoptosis protein, and pannexin 1 is expressed in neurons of the cerebral cortex. Moderate parasagittal fluid-percussion injury (FPI) induced processing of interleukin-1β, activation of caspase-1, cleavage of X-linked inhibitor of apoptosis protein, and promoted assembly of the NLRP1 inflammasome complex. Anti-ASC neutralizing antibodies administered immediately after fluid-percussion injury to injured rats reduced caspase-1 activation, X-linked inhibitor of apoptosis protein cleavage, and processing of interleukin-1β, resulting in a significant decrease in contusion volume. These studies show that the NLRP1 inflammasome constitutes an important component of the innate central nervous system inflammatory response after traumatic brain injury and may be a novel therapeutic target for reducing the damaging effects of posttraumatic brain inflammation.Journal of Cerebral Blood Flow & Metabolism (2009) 29, 1251–1261; doi:10.1038/jcbfm.2009.46; published online 29 April 2009 [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF) plays a critical role in pathomechanisms associated with secondary damage after traumatic brain injury (TBI). The TNF ligand-receptor system stimulates inflammation by activation of gene transcription through the IκB kinase (IKK)–NF-κB and c-Jun NH2-terminal kinase (JNK)–AP-1 signaling cascades. TNF signaling following TBI involves both cell survival and apoptotic pathways, but the mechanism that accounts for the dual role of TNF remains unclear. Multiple studies have reported hypothermia to be protective following TBI, but the precise mechanism has not been clearly defined. Here, TNFR1 signaling pathways were investigated in the cerebral cortex of adult male Sprague–Dawley rats subjected to moderate fluid-percussion TBI and of naïve controls. Another group was subjected to moderate TBI with 30 min of pre- and post-traumatic hypothermia (33 °C). Rapid and marked increases in protein expression of TNFR1 and signaling intermediates in both the IKK–NF-κB and JNK pathways were induced in traumatized cortices. Hypothermia decreased TNFR1 protein expression acutely in traumatized cortices and stimulated early activation of signaling intermediates in the JNK, but not the IKK–NF-κB, signaling pathways. Hypothermia promoted a rapid activation of caspase-3 acutely after injury but suppressed caspase-3 activation at later time points. Moreover, hypothermia treatment suppressed cleavage of X-linked inhibitor of apoptosis protein (XIAP) into fragments induced by TBI. These data suggest that hypothermia may regulate both the JNK signaling cascade via XIAP and the preconditioning pathways that activate caspases. Thus, hypothermia mediates TNFR1 responses via early activation of the JNK signaling pathway and caspase-3, leading to endogenous neuroprotective events. [ABSTRACT FROM AUTHOR]

The tumor necrosis factor (TNF) ligand-receptor system plays an essential role in apoptosis that contributes to secondary damage after traumatic brain injury (TBI). TNF also stimulates inflammation by activation of gene transcription through the IκB kinase (IKK)/NF-κB and INK (c-Jun N-terminal protein kinase)/AP-1 signaling cascades. The mechanism by which TNF signals between cell death and survival and the role of receptor localization in the activation of downstream signaling events are not fully understood. Here, TNF receptor 1 (TNFR1) signaling complexes in lipid rafts were investigated in the cerebral cortex of adult male Sprague Dawley rats subjected to moderate (1.8-2.2 atmospheres) fluid-percussion TBI and naive controls. In the normal rat cortex, a portion of TNFR1 was present in lipid raft microdomains, where it associated with the adaptor proteins TRADD (TNF receptor-associated death domain), TNF receptor-associated factor-2 (TRAF-2), the Ser/Thr kinase RIP (receptor-interacting protein), TRAF1, and cIAP-1 (cellular inhibitor of apoptosis protein-1), forming a survival signaling complex. Moderate TBI resulted in rapid recruitment of TNFR1, but not TNFR2 or Fas, to lipid rafts and induced alterations in the composition of signaling intermediates. TNFR1 and TRAF1 were polyubiquitinated in lipid rafts after TBI. Subsequently, the signaling complex contained activated caspase-8, thus initiating apoptosis. In addition, TBI caused a transient activation of NF-κB, but receptor signaling interacting proteins IKKα and IKKβ were not detected in raft-containing fractions. Thus, redistribution of TNFR1 in lipid rafts and nonraft regions of the plasma membrane may regulate the diversity of signaling responses initiated by these receptors in the normal brain and after TBI. [ABSTRACT FROM AUTHOR]

The inhibitor of apoptosis (IAP) gene family prevents cell death by binding to and inhibiting caspases. In addition to negatively regulating apoptosis, IAPs bind to signaling intermediates and receptors, and participate in diverse cellular functions. Here, we describe the physiological functions of IAPs and their participation in injury and disease processes. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) patients frequently develop cardiopulmonary system complications such as acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). However, the mechanism by which TBI causes ALI/ARDS is not fully understood. Here, we used a severe TBI model to examine the effects of a low-molecular-weight heparin, enoxaparin, on inflammasome activation and lung injury damage. We investigated whether enoxaparin inhibits ALI and inflammasome signaling protein expression in the brain and lungs after TBI in mice. C57/BL6 mice were subjected to severe TBI and were treated with vehicle or 1 mg/kg of enoxaparin 30 min after injury. Lung and brain tissue were collected 24 h post-TBI and were analyzed by immunoblotting for expression of the inflammasome proteins, caspase-1 and interleukin (IL)-1β. In addition, lung tissue was collected for histological analysis to determine ALI scoring and neutrophil and macrophage infiltration post-injury. Our data show that severe TBI induces increased expression of inflammasome proteins caspase-1 and IL-1β in the brain and lungs of mice after injury. Treatment with enoxaparin attenuated inflammasome expression in the brain and lungs 24 h after injury. Enoxaparin significantly decreased ALI score as well as neutrophil and macrophage infiltration in lungs at 24 h after injury. This study demonstrates that enoxaparin attenuates ALI and inhibits inflammasome expression in the brain and lungs after TBI. These findings support the hypothesis that inhibition of the neural-respiratory inflammasome axis that is activated after TBI may have therapeutic potential. [ABSTRACT FROM AUTHOR]

Approximately 20–25% of traumatic brain injury (TBI) subjects develop acute lung injury (ALI), but the pathomechanisms of TBI-induced ALI remain poorly defined. Our previous work has shown that the inflammasome plays a critical role in TBI-induced secondary pathophysiology and that inflammasome proteins are released in extracellular vesicles (EV) after TBI. Here we investigated whether EV-mediated inflammasome signaling contributed to the etiology of TBI-induced ALI. C57/BL6 male mice were subjected to controlled cortical impact (CCI), and the brains and lungs were examined for inflammasome activation and ALI at 4 and 24 h after TBI. We show that TBI releases EV containing inflammasome proteins into serum that target the lung to cause ALI, supporting activation of a neural-respiratory-inflammasome axis. Administration of a low-molecular-weight heparin (enoxaparin, a blocker of EV uptake) or treatment with a monoclonal antibody against apoptosis speck-like staining protein containing a caspase recruitment domain (anti-ASC) after adoptive transfer of EV isolated from TBI-injured mice significantly inhibited inflammasome activation in the lungs of recipient mice resulting in improved ALI scores.This axis constitutes an important arm of the innate inflammatory response in lung pathology after TBI and targeting this axis represents a novel therapeutic treatment for TBI-induced ALI. [ABSTRACT FROM AUTHOR]

Abstract: Sex influences histological and behavioral outcomes following traumatic brain injury (TBI), but the underlying sex-dependent pathomechanisms regulating outcome measures remain poorly defined. Here, we investigated the TBI-induced regulation of the X-linked inhibitor of apoptosis protein (XIAP) that, in addition to suppressing cell death by inhibition of caspases, is involved in signaling cascades, including immune regulation and cell migration. Since estrogen has been shown to have anti-apoptotic properties, we specifically examined sex differences and the influence of estrogen on XIAP processing after TBI. Sprague–Dawley male (TBI-M), female (TBI-F), ovariectomized female (TBI-OVX) and ovariectomized females supplemented with estrogen (TBI-OVX+EST) were subjected to moderate (1.7–2.2atm) fluid percussion (FP) injury. Animals were sacrificed 24h after FP injury; cortical tissue (ipsilateral and contralateral) was dissected and analyzed for XIAP processing by immunoblot analysis (n =6–7/group) or confocal microscopy (n =2–3/group). Significant differences in XIAP cleavage products in the ipsilateral cortex were found between groups (p <0.03). Post hoc analysis showed an increase in XIAP processing in both TBI-F and TBI-OVX+EST compared to TBI-M and TBI-OVX (p <0.05), indicating that more XIAP is cleaved following injury in intact females and TBI-OVX+EST than in TBI-M and TBI-OVX groups. Co-localization of XIAP within neurons also demonstrated sex-dependent changes. Based on these data, it appears that the processing of XIAP after injury is different between males and females and may be influenced by exogenous estrogen treatment. [Copyright &y& Elsevier]

Pulmonary dysfunction often occurs in patients with severe Traumatic Brain Injury (TBI). Acute lung injury (ALI) is one of the most common respiratory complications after TBI, which is seen in 20-25% of patients with TBI. The DNA binding protein, high mobility group box protein 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that is immediately released after TBI and has been implicated in TBI-induced pulmonary dysfunction. However, the precise underlying molecular mechanisms remain unclear. HMGB1 is also known to activate the DNA sensing inflammasome, absent in melanoma (AIM2) and may be involved in the pathomechanisms of TBI-induced ALI. We hypothesized that the AIM2 inflammasome plays a central role in innate immune signaling after TBI, thus resulting in the development of ALI. C57/BL-6 mice were injured at 6m/s using a Controlled Cortical Impact (CCI) injury and were sacrificed at 4 and 24 hours post-injury. Mouse lung and brain tissue lysates were collected for inflammasome protein expression using Western Blot analysis. Mouse lungs were perfused intratracheally for immunohistochemical analysis. Lungs were sectioned and stained with hematoxylin and eosin (H&E) for evidence of alveolar edema and inflammatory cell infiltration. At 4 and 24 hours after TBI there was a significant increase in the levels of inflammasome proteins caspase-1, apoptosis speck like protein containing a caspase recruiting domain (ASC), Interleukin-18, AIM2, and HMGB1 in mouse brain and lung lysates. Lungs showed changes in alveolar morphology changes at 4 and 24 hours, which were more pronounced at 4 hours. In addition, lung sections showed co-localization of caspase-1 and ASC with surfactant protein C (SPC), a type II alveolar epithelial cell marker, at 4 and 24 hours post-TBI. Our results support the hypothesis that inflammasome signaling plays a role in the pathophysiology of ALI after TBI, and may identify novel targets for therapeutic intervention in TBI-induced ALI and pulmonary dysfunction. [ABSTRACT FROM AUTHOR]