Pharmacological inhibition of STING reduces neuroinflammation-mediated damage post-traumatic brain injury.

BACKGROUND AND PURPOSE: Traumatic brain injury (TBI) remains a major public health concern worldwide with unmet effective treatment. Stimulator of interferon genes (STING) and its downstream type-I interferon (IFN) signalling are now appreciated to be involved in TBI pathogenesis. Compelling evidence have shown that STING and type-I IFNs are key in mediating the detrimental neuroinflammatory response after TBI. Therefore, pharmacological inhibition of STING presents a viable therapeutic opportunity in combating the detrimental neuroinflammatory response after TBI. EXPERIMENTAL APPROACH: This study investigated the neuroprotective effects of the small-molecule STING inhibitor n-(4-iodophenyl)-5-nitrofuran-2-carboxamide (C-176) in the controlled cortical impact mouse model of TBI in 10- to 12-week-old male mice. Thirty minutes post-controlled cortical impact surgery, a single 750-nmol dose of C-176 or saline (vehicle) was administered intravenously. Analysis was conducted 2 h and 24 h post-TBI. KEY RESULTS: Mice administered C-176 had significantly smaller cortical lesion area when compared to vehicle-treated mice 24 h post-TBI. Quantitative temporal gait analysis conducted using DigiGait™ showed C-176 administration attenuated TBI-induced impairments in gait symmetry, stride frequency and forelimb stance width. C-176-treated mice displayed a significant reduction in striatal gene expression of pro-inflammatory cytokines Tnf-α, Il-1ß and Cxcl10 compared to their vehicle-treated counterparts 2 h post-TBI. CONCLUSION AND IMPLICATIONS: This study demonstrates the neuroprotective activity of C-176 in ameliorating acute neuroinflammation and preventing white matter neurodegeneration post-TBI. This study highlights the therapeutic potential of small-molecule inhibitors targeting STING for the treatment of trauma-induced inflammation and neuroprotective potential.

The use of bioactive matrices in regenerative therapies for traumatic brain injury.

Functional deficits due to neuronal loss are a common theme across multiple neuropathologies, including traumatic brain injury (TBI). Apart from mitigating cell death, another approach to treating brain injuries involves re-establishing the neural circuitry at the lesion site by utilizing exogeneous and/or endogenous stem cells to achieve functional recovery. While there has been limited success, the emergence of new bioactive matrices that promote neural repair introduces new perspectives on the development of regenerative therapies for TBI. This review briefly discusses current development on cell-based therapies and the use of bioactive matrices, hydrogels in particular, when incorporated in regenerative therapies. Desirable characteristics of bioactive matrices that have been shown to augment neural repair in TBI models were identified and further discussed. Understanding the relative outcomes of newly developed biomaterials implanted in vivo can better guide the development of biomaterials as a therapeutic strategy, for biomaterial-based cellular therapies are still in their nascent stages. Nonetheless, the value of bioactive matrices as a treatment for acute brain injuries should be appreciated and further developed. STATEMENT OF SIGNIFICANCE: Cell-based therapies have received attention as an alternative therapeutic strategy to improve clinical outcome post-traumatic brain injury but have achieved limited success. Whilst the incorporation of newly developed biomaterials in regenerative therapies has shown promise in augmenting neural repair, studies have revealed new hurdles which must be overcome to improve their therapeutic efficacy. This review discusses the recent development of cell-based therapies with a specific focus on the use of bioactive matrices in the form of hydrogels, to complement cell transplantation within the injured brain. Moreover, this review consolidates in vivo animal studies that demonstrate relative functional outcome upon the implantation of different biomaterials to highlight their desirable traits to guide their development for regenerative therapies in traumatic brain injury.

Targeting high-mobility group box protein 1 (HMGB1) in pediatric traumatic brain injury: Chronic neuroinflammatory, behavioral, and epileptogenic consequences.

High mobility group box protein-1 (HMGB1) has been implicated as a key mediator of neuroinflammation and neurodegeneration in a range of neurological conditions including traumatic brain injury (TBI) and epilepsy. To date, however, most studies have examined only acute outcomes, and the adult brain. We have recently demonstrated HMGB1 release after experimental TBI in the pediatric mouse. This study therefore examined the chronic consequences of acute HMGB1 inhibition in the same model, to test the hypothesis that HMGB1 is a pivotal mediator of neuropathological, neurobehavioral, and epilepsy outcomes in pediatric TBI. HMGB1 was inhibited by treatment with 50 mg/kg i.p. Glycyrrhizin (Gly), compared to vehicle controls, commencing 1 h prior to moderate TBI or sham surgery in post-natal day 21 mice. We first demonstrated that Gly reduced brain HMGB1 levels and brain edema at an acute time point of 3 days post-injury. Subsequent analysis over a chronic time course found that pediatric TBI resulted in short-term spatial memory and motor learning deficits alongside an apparent increase in hippocampal microglial reactivity, which was prevented in Gly-treated TBI mice. In contrast, Gly treatment did not reduce the severity of evoked seizures, the proportion of animals exhibiting chronic spontaneous seizure activity, or cortical tissue loss. Together, our findings contribute to a growing appreciation for HMGB1's role in neuropathology and associated behavioral outcomes after TBI. However, further work is needed to fully elucidate the contribution of HMGB1 to epileptogenesis in this context.

Age-dependent release of high-mobility group box protein-1 and cellular neuroinflammation after traumatic brain injury in mice.

Accumulating research suggests that children may be more vulnerable to poor long-term outcomes after traumatic brain injury (TBI) compared to adults. The neuroinflammatory response, known to contribute to neuropathology after TBI, appears to differ depending upon age-at-insult, although this response has not been well-characterized. Elevated levels of a key initiator of inflammation, high-mobility group box protein 1 (HMGB1), have been associated with worsened outcomes after TBI in young patients. This study therefore aimed to characterize the acute time course of key mediators of the inflammatory cascade, including HMGB1, after pediatric and adult TBI. Male C57Bl/6 mice were subjected to severe controlled cortical impact or a sham control surgery, at either early adulthood (8-10 weeks) or a pediatric age (3 weeks). Cortical tissue was collected for Western blot detection of astrocyte and microglial activation (GFAP and CD68) and HMGB1 at 2 hr, 6 hr, 24 hr, 3 days, and 7 days postinjury, and serum was collected for enzyme-linked immunoassays to quantify peripheral HMGB1. An additional cohort of brains was harvested at 3 day postinjury for immunofluorescence staining. Results demonstrated a temporal profile of CD68, GFAP, and HMGB1 after TBI relative to sham, which differed between the adult and pediatric cohorts. An increase in peripheral HMGB1 was found in serum from pediatric TBI mice, which was not evident in adult serum. Together, these findings demonstrate that HMGB1 and the downstream cellular inflammatory response are influenced by age-at-insult, which may be an important consideration for treatment strategies aiming to ameliorate this response after TBI.

High-throughput screening for small molecule inhibitors of the type-I interferon signaling pathway.

Interferons (IFNs) are cytokines with fundamental roles in resistance to infections, cancer and other diseases. Type-I IFNs, interferon α (IFN-α) and interferon ß (IFN-ß), act through a shared receptor complex (IFNAR) comprised of IFNAR1 and IFNAR2 subunits. Binding of type-I IFN to IFNAR1 will robustly activate Janus activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Aberrant activation of the type-I IFN response results in a spectrum of disorders called interferonopathies. The purpose of this research is to develop an assay for high-throughput screening (HTS) of small molecule inhibitors of the type-I IFN signaling pathway. Inhibition of type-I IFN signaling can be beneficial in terms of therapeutic use and understanding the underlying mechanism of action. We report here a HTS campaign with the secreted embryonic alkaline phosphatase (SEAP) reporter gene assay against 32,000 compounds which yielded 25 confirmed hits. These compounds were subsequently characterized for their cytotoxicity, effects on STAT phosphorylation and activities in IFN regulatory factor (IRF) transcription.

STING-mediated type-I interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) represents a major cause of disability and death worldwide with sustained neuroinflammation and autophagy dysfunction contributing to the cellular damage. Stimulator of interferon genes (STING)-induced type-I interferon (IFN) signalling is known to be essential in mounting the innate immune response against infections and cell injury in the periphery, but its role in the CNS remains unclear. We previously identified the type-I IFN pathway as a key mediator of neuroinflammation and neuronal cell death in TBI. However, the modulation of the type-I IFN and neuroinflammatory responses by STING and its contribution to autophagy and neuronal cell death after TBI has not been explored. METHODS: C57BL/6J wild-type (WT) and STING-/- mice (8-10-week-old males) were subjected to controlled cortical impact (CCI) surgery and brains analysed by QPCR, Western blot and immunohistochemical analyses at 2 h or 24 h. STING expression was also analysed by QPCR in post-mortem human brain samples. RESULTS: A significant upregulation in STING expression was identified in late trauma human brain samples that was confirmed in wild-type mice at 2 h and 24 h after CCI. This correlated with an elevated pro-inflammatory cytokine profile with increased TNF-α, IL-6, IL-1ß and type-I IFN (IFN-α and IFN-ß) levels. This expression was suppressed in the STING-/- mice with a smaller lesion volume in the knockout animals at 24 h post CCI. Wild-type mice also displayed increased levels of autophagy markers, LC3-II, p62 and LAMP2 after TBI; however, STING-/- mice showed reduced LAMP2 expression suggesting a role for STING in driving dysfunctional autophagy after TBI. CONCLUSION: Our data implicates a detrimental role for STING in mediating the TBI-induced neuroinflammatory response and autophagy dysfunction, potentially identifying a new therapeutic target for reducing cellular damage in TBI.

Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer's Disease Subjects.

Ferroxidase activity has been reported to be altered in various biological fluids in neurodegenerative disease, but the sources contributing to the altered activity are uncertain. Here we assay fractions of serum and cerebrospinal fluid with a newly validated triplex ferroxidase assay. Our data indicate that while ceruloplasmin, a multicopper ferroxidase, is the predominant source of serum activity, activity in CSF predominantly derives from a <10 kDa component, specifically from polyanions such as citrate and phosphate. We confirm that in human biological samples, ceruloplasmin activity in serum is decreased in Alzheimer's disease, but in CSF a reduction of activity in Alzheimer's disease originates from the polyanion component.

COPD and stroke: are systemic inflammation and oxidative stress the missing links?

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and loss of lung function, and is currently the third largest cause of death in the world. It is now well established that cardiovascular-related comorbidities such as stroke contribute to morbidity and mortality in COPD. The mechanisms linking COPD and stroke remain to be fully defined but are likely to be interconnected. The association between COPD and stroke may be largely dependent on shared risk factors such as aging and smoking, or the association of COPD with traditional stroke risk factors. In addition, we propose that COPD-related systemic inflammation and oxidative stress may play important roles by promoting cerebral vascular dysfunction and platelet hyperactivity. In this review, we briefly discuss the pathogenesis of COPD, acute exacerbations of COPD (AECOPD) and cardiovascular comorbidities associated with COPD, in particular stroke. We also highlight and discuss the potential mechanisms underpinning the link between COPD and stroke, with a particular focus on the roles of systemic inflammation and oxidative stress.

Ablation of Type-1 IFN Signaling in Hematopoietic Cells Confers Protection Following Traumatic Brain Injury.

Type-1 interferons (IFNs) are pleiotropic cytokines that signal through the type-1 IFN receptor (IFNAR1). Recent literature has implicated the type-1 IFNs in disorders of the CNS. In this study, we have investigated the role of type-1 IFNs in neuroinflammation following traumatic brain injury (TBI). Using a controlled cortical impact model, TBI was induced in 8- to 10-week-old male C57BL/6J WT and IFNAR1(-/-) mice and brains were excised to study infarct volume, inflammatory mediator release via quantitative PCR analysis and immune cell profile via immunohistochemistry. IFNAR1(-/-) mice displayed smaller infarcts compared with WT mice after TBI. IFNAR1(-/-) mice exhibited an altered anti-inflammatory environment compared with WT mice, with significantly reduced levels of the proinflammatory mediators TNFα, IL-1ß and IL-6, an up-regulation of the anti-inflammatory mediator IL-10 and an increased activation of resident and peripheral immune cells after TBI. WT mice injected intravenously with an anti-IFNAR1 blocking monoclonal antibody (MAR1) 1 h before, 30 min after or 30 min and 2 d after TBI displayed significantly improved histological and behavioral outcome. Bone marrow chimeras demonstrated that the hematopoietic cells are a peripheral source of type-1 IFNs that drives neuroinflammation and a worsened TBI outcome. Type-1 IFN mRNA levels were confirmed to be significantly altered in human postmortem TBI brains. Together, these data demonstrate that type-1 IFN signaling is a critical pathway in the progression of neuroinflammation and presents a viable therapeutic target for the treatment of TBI.

Ceruloplasmin and ß-amyloid precursor protein confer neuroprotection in traumatic brain injury and lower neuronal iron.

Traumatic brain injury (TBI) is in part complicated by pro-oxidant iron elevation independent of brain hemorrhage. Ceruloplasmin (CP) and ß-amyloid protein precursor (APP) are known neuroprotective proteins that reduce oxidative damage through iron regulation. We surveyed iron, CP, and APP in brain tissue from control and TBI-affected patients who were stratified according to time of death following injury. We observed CP and APP induction after TBI accompanying iron accumulation. Elevated APP and CP expression was also observed in a mouse model of focal cortical contusion injury concomitant with iron elevation. To determine if changes in APP or CP were neuroprotective we employed the same TBI model on APP(-/-) and CP(-/-) mice and found that both exhibited exaggerated infarct volume and iron accumulation postinjury. Evidence supports a regulatory role of both proteins in defence against iron-induced oxidative damage after TBI, which presents as a tractable therapeutic target.

Type-1 interferon signaling mediates neuro-inflammatory events in models of Alzheimer's disease.

A neuro-inflammatory response has been implicated in human patients and animal models of Alzheimer's disease (AD). Type-1 interferons are pleiotropic cytokines involved in the initiation and regulation of the pro-inflammatory response; however, their role in AD is unknown. This study investigated the contribution of type-1 IFN signaling in the neuro-inflammatory response to amyloid-beta (Aß) in vitro and in the APP/PS1 transgenic mouse model of AD. Enzyme-linked immunosorbent assay confirmed a 2-fold increase in IFNα in APP/PS1 brains compared with control brains. Quantitative polymerase chain reaction also identified increased IFNα and IFNß expression in human pre-frontal cortex from AD patients. In vitro studies in primary neurons demonstrated Aß-induced type-1 IFN expression preceded that of other classical pro-inflammatory cytokines, IL1-ß, and IL-6. Significantly, ablation of type-1 interferon-α receptor 1 expression in BE(2)M17 neuroblastoma cells and primary neurons afforded protection against Aß-induced toxicity. This study supports a role for type-1 interferons in the pro-inflammatory response and neuronal cell death in AD and suggests that blocking type-1 interferon-α receptor 1 maybe a therapeutic target to limit the disease progression.

MyD88 is a critical regulator of hematopoietic cell-mediated neuroprotection seen after stroke.

Neuroinflammation is critical in the neural cell death seen in stroke. It has been shown that CNS and peripheral responses drive this neuroinflammatory response in the brain. The Toll-like receptors (TLRs) are important regulators of inflammation in response to both exogenous and endogenous stressors. Taking advantage of a downstream adapter molecule that controls the majority of TLR signalling, this study investigated the role of the TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the control of CNS and peripheral inflammation. Reversible middle-cerebral artery occlusion was used as the model of stroke in vivo; in vitro primary cultured neurons and glia were subject to four hours of oxygen and glucose deprivation (OGD). Both in vitro and in vivo Myd88(-/-) animals or cells were compared with wild type (WT). We found that after stroke Myd88(-/-) animals have a larger infarct volume compared to WT animals. Interestingly, in vitro there was no difference between the survival of Myd88(-/-) and WT cells following OGD, suggesting that peripheral responses were influencing stroke outcome. We therefore generated bone marrow chimeras and found that Myd88(-/-) animals have a smaller stroke infarct than their radiation naive counterparts if their hematopoietic cells are WT. Furthermore, WT animals have a larger stroke than their radiation naive counterparts if the hematopoietic cells are Myd88(-/-) . We have demonstrated that MyD88-dependent signalling in the hematopoietic cell lineage reduces infarct size following stroke and that infiltrating cells to the site of neuroinflammation are neuroprotective following stroke.

Glutathione peroxidase-1 primes pro-inflammatory cytokine production after LPS challenge in vivo.

Reactive oxygen species produced during the innate immune response to LPS are important agents of anti-pathogen defence but may also cause oxidative lung damage. Glutathione peroxidase-1 (gpx-1) is an anti-oxidant enzyme that may protect lungs from such damage. We assessed the in vivo importance of gpx-1 in LPS-induced lung inflammation. Male wild-type (WT) or gpx-1 deficient (gpx-1(-/-)) mice were treated intranasally with PBS or 10 µg LPS and killed 3 and 24 h post LPS. Lungs were lavaged with PBS and then harvested for inflammatory marker expression. LPS caused an intense neutrophilia in WT BALF evident 3 and 24 h post challenge that was reduced in gpx-1(-/-) mice. In addition, LPS-treated gpx-1(-/-) mice had significantly fewer macrophages than LPS-treated WT mice. To understand the basis for this paradoxical reduction we assessed inflammatory cytokines and proteases at protein and transcript levels. MMP-9 expression and net gelatinase activity in BALF of gpx-1(-/-) mice treated with LPS for 3 and 24 h was no different to that found in LPS-treated WT mice. BALF from LPS-treated gpx-1(-/-) mice (3 h) had less TNF-α, MIP-2 and GM-CSF protein than LPS-treated WT mice. In contrast, LPS-induced increases in TNF-α, MIP-2 and GM-CSF mRNA expression in WT mice were similar to those observed in gpx-1(-/-) mice. These attenuated protein levels were unexpectedly not mirrored by reduced mRNA transcripts but were associated with increased 20S proteasome expression. Thus, these data suggest that gpx-1 primes pro-inflammatory cytokine production after LPS challenge in vivo.

Insulin-regulated aminopeptidase deficiency provides protection against ischemic stroke in mice.

Recent studies have demonstrated that angiotensin IV (Ang IV) provides protection against brain injury caused by cerebral ischemia. Ang IV is a potent inhibitor of insulin-regulated aminopeptidase (IRAP). Therefore, we examined the effect of IRAP gene inactivation on neuroprotection following transient middle cerebral artery occlusion (MCAo) in mice. IRAP knockout mice and wild-type controls were subjected to 2 h of transient MCAo using the intraluminal filament technique. Twenty-four hours after reperfusion, neurological deficits of the stroke-induced mice were assessed and infarct volumes were measured by TTC staining. The cerebral infarct volume was significantly reduced in the IRAP knockout mice compared to wild-type littermates with corresponding improvement in neurological performance at 24 h post-ischemia. An increase in compensatory cerebral blood flow during MCAo was observed in the IRAP knockout animals with no differences in cerebral vascular anatomy detected. The current study demonstrates that deletion of the IRAP gene protects the brain from ischemic damage analogous to the effect of the IRAP inhibitor, Ang IV. This study indicates that IRAP is potentially a new therapeutic target for the development of treatment for ischemic stroke.

A global transcriptomic view of the multifaceted role of glutathione peroxidase-1 in cerebral ischemic-reperfusion injury.

Transient cerebral ischemia often results in secondary ischemic/reperfusion injury, the pathogenesis of which remains unclear. This study provides a comprehensive, temporal description of the molecular events contributing to neuronal injury after transient cerebral ischemia. Intraluminal middle cerebral artery occlusion (MCAO) was performed to induce a 2-h ischemia with reperfusion. Microarray analysis was then performed on the infarct cortex of wild-type (WT) and glutathione peroxidase-1 (a major antioxidant enzyme) knockout (Gpx1(-/-)) mice at 8 and 24h postreperfusion to identify differential gene expression profile patterns and potential alternative injury cascades in the absence of Gpx1, a crucial antioxidant enzyme, in cerebral ischemia. Genes with at least ±1.5-fold change in expression at either time point were considered significant. Global transcriptomic analyses demonstrated that 70% of the WT-MCAO profile overlapped with that of Gpx1(-/-)-MCAO, and 28% vice versa. Critical analysis of the 1034 gene probes specific to the Gpx1(-/-)-MCAO profile revealed regulation of additional novel pathways, including the p53-mediated proapoptotic pathway and Fas ligand (CD95/Apo1)-mediated pathways; downplay of the Nrf2 antioxidative cascade; and ubiquitin-proteasome system dysfunction. Therefore, this comparative study forms the foundation for the establishment of screening platforms for target definition in acute cerebral ischemia intervention.

Levosimendan preserves the contractile responsiveness of hypoxic human myocardium via mitochondrial K(ATP) channel and potential pERK 1/2 activation.

This study investigated the role of levosimendan, a mitochondrial K(ATP) channel opener, during hypoxia-reoxygenation injury in human isolated tissue. The activation of preconditioning pathways, and the release of mitochondrial cytochrome c were determined. Human right atrial trabeculae were mounted in an organ bath, electrically paced and contractile force measured. Tissue was subjected to hypoxia-reoxygenation, and isoproterenol concentration-response experiments were performed as an index of contractile viability. The intracellular activities of Akt, ERK 1/2, P38, caspase 3, and cytochrome c were assayed by western blot. Following hypoxia-reoxygenation, the maximal contractile response of trabeculae to isoproterenol was significantly increased with levosimendan pretreatment compared to the hypoxia-reoxygenation control (0.88±0.02 versus 0.60±0.01g, P<0.01). This enhanced response was blocked by 5-hydroxydecoanate (0.54±0.09g, P<0.01). A significant increase in both phosphorylated and total ERK 1/2 and P38 occurred at 60min reoxygenation, compared to control tissue. No difference was observed in phosphorylated or total Akt, though there was a trend for increased levels in hypoxic tissue. Cytochrome c was detected at 60min post reoxygenation, in both levosimendan treated and untreated tissue. No increase in cleaved-caspase 3 activity was observed. Our findings suggest that levosimendan preserves the contractile force to isoproterenol after hypoxia-reoxygenation, a response mediated via mK(ATP) channel activation. The significant increase in the activity of prosurvival mediators ERK 1/2 and P38 following hypoxia indicates a potential mechanism of action for levosimendan-induced cardioprotection.

Divergent roles of glutathione peroxidase-1 (Gpx1) in regulation of leukocyte-endothelial cell interactions in the inflamed cerebral microvasculature.

OBJECTIVE: The aim of this study was to assess the ability of Gpx1 to regulate leukocyte-endothelial cell interactions in the cerebral microcirculation under inflammatory conditions associated with oxidative stress. METHODS: To induce cerebral inflammation, wild-type and Gpx1(-/-) mice underwent systemic treatment with TNF or transient focal cerebral ischemia via MCAO. Leukocyte rolling and adhesion in cerebral postcapillary venules were assessed by intravital microscopy. RESULTS: Absence of Gpx1(-/-) resulted in increased cerebral oxidant production in response to TNF. Under these conditions, leukocyte rolling in cerebral venules was significantly elevated in Gpx1(-/-) mice, whereas leukocyte adhesion was lower than that in wild-type mice. Despite this, expression of key adhesion molecules did not differ between the strains. Following MCAO, Gpx1(-/-) mice displayed significant reductions in rolling and adhesion associated with severe blood flow restriction. In contrast, following treatment with the anti-oxidant ebselen to equalize postischemic cerebral blood flow in wild-type and Gpx1(-/-) mice, absence of Gpx1 was associated with significant elevations in leukocyte interactions. CONCLUSIONS: These data show that under some inflammatory conditions, Gpx1 regulates leukocyte-endothelial cell interactions in the cerebral microvasculature, but that this is affected by the nature of the inflammatory insult.

Glutathione peroxidase-1 protects against cigarette smoke-induced lung inflammation in mice.

Reactive oxygen species (ROS) produced from cigarette smoke cause oxidative lung damage including protein denaturation, lipid peroxidation, and DNA damage. Glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that may protect lungs from such damage. The aim of this study was to determine whether gpx-1 protects the lung against oxidative stress-induced lung inflammation in vivo. Male wild-type (WT) or gpx-1(-/-) mice were exposed to cigarette smoke generated from nine cigarettes per day for 4 days to induce oxidative stress and lung inflammation. The effect of the gpx mimetic ebselen on cigarette smoke-induced lung inflammation was evaluated when given prophylactically and therapeutically, i.e., during established inflammation. Mice were killed, and the lungs were lavaged with PBS and then harvested for genomic and proteomic analysis. Gpx-1(-/-) mice exposed to cigarette smoke had enhanced BALF neutrophils, macrophages, proteolytic burden, whole lung IL-17A, and MIP1alpha mRNA compared with WT mice. The gpx mimetic ebselen (10 and 100 microM) inhibited cigarette smoke extract-induced oxidation of MH-S cells in vitro and inhibited cigarette smoke-induced increases in BALF macrophages, neutrophils, proteolytic burden, and macrophage and neutrophil chemotactic factor gene expression when administered prophylactically. In addition, ebselen inhibited established BALF inflammation when administered therapeutically. These data show that gpx-1 protects against cigarette smoke-induced lung inflammation, and agents that mimic the actions of gpx-1 may have therapeutic utility in inflammatory lung diseases where cigarette smoke plays a role.

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells.

Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kappaB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice-induced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

Reactive oxygen species enhance insulin sensitivity.

Chronic reactive oxygen species (ROS) production by mitochondria may contribute to the development of insulin resistance, a primary feature of type 2 diabetes. In recent years it has become apparent that ROS generation in response to physiological stimuli such as insulin may also facilitate signaling by reversibly oxidizing and inhibiting protein tyrosine phosphatases (PTPs). Here we report that mice lacking one of the key enzymes involved in the elimination of physiological ROS, glutathione peroxidase 1 (Gpx1), were protected from high-fat-diet-induced insulin resistance. The increased insulin sensitivity in Gpx1(-/-) mice was attributed to insulin-induced phosphatidylinositol-3-kinase/Akt signaling and glucose uptake in muscle and could be reversed by the antioxidant N-acetylcysteine. Increased insulin signaling correlated with enhanced oxidation of the PTP family member PTEN, which terminates signals generated by phosphatidylinositol-3-kinase. These studies provide causal evidence for the enhancement of insulin signaling by ROS in vivo.