Katerina Akassoglou

NF-κB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1α

The hypoxic response is an ancient stress response triggered by low ambient oxygen (O2) (ref. 1) and controlled by hypoxia-inducible transcription factor-1 (HIF-1), whose α subunit is rapidly degraded under normoxia but stabilized when O2-dependent prolyl hydroxylases (PHDs) that target its O2-dependent degradation domain are inhibited. Thus, the amount of HIF-1α, which controls genes involved in energy metabolism and angiogenesis, is regulated post-translationally. Another ancient stress response is the innate immune response, regulated by several transcription factors, among which NF-κB plays a central role. NF-κB activation is controlled by IκB kinases (IKK), mainly IKK-β, needed for phosphorylation-induced degradation of IκB inhibitors in response to infection and inflammation. IKK-β is modestly activated in hypoxic cell cultures when PHDs that attenuate its activation are inhibited. However, defining the relationship between NF-κB and HIF-1α has proven elusive. Using in vitro systems, it was reported that HIF-1α activates NF-κB, that NF-κB controls HIF-1α transcription and that HIF-1α activation may be concurrent with inhibition of NF-κB. Here we show, with the use of mice lacking IKK-β in different cell types, that NF-κB is a critical transcriptional activator of HIF-1α and that basal NF-κB activity is required for HIF-1α protein accumulation under hypoxia in cultured cells and in the liver and brain of hypoxic animals. IKK-β deficiency results in defective induction of HIF-1α target genes including vascular endothelial growth factor. IKK-β is also essential for HIF-1α accumulation in macrophages experiencing a bacterial infection. Hence, IKK-β is an important physiological contributor to the hypoxic response, linking it to innate immunity and inflammation.

Suppression of Oxidative Stress by β-Hydroxybutyrate, an Endogenous Histone Deacetylase Inhibitor

Stress Protector During prolonged fasting, the oxidation of fatty acids leads to increased accumulation of d-β-hydroxybutyrate (βOHB) in the bloodstream. Such increased concentrations of βOHB inhibit class I histone deacetylases. Histone acetylation in turn influences transcriptional activity at various genes. Shimazu et al. (p. 211, published online 6 December; see the Perspective by Sassone-Corsi) found that among the genes showing increased transcription in animals treated with high concentrations of βOHB were two genes implicated in cellular responses to oxidative stress. When treated ahead of time with βOHB, mice were protected from the toxic effects of the oxidative stress causing poison paraquat. Ketone bodies, metabolites that accumulate during fasting, change gene expression by inhibiting histone deacetylases. [Also see Perspective by Sassone-Corsi] Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.

Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice models for multiple sclerosis with primary oligodendrogliopathy

The scientific dogma that multiple sclerosis (MS) is a disease caused by a single pathogenic mechanism has been challenged recently by the heterogeneity observed in MS lesions and the realization that not all patterns of demyelination can be modeled by autoimmune-triggered mechanisms. To evaluate the contribution of local tumor necrosis factor (TNF) ligand/receptor signaling pathways to MS immunopathogenesis we have analyzed disease pathology in central nervous system-expressing TNF transgenic mice, with or without p55 or p75TNF receptors, using combined in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling and cell identification techniques. We demonstrate that local production of TNF by central nervous system glia potently and selectively induces oligodendrocyte apoptosis and myelin vacuolation in the context of an intact blood-brain barrier and absence of immune cell infiltration into the central nervous system parenchyma. Interestingly, primary demyelination then develops in a classical manner in the presence of large numbers of recruited phagocytic macrophages, possibly the result of concomitant pro-inflammatory effects of TNF in the central nervous system, and lesions progress into acute or chronic MS-type plaques with axonal damage, focal blood-brain barrier disruption, and considerable oligodendrocyte loss. Both the cytotoxic and inflammatory effects of TNF were abrogated in mice genetically deficient for the p55TNF receptor demonstrating a dominant role for p55TNF receptor-signaling pathways in TNF-mediated pathology. These results demonstrate that aberrant local TNF/p55TNF receptor signaling in the central nervous system can have a potentially major role in the aetiopathogenesis of MS demyelination, particularly in MS subtypes in which oligodendrocyte death is a primary pathological feature, and provide new models for studying the basic mechanisms underlying oligodendrocyte and myelin loss.

Fibrinogen as a key regulator of inflammation in disease

The interaction of coagulation factors with the perivascular environment affects the development of disease in ways that extend beyond their traditional roles in the acute hemostatic cascade. Key molecular players of the coagulation cascade like tissue factor, thrombin, and fibrinogen are epidemiologically and mechanistically linked with diseases with an inflammatory component. Moreover, the identification of novel molecular mechanisms linking coagulation and inflammation has highlighted factors of the coagulation cascade as new targets for therapeutic intervention in a wide range of inflammatory human diseases. In particular, a proinflammatory role for fibrinogen has been reported in vascular wall disease, stroke, spinal cord injury, brain trauma, multiple sclerosis, Alzheimer’s disease, rheumatoid arthritis, bacterial infection, colitis, lung and kidney fibrosis, Duchenne muscular dystrophy, and several types of cancer. Genetic and pharmacologic studies have unraveled pivotal roles for fibrinogen in determining the extent of local or systemic inflammation. As cellular and molecular mechanisms for fibrinogen functions in tissues are identified, the role of fibrinogen is evolving from a marker of vascular rapture to a multi-faceted signaling molecule with a wide spectrum of functions that can tip the balance between hemostasis and thrombosis, coagulation and fibrosis, protection from infection and extensive inflammation, and eventually life and death. This review will discuss some of the main molecular links between coagulation and inflammation and will focus on the role of fibrinogen in inflammatory disease highlighting its unique structural properties, cellular targets, and signal transduction pathways that make it a potent proinflammatory mediator and a potential therapeutic target.

Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation

Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo. Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.

Resolving postoperative neuroinflammation and cognitive decline

Cognitive decline accompanies acute illness and surgery, especially in the elderly. Surgery engages the innate immune system that launches a systemic inflammatory response that, if unchecked, can cause multiple organ dysfunction. We sought to understand the mechanisms whereby the brain is targeted by the inflammatory response and how this can be resolved.

Chronic optical access through a polished and reinforced thinned skull

We present a method to form an optical window in the mouse skull that spans millimeters and is stable for months without causing brain inflammation. This enabled us to repeatedly image blood flow in cortical capillaries of awake mice and determine long-range correlations in speed. We also repeatedly imaged dendritic spines, microglia and angioarchitecture, as well as used illumination to drive motor output via optogenetics and induce microstrokes via photosensitizers.

The fibrin-derived γ377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease

Perivascular microglia activation is a hallmark of inflammatory demyelination in multiple sclerosis (MS), but the mechanisms underlying microglia activation and specific strategies to attenuate their activation remain elusive. Here, we identify fibrinogen as a novel regulator of microglia activation and show that targeting of the interaction of fibrinogen with the microglia integrin receptor Mac-1 (αMβ2, CD11b/CD18) is sufficient to suppress experimental autoimmune encephalomyelitis in mice that retain full coagulation function. We show that fibrinogen, which is deposited perivascularly in MS plaques, signals through Mac-1 and induces the differentiation of microglia to phagocytes via activation of Akt and Rho. Genetic disruption of fibrinogen–Mac-1 interaction in fibrinogen-γ390-396A knock-in mice or pharmacologically impeding fibrinogen–Mac-1 interaction through intranasal delivery of a fibrinogen-derived inhibitory peptide (γ377-395) attenuates microglia activation and suppresses relapsing paralysis. Because blocking fibrinogen–Mac-1 interactions affects the proinflammatory but not the procoagulant properties of fibrinogen, targeting the γ377-395 fibrinogen epitope could represent a potential therapeutic strategy for MS and other neuroinflammatory diseases associated with blood-brain barrier disruption and microglia activation.

Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-β after vascular damage

Scar formation in the nervous system begins within hours after traumatic injury and is characterized primarily by reactive astrocytes depositing proteoglycans that inhibit regeneration. A fundamental question in CNS repair has been the identity of the initial molecular mediator that triggers glial scar formation. Here we show that the blood protein fibrinogen, which leaks into the CNS immediately after blood–brain barrier (BBB) disruption or vascular damage, serves as an early signal for the induction of glial scar formation via the TGF-β/Smad signaling pathway. Our studies revealed that fibrinogen is a carrier of latent TGF-β and induces phosphorylation of Smad2 in astrocytes that leads to inhibition of neurite outgrowth. Consistent with these findings, genetic or pharmacologic depletion of fibrinogen in mice reduces active TGF-β, Smad2 phosphorylation, glial cell activation, and neurocan deposition after cortical injury. Furthermore, stereotactic injection of fibrinogen into the mouse cortex is sufficient to induce astrogliosis. Inhibition of the TGF-β receptor pathway abolishes the fibrinogen-induced effects on glial scar formation in vivo and in vitro. These results identify fibrinogen as a primary astrocyte activation signal, provide evidence that deposition of inhibitory proteoglycans is induced by a blood protein that leaks in the CNS after vasculature rupture, and point to TGF-β as a molecular link between vascular permeability and scar formation.

TNF-α transgenic and knockout models of CNS inflammation and degeneration

Tumour necrosis factor-alpha (TNF-alpha) plays a central role in inflammatory events including those taking place in the central nervous system (CNS), and has been implicated as a key pathogenic mediator in several human inflammatory, infectious and autoimmune CNS disorders. Using transgenic and gene knockout mice we have investigated the role of deregulated TNF-alpha production in the CNS. We show that the overexpression of wild-type murine or human TNF-alpha transgenes by resident CNS astrocytes or neurons in sufficient to trigger a neurological disorder characterised by ataxia, seizures and paresis, with histopathological features of chronic CNS inflammation and white matter degeneration. Furthermore, we show that transmembrane human TNF-alpha is sufficient to trigger CNS inflammation and degeneration when overexpressed by astrocytes but not by neurons, indicating that target cells mediating the neuroinflammatory activities of TNF-alpha localise in the vicinity of astrocytes rather than neurons. Our results establish that both soluble and transmembrane molecular forms of TNF-alpha can play critical roles in vivo in the pathogenesis of CNS inflammation and demyelination, and validate TNF-alpha transgenic and mutant mice as important models for the further study of related human CNS diseases.