Enrica Caterina Pietronigro

Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin

Inflammation is a pathological hallmark of Alzheimers disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimers disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-β (Aβ) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aβ42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimers disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimers disease–like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimers disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimers disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimers disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimers disease.

TIM-1 Glycoprotein Binds the Adhesion Receptor P-Selectin and Mediates T Cell Trafficking during Inflammation and Autoimmunity

Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease.

Inverse agonism of cannabinoid CB1 receptor blocks the adhesion of encephalitogenic T cells in inflamed brain venules by a protein kinase A-dependent mechanism

It is well known that the cannabinoid system has a significant role in the regulation of the immune responses. Cannabinoid receptors CB1 and CB2 are expressed on T lymphocytes and mediate the immunomodulatory effects of cannabinoids on T cell functions. Here we show that the treatment of proteolipid protein (PLP)139-151-specific T cells with SR141716A, a CB1 inverse agonist and prototype of the diarylpyrazoles series, induced a strong inhibition of firm adhesion in inflamed brain venules in intravital microscopy experiments. In contrast, SR144528, a potent CB2 inverse agonist, had no significant effect on both rolling and arrest of activated T cells. In addition, two analogs of SR141716A and CB1 inverse agonists, AM251 and AM281 inhibited encephalitogenic T cell adhesion suggesting that selective CB1 inverse agonism interfere with lymphocyte trafficking in the CNS. Flow cytometry experiments showed that CB1 inverse agonists have no effect on adhesion molecule expression suggesting that CB1 blockade interferes with signal transduction pathways controlling T cell adhesion in inflamed brain venules. In addition, integrin clustering was not altered after treatment with CB1 inverse agonists suggesting that adhesion blockade is not due to the modulation of integrin valency. Notably, the inhibitory effect exerted by AM251 and AM281 on the adhesive interactions was completely reverted in the presence of protein kinase A (PKA) inhibitor H89, suggesting that cAMP and PKA activation play a key role in the adhesion blockade mediated by CB1 inverse agonists. To further strengthen these results and unveil a previously unknown inhibitory role of cAMP on activated T cell adhesion in vivo in the context of CNS inflammation, we showed that intracellular increase of cAMP induced by treatment with Bt2cAMP, a permeable analog of cAMP, and phosphodiesterase (PDE) inhibitor theophylline efficiently blocked the arrest of encephalitogenic T cells in inflamed brain venules. Our data show that modulation of CB1 function has anti-inflammatory effects and suggests that inverse agonism of CB1 block signal transduction mechanisms controlling encephalitogenic T cells adhesion in inflamed brain venules by a PKA-dependent mechanism.

Imaging of Leukocyte Trafficking in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by a progressive decline of cognitive functions. The neuropathological features of AD include amyloid beta (Aβ) deposition, intracellular neurofibrillary tangles derived from the cytoskeletal hyperphosphorylated tau protein, amyloid angiopathy, the loss of synapses, and neuronal degeneration. In the last decade, inflammation has emerged as a key feature of AD, but most studies have focused on the role of microglia-driven neuroinflammation mechanisms. A dysfunctional blood–brain barrier has also been implicated in the pathogenesis of AD, and several studies have demonstrated that the vascular deposition of Aβ induces the expression of adhesion molecules and alters the expression of tight junction proteins, potentially facilitating the transmigration of circulating leukocytes. Two-photon laser scanning microscopy (TPLSM) has become an indispensable tool to dissect the molecular mechanisms controlling leukocyte trafficking in the central nervous system (CNS). Recent TPLSM studies have shown that vascular deposition of Aβ in the CNS promotes intraluminal neutrophil adhesion and crawling on the brain endothelium and also that neutrophils extravasate in the parenchyma preferentially in areas with Aβ deposits. These studies have also highlighted a role for LFA-1 integrin in neutrophil accumulation in the CNS of AD-like disease models, revealing that LFA-1 inhibition reduces the corresponding cognitive deficit and AD neuropathology. In this article, we consider how current imaging techniques can help to unravel new inflammation mechanisms in the pathogenesis of AD and identify novel therapeutic strategies to treat the disease by interfering with leukocyte trafficking mechanisms.

NETosis in Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions. Its neuropathological features include amyloid-β (Aβ) accumulation, the formation of neurofibrillary tangles, and the loss of neurons and synapses. Neuroinflammation is a well-established feature of AD pathogenesis, and a better understanding of its mechanisms could facilitate the development of new therapeutic approaches. Recent studies in transgenic mouse models of AD have shown that neutrophils adhere to blood vessels and migrate inside the parenchyma. Moreover, studies in human AD subjects have also shown that neutrophils adhere and spread inside brain vessels and invade the parenchyma, suggesting these cells play a role in AD pathogenesis. Indeed, neutrophil depletion and the therapeutic inhibition of neutrophil trafficking, achieved by blocking LFA-1 integrin in AD mouse models, significantly reduced memory loss and the neuropathological features of AD. We observed that neutrophils release neutrophil extracellular traps (NETs) inside blood vessels and in the parenchyma of AD mice, potentially harming the blood–brain barrier and neural cells. Furthermore, confocal microscopy confirmed the presence of NETs inside the cortical vessels and parenchyma of subjects with AD, providing more evidence that neutrophils and NETs play a role in AD-related tissue destruction. The discovery of NETs inside the AD brain suggests that these formations may exacerbate neuro-inflammatory processes, promoting vascular and parenchymal damage during AD. The inhibition of NET formation has achieved therapeutic benefits in several models of chronic inflammatory diseases, including autoimmune diseases affecting the brain. Therefore, the targeting of NETs may delay AD pathogenesis and offer a novel approach for the treatment of this increasingly prevalent disease.

INHIBITION OF PROTEIN ARGININE DEIMINASES IMPROVES COGNITION AND REDUCES NEUROPATHOLOGICAL CHANGES IN MOUSE MODELS OF ALZHEIMER’S DISEASE

highly specific <25mM concentration. 2-PMAP does not alter APP mRNA level, while its dose-dependent effect on APP translation was directly showed in pulse-chase studies, which also confirmed absence of 2-PMAP effect on APPmaturation, processing, and degradation. 2-PMAPhasnoeffect onAPPcleavage as shownusing specific functional assays of APP secretases. 2-PMAP is BBB penetrant after oral and intravenous dosing and when given systemically to APPSW/ PS1dE9 AD transgenic mice it reduced levels of full length APP, APP C-terminal fragments (CTFs) and levels of soluble Abx-40 and Abx-42 in the brain. A chronic, four-month treatment of APPSW/ PS1dE9micewith 2-PMAP produced significant reduction inAb brain deposition and did not reveal any evidence of observable toxicity, while rescuing the mice from memory deficit. Conclusions: 2-PMAP is a small molecule therapeutic effectively ameliorating Ab pathology through targeting APP translation. 2-PMAP is unlikely to evoke off target effects associated with APP secretases inhibition and provides additional benefit in lowering levels of cytotoxic APP CTFs.

TREATMENT WITH CALCIUM DOBESILATE REDUCES NEUROINFLAMMATION AND IMPROVES COGNITION IN A MOUSE MODEL OF ALZHEIMER’S DISEASE

activated in brains following TBI. We show here that PERK activation following injury is time dependent and cell specific. We also show inhibition of PERK following traumatic brain injury in two month old mice affected tau pathology, overall protein synthesis, broad neuronal function, cognition, and gliosis. Conclusions: Our data provide novel insight into the physiological mechanisms of TBI, and suggest that PERK plays an important role in injury progression and cellular response. These data also suggest that the timing of PERK inhibition is critical during recovery. Overall, we show that the exploration of PERK inhibition as a therapeutic treatment for traumatic brain injury is an attractive option.

BLOCKADE OF ALPHA4 INTEGRINS AMELIORATES COGNITIVE DYSFUNCTION AND NEUROPATHOLOGICAL CHANGES IN TRANSGENIC ANIMALS WITH ALZHEIMER'S-LIKE DISEASE

Background: Blood-derived leukocyte subpopulations, including lymphocytes, monocytes and neutrophils, have been identified in the brains of patients with Alzheimer’s disease (AD) and in corresponding animal models, but their role in disease pathogenesis is unclear. We have recently demonstrated that neutrophils infiltrate the AD brain and that neutrophil depletion has therapeutic effect in AD transgenic mouse models. However, leukocyte trafficking mechanisms in the central nervous system (CNS) during AD are largely unknown and our goal was to determine the role of alpha4 integrins in disease pathogenesis. Methods: Intravital microscopy studies were performed to visualize and analyze leukocyte-vascular interactions in 3xTg-AD mice, which present both amyloid and tau pathology. Contextual fear conditioning and Ymaze tests were performed to analyze the cognitive deficit. Neuropathological studies were performed to evaluate amyloid beta deposition, tau hyperphosphorylation, microglial activation and expression of synaptic proteins. Results: Intravital microscopy studies performed in the CNS microcirculation of 3xTg-AD mice showed that blockade of alpha4 integrins prevents activated T cell rolling and firm adhesion on brain endothelium. Treating 3xTg-AD mice with an anti-alpha4 integrin antibody starting during early disease phases in mice already presenting cognitive deficits clearly improved memory function compared to mice treated with a control antibody in Y maze and contextual fear conditioning tests. These findings were supported by neuropathological data showing a reduction in Abeta deposition and lower density and activation state of microglia in the cortex and hippocampus of 3xTg-ADmice treated with anti-alpha4 antibody compared to animals treated with an isotype control antibody. Moreover, anti-apha4 treatment reduced tau hyperphosphorylation and restored synaptic protein expression compared to control animals. Notably, restoration of cognitive function in mice with temporary anti-alpha4 treatment during early disease was maintained also at later time points in aged animals, suggesting that therapeutic blockade of leukocyte adhesion during the early stages of disease provides a long-term beneficial effect on cognition in older mice. Conclusions: Antibodies anti-alpha4 integrin are currently used for the treatment of patients with autoimmune diseases and our data suggest that blockade of alpha4 integrins may represent a novel therapeutic approach in AD that has the potential for rapid translation into the clinic.

Targeting leukocyte integrins has therapeutic effect in Alzheimer's-like disease

demonstrated that the classic TSPO ligands, Ro5-4864 and PK11195, increase brain steroid levels, reduce Ab accumulation, and improve cognition in a mouse model of AD. Here we evaluated the steroidogenic efficacy and acute behavioral effects of three TSPO imidazopyridine ligands, CLINDE, PBR175, and PBR162, which we have previously shown to be non-toxic in vitro. CLINDE, which exhibited the most favorable behavioral and pharmacokinetic profile, was investigated further for its specificity and binding affinity in human brain. Methods: The acute behavioural effects of the three novel TSPO ligands were compared with Ro5-4864, in 3 month old castrated C57BL/6J mice. Two hours after ligand injection (3 mg/kg i.p.), anxiety-, depression-, and memory-related behaviors were assessed in the elevated-plus maze, open field maze, tail-suspension test, and object recognition tests respectively. Brain steroids levels were measured by LC-MS/MS. To determine if the behavioral effects of TSPO ligands were mediated via increased neurosteroidogenesis, mice were pretreated with a steroidogenesis inhibitor, aminogluthetimide (10mg/ kg). The pharmacokinetic properties of the novel ligands were assessed by competitive PET imaging [11C]PK-11195. The specificity of action of CLINDE was determined in TSPO knockout mice, and the affinity for TSPO (Ki value) in human brain homogenate was investigated in vitro by [11C] PK-11195 binding assay. Results: CLINDE and PBR175 improved learning and memory performance equally well as Ro5-4864. These improvements were completely ablated by aminogluthetimide, confirming that the beneficial effects were mediated through an increase in neurosteroidogenesis. Anxietyand depression-related behaviors were unaffected by the TSPO ligands. Competitive PET studies indicated that CLINDE showed the most long lasting binding to TSPO. The specificity of behavioral effects of CLINDE was confirmed. The affinity of CLINDE in high-affinity binder (HAB) and low-affinity binder (LAB) subjects were 5.5 and 29.7 respectively. Conclusions:These findings indicate that CLINDE and PBR175 rapidly promote neurosteroidogenesis, thereby enhancing learning and memory in vivo. These second generation TSPO ligands are promising therapeutic candidates with improved pharmacokinetic properties compared to classic TSPO ligands.

Krebs cycle potentiation has immunosuppressive effects and inhibits experimental autoimmune encephalomyelitis development

Background: Immunometabolism represents a novel field of investigation that links immune cell functions with the biochemical changes correlated to their intracellular metabolic state. Recent studies suggest that immune responses can be regulated by modulating immune cell metabolism, and that metabolic intervention can affect autoimmune disease development in animalmodels. Pantethine (PTTH) is ametabolic precursor of coenzyme A and a Krebs cycle stimulator, andwas successfully used in cardiovascular riskpatients. The aimof our studywas to investigate the effect ofmetabolic interventionwith pantethine on the effector functions of encephalitogenic T cells and on the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the murine model of human multiple sclerosis (MS). Results: Metabolomic and phosphoproteomic studies followed by bioinformatics computational analysis shown that, compared to naïve T cells, PLP139–151-specific encephalitogenic T cells undergo a metabolic reorganization known as Warburg effect, with reduced fatty acid oxidation (FAO) and Krebs cycle rate, strongly increased aerobic glycolysis and shunt of glycolysis intermediates to the pentose phosphate pathway (PPP). This metabolic shift led to increased synthesis of nucleotide, lipid andamino acid building blocks necessary for cell growthandproliferation. In vitro treatment with PTTH rapidly induced Krebs cycle potentiation in encephalitogenic T cells, followed by increased FAO, reduced glycolysis, block of PPP and inhibition of macromolecule biosynthesis. This metabolic tuning strongly affected antigen-specific proliferation and cytokine production by encephalitogenic T cells and inhibited integrin-mediated T cell adhesion in vitro. Accordingly, treatment with PTTH during the preclinical phase of disease prevented the development of both chronic (C-EAE) and relapsing–remitting (RR-EAE) EAE models, by blocking T cell activation and leukocyte adhesion in pial venules, leading to reduced pathogenic cell trafficking to the central nervous system. Notably, therapeutic treatment with PTTH significantly reduced the severity of established EAE in both C-EAE and RR-EAE models. Conclusions: Our data demonstrate that metabolic regulation of encephalitogenic T cell effector functions with pantethine has therapeutic effect on EAE and may represent a novel therapeutic strategy for MS. This work was supported by the European Research Council (ERC) grant Neurotrafficking (to G.C.).