Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury.

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).

Neuropathological changes in the TASTPM mouse model of Alzheimer's disease and their relation to hyperexcitability and cortical spreading depolarization.

Although Alzheimer's disease (AD) is characterized by distinct pathological changes, their precise impact on cortical functions are not well understood. Here we used TASTPM mice as an AD model and asked whether the development of neurodegenerative changes has an impact on the extracellular space (ECS) and neuronal excitability, in particular cortical spreading depolarization (CSD) which requires intact neuron and glial functions. We studied wildtype (WT) and TASTPM mice (3, 6, and 12 months old). TASTPM mice showed progressive proliferation of neocortical Amyloid-beta (Aß) plaques between 3 and 12 months (more deposits in females than in males) and Aß accumulation in cortical vessels. As plaques proliferated, neuroinflammatory microglial reaction (CD68, CD39 and Galectin-3) and astrogliosis (GFAP) developed progressively. The cortical ECS volume shrank significantly to about half the size of the WT. CSD in both WT and TASTPM mice showed considerable heterogeneity but did not correlate with the histological changes. However, CSDs were easier to elicit in TASTPM than in WT mice at 3 months, and also compared to older TASTPM mice. Moreover, TASTPM mice showed more hyperexcitability manifested as clonic-tonic behavior after sodium thiopental anesthesia. Thus, AD pathology was associated with abnormal hyperexcitability but did not homogenously alter CSD susceptibility.

Loss of ceramide synthase 5 inhibits the development of experimentally induced aortic valve stenosis.

AIM: Inflammation and calcification are hallmarks in the development of aortic valve stenosis (AVS). Ceramides mediate inflammation and calcification in the vascular tissue. The highly abundant d18:1,16:0 ceramide (C16) has been linked to increased cardiovascular mortality and obesity. In this study, we investigate the role of ceramide synthase 5 (CerS5), a critical enzyme for C16 ceramide synthesis, in the development of AVS, particularly in conjunction with a high-fat/high-cholesterol diet (Western diet, WD). METHODS: We used wild-type (WT) and CerS5-/- mice on WD or normal chow in a wire injury model. We measured the peak velocity to determine AVS development and performed histological analysis of the aortic valve area, immune cell infiltration (CD68 staining), and calcification (von Kossa). In vitro experiments involved measuring the calcification of human aortic valvular interstitial cells (VICs) and evaluating cytokine release from THP-1 cells, a human leukemia monocytic-like cell line, following CerS5 knockdown. RESULTS: CerS5-/- mice showed a reduced peak velocity compared to WT only in the experiment with WD. Likewise, we observed reduced immune cell infiltration and calcification in the aortic valve of CerS5-/- mice, but only on WD. In vitro, calcification was reduced after knockdown of CerS5 in VICs, while THP-1 cells exhibited a decreased inflammatory response following CerS5 knockdown. CONCLUSION: We conclude that CerS5 is an important mediator for the development of AVS in mice on WD and regulates critical pathophysiological hallmarks of AVS formation. CerS5 is therefore an interesting target for pharmacological therapy and merits further investigation.

Proteomic analysis of peripheral nerve myelin during murine aging.

Aging of the peripheral nervous system (PNS) is associated with structural and functional changes that lead to a reduction in regenerative capacity and the development of age-related peripheral neuropathy. Myelin is central to maintaining physiological peripheral nerve function and differences in myelin maintenance, degradation, formation and clearance have been suggested to contribute to age-related PNS changes. Recent proteomic studies have elucidated the complex composition of the total myelin proteome in health and its changes in neuropathy models. However, changes in the myelin proteome of peripheral nerves during aging have not been investigated. Here we show that the proteomes of myelin fractions isolated from young and old nerves show only subtle changes. In particular, we found that the three most abundant peripheral myelin proteins (MPZ, MBP, and PRX) do not change in old myelin fractions. We also show a tendency for high-abundance myelin proteins other than these three to be downregulated, with only a small number of ribosome-related proteins significantly downregulated and extracellular matrix proteins such as collagens upregulated. In addition, we illustrate that the peripheral nerve myelin proteome reported in this study is suitable for assessing myelin degradation and renewal during peripheral nerve degeneration and regeneration. Our results suggest that the peripheral nerve myelin proteome is relatively stable and undergoes only subtle changes in composition during mouse aging. We proffer the resultant dataset as a resource and starting point for future studies aimed at investigating peripheral nerve myelin during aging. Said datasets are available in the PRIDE archive under the identifier PXD040719 (aging myelin proteome) and PXD041026 (sciatic nerve injury proteome).

Cortical spreading depolarization is a potential target for rat brain excitability modulation by Galanin.

The inhibitory neuropeptide Galanin (Gal) has been shown to mediate anticonvulsion and neuroprotection. Here we investigated whether Gal affects cortical spreading depolarization (CSD). CSD is considered the pathophysiological neuronal mechanism of migraine aura, and a neuronal mechanism aggravating brain damage upon afflictions of the brain. Immunohistochemistry localized Gal and the Gal receptors 1-3 (GalR1-3) in native rat cortex and evaluated microglial morphology after exposure to Gal. In anesthetized rats, Gal was applied alone and together with the GalR antagonists M40, M871, or SNAP 37889 locally to the exposed cortex. The spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection were measured. In rat cortex, Gal was present in all neurons of all cortical layers, but not in astrocytes, microglia and vessels. GalR2 and GalR3 were expressed throughout all neurons, whereas GalR1 was preponderantly located at neurons in layers IV and V, but only in about half of the neurons. In susceptible rats, topical application of Gal on cortex decreased CSD amplitude, slowed CSD propagation velocity, and increased the threshold for KCl to ignite CSD. In some rats, washout of previously applied Gal induced periods of epileptiform patterns in the electrocorticogram. Blockade of GalR2 by M871 robustly prevented all Gal effects on CSD, whereas blockade of GalR1 or GalR3 was less effective. Although microglia did not express GalRs, topical application of Gal changed microglial morphology indicating microglial activation. This effect of Gal on microglia was prevented by blocking neuronal GalR2. In conclusion, Gal has the potential to ameliorate CSD thus reducing pathophysiological neuronal events caused by or associated with CSD.

Autism Spectrum Disorder: A Neuro-Immunometabolic Hypothesis of the Developmental Origins.

Fetal neuroinflammation and prenatal stress (PS) may contribute to lifelong neurological disabilities. Astrocytes and microglia, among the brain's non-neuronal "glia" cell populations, play a pivotal role in neurodevelopment and predisposition to and initiation of disease throughout lifespan. One of the most common neurodevelopmental disorders manifesting between 1-4 years of age is the autism spectrum disorder (ASD). A pathological glial-neuronal interplay is thought to increase the risk for clinical manifestation of ASD in at-risk children, but the mechanisms remain poorly understood, and integrative, multi-scale models are needed. We propose a model that integrates the data across the scales of physiological organization, from genome to phenotype, and provides a foundation to explain the disparate findings on the genomic level. We hypothesize that via gene-environment interactions, fetal neuroinflammation and PS may reprogram glial immunometabolic phenotypes that impact neurodevelopment and neurobehavior. Drawing on genomic data from the recently published series of ovine and rodent glial transcriptome analyses with fetuses exposed to neuroinflammation or PS, we conducted an analysis on the Simons Foundation Autism Research Initiative (SFARI) Gene database. We confirmed 21 gene hits. Using unsupervised statistical network analysis, we then identified six clusters of probable protein-protein interactions mapping onto the immunometabolic and stress response networks and epigenetic memory. These findings support our hypothesis. We discuss the implications for ASD etiology, early detection, and novel therapeutic approaches. We conclude with delineation of the next steps to verify our model on the individual gene level in an assumption-free manner. The proposed model is of interest for the multidisciplinary community of stakeholders engaged in ASD research, the development of novel pharmacological and non-pharmacological treatments, early prevention, and detection as well as for policy makers.

Training vs. Tolerance: The Yin/Yang of the Innate Immune System.

For almost nearly a century, memory functions have been attributed only to acquired immune cells. Lately, this paradigm has been challenged by an increasing number of studies revealing that innate immune cells are capable of exhibiting memory-like features resulting in increased responsiveness to subsequent challenges, a process known as trained immunity (known also as innate memory). In contrast, the refractory state of endotoxin tolerance has been defined as an immunosuppressive state of myeloid cells portrayed by a significant reduction in the inflammatory capacity. Both training as well tolerance as adaptive features are reported to be accompanied by epigenetic and metabolic alterations occurring in cells. While training conveys proper protection against secondary infections, the induction of endotoxin tolerance promotes repairing mechanisms in the cells. Consequently, the inappropriate induction of these adaptive cues may trigger maladaptive effects, promoting an increased susceptibility to secondary infections-tolerance, or contribute to the progression of the inflammatory disorder-trained immunity. This review aims at the discussion of these opposing manners of innate immune and non-immune cells, describing the molecular, metabolic and epigenetic mechanisms involved and interpreting the clinical implications in various inflammatory pathologies.

Targeted Interleukin-9 delivery in pulmonary hypertension: Comparison of immunocytokine formats and effector cell study.

AIMS: Pulmonary hypertension (PH) is accompanied by pulmonary vascular remodelling. By targeted delivery of Interleukin-9 (IL9) via the immunocytokine F8IL9, beneficial effects could be demonstrated in a mouse model of PH. This study aimed to compare two immunocytokine formats (single-chain Fv and full IgG) and to identify potential target cells of IL9. METHODS: The Monocrotaline mouse model of PH (PH, n = 12) was chosen to evaluate the treatment effects of F8IL9F8 (n = 12) and F8IgGIL9 (n = 6) compared with sham-induced animals (control, n = 10), the dual endothelin receptor antagonist Macitentan (MAC, n = 12) or IL9-based immunocytokines with irrelevant antigen specificity (KSFIL9KSF, n = 12; KSFIgGIL9 n = 6). Besides comparative validation of treatment effects, the study was focused on the detection and quantification of mast cells (MCs) and regulatory T cells (Tregs). RESULTS: There was a significantly elevated systolic right ventricular pressure (104 ± 36 vs. 45 ± 17 mmHg) and an impairment of right ventricular echocardiographic parameters (RVbasal: 2.52 ± 0.25 vs. 1.94 ± 0.13 mm) in untreated PH compared with controls (p < 0.05). Only the groups treated with F8IL9, irrespective of the format, showed consistent beneficial effects (p < 0.05). Moreover, F8IL9F8 but not F8IgGIL9 treatment significantly reduced lung tissue damage compared with untreated PH mice (p < 0.05). There was a significant increase in Tregs in F8IL9-treated compared with control animals, the untreated PH and the MAC group (p < 0.05). CONCLUSIONS: Beneficial treatment effects of targeted IL9 delivery in a preclinical model of PH could be convincingly validated. IL9-mediated recruitment of Tregs into lung tissue might play a crucial role in the induction of anti-inflammatory and anti-proliferative mechanisms potentially contributing to a novel disease-modifying concept.

Ezrin deficiency triggers glial fibrillary acidic protein upregulation and a distinct reactive astrocyte phenotype.

Astrocytes are increasingly being recognized as contributors to physiological brain function and behavior. Astrocytes engage in glia-synaptic interactions through peripheral astrocyte processes, thus modulating synaptic signaling, for example, by handling glutamate removal from the synaptic cleft and (re)provision to axonal terminals. Peripheral astrocyte processes are ultrafine membrane protrusions rich in the membrane-to-actin cytoskeleton linker Ezrin, an essential component of in vitro filopodia formation and in vivo peripheral astrocyte process motility. Consequently, it has been postulated that Ezrin significantly contributes to neurodevelopment as well as astrocyte functions within the adult brain. However, while Ezrin has been studied in vitro within cultured primary astrocytes, in vivo studies on the role of Ezrin in astrocytes remain to be conducted and consequences of its depletion to be studied. Here, we investigated consequences of Ezrin deletion in the mouse brain starting from early neuronal specification. While Ezrin knockout did not impact prenatal cerebral cortex development, behavioral phenotyping depicted reduced exploratory behavior. Starting with postnatal appearance of glia cells, Ezrin was verified to remain predominantly expressed in astrocytes. Proteome analysis of Ezrin deficient astrocytes revealed alterations in glutamate and ion homeostasis, metabolism and cell morphology - important processes for synaptic signal transmission. Notably, Ezrin deletion in astrocytes provoked (GFAP) glial fibrillary acidic protein upregulation - a marker of astrocyte activation and reactive astrogliosis. However, this spontaneous, reactive astrogliosis exhibited proteome changes distinct from ischemic-induced reactive astrogliosis. Moreover, in experimental ischemic stroke, Ezrin knockout mice displayed reduced infarct volume, indicating a protective effect of the Ezrin deletion-induced changes and astrogliosis.

Drosophila Lipase 3 Mediates the Metabolic Response to Starvation and Aging.

The human LIPA gene encodes for the enzyme lysosomal acid lipase, which hydrolyzes cholesteryl ester and triacylglycerol. Lysosomal acid lipase deficiency results in Wolman disease and cholesteryl ester storage disease. The Drosophila genome encodes for two LIPA orthologs, Magro and Lipase 3. Magro is a gut lipase that hydrolyzes triacylglycerides, while Lipase 3 lacks characterization based on mutant phenotypes. We found previously that Lipase 3 transcription is highly induced in mutants with defects in peroxisome biogenesis, but the conditions that allow a similar induction in wildtypic flies are not known. Here we show that Lipase 3 is drastically upregulated in starved larvae and starved female flies, as well as in aged male flies. We generated a lipase 3 mutant that shows sex-specific starvation resistance and a trend to lifespan extension. Using lipidomics, we demonstrate that Lipase 3 mutants accumulate phosphatidylinositol, but neither triacylglycerol nor diacylglycerol. Our study suggests that, in contrast to its mammalian homolog LIPA, Lipase 3 is a putative phospholipase that is upregulated under extreme conditions like prolonged nutrient deprivation and aging.

The ER protein Creld regulates ER-mitochondria contact dynamics and respiratory complex 1 activity.

Dynamic contacts are formed between endoplasmic reticulum (ER) and mitochondria that enable the exchange of calcium and phospholipids. Disturbed contacts between ER and mitochondria impair mitochondrial dynamics and are a molecular hallmark of Parkinson's disease, which is also characterized by impaired complex I activity and dopaminergic neuron degeneration. Here, we analyzed the role of cysteine-rich with EGF-like domain (Creld), a poorly characterized risk gene for Parkinson's disease, in the regulation of mitochondrial dynamics and function. We found that loss of Creld leads to mitochondrial hyperfusion and reduced ROS signaling in Drosophila melanogaster, Xenopus tropicalis, and human cells. Creld fly mutants show differences in ER-mitochondria contacts and reduced respiratory complex I activity. The resulting low-hydrogen peroxide levels are linked to disturbed neuronal activity and lead to impaired locomotion, but not neurodegeneration, in Creld mutants. We conclude that Creld regulates ER-mitochondria communication and thereby hydrogen peroxide formation, which is required for normal neuron function.

From spreading depolarization to epilepsy with neuroinflammation: The role of CGRP in cortex.

CGRP release plays a major role in migraine pain by activating the trigeminal pain pathways. Here we explored putative additional effects of CGRP on cortical circuits and investigated whether CGRP affects cortical excitability, cortical spreading depolarization (CSD), a phenomenon associated with migraine aura, blood-brain-barrier (BBB) and microglial morphology. We used immunohistochemistry to localize CGRP and the CGRP receptor (CGRP-R) in native cortex and evaluated morphology of microglia and integrity of the BBB after exposure to CGRP. In anesthetized rats we applied CGRP and the CGRP-R antagonist BIBN4096BS locally to the exposed cortex and monitored the spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection. In mouse brain slices CGRP effects on neuronal activity were explored by multielectrode array. CGRP immunoreactivity was detectable in intracortical vessels, and all cortical neurons showed CGRP-R immunoreactivity. In rat cortex in vivo, topical CGRP induced periods of epileptiform discharges, however, also dose-dependently reduced CSD amplitudes and propagation velocity. BIBN4096BS prevented these effects. CGRP evoked synchronized bursting activity in mouse cortical but not in cerebellar slices. Topical application of CGRP to rat cortex induced plasma extravasation and this was associated with reduced ramification of microglial cells. From these findings we conclude that CGRP induces a pathophysiological state in the cortex, consisting in neuronal hyperexcitability and neuroinflammation. Thus, CGRP may have a pronounced impact on brain functions during migraine episodes supporting the benefit of CGRP antagonists for clinical use. However, increased cortical CGRP may end the CSD-induced aura phase of migraine.

Long-chain ceramides are cell non-autonomous signals linking lipotoxicity to endoplasmic reticulum stress in skeletal muscle.

The endoplasmic reticulum (ER) regulates cellular protein and lipid biosynthesis. ER dysfunction leads to protein misfolding and the unfolded protein response (UPR), which limits protein synthesis to prevent cytotoxicity. Chronic ER stress in skeletal muscle is a unifying mechanism linking lipotoxicity to metabolic disease. Unidentified signals from cells undergoing ER stress propagate paracrine and systemic UPR activation. Here, we induce ER stress and lipotoxicity in myotubes. We observe ER stress-inducing lipid cell non-autonomous signal(s). Lipidomics identifies that palmitate-induced cell stress induces long-chain ceramide 40:1 and 42:1 secretion. Ceramide synthesis through the ceramide synthase 2 de novo pathway is regulated by UPR kinase Perk. Inactivation of CerS2 in mice reduces systemic and muscle ceramide signals and muscle UPR activation. The ceramides are packaged into extracellular vesicles, secreted and induce UPR activation in naïve myotubes through dihydroceramide accumulation. This study furthers our understanding of ER stress by identifying UPR-inducing cell non-autonomous signals.

Distinct Actions of the Thyroid Hormone Transporters Mct8 and Oatp1c1 in Murine Adult Hippocampal Neurogenesis.

Inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) result in Allan-Herndon-Dudley Syndrome, a severe form of psychomotor retardation, while inactivating mutations in another TH transporter, organic anion transporting polypeptide 1c1 (OATP1C1), are linked to juvenile neurodegeneration. These diseases point to essential roles for TH transporters in CNS function. We recently defined the presence of Mct8 in adult hippocampal progenitors and mature granule cell neurons and unraveled cell-autonomous and indirect requirements for Mct8 in adult hippocampal neurogenesis. Here, we investigated whether Oatp1c1 is involved in the hippocampal neurogenic process in concert with Mct8. We detected Oatp1c1 gene expression activity and transcripts in subsets of progenitors, neurons and niche cells in the dentate gyrus. Absence of Oatp1c1 resulted in increased neuroblast and reduced immature neuron numbers in 6-month-old Oatp1c1ko and Mct8/Oatp1c1 double knockout (M/Odko) mice. Reduced EdU-label retention in Mct8ko and M/Odko mice confirmed the impact of Mct8 on neuron formation. In contrast, no significant effect of Oatp1c1 loss on granule cell neuron production and anxiety-like behavior in the open field arena were seen. Together, our results reinforce that distinct actions of each TH transporter are required at multiple stages to ensure proper adult hippocampal neurogenesis.

PI3Kγ Mediates Microglial Proliferation and Cell Viability via ROS.

(1) Background: Rapid microglial proliferation contributes to the complex responses of the innate immune system in the brain to various neuroinflammatory stimuli. Here, we investigated the regulatory function of phosphoinositide 3-kinase γ (PI3Kγ) and reactive oxygen species (ROS) for rapid proliferation of murine microglia induced by LPS and ATP. (2) Methods: PI3Kγ knockout mice (PI3Kγ KO), mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) and wild-type mice were assessed for microglial proliferation using an in vivo wound healing assay. Additionally, primary microglia derived from newborn wild-type, PI3Kγ KO and PI3Kγ KD mice were used to analyze PI3Kγ effects on proliferation and cell viability, senescence and cellular and mitochondrial ROS production; the consequences of ROS production for proliferation and cell viability after LPS or ATP stimulation were studied using genetic and pharmacologic approaches. (3) Results: Mice with a loss of lipid kinase activity showed impaired proliferation of microglia. The prerequisite of induced microglial proliferation and cell viability appeared to be PI3Kγ-mediated induction of ROS production. (4) Conclusions: The lipid kinase activity of PI3Kγ plays a crucial role for microglial proliferation and cell viability after acute inflammatory activation.

Update to the dataset of cerebral ischemia in juvenile pigs with evoked potentials.

We expand from a spontaneous to an evoked potentials (EP) data set of brain electrical activities as electrocorticogram (ECoG) and electrothalamogram (EThG) in juvenile pig under various sedation, ischemia and recovery states. This EP data set includes three stimulation paradigms: auditory (AEP, 40 and 2000 Hz), sensory (SEP, left and right maxillary nerve) and high-frequency oscillations (HFO) SEP. This permits derivation of electroencephalogram (EEG) biomarkers of corticothalamic communication under these conditions. The data set is presented in full band sampled at 2000 Hz. We provide technical validation of the evoked responses for the states of sedation, ischemia and recovery. This extended data set now permits mutual inferences between spontaneous and evoked activities across the recorded modalities. Future studies on the dataset may contribute to the development of new brain monitoring technologies, which will facilitate the prevention of neurological injuries.

Creld2 function during unfolded protein response is essential for liver metabolism homeostasis.

The unfolded protein response (UPR) is associated with hepatic metabolic function, yet it is not well understood how endoplasmic reticulum (ER) disturbance might influence metabolic homeostasis. Here, we describe the physiological function of Cysteine-rich with EGF-like domains 2 (Creld2), previously characterized as a downstream target of the ER-stress signal transducer Atf6. To this end, we generated Creld2-deficient mice and induced UPR by injection of tunicamycin. Creld2 augments protein folding and creates an interlink between the UPR axes through its interaction with proteins involved in the cellular stress response. Thereby, Creld2 promotes tolerance to ER stress and recovery from acute stress. Creld2-deficiency leads to a dysregulated UPR and causes the development of hepatic steatosis during ER stress conditions. Moreover, Creld2-dependent enhancement of the UPR assists in the regulation of energy expenditure. Furthermore, we observed a sex dimorphism in human and mouse livers with only male patients showing an accumulation of CRELD2 protein during the progression from non-alcoholic fatty liver disease to non-alcoholic steatohepatitis and only male Creld2-deficient mice developing hepatic steatosis upon aging. These results reveal a Creld2 function at the intersection between UPR and metabolic homeostasis and suggest a mechanism in which chronic ER stress underlies fatty liver disease in males.

Targeted delivery of a phosphoinositide 3-kinase γ inhibitor to restore organ function in sepsis.

Jaundice, the clinical hallmark of infection-associated liver dysfunction, reflects altered membrane organization of the canalicular pole of hepatocytes and portends poor outcomes. Mice lacking phosphoinositide 3-kinase-γ (PI3Kγ) are protected against membrane disintegration and hepatic excretory dysfunction. However, they exhibit a severe immune defect that hinders neutrophil recruitment to sites of infection. To exploit the therapeutic potential of PI3Kγ inhibition in sepsis, a targeted approach to deliver drugs to hepatic parenchymal cells without compromising other cells, in particular immune cells, seems warranted. Here, we demonstrate that nanocarriers functionalized through DY-635, a fluorescent polymethine dye, and a ligand of organic anion transporters can selectively deliver therapeutics to hepatic parenchymal cells. Applying this strategy to a murine model of sepsis, we observed the PI3Kγ-dependent restoration of biliary canalicular architecture, maintained excretory liver function, and improved survival without impairing host defense mechanisms. This strategy carries the potential to expand targeted nanomedicines to disease entities with systemic inflammation and concomitantly impaired barrier functionality.

The Role of the Pathogen Dose and PI3Kγ in Immunometabolic Reprogramming of Microglia for Innate Immune Memory.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.