Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke

Neuroinflammation is strongly induced by cerebral ischemia. The early phase after the onset of ischemic stroke is characterized by acute neuronal injury, microglial activation, and subsequent infiltration of blood-derived inflammatory cells, including macrophages. Therefore, modulation of the microglial/macrophage responses has increasingly gained interest as a potential therapeutic approach for the ischemic stroke. In our study, we investigated the effects of peripherally administered interleukin 13 (IL-13) in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Systemic administration of IL-13 immediately after the ischemic insult significantly reduced the lesion volume, alleviated the infiltration of CD45(+) leukocytes, and promoted the microglia/macrophage alternative activation within the ischemic region, as determined by arginase 1 (Arg1) immunoreactivity at 3 days post-ischemia (dpi). Moreover, IL-13 enhanced the expression of M2a alternative activation markers Arg1 and Ym1 in the peri-ischemic (PI) area, as well as increased plasma IL-6 and IL-10 levels at 3 dpi. Furthermore, IL-13 treatment ameliorated gait disturbances at day 7 and 14 and sensorimotor deficits at day 14 post-ischemia, as analyzed by the CatWalk gait analysis system and adhesive removal test, respectively. Finally, IL-13 treatment decreased neuronal cell death in a coculture model of neuroinflammation with RAW 264.7 macrophages. Taken together, delivery of IL-13 enhances microglial/macrophage anti-inflammatory responses in vivo and in vitro, decreases ischemia-induced brain cell death, and improves sensory and motor functions in the pMCAo mouse model of cerebral ischemia.Peer reviewe

Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury

Rationale The recently discovered meningeal lymphatic vessels (mLVs) have been proposed to be the missing link between the immune and the central nervous system. The role of mLVs in modulating the neuro-immune response following a traumatic brain injury (TBI), however, has not been analyzed. Parenchymal T lymphocyte infiltration has been previously reported as part of secondary events after TBI, suggestive of an adaptive neuro-immune response. The phenotype of these cells has remained mostly uncharacterized. In this study, we identified subpopulations of T cells infiltrating the perilesional areas 30 days post-injury (an early-chronic time point). Furthermore, we analyzed how the lack of mLVs affects the magnitude and the type of T cell response in the brain after TBI. Methods TBI was induced in K14-VEGFR3-Ig transgenic (TG) mice or in their littermate controls (WT; wild type), applying a controlled cortical impact (CCI). One month after TBI, T cells were isolated from cortical areas ipsilateral or contralateral to the trauma and from the spleen, then characterized by flow cytometry. Lesion size in each animal was evaluated by MRI. Results In both WT and TG-CCI mice, we found a prominent T cell infiltration in the brain confined to the perilesional cortex and hippocampus. The majority of infiltrating T cells were cytotoxic CD8+ expressing a CD44(hi)CD69+ phenotype, suggesting that these are effector resident memory T cells. K14-VEGFR3-Ig mice showed a significant reduction of infiltrating CD4+ T lymphocytes, suggesting that mLVs could be involved in establishing a proper neuro-immune response. Extension of the lesion (measured as lesion volume from MRI) did not differ between the genotypes. Finally, TBI did not relate to alterations in peripheral circulating T cells, as assessed one month after injury. Conclusions Our results are consistent with the hypothesis that mLVs are involved in the neuro-immune response after TBI. We also defined the resident memory CD8+ T cells as one of the main population activated within the brain after a traumatic injury.Peer reviewe

Activation of P2X7 Receptors in Peritoneal and Meningeal Mast Cells Detected by Uptake of Organic Dyes: Possible Purinergic Triggers of Neuroinflammation in Meninges

Extracellular ATP activates inflammasome and triggers the release of multiple cytokines in various immune cells, a process primarily mediated by P2X7 receptors. However, the expression and functional properties of P2X7 receptors in native mast cells in tissues such as meninges where migraine pain originates from have not been explored. Here we report a novel model of murine cultured meningeal mast cells and using these, as well as easily accessible peritoneal mast cells, studied the mechanisms of ATP-mediated mast cell activation. We show that ATP induced a time and dose-dependent activation of peritoneal mast cells as analyzed by the uptake of organic dye YO-PRO1 as well as 4,6-diamidino-2-phenylindole (DAPI). Both YO-PRO1 and DAPI uptake in mast cells was mediated by the P2X7 subtype of ATP receptors as demonstrated by the inhibitory effect of P2X7 antagonist A839977. Consistent with this, significant YO-PRO1 uptake was promoted by the P2X7 agonist 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP). Extracellular ATP-induced degranulation of native and cultured meningeal mast cells was shown with Toluidine Blue staining. Taken together, these data demonstrate the important contribution of P2X7 receptors to ATP-driven activation of mast cells, suggesting these purinergic mechanisms as potential triggers of neuroinflammation and pain sensitization in migraine.Peer reviewe

Bim/Bcl-2 balance is critical for maintaining naive and memory T cell homeostasis

We examined the role of the antiapoptotic molecule Bcl-2 in combating the proapoptotic molecule Bim in control of naive and memory T cell homeostasis using Bcl-2−/− mice that were additionally deficient in one or both alleles of Bim. Naive T cells were significantly decreased in Bim+/−Bcl-2−/− mice, but were largely restored in Bim−/−Bcl-2−/− mice. Similarly, a synthetic Bcl-2 inhibitor killed wild-type, but not Bim−/−, T cells. Further, T cells from Bim+/−Bcl-2−/− mice died rapidly ex vivo and were refractory to cytokine-driven survival in vitro. In vivo, naive CD8+ T cells required Bcl-2 to combat Bim to maintain peripheral survival, whereas naive CD4+ T cells did not. In contrast, Bim+/−Bcl-2−/− mice generated relatively normal numbers of memory T cells after lymphocytic choriomeningitis virus infection. Accumulation of memory T cells in Bim+/−Bcl-2−/− mice was likely caused by their increased proliferative renewal because of the lymphopenic environment of the mice. Collectively, these data demonstrate a critical role for a balance between Bim and Bcl-2 in controlling homeostasis of naive and memory T cells

Granulocyte colony stimulating factor attenuates inflammation in a mouse model of amyotrophic lateral sclerosis

<p>Abstract</p> <p>Background</p> <p>Granulocyte colony stimulating factor (GCSF) is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery.</p> <p>Methods</p> <p>Human mutant G93A superoxide dismutase (SOD1) ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed <it>in vitro</it>.</p> <p>Results</p> <p>Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS.</p> <p>Conclusions</p> <p>GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS.</p

Dysfunction of Cellular Proteostasis in Parkinson’s Disease

Despite decades of research, current therapeutic interventions for Parkinson’s disease (PD) are insufficient as they fail to modify disease progression by ameliorating the underlying pathology. Cellular proteostasis (protein homeostasis) is an essential factor in maintaining a persistent environment for neuronal activity. Proteostasis is ensured by mechanisms including regulation of protein translation, chaperone-assisted protein folding and protein degradation pathways. It is generally accepted that deficits in proteostasis are linked to various neurodegenerative diseases including PD. While the proteasome fails to degrade large protein aggregates, particularly alpha-synuclein (α-SYN) in PD, drug-induced activation of autophagy can efficiently remove aggregates and prevent degeneration of dopaminergic (DA) neurons. Therefore, maintenance of these mechanisms is essential to preserve all cellular functions relying on a correctly folded proteome. The correlations between endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) that aims to restore proteostasis within the secretory pathway are well-established. However, while mild insults increase the activity of chaperones, prolonged cell stress, or insufficient adaptive response causes cell death. Modulating the activity of molecular chaperones, such as protein disulfide isomerase which assists refolding and contributes to the removal of unfolded proteins, and their associated pathways may offer a new approach for disease-modifying treatment. Here, we summarize some of the key concepts and emerging ideas on the relation of protein aggregation and imbalanced proteostasis with an emphasis on PD as our area of main expertise. Furthermore, we discuss recent insights into the strategies for reducing the toxic effects of protein unfolding in PD by targeting the ER UPR pathway

Stormbreaker8 and A3Wally Bacteriophage Genome Annotations

Stormbreaker8 and A3Wally are two novel bacteriophages isolated and purified on Microbacterium foliorum NRRL B-24224 by students in the Fall 2020 Discovery course. Stormbreaker8, an EA1 cluster lytic phage, was isolated from soil collected in Orange City, IA. Its circular permuted genome contains 41,751 base-pairs with 63.4% GC content. A3Wally, a GD cluster phage, was isolated from soil collected in Sioux Center, IA. Its genome is 60.1% GC, contains 194,724 base-pairs, and its ends are direct terminal repeats. Spring 2021 Genetics students annotated the genomes using bioinformatics software

An arylthiazyne derivative is a potent inhibitor of lipid peroxidation and ferroptosis providing neuroprotection in vitro and in vivo

Lipid peroxidation-initiated ferroptosis is an iron-dependent mechanism of programmed cell death taking place in neurological diseases. Here we show that a condensed benzo[b]thiazine derivative small molecule with an arylthiazine backbone (ADA-409-052) inhibits tert-Butyl hydroperoxide (TBHP)-induced lipid peroxidation (LP) and protects against ferroptotic cell death triggered by glutathione (GSH) depletion or glutathione peroxidase 4 (GPx4) inhibition in neuronal cell lines. In addition, ADA-409-052 suppresses pro-inflammatory activation of BV2 microglia and protects N2a neuronal cells from cell death induced by pro-inflammatory RAW 264.7 macrophages. Moreover, ADA-409-052 efficiently reduces infarct volume, edema and expression of pro-inflammatory genes in a mouse model of thromboembolic stroke. Targeting ferroptosis may be a promising therapeutic strategy in neurological diseases involving severe neuronal death and neuroinflammation.Peer reviewe

Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro

ObjectiveNatural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro.MethodsNK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co‑cultures with the glioma cell lines U‑87 MG, U‑87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell‑selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL‑21/15, IL‑27/18/15 and IL-12/18/15).ResultsThe expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL‑12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15.ConclusionAccording to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing

NF-E2-related factor 2 activation boosts antioxidant defenses and ameliorates inflammatory and amyloid properties in human Presenilin-1 mutated Alzheimer's disease astrocytes

Alzheimer's disease (AD) is a common dementia affecting a vast number of individuals and significantly impairing quality of life. Despite extensive research in animal models and numerous promising treatment trials, there is still no curative treatment for AD. Astrocytes, the most common cell type of the central nervous system, have been shown to play a role in the major AD pathologies, including accumulation of amyloid plaques, neuroinflammation, and oxidative stress. Here, we show that inflammatory stimulation leads to metabolic activation of human astrocytes and reduces amyloid secretion. On the other hand, the activation of oxidative metabolism leads to increased reactive oxygen species production especially in AD astrocytes. While healthy astrocytes increase glutathione (GSH) release to protect the cells, Presenilin‐1‐mutated AD patient astrocytes do not. Thus, chronic inflammation is likely to induce oxidative damage in AD astrocytes. Activation of NRF2, the major regulator of cellular antioxidant defenses, encoded by the NFE2L2 gene, poses several beneficial effects on AD astrocytes. We report here that the activation of NRF2 pathway reduces amyloid secretion, normalizes cytokine release, and increases GSH secretion in AD astrocytes. NRF2 induction also activates the metabolism of astrocytes and increases the utilization of glycolysis. Taken together, targeting NRF2 in astrocytes could be a potent therapeutic strategy in AD.</p