Culture Activation Research Articles

Elevated levels of peripheral and central nervous system immune markers reflect innate immune dysregulation in autism spectrum disorder

BackgroundEvidence suggests dysregulated immune functions in the pathophysiology of Autism spectrum disorder (ASD), although specific immune mechanisms are yet to be identified. MethodsWe assessed circulating levels of 25 immune/neuroinflammatory markers in a large ASD sample (n = 151) and matched controls (n = 72) using linear models. In addition, we performed global brain transcriptomics analyses of relevant immune-related genes. We also assessed the expression and function of factors and pathway elements of the inflammasome system in peripheral blood mononuclear cells (PBMC) isolated from ASD and controls using in vitro methods. ResultsWe found higher circulating levels of IL-18 and adhesion factors (ICAM-1, MADCAM1) in individuals with ASD relative to controls. Consistent with this, brain levels of ICAM1 mRNA were also higher in ASD compared to controls. Furthermore, we found higher expression/activity of Caspase-1 and the inflammasome sensor NLRP3 in PBMCs in ASD, both at baseline and following inflammatory challenge. This corresponded with higher levels of secreted IL-18, IL-1β, and IL-8, as well as increased expression of adhesion factors following inflammasome activation in ASD PBMC cultures. Inhibition of the NLRP3-inflammasome rescued the observed immune phenotype in ASD in vitro. ConclusionOur results suggest a role for inflammasome dysregulation in ASD pathophysiology.

Direct M2 macrophage co-culture overrides viscoelastic hydrogel mechanics to promote fibroblast activation.

Fibroblast activation drives fibrotic diseases such as pulmonary fibrosis. However, the complex interplay of how tissue mechanics and macrophage signals combine to influence fibroblast activation is not well understood. Here, we use hyaluronic acid hydrogels as a tunable cell culture system to mimic lung tissue stiffness and viscoelasticity. We applied this platform to investigate the influence of macrophage signaling on fibroblast activation. Fibroblasts cultured on stiff (50 kPa) hydrogels mimicking fibrotic tissue exhibit increased activation as measured by spreading as well as type I collagen and cadherin-11 expression compared to fibroblasts cultured on soft (1 kPa) viscoelastic hydrogels mimicking normal tissue. These trends were unchanged in fibroblasts cultured with macrophage-conditioned media. However, fibroblasts directly co-cultured with M2 macrophages show increased activation, even on soft viscoelastic hydrogels that normally suppress activation. Inhibition of interleukin 6 (IL6) signaling does not change activation in fibroblast-only cultures but ameliorates the pro-fibrotic effects of M2 macrophage co-culture. These results underscore the ability of direct M2 macrophage co-culture to override hydrogel viscoelasticity to promote fibroblast activation in an IL6-dependent manner. This work also highlights the utility of using hydrogels to deconstruct complex tissue microenvironments to better understand the interplay between microenvironmental mechanical and cellular cues.

Mechanism of Alzheimer type II astrocyte development in hepatic encephalopathy

Type C hepatic encephalopathy (Type C HE) is a major and complex neurological condition that occurs following chronic liver failure. The molecular basis of Type C HE remains elusive. Type C HE is characterized by mental confusion, cognitive and motor disturbances. The presence of Alzheimer type II astrocytes (AT2A) is the key histopathological finding observed in Type C HE. However, nothing is currently known regarding AT2A development and its involvement in cognitive, and motor deficits in Type C HE. We, therefore, examined in rats the mechanisms by which liver failure contributes to the progression of AT2A, and its role in the development of cognitive and motor deficits in thioacetamide (TAA) model of Type C HE. We and others earlier reported increased oxidative/nitrosative stress (ONS), JNK1/2, and cMyc activation in ammonia-treated astrocyte cultures, as well as in brains from chronic liver failure. We now found increased levels of astrocytic glia maturation factor (GMF, a factor strongly implicated in neuroinflammation), as well as various inflammatory factors (IL-1β, TNF-α, IL-6, MMP-3, COX2, CXCL1, and PGE2), and reduced levels of GFAP and increased levels of aggregated nuclear protein Lamin A/C in rat brain cortex post-chronic liver failure. We also found increased levels of GMF and inflammatory factors (MMP-3, COX2, CXCL1, and PGE2) in astrocytes post-ammonia treatment in vitro. Additionally, pharmacological inhibition of upstream signaling of GMF (ONS, JNK1/2, and cMyc) or GMF inhibitors W-7 and trifluoperazine significantly reduced the levels of inflammatory factors, the number of AT2A cells, as well as the cognitive and motor deficits in TAA-treated rats. Increased levels of GMF were also identified in human post-mortem brain sections. These findings strongly suggest that increased levels of astrocytic GMF due to elevated levels of ONS, JNK1/2, and cMyc and the subsequent inflammation contribute to the development of AT2A and the consequent cognitive, and motor deficits in chronic liver failure.

Tumor Cell Spatial Organization Directs EGFR/RAS/RAF Pathway Primary Therapy Resistance through YAP Signaling.

Non-small cell lung cancers (NSCLC) harboring common mutations in EGFR and KRAS characteristically respond transiently to targeted therapies against those mutations, but invariably, tumors recur and progress. Resistance often emerges through mutations in the therapeutic target or activation of alternative signaling pathways. Mechanisms of acute tumor cell resistance to initial EGFR (EGFRi) or KRASG12C (G12Ci) pathway inhibition remain poorly understood. Our study reveals that acute response to EGFR/RAS/RAF-pathway inhibition is spatial and culture context specific. In vivo, EGFR mutant tumor xenografts shrink by > 90% following acute EGFRi therapy, and residual tumor cells are associated with dense stroma and have increased nuclear YAP. Interestingly, in vitro EGFRi induced cell cycle arrest in NSCLC cells grown in monolayer, while 3D spheroids preferentially die upon inhibitor treatment. We find differential YAP nuclear localization and activity, driven by the distinct culture conditions, as a common resistance mechanism for selective EGFR/KRAS/BRAF pathway therapies. Forced expression of the YAPS127A mutant partially protects cells from EGFR-mediated cell death in spheroid culture. These studies identify YAP activation in monolayer culture as a non-genetic mechanism of acute EGFR/KRAS/BRAF therapy resistance, highlighting that monolayer vs spheroid cell culture systems can model distinct stages of patient cancer progression.

Cytokine priming enhances the antifibrotic effects of human adipose derived mesenchymal stromal cells conditioned medium

BackgroundFibrosis is a pathological scarring process characterized by persistent myofibroblast activation with excessive accumulation of extracellular matrix (ECM). Fibrotic disorders represent an increasing burden of disease-associated morbidity and mortality worldwide for which there are limited therapeutic options. Reversing fibrosis requires the elimination of myofibroblasts, remodeling of the ECM, and regeneration of functional tissue. Multipotent mesenchymal stromal cells (MSC) have antifibrotic properties mediated by secreted factors present in their conditioned medium (MSC-CM). However, there are no standardized in vitro assays to predict the antifibrotic effects of human MSC. As a result, we lack evidence on the effect of cytokine priming on MSC’s antifibrotic effects. We hypothesize that the MSC-CM promotes fibrosis resolution in vitro and that this effect is enhanced following MSC cytokine priming.MethodsWe compared the antifibrotic effects of resting versus interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) primed MSC-CM in four in vitro assays: prevention of fibroblast activation, myofibroblasts deactivation, ECM degradation and fibrosis resolution in lung explant cultures. Furthermore, we performed transcriptomic analysis of myofibroblasts treated or not with resting or primed MSC-CM and proteomic characterization of resting and primed MSC-CM.ResultsWe isolated MSC from adipose tissue of 8 donors, generated MSC-CM and tested each MSC-CM independently. We report that MSC-CM treatment prevented TGF-β induced fibroblast activation to a similar extent as nintedanib but, in contrast to nintedanib, MSC-CM reduced fibrogenic myofibroblasts (i.e. transcriptomic upregulation of apoptosis, senescence, and inflammatory pathways). These effects were larger when primed rather than resting MSC-CM were used. Priming increased the ability of MSC-CM to remodel the ECM, reducing its content of collagen I and fibronectin, and reduced the fibrotic load in TGF-β treated lung explant cultures. Priming increased the following antifibrotic proteins in MSC-CM: DKK1, MMP-1, MMP-3, follistatin and cathepsin S. Inhibition of DKK1 reduced the antifibrotic effects of MSC-CM.ConclusionsIn vitro, MSC-CM promote fibrosis resolution, an effect enhanced following MSC cytokine priming. Specifically, MSC-CM reduces fibrogenic myofibroblasts through apoptosis, senescence, and by enhancing ECM degradation. Future studies will establish the in vivo relevance of MSC priming to fibrosis resolution.

CD11b-NOX2 mutual regulation-mediated microglial exosome release contributes to rotenone-induced inflammation and neurotoxicity in BV2 microglia and primary cultures

Epidemiological studies have revealed a potent association between chronic exposure to rotenone, a commonly used pesticide, in individuals and the incidence of Parkinson's disease (PD). We previously identified the contribution of the activation of microglial NADPH oxidase (NOX2) in rotenone-induced neurotoxicity. However, the regulation of NOX2 activation remains unexplored. Integrins are known to be bidirectionally regulated in the plasma membrane through the inside-out and outside-in signaling. CD11b is the α-chain of integrin macrophage antigen complex-1. This study aimed to investigate whether CD11b mediates rotenone-induced NOX2 activation. We observed that rotenone exposure increased NOX2 activation in BV2 microglia, which was associated with elevated CD11b expression. Silencing CD11b significantly reduced rotenone-induced ROS production and p47phox phosphorylation, a key step for NOX2 activation. Furthermore, the Src-FAK-PKB and Syk-Vav1-Rac1 signaling pathways downstream of CD11b were found to be essential for CD11b-mediated NOX2 activation in rotenone-intoxicated microglia. Interestingly, we also found that inhibition of NOX2 decreased rotenone-induced CD11b expression, indicating a crosstalk between CD11b and NOX2. Subsequently, the inhibition of the CD11b-NOX2 axis suppressed rotenone-induced microglial activation and exosome release. Furthermore, inhibiting exosome synthesis in microglia blocked rotenone-induced gene expression of proinflammatory factors and related neurotoxicity. Finally, blocking the CD11b-NOX2 axis and exosome synthesis or endocytosis mitigated microglial activation and dopaminergic neurodegeneration in rotenone-intoxicated midbrain primary cultures. Our findings highlight the crucial involvement of the CD11b-NOX2 axis in rotenone-induced inflammation and neurotoxicity, offering fresh perspectives on the underlying mechanisms of pesticide-induced neuronal damage.

Fate, inducibility, and behavior of Latilactobacillus curvatus temperate phage TMW 1.591 P1 during sausage fermentation.

Temperate phages insert their genome into the host's chromosome. As prophages, they remain latent in the genome until an induction event leads to lytic phage production. When this occurs in a starter culture that has been added to food fermentation, this can impair the fermentation success. This study aimed to analyze prophage inducibility in the Latilactobacillus curvatus TMW 1.591 strain during meat fermentation and investigate whether an induction signal before cryopreservation is maintained during storage and can lead to phage-induced lysis after culture activation. A prophage-free isogenic derivative of the model starter organism, L. curvatus TMW 1.591, was developed as a negative control (L. curvatus TMW 1.2406). Raw meat fermentation was performed with the wild-type (WT) and phage-cured strains. The WT strain produced high numbers of phages (5.2±1.8×107 plaque-forming units g-1) in the meat batter. However, the prophage did not significantly affect the meat fermentation process. Induction experiments suggested an acidic environment as a potential trigger for prophage induction. Phage induction by ultraviolet light before strain cryopreservation remains functional for at least 10 weeks of storage. Intact prophages are active during meat fermentation. However, in this study, this has no measurable consequences for fermentation, suggesting a high resiliency of meat fermentation against phages. Inadequate handling of lysogenic starter strains, even before preservation, can lead to phage introduction into food fermentation and unintended host lysis.

Synthetic Peptides Capable of Potent Multigroup Staphylococcal Quorum Sensing Activation and Inhibition in Both Cultures and Biofilm Communities.

The pathogen Staphylococcus epidermidis uses a chemical signaling process, i.e., quorum sensing (QS), to form robust biofilms and cause human infection. Many questions remain about QS in S. epidermidis, as it uses this intercellular communication pathway to both negatively and positively regulate virulence traits. Herein, we report synthetic multigroup agonists and antagonists of the S. epidermidis accessory gene regulator (agr) QS system capable of potent superactivation and complete inhibition, respectively. These macrocyclic peptides maintain full efficacy across the three major agr specificity groups, and their activity can be "mode-switched" from agonist to antagonist via subtle residue-specific structural changes. We describe the design and synthesis of these non-native peptides and demonstrate that they can appreciably decrease biofilm formation on abiotic surfaces, underscoring the potential for agr agonism as a route to block S. epidermidis virulence. Additionally, we show that both the S. epidermidis agonists and antagonists are active in S. aureus, another common pathogen with a related agr system, yet only as antagonists. This result not only revealed one of the most potent agr inhibitors known in S. aureus but also highlighted differences in the mechanisms of agr agonism and antagonism between these related bacteria. Finally, our investigations reveal unexpected inhibitory behavior for certain S. epidermidis agr agonists at sub-activating concentrations, an observation that can be leveraged for the design of future probes with enhanced potencies. Together, these peptides provide a powerful tool set to interrogate the role of QS in S. epidermidis infections and in Staphylococcal pathogenicity in general.

LP340, a novel histone deacetylase inhibitor, decreases liver injury and fibrosis in mice: role of oxidative stress and microRNA-23a.

Effective therapy for liver fibrosis is lacking. Here, we examined whether LP340, the lead candidate of a new-generation of hydrazide-based HDAC1,2,3 inhibitors (HDACi), decreases liver fibrosis. Liver fibrosis was induced by CCl4 treatment and bile duct ligation (BDL) in mice. At 6 weeks after CCl4, serum alanine aminotransferase increased, and necrotic cell death and leukocyte infiltration occurred in the liver. Tumor necrosis factor-α and myeloperoxidase markedly increased, indicating inflammation. After 6 weeks, α-smooth muscle actin (αSMA) and collagen-1 expression increased by 80% and 575%, respectively, indicating hepatic stellate cell (HSC) activation and fibrogenesis. Fibrosis detected by trichrome and Sirius-red staining occurred primarily in pericentral regions with some bridging fibrosis in liver sections. 4-Hydroxynonenal adducts (indicator of oxidative stress), profibrotic cytokine transforming growth factor-β (TGFβ), and TGFβ downstream signaling molecules phospho-Smad2/3 also markedly increased. LP340 attenuated indices of liver injury, inflammation, and fibrosis markedly. Moreover, Ski-related novel protein-N (SnoN), an endogenous inhibitor of TGFβ signaling, decreased, whereas SnoN expression suppressor microRNA-23a (miR23a) increased markedly. LP340 (0.05mg/kg, ig., daily during the last 2weeks of CCl4 treatment) decreased 4-hydroxynonenal adducts and miR23a production, blunted SnoN decreases, and inhibited the TGFβ/Smad signaling. By contrast, LP340 had no effect on matrix metalloproteinase-9 expression. LP340 increased histone-3 acetylation but not tubulin acetylation, indicating that LP340 inhibited Class-I but not Class-II HDAC in vivo. After BDL, focal necrosis, inflammation, ductular reactions, and portal and bridging fibrosis occurred at 2 weeks, and αSMA and collagen-1 expression increased by 256% and 560%, respectively. LP340 attenuated liver injury, ductular reactions, inflammation, and liver fibrosis. LP340 also decreased 4-hydroxynonenal adducts and miR23a production, prevented SnoN decreases, and inhibited the TGFβ/Smad signaling after BDL. In vitro, LP340 inhibited immortal human hepatic stellate cells (hTERT-HSC) activation in culture (αSMA and collagen-1 expression) as well as miR23a production, demonstrating its direct inhibitory effects on HSC. In conclusions, LP340 is a promising therapy for both portal and pericentral liver fibrosis, and it works by inhibiting oxidative stress and decreasing miR23a.

DPP-4 inhibitors sitagliptin and PF-00734,200 mitigate dopaminergic neurodegeneration, neuroinflammation and behavioral impairment in the rat 6-OHDA model of Parkinson’s disease

Epidemiological studies report an elevated risk of Parkinson's disease (PD) in patients with type 2 diabetes mellitus (T2DM) that is mitigated in those prescribed dipeptidyl peptidase 4 (DPP-4) inhibitors. With an objective to characterize clinically translatable doses of DPP-4 inhibitors (gliptins) in a well-characterized PD rodent model, sitagliptin, PF-00734,200 or vehicle were orally administered to rats initiated either 7-days before or 7-days after unilateral medial forebrain bundle 6-hydroxydopamine (6-OHDA) lesioning. Measures of dopaminergic cell viability, dopamine content, neuroinflammation and neurogenesis were evaluated thereafter in ipsi- and contralateral brain. Plasma and brain incretin and DPP-4 activity levels were quantified. Furthermore, brain incretin receptor levels were age-dependently evaluated in rodents, in 6-OHDA challenged animals and human subjects with/without PD. Cellular studies evaluated neurotrophic/neuroprotective actions of combined incretin administration. Pre-treatment with oral sitagliptin or PF-00734,200 reduced methamphetamine (meth)-induced rotation post-lesioning and dopaminergic degeneration in lesioned substantia nigra pars compacta (SNc) and striatum. Direct intracerebroventricular gliptin administration lacked neuroprotective actions, indicating that systemic incretin-mediated mechanisms underpin gliptin-induced favorable brain effects. Post-treatment with a threefold higher oral gliptin dose, likewise, mitigated meth-induced rotation, dopaminergic neurodegeneration and neuroinflammation, and augmented neurogenesis. These gliptin-induced actions associated with 70–80% plasma and 20–30% brain DPP-4 inhibition, and elevated plasma and brain incretin levels. Brain incretin receptor protein levels were age-dependently maintained in rodents, preserved in rats challenged with 6-OHDA, and in humans with PD. Combined GLP-1 and GIP receptor activation in neuronal cultures resulted in neurotrophic/neuroprotective actions superior to single agonists alone. In conclusion, these studies support further evaluation of the repurposing of clinically approved gliptins as a treatment strategy for PD.

Abstract 2909: ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms

Abstract CCAAT/Enhancer Binding Protein β (C/EBPβ) is a transcription factor that is an established driver of cellular transformation in several cancer types with poor prognosis, including glioblastoma (GBM), triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). These cancers’ growth is often supported by an immunosuppressive (IS) tumor microenvironment (TME), characterized by high expression of TAMs, MDSCs, Tregs, and in the case of GBM, IS microglia. In addition to its cell-autonomous role of promoting survival and proliferation in cancer cells, C/EBPβ is also critical for IS M2 macrophage polarization, while its role in other TME populations is less understood. ST101 is a peptide antagonist of C/EBPβ that is being evaluated in a Phase 2 clinical study in patients with recurrent and newly diagnosed glioblastoma (NCT04478279). Here we explored the impact of ST101 on IS TME cell populations and on activation of cytotoxic T-cells in macrophage co-culture systems. ST101 increased the M1:M2 ratio up to 40-fold in hPBMC-derived macrophages cultured under conditions that typically drive M2 polarization. Further, ST101 promoted M2 repolarization to the M1 cell type, demonstrating that C/EBPβ is required for maintenance of the M2 program. When added to co-cultures of T cells with M2 macrophages, ST101 induced a 3-4-fold increase in intracellular IFN-γ staining indicating enhanced T cell activation. Notably, in M1 cultures ST101 further enhanced expression of the immune activity marker CD80 and T cell activation. Similar to its effect on M2 repolarization, ST101 induced activation of iPs-derived IS human microglia cultures toward an M1-like program. ST101 also impacted IS cell populations in vivo, as demonstrated in an orthotopic TNBC model in which ST101 exposure was associated with a reduction of the TAM fraction, increased intratumoral M1/M2 ratios, reduction of Tregs, increase of CD8:Treg fraction, and enhanced activity in combination with anti-PD-1. Similar observations were observed in clinical samples, where following ST101 exposure a significant decrease in expression of factors involved in M2 polarization, including CD209, SIGLEC5, and IL-24, and T cell suppression, including FoxP3 and inhibitory KIR proteins, were observed. Gene expression analysis suggests that ST101 induced a decrease in the intratumoral Treg:TIL ratio, indicating a shift towards a more immune-active TME. Overall, these results provide novel evidence for the contribution of C/EBPβ to the expression of multiple IS cancer-associated cell types and support the use of ST101 to antagonize C/EBPβ and promote an immune-active TME. Further, these data provide rational for evaluating the clinical impact of ST101 in combinations with immune checkpoint inhibitors. Citation Format: Claudio Scuoppo, Julia Diehl, Ricardo Ramirez, Mark Koester, Erin Gallagher, Siok Leong, Jerel Gonzales, Karen Mendelson, Zach Mattes, Lila Ghamsari, Gene Merutka, Abi Vainstein-Haras, Barry J. Kappel, Jim A. Rotolo. ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2909.

Methamphetamine Enhancement of HIV-1 gp120-Mediated NLRP3 Inflammasome Activation and Resultant Proinflammatory Responses in Rat Microglial Cultures.

Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders (HANDs) remain prevalent in HIV-1-infected individuals despite the evident success of combined antiretroviral therapy (cART). The mechanisms underlying HAND prevalence in the cART era remain perplexing. Ample evidence indicates that HIV-1 envelope glycoprotein protein 120 (gp120), a potent neurotoxin, plays a pivotal role in HAND pathogenesis. Methamphetamine (Meth) abuse exacerbates HANDs, but how this occurs is not fully understood. We hypothesize that Meth exacerbates HANDs by enhancing gp120-mediated neuroinflammation. To test this hypothesis, we studied the effect of Meth on gp120-induced microglial activation and the resultant production of proinflammatory cytokines in primary rat microglial cultures. Our results show that Meth enhanced gp120-induced microglial activation, as revealed by immunostaining and Iba-1 expression, and potentiated gp120-mediated NLRP3 expression and IL-1β processing and release, as assayed by immunoblotting and ELISA. Meth also augmented the co-localization of NLRP3 and caspase-1, increased the numbers of NLRP3 puncta and ROS production, increased the levels of iNOS expression and NO production, and increased the levels of cleaved gasderminD (GSDMD-N; an executor of pyroptosis) in gp120-primed microglia. The Meth-associated effects were attenuated or blocked by MCC950, an NLRP3 inhibitor, or Mito-TEMPO, a mitochondrial superoxide scavenger. These results suggest that Meth enhances gp120-associated microglial NLRP3 activation and the resultant proinflammatory responses via mitochondria-dependent signaling.

Abstract 5287: Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids

Abstract Gliomas, a type of brain tumor originating from glial cells, comprise a heterogenous group of neoplasms. High-grade gliomas, in particular, present significant therapeutic challenges in the clinic. Unlike other solid tumors, the heterogenous glioma microenvironment is heavily enriched with tumor associated suppressor cells including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which can account for 30-40% of the cellular composition1. These pro-tumorigenic myeloid cells are thought to contribute to the resistance of glioma to immunotherapy. Efforts to target the suppressive nature of these dominating cell types are in development to yield more therapeutic options for patients with these devastating diseases. Constitutive activation of STAT3 in gliomas has been associated with increased tumor progression and reduced immune response. We have observed in other cancer models that a phenotypic switch occurs from STAT3 pathway activation in 3D spheroid culture to STAT3 inactivation when cultured in 2D. Therefore, we sought to determine the role of STAT3 activation and its role in the interplay between glioma and their immune suppressive environment using a 3D spheroid model. We modified our commercially available test platform, 3D-Predict™ Glioma, such that it is conducive to monitoring immune cell function. Newly diagnosed treatment naive primary samples from HGG patients were dissociated and cryogenically preserved and banked. Samples were profiled for the abundance of tumor associated suppressive myeloid cells using flow cytometry. We determined that relative proportions of tumor suppressor cells were detectable and varied across HGG primary patient samples with TAMs being the predominate immune cell type identified. Pro-tumorigenic TAM markers including CD163 and CD206 were correlated with temozolomide response and were associated with poor patient prognosis. When we evaluated the role of p-STAT in HGG primary samples, p-STAT was detected at baseline and abundance was related back to the presence of tumor associated immune suppressor cells. The impact of STAT3 signaling blockade by the inhibitor WP1066 was determined ex vivo, and efficacy was related back to MGMT promotor methylation status and patient clinical outcome. We detected shifts in cytokine signatures following treatment of HGG 3D spheroid models with WP1066 which are known to target tumor associated suppressor cell functions. This work demonstrates the critical role suppressive immune cells play in HGG clinical response. Given p-STAT can be altered in 2D culture compared to 3D culture, ex vivo therapeutic response profiling of HGG samples is necessary using the more physiologically relevant spheroid culture model. 1.Hambardzumyan et al. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 19, 20-27 (2016). Citation Format: Katy A. Lassahn, Ashley K. Elrod, Teresa M. DesRochers, Kathryn M. Appleton. Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5287.

Intestinal tissue-resident memory T cells maintain distinct identity from circulating memory T cells after in vitro restimulation.

Resident memory T (TRM) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that TRM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro TRM expansion to apply in an immunotherapy setting. However, it has also been shown that TRM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how TRM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that TRM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded TRM retained their phenotype, including the expression of key TRM markers CD69 and CD103 (ITGAE). The optimal culture of TRM required low O2 pressure to maintain the expression of these and other TRM-associated molecules. Expanded TRM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed TRM profile retention, including expression of TRM-related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of TRM cells that maintain their TRM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of TRM.

Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin.

Long-read transcriptome sequencing provides us with a convenient tool for the thorough study of biological processes such as neuronal plasticity. Here, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome the limitations of short-read RNA-Seq approaches, we performed an Oxford Nanopore Technologies MinION-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation. We used a specific approach to exclude uncapped mRNAs during sample preparation. Overall, we found 23,652 novel transcripts in comparison to reference annotations, out of which ~6000 were entirely novel and mostly transposon-derived loci. Analysis of differentially expressed genes (DEG) showed that 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points, respectively. Most notably, multiple genes encoding ribosomal proteins, with a high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms observed in this study may help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity.

Efficient material-induced activation of monocyte-derived dendritic cells releasing surface molecules, matrix metalloproteinases, and growth factors needed for regenerative tissue remodeling

New experimental approaches for tissue repair have recently been proposed and include the application of natural or synthetic biomaterials and immune cells. Herein, fully synthetic poly(glycidyl ether) (PGE) copolymer coatings are evaluated as bioinstructive materials for the in vitro culture and intrinsic activation of human immune cells. Immature monocyte-derived dendritic cells (moDCs) are exposed to PGE brush and gel coatings of varying copolymer composition, wettability, and deformability immobilized on polystyrene culture dishes. Compared to moDCs cultured on standard tissue culture-treated polystyrene, activation marker levels on the cell surface are strongly enhanced on PGE substrates. Thereby, moDCs undergo a distinct morphological change and reach levels of activation comparable to those achieved by toll-like receptor (TLR) ligand liposaccharide (LPS), specifically for the expression of costimulatory molecules CD86 and CD40 as well as human leukocyte antigen (HLA)-DR. In addition, PGE coatings induce a significantly enhanced level of programmed cell death ligands 1 and 2 (PD-L1/-L2) on the moDC surface, two molecules crucially involved in maintaining immune tolerance. In addition, an increased release of matrix metalloproteinases MMP-1 and MMP-7, as well as transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) was observed in moDCs cultured on PGE substrates. As fully synthetic biomaterials, PGE coatings demonstrate intrinsic functional competence in instructing immature human moDCs for phenotypic activation in vitro, accompanied by the secretion of bioactive molecules, which are known to be crucial for tissue regeneration. Hence, PGE coatings hold strong potential for immune-modulating implant coatings, while PGE-activated moDCs are promising candidates for future clinical cell-based immunoengineering therapies.

Cholesterol 24-hydroxylase at the choroid plexus contributes to brain immune homeostasis

The choroid plexus (CP) plays a key role in remotely controlling brain function in health, aging, and disease. Here, we report that CP epithelial cells express the brain-specific cholesterol 24-hydroxylase (CYP46A1) and that its levels are decreased under different mouse and human brain conditions, including amyloidosis, aging, and SARS-CoV-2 infection. Using primary mouse CP cell cultures, we demonstrate that the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, downregulates inflammatory transcriptomic signatures within the CP, found here to be elevated across multiple neurological conditions. Invitro, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) downregulates CYP46A1 expression, while overexpression of CYP46A1 or its pharmacological activation in mouse CP organ cultures increases resilience to TNF-α. Invivo, overexpression of CYP46A1 in the CP in transgenic mice with amyloidosis is associated with better cognitive performance and decreased brain inflammation. Our findings suggest that CYP46A1 expression in the CP impacts the role of this niche as a guardian of brain immune homeostasis.

Pharmacological blockade of cannabinoid receptor 2 signaling does not affect LPS/IFN-γ-induced microglial activation

Cannabinoid receptor 2 (CB2) signaling modulates microglial responses to inflammatory stimuli. Our previous studies demonstrated that genetic deletion of CB2 inhibits microglial activation during inflammatory stimulation of toll-like receptors (TLRs) or in neurodegenerative conditions. However, we cannot exclude developmental effects of the constitutive CB2 knockout (CB2−/−), which could mediate compensatory outcomes in CB2−/− mice. In the present study, we therefore tested whether acute pharmacological inhibition of CB2 receptor has a similar effect on microglial activation as in CB2−/− in response to inflammatory stimulation. Our findings suggest that the CB2-specific antagonist SR144528 has little or no effect on LPS/IFN-γ-induced activation in primary microglia or organotypic hippocampal slice cultures at nanomolar concentrations. We show that SR144528 did not alter LPS/IFN-γ-mediated microglial cytokine secretion, Iba1 and CD68 staining intensity or morphology at 1 and 10 nM. Although SR144528 suppressed LPS/IFN-γ-induced microglial activation at 1 µM, this anti-inflammatory effect was not dependent on CB2 receptors and exceeded the Ki on CB2 receptors by more than a thousand-fold. Thus, SR144528 does not mimic the anti-inflammatory effects observed in the CB2−/− microglia after LPS/IFN-γ stimulation. Therefore, we propose that the deletion of CB2 probably triggered an adaptive mechanism, making microglia less responsive to inflammatory stimulation.

Transient receptor potential ankyrin 1 (TRPA1) mediates reactive oxygen species-induced Ca2+ entry, mitochondrial dysfunction, and caspase-3/7 activation in primary cultures of metastatic colorectal carcinoma cells

Colorectal carcinoma (CRC) represents the fourth most common cancer worldwide and is the third most common cause of malignancy-associated mortality. Distant metastases to the liver and lungs are the main drivers of CRC-dependent death. Pro-oxidant therapies, which halt disease progression by exacerbating oxidative stress, represent an antitumour strategy that is currently exploited by chemotherapy and ionizing radiation. A more selective strategy to therapeutically exploit reactive oxygen species (ROS) signaling would consist in targeting a redox sensor that is up-regulated in metastatic cells and is tightly coupled to the stimulation of cancer cell death programs. The non-selective cation channel, Transient Receptor Potential Ankyrin 1 (TRPA1), serves as a sensor of the cellular redox state, being activated to promote extracellular Ca2+ entry by an increase in oxidative stress. Recent work demonstrated that TRPA1 channel protein is up-regulated in several cancer types and that TRPA1-mediated Ca2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca2+ dysfunction and apoptosis. Herein, we sought to assess for the first time the outcome of TRPA1 activation by ROS on primary cultures of metastatic colorectal carcinoma (mCRC cells). We found that TRPA1 channel protein is up-regulated and mediates enhanced hydrogen peroxide (H2O2)-induced Ca2+ entry in mCRC cells as compared to non-neoplastic control cells. The lipid peroxidation product 4-hydroxynonenal (4-HNE) is the main ROS responsible for TRPA1 activation upon mCRC cell exposure to oxidative stress. TRPA1-mediated Ca2+ entry in response to H2O2 and 4-HNE results in mitochondrial Ca2+ overload, followed by mitochondrial depolarization and caspase-3/7 activation. Therefore, targeting TRPA1 could represent an alternative strategy to eradicate metastatic CRC by enhancing its sensitivity to oxidative stress.

T-cell-derived TNF-α and a cluster of immunological parameters from plasma allow a separation between SARS-CoV-2 convalescent versus vaccinated elite athletes.

Guidelines for medical clearing after SARS-CoV-2 infection in elite athletes do not include T-cell immunity aspects despite its relevance in the course of COVID-19 disease. Therefore, we aimed to analyze T-cell-related cytokines before and after in-vitro activation of CD4+ T-cells. We sampled professional indoor sports athletes at medical clearing after SARS-CoV-2 infection obtaining clinical, fitness data, and serological data including CD4+ T-cell cytokines. All data were analyzed by principal component analysis and 2 × 2 repeated measures ANOVA. CD4+ T-cells were sampled for cell culture activation with anti-CD3/anti-CD28 tetramers. At medical clearing, CD4+ T-cells from convalescent athletes secreted increased levels of TNF-α 72h after in-vitro activation compared to vaccinated athletes. IL-18 levels in plasma were elevated and a cluster of parameters differentiated convalescent from vaccinated athletes by 13 parameters at the timepoint of medical clearing. All clinical data indicate infection is resolved, while increased TNF-α may reflect altered proportions of peripheral T-cells as a hangover of infection.