Dysregulated functional and metabolic response in multiple sclerosis patient macrophages correlate with a more inflammatory state, reminiscent of trained immunity

AbstractIn multiple sclerosis (MS), immune cells invade the central nervous system and destroy myelin. Macrophages contribute to demyelination and myelin repair, and their role in each process depends on their ability to acquire specific phenotypes in response to external signals. Here, we assess whether defects in MS patient macrophage responses may lead to increased inflammation or lack of neuro-regenerative effects.To test this hypothesis, CD14+CD16- monocytes from MS patients and healthy controls were activated in vitro to obtain homeostatic-like, pro-inflammatory and pro-regenerative macrophages. Myelin phagocytic capacity and surface molecule expression of CD14, CD16 and HLA-DR were evaluated with flow cytometry. In parallel, macrophages were assessed through RNA sequencing and metabolomics.We observed that MS patient monocytes ex vivo recapitulate their preferential activation toward a CD16+ phenotype, a subset of pro-inflammatory cells present in MS lesions. Even in the absence of pro-inflammatory stimuli, MS patient macrophages exhibit a pro-inflammatory transcriptomic profile with higher levels of cytokine/chemokine suggesting increased recruitment capacities. Interestingly, MS patient macrophages exhibit a specific metabolic signature with a mitochondrial energy metabolism blockage resulting in a shift from oxidative phosphorylation to glycolysis. Furthermore, we observe a failure to up-regulate apoptosis effector genes in the pro inflammatory state suggesting a longer-lived pro-inflammatory macrophage population.Our results highlight an intrinsic defect of MS patient macrophages that provide evidence of innate immune cell memory in MS.

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Towards a ‘hot’ tumor phenotype: DKN-01 sensitizes the tumor micro-environment via pro-immune cell cytokine release in vitro and ex vivo

Gynecologic Oncology ◽

10.1016/s0090-8258(21)00670-3 ◽

2021 ◽

Vol 162 ◽

pp. S12

Author(s):

Jhalak Dholakia ◽

Jaclyn Wall ◽

Carly Bess Scalise ◽

Ashwini Katre ◽

Rebecca Arend

Keyword(s):

Immune Cell ◽

Ex Vivo ◽

Cytokine Release ◽

Tumor Phenotype ◽

Release In Vitro

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Using ex vivo culture to assess dynamic phenotype changes in human prostate macrophages following exposure to therapeutic drugs

Scientific Reports ◽

10.1038/s41598-021-98903-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Clovis Boibessot ◽

France-Hélène Joncas ◽

Aerin Park ◽

Zohra Berrehail ◽

Jean-François Pelletier ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Cell ◽

Ex Vivo ◽

Cell Phenotype ◽

Gleason Grade ◽

Tumor Resistance ◽

Grade Group ◽

In Vivo Models ◽

Novel Associations

AbstractWithin the prostate tumor microenvironment (TME) there are complex multi-faceted and dynamic communication occurring between cancer cells and immune cells. Macrophages are key cells which infiltrate and surround tumor cells and are recognized to significantly contribute to tumor resistance and metastases. Our understanding of their function in the TME is commonly based on in vitro and in vivo models, with limited research to confirm these model observations in human prostates. Macrophage infiltration was evaluated within the TME of human prostates after 72 h culture of fresh biopsies samples in the presence of control or enzalutamide. In addition to immunohistochemistry, an optimized protocol for multi-parametric evaluation of cellular surface markers was developed using flow cytometry. Flow cytometry parameters were compared to clinicopathological features. Immunohistochemistry staining for 19 patients with paired samples suggested enzalutamide increased the expression of CD163 relative to CD68 staining. Techniques to validate these results using flow cytometry of dissociated biopsies after 72 h of culture are described. In a second cohort of patients with Gleason grade group ≥ 3 prostate cancer, global macrophage expression of CD163 was unchanged with enzalutamide treatment. However, exploratory analyses of our results using multi-parametric flow cytometry for multiple immunosuppressive macrophage markers suggest subgroup changes as well as novel associations between circulating biomarkers like the neutrophil to lymphocyte ratio (NLR) and immune cell phenotype composition in the prostate TME. Further, we observed an association between B7–H3 expressing tumor-associated macrophages and the presence of intraductal carcinoma. The use of flow cytometry to evaluate ex vivo cultured prostate biopsies fills an important gap in our ability to understand the immune cell composition of the prostate TME. Our results highlight novel associations for further investigation.

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ER stress-mediated BK dysfunction in the DRG underlies pain in a model of multiple sclerosis

10.1101/2020.01.22.915546 ◽

2020 ◽

Author(s):

Muhammad Saad Yousuf ◽

Samira Samtleben ◽

Shawn M. Lamothe ◽

Timothy Friedman ◽

Ana Catuneanu ◽

...

Keyword(s):

Multiple Sclerosis ◽

Er Stress ◽

Treatment Options ◽

Inflammatory Cells ◽

Bk Channels ◽

Resting Membrane Potential ◽

Common Symptom ◽

Chemical Chaperone ◽

Pain Hypersensitivity

ABSTRACTNeuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went to assess disease-mediated changes in the functional properties of Ca2+-sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.

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Anti-CD20 therapy depletes activated myelin-specific CD8+T cells in multiple sclerosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915309116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25800-25807 ◽

Cited By ~ 17

Author(s):

Joseph J. Sabatino ◽

Michael R. Wilson ◽

Peter A. Calabresi ◽

Stephen L. Hauser ◽

Jonathan P. Schneck ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Antibody Therapy ◽

Myelin Proteins ◽

T Cell Epitopes ◽

Control Subjects ◽

Anti Cd20

CD8+T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential autoantigenic targets, prior studies of myelin-reactive CD8+T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8+T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8+T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8+T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8+T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8+T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8+T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20+CD8+T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8+T cells in MS, indicates these cells may be attractive targets in MS therapy.

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Topical Pioglitazone Nanoformulation for the Treatment of Atopic Dermatitis: Design, Characterization and Efficacy in Hairless Mouse Model

Pharmaceutics ◽

10.3390/pharmaceutics12030255 ◽

2020 ◽

Vol 12 (3) ◽

pp. 255 ◽

Cited By ~ 3

Author(s):

Lupe Carolina Espinoza ◽

Rodrigo Vera-García ◽

Marcelle Silva-Abreu ◽

Òscar Domènech ◽

Josefa Badia ◽

...

Keyword(s):

Atopic Dermatitis ◽

Ex Vivo ◽

In Vitro Release ◽

Skin Barrier ◽

Inflammatory Cells ◽

Hairless Mouse ◽

Skin Barrier Function ◽

Inflammatory Parameters ◽

Therapeutic Benefits

Pioglitazone (PGZ) is a drug used to treat type 2 diabetes mellitus that has been reported to show additional therapeutic activities on diverse inflammatory parameters. The aim of this study was to optimize a topical PGZ-loaded nanoemulsion (PGZ-NE) in order to evaluate its effectiveness for treating atopic dermatitis (AD). The composition of the nanoformulation was established by pseudo-ternary diagram. Parameters such as physical properties, stability, in vitro release profile, and ex vivo permeation were determined. The efficacy study was carried out using oxazolone-induced AD model in hairless mice. PGZ-NE released the drug following a hyperbolic kinetic. Additionally, its properties provided high retention potential of drug inside the skin. Therapeutic benefits of PGZ-NE were confirmed on diverse events of the inflammatory process, such as reduction of lesions, enhancement of skin barrier function, diminished infiltration of inflammatory cells, and expression of pro-inflammatory cytokines. These results were reinforced by atomic force microscope (AFM), which demonstrated the ability of the formulation to revert the rigidification caused by oxazolone and consequently improve the elasticity of the skin. These results suggest that PGZ-NE may be a promising treatment for inflammatory dermatological conditions such as AD.

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The spectrum of spinal cord lesions in a primate model of multiple sclerosis

Multiple Sclerosis Journal ◽

10.1177/1352458518822408 ◽

2019 ◽

Vol 26 (3) ◽

pp. 284-293 ◽

Cited By ~ 2

Author(s):

Jennifer A Lefeuvre ◽

Joseph R Guy ◽

Nicholas J Luciano ◽

Seung-Kwon Ha ◽

Emily Leibovitch ◽

...

Keyword(s):

Multiple Sclerosis ◽

Spinal Cord ◽

Nonhuman Primate ◽

Ex Vivo ◽

Inflammatory Cells ◽

Common Marmoset ◽

Autoimmune Encephalomyelitis ◽

Primate Model ◽

Magnetic Resonance Imaging Mri ◽

Experimental Autoimmune

Background: Experimental autoimmune encephalomyelitis (EAE) in the common marmoset is a nonhuman primate model of multiple sclerosis (MS) that shares numerous clinical, radiological, and pathological features with MS. Among the clinical features are motor and sensory deficits that are highly suggestive of spinal cord (SC) damage. Objective: To characterize the extent and nature of SC damage in symptomatic marmosets with EAE using a combined magnetic resonance imaging (MRI) and histopathology approach. Materials and Methods: SC tissues from five animals were scanned using 7 T MRI to collect high-resolution ex vivo images. Lesions were segmented and classified based on shape, size, and distribution along the SC. Tissues were processed for histopathological characterization (myelin and microglia/macrophages). Statistical analysis, using linear mixed-effects models, evaluated the association between MRI and histopathology. Results: Marmosets with EAE displayed two types of SC lesions: focal and subpial lesions. Both lesion types were heterogeneous in size and configuration and corresponded to areas of marked demyelination with high density of inflammatory cells. Inside the lesions, the MRI signal was significantly correlated with myelin content ( p < 0.001). Conclusions: Our findings underscore the relevance of this nonhuman primate EAE model for better understanding mechanisms of MS lesion formation in the SC.

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Impact of Exercise on Immunometabolism in Multiple Sclerosis

Journal of Clinical Medicine ◽

10.3390/jcm9093038 ◽

2020 ◽

Vol 9 (9) ◽

pp. 3038 ◽

Cited By ~ 1

Author(s):

Remsha Afzal ◽

Jennifer K Dowling ◽

Claire E McCoy

Keyword(s):

Multiple Sclerosis ◽

Cell Function ◽

Immune Cell ◽

Therapeutic Benefits ◽

Demyelinating Lesions ◽

Inflammatory State ◽

Autoimmune Condition ◽

The Central Nervous System ◽

Axonal Degradation

Multiple Sclerosis (MS) is a chronic, autoimmune condition characterized by demyelinating lesions and axonal degradation. Even though the cause of MS is heterogeneous, it is known that peripheral immune invasion in the central nervous system (CNS) drives pathology at least in the most common form of MS, relapse-remitting MS (RRMS). The more progressive forms’ mechanisms of action remain more elusive yet an innate immune dysfunction combined with neurodegeneration are likely drivers. Recently, increasing studies have focused on the influence of metabolism in regulating immune cell function. In this regard, exercise has long been known to regulate metabolism, and has emerged as a promising therapy for management of autoimmune disorders. Hence, in this review, we inspect the role of key immunometabolic pathways specifically dysregulated in MS and highlight potential therapeutic benefits of exercise in modulating those pathways to harness an anti-inflammatory state. Finally, we touch upon current challenges and future directions for the field of exercise and immunometabolism in MS.

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Studies to Elucidate the Mechanism of Cardio Protective and Hypotensive Activities of Anogeissus acuminata (Roxb. ex DC.) in Rodents

Molecules ◽

10.3390/molecules25153471 ◽

2020 ◽

Vol 25 (15) ◽

pp. 3471

Author(s):

Fatima Saqib ◽

Muhammad Arif Aslam ◽

Khizra Mujahid ◽

Luigi Marceanu ◽

Marius Moga ◽

...

Keyword(s):

Creatine Kinase ◽

Ex Vivo ◽

Inflammatory Cells ◽

Left Ventricular ◽

Guanosine Monophosphate ◽

Sprague Dawley ◽

Positive Effects ◽

Creatine Kinase Mb

Anogeissus acuminata (Roxb. ex DC.) is a folkloric medicinal plant in Asia; including Pakistan; used as a traditional remedy for cardiovascular disorders. This study was planned to establish a pharmacological basis for the trivial uses of Anogeissus acuminata in certain medical conditions related to cardiovascular systems and to explore the underlying mechanisms. Mechanistic studies suggested that crude extract of Anogeissus acuminata (Aa.Cr) produced in vitro cardio-relaxant and vasorelaxant effects in isolated paired atria and aorta coupled with in vivo decrease in blood pressure by invasive method; using pressure and force transducers connected to Power Lab Data Acquisition System. Moreover; Aa.Cr showed positive effects in left ventricular hypertrophy in Sprague Dawley rats observed hemodynamically by a decrease in cardiac cell size and fibrosis; along with absence of inflammatory cells; coupled with reduced levels of angiotensin converting enzyme (ACE) and renin concentration along with increased concentrations of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP). In Acute Myocardial Infarction (AMI) model; creatine kinase (CK), creatine kinase-MB (CK-MB) and lactic acid dehydrogenase (LDH levels) were found to be decreased; along with decreased necrosis; edema and recruitment of inflammatory cells histologically. In vivo and ex vivo studies of Anogeissus acuminata provided evidence of vasorelaxant; hypotensive and cardioprotective properties facilitated through blockage of voltage-gated Ca++ ion channel; validating its use in cardiovascular diseases

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GABAA receptor α4-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00107.2017 ◽

2017 ◽

Vol 313 (2) ◽

pp. L406-L415 ◽

Cited By ~ 14

Author(s):

Gene T. Yocum ◽

Damian L. Turner ◽

Jennifer Danielsson ◽

Matthew B. Barajas ◽

Yi Zhang ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Immune Cells ◽

Cell Activation ◽

Cell Function ◽

Immune Cell ◽

Therapeutic Potential ◽

Ex Vivo ◽

Asthma Model

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

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Interferon-beta affects mitochondrial activity in CD4+lymphocytes: Implications for mechanism of action in multiple sclerosis

Multiple Sclerosis Journal ◽

10.1177/1352458514561909 ◽

2014 ◽

Vol 21 (10) ◽

pp. 1262-1270 ◽

Cited By ~ 4

Author(s):

Aiden Haghikia ◽

Simon Faissner ◽

Derek Pappas ◽

Bartosz Pula ◽

Denis A Akkad ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

Energy Metabolism ◽

Mitochondrial Function ◽

Interferon Beta ◽

Mononuclear Cells ◽

Ex Vivo ◽

Mitochondrial Activity ◽

Mitochondrial Energy Metabolism ◽

The Impact

Background:Whereas cellular immune function depends on energy supply and mitochondrial function, little is known on the impact of immunotherapies on cellular energy metabolism.Objective:The objective of this paper is to assess the effects of interferon-beta (IFN-β) on mitochondrial function of CD4+T cells.Methods:Intracellular adenosine triphosphate (iATP) in phytohemagglutinin (PHA)-stimulated CD4+cells of multiple sclerosis (MS) patients treated with IFN-β and controls were analyzed in a luciferase-based assay. Mitochondrial-transmembrane potential (ΔΨm) in IFN-β-treated peripheral blood mononuclear cells (PBMCs) was investigated by flow cytometry. Expression of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) in CD4+cells of IFN-β-treated individuals and correlations between genetic variants in the key metabolism regulator PGC-1α and IFN-β response in MS were analyzed.Results:IFN-β-treated MS patients exhibited a dose-dependent reduction of iATP levels in CD4+T cells compared to controls ( p < 0.001). Mitochondrial effects were reflected by depolarization of ΔΨm. Expression data revealed changes in the transcription of OXPHOS-genes. iATP levels in IFN-β-responders were reduced compared to non-responders ( p < 0.05), and the major T allele of the SNP rs7665116 of PGC-1α correlated with iATP-levels.Conclusion:Reduced iATP-synthesis ex vivo and differential expression of OXPHOS-genes in CD4+T cells point to unknown IFN-β effects on mitochondrial energy metabolism, adding to potential pleiotropic mechanisms of action.

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