Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 and Spike Protein Subunit 1

A hallmark of COVID-19 is a hyperinflammatory state associated with severity. Monocytes undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism associated with production of pro-inflammatory cytokines. This response was dependent on hypoxia-inducible factor-1α, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production and metabolic reprogramming. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.

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Metformin Suppresses Monocyte Inflammation and Metabolic Reprogramming by SARS-CoV-2 Spike Protein

Innovation in Aging ◽

10.1093/geroni/igab046.1284 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. 332-332

Author(s):

Theodore Cory ◽

Russell Emmons ◽

Johnathan Yarbro ◽

Brandt Pence

Keyword(s):

Older Adults ◽

Inflammatory Cytokines ◽

Cytokine Production ◽

Immune Cell ◽

Mitochondrial Metabolism ◽

Metabolic Reprogramming ◽

Spike Protein ◽

Glycolytic Metabolism ◽

Protein Subunit ◽

Necrosis Factor Alpha

Abstract COVID-19 disproportionately affects older adults, and a hallmark of the disease is a hyperinflammatory state that is associated with severity. Various anti-inflammatory therapeutics have shown mixed efficacy in treating COVID-19, and the mechanisms by which hyperinflammation occurs are not well understood. Previous research indicated that monocytes, a key innate immune cell, undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism – including the geroprotector drug metformin – could inhibit the inflammatory response in monocytes. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism that was associated with production of pro-inflammatory cytokines including interleukin-6 and tumor necrosis factor-alpha. This response was dependent on hypoxia-inducible factor-1alpha, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production in monocytes, and abrogated glycolytic and mitochondrial metabolism. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by treatments that interfere with glycolytic activation, including metformin. This has potential implications for the treatment of hyperinflammation during COVID-19, which disproportionately affects older adults.

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Modulation of monocyte responses by progressive multiple sclerosis therapy.

10.26686/wgtn.17148233.v1 ◽

2021 ◽

Author(s):

◽

Carl Beyers

Keyword(s):

Multiple Sclerosis ◽

Inflammatory Cytokines ◽

Immune Cell ◽

Inflammatory Responses ◽

Neurodegenerative Disorder ◽

Cell Activity ◽

Cell Subset ◽

Disease Heterogeneity ◽

Receptor Affinity ◽

Anti Inflammatory

<p>Multiple sclerosis (MS) is an immune-mediated neurodegenerative disorder that is distinguished by neuroinflammation and demyelination. MS is severely debilitating and remains the most common cause of disability arising from non-traumatic brain and CNS damage in adults. In its progressive phase there are no effective treatments, so new therapy options are an urgent research priority. Extensive work has been done on the role of the adaptive immune system in contributing to the disease pathology and on the effects of therapies targeting lymphocytes in relapsing-remitting MS. Fewer studies have examined innate immune cells in people with progressive MS. This thesis addresses that gap by profiling monocyte phenotype and function in response to new and repurposed drugs that may provide benefit in progressive MS. This was achieved by modelling the drugs’ effects in vitro using peripheral blood cells from people with progressive MS and healthy subjects. Clozapine is an atypical antipsychotic with broad receptor affinity that is primarily used to treat refractory schizophrenia. In addition to is antipsychotic action through dopamine receptor (DR) D2, its broad neuro-immune receptor affinity is thought to dampen inflammatory responses in the CNS. This thesis highlights clozapine’s anti-inflammatory effect by demonstrating a reduction in the expression of pro-inflammatory cytokines that are associated with MS pathology in treated monocytes. Clozapine also induced a significant increase in the expression of D1. We observed that D1 expression changes happened alongside alterations to immune cell activity and that MS participant monocytes were much more susceptible to DR expression changes compared to healthy people. Together this data substantiates clozapine as a potential treatment for progressive disease. MIS416 is a large, non-soluble microparticle suspension that induces nuclear factor kappa B (NFB) dependent cytokine induction. We show here that monocytes are key cytokine responder cells to MIS416 and explore the molecular mechanism by demonstrating its effects on transcription factor activity. Our data showing increased production of cytokines by MIS416 suggests a route of treatment efficacy through tolerisation mechanisms, and by reducing inflammation through upregulation of anti-inflammatory cytokines and negative feedback from pro-inflammatory cytokine release. Furthermore, we demonstrate how disease heterogeneity, phenotype, and genotype could significantly affect drug response outcomes in patients who received the drug as part of a phase 2 clinical trial. Much of this work was done using new spectral cytometer technology. Its use allowed for the novel approach that enabled the subtraction of autofluorescent noise from out data, and we demonstrate its efficient functioning, ease of use, and utility in acquiring high dimensional datasets. The resulting large dataset allowed us the opportunity to interrogate it using bioinformatics tools, and we show their utility as adjunct tools to conventional methods of gating and statistical analysis. These analyses help demonstrate that monocytes are a heterogenous immune cell subset that is functionally distinct in people with progressive MS when compared to monocytes from healthy individuals.</p>

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Expression Suppression and Activity Inhibition of TRPM7 Regulate Cytokine Production and Multiple Organ Dysfunction Syndrome During Endotoxemia: a New Target for Sepsis

Current Molecular Medicine ◽

10.2174/1566524019666190709181726 ◽

2019 ◽

Vol 19 (8) ◽

pp. 547-559 ◽

Cited By ~ 2

Author(s):

Sebastian Gatica ◽

Felipe Eltit ◽

Juan F. Santibanez ◽

Diego Varela ◽

Claudio Cabello-Verrugio ◽

...

Keyword(s):

Ion Channel ◽

Inflammatory Cytokines ◽

Transient Receptor Potential ◽

Cytokine Production ◽

Inflammatory Responses ◽

Receptor Potential ◽

Multiple Organ ◽

Blood Levels ◽

Transient Receptor ◽

Pro Inflammatory Cytokines

Background:Main pathological features detected during sepsis and endotoxemia include over-secretion of pro-inflammatory cytokines and multiorgan dysfunction syndrome (MODS). Unfortunately, current clinical efforts to treat sepsis are unsatisfactory, and mortality remains high. Interestingly, transient receptor potential (TRP) melastatin 7 (TRPM7) ion channel controlling Ca2+ and Mg2+ permeability is involved in cytokine production and inflammatory response. Furthermore, TRPM7 downregulation has been shown to alleviate local symptoms in some models of sepsis, but its effects at a systemic level remain to be explored.Objective:To test whether TRPM7 mediates cytokine production and MODS during endotoxemia.Method:Endotoxemic and sham-endotoxemic rats were subjected to pharmacological inhibition of TRPM7 using carvacrol, or to expression suppression by adenovirus delivery of shRNA (AdVshTRPM7). Then, cytokine and MODS levels in the blood were measured.Results:Inhibition of TRPM7 with carvacrol and suppression with AdVshTRPM7 were both efficient in inhibiting the over-secretion of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-12, in endotoxemic rats, without inducing downregulation in blood levels of antiinflammatory cytokines IL-10 and IL-4. Additionally, the use of carvacrol and AdVshTRPM7 significantly prevented liver and pancreas dysfunction, altered metabolic function, and hypoglycemia, induced by endotoxemia. Furthermore, muscle mass wasting and cardiac muscle damage were also significantly reduced by the use of carvacrol and AdVshTRPM7 in endotoxemic rats.Conclusion:Our results indicate TRPM7 ion channel as a key protein regulating inflammatory responses and MODS during sepsis. Moreover, TRPM7 appears as a novel molecular target for the management of sepsis.

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Modulation of monocyte responses by progressive multiple sclerosis therapy.

10.26686/wgtn.17148233 ◽

2021 ◽

Author(s):

◽

Carl Beyers

Keyword(s):

Multiple Sclerosis ◽

Inflammatory Cytokines ◽

Immune Cell ◽

Inflammatory Responses ◽

Neurodegenerative Disorder ◽

Cell Activity ◽

Cell Subset ◽

Disease Heterogeneity ◽

Receptor Affinity ◽

Anti Inflammatory

<p>Multiple sclerosis (MS) is an immune-mediated neurodegenerative disorder that is distinguished by neuroinflammation and demyelination. MS is severely debilitating and remains the most common cause of disability arising from non-traumatic brain and CNS damage in adults. In its progressive phase there are no effective treatments, so new therapy options are an urgent research priority. Extensive work has been done on the role of the adaptive immune system in contributing to the disease pathology and on the effects of therapies targeting lymphocytes in relapsing-remitting MS. Fewer studies have examined innate immune cells in people with progressive MS. This thesis addresses that gap by profiling monocyte phenotype and function in response to new and repurposed drugs that may provide benefit in progressive MS. This was achieved by modelling the drugs’ effects in vitro using peripheral blood cells from people with progressive MS and healthy subjects. Clozapine is an atypical antipsychotic with broad receptor affinity that is primarily used to treat refractory schizophrenia. In addition to is antipsychotic action through dopamine receptor (DR) D2, its broad neuro-immune receptor affinity is thought to dampen inflammatory responses in the CNS. This thesis highlights clozapine’s anti-inflammatory effect by demonstrating a reduction in the expression of pro-inflammatory cytokines that are associated with MS pathology in treated monocytes. Clozapine also induced a significant increase in the expression of D1. We observed that D1 expression changes happened alongside alterations to immune cell activity and that MS participant monocytes were much more susceptible to DR expression changes compared to healthy people. Together this data substantiates clozapine as a potential treatment for progressive disease. MIS416 is a large, non-soluble microparticle suspension that induces nuclear factor kappa B (NFB) dependent cytokine induction. We show here that monocytes are key cytokine responder cells to MIS416 and explore the molecular mechanism by demonstrating its effects on transcription factor activity. Our data showing increased production of cytokines by MIS416 suggests a route of treatment efficacy through tolerisation mechanisms, and by reducing inflammation through upregulation of anti-inflammatory cytokines and negative feedback from pro-inflammatory cytokine release. Furthermore, we demonstrate how disease heterogeneity, phenotype, and genotype could significantly affect drug response outcomes in patients who received the drug as part of a phase 2 clinical trial. Much of this work was done using new spectral cytometer technology. Its use allowed for the novel approach that enabled the subtraction of autofluorescent noise from out data, and we demonstrate its efficient functioning, ease of use, and utility in acquiring high dimensional datasets. The resulting large dataset allowed us the opportunity to interrogate it using bioinformatics tools, and we show their utility as adjunct tools to conventional methods of gating and statistical analysis. These analyses help demonstrate that monocytes are a heterogenous immune cell subset that is functionally distinct in people with progressive MS when compared to monocytes from healthy individuals.</p>

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Vitamin D3 controls TLR4- and TLR2-mediated inflammatory responses of endometrial cells

10.21203/rs.2.19548/v1 ◽

2019 ◽

Author(s):

Alireza Ghanavatinejad ◽

Nesa Rashidi ◽

Mahroo Mirahmadian ◽

Simin Rezania ◽

Mahdokht Mosalaei ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Vitamin D3 ◽

Inflammatory Responses ◽

Female Reproductive Tract ◽

Endometrial Stromal Cells ◽

Endometrial Cells ◽

Tnf Α ◽

Pre Treatment ◽

Tract Infections ◽

Pro Inflammatory Cytokines

Abstract Background: There is a significant association between intrauterine infection-associated inflammatory responses and such pregnancy complications as abortion and preterm labor. Here, we aimed to investigate anti-inflammatory effects of 1,25 (OH)2 D3 on pro-inflammatory cytokines secretion and expression of TLR2, TLR4 and MyD88 in endometrial stromal cells (ESCs) and whole endometrial cells (WECs). Method: WECs were treated with either lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and ESCs were treated with LPS. IL-6, IL8 and TNF-α were quantified using ELISA technique. TLR2, TLR4 and MyD88 expression were assessed by RT-qPCR. TLR4 expression at the protein level was studied by Western blot technique. Results: 1,25 (OH)2 D3 significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pre-treatment increased production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pre-treatment markedly reduced LPS-induced TLR-4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pre-treated with 1,25 (OH)2 D3 before stimulation with LPS. Conclusion: 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintenance of endometrial immune homeostasis through controlling potentially harmful inflammatory responses associated with female reproductive tract infections. Key words: Vitamin D3, Endometrium, Inflammation, Toll like receptors, Pro-inflammatory cytokines

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DSS-induced inflammation in the colon drives a pro-inflammatory signature in the brain that is ameliorated by prophylactic treatment with the S100A9 inhibitor paquinimod

10.1101/2021.09.22.461244 ◽

2021 ◽

Author(s):

Sarah Talley ◽

Rasa Valiauga ◽

Lillian Anderson ◽

Abigail R Cannon ◽

Mashkoor A Choudhry ◽

...

Keyword(s):

Rna Sequencing ◽

Inflammatory Cytokines ◽

Immune Cell ◽

Inflammatory Responses ◽

Light Sheet ◽

Positive Control ◽

Light Sheet Microscopy ◽

Cytokines And Chemokines ◽

Organ Systems ◽

The Brain

Background: Inflammatory Bowel Disease (IBD) is established to drive pathological sequelae in organ systems outside the intestine, including the central nervous system (CNS). Many patients exhibit cognitive deficits, particularly during disease flare. The connection between colonic inflammation and neuroinflammation remains unclear and characterization of the neuroinflammatory phenotype in the brain during colitis is ill-defined. Methods: Transgenic mice expressing a bioluminescent reporter of active caspase-1 were treated with 2% Dextran Sodium Sulfate (DSS) for 7 days to induce acute colitis, and colonic, systemic and neuroinflammation were assessed. In some experiments, mice were prophylactically treated with paquinimod (ABR-215757) to inhibit S100A9 inflammatory signaling. As a positive control for peripheral-induced neuroinflammation, mice were injected with lipopolysaccharide (LPS). Colonic, systemic and brain inflammatory cytokines and chemokines were measured by cytokine bead array (CBA) and Proteome profiler mouse cytokine array. Bioluminescence was quantified in the brain and caspase activation was confirmed by immunoblot. Immune cell infiltration into the CNS was measured by flow cytometry, while light sheet microscopy was used to monitor changes in resident microglia localization in intact brains during DSS or LPS-induced neuroinflammation. RNA sequencing was performed to identify transcriptomic changes occurring in the CNS of DSS-treated mice. Expression of inflammatory biomarkers were quantified in the brain and serum by qRT-PCR, ELISA and WB. Results: DSS-treated mice exhibited clinical hallmarks of colitis, including weight loss, colonic shortening and inflammation in the colon. We also detected a significant increase in inflammatory cytokines in the serum and brain, as well as caspase and microglia activation in the brain of mice with ongoing colitis. RNA sequencing of brains isolated from DSS-treated mice revealed differential expression of genes involved in the regulation of inflammatory responses. This inflammatory phenotype was similar to the signature detected in LPS-treated mice, albeit less robust and transient, as inflammatory gene expression returned to baseline following cessation of DSS. Pharmacological inhibition of S100A9, one of the transcripts identified by RNA sequencing, attenuated colitis severity and systemic and neuroinflammation. Conclusions: Our findings suggest that local inflammation in the colon drives systemic inflammation and neuroinflammation, and this can be ameliorated by inhibition of the S100 alarmin, S100A9.

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Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses

Vaccines ◽

10.3390/vaccines8030433 ◽

2020 ◽

Vol 8 (3) ◽

pp. 433

Author(s):

Seiki Shirai ◽

Atsushi Kawai ◽

Meito Shibuya ◽

Lisa Munakata ◽

Daiki Omata ◽

...

Keyword(s):

Immune Responses ◽

Cytokine Production ◽

Immune Cell ◽

Inflammatory Responses ◽

Influenza Viruses ◽

Inflammatory Cytokine Production ◽

Virus Challenge ◽

Aluminum Salts ◽

Lipid Nanoparticle

Vaccination is a critical and reliable strategy for controlling the spread of influenza viruses in populations. Conventional seasonal split vaccines (SVs) for influenza evoke weaker immune responses than other types of vaccines, such as inactivated whole-virion vaccines, although SVs are highly safe compared to other types. Here, we assessed the potential of the lipid nanoparticle (LNP) we developed as an adjuvant for conventional influenza SV as an antigen in mice. The LNP did not induce the production of cytokines such as interleukin-6 (IL-6) and IL-12 p40 by dendritic cells or the expression of co-stimulatory molecules on these cells in vitro. In contrast, an SV adjuvanted with LNP improved SV-specific IgG1 and IgG2 responses and the Th1 response compared to the SV alone in mice. In addition, SV adjuvanted with an LNP gave superior protection against the influenza virus challenge over the SV alone and was as effective as SV adjuvanted with aluminum salts in mice. The LNP did not provoke inflammatory responses such as inflammatory cytokine production and inflammatory immune cell infiltration in mice, whereas aluminum salts induced inflammatory responses. These results suggest the potential of the LNP as an adjuvant without inflammatory responses for influenza SVs. Our strategy should be useful for developing influenza vaccines with enhanced efficacy and safety.

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Macrophages rely on extracellular serine to suppress aberrant cytokine production

Scientific Reports ◽

10.1038/s41598-021-90086-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kento Kurita ◽

Hiroya Ohta ◽

Ibuki Shirakawa ◽

Miyako Tanaka ◽

Yasuyuki Kitaura ◽

...

Keyword(s):

Cytokine Production ◽

Immune Cell ◽

De Novo ◽

Inflammatory Responses ◽

Culture Media ◽

Expression Patterns ◽

Interleukin 10 ◽

Cell Functions ◽

Mitochondrial Ros

AbstractA growing body of evidence indicates that cellular metabolism is involved in immune cell functions, including cytokine production. Serine is a nutritionally non-essential amino acid that can be generated by de novo synthesis and conversion from glycine. Serine contributes to various cellular responses, but the role in inflammatory responses remains poorly understood. Here, we show that macrophages rely on extracellular serine to suppress aberrant cytokine production. Depleting serine from the culture media reduced the cellular serine content in macrophages markedly, suggesting that macrophages depend largely on extracellular serine rather than cellular synthesis. Under serine deprivation, macrophages stimulated with lipopolysaccharide showed aberrant cytokine expression patterns, including a marked reduction of anti-inflammatory interleukin-10 expression and sustained expression of interleukine-6. Transcriptomic and metabolomics analyses revealed that serine deprivation causes mitochondrial dysfunction: reduction in the pyruvate content, the NADH/NAD+ ratio, the oxygen consumption rate, and the mitochondrial production of reactive oxygen species (ROS). We also found the role of mitochondrial ROS in appropriate cytokine production. Thus, our results indicate that cytokine production in macrophages is tightly regulated by the nutritional microenvironment.

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SARS-CoV-2 Spike protein promotes hyper-inflammatory response that can be ameliorated by Spike-antagonistic peptide and FDA-approved ER stress and MAP kinase inhibitors in vitro

10.1101/2020.09.30.317818 ◽

2020 ◽

Author(s):

Alan C-Y. Hsu ◽

Guoqiang Wang ◽

Andrew T. Reid ◽

Punnam Chander Veerati ◽

Prabuddha S. Pathinayake ◽

...

Keyword(s):

Lung Injury ◽

Er Stress ◽

Map Kinase ◽

Inflammatory Cytokines ◽

Kinase Inhibitors ◽

Spike Protein ◽

Protein Subunit ◽

Epithelial Damage ◽

Antagonistic Peptide ◽

Pro Inflammatory Cytokines

SummarySARS-CoV-2 infection causes an inflammatory cytokine storm and acute lung injury. Currently there are no effective antiviral and/or anti-inflammatory therapies. Here we demonstrate that 2019 SARS-CoV-2 spike protein subunit 1 (CoV2-S1) induces high levels of NF-κB activations, production of pro-inflammatory cytokines and mild epithelial damage, in human bronchial epithelial cells. CoV2-S1-induced NF-κB activation requires S1 interaction with human ACE2 receptor and early activation of endoplasmic reticulum (ER) stress, and associated unfolded protein response (UPR), and MAP kinase signalling pathways. We developed an antagonistic peptide that inhibits S1-ACE2 interaction and CoV2-S1-induced productions of pro-inflammatory cytokines. The existing FDA-approved ER stress inhibitor, 4-phenylburic acid (4-PBA), and MAP kinase inhibitors, trametinib and ulixertinib, ameliorated CoV2-S1-induced inflammation and epithelial damage. These novel data highlight the potentials of peptide-based antivirals for novel ACE2-utilising CoVs, while repurposing existing drugs may be used as treatments to dampen elevated inflammation and lung injury mediated by SARS-CoV-2.

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