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Clinical Infection & Immunity

Short Communication

Volume 6, Number 3, September 2021, pages 82-85

Metformin Regulates the Inflammatory Response of Human Monocytes to SARS-CoV-2 Spike Glycoprotein

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel type of coronavirus that causes coronavirus disease 2019 (COVID-19). The World Health Organization characterized the global outbreak of COVID-19 as a pandemic on March of 2020. Critical cases of COVID-19 are associated with high mortality. Severity in COVID-19 involves an excessive inflammatory response, known as a cytokine storm [1-3], which includes increased secretion of interleukin (IL)-1β, IL-18, IL-6, and IL-8 [3].

Metformin (MTF) is currently used as the standard treatment for type 2 diabetes mellitus (T2DM) as it decreases glucose production in the liver. It was originally an antiviral drug used in influenza, and its hypoglycemic properties were just only one of its side effects [4]. T2DM is associated with greater disease risk and mortality by COVID-19 [5]. Studies indicate that among patients with both T2DM and COVID-19, mortality is lower among patients with T2DM who use MTF compared to non-users [1, 6]. MTF also exhibits an anti-inflammatory effect, regardless of diabetes status, in patients with COVID-19 [1, 2, 7]. Identifying a therapeutic role in MTF could be beneficial for developing an adjuvant treatment for COVID-19 [1].

Type I interferons (IFNs), such as IFN-β1, have been noted to have antiviral effects against RNA viruses, and play a key role in the defense against SARS-CoV-2 [2, 8].

We here study the effect of MTF on the response of human monocytes to SARS-CoV-2 spike (S) glycoprotein.

THP1-Dual monocytes (InvivoGen) were treated with 2 mM of MTF and incubated for 6 h. Cells were then stimulated with 11.2 nM SARS-CoV-2 S proteins for 4 h. These included SARS-CoV-2 S glycoprotein ectodomains (NR-52397, NR-52724, and NR-53589), receptor biding domain (BR-52946) and a stabilized trimer (NR-53524) (BEI Resources). Detection of transcription factor nuclear factor kappa B (NF-κB) and interferon regulatory factor (IRF) activity was performed according to manufacturers’ instructions (InvivoGen). NF-κB and IRF activation was expressed as a response ratio for each stimulus relative to activity in unstimulated cells. Lipopolysaccharide (LPS) was used as control at a concentration of 0.2 µg/mL. Plates were read in a Synergy Microplate reader (Biotek) after 24 h stimulation.

RNA was extracted from the cells after MTF treatment and stimulation with SARS-CoV-2 S glycoprotein stabilized trimer (NR-53524) (BEI Resources), and then converted into cDNA which was used for RT-PCR. RT-PCR was conducted according to SYBR Green Supermix protocol. Commercially available primers for the amplification of human IFN-B1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used (Applied Biosystems). Cycle threshold (CT) values of IFN-β1 were compared to the control GAPDH CT values. The 2^-ΔΔCT method was used to quantify the relative fold gene expression of IFN-β [9].

Stimulation of human monocytes with various portions and forms of the SARS-CoV-2 S glycoprotein elicited different levels of IRF activation (Fig. 1, white bars). S glycoprotein ectodomains (NR-52397 and NR-53589), and a stabilized trimer (NR-53524) induced activation of IRF in untreated cells. NF-κB was almost inexistent. In fact only the SARS-CoV-2 S glycoprotein stabilized trimer (NR-53524) was able to induce activation of NF-κB in human monocytes (Fig. 1b).

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Figure 1. Metformin (MTF) decreases interferon regulatory factor (IRF) activation in human monocytes stimulated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoproteins. (a) IRF and (b) nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in human monocytes were treated with 2 mM of MTF for 6 h, and then stimulated 11.2 nM of the following SARS-CoV-2 S proteins for 4 h: NR-52397: 1,196 residues (ectodomain) of the SARS-CoV-2 S glycoprotein; NR-52724: 1,194 residues (ectodomain) of the SARS-CoV-2 S glycoprotein; NR-52946: 223 residues of the SARS-CoV-2 S glycoprotein receptor binding domain; NR-53524: 1,194 residues (ectodomain) of the SARS-CoV-2 S glycoprotein (stabilized trimer); NR-53589: 1,194 residues (ectodomain) of the SARS-CoV-2 S glycoprotein. No statistically significant differences between untreated vs. MTF-treated (Mann-Whitney test, on four biological replicates).

Pre-treatment of human monocytic cells with MTF did not affect the activation of transcription factor NF-κB in response to SARS-CoV-2 S glycoprotein (Fig. 1b). However, IRF activation, if present, was decreased if cells had been treated with MTF (Fig. 1a).

This result indicates that MTF inhibits the expression of IRF. Since IRF modulates the downstream expression of type I IFN signaling pathways, the expression of IFN-β1was investigated.

We resourced to using RT-PCR in order to determine the expression of IFN-β1 in monocytes treated with MTF and stimulated with SARS-CoV-2 S protein stabilized trimer. The trimeric form of SARS-CoV-2 S glycoprotein upregulated the transcription of IFN-β, and this was abrogated if the cells have been pre-treated with MTF (Fig. 2).

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Figure 2. Metformin (MTF) decreases transcription of interferon (IFN)-beta in monocytes upon stimulation with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein stabilized trimer. Human monocytes were treated with 2 mM of MTF for 6 h, and then stimulated 11.2 nM of a stabilized trimer of the SARS-CoV-2 S glycoprotein proteins for 4 h. Expression levels for IFN-beta transcripts were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). *P < 0.05 unpaired t-test on three biological replicates.

MTF, a widely used drug to treat T2DM and metabolic syndrome, has immunomodulatory activity that reduces the production of pro-inflammatory cytokines by macrophages and neutrophils [7]. Given their pivotal function in host defense to COVID-19, understanding monocytes functions is warranted [10]. In vitro studies in monocytes, as well as clinical data have shown that elevated glucose concentrations lead to increased SARS-CoV-2 viral load, angiotensin-converting enzyme 2, and increased cytokine profiles [5, 6].

IRFs are transcription factors which control the production of type I IFNs, which play a key role in the induction of antiviral immune responses in coronavirus infections [2]. An imbalance of IRF can alter immune status and disease progression through its regulation of type I IFNs [11]. Our findings show a slight decrease in IRF activation upon stimulation with SARS-CoV-2 S glycoproteins in MTF-treated monocytes. The transcription of type I IFN, IFN-β1, however, was greatly increased by SARS-CoV-2 stabilized timer stimulation, and almost completely abrogated by MTF. MTF has previously been shown to decrease type I IFN, IFN-α pathways in the context of influenza vaccination [12]. IFN-α plays a vital role in antibody response and protection against viral infection, and its downregulation by MTF was mediated via mammalian target of rapamycin (mTOR) signaling inhibition [13]. Genetic studies on life-threatening COVID-19 patients have shown multiple receptors and regulators associated with type I immunity such as TLR3, TICAM1, TBK1, IRF3, UNC93B1, IRF7, IFNAR1, and IFNAR2 [13]. Although much has been described on the role of type I IFNs in alveolar macrophages (AMs), most of the effects on monocytes reported appear secondary, such as monocyte recruitment by monocyte chemoattractant protein-1 (MCP1) secreted by AMs [10]. Type I IFNs regulate the expression of other mediators through interferon-stimulated genes (ISGs). Some of these are downregulated or upregulated, with the purpose of switching the immune response away from neutrophil-mediated inflammation towards a lymphocyte-mediated response [14]. Myeloid cells of severe COVID-19 patients showed higher expression of pro-inflammatory cytokines and chemokines such as cxcl8 [15]. The levels of CXCL8 were shown to be elevated in both the blood and alveolar spaces in SARS-CoV patients early after disease onset [16]. MTF suppresses the expression of cxcl8 by monocytes [14, 17], so its anti-inflammatory effect may be through ISGs rather than a direct effect on NF-κB activation, which we did no observe. Corticosteroid treatment has been shown to reduce the expression of CXCL8 in SARS-CoV-infected human cells [18].

Besides its anti-inflammatory properties, MTF antioxidant, immunomodulatory, and antiviral capabilities could also offer protection in COVID-19, and help explain the significant reduction in disease severity and mortality in treated individuals.

Acknowledgments

None to declare.

Financial Disclosure

None to declare.

Conflict of Interest

None to declare.

Informed Consent

Not applicable.

Author Contributions

JC conceptualized the study. MP, JB, MM, and AC performed the experiments. JC, AC, and MM wrote the manuscript.

Data Availability

The authors declare that data supporting the findings of this study are available within the article.

1. Scheen AJ. Metformin and COVID-19: From cellular mechanisms to reduced mortality. Diabetes Metab. 2020;46(6):423-426.
   doi pubmed
2. Bagheri A, Moezzi SMI, Mosaddeghi P, Nadimi Parashkouhi S, Fazel Hoseini SM, Badakhshan F, Negahdaripour M. Interferon-inducer antivirals: Potential candidates to combat COVID-19. Int Immunopharmacol. 2021;91:107245.
   doi pubmed
3. Vabret N, Britton GJ, Gruber C, Hegde S, Kim J, Kuksin M, Levantovsky R, et al. Immunology of COVID-19: Current State of the Science. Immunity. 2020;52(6):910-941.
   doi pubmed
4. Amin S, Lux A, O'Callaghan F. The journey of metformin from glycaemic control to mTOR inhibition and the suppression of tumour growth. Br J Clin Pharmacol. 2019;85(1):37-46.
   doi pubmed
5. Lim S, Bae JH, Kwon HS, Nauck MA. COVID-19 and diabetes mellitus: from pathophysiology to clinical management. Nat Rev Endocrinol. 2021;17(1):11-30.
   doi pubmed
6. Varghese E, Samuel SM, Liskova A, Kubatka P, Busselberg D. Diabetes and coronavirus (SARS-CoV-2): Molecular mechanism of Metformin intervention and the scientific basis of drug repurposing. PLoS Pathog. 2021;17(6):e1009634.
   doi pubmed
7. Chen X, Guo H, Qiu L, Zhang C, Deng Q, Leng Q. Immunomodulatory and antiviral activity of metformin and its potential implications in treating coronavirus disease 2019 and lung injury. Front Immunol. 2020;11:2056.
   doi pubmed
8. Luo P, Qiu L, Liu Y, Liu XL, Zheng JL, Xue HY, Liu WH, et al. Metformin treatment was associated with decreased mortality in COVID-19 patients with diabetes in a retrospective analysis. Am J Trop Med Hyg. 2020;103(1):69-72.
   doi pubmed
9. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-408.
   doi pubmed
10. Knoll R, Schultze JL, Schulte-Schrepping J. Monocytes and macrophages in COVID-19. Front Immunol. 2021;12:720109.
    doi pubmed
11. Jefferies CA. Regulating IRFs in IFN Driven Disease. Front Immunol. 2019;10:325.
    doi pubmed
12. Saenwongsa W, Nithichanon A, Chittaganpitch M, Buayai K, Kewcharoenwong C, Thumrongwilainet B, Butta P, et al. Metformin-induced suppression of IFN-alpha via mTORC1 signalling following seasonal vaccination is associated with impaired antibody responses in type 2 diabetes. Sci Rep. 2020;10(1):3229.
    doi pubmed
13. Zhang Q, Bastard P, Liu Z, Le Pen J, Moncada-Velez M, Chen J, Ogishi M, et al. Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. Science. 2020;370(6515):eabd4570.
14. Glennon-Alty L, Moots RJ, Edwards SW, Wright HL. Type I interferon regulates cytokine-delayed neutrophil apoptosis, reactive oxygen species production and chemokine expression. Clin Exp Immunol. 2021;203(2):151-159.
    doi pubmed
15. Park JH, Lee HK. Re-analysis of Single Cell Transcriptome Reveals That the NR3C1-CXCL8-Neutrophil Axis Determines the Severity of COVID-19. Front Immunol. 2020;11:2145.
    doi pubmed
16. Khalil BA, Elemam NM, Maghazachi AA. Chemokines and chemokine receptors during COVID-19 infection. Comput Struct Biotechnol J. 2021;19:976-988.
    doi pubmed
17. Cory TJ, Emmons RS, Yarbro JR, Davis KL, Pence BD. Metformin suppresses monocyte immunometabolic activation by SARS-CoV-2 and spike protein subunit 1. bioRxiv. 2021.
    doi
18. Cinatl J, Jr., Michaelis M, Morgenstern B, Doerr HW. High-dose hydrocortisone reduces expression of the pro-inflammatory chemokines CXCL8 and CXCL10 in SARS coronavirus-infected intestinal cells. Int J Mol Med. 2005;15(2):323-327.
    doi pubmed

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Clinical Infection and Immunity is published by Elmer Press Inc.