SARS-CoV-2 Causes Lung Inflammation through Metabolic Reprogramming and RAGE

Abstract

Clinical studies indicate that patients infected with SARS-CoV-2 develop hyperinflammation, which correlates with increased mortality. The SARS-CoV-2/COVID-19-dependent inflammation is thought to occur via increased cytokine production and hyperactivity of RAGE in several cell types, a phenomenon observed for other disorders and diseases. Metabolic reprogramming has been shown to contribute to inflammation and is considered a hallmark of cancer, neurodegenerative diseases, and viral infections. Malfunctioning glycolysis, which normally aims to convert glucose into pyruvate, leads to the accumulation of advanced glycation end products (AGEs). Being aberrantly generated, AGEs then bind to their receptor, RAGE, and activate several pro-inflammatory genes, such as IL-1b and IL-6, thus, increasing hypoxia and inducing senescence. Using the lung epithelial cell (BEAS-2B) line, we demonstrated that SARS-CoV-2 proteins reprogram the cellular metabolism and increase pyruvate kinase muscle isoform 2 (PKM2). This deregulation promotes the accumulation of AGEs and senescence induction. We showed the ability of the PKM2 stabilizer, Tepp-46, to reverse the observed glycolysis changes/alterations and restore this essential metabolic process.

Keywords: RAGE; SARS-CoV-2; Tepp-46; glycolysis; inflammation; metabolic reprogramming; mitochondria.

Figure 1

Long COVID-19 or post-acute sequelae. Schematic representation of persistent symptoms post-acute COVID-19. The figure was created using the BioRender.com website accessed on 15 March 2022.

Figure 2

SARS-CoV-2 proteins alter mitochondrial maximal respiration. (A,B). Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BEAS-2B cells untreated or transfected with SARS-CoV-2 proteins for 48 h using a Seahorse XFe96 analyzer and a Mito stress test kit from Seahorse. Oligomycin, FCCP, and rotenone/antimycin (arrows) were added at 23, 50, and 75 min, respectively. (C,D). Graphs displaying the maximal respiration and spare capacity measured (pmol/min).

Figure 3

Changes in Proteins Responsible for Metabolic Reprogramming. Expression of PTBP1 (A) and PKM2 (B) proteins isolated from untreated or BEAS-2B transfected as indicated. Quantification of the relative protein levels was determined from the band intensity using ImageJ software and normalized relative to the H3. PTBP (C), PKM1 (D), and PKM2 (E) mRNA expression was obtained by qPCR using BEAS-2B transfected with all SARS-CoV-2 plasmids except for the membrane. Bar graphs represent the means ± S.D. of at least two independent experiments. Data represent the mean ± S.D. Results were judged statistically significant by ANOVA (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Figure 4

(A). Concentration of intercellular pyruvate in BEAS-2B cells transfected with SARS-CoV-2 plasmids as indicated (Envelope (Env.), Spike, or All (all SARS-CoV-2 plasmids except for the membrane). (B). Cellular concentration of AGEs (ng/mL) was measured by ELISA using BEAS-2B transfected with all SARS-CoV-2 plasmids except for the membrane. (C,D). mRNA expression of RAGE and HIF-1α. mRNA measured by qPCR using BEAS-2B transfected with all SARS-CoV-2 plasmids except for the membrane. Results were judged statistically significant by ANOVA (ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 5

Persistence of Inflammation. (A). Expression of p65 subunit of NF-kB proteins isolated from untreated or BEAS-2B transfected as indicated by Western blot analysis. (B,C). mRNA expression of IL-1β and IL-8 as obtained by qPCR using BEAS-2B transfected then treated with 10 nM of Tepp-46 as indicated. Results were judged statistically significant by ANOVA (ns = not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (D), Nsp2, Nsp4 (modest), Nsp5, and the Spike proteins promote senescence in BEAS-2B as obtained using β-gal assay compared to the Mock untransfected control. (E). The addition of Nsp4 and NNsp5 to BEAS-2B causes a strong Golgi apparatus fragmentation compared to the Mock or the spike alone.

Figure 6

Schematic representation of metabolic reprograming leading to RAGE signaling and inflammation pathways in post-acute COVID-19 (black arrows). PKM2 stabilizer, Tepp-46, reverses this metabolic reprogramming and inhibits RAGE signaling (green arrows).