A New Butyrate Releaser Exerts a Protective Action against SARS-CoV-2 Infection in Human Intestine

Abstract

Butyrate is a major gut microbiome metabolite that regulates several defense mechanisms against infectious diseases. Alterations in the gut microbiome, leading to reduced butyrate production, have been reported in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A new butyrate releaser, useful for all the known applications of butyrate, presenting physiochemical characteristics suitable for easy oral administration, (N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), has been recently developed. We investigated the protective action of FBA against SARS-CoV-2 infection in the human small intestine and enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), neuropilin-1 (NRP1), pro-inflammatory cytokines expression, genes involved in the antiviral response and the activation of Nf-kB nuclear factor (erythroid-derived 2-like) 2 (Nfr2) pathways. We found that FBA positively modulates the crucial aspects of the infection in small intestinal biopsies and human enterocytes, reducing the expression of ACE2, TMPRSS2 and NRP1, pro-inflammatory cytokines interleukin (IL)-15, monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and regulating several genes involved in antiviral pathways. FBA was also able to reduce the number of SARS-CoV-2-infected cells, and ACE2, TMPRSS2 and NRP1 expression. Lastly, through the inhibition of Nf-kB and the up-regulation of Nfr2, it was also able to reduce the expression of pro-inflammatory cytokines IL-15, MCP-1 and TNF-α in human enterocytes. The new butyrate releaser, FBA, exerts a preventive action against SARS-CoV-2 infection. It could be considered as an innovative strategy to limit COVID-19.

Keywords: COVID-19; angiotensin-converting enzyme-2; intestinal models; transmembrane protease serine 2; viral infection.

Figure 1

Effect of FBA on the main molecular players of SARS-CoV-2 infection and inflammatory cytokines expression in ex vivo and in vitro models. (A) Small intestinal biopsies from 5 healthy subjects were treated with 2 mM FBA for 24 h. Cells were collected for RNA extraction. FBA was able to reduce ACE2, TMPRSS2 and NRP1 (panel above) and IL-15, MCP-1, and TNF-α expression (panel below). (B) Caco-2 cells were treated with 2 mM FBA for 24 h. Cells were collected for RNA extraction. The treatment with FBA reduced ACE2, TMPRSS2 and NRP1 (panel above) and IL-15, MCP-1, and TNF-α expression (panel below). Data represent the means with ±SD of 3 independent experiments, each performed in triplicate. Data were analyzed using a paired t-test. * p < 0.05 vs. only medium.

Figure 2

Effects of FBA on several genes involved in the anti-viral pathways. Heatmap reporting the average relative expression of different genes between untreated cells (NT) and 2 mM FBA-stimulated human enterocytes (administered FBA for 24 h). Differentially expressed genes were (i) those with a Log2 fold change above 0.3 or below −0.3, and an adjusted p-value < 0.05 (here shown as −Log10 FDR).

Figure 3

FBA prevented SARS-CoV-2 infection through Nf-Kb and nuclear factor (erythroid-derived 2-like) 2 (Nfr2) regulation. (A) Caco-2 cells were infected with 0.1 MOI of WT SARS-CoV-2 that was pretreated with 2 mM FBA for 24 h. Cells were collected for RNA and protein extraction and processed for immunofluorescence. The N protein was visualized using N Alexa Fluor-594 (green) and nuclei were stained with DAPI (blue). Cells were observed through confocal microscopy (Left panel). SARS-CoV-2 infection in Caco-2 cells was confirmed by the quantification of N proteins by immunofluorescence staining. The pretreatment with FBA resulted in a reduction in the number of SARS-CoV-2-infected enterocytes. Representative imagines are reported in Figure 3A. Scale bar, 20 µm. (Right panel) The quantification of the N gene of viral RNA by RT-PCR analysis. The pretreatment with FBA inhibited N gene transcription in SARS-CoV-2-infected cells. (B) Pre-incubation with FBA significantly down-regulated ACE2, TMPRSS2 and NRP1 expression in Caco-2 cells exposed to WT SARS-CoV-2. (C) Pre-incubation with FBA significantly reduced the IL-15, MCP-1 and TNF-α expression in Caco-2 cells exposed to WT SARS-CoV-2. (D) Pre-incubation with FBA inhibited Nf-kB activation and increased antioxidant nuclear factor Nfr2. The amounts of these proteins and of β-actin were measured via Western blot. The histogram below shows optical density of the proteins, obtained with Image Lab software. The relative quantification of proteins was normalized against β-actin proteins and was calculated using the ratio between phosphorylated and total proteins. The figure shows representative images of three experiments that are qualitatively similar. The expression data are normalized against non-infected cells (NI). “+” or “–“ mean the presence or the absence of a particular compound. Data represent the means with ±SD of 3 independent experiments, each performed in triplicate. Data were analyzed using a paired t-test. * p < 0.05.

Figure 4

The molecular structure of N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA).