Gut probiotic Lactobacillus rhamnosus attenuates PDE4B-mediated interleukin-6 induced by SARS-CoV-2 membrane glycoprotein

 Membrane glycoprotein is the most abundant protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but its role in COVID-19 (COVID-2019) has not been fully characterized. Here, we investigated the role of this protein in the cytokine storm and found that it can down-regulate PDE4B expression and IL-6 secretion.

Critical pneumonia in patients with coronavirus disease 2019 (COVID-19) can be associated with elevated inflammatory cytokines, a phenomenon known as a cytokine storm or macrophage activation syndrome (MAS)

• The hyper-induction of cytokines aggravated the auto-exaggerating inflammatory cascade, followed by multiple organ failure and lymphocytopenia, leading to dysregulated immunity
• Although there are many designated modalities such as corticosteroids, anti-viral or anti-malarial drugs to clinically control SARS-CoV-2, no evidence to date for the fully effective treatments to cure or alleviate the cytokine crisis
• An alternative approach may provide a great assistance in controlling the emergence of this pandemic
• Probiotic bacteria may propose a solution by delivering the fermentation metabolites to the lung via the bloodstream
• Short-chain fatty acids (SCFAs) such as butyric acid produced by gut probiotic can bind to free fatty acid receptors and dampen inflammation responses

Ethics statement

• Experiments of using Institute of Cancer Research (ICR) and PDE4B knockout mice were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC).
• ICR mice (female, 8-9 weeks old) were purchased from the National Laboratory Animal Center Taipei, Taiwan.
• Five mice per group were used in each experiment.

Methodology

• A plasmid carrying a gene encoding membrane glycoprotein (YP_009724393.1) of SARS-CoV-2 (Genebank accession: NC_045512.2) was transformed into Escherichia coli (E. coli) BL21 competent cells (Yeastern Biotech Co., Ltd, Taiwan) by enzymatic transformation.
• Proteins in E. coli BL21 were expressed by addition of 1 mM isopropyl β-D-thiogalactopyranoside at 20°C for 4 h, purified by a ProBondTM Purification System, and visualized in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels stained by Coomassie Brilliant Blue R-250.

Detection of IL-6 in BALF

• Recombinant proteins (100 µg) including SARS-CoV-2 membrane glycoprotein or GFP in phosphate buffered saline (PBS) (60 µL) were intranasally inoculated to mice.

Real-time polymerase chain reaction (RT PCR)

• After intranasal inoculation of recombinant membrane glycoprotein or GFP, lung of ICR mice was homogenized in T-PER™ Tissue Protein Extraction Reagent (ThermoFisher Scientific, Waltham, MA, USA) supplemented with an ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA).
• RNA (1 ng) was converted into cDNA using an iScript cDNA Synthesis kit.
• Primers were designed using the Primer-Blast tool from the National Center for Biotechnology Information (NCBI). The reaction of using cDNA (50 ng/µL) as a template was set for 40 cycles as follows: 95°C for 10 min, followed by a rapid-fire (RT) reaction in which the temperature was increased by 30 degrees Celsius.

Mycelium preparation

• The inner portion of the basidiomata of P. pulmonarius, an oyster mushroom, was excised by a flamed scalpel blade under sterile conditions and placed onto on malt extract agar medium (MEA)

Mycelium fermentation of L. rhamnosus EH8

• in Lactobacilli MRS broth (55.15 g/L) in the absence or presence of 2% mycelium powder at 37°C for 24 h.
• Phenol red [0.001% (w/v) (pH 7.4), Sigma-Aldrich] in MRS-flavoured broth served as an indicator of fermentation, converting from red to orange-yellow when fermentation occurred.

Quantification of L. rhamnosus in feces

• Five mice per group were used in each experiment. Two weeks after feeding, fecal was collected and dissolved 1:10 in 50 mM EDTA containing 5 mg lysozyme (Sigma-Aldrich).
• DNA in the pellet was extracted by using a heat lysis protocol as described above.

Cell culture J774 macrophage cell lines (105 cells/well) were cultured in Dulbecco's Modified Eagle Medium (DMEM, ThermoFisher Scientific) in 12-well cell culture plates.

• Cells were treated with 100 µM butyric acid or distilled H2O (control) for 10 min before addition of 50 µg recombinant membrane glycoprotein at 37°C, 5% CO2 for 12 h. After centrifugation, the level of IL-6 in supernatants was quantified by ELISA.

Detection of butyric acid by high performance liquid chromatography (HPLC)

• L. rhamnosus EH8 (107 CFU/mL) was cultured in MRS broth in the presence or absence of 2% mycelium powder at 37°C for 24 h.
• Culture media was collected and filtered through a 0.22 µm microfiltration membrane to remove bacteria and insoluble particles, and samples were added with concentrated HCl (100 µL) and extracted for 20 min by gently rolling using 5 ml diethyl ether.

Inhibition of Ffar2 ICR

• 200 µL of GLPG-0974 (5 µM) was orally administrated every day for 3 days 10 min before mice were fed with L. rhamnosus EH8 in the presence or absence of mycelium powder.
• After two-week feeding, mice were inoculated with recombinant membrane glycoprotein (100 µg) for 6 h and the level of IL-6 was measured by ELISA.

Importance of PDE4B

• IL-6 is a chief component of excessive cytokines released by activated macrophages observed in severe COVID-19 patients
• The membrane glycoprotein (YP_009724393.1) of SARS-CoV-2 was cloned into E. coli BL21 competent cells to obtain recombinant proteins, which were intranasally inoculated into ICR mice for 6 h followed by ELISA.
• Infection of recombinant SARS with recombinant protein produced an approximately 2-fold increase in the mRNA expression of the protein, whereas inoculation with control GFP produced a decrease in mRNA expression.

Mycelium fermentation of L. rhamnosus EH8 mitigates SARS-CoV-2 membrane glycoprotein-induced IL-6

• Many natural extracts as prebiotics, containing high content of polysaccharides, can withstand digestion and absorption in the small intestine and can be selectively fermented by probiotic gut bacteria.
• To examine the prebiotic property of fungal mycelia, a commensal bacterial strain isolated from human ****** was incubated in phenol red-containing Lactobacilli MRS broth in the presence of 2% mycelium powder under anaerobic conditions for 24 h.

Butyric acid was produced by mycelium fermentation of L. rhamnosus EH8

• It reduced IL-6 secretion and PDE4B expression in macrophages.
• The anti-inflammatory property of the acid has been demonstrated by previous studies
• In addition, it inhibited the expression of SARS-CoV-2 membrane glycoprotein, which is known to cause lung inflammation.

Ffar2 mediates the effect of mycelium fermentation of L. rhamnosus EH8 on reduction of PDE4B and IL-6

• GLPG-0974 in DMSO was given to mice to antagonize the Ffar 2 before feeding mice with L.rhμnosus eH8
• Two weeks after feeding the mice with the powder every day, the mice were intranasally inoculated with SARS-CoV-2 membrane glycoprotein for 6 h.
• Compared to the control, mice given the drug resulted in a substantial increase in the expression of the enzyme in the lung compared to those given the control (Fig. 4A).
• Treatment with GLPG also significantly raised the level (8.44 ± 0.82 pg/mg) of IL in BALF compared to IL in mice treated with control.

Discussion

• SARS-CoV-2 enters host cells via binding of the spike protein to angiotensin-converting enzyme 2 (ACE2), which can induce hyper-activation of NF-κB pathway leading to the induction of a variety of pro-inflammatory cytokines, including IL-6, TNF-α, and chemokines.
• Literature has revealed that nucleocapsid protein of SARS coronavirus HB can bind directly to the NF-Kappa recognition elements on the IL-600 promoter, which can regulate the expression of inflammatory cytokines (i.e., IL-56).
• The relationship between the production of anti-viral type I and III interferon (IFN) cytokines by targeting the signalling mediated by retinoic acid- inducible gene I (RIG-I), mitochondrial antiviral signaling (MAVS), and TANK-binding kinase 1 (TBK1), blocking the phosph- orylation, nuclear translocation, and activation of phosphorylated Interferon regulatory factor 3 (IRF3), a transcription factor which can be translocated to the nucleus and activate genes encoding IFN, has not been elucidated.

Reference

10.1016/j.jnutbio.2021.108821