key: cord-0737323-8qqf81gz authors: Pham, Minh Tan; Yang, Albert Jackson; Kao, Ming-Shan; Gankhuyag, Uuganbayar; Zayabaatar, Enkhbat; Jin, Shiow-Lian Catherine; Huang, Chun-Ming title: Gut probiotic Lactobacillus rhamnosus attenuates PDE4B-mediated interleukin-6 induced by SARS-CoV-2 membrane glycoprotein date: 2021-07-14 journal: J Nutr Biochem DOI: 10.1016/j.jnutbio.2021.108821 sha: 4a88cbc5634db5d5f72a0050d0affb998d20322c doc_id: 737323 cord_uid: 8qqf81gz Membrane glycoprotein is the most abundant protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but its role in coronavirus disease 2019 (COVID-19) has not been fully characterized. Mice intranasally inoculated with membrane glycoprotein substantially increased the interleukin (IL)-6, a hallmark of the cytokine storm, in bronchoalveolar lavage fluid (BALF), compared to mice inoculated with green fluorescent protein (GFP). The high level of IL-6 induced by membrane glycoprotein was significantly diminished in phosphodiesterase 4 (PDE4B) knockout mice, demonstrating the essential role of PDE4B in IL-6 signaling. Mycelium fermentation of Lactobacillus rhamnosus (L. rhamnosus) EH8 strain yielded butyric acid, which can down-regulate the PDE4B expression and IL-6 secretion in macrophages. Feeding mice with mycelia increased the relative abundance of commensal L. rhamnosus. Two-week supplementation of mice with L. rhamnosus plus mycelia considerably decreased membrane glycoprotein-induced PDE4B expression and IL-6 secretion. The probiotic activity of L. rhamnosus plus mycelia against membrane glycoprotein was abolished in mice treated with GLPG-0974, an antagonist of free fatty acid receptor 2 (Ffar2). Activation of Ffar2 in the gut-lung axis for down-regulation of the PDE4B-IL-6 signalling may provide targets for development of modalities including probiotics for treatment of the cytokine storm in COVID-19. Critical pneumonia in patients with coronavirus disease 2019 can be associated with elevated inflammatory cytokines, a phenomenon known as a cytokine storm or macrophage activation syndrome (MAS) [1] . The hyperinduction of cytokines aggravated the auto-exaggerating inflammatory cascade, followed by multiple organ failure and lymphocytopenia, leading to dysregulated immunity [2] . In severe COVID-19 cases, the storm of interleukin-6 (IL)-6 can enhance the massive release of various inflammatory cytokines [3] . It has been documented that severe acute respiratory syndrome coronavirus (SARS-CoV) directly triggered IL-6 induction in human epithelial cells by binding to nuclear factor kappa-light-chain (NF-κB) of activated B cells and up-regulated gene expression of IL-6 [4] . Recent reports demonstrated that administration of phosphodiesterase 4 (PDE4) inhibitors, such as roflumilast with a higher affinity to PDE4B than PDE4A, C and D [5] , acting on cyclic adenosine monophosphate (cAMP) pathway, represented a potential and effective therapy for COVID-19. Especially, PDE4 hydrolyzed cAMP to AMP by blocking cAMP hydrolysis, resulting in accumulating intracellular cAMP to positively regulate cytokine releases in macrophages [6] . Although there are many designated modalities such as corticosteroids, antiviral 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 storm in COVID-19 patients [7] . While the number of COVID-19 positive cases is rapidly surging, the vaccine development has faced many challenges including the antigenic selection and drift, adverse effect, manufacture, and global distribution [8] . Therefore, an alternative approach may provide a great assistance in controlling the emergence of COVID-19 pandemic. The use of probiotic may propose a valuable solution against SARS-CoV-2 by generation of beneficial molecules to hinder the viral spread and suppress the massive inflammatory responses [9] . Since the discovery of the gut-lung axis, evidence have revealed the advantages of probiotic bacteria to pulmonary health by delivering the fermentation metabolites to the lung via the bloodstream [10, 11] . Short-chain fatty acids (SCFAs) such as butyric acid produced by gut probiotic can bind to free fatty acid receptors (Ffar) to dampen inflammation responses [12, 13] . Lactobacillus rhamnosus (L. rhamnosus), a Gram-positive bacterium, is a commensal intestinal bacterial strain in mice and human [14] . Several studies have provided evidence for the protective role of lactic acid-producing bacteria in the defense against infections of viruses, including Ebola virus and cytomegalovirus [15] . Lactobacillus paracasei and Lactobacillus plantarum attenuated the secretion of pro-inflammatory IL-6 and IL-8 cytokines [16] . L. rhamnosus GG GR-1, and 4B15 strains substantially inhibited the release of tumor necrosis factor-alpha (TNF-α) in mouse macrophages [17, 18] . Moreover, feeding mice with Lactobacillus mucosae or fermentum down-regulated IL-6 and TNF-α in both acute and chronic inflammation [19] . In this study, we induced the fermentation of L. rhamnosus by the powder of mycelia, the vegetative part with high content of carbohydrates of a fungus [20] , as a prebiotic. Our results demonstrated for the first time that the secretion of IL-6 and expression of PDE4B induced by SARS-CoV-2 membrane glycoprotein in the lung were considerably reduced when mice fed with L. rhamnosus plus fungal mycelia. Experiments of using Institute of Cancer Research (ICR) and PDE4B knockout mice [21] housed in an animal facility at National Central University (NCU) were conducted in accordance with a protocol ( Recombinant proteins (100 μg) including SARS-CoV-2 membrane glycoprotein or GFP in phosphate buffered saline (PBS) (60 μL) were intranasally inoculated to mice. A small incision was made to insert a cannula into trachea. Bronchoalveolar lavage fluid (BALF) was collected by lavaging the lungs 3 times with 1 mL PBS 6 h after protein inoculation. The enzyme-linked immunosorbent assay (ELISA) using a Quantikine mouse IL-6 set (R&D Systems, Minneapolis, MN, USA) was carried out to quantified the level of IL-6 which was normalized to the concentration of total proteins in lung lavage. After intranasal inoculation of recombinant membrane glycoprotein or GFP, and reverse 5' GATGCCTGCTTCACCACCTT 3', respectively. The inner portion of the basidiomata of P. pulmonarius, an oyster mushroom, ICR mice were fed with 200 µl PBS or 2% mycelium powder in PBS daily. Two weeks after feeding, fecal was collected and dissolved 1:10 in 50 mM EDTA containing 5 mg lysozyme (Sigma-Aldrich). After centrifugation at 14,000 g for 2 min, DNA in the pellet was extracted by using a heat lysis protocol as described [22] . A StepOnePlus Real-time PCR System (ThermoFisher Scientific) using Power SYBR Green and PCR Master Mix (ThermoFisher Scientific) was used to quantify the abundance of L. rhamnosus in mouse feces. The reaction conditions of RT PCR was described above. Primers for L. rhamnosus detection were forward 5' CGCCCTTAACAGCAGTCTTC 3' and reverse 5' GCCCTCCGTATGCTTAAACC 3'. Five mice per group were used in each experiment. GraphPad Prism ® software was utilized for data analysis by unpaired t-test. The statistical significance was considered as: P-values of <0.05 (*), <0.01 (**), and <0.001 (***). The mean ± standard deviation (SD) was calculated from results obtained from at least three independent experiments. (Fig. 1B) . Furthermore, the increase in IL-6 secretion was completely suppressed when SARS-CoV-2 membrane glycoprotein was intranasally inoculated into the PDE4B knockout mice (Fig. 1C) , suggesting that PDE4B plays an essential role in SARS-CoV-2 membrane glycoprotein-induced IL-6 secretion in the lung. 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 [24] . Mycelia, which are rich in polysaccharides, were isolated from Pleurotus pulmonarius (P. pulmonarius) [25] . To examine the prebiotic property of fungal mycelia, the L. rhamnosus. Results in Fig. 2A -C suggested that mycelium powder may promote L. rhamnosus fermentation to reduce SARS-CoV-2 membrane glycoproteininduced IL-6. We next fed mice with L. rhamnosus EH8 in the presence or absence of mycelium powder every day for 2 weeks. Feeding mice with L. rhamnosus EH8 plus mycelium powder markedly lowered the SARS-CoV-2 membrane glycoprotein-induced IL-6 in BALF compared to mice fed with L. rhamnosus EH8 alone (Fig. 2D ). Since the anti-inflammatory property of butyric acid has been demonstrated Since, from four PDE4 isoforms, PDE4B is the only one expressed in macrophages [28] , we cultured the J774 macrophages, a murine cell line that was widely used for studying of respiratory infection [29] , to examine the effect of butyric acid on the PDE4 mRNA expression and IL-6 secretion. As shown in Ffar2 has high affinity for butyric acid and is expressed by various cells including macrophages and epithelial cells in nasal cavities and lung [30] . To assess the engagement of Ffar2 on the effect of mycelium fermentation of L. rhamnosus EH8, GLPG-0974 in DMSO was given to mice to antagonize the Ffar2 before feeding mice with mycelium fermentation of L. rhamnosus EH8. Mice given DMSO served as a control. Two weeks after feeding mice with L. rhamnosus EH8 plus mycelium powder every day, mice were intranasally inoculated with SARS-CoV-2 membrane glycoprotein for 6 h. Compared to PDE4B mRNA expression in mice given DMSO control, mice given GLPG-0974 resulted in a substantial increase in the PDE4B expression in the lung (Fig. 4A) . Similarly, treatment with GLPG-0974 significantly raised the level (8.44 ± 0.82 pg/mg) of IL-6 in BALF compared to IL-6 content (1.00 ± 0.25 pg/mg) in mice treated with control (Fig. 4B) . Cumutatively, these data demonstrate that mycelium fermentation of L. rhamnosus EH8 required Ffar2 to weaken the aggravation of PDE4B mRNA expression and IL-6 secretion induced by SARS-CoV-2 membrane glycoprotein. The envelopes of coronaviruses, a group of positive-strand RNA viruses, contain three main proteins: the spike protein, membrane glycoprotein, and envelope protein. a non-glycosylated protein [31] . The RNA is packaged by the nucleocapsid protein into a helical nucleocapsid. SARS-CoV-2 enters host cells via binding of the spike protein to angiotensin-converting enzyme 2 (ACE2). The binding of spike protein to ACE2 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 [32] . Literature has revealed that nucleocapsid protein of SARS coronavirus HB can bind directly to the NF-κB recognition elements on the IL-6 promoter and regulate IL-6 expression [4] . four PDE4 isoforms expressed in macrophages [28] . Although many cells including macrophages and endothelial cells [35] in the respiratory system can produce IL-6, we demonstrated that SARS-CoV-2 membrane glycoprotein can increase the mRNA expression of PDE4B and trigger release of IL-6 from J774 macrophage cells (Fig. S3) . Furthermore, the PDE4B is necessary for the secretion of IL-6 induced by membrane glycoprotein, since there is no difference in IL-6 content in PDE4B knockout mice intranasally inoculated with recombinant membrane glycoprotein or GFP. The result indicated that SARS-CoV-2 membrane glycoprotein may activate PDE4B to induce IL-6 secretion. Regulation of cAMP signalling by PDE4 to modulate the inflammatory responses in airway cells has been illustrated [36] . PDE4 inhibitor roflumilast has been approved as anti-inflammatory therapy [37] . Our results support the previous concept of using PDE4 inhibitors as potential drugs to target the cytokine storm in COVID-19 [6] . Although the clinical cut-off value for IL-6 as a biomarker for cytokine storm has not yet been determined, it has been reported that an IL-6 threshold greater than 80 pg/mL in sera carried prognostic value to predict respiratory failure COVID-19 [38] . An IL-6 level above 100 pg/mL in sera of COVID-19 patients was found to correlate with mortality [39] . Although the process of collecting BALF dilutes the specimen, it is still unknown what real level of IL-6 in the lung can trigger a local cytokine storm. After normalization to the concentration of total proteins in lung lavage, the levels of IL-6 ranging from approximately 40 to 400 pg/mg in BALF of mice inoculated with SARS-CoV-2 membrane glycoprotein for 6 h were detected (Figs. 1-4) . The fact of varying amounts of IL-6 in BALF were detected may be due to the differences in the molecules that were intranasally inoculated into mice. Cytokine storm can cause multiple-organ failure in COVID-19 infections [40] . Although inoculation with membrane glycoprotein for 6 h led to 23.1% higher level of IL-6 in BALF when compared to inoculation with GFP (Fig. 1A) , cell death (55 ± 6%) in the lung inoculated with membrane glycoprotein was considerably higher than that (16 ± 3%) in the lung inoculated with GFP (Fig. S4) . The result indicated that elevation of IL-6 may correlate with massive cell death and organ dysfunction. The understanding of host-microbe cross-talk along the gut-lung axis highlights the significant role of SCFAs in shaping and promoting both innate and adaptive immunity in bone marrow to resolve airway inflammation [41] . Our results in Fig. 3 demonstrated that more than 20 mM butyric acid can be produced by the mycelium fermentation of L. rhamnosus EH8. Glucans in P. pulmonarius mycelium extracts exhibited the anti-inflammatory effect by blocking TNF-α-induced NF-κB nuclear translocation [42] . In addition, ACE inhibitory proteins have been identified in mycelium of P. pulmonarius [43] . Although glucose and galactose are prominent polysaccharides in fungal mycelium [44] , it is not determined which carbon-rich molecules in P. pulmonarius mycelium act as prebiotics to induce the fermentation of L. rhamnosus EH8. Feeding mice with mycelium powders increased the relative abundance of L. rhamnosus in fecal samples (Fig. 2C) . Augmentation of L. rhamnosus in mouse gut may explain why feeding mice with mycelium can suppress an elevation of IL-6 induced by SARS-CoV-2 membrane glycoprotein ( Fig. 2B) . Future studies will include the quantification of SCFAs in the gut after feeding mice with mycelium powders. Studies are ongoing to test the notion of using probiotics to flatten the curve of COVID-2019 pandemic [45] . It has been reported that 3-60-day supplementation of probiotic L rhamnosus GG lowered the incidence of the respiratory tract infections of viruses by 2-to 3-fold compared to the placebo [46] . Inhibition of Ffar2 by GLPG-0974 dramatically blocked the action of L. rhamnosus EH8 plus mycelium to reduce SARS-CoV-2 membrane glycoprotein-induced PDE4B mRNA expression and IL-6 secretion (Fig. 4) , demonstrating the essential role of Ffar2 in the probiotic activity of L. rhamnosus EH8 against the virulence of SARS-CoV-2 membrane glycoprotein. GLPG-0974 can be quickly absorbed with peak plasma concentrations reached between 2 and 4 h after oral administration in humans [47] , indicating that oral administration of GLPG-0974 can effectively inhibit the Ffar2 in intestinal epithelium [48] and alveolar macrophages [49] . A knockout of Ffar2 which abundantly expressed on intestinal cells in mice exacerbated the colitis and intestinal inflammation [50] . Treatment with probiotic bacteria can upsurge the abundance of beneficial microbes and the levels of SCFAs including butyric acid, while down-regulating the production of pro-inflammatory IL-6 cytokine in aging mice [51] . Results from recent studies have demonstrated that sodium butyrate can suppress ACE2 expression in gut epithelial cells, alleviating the gastrointestinal inflammation associated with COVID-19 [52] . Cytokine storm in COVID-19 is triggered by the release of IL-6 by alveolar macrophages [53] . Mycelium fermentation of L. rhamnosus EH8 yielded substantial amounts of butyric acid ( Fig. 2A, B) , which can lower both PDE4B mRNA expression and IL-6 secretion in J774 macrophage cell lines (Fig. 2C, D) . Thus, it is worth investigating whether activation of Ffar2 on alveolar macrophages by butyric acid or its derivatives can efficiently inhibit the SARS-CoV-2 membrane glycoprotein-induced IL-6 secretion. Inhibition of PDE4 increased intracellular cAMP and suppressed the release of pro-inflammatory cytokines [54] . We thus envision that PDE4B-mediated cAMP signalling in alveolar macrophages modulated the SARS-CoV-2 membrane glycoprotein-induced IL-6 secretion. The study was supported by the Ministry of Science and Technology (MOST) Grants (108-2622-B-008-001-CC1; 108-2314-B-008-003-MY3, and 107-2923-B-008-001-MY3) and 106/107/108-Landseed Hospital-NCU joint grants. The authors declare no conflict of interest. rhamnosus EH8 in the presence (L.r+FM) or absence (L.r) of 2% fungal mycelia. Data are the mean ± SD of three independent experiments. **p < 0.01. ***p < 0.001 (two-tailed t-test). Five mice per group were used in each experiment. Ffar2 antagonist, or its DMSO solvent as a control by gastric gavage every day for 3 days, then subsequently fed with L. rhamnosus EH8 plus fungal mycelium every day. Two weeks after feeding, mice were intranasally inoculated with SARS-CoV-2 membrane glycoprotein (100 μg/60µL PBS per mouse). The mRNA expression of PDE4B (A) and levels of IL-6 (B) in BALF were detected by RT PCR and ELISA, respectively. Data are the mean ± SD of experiments performed in triplicate. ***p < 0.001 (two-tailed t-test). Cytokine storm induced by SARS-CoV Can we use interleukin-6 (IL-6) blockade for coronavirus disease 2019 (COVID-19)-induced cytokine release syndrome (CRS)? 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We also thank Syifa Labibah, Nguyen Mai Trinh Tang, Arun Balasubramaniam, and Shinta Marito for protein purification and cell culture.