Babaodan controls excessive immune responses and may represent a cytokine-targeted agent suitable for COVID-19 treatment

Abstract

It has become evident that the actions of pro-inflammatory cytokines and/or the development of a cytokine storm are responsible for the occurrence of severe COVID-19 during SARS-CoV-2 infection. Although immunomodulatory mechanisms vary among viruses, the activation of multiple TLRs that occurs primarily through the recruitment of adapter proteins such as MyD88 and TRIF contributes to the induction of a cytokine storm. Based on this, controlling the robust production of pro-inflammatory cytokines by macrophages may be applicable as a cellular approach to investigate potential cytokine-targeted therapies against COVID-19. In the current study, we utilized TLR2/MyD88 and TLR3/TRIF co-activated macrophages and evaluated the anti-cytokine storm effect of the traditional Chinese medicine (TCM) formula Babaodan (BBD). An RNA-seq-based transcriptomic approach was used to determine the molecular mode of action. Additionally, we evaluated the anti-inflammatory activity of BBD in vivo using a mouse model of post-viral bacterial infection-induced pneumonia and seven severely ill COVID-19 patients. Our study reveals the protective role of BBD against excessive immune responses in macrophages, where the underlying mechanisms involve the inhibition of the NF-κB and MAPK signaling pathways. In vivo, BBD significantly inhibited the release of IL-6, thus resulting in increased survival rates in mice. Based on limited data, we demonstrated that severely ill COVID-19 patients benefited from BBD treatment due to a reduction in the overproduction of IL-6. In conclusion, our study indicated that BBD controls excessive immune responses and may thus represent a cytokine-targeted agent that could be considered to treating COVID-19.

Keywords: Babaodan; COVID-19; Cytokine storm; Inflammation; Traditional Chinese medicine.

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Figures

Graphical abstract

Fig. 1

Babaodan (BBD) inhibits TLR1/2 and TLR3 dual stimulation-induced cytokine expression in Raw 264.7 macrophages. Raw 264.7 cells were grown on 12-well plates and incubated with different doses of BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) for 24 h. Supernatants were collected, and TNF-α and IL-6 concentrations were analyzed using the corresponding ELISA kits. *P < 0.05, **P < 0.01，***P < 0.001 vs. model group.

Fig. 2

BBD abolishes IL-6 and TNF-α production after optimized TLR1/2 and TLR3 dual activation of Raw264.7 macrophages. (A) Raw 264.7 cells were grown on 12-well plates and incubated with different combinations of Poly(I:C) and Pam3CSK4 for 24 h. Supernatants were collected, and TNF-α and IL-6 concentrations were determined using the corresponding ELISA kits. (B) Raw 264.7 cells were grown on 6-well plates and incubated with 1 mg/mL of BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) for 8 h. IL-6 and TNF-α mRNA levels were detected by RT-qPCR. *** P < 0.001 vs. model group.

Fig. 3

Gene expression profiling by RNA-seq. Raw 264.7 cells grown on 6-well plates were treated with a combination of 10 μg/mL Poly(I:C) and 100 ng/mL Pam3CSK4 (model) for 8 h with or without pre-treatment with 1 mg/mL of Babaodan (BBD) for 24 h. RNA samples were collected for RNA-seq analysis. Two samples were analyzed per group. Differentially expressed genes (DEG) are identified by absolute log2 (FC) > 2 and P < 0.05 values. (A) Number of up- and downregulated genes in the model and treatment groups. (B) Venn diagram summary of DEGs between the control vs. model and model vs. BBD groups. (C) Top 15 KEGG pathway enrichment scores of co-regulated DEGs in the model vs. control and BBD vs. model groups.

Fig. 4

Transcriptional profiling changes in inflammatory DEG result from BBD treatment. (A) Heatmap of relative gene expression (log2[FC]) for the 124 inflammatory genes that were upregulated and the 37 inflammatory genes that were downregulated in the model vs. control groups. BBD regulates the expression of 84 and 9 of these categories of genes, respectively, compared to the control group. Blue, downregulation; red, upregulation. (B) Transcription factor (TF) enrichment analysis was performed for the 84 downregulated inflammatory DEGs, and the top 10 TFs with the highest importance are listed.

Fig. 5

BBD inhibits NF-κB and MAPK signaling in macrophages promoting excessive inflammatory processes. Raw 264.7 cells were grown on 6-well plates and incubated with 1 mg/mL BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) (P2P) for 15, 30, 60, and 120 min. The cells were harvested at the indicated time points after stimulation. Cell lysates were resolved by SDS-PAGE, blotted, and analyzed using the respective antibodies. One representative experiment of three is shown.

Fig. 6

BBD attenuates lung inflammation in mice with acute lung injury. C57BL/6 mice were infected with 200 PFU of influenza A PR/8/34 or vehicle for 5 days before being challenged with Staphylococcus aureus (SA) at the indicated CFU in 50 μL: 5 × 107 for A-B and 2.5 × 107 for C-D. (A) Survival curves; n = 16, model group; n = 17, BBD treatment group. (B) Representative histological sections of lungs at 12 h post SA challenge (n = 4–5 for each group). (C) CFU counts in LH and BAL samples (n = 4–5 for each group). (D) Concentrations of TNF-α and IL-6 in BALF (n = 3–5 for each group). * P < 0.05 vs. model group. CFU, colony-forming units; PFU, plaque-forming units; BALF, bronchoalveolar lavage fluid.

Fig. 7

Outcomes of CT scan image and inflammation analyses in a severely ill COVID-19 patient (PT-7) following BBD treatment. (A) Lung CT scan images for the patient at 2, 5, 7, and 11 days after BBD treatment prior to being discharged. While the bilateral pneumonia continued to develop at 2, 5, and 7 days after BBD treatment, improvement occurred on the 11th day after BBD treatment. (B-C) The dynamics of serum IL-6 and CRP levels were monitored.