Evaluation of the immunomodulatory effects of anti-COVID-19 TCM formulae by multiple virus-related pathways

Dear Editor, The coronavirus infectious disease 2019 (COVID-19) outbreak is seriously endangering human health. Most patients with severe COVID-19 are characterized by sustained cytokine production and hyper-inflammation, which is known as cytokine storm syndrome. Elaborating the anti-inflammatory response is crucial to these patients, and interleukin-6 (IL6) inhibitors and steroids have been recommended in clinical practice. However, after the cytokine storm phase, the host immune response to sepsis may develop into a protracted immunosuppressive phase. Agents enhancing host immunity administered to patients in the immunosuppressive phase of sepsis could improve survival. Therefore, different treatments should be provided to different COVID-19 patients with cytokine storm phase or immunosuppression phase. Traditional Chinese medicine (TCM) has been widely used to treat over 90% of the patients with COVID-19 in Chinese hospitals and among all patients with mild and moderate symptoms in Wuhan, none have generated severe symptoms when they were only treated with TCM. It is essential to evaluate their efficacy and select the most appropriate ones in a specific context. We collected 125 anti-COVID-19 TCM formulae from public sources (The formulae and their sources are summarized in Table S1) containing 196 TCMs (Table S2), 166 of which were included in our study. To establish gene signature profiles of 166 TCMs, we performed high-throughput sequencing-based high-throughput screening (HTS) on phorbol-12-myristate-13-acetate-induced THP-1 cells. A set of 3267 genes were detected and these genes were derived from 139 pathways related to virus infection, immunity, inflammation, metabolism, cell proliferation, apoptosis, and migration (Table S3). A gene set enrichment analysis (GSEA) was used to perform pathway enrichment analyses of 11 virus-related pathways in cells exposed to the 166 TCMs (Fig. S1 and Table S4). The following 11 pathways were included: human papillomavirus infection, Kaposi sarcoma-associated herpesvirus infection, human T-cell leukemia virus 1 infection, Epstein-Barr virus infection, human immunodeficiency virus 1 infection, influenza A, human cytomegalovirus infection, hepatitis C, herpes simplex virus 1 infection, hepatitis B and measles. Most virus-related pathways involved the Toll-like receptor signaling pathway, JAK-STAT signaling pathway, NF-κB signaling pathway, RIG-I-like receptor signaling pathway, and antigen processing and presentation, suggesting an immune response after viral infection. The efficacy of each individual TCM against each virusrelated pathway was represented by the normalized enrichment score (NES). A positive NES indicates that the pathway is enriched in upregulated genes, while a negative NES indicates enrichment in downregulated genes. The efficacy of each anti-COVID-19 TCM formula was calculated as the sum of the NESs of each TCM in the formula for the pathways. In our study, we evaluated the efficacy of the TCM formulae from two aspects. For patients with cytokine storm syndrome, we proposed TCM formulae with negative NESs for virus-related pathways; and for immunosuppression, we proposed TCM formulae with positive NESs. In addition, representative transcriptome datasets related to COVID-19 were analyzed, including leukocytes, macrophages, and lung tissues from COVID-19 patients, and SARS-CoV-2-infected cell lines. The NESs for the 11 virus-related pathways were positive in all 7 SARS-CoV-2-infected samples (Table S5), suggesting that the immune response was induced after viral infection. Hierarchical clustering was performed for the 132 samples (125 formulae and 7 SARS-CoV-2-infected samples) (Fig. 1a) by considering the 11 virus-related pathways, and three groups were identified. The formulae in the same group have similar effects on 11 virusrelated pathways, which may be a result of similar TCM composition and they may be suitable for patients with similar symptoms. The first group contained 22 formulae and 7 SARS-CoV-2-infected samples, which had positive NESs in most pathways and may work to activate the immune response (Fig. 1a, cluster 3). There were 45.5% and 40.9% of formulae in this group containing Citri Reticulatae Pericarpium (Chenpi) and Ophiopogonis Radix (Maidong), which were characterized as health-strengthening TCM and can be used to treat impaired type-I interferon and lymphopenia. The second group contained 21 formulae with negative NESs for most pathways, indicating anti-inflammatory effects (Fig. 1a, cluster 1). Maxing Shigan Decoction was contained in 28.6% of formulae in this group. Glycyrrhizae Radix et Rhizoma (Gancao) and Ephedrae Herba (Mahuang) were in 57.1% of formulae in this group respectively, which were recognized as TCMs relieving exterior syndrome and dispelling cold and can inhibit IL6 and alleviate inflammation. The third group contained the remaining formulae, which had mixed or relatively weak regulatory effects on the pathways (Fig. 1a, cluster 2). To quantitatively evaluate the efficacy of the TCM formulae, we defined the efficacy as a score by summing the NESs for each TCM formula on the 11 virus-related pathways (Table S5). The scores of all formulae ranged from −84.490 to 41.031. The scores of the SARS-CoV-2-infected samples ranged from 6.467 to 40.734. A total of 98 formulae had a negative NES, indicating their potential antiinflammatory effects. The top 3 formulae are listed in Fig. 1b: DTPC6-Qingfei Paidu Decoction, Handbook-Jiuwei Qianghuo Decoction-Shenshou Taiyi Powder, and Yongyan Wang3-Guizhi Decoction-Mahuang Fuzi Xixin Decoction. The core components of these three formulae are Asari Radix et Rhizoma (Xixin), Cinnamomi Ramulus (Guizhi), Ephedrae Herba, and Glycyrrhizae Radix et Rhizoma. These TCMs can reverse multiple over-activated virus-related pathways of SARS-CoV-2-infected samples, for example, influenza A (Fig. 1c and d). In addition, Asari Radix et Rhizoma, Cinnamomi Ramulus and Glycyrrhizae Radix et Rhizoma downregulated NF-κB signaling pathway (Fig. 1e), which is a crucial pathway of cytokine storm in severe COVID-19. In contrast, 26 formulae had a positive NES for virus-related pathways, indicating an immune-activating effect. DTPC4-Internal Blockage And External Desertion-Shengmai Yin, DTPC4-Lung-Spleen Qi


This PDF file includes:
Materials and Methods Figure. S1 Captions for Data S1 to S5

Other Supplementary Materials for this manuscript include the following:
Data S1 to S5 Table S1. A set of 125 anti-COVID-19 TCM formulae was collected from public sources. Table S2. The set of 196 TCMs constituting the 125 anti-COVID-19 TCM formulae. Table S3. The list of 139 pathways and 3267 genes. Table S4. The number of DEGs and NES for 166 TCMs on 11 virus-related pathways. Table S5. The total pathway scores and NES for each pathway of the 125 formulae and 7 COVID-19-related samples.

Materials and Methods
Cell culture THP-1 (National Infrastructure of Cell Line Resource) cells were cultured in RPMI1640 (HyClone, Logan, UT) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (HyClone) at 37°C and 5% CO2 in a humidified atmosphere.
Probe design for high-throughput sequencing-based high-throughput screening (HTS 2 ) A pair of probes was designed to hybridize with the 3' side and the 5' side of the transcript, which were designated the acceptor and donor. The acceptor comprises the common index primer and the 20-nt complementary sequence of the target. The donor comprises the specific 20-nt complementary sequence of the target and the P5 primer. Probes are preferentially designed against exon-exon junctions. Probes for a set of 3267 genes related to virus infection, immunity, inflammation, metabolism, cell proliferation, apoptosis, and migration were designed for HTS 2 .

Construction of Traditional Chinese medicine (TCM) extract bank
A total of 166 TCMs from the 2015 Chinese Pharmacopeia were collected and enrolled in the TCM extract bank. TCMs were purchased from Anguo Changda Chinese Herbal Pieces Ltd. (Hebei, China) and Beijing Tongrentang Medicine Corporation Ltd. (Beijing, China). TCMs (10 g) were extracted and refluxed with 150 mL of 90% (v/v) ethanol for 3 hours and concentrated for 20 min using a Soxhlet apparatus (BUCHI Labortechnik AG, Flawil, Switzerland). All the extracts were further freeze dried and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/mL. There were some exceptions, i.e., Gypsum Fibrosum (Shigao) (10 g) was immersed in 100 mL water and refluxed for 2 hours, the precipitate was obtained by centrifugation and subjected to the second reflux in 80 mL water for 1.5 hours followed by centrifugation. The supernatants collected from twice centrifugations were concentrated, freeze dried, and dissolved in RPMI1640 complete medium at a concentration of 200 μg/mL. Borneolum Syntheticum (Bingpian), Styrax (Suhexiang), Moschus (Shexiang), Realgar (Xionghuang) were directly smashed and dissolved in DMSO, and Natrii Sulfas (Mangxiao) and Natrii Sulfas Exsiccatus (Xuanmingfen) were directly dissolved in water.

HTS 2
THP-1 cells (4,000 per well) were seeded in 384-well plates (Corning Inc., Corning, NY) and incubated with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) for 48 hours, followed by treatment with TCM crude extracts at 100 μg/mL or DMSO for another 24 hours. Cells were lysed and kept at −80°C until performing an RNA-mediated oligonucleotide annealing, selection, and ligation (RASL) assay. The RASL assay has been reported previously. Briefly, samples were heated at 65°C for 8 min to denature RNA. Then, the samples were incubated at 45°C for 60 min so that probes were annealed to total RNA and were paired with target mRNA. Biotinylated oligo (dT) and streptavidin-coated magnetic beads were used to capture mRNA so that the aligned probes could resist washing. After washing, the aligned probes were ligated by T4 ligase at 37°C for 60 min. During polymerase chain reaction (PCR) amplification, products from a single sample were indexed by limited PCR amplification with a set of barcode primers. The products were finally combined, purified, quantified, and subjected to high-throughput sequencing using the HiSeq Xten PE150 platform (Illumina, San Diego, CA). The sequencing data of HTS 2 were mapped with Bowtie2. Then, the value of log2 fold change (log2FC) of TCM (n=3) in comparison with DMSO (n=16) after PMA treatment was normalized and calculated by DEseq2. The differentially expressed genes were identified with a FC ≥ 1.5 or FC ≤ 0.67 and p-value ≤ 0.05.

Pathway enrichment analysis
A gene set enrichment analysis (GSEA) was utilized to perform pathway enrichment analysis by using GSEA software 3.0 from the Broad Institute and University of California, San Diego. The GseaPreRanked tool (nperm=1,000, set_min=5, set_max=500, scoring_scheme=classic, random.seed=666) was used to analyze 3267 genes ranked by log2FC after treatment. Significantly enriched pathways were determined with a cutoff of false discovery rate (FDR) < 0.25. The hierarchical clustering of pathways and samples was utilized to analyze the enriched pathway profiles of TCMs based on the normalized enrichment score (NES).

Pathway simulation of the anti-COVID-19 formula
The NES of an anti-COVID-19 TCM formula for each pathway was simulated with the sum of the NES of each constitutive TCM from the formula for this pathway. The total pathway score of a formula was calculated by adding the NES of this formula on 11 virus-related pathways. Although 196 TCMs were included in the 125 formulae, 30 were excluded because of unfavorable accessibility or unsuccessful extraction. We computed the pathway score for each formula as follows: where i is the number of TCMs contained in a formula and j is the number of pathways (j=11 in this study).   The total pathway scores and NES for each pathway of the 125 formulae and 7 COVID-19related samples. The total pathway scores and NES for individual pathways for each formula or COVID-19-related sample are listed. The formula or sample is ordered as they appear in the heatmap of Fig. 1a.