Pharmacological potential of Withania somnifera (L.) Dunal and Tinospora cordifolia (Willd.) Miers on the experimental models of COVID-19, T cell differentiation, and neutrophil functions

Abstract

Cytokine release syndrome (CRS) due to severe acute respiratory coronavirus-2 (SARS-CoV-2) infection leads to life-threatening pneumonia which has been associated with coronavirus disease (COVID-19) pathologies. Centuries-old Asian traditional medicines such as Withania somnifera (L.) Dunal (WS) and Tinospora cordifolia (Willd.) Miers (TC) possess potent immunomodulatory effects and were used by the AYUSH ministry, in India during the COVID-19 pandemic. In the present study, we investigated WS and TC's anti-viral and immunomodulatory efficacy at the human equivalent doses using suitable in vitro and in vivo models. While both WS and TC showed immuno-modulatory potential, WS showed robust protection against loss in body weight, viral load, and pulmonary pathology in the hamster model of SARS-CoV2. In vitro pretreatment of mice and human neutrophils with WS and TC had no adverse effect on PMA, calcium ionophore, and TRLM-induced ROS generation, phagocytosis, bactericidal activity, and NETs formation. Interestingly, WS significantly suppressed the pro-inflammatory cytokines-induced Th1, Th2, and Th17 differentiation. We also used hACE2 transgenic mice to further investigate the efficacy of WS against acute SARS-CoV2 infection. Prophylactic treatment of WS in the hACE2 mice model showed significant protection against body weight loss, inflammation, and the lung viral load. The results obtained indicate that WS promoted the immunosuppressive environment in the hamster and hACE2 transgenic mice models and limited the worsening of the disease by reducing inflammation, suggesting that WS might be useful against other acute viral infections. The present study thus provides pre-clinical efficacy data to demonstrate a robust protective effect of WS against COVID-19 through its broader immunomodulatory activity.

Keywords: COVID-19; SARS-CoV-2; T cells; Tinospora cordifolia; Withania somnifera (Ashwagandha); and hACE2 transgenic mice; hamster model; neutrophils.

Figure 1

Prophylactic efficacy of selected herbal extract on the SARS-CoV-2 infected hamsters. (A) Schematic representation of dosing regimen for prophylactic treatment of WS, TC or TC in combination with PL, positive control remdesivir (RDV), infection control (I) or uninfected hamster group. All the animals except the uninfected control was intranasally challenged with 10⁵ pfu SARS-CoV-2 on day 0 and sacrificed on 4-day post infection (dpi). (B) Body mass of the animals were monitored post challenge and was plotted as %age change as compared to its day 0 body mass. (C) Representative images of harvested lungs post necropsy. (D) Relative viral load by N gene expression by qPCR shown as bar graph mean ± SEM. Histological analysis of left lung lower lobe was carried out post necropsy. The samples were fixed in 10% neutral formalin solution, paraffin embedded, sectioned and hematoxylin (H) & eosin (E) stained. Stained sections were then imaged at 10X and assessed by trained pathologist for histological features. (E) Representative images of HE stained lungs showing pneumonitis (blue), bronchitis (red), epithelial injury (green) and inflammation (yellow). (F) Blinded pathological score for pneumonitis, bronchitis, lung injury, epithelial injury and inflammation as assessed by trained pathologist. (G) mRNA expression of key genes involved in cellular injury of lungs. For each experiment N=5. One way-Anova using non-parametric Kruskal-Wallis test for multiple comparison. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Immunomodulatory effects of WS or TC on the infected hamsters. Immunomodulatory activity of prophylactic treatment of WS or TC on infected vs uninfected hamsters were studied. (A) representative spleen images harvested post necropsy. (B) changes in spleen mass to body mass ratio for different groups (C) modulation in the mRNA expression of pro-inflammatory cytokines and (D) transcription factors. For each experiment N=5. One way-Anova using non-parametric Kruskal-Wallis test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

Effect of WS and TC on TRLM primed-PMA/ionomycin induced NETs formation in human PMNs. After pre-incubation with different concentrations TC and WS, PMNs were treated with TRLM (10 µg/ml) for 30 min and stimulated with sub-maximal concentration of PMA (12.5 nM) and ionomycin (2 µM) for 30 min. SYTOX Green (100 nM) was used to monitor extracellular DNA release using a plate reader (A, B: TC; C, D: WS). Total MFI in each experimental condition is expressed as Mean ± SEM of min 3 experiments. NETosis in human PMNs was also monitored using immunofluorescence imaging with DAPI (blue), anti-MPO antibody (green), and anti-H4Cit3 antibody (red, E–H). Representative fields are shown at 100X with a scale bar of 10 µm. Bar diagram represents quantification of percent NETs forming cells as calculated from five transects from three independent experiments. Statistical analysis consisted of one-way ANOVA followed by Bonferroni’s test (*p < 0.05, **p < 0.01, vs respective control groups; C, control; V, VAS2870; D, Diltiazem; WS100, WS 100 μg/ml; WS300, WS 300 μg/ml; TC100, TC 100 μg/ml; TC300, TC 300 μg/ml.

Figure 4

Effect of WS and TC on TRLM primed-PMA/ionomycin induced cytosolic ROS and mtROS production in human PMNs. PMNs pre-incubated at different concentrations of TC and WS were treated with TRLM (10 µg/ml) for 30 min and stimulated with sub-maximal concentration of PMA (12.5 nM) and ionomycin (2 µM) for 30 min. DCF-DA (10 µM) and MitoSOX (10 µM) were used for cytosolic ROS and mtROS detection, respectively using flow cytometry. All the data are represented as Mean ± SEM, n = min 3 per group, and statistical analysis consisted of one-way ANOVA followed by Bonferroni’s test (A–F) (**p < 0.01, vs respective control groups;. C, control; N, N-acetyl cysteine; MT, MitoTEMPO; WS50, WS 50 μg/ml; WS100, WS 100 μg/ml; TC50, TC 50 μg/ml; TC100, TC 100 μg/ml.

Figure 5

Dose kinetics of WS response on in vitro differentiation of Th1, Th2 and Th17 cells from naïve CD4+ T cells. Sorted naïve CD4+ T cells from mouse spleen and lymph nodes were activated using soluble anti-CD3 antibody and differentiated into helper T (Th)2 (A, B), Th17 cells (D, E) and Th1 subtypes (G, H) by using different cytokines viz recombinant mouse IL-4; TGF-β + IL-6 and IL-12 cytokines respectively. WS was added in concentrations ranging from 10ug/ml to 1000ug/ml initially at the time of cell seeding. After 72 h of incubation IL-4, Il-17 and IFN-gamma production was measured respectively for Th2, Th17 and Th1 cells by intracellular cytokine staining. IC50 values were calculated using Graph pad prism software (C, F, I). ****P < 0.0001 by one-way ANOVA.

Figure 6

Assessment of protective efficacy of WS in acute SARS-CoV-2 infection model of hACE2 transgenic mice. To evaluate the effect of prophylactic treatment of WS on severe SARS-CoV-2 infection, we used hACE2 mice model for acute infection and compared it with RDV control. (A) Schematic representation showing treatment regimen for WS and RDV. Mice were intranasally infected with SARS-CoV-2 and (B) %age changes in body mass and (C) mortality was monitored and plotted. (D) Representative excised lung images 6 days post infection (E) Lung viral load presented as Log10 N copy number (F) Lower lung lobe was used for HE staining (G) and assessed for pathological features by blinded scoring by trained pathologist. For each experiment N=5. One way-Anova using non-parametric Kruskal-Wallis test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Changes in the major immune populations of infected hACE2 mice with or without treatment. Flow cytometry-based quantitation was done to evaluate changes in the major immune population in the lymph nodes of sacrificed animals at 6 dpi. The % age frequency was plotted as bar graph along with the representative contour plot (A) CD45+ population (B) CD3+ T lymphocytes, NK cells and NKT cells (C) CD4+ T helper cells and CD8+ T cytotoxic cells (D) Macrophages (E) Monocytes, neutrophils and MDSCs population. For each experiment N=5. One way-Anova using non-parametric Kruskal-Wallis test for multiple comparisons. *P < 0.05, **P < 0.01.

Figure 8

Changes in the effector cytokines of CD4+ T cells of infected hACE2 mice with or without treatment. Flow cytometry-based quantitation was done to evaluate changes in the major immune population in the lymph nodes of sacrificed animals at 6 dpi. The % age frequency was plotted as bar graph along with the representative contour plot (A) CD4+IFNγ+ cells (B) CD4+IL4+ cells (C) CD4+IL17A+ cells (D) CD4+TNFα+ cells for each experiment N=5. One way-Anova using non-parametric Kruskal-Wallis test for multiple comparison. *P < 0.05, **P < 0.01.

Figure 9

Summary figure highlighting the study design and novel findings from the study.