Immunomodulatory effects of new phytotherapy on human macrophages and TLR4- and TLR7/8-mediated viral-like inflammation in mice

Abstract

Background: While all efforts have been undertaken to propagate the vaccination and develop remedies against SARS-CoV-2, no satisfactory management of this infection is available yet. Moreover, poor availability of any preventive and treatment measures of SARS-CoV-2 in economically disadvantageous communities aggravates the course of the pandemic. Here, we studied a new immunomodulatory phytotherapy (IP), an extract of blackberry, chamomile, garlic, cloves, and elderberry as a potential low-cost solution for these problems given the reported efficacy of herbal medicine during the previous SARS virus outbreak.

Methods: The key feature of SARS-CoV-2 infection, excessive inflammation, was studied in in vitro and in vivo assays under the application of the IP. First, changes in tumor-necrosis factor (TNF) and lnteurleukin-1 beta (IL-1β) concentrations were measured in a culture of human macrophages following the lipopolysaccharide (LPS) challenge and treatment with IP or prednisolone. Second, chronically IP-pre-treated CD-1 mice received an agonist of Toll-like receptors (TLR)-7/8 resiquimod and were examined for lung and spleen expression of pro-inflammatory cytokines and blood formula. Finally, chronically IP-pre-treated mice challenged with LPS injection were studied for "sickness" behavior. Additionally, the IP was analyzed using high-potency-liquid chromatography (HPLC)-high-resolution-mass-spectrometry (HRMS).

Results: LPS-induced in vitro release of TNF and IL-1β was reduced by both treatments. The IP-treated mice displayed blunted over-expression of SAA-2, ACE-2, CXCL1, and CXCL10 and decreased changes in blood formula in response to an injection with resiquimod. The IP-treated mice injected with LPS showed normalized locomotion, anxiety, and exploration behaviors but not abnormal forced swimming. Isoquercitrin, choline, leucine, chlorogenic acid, and other constituents were identified by HPLC-HRMS and likely underlie the IP immunomodulatory effects.

Conclusions: Herbal IP-therapy decreases inflammation and, partly, "sickness behavior," suggesting its potency to combat SARS-CoV-2 infection first of all via its preventive effects.

Keywords: SARS-CoV-2; inflammation; mice; pro-inflammatory cytokines; toll-like receptors.

Figure 1

Schematic of in vivo tests performed and the outcome from the in vitro assay. In the in vivo experiments, animals were exposed to a daily administration of herbal drops or vehicle during the 14 days. On the day of the experiment, mice received an intraperitoneal injection (A) with resiquimod or vehicle and killed 6 h post-challenge, (B) with LPS or vehicle and 6 h post-challenge completed the novel cage, open field, and the forced swim test. In the in vitro assay, herbal drops or prednisolone was applied in the human macrophage cell culture that was treated with LPS. In comparison with the non-stimulated samples, there was a significant increase in the concentrations of (C) IL-1β and (D) TNF in all LPS-challenged samples. Significant group differences: *vs. non-challenged samples, (one-way ANOVA and Tukey's test). Data presented as mean ± SEM.

Figure 2

Pro-inflammatory resiquimod-induced gene expression changes in the liver and spleen are ameliorated with the herbal IP treatment. (A) Resiquimod-challenged groups showed a significant increase in the normalized liver expression of SAA-2mRNA in comparison to a 100%-level. This measure was lower in the liver of IP–treated animals. No such differences were shown for the spleen. (B) As compared to a 100%-level, there was a significant decrease in the normalized ACE-2mRNA expression in the liver of the IP-treated group but not in the non-treated resiquimod-challenged mice that was not found for the spleen (C) Both resiquimod-challenged groups showed a significant increase in liver CXCL1mRNA normalized concentrations as compared to 100%; the IP-treated group subjected to the resiquimod injection had a significantly decreased normalized liver CXCL1mRNA expression compared with resiquimod-injected animals. In the spleen, compared to 100%, this measure was decreased in the IP-treated resiquimod-challenged group. (D) We found a significant increase in the normalized CXCL10mRNA expression in the liver and spleen compared to 100% in both groups, while this measure was lower in the IP-treated group than in resiquimod-challenged mice without treatment; no group difference in this measure was found for the spleen. (E) Elevated normalized IL-1βmRNA levels compared to 100% in the liver and spleen were shown for both groups; no other differences were found. (F) Normalized mRNA expression of IL-6 was similarly elevated in two groups in the liver and spleen compared to the 100% level. No other differences were found. *vs. 100% level, # vs. the challenged non-treated group (Welch's test and t-test, see the text). Data presented as mean ± SEM.

Figure 3

Pro-inflammatory effects of resiquimod on blood formula are reduced in mice treated with the herbal IP composition. Normalized counts of (A) neutrophils, (B) monocytes, (C) eosinophils were significantly decreased in IP-treated resiquimod-stimulated mice. (D) An increase in basophil counts was similar in the two challenged groups. (E) No changes were found in the lymphocyte counts of the resiquimod-injected groups; *vs. 100% level, #vs. the challenged non-treated group (Welch's test and t-test, see the text). Data presented as mean ± SEM.

Figure 4

LPS induces a sickness behavior phenotype, which is partly normalized by the herbal IP. In comparison to non-treated challenged animals, IP-treated LPS-challenged mice displayed (A) a normalized number of exploratory rears, (B) a number of peripheral and (C) central crossings in the open field (two-way ANOVA and Tukey's test). There were no such differences (D) in the latency to float and (E) duration of the floating in the forced swim test; *vs. the challenged non-treated group. Data presented as mean ± SEM.