Two new and effective food-extracted immunomodulatory agents exhibit anti-inflammatory response activity in the hACE2 acute lung injury murine model of COVID-19

Abstract

Objective: The coronavirus disease 2019 (COVID-19) spread rapidly and claimed millions of lives worldwide. Acute respiratory distress syndrome (ARDS) is the major cause of COVID-19-associated deaths. Due to the limitations of current drugs, developing effective therapeutic options that can be used rapidly and safely in clinics for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is necessary. This study aims to investigate the effects of two food-extracted immunomodulatory agents, ajoene-enriched garlic extract (AGE) and cruciferous vegetables-extracted sulforaphane (SFN), on anti-inflammatory and immune responses in a SARS-CoV-2 acute lung injury mouse model.

Methods: In this study, we established a mouse model to mimic the SARS-CoV-2 infection acute lung injury model via intratracheal injection of polyinosinic:polycytidylic acid (poly[I:C]) and SARS-CoV-2 recombinant spike protein (SP). After the different agents treatment, lung sections, bronchoalveolar lavage fluid (BALF) and fresh faeces were harvested. Then, H&E staining was used to examine symptoms of interstitial pneumonia. Flow cytometry was used to examine the change of immune cell populations. Multiplex cytokines assay was used to examine the inflammatory cytokines.16S rDNA high-throughput sequencing was used to examine the change of gut microbiome.

Results: Our results showed that AGE and SFN significantly suppressed the symptoms of interstitial pneumonia, effectively inhibited the production of inflammatory cytokines, decreased the percentage of inflammatory cell populations, and elevated T cell populations in the mouse model. Furthermore, we also observed that the gut microbiome of genus Paramuribaculum were enriched in the AGE-treated group.

Conclusion: Here, for the first time, we observed that these two novel, safe, and relatively inexpensive immunomodulatory agents exhibited the same effects on anti-inflammatory and immune responses as neutralizing monoclonal antibodies (mAbs) against interleukin 6 receptor (IL-6R), which have been suggested for treating COVID-19 patients. Our results revealed the therapeutic ability of these two immunomodulatory agents in a mouse model of SARS-CoV-2 acute lung injury by promoting anti-inflammatory and immune responses. These results suggest that AGE and SFN are promising candidates for the COVID-19 treatment.

Keywords: COVID-19; ajoene-enriched garlic extract; anti-inflammatory; hACE2; sulforaphane.

Figure 1

Poly(I:C) and SP-induced acute lung injury and CRS in hACE2 mice. (A) Schematic diagram showing the construction of an acute lung injury mouse model via intratracheal injection with 20 mg/kg poly(I:C) and 5 μg SARS-CoV-2 recombinant spike protein (SP). (B) Hematoxylin and eosin (HE) staining of histological lung sections of representative normal and injected mice at different time points. Regions of the lung anatomy where alveolar and peribronchiolar inflammation were assessed are highlighted in boxes. Images show low- (up panels) and high-power magnification (down panels) of the same tissue section. (C) The inflammation cytokines derived from BALF of the ARDS mouse model at different time points were measured using multiplex cytokines assays. (D–E) The percentage of CD11b⁺/GR1⁺ cells derived from the BALF of normal and injected mice was measured using flow cytometry. (n=5). *p <0.05, **p <0.01, and ***p <0.001. ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; hACE2, humanized angiotensin-converting enzyme-2; poly(I:C), polyinosinic:polycytidylic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.

Figure 2

Anti-inflammatory effects of AGE and SFN in the SARS-CoV-2 acute lung injury mouse model. (A) Schematic diagram showing the treatment of AGE, SFN, and IL-6R antagonists in a SARS-CoV-2 acute lung injury mouse model. (B) HE staining of histological lung sections of the representative injected untreated mice and mice treated with the IL-6R neutralizing antibodies, AGE, and SFN at 48 h and 5 dpi. (C) Histopathological severity scoring was evaluated according to the pathological changes. Data from one independent experiment: injected untreated, injected treated. *p <0.05 and ***p <0.001. AGE, ajoene-enriched garlic extract; dpi, days postinoculation; IL-6R, interleukin 6 receptor; HE, hematoxylin and eosin; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; SFN, sulforaphane.

Figure 3

(A–F) The inflammatory cytokines changes in the SARS-CoV-2 acute lung injury mouse model BALF after AGE and SFN treatment. After the treatment with AGE, SFN, and IL-6R neutralizing antibodies, the inflammation cytokines from BALF were measured using a multiplex cytokines assay. *p <0.05, **p <0.01, and ***p <0.001. AGE, ajoene-enriched garlic extract; BALF, bronchoalveolar lavage fluid; IL-6R, interleukin 6 receptor; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; SFN, sulforaphane.

Figure 4

Effects of AGE and SFN treatment on immune response. The percentage of CD11b⁺GR1⁺, CD4⁺T, and CD8⁺T cells in BALF at 24 h (A–C) and 48 h (D–F) after AGE and SFN treatment was examined using flow cytometry. *p <0.05, **p <0.01, and ***p <0.001. AGE, ajoene-enriched garlic extract; BALF, bronchoalveolar lavage fluid; SFN, sulforaphane.

Figure 5

AGE treatment induces intestinal microbiome changes. (A) Chao1 indices were observed, and Shannon and Simpson indices were analyzed in the AGE-treated and control groups. (B) PCoA of β-diversity using the Bray–Curtis dissimilarity. (C, D). Overall exhibition of effect size (LefSe) analysis using a cladogram in the AGE and NC groups. (E–G). The genus present in the AGE and NC groups was analyzed using a heat map and Sankey diagram. AGE, ajoene-enriched garlic extract; NC, negative control; PCoA, principal coordinate analysis.