Protective Effect of Indole-3-Aldehyde in Murine COVID-19-Associated Pulmonary Aspergillosis

Abstract

Aspergillus fumigatus is an environmental fungus recently included in the fungal high-priority pathogens by the World Health Organization. While immunodeficiency and/or pre-existing lung damage represent a well-recognized fertile ground for fungal growth, it is increasingly being recognized that severe viral infections may similarly favor A. fumigatus colonization and infection, as recently experienced in the Coronavirus disease 2019 (COVID-19) pandemic. Herein, in a murine model of COVID-19-associated pulmonary aspergillosis (CAPA), obtained by the concomitant exposure to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike protein and A. fumigatus conidia, we found that the microbial compound indole-3-aldehyde (3-IAld) was able to ameliorate CAPA by working at multiple levels during viral infection and fungal superinfection, including epithelial barrier protection, promotion of antiviral responses, and limiting viral replication. As a consequence, 3-IAld limited the pathogenic sequelae of fungal superinfection as revealed by the controlled fungal burden and restrained inflammatory pathology. These results point to indole compounds as potential agents to prevent CAPA.

Keywords: CAPA; SARS-CoV-2; aspergillosis; indole-3-aldehyde.

Figure 1

SARS-CoV-2 Spike protein worsens Aspergillus infection in murine CAPA. BALB/c mice were intratracheally infected with Aspergillus fumigatus conidia and treated with the Spike protein from the wild-type (SPwt) or the Delta (SPΔ) variant and siRNA of Ace2 (SiAce2) as indicated in (A). Mice were evaluated for (B) fungal growth (log₁₀ CFU, mean ± SEM); (C) expression of Ace2 in the lung by RT-PCR; (D) lung histopathology and neutrophil recruitment (% in the BAL, in the inset); and (E) IL-1β production (by ELISA). Images were taken with a high-resolution microscope (Olympus BX51), 20× magnification (scale bars, 200 μm). Data are presented as mean ± SD of one representative out of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Aspergillus-infected mice treated with the SPwt and/or SPΔ, with or without SiAce2 vs. Aspergillus-infected mice, one-way ANOVA, Bonferroni post hoc test.

Figure 2

3-IAld protects against CAPA. BALB/c mice were intratracheally infected with Aspergillus fumigatus conidia, exposed to the wild-type Spike protein (SPwt), and treated with 3-IAld administered orally at a dose of 18 mg/kg, a day before and a day after the infection, as illustrated in (A). Mice were evaluated for (B) fungal growth (log₁₀ CFU, mean ± SEM); (C,D) lung (neutrophil recruitment in the BAL, in the inset) and intestine histopathology, and TUNEL staining; (E) IL-1β production (ELISA) in lung homogenates; and (F) expression of Ahr, Cyp1b1, and Ahrr in the lung by RT-PCR. Images were taken with a high-resolution microscope (Olympus BX51), 20× magnification (scale bars, 200 μm). Data are presented as mean ± SD of one representative out of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, Aspergillus-infected mice treated with SPwt and 3-IAld vs. Aspergillus-infected mice treated only with SPwt. One-way ANOVA, Bonferroni post hoc test.

Figure 3

3-IAld counteracts the SARS-CoV-2 immunomodulatory effects in nasal epithelial cells. RPMI cells were pre-treated for 1 h with 10 μM of 3-IAld in DMSO or 50 μM of I3C, exposed to the SARS-CoV-2, B.1.1.7 variant of concern, and evaluated for (A) morphology and expression of (B) IL-1β and IL-10 cytokines genes (C), antiviral type I IFNs and OAS1 genes, and (D) PPARG, NQO1, and HMOX-1 oxidative stress genes. Data are presented as mean ± SD of two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, treated with I3C and 3-IAld vs. untreated virus-exposed cells, one-way ANOVA, Bonferroni post hoc test.

Figure 4

3-IAld exerts direct antiviral effects in vitro. Vero E6 cells were infected with the SARS-CoV-2 strain and exposed to 10 μM of 3-IAld, 50 μM of I3C in DMSO, or 10 μM of Remdesivir, either 1 h before (P, prophylaxis) or 1 h after (T, therapy) SARS-CoV-2 infection. The compounds were dissolved to 10 mM in DMSO and then diluted in culture medium. DMSO (1 and 0.01% (v/v)) was used as control. Cells were assessed for (A) viability by the standard crystal violet staining assay, measuring the optical density (OD) at 595 nm; (B) viral titer as plaque-forming units per ml; (C) expression of Orf8 and RdRp by RT-PCR. Data are presented as mean ± SD of two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, treated with I3C and 3-IAld vs. untreated virus-exposed cells, one-way ANOVA, Bonferroni post hoc test. ND, not determined.