NRF2 agonists 4-octyl-itaconate and dimethyl fumarate reduce human and bovine RSV proliferation and RSV disease in a murine model

Abstract

Human and bovine respiratory syncytial virus (RSV) are significant causes of morbidity and mortality in human and cattle populations worldwide, respectively. RSV disease is characterized by deleterious inflammatory immune responses as well as generation of radical oxygen species in the airways. Recent reports have shown antiviral and anti-inflammatory activity of NRF2 agonists and immunometabolite derivatives 4-octyl-itaconate (4-OI) and dimethyl fumarate (DMF), suggesting their potential to protect against viral-induced inflammation. Here, we evaluated whether 4-OI or DMF impact human and bovine RSV replication and its associated inflammatory response in vitro and the efficacy of these NRF2 agonists in preventing RSV disease in a murine model. We observed that 4-OI and DMF inhibited the early inflammatory response to RSV as well as reduced infectious titers in epithelial cells. Moreover, mice treated with 4-OI or DMF were partially protected against RSV-induced weight loss and airway inflammation and showed reduced viral loads and interleukin-6 levels in the lung. Overall, these results support the use of NRF2 agonists 4-OI and DMF in the prevention of RSV disease in target populations.

Keywords: large animals; lung; rodent; viral.

Figure 1.

4-OI and DMF modulate transcription of nrf2 and NRF2-ARE–induced enzymes in BT cells during bRSV infection. BT cells were pretreated with DMF (50, 100 µM) or 4-OI (100, 200 µM) and infected 6 h later with bRSV at an MOI of 0.1. Twelve hours later, RNA was isolated and reverse transcribed to cDNA, and relative expression of (A) nrf2, (B) nqo1, and (C) hmox1 was determined using the 2^−ΔΔCt method. Expression level was normalized to vehicle-treated, uninfected cells, and rps9 was used as housekeeping gene. Data show the mean ± SEM from 2 independent experiments (n = 3–9), and all data points were plotted. P values were obtained from a 1-way analysis of variance followed by Dunnett’s multiple comparison test.

Figure 2.

4-OI and DMF strongly downregulate inflammatory responses to bRSV and hRSV infection and ifn1b transcription in respiratory ECs. (A–E) BT and (F–J) BEAS-2b cells were pretreated with DMF (50, 100 µM) or 4-OI (100, 200 µM) and infected 6 h later with bRSV or hRSV, respectively, at an MOI of 0.1. Treatments were repeated daily twice. After 72 h, RNA was isolated and reverse transcribed to cDNA, and relative expression of (A, F) il-6, (B, G) cxcl8, (C, H) ccl5, (D, I) il-1b, and (E, J) ifnb1 was determined using the 2^−ΔΔCt method. The expression level was normalized to vehicle-treated, uninfected cells, and rps9 was used as a housekeeping gene. Data show the mean ± SEM from 3 independent experiments (n = 6–9), and all data points were plotted. P values were obtained from a 1-way analysis of variance followed by Dunnett’s multiple comparison test.

Figure 3.

4-OI and DMF reduce hRSV and bRSV infectious titers in vitro. (A, B) BT and BEAS-2b cells were pretreated with DMF (50, 100 µM) or 4-OI (100, 200 µM) and infected 6 h later with bRSV or hRSV, respectively, at an MOI of 0.1. A second dose of DMF, 4-OI, or vehicle was administered 24 h after infection. (C, D) BT and BEAS-2b cells were infected with bRSV or hRSV. Then, 24 h later, cells were treated with DMF (50, 100 µM) or 4-OI (100, 200 µM). In both experiments, plates were frozen after 48 h, and infectious titers were determined by culture titration in BT (bRSV) or HEp-2 (hRSV) cells and calculated using the Reed and Muench method. Titers are expressed as TCID₅₀/mL. Data show the median ± range from 2 independent experiments (n = 5–6), and all data points were plotted. P values were obtained from a Kruskal-Wallis test. Diagram created in BioRender.

Figure 4.

Effects of repeated intranasal 4-OI and DMF administration in RSV disease in mice. (A) Experiment design. Mice were anesthetized with isoflurane and i.n. administered saline (vehicle), DMF at 80 μg, or 4-OI at 400 μg. Six hours later, mice were either mock infected (DMEM) or infected i.n. with 50 µL of 2.3 × 10⁸ TCID₅₀/mL hRSV A/1997. Animals were given drugs daily and were euthanized 72 h after infection. (B) Lungs (n = 7–8, all data points plotted) were collected and homogenized in TRIzol for RNA and cDNA preparation. N-hRSV and beta-actin copies were determined by RT-PCR. P values were obtained from a 1-way analysis of variance followed by Dunnett’s multiple comparisons test. (C) Body weights from mice (n = 9–12, all data points plotted) were recorded daily after infection and weight percentage relative to day 0 was calculated. P values were obtained from a 2-way analysis of variance with repeated measures and followed by Tukey’s multiple comparisons test. (D, E) Whole lungs from mice (n = 3–9) were formalin-fixed for histopathology scoring performed by blinded, trained pathologist with criteria specified in material and methods. P values obtained from a Kruskal-Wallis test. Data in panels D and E show median ± range from 3 independent experiments, and all data points were plotted. **P < 0.01. D, day. Diagram created in BioRender.

Figure 5.

Effects of intranasal 4-OI and DMF administration in the inflammatory response and H₂O₂ production in RSV infected mice lungs. (A) RNA and cDNA were processed from mice lungs (n = 9) and transcription of inflammatory cytokines was determined through RT-PCR. Expression level was normalized to vehicle-treated, uninfected cells, and beta-actin (mouse) was used as a housekeeping gene. Statistical analyses results are shown in Figure S3. (B) IL-6 protein levels were quantified in mice lung lysates (n = 8–11) through an indirect ELISA. P values were obtained from a 1-way analysis of variance followed by Dunnett’s multiple comparisons test. For A and B, data show the mean ± SEM 3 independent experiments. (C) H₂O₂ levels were measured in mice lung lysates (n = 6) using a colorimetric assay. P values were obtained from a Kruskal-Wallis test. Data show the median ± range from 2 dependent experiments. All data points were plotted.