Dietary abscisic acid ameliorates influenza-virus-associated disease and pulmonary immunopathology through a PPARγ-dependent mechanism

Abstract

The anti-inflammatory phytohormone abscisic acid (ABA) modulates immune and inflammatory responses in mouse models of colitis and obesity. ABA has been identified as a ligand of lanthionine synthetase C-like 2, a novel therapeutic target upstream of the peroxisome proliferator-activated receptor γ (PPARγ) pathway. The goal of this study was to investigate the immune modulatory mechanisms underlying the anti-inflammatory efficacy of ABA against influenza-associated pulmonary inflammation. Wild-type (WT) and conditional knockout mice with defective PPARγ expression in lung epithelial and hematopoietic cells (cKO) treated orally with or without ABA (100 mg/kg diet) were challenged with influenza A/Udorn (H3N2) to assess ABA's impact in disease, lung lesions and gene expression. Dietary ABA ameliorated disease activity and lung inflammatory pathology, accelerated recovery and increased survival in WT mice. ABA suppressed leukocyte infiltration and monocyte chemotactic protein 1 mRNA expression in WT mice through PPARγ since this effect was abrogated in cKO mice. ABA ameliorated disease when administered therapeutically on the same day of the infection to WT but not mice lacking PPARγ in myeloid cells. We also show that ABA's greater impact is between days 7 and 10 postchallenge when it regulates the expression of genes involved in resolution, like 5-lipoxygenase and other members of the 5-lipoxygenase pathway. Furthermore, ABA significantly increased the expression of the immunoregulatory cytokine interleukin-10 in WT mice. Our results show that ABA, given preventively or therapeutically, ameliorates influenza-virus-induced pathology by activating PPARγ in pulmonary immune cells, suppressing initial proinflammatory responses and promoting resolution.

Figure 1

Peroxisome proliferator-activated receptor (PPAR)γ protein expression during influenza virus infection in cells obtained by bronchoalveolar lavage fluid (BALF) (top), and lung (bottom). WT mice were infected with 5 × 10⁴ tissue culture infectious dose 50 (TCID₅₀) of influenza A/Udorn (H3N2) and sacrificed 1,3,5,7 or 9 days post-challenge. Non-infected mice (0) were used to assess baseline PPAR γ expression at day 0. Influenza virus infection upregulated PPAR γ protein, reaching highest expression at day one post-infection in whole lung and on day 9 post-infection in cells obtained from the bronchoalveolar space. Results from BALF correspond to cells pooled from at least 3 mice at each time point. Results from lung correspond to a sample obtained from one mouse at each time point and the picture is representative of 4 replicates with identical results.

Figure 2

Effect of dietary abscisic acid (ABA)-supplementation on weight loss (A), survival rates (B), and lung viral loads(C) following infection with influenza virus. Wild-type (WT) or immune/epithelial cell-specific PPAR γ null mice (cKO) mice were fed either a control or an ABA-supplemented diet (100 mg/ABA kg of diet) for 36 days and then challenged with 5 × 10⁴ tissue culture infectious dose 50 (TCID₅₀) of influenza A/Udorn (H3N2). Body weight results indicate that ABA treatment prevented weight loss associated with influenza virus infection in the WT but not in the cKO mice, suggesting a PPAR γ-dependent mechanism of action (A). A similar beneficial pattern was observed in the survival rates for the ABA-treated mice (B). For the weight loss data, Asterisks denote statistically significant differences between the ABA-treated WT and the myeloid KO mice. Pound signs denote statistically significant differences between the ABA-treated WT and the control WT (n=10 mice per treatment and genotype). These effects were not associated to differences in lung viral load due to ABA treatment on day 4 post-infection (C).

Figure 3

Effect of dietary ABA-supplementation on lung histopathology. Wild-type (WT) or cKO mice were fed either a control or pre-treated with ABA and infected with influenza 10⁴ TCID₅₀ of A/Udorn (H3N2) virus. ABA treatment did not ameliorate epithelial necrosis (A), but it diminished perivascular infiltration (C) as well as the infiltration of the terminal respiratory airways mucosa and submucosa (D) when compared to control-fed mice. The lower number of inflammatory cells in the lungs of ABA-treated WT mice correlated with downregulation of monocyte chemo attractant protein-1 (MCP-1) mRNA following infection when compared to control-fed mice(E). Data points with an asterisk (P<0.05) are significantly different (n=10 mice per treatment and genotype).

Figure 4

Effect of dietary ABA-supplementation on pulmonary expression of PPAR γ and monocyte chemotactic protein 1 (MCP-1) in broncholveolar lavage-derived cells. Cells were collected from the airway of WT or cKO mice 4 days post-infection. Dietary treatment and infection were as described before. ABA treatment increased PPAR γ mRNA expression in healthy non-infected WT mice (A) although following infection WT mice fed the control diet had significantly higher levels of PPAR γ, which correlated with higher mRNA expression of MCP-1 and TNFα, compared to the rest of the groups. Data points with different letters (P<0.05) are significantly different (n=10 mice per treatment and genotype).

Figure 5

Effect ABA treatment on vascular infiltration (A) and airway infiltration (B) in the lung following infection with influenza virus. Wild-type (WT) or myeloid-specific PPAR γ null mice (myeloid KO) mice were challenged with 5 x TCID₅₀ of influenza A/Udorn (H3N2) and then treated with either PBS or ABA (100 mg/ABA kg body weight) by oral gavage for 10 days starting on the day of infection. Asterisks denote statistically significant differences between the ABA-treated WT and the myeloid KO mice. Pound signs denote statistically significant differences between the ABA-treated WT and the control WT (n=10 mice per treatment and genotype).

Figure 6

Effect of ABA treatment on pulmonary gene expression following infection with influenza virus.(A) PPAR γ, (B) LANCL2, (C) angiopoietin-like 4, (D) iPLA2, (E) LOX5, (F) FLAP, (G) IL-10 and (H) TNF-α. WT or myeloid KO mice were challenged with 5 × 10⁴ TCID₅₀ of influenza A/Udorn (H3N2) and then treated with either PBS or ABA (100 mg/ABA kg body weight) by oral gavage for 10 days. Asterisks denote statistically significant differences between the ABA-treated WT and the myeloid KO mice. Pound signs denote statistically significant differences between the ABA-treated WT and the control WT (n=10 mice per treatment and genotype).