Aflatoxin B1 Promotes Influenza Replication and Increases Virus Related Lung Damage via Activation of TLR4 Signaling

Abstract

Aflatoxin B₁ (AFB₁), which alters immune responses to mammals, is one of the most common mycotoxins in feeds and food. Swine influenza virus (SIV) is a major pathogen of both animals and humans. However, there have been few studies about the relationship between AFB₁ exposure and SIV replication. Here, for the first time, we investigated the involvement of AFB₁ in SIV replication <i>in vitro</i> and <i>in vivo</i> and explored the underlying mechanism using multiple cell lines and mouse models. <i>In vitro</i> studies demonstrated that low concentrations of AFB₁ (0.01-0.25 μg/ml) markedly promoted SIV replication as revealed by increased viral titers and matrix protein (M) mRNA and nucleoprotein (NP) levels in MDCK cells, A549 cells and PAMs. <i>In vivo</i> studies showed that 10-40 μg/kg of AFB₁ exacerbated SIV infection in mice as illustrated by significantly higher lung virus titers, viral M mRNA levels, NP levels, lung indexes and more severe lung damage. Further study showed that AFB₁ upregulated TLR4, but not other TLRs, in SIV-infected PAMs. Moreover, AFB₁ activated TLR4 signaling as demonstrated by the increases of phosphorylated NFκB p65 and TNF-α release in PAMs and mice. In contrast, TLR4 knockdown or the use of BAY 11-7082, a specific inhibitor of NFκB, blocked the AFB₁-promoted SIV replication and inflammatory responses in PAMs. Furthermore, a TLR4-specific antagonist, TAK242, and TLR4 knockout both attenuated the AFB₁-promoted SIV replication, inflammation and lung damage in mice. We therefore conclude that AFB₁ exposure aggravates SIV replication, inflammation and lung damage by activating TLR4-NFκB signaling.