Inactivation of glutathione <i>S</i> -transferase alpha 4 blocks <i>Enterococcus faecalis</i> -induced bystander effect by promoting macrophage ferroptosis

<i>Enterococcus faecalis</i>-infected macrophages produce 4-hydroxynonenal (4-HNE) that mediates microbiota-induced bystander effect (MIBE) leading to colorectal cancer (CRC). Glutathione <i>S</i>-transferase alpha 4 (Gsta4), a specific detoxifying enzyme for 4-HNE, is overexpressed in human CRC and <i>E. faecalis</i>-induced murine CRC. However, the roles of Gsta4 in <i>E. faecalis</i>-induced colitis and CRC remain unclear. Herein, we demonstrate that Gsta4 is essential for MIBE by protecting macrophages from <i>E. faecalis</i>-induced ferroptosis. <i>E. faecalis</i> OG1RFSS was used to induce colitis in <i>Gsta4</i>^-/- and <i>Il10</i>^-/-<i>/Gsta4^-/-</i> mice by orogastric gavage. Ferroptosis was assessed in Gsta4-deficient murine macrophages. We found that, unlike <i>Il10</i>^-/- mice, <i>Gsta4^-/-</i> and <i>Il10</i>^-/-/<i>Gsta4</i>^-/- mice colonized with <i>E. faecalis</i> failed to develop colitis or CRC. Immunofluorescent staining showed a reduction of macrophages in the lamina propria of <i>E. faecalis</i>-colonized <i>Il10</i>^-/-/<i>Gsta4</i>^-/- mice, as well as decreased Gpx4 expression, indicating the occurrence of ferroptosis. Ferroptosis was further confirmed in <i>Gsta4</i>-deficient murine macrophages infected with <i>E. faecalis</i>. Moreover, Gsta4 inactivation induced the upregulation of Hmox1 and phosphorylated c-Jun while blocked Nos2 expression, leading to the accumulation of intracellular ferrous iron, lipid peroxidation and, eventually, ferroptosis. Finally, Mapk8, as a ferroptosis driver, was remarkably elevated in <i>E. faecalis</i>-infected <i>Gsta4</i>-deficient macrophages. These results suggest that Gsta4 inactivation blocks MIBE by eliminating macrophages, thereby attenuates <i>E. faecalis</i>-induced colitis and CRC.