P004 Activation of the GCN2/eIF2alpha/ATF4 signaling pathway triggers autophagy response to infection with Crohn's disease-associated adherent-invasive Escherichia coli
A. Bretin1, J. Carrière1, G. Dalmasso1, N. Barnich1, A. Bergougnoux1, A.-C. Maurin1, A. Bruhat2, H. Nguyen*3
1UMR 1071 Inserm/Université d'Auvergne; USC-INRA 2018, Microbes, Intestine, Inflammation et Susceptibility of the host, Clermont-Ferrand, France, 2INRA, Human Nutrition Unit (UNH), Theix, France, 3University of Auvergne, UMR 1071/Inserm, Clermont-Ferrand, France
A high prevalence of the adherent-invasive E. coli (AIEC) in the intestinal mucosa of Crohn's disease patients has been shown. We previously showed that upon AIEC infection, autophagy is induced in host cells to restrain AIEC intracellular replication. The mechanism underlying such autophagy induction, however, remains largely unknown. Here, we investigated the role of the GCN2/eIF2α/ATF4 pathway in autophagy response to AIEC infection.
Autophagic activity was assessed by Western blot and immunofluorescent labelling of LC3. Intracellular bacterial number was determined by bacterial invasion assay and confocal microscopy. Binding of ATF4 to autophagy gene promoters was assessed by Chromatin immunoprecipitation (ChIP) assay. Wild type (WT) and GCN2 knockout (KO) mice were infected with an AIEC reference strain LF82 by gavage.
Median (range) drugs levels at birth according to time of cessation of anti-TNF
|IFX level (mcg/ml)||ADA level (mcg/ml)|
|Last infusion prior to GW 30||Last infusion at or after GW 30||P value||Last injection prior to GW 30||Last injection at or after GW 30||P value|
|Mother||0.6 (0.0–3.3)||4.0 (0.0–22.2)||< 0.001||0.3 (0.0–0.7)||2.0 (0.0–10.0)||< 0.004|
|Cord Blood||1.9 (0.1–8.9)||9.6 (1.9–28.7)||0.0001||0.5 (0.0–1.2)||2.4 (0.0–12.1)||< 0.02|
Infection of human intestinal epithelial T84 cells with the AIEC LF82 strain activated the GCN2/eIF2α/ATF4 pathway as shown by increased phospho-GCN2 and phospho-eIF2α levels, enhanced ATF4 protein expression, and upregulated mRNA expression levels of ATF4 target genes. To explore the role of this pathway in host responses to AIEC infection, we used GCN2-deficient mouse embryonic fibroblasts (GCN2-/- MEF). GCN2 depletion suppressed eIF2α activation and inhibited the increase in ATF4 protein level induced by LF82 infection. mRNA expression levels of the autophagy genes p62, MAP1lc3, Beclin1, atg3 and atg7 were significantly increased in WT MEF upon LF82 infection, and this was blocked in GCN2-/- MEF. ChIP assay showed that GCN2 depletion inhibited the LF82-induced binding of ATF4 to the promoters of these autophagy genes. Consequently, autophagy induction upon LF82 infection was suppressed in GCN2-/- MEF, leading to increased LF82 intracellular replication and elevated pro-inflammatory cytokine production, compared to WT MEF. In vivo study consistently showed that LF82 infection activated the GCN2/eIF2α/ATF4 pathway in enterocytes from WT mice, but not GCN2 KO mice. In response to AIEC infection, autophagy was induced in WT mouse-derived enterocytes, and this was not observed in KO mice. LF82 persistence in the gut was increased in KO mice, leading to aggravated intestinal inflammation, compared to that in WT mice. Depletion of GCN2 did not affect susceptibility of mice to DSS-induced colitis, indicating that the effects obtained were not a consequence of inflammation and were specific for AIEC infection.
The GCN2/eIF2α/ATF4 pathway is activated in host cells during AIEC infection, which is served as a defense mechanism to induce a functional autophagy to control AIEC intracellular replication.