Adherent and invasive Escherichia coli (AIEC) associated with Crohn's disease are able to survive and to replicate extensively in active phagolysosomes within macrophages. AIEC-infected macrophages release large amounts of tumour necrosis factor-alpha (TNF-alpha) and do not undergo cell death. The aim of the present study was to determine what benefit AIEC bacteria could gain from inducing the release of large amounts of TNF-alpha by infected macrophages and to what extent the neutralization of TNF-alpha could affect AIEC intramacrophagic replication. Our results showed that the amount of TNF-alpha released by infected macrophages is correlated with the load of intramacrophagic AIEC bacteria and their intracellular replication. TNF-alpha secretion was not related to the number of bacteria entering host cells because when the number of bacteria internalized in macrophage was decreased by blocking lipid raft-dependent and clathrin-coated pits-dependent endocytosis, the amount of TNF-alpha secreted by infected macrophages was not modified. Interestingly, dose-dependent increases in the number of intracellular AIEC LF82 bacteria were observed when infected macrophages were stimulated with exogenous TNF-alpha, and neutralization of TNF-alpha secreted by AIEC-infected macrophages using anti-TNF-alpha antibodies induced a significant decrease in the number of intramacrophagic bacteria. These results indicate that AIEC bacteria use TNF-alpha as a Trojan horse to ensure their intracellular replication because replication of AIEC bacteria within macrophages induces the release of TNF-alpha, which in turn increases the intramacrophagic replication of AIEC. Neutralizing TNF-alpha secreted by infected macrophages may represent an effective strategy to control AIEC intracellular replication. Laboratory Investigation (2012) 92, 411-419; doi:10.1038/labinvest.2011.156; published online 31 October 2011