Restriction of L egionella pneumophila Replication in Macrophages Requires Concerted Action of the Transcriptional Regulators Irf1 and Irf8 and Nod-Like Receptors Naip5 and Nlrc4

Abstract

ABSTRACT The unique permissiveness of A/J mouse macrophages for replication of Legionella pneumophila is caused by a deficiency in the Nod-like receptor (NLR) protein and intracellular sensor for L. pneumophila flagellin ( Naip5 ). The signaling pathways and proteins activated by Naip5 sensing in macrophages were investigated. Transcript profiling of macrophages from susceptible A/J mice and from resistant A/J mice harboring a transgenic wild-type copy of Naip5 at 4 h following L. pneumophila infection suggested that two members of the Irf transcriptional regulator family, Irf1 and Irf8, are regulated in response to Naip5 sensing of L. pneumophila . We show that macrophages having defective alleles of either Irf1 ( Irf1 − / − ) or its heterodimerization partner gene Irf8 ( Irf8 R294C ) become permissive for L. pneumophila replication, indicating that both the Irf1 and Irf8 proteins are essential for macrophage defense against L. pneumophila . Moreover, macrophages doubly heterozygous ( Naip5 AJ/WT Irf8 R294C/WT or Nlrc4 −/+ Irf8 R294C/WT ) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila , indicating that there is a strong genetic interaction between Irf8 and the NLR protein family in the macrophage response to L. pneumophila. Legionella -containing phagosomes (LCPs) formed in permissive Irf1 − / − or Irf8 R294C macrophages behave like LCPs formed in Naip5 -insufficient and Nlrc4 -deficient macrophages which fail to acidify. These results suggest that, in addition to Naip5 and Nlrc4 , Irf1 and Irf8 play a critical role in the early response of macrophages to infection with L. pneumophila , including antagonizing the ability of L. pneumophila to block phagosome acidification. They also suggest that flagellin sensing by the NLR proteins Naip5 and Nlrc4 may be coupled to Irf1-Irf8-mediated transcriptional activation of key effector genes essential for macrophage resistance to L. pneumophila infection.