Results Induction of IFN- by L. monocytogenes Requires IRF3. The transcription factor IRF3 has been shown to mediate the induction of the IFN- gene in response to both viruses and Toll-like receptor signaling (6, 19). To determine if induc- tion of type I IFNs by L. monocytogenes also involves IRF3, primary murine BMMs were challenged with L. monocyto- genes and IRF3 activation was assayed by nuclear transloca- tion. As shown in Fig. 1 A, L. monocytogenes can induce strong IRF3 nuclear translocation by 4 h after infection. We also found that L. monocytogenes induced nuclear translocation of the NF- B subunit p65 by 1 h, whereas USF-2 was mon- itored as a loading control (Fig. 1 A). Next, we sought to as- sess whether IRF3 is required for the induction of the type I IFN system. L. monocytogenes induction of IFN- occurred normally in BMMs deficient in the Toll-like receptor and IL-1R adaptor molecule MyD88, which has been shown to play an important protective role during an L. monocytogenes infection in vivo (Fig. 1 B; references 20 and 21). However, IRF3 / BMMs infected with L. monocytogenes were unable to induce expression of IFN- . As a control, we found that IFN- production in BMMs deficient in the IFNAR sub- unit IFNAR1 was similar to wild-type. Once produced, type I IFNs can signal in an autocrine and paracrine manner leading to the phosphorylation- dependent activation of STAT1 through IFNAR (2). Con- sistent with our observations that IRF3 is required for L. monocytogenes–induced IFN- production, STAT1 phos- phorylation also required IRF3 (Fig. 1 C). In contrast, L. monocytogenes–challenged MyD88 / BMMs, which were capable of IFN- production after L. monocytogenes infec- tion, induced STAT1 phosphorylation similar to wild-type controls (Fig. 1 C). STAT1 phosphorylation was dependent upon type I IFNs, as BMMs deficient in IFNAR1 did not activate STAT1 (Fig. 1 C). Collectively, these findings indi- cate that like some viruses, L. monocytogenes induction of IFN- and subsequent STAT1 activation also occur through an IRF3-dependent, MyD88-independent pathway. Mice Deficient in IRF3 Have an Enhanced Ability to Clear Infection by L. monocytogenes. IRF3 plays a protective role during viral infection (19). To assess the functional role of IRF3 activation during a bacterial infection, we inoculated both wild-type and IRF3-deficient mice with a strain of L. monocytogenes expressing an epitope from the NP protein (see Materials and Methods). Surprisingly, we found that 56% of IRF3-deficient mice survived a dose of L. monocyto- genes that was lethal for all mice in the wild-type control group (Fig. 2 A). Although bacterial titers were roughly equivalent on day 1 (not depicted), IRF3-deficient mice had 100-fold fewer bacteria compared with wild-type controls in both the spleen and liver by day 3 after a sublethal L. monocytogenes infection (Fig. 2 B). A similar pattern was noted when using a dose of L. monocytogenes that was ap- 439 O’Connell et al. Figure 1. Induction of IFN- by L. monocytogenes re- quires IRF3. (A) BMMs from C57BL/6 wild-type mice were infected with L. monocytogenes at a multiplicity of in- fection of 1 and IRF3 and p65 nuclear translocation were assayed at the indicated times by immunoblotting using nu- clear extract. USF-2 was also assayed as a loading control. (B) MyD88 / , IRF3 / , IFNAR1 / , and wild-type BMMs were infected with L. monocytogenes as described above and assayed at the indicated time points for IFN- gene expression by Q-PCR and (C) phospho-STAT1 / , or total STAT1 / by immunoblotting. Results shown are representative of at least two independent experiments, and Q-PCR results are expressed in relative expression units and have been normalized to L32 mRNA levels.