Cutting edge: Cytosolic bacterial DNA activates the inflammasome via Aim2

Abstract

Pathogens are detected by pattern recognition receptors that, upon activation, orchestrate an appropriate immune response. The TLRs and the nucleotide-binding oligomerization domain-like receptors (NLRs) are prototypic pattern recognition receptors that detect extracellular and cytosolic pathogens, respectively. Listeria monocytogenes has both extracellular and cytosolic phases and is detected in the cytosol by members of the NLR family. These include two NLR members, NLRC4 and NLRP3, that, upon detection of cytosolic L. monocytogenes, induce the assembly of the inflammasome. Inflammasomes serve as platforms for the activation of the protease caspase 1, which mediates the processing and secretion of pro-IL-1beta and pro-IL-18. We previously provided evidence that L. monocytogenes is also detected by a third inflammasome. We now use biochemical and genetic approaches to demonstrate that the third detector senses bacterial DNA and identify it as Aim2, a receptor that has previously been shown to detect viral DNA.

FIGURE 1

A third sensor activates IL-1β secretion following L. monocytogenes infection. LPS-primed macrophages were infected with L. monocytogenes at a multiplicity of infection 5 for 1 h (A), 4 h (B), or 8 h (C), and IL-1β secretion was determined by ELISA. D, IL-1β processing was determined by Western blot 4 h postinfection.

FIGURE 2

L. monocytogenes DNA induces IL-1β secretion. A, L. monocytogenes lysate was mock treated or digested with trypsin or DNase before delivery to LPS-primed NLRP3/NLRC4^DKO macrophages by lipid transfection. IL-1β secretion was measured after 2 h. Indicated microliters of lysate were transfected into LPS primed macrophages. B, Genomic DNA from L. monocytogenes was purified and delivered to LPS-primed macrophages by lipid transfection. IL-1β secretion was determined after 4 h. *p < 0.05; **p < 0.01.

FIGURE 3

L. monocytogenes DNA colocalizes with ASC specks. NLRP3/NLRC4^DKO macrophages expressing ASC-V5 were infected with L. monocytogenes. A, Hoechst-stained WT or ΔLLO L. monocytogenes DNA (blue) and ASC (green). B, DAPI-stained or unstained L. monocytogenes DNA (blue) and ASC (green). C, DAPI-stained L. monocytogenes DNA (blue), L. monocytogenes cell wall (red), and ASC (green). Enlarged merged images of L. monocytogenes DNA with cell wall from (1) an intact bacterium and (2) a lysed bacterium are shown without ASC green overlay. Corresponding phase images are shown. Scale bars, 15 μm. Original magnification ×60.

FIGURE 4

Aim2 responds to cytosolic bacteria. Control or Aim2 shRNA expressing NLRP3/NLRC4^DKO (A) or WT macrophages (B) were generated by retroviral transduction and infected with L. monocytogenes at a multiplicity of infection of 10, and IL-1β secretion was determined after 4 h. C, Control or Aim2 shRNA expressing NLRP3/NLRC4^DKO macrophages were infected with Francisella novicida for 8 h, and IL-1β secretion was determined. *p < 0.05.