Innate recognition of bacteria by a macrophage cytosolic surveillance pathway

Abstract

Host recognition of bacterial pathogens is a critical component of the immune response. Intracellular bacterial pathogens are able to evade the humoral immune system by residing within the host cell. Here we show the existence of an innate host surveillance mechanism in macrophages that specifically distinguishes bacteria in the cytosol from bacteria in the vacuole. Recognition of Gram-positive and Gram-negative bacterial products by this surveillance system results in transcription of the ifnb gene. The activation of cytosol-specific signaling is associated with translocation of NF-kappaB into the nucleus and phosphorylation of the p38 mitogen-activated protein (MAP) kinase. Activation of the p38 kinase is required for the induction of gene expression by the cytosolic surveillance pathway. Our studies suggest that infection by intracellular bacterial pathogens results in an immune response distinct from that of infection by extracellular bacterial pathogens.

Figure 1

Infection of BMDM by wild-type L. monocytogenes, but not an LLO⁻ strain, results in up-regulation of ifnb gene expression. (A) C57BL/6 macrophages were infected at a moi resulting in 1–5 bacteria per cell with L. monocytogenes or treated with E. coli LPS (1 ng/ml), or B. subtilis LTA (1 μg/ml). RNA was isolated from macrophage monolayers at 6 h.p.i. and analyzed by RT-PCR. Actin reactions shown were amplified for 15 cycles; ifnb reactions shown were amplified for 30 cycles. (B) C57BL/6 macrophages were infected as described above and RNA was isolated from macrophage monolayers. Actin reactions shown were amplified for 15 cycles; ifnb reactions shown were amplified for 30 cycles.

Figure 2

LLO is not required for ifnb gene expression in HeLa cells. (A) Indirect immunofluorescence was performed on HeLa cells at 4 h.p.i. Coverslips were incubated with a polyclonal antibody directed against L. monocytogenes and rhodamine-phalloidin. White arrowheads mark bacteria colocalizing with F-actin; open arrowheads mark bacteria that are not colocalized with F-actin. (B) HeLa cells were infected with L. monocytogenes and treated with cycloheximide at 1.5 h.p.i. RNA was isolated at 6 h.p.i. and subjected to RT-PCR analysis. Actin reactions shown were amplified for 15 cycles; ifnb reactions shown were amplified for 30 cycles.

Figure 3

E. coli and B. subtilis in the cytosol of macrophages up-regulate ifnb expression, but LPS and LTA do not. RT-PCR analysis was performed on C3H/HeJ BMDM treated with liposomes, labeled as lip(), or infected with the E. coli, B. subtilis, or L. monocytogenes strains indicated. Macrophages treated with poly(I)⋅poly(C) (100 μg/ml) were isolated as a positive control for ifnb induction. RNA was isolated at 6 h.p.i. or 6 h post treatment and analyzed by RT-PCR. Actin reactions shown were amplified for 15 cycles; ifnb reactions shown were amplified for 30 cycles.

Figure 4

NF-κB p65 is translocated to the nucleus when bacterial products are present in the cytosol of macrophages. Indirect immunofluorescence was performed on C3H/HeJ BMDM infected with E. coli expressing OVA with or without LLO by using monoclonal antibody specific for p65 (1:200) and subsequently with a FITC-conjugated secondary goat anti-mouse antibody (1:100).

Figure 5

The p38 MAP kinase is phosphorylated when L. monocytogenes is present in the cytosol. (A) C57BL/6 BMDM were infected with wild-type or the LLO⁻ strain of L. monocytogenes or treated with 1 μg/ml LPS as a positive control. Cells were harvested at 1.5 h.p.i. and subjected to SDS/PAGE and Western blot analysis. A phospho-specific antibody was used to detect the phosphorylated form of p38 (Pp38). An antibody against p38 was used to detect total cellular pools of p38 by using the same whole cell lysates. (B) C57BL/6 BMDM were pretreated with p38 inhibitor, SB202190 (SB), or MEK-1 inhibitor, U0126 (U), for 30 min before infection by L. monocytogenes, or treated with LPS (1 ng/ml) or anisomycin (30 μg/ml). RNA was isolated at 6 h.p.i. or 6 h post treatment and analyzed by RT-PCR. Actin reactions shown were amplified for 15 cycles; ifnb reactions shown were amplified for 30 cycles.