LIMP-2 links late phagosomal trafficking with the onset of the innate immune response to Listeria monocytogenes: a role in macrophage activation

Abstract

The innate immune response to Listeria monocytogenes depends on phagosomal bacterial degradation by macrophages. Here, we describe the role of LIMP-2, a lysosomal type III transmembrane glycoprotein and scavenger-like protein, in Listeria phagocytosis. LIMP-2-deficient mice display a macrophage-related defect in Listeria innate immunity. They produce less acute phase pro-inflammatory cytokines/chemokines, MCP-1, TNF-α, and IL-6 but normal levels of IL-12, IL-10, and IFN-γ and a 25-fold increase in susceptibility to Listeria infection. This macrophage defect results in a low listericidal potential, poor response to TNF-α activation signals, impaired phago-lysosome transformation into antigen-processing compartments, and uncontrolled LM cytosolic growth that fails to induce normal levels of acute phase pro-inflammatory cytokines. LIMP-2 transfection of CHO cells confirmed that LIMP-2 participates in the degradation of Listeria within phagosomes, controls the late endosomal/lysosomal fusion machinery, and is linked to the activation of Rab5a. Therefore, the role of LIMP-2 appears to be connected to the TNF-α-dependent and early activation of Listeria macrophages through internal signals linking the regulation of late trafficking events with the onset of the innate Listeria immune response.

FIGURE 1.

LIMP-2^−/− but not LAMP-1^−/− mice were highly permissive to LM infection and displayed an impaired LM innate immunity. WT mice (white bars), LAMP-1^−/− (gray bars), or LIMP-2^−/− littermates (black bars) were infected with 5 × 10⁴ LM for different times. Next, CFU were quantified in homogenized organs. The experiments were performed at least three times. A, CFU analysis of spleen from LM infected LIMP-2^−/−, LAMP-1^−/−, and wild-type mice at 3 and 7 dpi. The results are expressed as the CFU × 10⁵ ± S.D. B, columns 1–5 represent different experiments of five mice/genotype in each experiment. CFU analysis of liver and spleen of LM infected LIMP-2^−/− and WT mice at 3 dpi. The results are expressed as the CFU × 10⁶ ± S.D. in liver and as CFU × 10⁵ ± S.D. in spleen of triplicate samples. C, spleen from wild-type mice (labeled as +/+), LAMP-1^−/−, or LIMP-2^−/− infected, or not, with 5 × 10⁴ LM for 7 days. Homogenates were incubated with 10 μCi of ³⁵S-translabel for 2 h. Next, the spleen cells were incubated with 2% dialyzed FCS-DMEM-Met/Cys-free for 14 h. The cells were lysed and immunoprecipitated with Y3P antibody to capture the newly synthesized MHC II molecules. The immunoprecipitates were run under SDS-PAGE, and the gels were exposed to x-ray films.

FIGURE 2.

The activation of LIMP-2^−/− BM-DM was impaired, and they failed to control cytosolic LM growth. A, BM-DM from WT or LIMP-2^−/− littermates were infected with LM for 0, 6, or 12 h. The results are expressed as CFU × 10⁴ ± S.D. of triplicate samples (“Experimental Procedures”). B, BM-DM were incubated with streptavidin-HRP to label early endosomes, late endosomes, or lysosomes and next infected with biotinylated LM to isolate phagosomes according to the protocol described under “Experimental Procedures.” The HRP enzymatic activity was measured by the absorbance at A_{450 nm}. The results are expressed as the percentages of total internalized HRP activity and reflect the percentage of fusion of phagosomes with either early, late endosomes or lysosomes. C, LM phagosomes from BM-DM pretreated with mIFN-γ before infection (IFN lanes) or nontreated (NT lanes) were isolated as described under “Experimental Procedures,” and 30 μg of phagosomal proteins were loaded per lane. Western blots indicate the inactive and membrane-bound Ctsd forms (mCtsd) and the active and soluble Ctsd bands (Ctsd), developed with a rabbit anti-Ctsd antibody. The Rab5a bands were developed with a mouse monoclonal anti-Rab5a antibody and the ASMase bands with a goat anti-mouse ASMase antibody. D, LM phagosomes from BM-DM labeled with 10 μCi of ³⁵S-translabel, as in Fig. 4C, were isolated as under “Experimental Procedures,” and 30 μg of phagosomal proteins were boiled (B lanes) or nonboiled (NB lanes) before loading. The labels show stable αβ dimers and unstable α and β chains as reported (32, 33). E, WT or LIMP-2^−/− BM-DM were infected with GFP-LM for 1 h, and MIIC compartments were labeled with biotinylated anti-IA^b antibody followed by streptavidin-phycoerythrin. Co-localization images appearing as yellow fluorescence were analyzed by confocal microscopy. The scale bars correspond with 5 μm. F, different BM-DM pretreated with IFN-γ (+IFN-γ samples), TNF-α (+TNF-α samples), or untreated (NT samples) were infected with GFP-LM (ratio 10:1, bacteria:cell) for 12 h, and fluorescent images were analyzed by conventional fluorescent microscopy. The scale bars correspond with 6 μm. CFU values at 12 h were 10 × 10⁴ (WT and untreated images), 2 × 10² (WT and +IFN-γ images), 3 × 10² (WT and +TNF-α images), 65 × 10⁴ (LIMP-2^−/− and untreated images), 13 × 10² (LIMP-2^−/− and +IFN-γ images), and 56 × 10⁴ (LIMP-2^−/− and TNF-α images).

FIGURE 3.

Linked actions of Rab5a activation and LIMP-2 degrade LM and promote lysosome fusion. LIMP-2 transfectants and active Rab5a: Q79L (AQ) or inactive Rab5a: S34N (AS) co-transfectants were infected with LM, and different trafficking parameters were analyzed. A, LM replication indices at 6 h post-infection in CHO control cells, LIMP-2, LIMP-2/AQ, and LIMP-2/AS co-transfectants. The results are expressed as RI ± S.D. of triplicate samples. B, degradation of [³⁵S]HKLM (500,000 cpm/sample) added to 4 × 10⁵ CHO cells/well in 96-well plates, centrifuged to synchronize infection, and internalized for 20 min. The cells were washed and cell-associated, and supernatant radioactivities were recorded after a 60-min incubation (14). The results are expressed as percentages of cell-associated (black bars) or supernatant (gray bars) compared with internalized radioactivity ± S.D. of triplicate samples. C, percentages of phagosome-lysosome fusion using HRP-loaded lysosomes transfer fusion assays (14). The results correspond to percentages ± S.D. of triplicate experiments. D, conventional fluorescent patterns of LIMP-2 transfectants, LIMP-2/AQ, and LIMP-2/AS co-transfectants. Scale bars, 4 μm.

FIGURE 4.

Model for the role of LIMP-2 in LM-primed MØ activity. Step 1, LM binds to different TLRs or scavenger receptors and is internalized into phagosomes. Step 2, the early phagosomal steps include Rab5a-GTP-mediated fusion with endosomes, the transport of membrane-bound and inactive Ctsd (mCtsd) and the activation of Ctsd into a listericidal, soluble, and active enzyme (step 2.1). LIMP-2 action on the phagosomal membrane requires Rab5a-GTP and promotes fusion with late endosomes/lysosomes, including late endocytic vesicles containing MHC II molecules (step 2.2). This overall process allows the transformation of phagosomes into phago-lysosomes with bactericidal abilities and features of MIIC (MIIC-Phg) (step 2.3). LIMP-2 regulation of this phagosomal transformation process influences the LM membrane permeability and confines the bacteria within phagosomes. Therefore, the number of cytosolic LM is limited (step 2.4). Step 3, the low numbers of cytosolic LM are sensed by NOD-2 receptors in the cytosol that activate the production of the acute phase pro-inflammatory cytokines/chemokines: MCP-1, TNF-α, and IL-6. Step 4, the release of these cytokines/chemokines such as TNF-α acts as a feedback loop that promotes the full activation of MØ, making them more efficient at degradation of LM.