Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes

Abstract

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.

FIGURE 1.

Human monocytes do not activate caspase-1 or IL-1β in response to L. pneumophila infection. Primary human monocytes were infected or not (NT) with L. pneumophila (Leg), L. monocytogenes (Lm) for 8 h, or S. typhimurium (Sal) for 4 h. A, cell culture supernatants were collected and analyzed by Western blot with anti-caspase-1 antibody. B, culture supernatants of primary human monocytes infected with L. pneumophila or S. typhimurium were analyzed for active IL-1β by ELISA.

FIGURE 2.

Effect of L. pneumophila infection on the expression of caspase-1, IL-1β, NLRC4, and ASC in human monocytes. Primary human monocytes were infected or not (NT) with L. pneumophila for 4 and 24 h at a multiplicity of infection of 1. The expression of caspase-1 (A), IL-1β (B), NLRC4 (C), and PYCARD/ASC (D), mRNA (upper panels), and protein levels (lower panels) were assessed using quantitative RT-PCR and Western blotting, respectively. The results are displayed as the mean of three independent experiments ± S.D. (*, p ≤ 0.05; **, p ≤ 0.01). Actin was used as a loading control in all panels.

FIGURE 3.

Exogenous expression of ASC in human monocytes restores caspase-1 activation. A, primary human monocytes were nucleofected with a plasmid coding ASC tagged with YFP (Y-ASC). A plasmid encoding the green florescent protein (GFP) was used as a control. Twenty four hours after nucleofection, cells were lysed, and Y-ASC (52 kDa) and normal ASC (25 kDa) were assessed using Western blotting. B, human monocytes were nucleofected with a plasmid encoding GFP or Y-ASC and then they were either left untreated (NT) or infected with L. pneumophila (Leg) for 24 h at a multiplicity of infection of 1. Supernatant was collected, and cleaved caspase-1 (p-20) was assessed via immunoblotting.

FIGURE 4.

Exogenous expression of ASC in THP-1 cells restricts L. pneumophila infection, increases apoptosis, and deters NF-κB activation. A, THP-1 cells overexpressing Y-ASC and normal THP-1 were infected with L. pneumophila, and bacterial growth was assessed at 1 and 24 h after infection by counting the CFUs. The bacterial count is displayed as log CFU/ml. The results are displayed as the mean of three independent experiments ± S.D. B, THP-1 cells overexpressing Y-ASC and normal THP-1 were infected or not (NT) with L. pneumophila (Leg), and apoptosis was evaluated after 24 h of infection. The results are displayed as the mean of three independent experiments ±S.D. (*, p ≤ 0.05; **, p ≤ 0.01.) C, THP-1 overexpressing Y-ASC and normal THP-1 were infected or not (0) with L. pneumophila for 20, 40, or 60 min. Levels of phosphorylation of IKKαβ and p65 were detected using immunoblots with corresponding antibodies. Actin was used as loading control.

FIGURE 5.

Depletion of ASC in primary human monocytes with specific siRNA allows more L. pneumophila growth and improves NF-κB activation. A, human monocytes were transfected with ASC-specific or control siRNA and then the expression of ASC was examined by quantitative PCR and by Western blot. B, transfected cells described in A were infected with L. pneumophila, and bacterial growth was evaluated by scoring CFU. The results are displayed as log CFU/ml, the mean of three independent experiments ± S.D. (**, p ≤ 0.01). Primary human monocytes transfected with siRNA targeting ASC (siASC) or control siRNA (siCTR) were infected or not (0) with L. pneumophila for 20, 40, or 60 min. The phosphorylation of IKKαβ and p65 was examined by Western blots using corresponding antibodies. Actin was used as loading control.