Asc-dependent and independent mechanisms contribute to restriction of legionella pneumophila infection in murine macrophages

Abstract

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase(-1-/-) and Nlrc4(-/-) allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc(-/-) macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.

Keywords: Asc; Legionella pneumophila; caspase-1; inflammasome.

Figure 1

Asc controls Legionella pneumophila replication in caspase-1^−/− macrophages. (A) Caspase-1^−/− (casp-1^−/−) macrophages and (B) WT macrophages were either left untreated (NT) or transfected with Asc specific siRNA (siAsc) or control siRNA (siCTR). After 48 h transfection Asc, levels were assessed using Western blotting. Actin was used as a loading control. Casp-1^−/− macrophages (C) and wild type macrophages (D) were transfected or not (NT) with siAsc or siCTR and 48 h after transfection cells were infected with L. pneumophila (Leg) at MOI = 0.1 and the bacterial replication was assessed by counting the CFU after 1, 24, 48, and 72 h. Results are displayed as mean ± SD of three independent wells. **P ≤ 0.01. The data shown in (C,D) are representative of four independent experiments showing the same results.

Figure 2

Asc hinders the activation of NF-κB induced by L. pneumophila. (A) Caspase-1^−/− (casp-1^−/−) macrophages were transfected with Asc specific siRNA (siAsc) or control siRNA (siCTR) and 48 h after transfection cells were infected or not (NT) with L. pneumophila (Leg) at MOI = 0.5 for 1, 4, and 8 h. Afterward, NF-κB activation was examined using electrophoretic mobility shift assay (EMSA). (B) WT and Asc^−/− mouse macrophages were infected or not (NT) with L. pneumophila (Leg) at MOI = 0.5 for 1 and 8 h. Subsequently, NF-κB activation was examined using EMSA.

Figure 3

A fraction of caspase-1 is activated in the cytosol of murine macrophages lacking Asc. (A) WT and Asc^−/− mouse macrophages were either not treated (NT) or infected with L. pneumophila (Leg) at MOI = 0.5 for 4 and 8 h. Then active caspase-1 (p-20) was detected in the cell extracts (A) and the supernatants (B) by Western blotting. Actin was used as a loading control.

Figure 4

Depletion of Asc decreases caspase-1 activation in response to L. pneumophila infection. (A,B,C) WT mouse macrophages were transfected or not (NT) with Asc specific siRNA (siAsc) or control siRNA (siCtr) and 48 h after transfection cells were infected with L. pneumophila (Leg). Then, active caspase-1 (p-20) was detected in the cell extracts (A) and in the supernatants (B) of 8 h infected samples by Western blotting. Active IL-1β was detected in supernatants using ELISA (C). The results displayed as mean ± SD (**P ≤ 0.01) of three independent wells.

Figure 5

Asc^−/− macrophages are permissive to flagellin mutant. (A) WT macrophages were infected with L. pneumophila (Leg), with corresponding flagellin mutant (Fla), or left untreated (NT). Active caspase-1 (p-20) was then detected in the cell extracts. (B) WT and Asc^−/− macrophages were infected with Fla mutant and CFUs were scored 72 h after infection. (C) WT and Asc^−/− macrophages were infected with Fla mutant for 1, 4, and 8 h then, nuclear extracts were processed for determination of NF-κB activation using electrophoretic mobility shift assay (EMSA). (D) WT and Asc macrophages were infected with Leg or Fla for 24 h then LDH release was determined and presented as percent cell death on the Y axis.

Figure 6

(A) Lysates from WT macrophages infected with L. pneumophila (Leg), the Fla mutant, or the type IV secretion mutant DotA were used to determine caspase-3 activity by the DEVD-AFC assay, or inactive full-length (FL-casp-3) and cleaved caspase-3 (cleaved casp-3) by immunoblotting. (B) WT and Asc^−/− macrophages were infected with Fla mutant for 1, 2, and 5 h then cell lysates were obtained and used to determine cleaved caspase-3 by Western Blotting.

Figure A1

(A) Wild type (WT) mouse macrophages and heterozygous Asc (Asc^−/+) were infected or not (NT) with L. pneumophila (Leg) at MOI = 0.5 for 1, 2, and 4 h. Total active caspase-1 (p-20) was detected in combined lysate and supernatants by Western blotting. (B) WT and caspase-1^−/− macrophages were infected with L. pneumophila (Leg) at MOI = 0.1 and the bacterial replication was assessed by counting the colony-forming units (CFU) after 1, 24, 48, and 72 h. Results are displayed as mean ± SD of three independent wells. **P ≤ 0.01.

Figure A2

Legionella pneumophila differentially regulates the expression of several components of the inflammasome. WT mouse macrophages were infected or not (NT) with Legionella pneumophila (Leg) at MOI = 0.5 for 4 and 24 h. then the expressions of (A) pro-caspase-1, (B) pro-Il-1β, (C) Pycard (Asc), and (D) Nlrc4 (Ipaf) were then assessed on both mRNA (upper panels) and protein levels (lower panels) using RT-PCR and Western blots, respectively. The data of RT-PCR are displayed as mean relative copy numbers (RCN) ± SD of three independent experiments. *P ≤ 0.05, **P ≤ 0.01. Actin was used as loading control.