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. 2014 Dec 9;111(49):17588-93.
doi: 10.1073/pnas.1419925111. Epub 2014 Nov 24.

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection

Si Ming Man  1 , Andrew Ekpenyong  2 , Panagiotis Tourlomousis  1 , Sarra Achouri  3 , Eugenia Cammarota  3 , Katherine Hughes  1 , Alessandro Rizzo  1 , Gilbert Ng  2 , John A Wright  1 , Pietro Cicuta  3 , Jochen R Guck  2 , Clare E Bryant  4
Affiliations

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection

Si Ming Man et al. Proc Natl Acad Sci U S A. .

Abstract

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

Keywords: ASC; ROS; caspase-1; cytoskeleton; innate immunity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The NLRC4–caspase-1 axis restricts high levels of intracellular S. Typhimurium numbers in macrophages. (A) Unprimed primary BMMs were infected with S. Typhimurium (MOI 1) for 1 h. Following 1-h infection, BMMs were treated with gentamicin (50 μg/mL) for 1 h to kill extracellular bacteria. Host cell lysates were plated onto LB agar and the number of viable intracellular bacteria was enumerated. (B) Unprimed BMMs were infected with S. Typhimurium expressing GFP (MOI 10) for 1 h, followed by gentamicin treatment (50 μg/mL) for 1 h to kill extracellular bacteria. The percentages of BMMs harboring different number of bacteria were determined by microscopy (WT, n = 1,134; Nlrc4−/−, n = 765; Casp1/11−/−, n = 708; Nlrp3−/−, n = 663; Asc−/−, n = 731). (C) Unprimed BMMs were infected with ΔfliCΔfljBΔprgJ S. Typhimurium (MOI 1) for 1 h and the number of viable intracellular bacteria was enumerated. Data are the mean of three independent experiments and error bars represent SEM. (A) One-way ANOVA with a Dunnett’s multiple comparisons test. (C) Two-way ANOVA with a Tukey’s multiple comparisons test. ***P < 0.001; ns, no statistical significance.
Fig. 2.
Fig. 2.
NLRC4 regulates mitochondrial ROS and H2O2 production to restrict bacterial replication by Salmonella in the SCV. (A) The bacterial growth rate in each SCV was determined in WT and Nlrc4−/− BMMs using live confocal imaging of SCVs (WT, n = 11; Nlrc4−/−, n = 30) over 17 h following inoculation. The growth rate of S. Typhimurium was calculated using the formula ∆n/t, where ∆n is the number of bacteria in a SCV immediately before the death of a macrophage minus the number of bacteria following the formation of a SCV, and t is the length of time that the macrophage had survived over the course of the infection. (B and C) Unprimed BMMs were infected with S. Typhimurium (MOI 10) for 30 min and stained with MitoSOX stain or CM-H2DCFDA for 30 min. (B) Levels of mROS and (C) H2O2 were measured using flow cytometry. (D) WT BMMs were treated with NAC for 1.5 h and then infected with S. Typhimurium (MOI 1) for 1 h, followed by gentamicin treatment for a total of 2 or 6 h. Lysates from BMMs were plated on LB agar and the number of viable intracellular bacteria was enumerated. Data are representative of three (C and D) or four (B) independent experiments. Error bars indicate SEM. Two-tailed t test, *P < 0.05; **P < 0.01.
Fig. 3.
Fig. 3.
NLRC4 inflammasome functions are linked to the actin cytoskeleton. (A) Unprimed BMMs were infected with S. Typhimurium expressing GFP (MOI 10) and tracked by confocal live imaging for 1 h. The number of bacteria internalized into each macrophage was counted every 60 s. (B) The number of viable intracellular bacteria recovered from WT BMMs infected with S. Typhimurium in the presence of the vehicle control DMSO, cytochalasin D (0.2, 2, and 20 µM) or colchicine (1, 10, and 100 µM) was counted. (C) Host cell viability and (D) levels of IL-1β secreted from LPS-primed WT BMMs infected with S. Typhimurium for 1 h in the presence of the vehicle control DMSO, cytochalasin D, or colchicine. (E) Unprimed WT BMMs were infected with S. Typhimurium for 1 h and stained for ASC (red) and DNA (blue). Data are the mean of three independent experiments and error bars represent SEM. One-way ANOVA with a Dunnett’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not statistically significant.
Fig. 4.
Fig. 4.
NLRC4 activation induces physical stiffening of macrophages by limiting actin availability for bacterial uptake and impairs bacterial dissemination. (A) The principle and setup of the optical stretcher. Two diverging and counterpropagating laser beams emanating from single-mode optical fibers are used to trap and deform single suspended cells. A 50:50 intensity-ratio fiber coupler (FC) is used to split the output of a fiber laser into the two optical fibers. A personal computer (PC) is used for laser control and data acquisition by video microscopy via a CCD camera. (B) Unprimed primary BMMs were infected with GFP-expressing S. Typhimurium (MOI 10) for 10 min. BMMs were then serially trapped and stretched outwardly along the laser beam axis. Shown are (Left) the average creep compliance responses of the various cells during the 4 seconds of stretching (indicated by darker red) and (Right) box plots of the average compliance during the stretch. (C) A 3D reconstruction of phalloidin-stained F-actin cytoskeleton (red) in unprimed immortalized BMMs infected with GFP-expressing S. Typhimurium (MOI of 10) for 5 min. (D) Unprimed primary BMMs were imaged for 3 h and then infected with S. Typhimurium (MOI 10) and imaged for 30 min. For each cell, the movies were divided in sections of 10 min and cell movement was analyzed. Data shown are from uninfected cells (over the 3-h time period) and from cells infected with S. Typhimurium for 20–30 min. The mean square displacement (MSD) (MSD(τ)=〈(x(t+τ)−x(t))2〉t) of infected cells compared with uninfected cells (τ = 2–10 min) is fitted with a power law Deff⋅tα. From the resulting fit, the value of the MSD at 5 min is calculated. In the box plot, the red bars represent the median of the distribution, the blue edges of the box indicate the 25th and 75th percentile, and dashed bars indicate the extreme points. (E) Histopathology of livers from WT (n = 4) and Nlrc4−/− (n = 5) mice i.v. infected with S. Typhimurium for 7 d. Dashed circles indicate extent of lesions. (F) Livers were fixed and lesions were counted from four liver sections from each mouse. Two-tailed t test, ***P < 0.001; ****P < 0.0001; ns, not statistically significant.
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