Type IV secretion-dependent activation of host MAP kinases induces an increased proinflammatory cytokine response to Legionella pneumophila

Abstract

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-kappaB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.

Figure 1. Increased cytokine production in response to L. pneumophila containing a functional T4SS compared to dotA mutants.

(A) ELISA measurements of cytokine levels in the BALFs of WT mice 24 hours following intranasal infection with PBS vehicle control or 5×10⁶ CFUs of WT, ΔflaA, or ΔdotA L. pneumophila on the thyA background. Each point represents an individual mouse. Lines indicate the mean cytokine levels for each group of mice. (B) ELISA measurement of cytokine production in WT bone marrow-derived macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila on the thyA background at an MOI = 5 for 24 hours. Data represent the mean±standard error of the mean (SEM) of the assay performed in triplicate and are representative of at least three independent experiments. P-values derived from two-tailed student's T test. * represents p<0.05. ** represents p<0.01. (C) Immunoblot analysis of pro-IL-1β production in WT bone marrow-derived macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila on the thyA background at an MOI = 5 for 24 hours. Blots were reprobed for analysis of total actin (loading control). Data are representative of at least two independent experiments.

Figure 2. TLR-dependent signaling synergizes with TLR-independent, T4SS-dependent signaling to induce an increased cytokine response.

(A) ELISA measurements of IL-1α, IL-6, IL-12 p40/p70, CXCL1, and TNF production in WT and Tlr2 ^−/− bone marrow-derived macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila on the thyA background at an MOI = 5 for 24 hours. Data are represented as the mean±SEM of the assay performed in triplicate and are representative of at least two independent experiments. (B) Quantitative RT-PCR analysis of WT or Myd88 ^−/− macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila at an MOI = 25 for four hours. Data represent the mean fold induction±SEM relative to uninfected macrophages of the assay performed in triplicate and are representative of at least two independent experiments.

Figure 3. RIP2-dependent NF-κB signaling in response to L. pneumophila type IV secretion is not required for T4SS-dependent cytokine production.

(A) Immunoblot analysis of IκB degradation in WT, Myd88 ^−/−, Rip2 ^−/−, or Myd88 ^−/− Rip2 ^−/− macrophages infected with WT or ΔdotA L. pneumophila at an MOI = 50. Blots were reprobed for analysis of total actin (loading control). Data are representative of at least three independent experiments. (B) ELISA measurements of cytokine production in WT and Rip2 ^−/− macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila on the thyA background at an MOI = 5 for 24 hours. Data represent the mean±SEM of the assay performed in triplicate and are representative of at least two independent experiments. (C) Immunoblot analysis of pro-IL-1β production in WT and Rip2 ^−/− bone marrow-derived macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila on the thyA background at an MOI = 5 for 24 hours. (D) Quantitative RT-PCR analysis of WT, Rip2^−/−, Myd88 ^−/−, or Myd88^−/−Rip2^−/− macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila at an MOI = 25 for four hours. Data are represented as the mean fold induction±SEM relative to uninfected macrophages of the assay performed in triplicate and are representative of at least two independent experiments.

Figure 4. L. pneumophila type IV secretion induces a MyD88- and RIP2-independent transcriptional response.

(A) Venn diagram showing genes transcriptionally induced two-fold or more in response to the T4SS in Myd88^−/−Trif^−/− or Myd88^−/−Rip2^−/− macrophages infected for four hours with WT L. pneumophila compared to those infected with the ΔdotA mutant on the thyA background at an MOI = 25 versus genes transcriptionally induced two-fold or more at four hours following ISD transfection of Myd88^−/−Trif^−/− macrophages . (B) Graph representing the percentage of genes transcriptionally induced two-fold or more upon infection with WT L. pneumophila compared to the ΔdotA mutant on the thyA background at an MOI = 25 for four hours in both Myd88 ^−/− Trif ^−/− and Myd88 ^−/− Rip2 ^−/− macrophages and that belong to various functional classes. (C) Quantitative RT-PCR analysis of Myd88 ^−/− Rip2 ^−/− macrophages infected with WT or ΔdotA mutant L. pneumophila at an MOI = 25 for four hours. Data are representative of at least two independent experiments.

Figure 5. L. pneumophila type IV secretion induces p38 and SAPK/JNK MAPK activation independently of TLR, Nod1, and Nod2 signaling.

(A) Immunoblot analysis of p-ERK1/2, p-p38, and p-SAPK/JNK MAPKs in WT, Myd88 ^−/− Trif ^−/−, and Myd88 ^−/− Rip2 ^−/− macrophages infected with WT or ΔdotA L. pneumophila at an MOI = 50. Total p38 MAPK is shown as a loading control. Data are representative of at least three independent experiments. (B) Immunoblot analysis of p-p38 and p-SAPK/JNK MAPKs in Myd88 ^−/− Rip2 ^−/− macrophages infected with WT, ΔdotA, or ΔflaA L. pneumophila at an MOI = 50. Total p38 MAPK is shown as a loading control. Data are representative of at least two independent experiments. (C) Immunoblot analysis of p-MKK4, p-MKK3/6, p-p38, p-SAPK/JNK, and p-c-Jun in Myd88 ^−/− Rip2 ^−/− macrophages infected with WT or ΔdotA L. pneumophila at an MOI = 50. Total p38 MAPK is shown as a loading control. Data are representative of at least two independent experiments. (D) Immunoblot analysis of p-p38 and p-SAPK/JNK in Myd88 ^−/− macrophages infected with WT, ΔdotA, or ΔicmS L. pneumophila at an MOI = 50. Total p38 MAPK is shown as a loading control. Data are representative of at least three independent experiments. (E) Quantitative RT-PCR analysis of Myd88 ^−/− Rip2^−/− macrophages infected with WT, ΔdotA, or ΔicmS L. pneumophila at an MOI = 25 for four hours. Data represent the mean fold induction±SEM relative to uninfected macrophages of the assay performed in triplicate and are representative of at least three independent experiments. (F) Immunoblot analysis of p-p38 and p-SAPK/JNK in Myd88 ^−/− Rip2 ^−/− macrophages pretreated with or without chloramphenicol (25 µg/mL) for 30 minutes prior to infection with WT or ΔdotA L. pneumophila at an MOI = 50. Total p38 is shown as a loading control.

Figure 6. MyD88-dependent and T4SS-dependent p38 and SAPK/JNK MAPK signaling collaborate to induce a maximal transcriptional response.

(A) Immunoblot analysis of p-p38 and p-SAPK/JNK MAPKs in WT and Myd88^−/− macrophages infected with WT and ΔdotA L. pneumophila at an MOI = 50. Total p38 is shown as a loading control. Shown on the right is a graphical representation of of the immunoblot analysis depicting the ratio of p-p38 intensity to total p38 intensity versus time. Immunoblots were quantified using ImageJ. Data are representative of at least two independent experiments. (B) Quantitative RT-PCR analysis of WT and Myd88 ^−/− macrophages. Macrophages were first infected with WT and ΔdotA mutant bacteria at an MOI = 25, then treated two hours later with 10 µM SB202190, 10 µM JNK II, 10 µM SB202190 plus 10 µM JNK II, or an equal volume of DMSO (vehicle control), and RNA harvested four hours after infection. Data represented the mean fold induction±SEM relative to uninfected cells of the assay performed in triplicate and are representative of at least two independent experiments.