Neutrophil and Alveolar Macrophage-Mediated Innate Immune Control of <i>Legionella pneumophila</i> Lung Infection via TNF and ROS

<div><p><i>Legionella pneumophila</i> is a facultative intracellular bacterium that lives in aquatic environments where it parasitizes amoeba. However, upon inhalation of contaminated aerosols it can infect and replicate in human alveolar macrophages, which can result in Legionnaires’ disease, a severe form of pneumonia. Upon experimental airway infection of mice, <i>L</i>. <i>pneumophila</i> is rapidly controlled by innate immune mechanisms. Here we identified, on a cell-type specific level, the key innate effector functions responsible for rapid control of infection. In addition to the well-characterized NLRC4-NAIP5 flagellin recognition pathway, tumor necrosis factor (TNF) and reactive oxygen species (ROS) are also essential for effective innate immune control of <i>L</i>. <i>pneumophila</i>. While ROS are essential for the bactericidal activity of neutrophils, alveolar macrophages (AM) rely on neutrophil and monocyte-derived TNF signaling via TNFR1 to restrict bacterial replication. This TNF-mediated antibacterial mechanism depends on the acidification of lysosomes and their fusion with <i>L</i>. <i>pneumophila</i> containing vacuoles (LCVs), as well as caspases with a minor contribution from cysteine-type cathepsins or calpains, and is independent of NLRC4, caspase-1, caspase-11 and NOX2. This study highlights the differential utilization of innate effector pathways to curtail intracellular bacterial replication in specific host cells upon <i>L</i>. <i>pneumophila</i> airway infection.</p></div

TNF mediates an antibacterial effect in macrophages via TNFR1, which is independent of NLRC4 and ROS.

<p><b>(A-B)</b> WT or knockout BMDM were infected with WT <i>L</i>. <i>pneumophila</i> at MOI 0.1. BMDM were either left untreated (left hand panels) or rTNF was added at the time of infection (right hand panels). 3 days p.i. BMDM were lysed and CFU were quantified on CYE agar plates. <b>(C)</b> WT BMDM were infected with WT <i>L</i>. <i>pneumophila</i> MOI 0.1, with or without the addition of TNFR1-Fc, anti-TNF Ab or anti-IL1β Ab. 3 days p.i. BMDM were lysed and CFU were quantified on CYE agar plates. Data are from 3–7 pooled experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to WT by Kruskal-Wallis test with Dunn's post test.</p

Entry of other Ebola viruses is CatB-independent.

<p>Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 µM; CatL inhibitor V 10, 5, 2.5, 1 µM) was added. After fixation on day 4, foci were stained with antibodies against VP40 (BEBOV = <i>Bundibugyo ebolavirus</i>, CIEBOV = <i>Côte d'Ivoire ebolavirus</i>, SEBOV = <i>Sudan ebolavirus</i>, strain Boniface) or NP (REBOV = <i>Reston ebolavirus</i>, strain Pennsylvania) and counted. The number of foci without inhibitor was set as 100%. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean.</p

TNF-mediated killing of <i>L</i>. <i>pneumophila</i> is associated with the fusion of LCVs with lysosomal compartments in macrophages.

<p><b>(A)</b> MN-TNF NAIP5^129S1 BMDM were pre-treated overnight with rTNF (rTNFѱ), rIFNγ (rIFNγѱ), or were left untreated, and then infected with <i>Lpn</i>-GFP or ΔT-GFP at MOI 5 with simultaneous addition of rTNF or rIFNγ where indicated. 1 hr or 3 hr p.i. co-localization of <i>Lpn</i>-GFP with lysosomes (stained with lysotracker Red) was analyzed via confocal microscopy, and at least 100 bacteria were counted per group. BMDM cell membranes were stained with Cholera toxin B AF647 (cy5). Data are representative of 2 experiments. <b>(B)</b> WT or TNFR1^-/- BMDM were pre-treated overnight with rTNF (rTNFѱ) or were left untreated, and then infected with ΔFlaA <i>Lpn</i>-GFP or ΔT-GFP at MOI 5 with simultaneous addition of rTNF where indicated. 1 hr p.i. co-localization of <i>Lpn</i>-GFP with lysosomes (stained with lysotracker Red) was analyzed via confocal microscopy as in A) and at least 100 bacteria were counted per group. Data is from 1 experiment.</p

<i>CatB^−/−</i> and <i>catL^−/−</i> mice succumb to Ebola virus but not to VSV infection.

<p>Groups of mice were i.p. infected with 10 ffu MA-ZEBOV (1,000 LD₅₀) or 1×10⁵ pfu VSV (serotype Indiana) and monitored daily for weight loss and other signs of illness. Survival (<b>A</b>) and weight curves (<b>B</b>) for MA-ZEBOV infection are shown. Body weights of VSV-infected mice are shown in (<b>C</b>). VSV antibodies were detected using ELISA to confirm infection (<b>D</b>).</p

TNF / TNFR1 signaling contributes to AM-mediated killing of <i>L</i>. <i>pneumophila</i>, while ROS are required for efficient neutrophil-mediated killing <i>in vivo</i>.

<p><b>(A-C)</b> Mixed BM chimeric mice reconstituted with 50% Ly5.1⁺ WT BM, and either 50% Ly5.2⁺ WT, TNFR1^-/- or CYBB^-/- BM were generated. <b>(A)</b> Chimeras were infected with WT <i>L</i>. <i>pneumophila</i>, and 2 days p.i. BALF was harvested and Ly5.1⁺ and Ly5.2⁺ AM and neutrophils were sorted. Cells were lysed and CFU were quantified on CYE agar plates. <b>(B)</b> Chimeras were infected with WT or ΔFlaA <i>L</i>. <i>pneumophila</i>, and CFU were quantified in AM as in A). <b>(C)</b> Chimeras were infected with <i>Lpn</i>-GFP or <i>Lpn</i>-GFPind (with IPTG induction) and BALF was analyzed by flow cytometry 38 hr p.i.. GFP⁺ neutrophils were normalized for the number of Ly5.1⁺ and Ly5.2⁺ neutrophils, respectively. Data are from 2–4 pooled experiments. *p<0.05, **p<0.01, ***p<0.001 by Wilcoxon test.</p

TNF / TNFR1 and ROS are important for clearance of <i>L</i>. <i>pneumophila in vivo</i>.

<p><b>(A-C)</b> WT or knockout mice were infected intranasally with WT <i>L</i>. <i>pneumophila</i>, and 5 days p.i. (A) or 3–7 days p.i. (C) BALF CFU were quantified on CYE agar plates. Data in panel A, B and C are from 15, 8, and 2 pooled experiments, respectively. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to WT by Kruskal-Wallis test with Dunn's post test.</p

<i>Zaire ebolavirus</i> growth is CatB and CatL-independent.

<p>(<b>A</b>) Vero E6 cells were seeded the night before infection in a 24-well-plate. One hour prior to infection cells were incubated with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (Baf A1). ZEBOVwt was added at an MOI of 1 and incubated for one hour at 37°C. After three washes the cells were covered with 1 ml medium containing 50 nM BafA1, 50 µM CA-074, 5 µM CatL inhibitor V or no inhibitor and incubated for 4 days. (B) and (C) MEF cell lines were seeded the night before infection in a 24-well-plate. Cells were infected for 1 hour with 0.2 ml ZEBOVwt (B) or MA-ZEBOV (C) at a MOI of 1. After three washes the cells were covered with 1 ml medium and incubated for 4 days. For all experiments, samples were collected at 0, 12, 24, 48, 72 and 96 hours post infection and infectious titers were determined. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean.</p

NAIP5^129S1 and TNF-deficiency in macrophages, monocytes and neutrophils are the genetic traits that render MN-TNF NAIP5^129S1 mice susceptible to <i>L</i>. <i>pneumophila</i> infection <i>in vitro</i> and <i>in vivo</i>.

<p><b>(A-B)</b> WT, TNF^-/-, MN-TNF NAIP5^129S1 mice were infected intranasally with WT <i>L</i>. <i>pneumophila</i>, and analyzed at the indicated time points. <b>(A)</b> TNF was quantified in the BALF via CBA assay. <b>(B)</b> BALF CFU were quantified on CYE agar plates. <b>(C)</b> WT or MN-TNF NAIP5^129S1 BMDM, or BMDM from the F2 offspring of MN-TNF NAIP5^129S1 x C57BL/6 intercrosses were infected with WT <i>L</i>. <i>pneumophila</i> at MOI 0.1. 3 days p.i. BMDM were lysed and CFU were quantified on CYE agar plates. <b>(D)</b> WT, TNF^-/-, MN-TNF NAIP5^129S1 mice, or the F2 offspring of MN-TNF NAIP5^129S1 x C57BL/6 intercrosses were infected intranasally with WT <i>L</i>. <i>pneumophila</i>, and 5 days p.i. BALF CFU were quantified on CYE agar plates. All of the F2 offspring shown are Cre⁺. Panel A is from 1 experiment, panels B-D are from 2–3 pooled experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to WT by Kruskal-Wallis test with Dunn's post test.</p

<i>Zaire ebolavirus</i> replicates to similar titers in knockout and control mice.

<p><i>CatB^−/−</i>, <i>catL^−/−</i> or control mice (n = 3) were i.p. infected with 1,000 LD₅₀ of MA-ZEBOV and euthanized at the indicated time point. Liver, spleen and blood samples were taken on day 3 (<b>A</b>) and day 7 (<b>B</b>) and viral titers were determined. A single 50% tissue culture infectious dose (TCID₅₀) value is depicted for each mouse. Bars indicate the mean value.</p