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

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

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

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

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

Unimpaired hematopoiesis in <i>Trem1^−/−</i> mice.

<p>(A) Representative dot plots show the FACS-based identification of lineage-depleted (lin⁻) Sca1⁺ c-kit^hi (LSK) cells and lin⁻ Sca1⁻ c-Kit^hi myeloid progenitors in <i>Trem1^+/+</i> (top panels) and <i>Trem1^−/−</i> (bottom panels) bone marrow (BM) following lineage depletion and depletion of lymphoid progenitors by MACS. Common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and megakaryocyte/erythrocyte progenitors (MEP) were further discriminated according to their expression of FcγR and CD34. Filled histograms show TREM-1 surface expression by LSK cells, CMP, GMP and MEP progenitors from <i>Trem1^−/−</i> mice in comparison to <i>Trem1^+/+</i> mice (lines). (B) Absolute cell numbers of total BM cells, lin⁻ BM cells, lin⁻ Sca1⁻ c-kit^hi myeloid progenitors, LSK cells, CMP, GMP and MEP and colony forming units (CFU) of hematopoietic precursors isolated from the BM of <i>Trem1^+/+</i> and <i>Trem1^−/−</i> mice were determined as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003900#s4" target="_blank">Materials and Methods</a> section. Mean values of n = 2 mice analysed are shown with error bars indicating the range. (C) Mixed BM chimeras were generated by i.v. transfer of 1∶1 mixed <i>Trem1^+/+ x GFP^+/+</i> and <i>Trem1^−/− x GFP^−/−</i> BM cells (white circles, dotted lines) into irradiated recipient mice. As control, and to account for potential interfering effects of the GFP expression, mixed BM from <i>Trem1^+/+ x GFP^+/+</i> and <i>Trem1^+/+ x GFP^−/−</i> mice (black circles and lines) was transferred into additional recipient mice. BM chimeras were analyzed after 10 and 31 weeks of chimerism. Neutrophils, Ly6C^hi and Ly6C^lo monocytes were identified in the peripheral blood according to the depicted gating strategy and the GFP⁻ : GFP⁺ cell ratio in the respective cell subsets was determined. Mean values of n = 4–5 mice analyzed per group are shown with error bars indicating the SEM. ns, no statistically significant difference. Data depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003900#ppat-1003900-g002" target="_blank">Figure 2</a> are representative of two independent experiments.</p

<i>Trem1^−/−</i> mice develop smaller inflammatory lesions and show decreased cellular infiltrates at <i>L. major</i> infection sites.

<p>(A) <i>Trem1^+/+</i> and <i>Trem1^−/−</i> mice were inoculated with 3×10⁶<i>L. major</i> promastigotes s.c. in the footpad and lesion development was measured over time. Each data point represents the mean lesion size ± SEM of n = 5 mice analysed per group. (B) Parasite load was assessed at 35 days post infection (p.i.) by limiting dilution analysis. (C) Infected footpads from <i>Trem1^+/+</i> and <i>Trem1^−/−</i> mice (n = 4–5 mice per group) were isolated 21 days p.i., digested and the cellular content was analysed by flow-cytometry. Data show mean values ± SEM and are representative of two independent experiments. (D) Draining lymph node cells from <i>Trem1^+/+</i> and <i>Trem1^−/−</i> mice (n = 4 mice per group) were isolated 35 days p.i.; the frequency of CD4⁺ IFNγ⁺ T cells was analysed by intracellular FACS staining or cells were re-stimulated with UV-treated <i>L. major</i> parasites and IFNγ levels in the supernatants were assessed by ELISA. Data show mean values ± SEM of triplicate measurements. Representative data from one out of three independent experiments are shown. ***, p<0.001; **, p<0.01; *, p<0.05.</p

<i>Trem1^−/− x Rag2^−/−</i> mice are protected from a CD4⁺ T cell-induced colitis.

<p>Colitis was induced in <i>Trem1^+/+ x Rag2^−/−</i> (filled circles) and <i>Trem1^−/− x Rag2^−/−</i> mice (white circles) by i.p. injection of 2×10⁵ CD4⁺ CD45RB^hi T cells. (A) Weight loss relative to the initial body weight. Mean values of n = 9 mice analysed per group are shown with error bars indicating the SEM. (B) Colon lengths were determined in individual mice (symbols). Lines show mean values for each group of mice. (C) Representative H&E-stained colonic tissue sections of a <i>Trem1^+/+ x Rag2^−/−</i> (histopathological score: 14) and <i>Trem1^−/− x Rag2^−/−</i> mouse (histopathological score: 2). (D) Total histopathological scores. Symbols show total scores for individual mice and lines indicate the mean value for each group of mice. Histopathological scores were determined for individual mice by a pathologist according to parameters defined in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003900#s4" target="_blank">Materials and Methods</a> section. (E) Individual parameters of histopathological scoring. Columns show mean values for n = 9 mice analysed per group and error bars indicate the SEM. ****, p<0.0001; ***, p<0.001; **, p<0.01. One representative experiment out of three independent experiments is shown.</p

Attenuated dextran sodium-sulfate (DSS)-induced colitis in <i>Trem1^−/−</i> mice.

<p>Colitis was induced in <i>Trem1^+/+</i> and <i>Trem1^−/−</i> mice by administration of 3% DSS in the drinking water for 5 days, followed by 4 days on regular tap water. (A) Weight loss relative to the initial body weight. Mean values of n = 17 (<i>Trem1^+/+</i>) and n = 16 (<i>Trem1^−/−</i>) mice are shown with error bars indicating the SEM. (B) Colon lengths were determined in individual DSS-treated and untreated control mice (symbols). Lines show mean values for each group of mice. (C) Representative H&E-stained colonic tissue sections of a <i>Trem1^+/+</i> (histopathological score: 13) and <i>Trem1^−/−</i> mouse (histopathological score: 5.5). (D) Total histopathological scores. Symbols show total scores for individual mice and lines indicate the mean value for each group of mice. Histopathological scores were determined for individual mice by a pathologist according to parameters defined in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003900#s4" target="_blank">Materials and Methods</a> section. (E) Individual parameters of histopathological scoring. Columns show mean values and error bars indicate the SEM. (F) Colonic tissues were assessed for the expression of pro-inflammatory mediators by qRT-PCR. Bars show mean values ± SEM for n = 7 mice per group from one independent experiment. (A, B, D) Pooled data from three independent experiments are shown. ****, p<0.001; ***, p<0.001; *, p<0.05.</p