Liquid–liquid phase separation of the Golgi matrix protein GM130

Golgins are an abundant class of peripheral membrane proteins of the Golgi. These very long (50–400 nm) rod-like proteins initially capture cognate transport vesicles, thus enabling subsequent SNARE-mediated membrane fusion. Here, we explore the hypothesis that in addition to serving as vesicle tethers, Golgins may also possess the capacity to phase separate and, thereby, contribute to the internal organization of the Golgi. GM130 is the most abundant Golgin at the cis Golgi. Remarkably, overexpressed GM130 forms liquid droplets in cells analogous to those described for numerous intrinsically disordered proteins with low complexity sequences, even though GM130 is neither low in complexity nor intrinsically disordered. Virtually pure recombinant GM130 also phase-separates into dynamic, liquid-like droplets in close to physiological buffers and at concentrations similar to its estimated local concentration at the cis Golgi

<i>In vivo</i>, neutrophils but not AM produce ROS and TNF in response to <i>L</i>. <i>pneumophila</i> infection.

(A) WT and CYBB-/- mice were infected intranasally with WT, ΔT or ΔFlaA L. pneumophila and BALF cells were harvested 24 hr p.i.. AM, neutrophils and monocytes were stained for ROS with Dihydroethidium (DHE) and analyzed by flow cytometry. (B) WT and TNF-/- mice were infected intranasally with WT, ΔT or ΔFlaA L. pneumophila and BALF cells were harvested 30 hr p.i.. AM, neutrophils and monocytes were stained for TNF and analyzed by flow cytometry. Data are from 2–3 pooled experiments. *p-/- or TNF-/- mice by Kruskal-Wallis test with Dunn's post test.</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

TNF-mediated restriction of <i>L</i>. <i>pneumophila</i> growth in macrophages is enhanced by cysteine-type cathepsins or calpains.

(A, C, D) WT, CtsB-/-, CtsH-/-, CtsL-/- or CtsS-/- BMDM were either pre-treated with rTNF overnight or left untreated, and then infected with ΔFlaA L. pneumophila at MOI 0.1. Where indicated rTNF and/or E-64d and/or Q-VD-OPh were added at the time of infection. 3 days p.i. BMDM were lysed and CFU were quantified on CYE agar plates. (B) Caspase-1/11-/- BMDM were infected with WT L. pneumophila at MOI 0.1. Where indicated rTNF and/or cathepsin D inhibitor pepstatin A were added at the time of infection. 3 days p.i. BMDM were lysed and CFU were quantified on CYE agar plates. (A) Data are from 2 pooled experiments. *p<0.05 compared to WT+rTNF by Kruskal-Wallis test with Dunn's post test. (B) Data are from 2 pooled experiments. **p<0.01 by Mann-Whitney test. (C) Data are from 2 pooled experiments. *p<0.05 by Mann-Whitney test (left panel) and *p<0.05 compared to WT by Kruskal-Wallis test with Dunn's post test. (D) Data are from 1 experiment. **p<0.01 compared to WT+rTNF+Q-VD-OPh by Kruskal-Wallis test with Dunn's post test.</p

TNF-mediated killing of <i>L</i>. <i>pneumophila</i> is dependent of lysosomal acidification and caspases other than caspase-1 and 11 in macrophages.

(A) Caspase-1/11-/- BMDM were infected with WT L. pneumophila at MOI 0.1. Where indicated rTNF and/or v-ATPase inhibitor bafilomycin A1 were added at the time of infection. 3 days p.i. BMDM were lysed and CFU were quantified on CYE agar plates. Data are from 2 pooled experiments. **p(B) WT BMDM were either pre-treated with rTNF overnight or left untreated, and then infected with ΔFlaA L. pneumophila at MOI 0.1. Where indicated rTNF and/or pan caspase inhibitor Q-VD-OPh were added at the time of infection. 3 days p.i. BMDM were lysed and CFU were quantified on CYE agar plates. Data are from 2 pooled experiments. **p<0.01, ***p<0.001 compared to WT+rTNF by Kruskal-Wallis test with Dunn's post test.</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

Mice Lacking γδ T Cells Exhibit Impaired Clearance of Pseudomonas aeruginosa Lung Infection and Excessive Production of Inflammatory Cytokines

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic and life-threatening infections in immunocompromised patients. A better understanding of the role that innate immunity plays in the control of P. aeruginosa infection is crucial for therapeutic development. Specifically, the role of unconventional immune cells like γδ T cells in the clearance of P. aeruginosa lung infection is not yet well characterized. </jats: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