Key mechanisms governing resolution of lung inflammation

Innate immunity normally provides excellent defence against invading microorganisms. Acute inflammation is a form of innate immune defence and represents one of the primary responses to injury, infection and irritation, largely mediated by granulocyte effector cells such as neutrophils and eosinophils. Failure to remove an inflammatory stimulus (often resulting in failed resolution of inflammation) can lead to chronic inflammation resulting in tissue injury caused by high numbers of infiltrating activated granulocytes. Successful resolution of inflammation is dependent upon the removal of these cells. Under normal physiological conditions, apoptosis (programmed cell death) precedes phagocytic recognition and clearance of these cells by, for example, macrophages, dendritic and epithelial cells (a process known as efferocytosis). Inflammation contributes to immune defence within the respiratory mucosa (responsible for gas exchange) because lung epithelia are continuously exposed to a multiplicity of airborne pathogens, allergens and foreign particles. Failure to resolve inflammation within the respiratory mucosa is a major contributor of numerous lung diseases. This review will summarise the major mechanisms regulating lung inflammation, including key cellular interplays such as apoptotic cell clearance by alveolar macrophages and macrophage/neutrophil/epithelial cell interactions. The different acute and chronic inflammatory disease states caused by dysregulated/impaired resolution of lung inflammation will be discussed. Furthermore, the resolution of lung inflammation during neutrophil/eosinophil-dominant lung injury or enhanced resolution driven via pharmacological manipulation will also be considered

Noncovalent Inhibitors of Mosquito Acetylcholinesterase 1 with Resistance Breaking Potency

Resistance development in insects significantly threatens the important benefits obtained by insecticide usage in vector control of disease-transmitting insects. Discovery of new chemical entities with insecticidal activity is highly desired in order to develop new insecticide candidates. Here, we present the design, synthesis, and biological evaluation of phenoxyacetamide-based inhibitors of the essential enzyme acetylcholinesterase 1 (AChE1). AChE1 is a validated insecticide target to control mosquito vectors of, e.g., malaria, dengue, and Zika virus infections. The inhibitors combine a mosquito versus human AChE selectivity with a high potency also for the resistance-conferring mutation G122S; two properties that have proven challenging to combine in a single compound. Structure–activity relationship analyses and molecular dynamics simulations of inhibitor–protein complexes have provided insights that elucidate the molecular basis for these properties. We also show that the inhibitors demonstrate in vivo insecticidal activity on disease-transmitting mosquitoes. Our findings support the concept of noncovalent, selective, and resistance-breaking inhibitors of AChE1 as a promising approach for future insecticide development

Toxin-Induced Necroptosis Is a Major Mechanism of <i>Staphylococcus aureus</i> Lung Damage

<div><p><i>Staphylococcus aureus</i> USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of <i>S</i>. <i>aureus</i> toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and <i>S</i>. <i>aureus</i> mutants lacking <i>agr</i>, <i>hla</i> or Hla pore formation, <i>lukAB</i> or <i>psms</i> were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1β production, suggesting a link to the inflammasome. <i>Rip3</i><i>^-/-</i> mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1β in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in <i>S</i>. <i>aureus</i> pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.</p></div

Macrophage expansion is limited during SA pneumonia.

<p>(<b>A-E</b>) Mice were infected intranasally with 10⁷ CFU/mouse MRSA USA300 and their lung homogenate analyzed by FACS for immune cell populations at 4 and 24 hours as compared to uninfected controls (UN) (n = 9 for UN, n = 9 for 4 h and n = 20 for 24 h for SA groups). (<b>A, B</b>) PMNs in BAL and lung quantified. (<b>C</b>) Percentages of immune cell populations at 4 and 24 hours as compared to uninfected controls (UN) were determined. (<b>D</b>) Natural killer cells (NKs) in BAL. (<b>E</b>) Macrophages in BAL and lung. (<b>F-I</b>) <i>Rip3-/-</i> and WT mice infected with SA for 18 hours (n = 3 for PBS and n = 8 per SA group). (<b>F, G</b>) Macrophages in BAL and lung of <i>Rip3-/-</i> and WT mice. (<b>H</b>) PMNs in BAL of <i>Rip3-/-</i> and WT mice. (<b>I</b>) Propidium iodide positive (PI⁺) macrophages in the BAL of WT and <i>Rip3-/-</i> mice. Data are pooled from three independent experiments. Each point in represents a mouse. Lines show median values. <i>p</i> values are indicated for significantly different comparisons (nonparametric Mann-Whitney test).</p

Blockade of necroptosis improves bacterial clearance.

<p>(<b>A</b>) C57BL/6J mice were treated with necrostatin-1 stable (Nec-1s) or DMSO and infected with MRSA USA300 (SA). CFU recovered in BAL and lung were quantified and normalized to DMSO controls (n = 2 for PBS and n = 10 per SA group). (<b>B</b>) <i>Cxcl1/Kc</i>, <i>Il-6</i> and <i>Tnf</i> expression in the lung homogenate quantified by real time PCR compared to control (ctrl). (<b>C,D</b>) Neutrophils (PMNs) in BAL and macrophages (Macs) in BAL and lung. (<b>E</b>) <i>Rip3-/-</i> or WT mice were infected with MRSA USA300 (SA) for 18 hours and CFU recovered in BAL and lung quantified (n = 3 for PBS and n = 8 per SA group). (<b>F</b>) Protein in the BAL fluid of WT and <i>Rip3-/-</i> mice. (<b>G</b>) Hematoxylin and eosin stain (H&E) staining of WT and <i>Rip3-/-</i> mice lungs (magnification of 100x; insert, magnification of 400x). (<b>H-L</b>) CXCL1/KC, IL-6, TNF, IL-1β and IL-1α levels in the BAL fluid measured by ELISA. (<b>M, N</b>) <i>Rip3-/-</i> or WT mice were infected with <i>agr</i> null or MRSA USA300 (USA300) for 18 hours and CFU recovered in BAL and lung quantified (n = 6 per group).Gating strategies for immune cells are in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004820#ppat.1004820.s002" target="_blank">S2E Fig</a>.</b> Data are pooled from two independent experiments. Each point represents a mouse. Lines show median values. <i>p</i> values were determined by nonparametric Mann-Whitney test.</p

Depletion of pulmonary macrophages contributes to poor outcome.

<p>(<b>A</b>) C57BL/6J mice were treated with clodronate- or PBS-loaded liposomes for 24 hours. Mice were infected intranasally with SA and numbers of macrophages in BAL and lung quantified 24 hours post-infection by flow cytometry (n = 4 for PBS groups, n = 10 for SA groups). (<b>B</b>) Numbers of SA (CFU) recovered from BAL and lung were quantified at 4 and 24 hours post-infection. (<b>C, D</b>) PMNs in BAL and lung 24 hours post-infection. (<b>E</b>) Ratios of CFU to PMNs in BAL and lung. (<b>F</b>) DCs in BAL and lung 24 post-infection. (<b>G</b>) NKs in BAL and lung 4 hours and 24 hours post-infection. (<b>H</b>) Protein in the BAL fluid 24 hours post-infection. (<b>I</b>) Hematoxylin and eosin stain (H&E) staining of mouse lung (magnification 100x; insert, magnification 400x). FACS blots showing depleted macrophages and unchanged PMNs after clodronate treatment are in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004820#ppat.1004820.s004" target="_blank">S4D Fig</a>.</b> Data are pooled from three independent experiments. Each point in represents a mouse. Lines show median values. <i>p</i> values obtained by nonparametric Mann-Whitney test.</p

Pulmonary macrophages from <i>Rip3-/-</i> mice have an anti-inflammatory phenotype.

<p>(<b>A-C</b>) <i>Rip3-/-</i> or WT mice were infected with SA for 18 hours. (<b>A</b>) The absolute numbers of CD54⁺ macrophages in BAL and lung quantified (n = 3 for PBS, n = 8 for SA groups). (<b>B</b>) CD86⁺ macrophages in BAL and lung (n = 4 for PBS, n = 6 for WT with SA, n = 7 for <i>Rip3-/-</i> with SA). (<b>C</b>) CD206⁺ macrophages in BAL and lung (n = 3 for PBS, n = 7 for WT with SA, n = 8 for <i>Rip3-/-</i> with SA). (<b>D</b>) CD200 receptor positive (CD200R⁺) macrophages in BAL and lung (n = 3 for PBS, n = 7 for WT with SA, n = 8 for <i>Rip3-/-</i> with SA). Gating strategies for macrophage markers are in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004820#ppat.1004820.s003" target="_blank">S3E Fig</a>.</b> Data are pooled from two independent experiments. Each point represents a mouse. Lines show median values. <i>p</i> values were determined by nonparametric Mann-Whitney test.</p