Neutrophils are potent immune cells with key antimicrobial functions. Previous in vitro work has shown that neutrophil effector functions are mainly fueled by intracellular glycolysis. Little is known about the state of neutrophils still in the circulation in patients during infection. Here, we combined flow cytometry, stimulation assays, transcriptomics, and metabolomics to investigate the link between inflammatory and metabolic pathways in blood neutrophils of patients with community-acquired pneumonia. Patients' neutrophils, relative to neutrophils from age- and sex- matched controls, showed increased degranulation upon ex vivo stimulation, and portrayed distinct upregulation of inflammatory transcriptional programs. This neutrophil phenotype was accompanied by a high-energy state with increased intracellular ATP content, and transcriptomic and metabolic upregulation of glycolysis and glycogenolysis. One month after hospital admission, these metabolic and transcriptomic changes were largely normalized. These data elucidate the molecular programs that underpin a balanced, yet primed state of blood neutrophils during pneumonia.
© 2023 The Authors.

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

In this study, we established a dynamic micromodel of urinary tract infection to analyze the impact of UT-segment-specific urinary outflow on the persistence of E. coli colonization. We found that the adherence of Dr+ E. coli to bladder T24 transitional cells and type IV collagen is maximal at lowest shear stress and is reduced by any increase in flow velocity. The analyzed adherence was effective in the whole spectrum of physiological shear stress and was almost irreversible over the entire range of generated shear force. Once Dr+ E. coli bound to host cells or collagen, they did not detach even in the presence of elevated shear stress or of chloramphenicol, a competitive inhibitor of binding. Investigating the role of epithelial surface architecture, we showed that the presence of budding cells-a model microarchitectural obstacle-promotes colonization of the urinary tract by E. coli. We report a previously undescribed phenomenon of epithelial cell "rolling-shedding" colonization, in which the detached epithelial cells reattach to the underlying cell line through a layer of adherent Dr+ E. coli. This rolling-shedding colonization progressed continuously due to "refilling" induced by the flow-perturbing obstacle. The shear stress of fluid containing free-floating bacteria fueled the rolling, while providing an uninterrupted supply of new bacteria to be trapped by the rolling cell. The progressive rolling allows for transfer of briefly attached bacteria onto the underlying monolayer in a repeating cascading event.

Monocytes and macrophages are key drivers in the pathogenesis of inflammatory diseases. Epigenetic targets have been shown to control the transcriptional profile and phenotype of these cells. Since histone deacetylase protein inhibitors demonstrate profound anti-inflammatory activity, we wanted to test whether HDAC inhibition within monocytes and macrophages could be applied to suppress inflammation in vivo. ESM technology conjugates an esterase-sensitive motif (ESM) onto small molecules to allow targeting of cells that express carboxylesterase 1 (CES1), such as mononuclear myeloid cells. This study utilized an ESM-HDAC inhibitor to target monocytes and macrophages in mice in both an acute response model and an atherosclerosis model. We demonstrate that the molecule blocks the maturation of peritoneal macrophages and inhibits pro-inflammatory cytokine production in both models but to a lesser extent in the atherosclerosis model. Despite regulating the inflammatory response, ESM-HDAC528 did not significantly affect plaque size or phenotype, although histological classification of the plaques demonstrated a significant shift to a less severe phenotype. We hereby show that HDAC inhibition in myeloid cells impairs the maturation and activation of peritoneal macrophages but shows limited efficacy in a model of atherosclerosis.
Copyright © 2019 Luque-Martin, Van den Bossche, Furze, Neele, van der Velden, Gijbels, van Roomen, Bernard, de Jonge, Rioja, Prinjha, Lewis, Mander and de Winther.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder. It is characterized by the presence of the Philadelphia (Ph) chromosome, t(9;22)(q34.1;q11.2), which carries the BCR-ABL1 fusion gene. Tyrosine kinase inhibitors (TKIs) have markedly changed the treatment approach of CML and have become the first-line agents for almost all CML patients. However, certain patients experience resistance to these medications, which occurs through several mechanisms, including the accumulation of TKI-resistant chromosomal abnormalities. The present study reports a case of a 27-year-old Saudi male with CML receiving TKI treatment, who presented with precursor B-cell lymphoblastic crisis demonstrating the presence of the novel combined chromosomal abnormalities; non-Ph der(22), i(9) and der(20), carrying the BCR-ABL1 fusion gene. This case report adds to the literature on novel TKI-resistance-conferring chromosomal abnormalities and links them to precursor B-cell lymphoblastic crisis.