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Summary: Phagocytosis, macropinocytosis and antigen presentation by dendritic cells (DC) requires reorganization of the actin cytoskeleton. Drebrin (Dbn1) is an actin binding and stabilizing protein with roles in endocytosis, formation of dendrite spines in neurons and coordinating cell–cell synapses in immune cells. However, its role in DC phagocytosis and antigen presentation is unknown. These studies now report that silencing of Dbn1 in DC resulted in restrained cell surface display of receptors, most notably MHC class I and II and co‐stimulatory molecules. This, as expected, resulted in impaired antigen‐specific T‐cell activation and proliferation. Studies additionally revealed that knockdown of Dbn1 in DC impaired macropinocytosis and phagocytosis. However, there was a concomitant increase in fluid‐phase uptake, suggesting that Dbn1 is responsible for the differential control of macropinocytosis versus micropinocytosis activities. Taken together, these findings now reveal that Dbn1 plays a major role in coordinating the actin cytoskeletal activities responsible for antigen presentation in DC. These studies now reveal that drebrin 1 (Dbn1) plays a major role in coordinating actin cytoskeletal activities in dendritic cells (DC) responsible for differential regulation of endocytosis. Silencing of Dbn1 in DC resulted in depressed cell surface receptor recycling and phagocytosis, coupled with increased micropinocytosis/fluid‐phase uptake. [ABSTRACT FROM AUTHOR]

Allograft Inflammatory Factor‐1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF‐hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage‐ and dendritic cell (DC)‐mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen‐specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL‐10 producing CD8+ CD122+ PD‐1+ regulatory T cells that suppressed neighboring immune effector responses through both IL‐10 and PD‐1‐dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen‐responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC‐based immunotherapies. Adaptive immune responses are impaired in CD8+ T cells primed by DC silenced for AIF1. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a significant obstacle to the treatment of bacterial infections, including in the context of Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). Lysogenic bacteriophages can integrate their genome into the bacterial chromosome and are known to promote genetic transfer between bacterial strains. However, the contribution of lysogenic phages to the incidence of AMR is poorly understood. Here, in a set of 187 clinical isolates of Pseudomonas aeruginosa collected from 82 patients with CF, we evaluate the links between prophages and both genomic and phenotypic resistance to five anti-pseudomonal antibiotics: tobramycin, colistin, ciprofloxacin, meropenem, aztreonam, and tazobactam. We find that P. aeruginosa isolates contain on average 3.06 +/-1.84 (SD) predicted prophages. We find no significant association between the number of prophages per isolate and the mean inhibitory concentration (MIC) for any of these antibiotics. We then investigate the relationship between particular prophages and AMR. We identify a single lysogenic phage that is associated with phenotypic resistance to the antibiotic tobramycin. Consistent with this association, we identify AMR genes associated with resistance to tobramycin in these strains and find that they are not encoded directly on prophage sequences. These findings suggest that prophages are infrequently associated with the AMR genes in clinical isolates of P. aeruginosa .

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings., Competing Interests: Competing Interest Statement: AO-B, JDB, RM are members of Felix Biotechnology, Inc., a phage therapy company. All other authors declare they have no competing interests.

Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% ( n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% ( n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca 2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8 + T cell effector responses. Silencing AIF1 expression in murine CD11c + DC suppressed antigen-specific CD8 + T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8 + T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8 + CD122 + PD-1 + regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4 + T cells by the CD8 + Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies., (©2018 Society for Leukocyte Biology.)