Infection with Influenzavirus A in a murine model induces epithelial bronchial lesions and distinct waves of innate immune-cell recruitment

IntroductionInflammatory lesions after Influenza A viruses (IAV) are potential therapeutic target for which better understanding of post-infection immune mechanisms is required. Most studies to evaluate innate immune reactions induced by IAV are based on quantitative/functional methods and anatomical exploration is most often non-existent. We aimed to study pulmonary damage and macrophage recruitment using two-photon excitation microscopy (TPEM) after IAV infection.MethodsWe infected C57BL/6 CD11c+YFP mice with A/Puerto Ricco/8/34 H1N1. We performed immune cell analysis, including flow cytometry, cytokine concentration assays, and TPEM observations after staining with anti-F4/80 antibody coupled to BV421. We adapted live lung slice (LLS) method for ex-vivo intravital microscopy to analyze cell motility.ResultsTPEM provided complementary data to flow cytometry and cytokine assays by allowing observation of bronchial epithelium lesions and spreading of local infection. Addition of F4/80-BV421 staining allowed us to precisely determine timing of recruitment and pulmonary migration of macrophages. Ex-vivo LLS preserved cellular viability, allowing us to observe acceleration of macrophage motility.ConclusionAfter IAV infection, we were able to explore structural consequences and successive waves of innate immune cell recruitment. By combining microscopy, flow cytometry and chemokine measurements, we describe novel and precise scenario of innate immune response against IAV

Early Circulating Edema Factor in Inhalational Anthrax Infection: Does It Matter?

Anthrax toxins are critical virulence factors of Bacillus anthracis and Bacillus cereus strains that cause anthrax-like disease, composed of a common binding factor, the protective antigen (PA), and two enzymatic proteins, lethal factor (LF) and edema factor (EF). While PA is required for endocytosis and activity of EF and LF, several studies showed that these enzymatic factors disseminate within the body in the absence of PA after intranasal infection. In an effort to understand the impact of EF in the absence of PA, we used a fluorescent EF chimera to facilitate the study of endocytosis in different cell lines. Unexpectedly, EF was found inside cells in the absence of PA and showed a pole-dependent endocytosis. However, looking at enzymatic activity, PA was still required for EF to induce an increase in intracellular cAMP levels. Interestingly, the sequential delivery of EF and then PA rescued the rise in cAMP levels, indicating that PA and EF may functionally associate during intracellular trafficking, as well as it did at the cell surface. Our data shed new light on EF trafficking and the potential location of PA and EF association for optimal cytosolic delivery

Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination

International audienceBackground: Through incorporation into virus particles, the HIV‑1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhance‑ ment of virus infectivity in established CD4‑positive cell lines via a nonenzymatic mechanism. Results: We report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV‑1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte‑derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2‑ and RPA32‑depleted T lymphocytes. Reciprocally, viruses produced in UNG2‑depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination. Conclusions: Our data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV‑1

Infection with Influenzavirus A in a murine model induces epithelial bronchial lesions and distinct waves of innate immune-cell recruitment

International audienceIntroduction Inflammatory lesions after Influenza A viruses (IAV) are potential therapeutic target for which better understanding of post-infection immune mechanisms is required. Most studies to evaluate innate immune reactions induced by IAV are based on quantitative/functional methods and anatomical exploration is most often non-existent. We aimed to study pulmonary damage and macrophage recruitment using two-photon excitation microscopy (TPEM) after IAV infection. Methods We infected C57BL/6 CD11c + YFP mice with A/Puerto Ricco/8/34 H1N1. We performed immune cell analysis, including flow cytometry, cytokine concentration assays, and TPEM observations after staining with anti-F4/80 antibody coupled to BV421. We adapted live lung slice (LLS) method for ex-vivo intravital microscopy to analyze cell motility. Results TPEM provided complementary data to flow cytometry and cytokine assays by allowing observation of bronchial epithelium lesions and spreading of local infection. Addition of F4/80-BV421 staining allowed us to precisely determine timing of recruitment and pulmonary migration of macrophages. Ex-vivo LLS preserved cellular viability, allowing us to observe acceleration of macrophage motility. Conclusion After IAV infection, we were able to explore structural consequences and successive waves of innate immune cell recruitment. By combining microscopy, flow cytometry and chemokine measurements, we describe novel and precise scenario of innate immune response against IAV

Biodiversité, évolution et fonctionnement des écosystèmes: Prospectives de l’Institut Ecologie et Environnement du CNRS,

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