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

Abstract

Introduction: 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.

Keywords: Influenza virus; cytokines; flow cytometry; immune response; live lung slice; two-photon excitation microscopy.

Figure 1

Flow cytometry data showing quantitative data and the evolution of innate immune cell recruitment in whole-lung tissues of 10 mice infected on D0 with influenza A (PR8 strain) versus that of two non-infected control animal(s) (mock-inf = mock-infected). Four types of cells were measured by flow cytometry: repartition in F4/80+ cells between CD11c+ (yellow bar) and CDIl c- (blue bar), *p<0.05.

Figure 2

Analyses of the concentrations (pg/mL) of nine proinflammatory cytokines and chemokines in whole-lung samples from n=10 mice experimentally infected at day 0 with influenza A (PR8) versus a/n=2 control animal(s) that did not receive any infection over a 4-days experiment. Cytokines: GM-CSF, IL-1a. Chemokines: CCL2, CCL12, CCL3 and CCL5. mock-infected = blue bar; Infected with PR8= red bar- *:p<0.05 (vs mock-infected).

Figure 3

Two-photon excitation micrographs showing the pulmonary bronchial epithelium three days after experimental infection with a modified influenza A/Puerto Rico/8/34/H1N1 strain that expresses red fluorescent protein. The replicating virus is shown in red and the epithelial cells in green. Image showing the normal bronchiolar epithelium of mock-infected mice and a zoom of normal epithelium versus a zoom of infected bronchiolar epithelium (A). Connective tissue (fiber in blue), macrophages (round cells in blue), CD11C⁺ cells (yellow). Recruitment to the mouse lung over four days following infection with a modified influenza A/Puerto Rico/8/34/H1N1 strain expressing red fluorescent protein. On D1 post-infection, resident macrophages can be seen (shown in blue) in the lung parenchyma. On D2, the macrophages have not yet moved into the perivascular areas. By D3, they can be seen in the perivascular area near the infected (red) bronchial epithelium. Finally, on D4, the macrophages have become homogeneously and diffusely distributed throughout the whole lung section. CD11c⁺ cells (yellow), bronchial and alveolar epithelium (green) (B).

Figure 4

Continuous successive images acquired by TPEM ex vivo of a mouse lung at D1 post-infection. Inter-alveolar motility of two round cells (macrophages), one macroscopically CD11c⁺ (image 4), the other CD11c^low (image 4), interacting with each other via direct contact.

Figure 5

Summary diagram of the pulmonary consequences after infection with Influenzavirus in a mouse model by compiling data from flow cytometry, cytokine assays, and TPEM on fixed and ex vivo lungs.