FiguresInfluenza in Phagocytic Cells: Direct Infection or Exogenous Antigen UptakeThomas Schmidt MHS, Jessica K. Fiege PhD, and Ryan A. Langlois PhD

University of Minnesota, Department of Microbiology and Immunology, Center for Immunology

AbstractInfluenza A virus (IAV), is an eight segmented, negative sense RNA virus.IAV is an extremely infectious virus that infects 9.2-35.6 million people inthe US each year1. Understanding the viral kinetics in the host is critical todeveloping an effective vaccination strategy. Phagocytic cells such asmacrophages and dendritic cells (DCs) are a vital link between the innateand adaptive immune systems, trafficking antigen from the site of infectionto the draining lymph node (dLN) and presenting antigen to T cells. Whilephagocytic cells have been reported to contain IAV antigen, it is unclear ifphagocytic cells obtain IAV antigen either through direct infection orphagocytosis. We developed an IAV expressing Cre recombinase(IAV_Cre) to specifically label infected cells in a Cre-inducible tdTomato(iRED) reporter mouse. Cre recombinase selectively removes a loxPflanked stop cassette and allows for the subsequent transcription of thefluorescent reporter tdTomato. This genetic alteration is irreversible andindelible, allowing us to track any cell that has ever been infected. Previousstudies have characterized tdTomato expression in epithelial cells in thelung after IAV infection2, while immune cells have not been studied in thissystem. While dendritic cells (DCs) and macrophages (MΦs) have beenreported to be infected by IAV, it is unclear in vivo how many of these cellsare actively infected, or have phagocytized IAV antigen. Previous studieshave shown a minority of CD45+ cells positive for IAV infection3, wehypothesize that the majority of reporter+ DCs and MΦs havephagocytozed antigen opposed to being directly infected. We sought to 1)identify what subsets of phagocytic cells are reporter+ at various time pointsafter IAV_Cre infection, 2) determine if phagocytic cells are directly infectedor take up exogenous tdTomato after IAV Cre infection of iRED mice. Todetermine the peak of tdTomato+ phagocytic cells, we harvested lungs,dLNs, and spleens on days 3, 5, 7, 10, and 21 post infection (dpi). Weobserved reporter+ phagocytic cells in the lung and dLNs at multiple timepoints after infection. Loss of reporter+ cells in the lung corresponded witha concordant gain of reporter+ phagocytic cells in the dLNs over the timecourse. To directly assess if phagocytic immune cells can obtain tdTomatovia phagocytosis, we used a bone marrow derived MΦs culture system. Wewere able to demonstrate that MΦs can successfully phagocytosetdTomato from IAV_Cre infected iRED fibroblasts. Using the bone marrowchimera mice, we were able to demonstrate MΦs and DCs canphagocytose tdTomato in vivo. These data demonstrate that phagocyticimmune cells can phagocytose IAV antigen and traffic to secondarylymphoid organs. These results have implications to IAV vaccinationdemonstrating that subunit vaccinations are a viable option for primingadaptive immune cells in secondary lymphoid organs.

Figure 3: Reporter+ cells are present in lung and draining lymph nodes

Acknowledgements

Future Directions• Replace WT bone marrow with iRED donor bone marrow to

determine the frequency of reporter+ phagocytic cells due to directIAV infection.

• Assess activation levels of CD80/86 in direct infection versusexogenous uptake of antigen

• In exogenous uptake of tdTomato, determine if changes in tdTomato+

populations are due to trafficking to and from tissues, degradation ofthe tdTomato+ fluorophore, or if phagocytic cells dying.

Lungs

Lymph nodes

SSC

-ASS

C-A

Conclusions• BMDM can phagocytose tdTomato fluorophore in vitro and in vivo• Reporter+ MΦs and DCs are at the greatest abundance at 5 days

post infection (dpi)• Reporter+ MΦs and DCs are barley detected past 10 dpi• Phagocytic immune cells can phagocytose IAV antigen and traffic to

secondary lymphoid organs. These results have implications to IAVvaccination demonstrating that subunit vaccinations are a viableoption for priming adaptive immune cells in secondary lymphoidorgans.

Figure 4: Phagocytic cells phagocytose tdTomato in vivo

A B

Stephanie AronTimmie BrittonElizabeth FayMatthew Markman

Yiping RenBarbara WaringLucy SjaastadIan Stone

Dan Mueller MDAndrea Stewart

Project Aims• Determine if MΦs phagocytose take up exogenous fluorophore.• Determine kinetics of reporter+ MΦs and DCs.• Determine if reporter+ MΦs and DCs are reporter+ due to direct

infection or exogenous uptake of fluorophore.

Figure 1: IAV_Cre permanently labels infected cells

Alveolar MΦs

PA_C

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CD11b+ DCs CD103+ cDCs

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Figure 3: Reporter+ cells are present in lungs and draining lymph nodes. A-C) Frequency of reporter+ cells from lungs (A) and draining lymph nodes (dLNs)(B) from naïve (blue), PA_Cre (red) or PR8 (green) mice. C) Representative flowcytometry plots from the lungs of PR8 (top) or PA_Cre (bottom) infected mice. D)Frequency of CD80/86+ cells in the lungs from naïve (blue), PR8 (purple),PA_Cre tdTomato+ (red), or PA_Cre tdTomato- (green).

Figure 4: MΦs and DCs phagocytose tdTomato in vivo. A)Experimental model: donor CD45.1 WT bone marrow is transplantedinto an irradiated CD45.2 iRED mouse. Mice recover for 6-8 weeks priorto infection with PR8 (control virus) or PA_Cre virus. B-C) Frequency ofdonor (CD45.1+) reporter+ cells in lungs (B) and dLNs (C) from PR8(blue) or PA_Cre (red) mice on day 5 post infection. E) Representativeflow cytometry plots from the lungs of PR8 (top) or PA_Cre (bottom)infected mice.

Funding• NIH T35 Medical Student Summer Research Program in Infection

and Immunity.• NIH, AI110581-01A1. “Harnessing microRNAs to explore influenza

virus immunity.”

References1. Estimating Seasonal Influenza-Associated Deaths in the United States. (2016,

December 09). Retrieved July 03, 2017, fromhttps://www.cdc.gov/flu/about/disease/us_flu-related_deaths.htm

2. Heaton, Nicholas S., et al. "Long-term survival of influenza virus infected club cellsdrives immunopathology." Journal of Experimental Medicine 211.9 (2014): 1707-1714.

3. Manicassamy, Balaji, et al. "Analysis of in vivo dynamics of influenza virus infectionin mice using a GFP reporter virus." Proceedings of the National Academy ofSciences 107.25 (2010): 11531-11536.

A

Figure 2: MΦs phagocytose tdTomato in vitro

BA

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Figure 2: MΦs phagocytose tdTomato in vitro A) Model of WT bone marrowderived MΦ (BMDM) culture conditions with: 1) killed debris from iRED cells, 2)killed debris from iRED cells infected with PR8, and 3) killed debris from iREDcells infected with PA_Cre. B) Frequency of MΦs that are reporter+ withconditions described in (A). C) Representative Flow cytometry plots of iRED cells(top) and BMDMs + killed debris from iRED cells (bottom)

Vector PR8 PA_Cre Ad_Cre

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Figure 1: A) Model demonstrating insertion of Cre recombinase (Cre)into IAV (IAV_Cre) genome and expression of tdTomato in reporter mice.B) Image of iRED mouse lung infected with PA_Cre.

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