Pneumoviruses infect eosinophils and elicit MyD88-dependent release of chemoattractant cytokines and interleukin-6 by Kimberly D. Dyer, Caroline M. Percopo, Elizabeth R. Fischer, Stanislaw J. Gabryszewski, and Helene F. Rosenberg Blood Volume 114(13):2649-2656 September 24, 2009 ©2009 by American Society of Hematology

PVM replicates in mouse bone marrow–derived eosinophils. PVM replicates in mouse bone marrow–derived eosinophils. Electron micrographs of mouse bone marrow–derived eosinophils (bmEos) documenting morphologic features typical of eosinophils, including the bilobed nucleus (N) and specific granules in the cytoplasm (sg). Magnification: (A) ×2500, bar = 2 μm; (B-C) ×5000 and ×12000, respectively; bar = 500 nm. (D) Q-RT-PCR detection of virus replication in bmEos. bmEos were inoculated with PVM (●) or heat-inactivated PVM (hiPVM; ○) at a multiplicity of infection (MOI) of 1; *P < .05. Data shown are representative of 2 experiments performed in triplicate. (E) PVM N protein is detected in infected bmEos at day 8 after inoculation. Total protein extracts from control, PVM-infected, or hiPVM-challenged bmEos were probed with anti-PVM N peptide antibody (αNPVM). Anti–human GAPDH (αGAPDH) antibody was used as a control for protein loading. (F) Interferon-γ–induced protein (IP-10/CXCL-10) released from bmEos on day 4 after inoculation in response to PVM infection, hiPVM-challenge, or control challenge. Results are representative of 2 experiments performed in triplicate, P values as indicated. (G) Infectious virions are released from PVM-infected bmEos. Supernatants from bmEos cultures at 2 or 8 days after inoculation were used to challenge cells of the RAW 264.7 cell line, which is highly permissive for PVM replication.14 Total RNA from the RAW 264.7 cells was harvested at 5 days after inoculation, and Q-RT-PCR was performed to determine virus copy number. Results are data combined from 3 experiments, each performed in triplicate, **P < .01. Data represent mean ± SEM. Kimberly D. Dyer et al. Blood 2009;114:2649-2656 ©2009 by American Society of Hematology

Accelerated replication of PVM in mouse bmEos from MyD88−/− mice. Accelerated replication of PVM in mouse bmEos from MyD88−/− mice. (A) Q-RT-PCR detection of virus titer in bmEos. Virus titer was determined in PVM-infected (filled symbols) and hiPVM-challenged (open symbols) wild-type (C57BL/6; circles) and MyD88−/− mouse bmEos (squares). Values shown are representative of 2 experiments performed in triplicate (data shown represent mean ± SEM); *P < .05. (B) Immunodetection of PVM in extracts of bmEos. PVM was detected at 9 days after inoculation of bmEos cultures on a Western blot probed with rabbit polyclonal anti-PVM N peptide antibody (αNPVM). Lanes 1 to 3 are extracts from bmEos from wild-type C57BL/6 mice; lanes 4 to 6, extracts from bmEos from MyD88−/− mice; lanes 1 and 4, from unchallenged bmEos (controls); lanes 2 and 5, from hiPVM-challenged bmEos; lanes 3 and 6, from PVM-infected bmEos. After probing with anti-PVM N antibody the blot was stripped and probed with anti–mouse GAPDH (αGAPDH) as a control for protein loading. Blot shown is single membrane, divided to omit intervening protein marker lanes. Kimberly D. Dyer et al. Blood 2009;114:2649-2656 ©2009 by American Society of Hematology

Cytokine release is MyD88-dependent. Cytokine release is MyD88-dependent. Differential release of (A) IP-10/CXCL10, (B) IL-6, (C) MIP-1α/CCL3, (D) MCP-1/CCL2, and (E) IFNα from PVM-infected wild-type (●) and MyD88−/− (■) mouse bmEos cultures. (A-D) Data were determined by multiplex bead assay, n = 3; *P < .05. (E) Data were results from enzyme-linked immunoabsorbent assay, n = 4. Data shown represent mean ± SEM. Kimberly D. Dyer et al. Blood 2009;114:2649-2656 ©2009 by American Society of Hematology

Recombinant IL-6 suppresses virus replication MyD88−/− bmEos. Recombinant IL-6 suppresses virus replication MyD88−/− bmEos. Cells were infected at MOI of 1 and resuspended in media with or without IL-6 (20 ng/mL). At time points indicated, RNA was isolated from the cells, and RT-PCR was performed to determine absolute virus copy number. Experimental conditions are as indicated, points for the wild-type C57BL/6 with and without IL-6 are superimposed over one another; n = 3 mice per experimental condition, *P < .01. Data shown represent mean ± SEM. Kimberly D. Dyer et al. Blood 2009;114:2649-2656 ©2009 by American Society of Hematology

Respiratory syncytial virus replicates in human eosinophils. Respiratory syncytial virus replicates in human eosinophils. (A) Green fluorescent protein detected in eosinophils after challenge with rgRSV. Shown are (i) phase contrast, (ii) fluorescent, and (iii) composite of panels i and ii, showing expression of green protein in eosinophils. Photographed with 32× objective in 6-well culture dish in media. (B) Replication of RSV in human eosinophils. Cells were challenged with respiratory syncytial virus (RSV; MOI = 1) for 2 hours, washed, and resuspended, and total RNA was isolated immediately (day 0) or 4 days later for determination of virus copy number. Results are pooled data from 3 separate experiments, ***P < .001. (C) IL-6 is released by RSV-infected eosinophils. Detection of IL-6 by multiplex bead assay in culture supernatants from unchallenged (control), RSV-infected, and hiRSV-challenged eosinophils. Each symbol represents an independent eosinophil donor; n = 3 to 4; *P < .02 and **P < .002. (D) Infectious virions are released from RSV-infected human eosinophils. Supernatants from human eosinophil cultures at 0 or 4 days after inoculation were used to challenge cells of the HEp-2 line, which is highly permissive for RSV replication. Total RNA from the HEp-2 cells was harvested at 7 days after inoculation, and Q-RT-PCR was performed to determine virus copy number. Results shown are a single experiment performed in triplicate, *P < .01. Data shown represent mean ± SEM. Kimberly D. Dyer et al. Blood 2009;114:2649-2656 ©2009 by American Society of Hematology