Activated Ly6G⁺ granulocytes protect from mortality during virus infection.

<p>(A) Cytokine gene expression in Ly6G⁺ enriched bone marrow cells. (B) Uptake of fluorescent latex beads by neutrophils (PI⁻CD45⁺Ly6G⁺CD11b⁺) in the lung of GCSF^+/+ and G-CSF^−/− mice four days after infection with SeV (n = 4). (C) Representative flow cytometry analysis of neutrophils (CD11b⁺Ly6c^int) and monocytes (CD11b⁺Ly6c^hi) in the blood of mice injected with a single dose of anti-Ly6G antibody (1A8) or an isotype control. Neutrophil depleted WT C57BL6 mice were infected with SeV and monitored for (D) weight loss (<i>p</i>>0.9 two way ANOVA for infected groups) and (E) survival (n = 10 for isotype treated, SeV infected and n = 20 for Ly6G depleted and SeV infected. Logrank test, <i>p</i><0.0001). Experiments shown are representative of two or three independent experiments.</p

G-CSF regulates the lung inflammatory response during virus infection.

<p>(A–B) Cytokine secretion in the lungs of G-CSF^−/− and G-CSF^+/+ mice infected with SeV was measured by multiplex ELISA (n = 4).</p

Adaptive immunity in the absence of G-CSF.

<p>(A) SeV specific antibodies in lung homogenate of G-CSF^+/+ and G-CSF^−/− mice measured by ELISA 7 days after infection (n = 4). Experiment shown is representative of two independent experiments. IgG2a <i>p</i>>0.15, IgG3 <i>p</i>>0.07, IgG2b <i>p</i><0.006, total IgG <i>p</i><0.03 between infected G-CSF^+/+ and infected G-CSF^−/− for all dilutions. (B) Mock and SeV infected G-CSF^+/+ or G-CSF^−/− mice were injected i.v. 7 days after infection with CFSE-labeled peptide-pulsed splenocytes. Splenocytes labeled with a high dose of CFSE were pulsed with SeV NP peptide and those labeled with a low dose of CFSE were pulsed with the irrelevant influenza virus (PR8) NP peptide. Empty histograms show CFSE staining on mock-infected animals 24 h after injection of labeled/pulsed splenocytes. Filled histograms correspond to CFSE staining on SeV infected mice 24 h after injection of labeled/pulsed splenocytes (n = 4 per group). Experiment shown is representative of two independent experiments. (C) Survival of G-CSF^+/+ and G-CSF^−/− mice infected with a sublethal dose of influenza virus ×31 or mock treated and challenged at day 19 with a normally lethal dose of influenza virus PR8. Survival was monitored daily (n = 14/16 mice in ×31 challenged groups, n = 5 in mock treated controls). Experiment shown is representative of two independent experiments.</p

G-CSF deficiency reduces lung infiltrate during virus infection.

<p>(A) Total cell counts in cytospins made with bronchoalveolar lavage (BAL) 8 days after infection with SeV. (B) Representative pictures of H&E-stained lung sections from naive and SeV infected G-CSF^+/+ and G-CSF^−/− C57BL/6 mice.</p

G-CSF regulates the cellular infiltrate during virus infection.

<p>(A) Cell numbers in the lungs of SeV infected mice calculated at the indicated time points from total cell counts and flow cytometry analysis. Populations were identified with the following markers: neutrophils (CD11b⁺Ly6c⁺Ly6G⁺), monocytes (CD11b⁺Ly6C⁺Ly6G⁻), pDCs (CD11b⁻B220⁺mPDCA⁺), CD4⁺ T cells (CD3⁺CD4⁺CD8⁻), CD8⁺ T cells (CD3⁺CD8⁺CD4⁻), and NK cells (CD11b⁺NK1.1⁺). Pre-gated on PI⁻CD45⁺ cells. Error bars represent standard deviation from the mean. Asterisks indicate <i>p</i> values (n = 3). (B) Ly6G⁺ granulocytes in the lung, BM and blood of G-CSF^−/− and G-CSF^+/+ mice mock treated or infected with SeV for 4 days. Pre-gated on PI⁻CD45⁺CD11b⁺ cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037334#s3" target="_blank">Results</a> are representative of more than two independent experiments.</p

Vital role for G-CSF produced in the lung of infected mice.

<p>(A) G-CSF measured in the sera of mice 3 days after infection with SeV or with influenza virus ×31 (IAV). (B) Percent weight change of G-CSF^+/+ and G-CSF^−/− mice infected with SeV (n = 6), influenza ×31 (n = 7) or mock treated controls. <i>P</i>>0.6 two-way ANOVA for infected groups. (C) Survival of G-CSF^+/+ and G-CSF^−/− mice monitored after infection with SeV (n = 6, <i>p</i> = 0.0009, logrank test) or influenza ×31 (n = 7, <i>p</i> = 0.012, logrank test). (D) SeV titer in the lungs of G-CSF^+/+ and G-CSF^−/− mice at the indicated time points (n = 8). (E) Viral titers in the lungs of G-CSF^−/− mice infected with SeV and treated with daily injections of H₂O or rhG-CSF four times before infection (Before) or continued treatment throughout the course of the experiment (Daily). ND: non-detected, NM: non-measured. Asterisks indicate <i>p</i> values. Data presented is representative of more than two independent experiments.</p

Defective Viral Genomes Arising <i>In Vivo</i> Provide Critical Danger Signals for the Triggering of Lung Antiviral Immunity

<div><p>The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections <i>in vivo</i> even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.</p></div

IAV deletion DVGs reduce virus virulence and are generated <i>in vivo</i> during infection.

<p>(<b>A</b>) PCR of standard and defective genomes in BMDCs infected for 6 h with a moi of 1.5 TCID₅₀/cell IAV PR8 HD or LD. PCR strategies and sequences of starred product are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003703#ppat.1003703.s007" target="_blank">Fig. S7</a>. (<b>B</b>) Weight loss of mice infected with 100 TCID₅₀ IAV PR8 HD or LD (n = 5) (**p<0.01; Two-way ANOVA with Bonferroni's post hoc test). (<b>C</b>) Quantification of IAV <i>Np</i> mRNA and (<b>D</b>) <i>Ifnb</i> mRNA from total lung homogenates by RT-qPCR. (<b>E</b>) Lungs from mice infected with IAV PR8 LD were analyzed at day 1 (D1) and day 3 (D3) post-infection for the presence of DVGs and genomic fragments by PCR. Results for two different D3 mice are shown. Sequences for starred products can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003703#ppat.1003703.s008" target="_blank">Fig. S8</a>. (<b>F</b>) Expression of <i>Ifnb</i> mRNA in whole lung homogenates analyzed by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes <i>Tuba1b</i> and <i>Rps11</i>. (*p<0.05, **p<0.01, ***p<0.001, ****p<0,0001; Unpaired, two tailed, t student test). Position of base pair size reference markers is indicated in each gel.</p

SeV DVGs reduce virulence <i>in vivo</i>.

<p>(<b>A–C</b>) Mice were infected with 10⁵ TCID₅₀/mouse of SeV Cantell HD (HD) or SeV Cantell LD (LD). (<b>A</b>) Weight loss (***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (<b>B</b>) Virus titers in the lung, (n = 11 for day 1, n = 6 for day 3), and (<b>C</b>) expression of <i>Ifnb</i> mRNA by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes <i>Tuba1b</i> and <i>Rps11</i> (***p<0.001, Unpaired, two tailed, t student test). (<b>D–F</b>) Mice were infected with 10⁴ TCID₅₀/mouse SeV Cantell LD alone, in the presence of 5,000 HA Units/mouse purified defective particles (DPs) or in the presence of UV-inactivated DPs (UVDP). Mice received DPs or UVDPs immediately following virus inoculation. (<b>D</b>) Weight loss, (†, mice sacrificed due to severe weight loss; ***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (<b>E</b>) lung lesion score at day 7 post-infection (n = 3)(*p<0.05, Mann-Whitney test). (<b>F</b>) Photos of the lung of a representative mouse at day 7 post-infection. Arrowhead indicates areas of lesions. (<b>G</b>) Lungs from mice infected with SeV Cantell LD alone, or in the presence of DPs or UVDPs were analyzed by flow cytometry for the expression of the SeV NP protein.</p

DVGs accumulate at a high rate in infected cells and are associated with the induction of the antiviral responses.

<p>(<b>A</b>) SeV Cantell LD RNA and DVG RNA were extracted from SeV Cantell LD stocks or <i>in vitro</i> transcribed DVG RNA respectively. RNAs were treated with calf intestinal phosphatase, RNase A, RNase V1, and/or RNase A/V1. LLC-MK2 cells were transfected with 250 ng of control or treated RNAs and analyzed 4 h after transfection by RT-qPCR. (<b>B</b>) LLC-MK2 cells were transfected with the indicated doses of genomic RNA and DVG RNA. After 4 h, total RNA was extracted and expression of <i>Ifnb</i> and <i>Il-6</i> was determined by RT-qPCR. (<b>C</b>) WT MEF cells were infected with SeV Cantell HD or SeV Cantell LD at the indicated mois. After 6 h, total RNA was extracted and the quantities of <i>Ifnb</i> mRNA, gSeV, and DVG were determined by RT-qPCR. (<b>D</b>) Total RNA was directly quantitated from SeV Cantell HD or LD solutions prepared to have the indicated infectious units. (<b>E</b>) WT MEF cells were infected with SeV Cantell HD at a moi of 3 TCID₅₀/cell and total RNA was analyzed by RT-qPCR at the indicated times post-infection. (<b>F</b>) Rate of gSeV and DVG replication calculated at 12 h post-infection. Gene expression is shown as copy number relative to the housekeeping genes <i>Tuba1b</i> and <i>Rps11</i>. Error bars indicate the standard deviation of triplicate measurements in a representative experiment (**p<0.01, ***p<0.001, ****p<0.001 by one-way ANOVA with Bonferroni post hoc test (A and C); ****p<0.001 by Unpaired, two tailed, t student test (F)).</p