GM-CSF intrinsically controls eosinophil accumulation in the setting of allergic airway inflammation.

ISSN:0091-6749ISSN:1097-682

GM-CSF instigates a dendritic cell–T-cell inflammatory circuit that drives chronic asthma development

Background Steroid-resistant asthma is often characterized by high levels of neutrophils and mixed TH2/TH17 immune profiles. Indeed, neutrophils are key drivers of chronic lung inflammation in multiple respiratory diseases. Their numbers correlate strongly with disease severity, and their presence is often associated with exacerbation of chronic lung inflammation. Objective What factors drive development of neutrophil-mediated chronic lung disease remains largely unknown, and we sought to study the role of GM-CSF as a potential regulator in chronic asthma. Methods Different experimental animal models of chronic asthma were used in combination with alveolar macrophage-reconstitution of global GM-CSF receptor knockout mice as well as cell-type–specific knockout animals to elucidate the role of GM-CSF signaling in chronic airway inflammation. Results We identify GM-CSF signaling as a critical factor regulating pulmonary accumulation of neutrophils. We show that although being not required for intrinsically regulating neutrophil migration, GM-CSF controls lung dendritic cell function, which in turn promotes T-cell–dependent recruitment of neutrophils to the airways. We demonstrate that GM-CSF regulates lung dendritic cell antigen uptake, transport, and TH2/TH17 cell priming in an intrinsic fashion, which in turn drives pulmonary granulocyte recruitment and contributes to development of airway hyperresponsiveness in chronic disease. Conclusions We identify GM-CSF as a potentially novel therapeutic target in chronic lung inflammation, describing a GM-CSF–dependent lung conventional dendritic cell-T-cell-neutrophil axis that drives chronic lung disease.ISSN:0091-6749ISSN:1097-682

PI3Kγ Is Critical for Dendritic Cell-Mediated CD8+ T Cell Priming and Viral Clearance during Influenza Virus Infection

Phosphoinositide-3-kinases have been shown to be involved in influenza virus pathogenesis. They are targeted directly by virus proteins and are essential for efficient viral replication in infected lung epithelial cells. However, to date the role of PI3K signaling in influenza infection in vivo has not been thoroughly addressed. Here we show that one of the PI3K subunits, p110γ, is in fact critically required for mediating the host’s antiviral response. PI3Kγ deficient animals exhibit a delayed viral clearance and increased morbidity during respiratory infection with influenza virus. We demonstrate that p110γ is required for the generation and maintenance of potent antiviral CD8+ T cell responses through the developmental regulation of pulmonary cross-presenting CD103+ dendritic cells under homeostatic and inflammatory conditions. The defect in lung dendritic cells leads to deficient CD8+ T cell priming, which is associated with higher viral titers and more severe disease course during the infection. We thus identify PI3Kγ as a novel key host protective factor in influenza virus infection and shed light on an unappreciated layer of complexity concerning the role of PI3K signaling in this context

PPARγ in dendritic cells and T cells drives pathogenic type-2 effector responses in lung inflammation

ISSN:0022-1007ISSN:1540-0069ISSN:1540-953

Frontline Science: Coincidental null mutation of Csf2rα in a colony of PI3Kγ-/- mice causes alveolar macrophage deficiency and fatal respiratory viral infection

ISSN:0741-5400ISSN:1938-367

Alveolar Macrophages Are Essential for Protection from Respiratory Failure and Associated Morbidity following Influenza Virus Infection

Alveolar macrophages (AM) are critical for defense against bacterial and fungal infections. However, a definitive role of AM in viral infections remains unclear. We here report that AM play a key role in survival to influenza and vaccinia virus infection by maintaining lung function and thereby protecting from asphyxiation. Absence of AM in GM-CSF-deficient (Csf2−/−) mice or selective AM depletion in wild-type mice resulted in impaired gas exchange and fatal hypoxia associated with severe morbidity to influenza virus infection, while viral clearance was affected moderately. Virus-induced morbidity was far more severe in Csf2−/− mice lacking AM, as compared to Batf3-deficient mice lacking CD8α+ and CD103+ DCs. Csf2−/− mice showed intact anti-viral CD8+ T cell responses despite slightly impaired CD103+ DC development. Importantly, selective reconstitution of AM development in Csf2rb−/− mice by neonatal transfer of wild-type AM progenitors prevented severe morbidity and mortality, demonstrating that absence of AM alone is responsible for disease severity in mice lacking GM-CSF or its receptor. In addition, CD11c-Cre/Ppargfl/fl mice with a defect in AM but normal adaptive immunity showed increased morbidity and lung failure to influenza virus. Taken together, our results suggest a superior role of AM compared to CD103+ DCs in protection from acute influenza and vaccinia virus infection-induced morbidity and mortality

<i>Csf2</i>^−/− mice succumb to influenza virus infection despite intact antiviral T and B cell responses.

<p>(A–I) Indicated groups of mice were infected i.t. with PR8 influenza virus (50 pfu unless otherwise specified). Loss of body weight (A) and temperature (B) was monitored. (C) Survival after infection with 100 pfu PR8. Values indicate mean ± SEM of 8–10 mice per group. (D) Virus titers in the lung at days 6 and 10 p.i. were measured by plaque-assay. (E–H) Comparison of immune responses in <i>Csf2</i>^−/−, <i>Batf3</i>^−/− and WT mice at day 10 post infection. (E) Percentages (upper panel) and total numbers (lower panel) of influenza NP34-specific CD8⁺ T cells in the BAL, lung and lung-draining LN. (F) Virus-specific IFNγ and TNFα production in CD8⁺ T cells of the BAL, lung and LN was analyzed by restimulation with virus-loaded BMDCs. (G and H) CD103⁺ DCs in the lung at d10 post-infection were gated on eF780⁻CD45⁺CD11c⁺MHCII⁺B220⁻Siglec-F⁻ cells. Dot plots show the frequencies of CD103⁺ DCs of individual mice representative for the group (G) and bar graphs display total numbers (H). Values indicate mean ± SD of 5–6 mice per group. (I) WT, <i>Csf2</i>^−/− and <i>Batf3</i>^−/− mice were monitored for body weight, body temperature and the survival during the course of infection. Data show mean ± SEM.</p

Selective restoration of AM development in <i>Csf2rb</i>^−/− mice prevents severe morbidity and mortality following influenza virus infection.

<p>Whole CD45⁺ cells containing progenitors of AM were sorted from lungs of CD45.1⁺ E18.5 embryos by flow cytometry and transferred intranasally into neonatal CD45.2⁺<i>Csf2rb</i>^−/− mice. (A) Six weeks after transfer, recipient mice were analyzed for the presence of donor-derived AM. (B) Bar graphs display the total AM cell number in the BAL and lung. (C) Dot plots depict the expression of CD103 and CD11b on CD11c⁺MHCII⁺Siglec-F⁻ lung DCs and frequencies of CD45.1⁺ (donor-derived) and CD45.2⁺ (recipient-derived) cells among CD103⁺ DCs are shown. (D–G) Eight weeks after transfer, recipient mice were infected with 50 pfu PR8 influenza virus. (D) Total protein concentration in the BAL at d5 after infection. (E) Loss of body weight and temperature and (F) survival was monitored during the course of infection (mean ± SEM of 6–9 mice per group). (G) Lung virus titer at day 5 p.i.</p

Influenza infection potently induces expression of interferon-regulated antiviral factors in AM.

<p>(A) Mice were infected with 50 pfu PR8 influenza virus. Intracellular NP expression was measured by flow cytometry in CD11c⁺autofluorescent AM isolated from BAL and lung 5 days after infection. (B) Mice were infected with 10⁶ pfu NS1-GFP virus <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004053#ppat.1004053-Manicassamy1" target="_blank">[51]</a> or 10³ pfu PR8. GFP expression was analyzed in AM isolated from BAL and lung 5 days after infection. (C) Microarray analysis of sorted AM from lungs of naive or influenza-infected animals at d5 post-infection with 50 pfu PR8. Bar graphs show relative expression levels of various interferon-induced genes plotted as log₂-fold change in AM from infected lungs compared to naïve. The mean of two microarray samples per condition is shown. For each sample, AM from two individual mice were pooled. Differences in expression levels were validated by qPCR for most of the depicted genes (i.e. <i>Ifitm3</i>, <i>Ifitm6</i>, <i>Ifit2</i>, <i>Ifit3</i> and <i>Ifi205</i>).</p

GM-CSF promotes development of CD103⁺ DCs and expression of CD103 in young and adult mice, respectively.

<p>Analysis of lung DC subsets in (A–C) 6–8 week-old and (D–E) 6-day-old mice. (A, D) Gated cells in dot plots show CD103⁺ and CD11b⁺ DCs with values indicating percentages among CD11c⁺Siglec-F⁻ lung DCs. Cells were pre-gated on CD45⁺eFluor780⁻ viable cells. (B,E) Bar graphs depict total number of CD103⁺ and CD11b⁺ DCs in the lung. (C) Mean fluorescence intensity (MFI) of CD103 expression on CD103⁺CD11b⁻ DCs. Values shown represent the mean ± SD (n = 3–4). (F) Analysis of lung DC subsets in mixed BM chimeras generated by transfer of 1∶1 mixture of CD45.1⁺WT∶CD45.2⁺<i>Csf2rb</i>^−/− or control CD45.1⁺WT∶CD45.2⁺WT into lethally irradiated recipients. Bar graphs show the percentage of CD45.2⁺WT and CD45.2⁺<i>Csf2rb</i>^−/− cells among CD103⁺ and CD11b⁺ DCs in the lung. The mean ± SD is shown (n = 3).</p