Publisher Correction: Exuberant fibroblast activity compromises lung function via ADAMTS4 (Nature, (2020), 587, 7834, (466-471), 10.1038/s41586-020-2877-5)

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. An amendment to this paper has been published and can be accessed via a link at the top of the paper

Exuberant fibroblast activity compromises lung function via ADAMTS4

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. Severe respiratory infections can result in acute respiratory distress syndrome (ARDS)1. There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS1,2. Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes—in particular the ECM protease ADAMTS4—and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections

Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases

Comparison of Ad5 viral loads between single adenovirus infected and lentiviruses co-infected cell cultures.

<p>Ad5 control indicates that single adenovirus infected 293A cells, and LV-CMV-IFNγ and LV-CMV-GFP indicate that LV-CMV-IFNγ or LV-CMV-GFP co-infected 293A cells, respectively. (<b>A</b>) Ad5 viral loads were quantified by QPCR from single adenovirus infected 293A cell control or 10⁸ copies of each lentivirus co-infected cell cultures in three groups of 10⁴, 10⁵ and 10⁶ IFU of Ad5 inocula after 72 h cultivation, and data were obtained from three representative tests. The curves were plotted by mean ± SD of Ad5 viral loads. <i>P</i> values were calculated by two-way ANOVA test for comparing differences of Ad5 viral loads between single virus and lentivirus co-infected cell cultures or between lentivirus co-infections. (<b>B</b>) Inhibition (%) of Ad5 replication was calculated as viral load reduction from lentivirus co-infection to single Ad5 control in each inoculum group of cell cultures.</p

Mean efficiency and fluorescent signal intensity of recombinant lentivirus transduced cells.

<p>Mean GFP expressing cell count and fluorescent signal intensity were measured by a flow cytometry in 72 h after lentivirus transduction to cells.</p

Characteristics of recombinant lentiviruses.

<p>(<b>A</b>) Schematic representation of lentiviral vector constructs. RRE: REV responsive element; cPPT: central polypurine tract; CMV: cytomegalovirus promoter; EF1α: human elongation factor 1 alpha promoter; EGFP: enhanced green fluorescence protein. (<b>B</b>) Morphology of passages from LV-CMV-GFP transduced 293A cells. The images were captured by a fluorescent or bright light microscope in 72 h after transduction or passaging over months.</p

Detection of GFP expression driven by three different promoters within lentiviruses transduced various cells.

<p>GFP expressing cell counts and fluorescent signal intensity were measured by a flow cytometry in 72 h after lentivirus transduction.</p

Infectivity of recombinant lentivirus to cells.

<p>(<b>A</b>) Amplification curve of IFNγ RNAs by SYBR-QPCR from lentiviruses transduced cells. The curves are representative of LV-CMV-IFNγ and IFNγ RNA positive control, respectively. (<b>B</b>) Dissociation curve (Derivative melting curve) of SYBR-QPCR was obtained from IFNγ RNA amplification. It is apparent that the point of infection (melting temperature of the amplicon) occurs at 85°C , and no contaminating product is present in this reaction. (<b>C</b>) Western Blot: lane 1, LV-CMV-IFNγ transduced cell lysate; lane 2, pTY-CMV-IFNγ plasmid transfected cell lysate; lane 3, LV-CMV-GFP transduced cell lysate control; lane 4, pTY-CMV-GFP plasmid transfected cell lysate control; lane 5, 293A cell lysate negative control. IFNγ was detected in 72 h after transduction or transfection to 293A cells.</p

Exuberant fibroblast activity compromises lung function via ADAMTS4

Severe respiratory infections can result in acute respiratory distress syndrome (ARDS) . There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS . Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes-in particular the ECM protease ADAMTS4-and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections