Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.

TLR3-Activated Monocyte-Derived Dendritic Cells Trigger Progression from Acute Viral Infection to Chronic Disease in the Lung.

Acute infection is implicated as a trigger for chronic inflammatory disease, but the full basis for this switch is uncertain. In this study, we examine this issue using a mouse model of chronic lung disease that develops after respiratory infection with a natural pathogen (Sendai virus). We investigate this model using a combination of TLR3-deficient mice and adoptive transfer of immune cells into these mice versus the comparable responses in wild-type mice. We found that acute and transient expression of TLR3 on monocyte-derived dendritic cells (moDCs) was selectively required to induce long-term expression of IL-33 and consequent type 2 immune-driven lung disease. Unexpectedly, moDC participation was not based on canonical TLR3 signaling and relied instead on a trophic effect to expand the alveolar epithelial type 2 cell population beyond repair of tissue injury and thereby provide an enriched and persistent cell source of IL-33 required for progression to a disease phenotype that includes lung inflammation, hyperreactivity, excess mucus production, and remodeling. The findings thereby provide a framework wherein viral infection activates TLR3 in moDCs as a front-line immune cell niche upstream of lung epithelial cells to drive the type 2 immune response, leading to chronic inflammatory diseases of the lung (such as asthma and chronic obstructive pulmonary disease in humans) and perhaps progressive and long-term postviral disease in general.

Group 2 Innate Lymphoid Cells Must Partner with the Myeloid-Macrophage Lineage for Long-Term Postviral Lung Disease.

Group 2 innate lymphoid cells (ILC2s) are implicated in host defense and inflammatory disease, but these potential functional roles need more precise definition, particularly using advanced technologies to better target ILC2s and engaging experimental models that better manifest both acute infection and chronic, even lifelong, disease. In this study, we use a mouse model that applies an improved genetic definition of ILC2s via IL-7r-conditional Rora gene targeting and takes advantage of a distinct progression from acute illness to chronic disease, based on a persistent type 2 immune response to respiratory infection with a natural pathogen (Sendai virus). We first show that ILC2s are activated but are not required to handle acute illness after respiratory viral infection. In contrast, we find that this type of infection also activates ILC2s chronically for IL-13 production and consequent asthma-like disease traits that peak and last long after active viral infection is cleared. However, to manifest this type of disease, the Csf1-dependent myeloid-macrophage lineage is also active at two levels: first, at a downstream level, this lineage provides lung tissue macrophages (interstitial macrophages and tissue monocytes) that represent a major site of Il13 gene expression in the diseased lung; and second, at an upstream level, this same lineage is required for Il33 gene induction that is necessary to activate ILC2s for participation in disease at all, including IL-13 production. Together, these findings provide a revised scheme for understanding and controlling the innate immune response leading to long-term postviral lung diseases with features of asthma and related progressive conditions.

Respiratory Enterovirus (like Parainfluenza Virus) Can Cause Chronic Lung Disease if Protection by Airway Epithelial STAT1 Is Lost.

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1f/f mice) to target the master IFN signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection because of mouse parainfluenza (Sendai) virus and human enterovirus D68 (EV-D68). Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1f/f mice and conventional Stat1-/- mice compared with wild-type mice. In concert, the chronic lung disease that develops after Sendai virus infection was also increased in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1-/ - mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk postinfection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1 -/- mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue.

Influenza A Virus Infection Causes Chronic Lung Disease Linked to Sites of Active Viral RNA Remnants.

Clinical and experimental observations suggest that chronic lung disease is linked to respiratory viral infection. However, the long-term aspect of this relationship is not yet defined using a virus that replicates at properly high levels in humans and a corresponding animal model. In this study, we show that influenza A virus infection achieves 1 × 106-fold increases in viral load in the lung and dose-dependent severity of acute illness in mice. Moreover, these events are followed by persistence of negative- and positive-strand viral RNA remnants for 15 wk and chronic lung disease for at least 26 wk postinfection. The disease is manifested by focal areas of bronchiolization and mucus production that contain increased levels of viral RNA remnants along with mucin Muc5ac and Il13 mRNA compared with uninvolved areas of the lung. Excess mucus production and associated airway hyperreactivity (but not fibrosis or emphysema) are partially attenuated with loss of IL-13 production or signaling (using mice with IL-13 or STAT6 deficiency). These deficiencies cause reciprocal increases in l17a mRNA and neutrophils in the lung; however, none of these disease endpoints are changed with IL-13/IL-17a compared with IL-13 deficiency or STAT6/IL-17a compared with STAT6 deficiency. The results establish the capacity of a potent human respiratory virus to produce chronic lung disease focally at sites of active viral RNA remnants, likely reflecting locations of viral replication that reprogram the region. Viral dose dependency of disease also implicates high-level viral replication and severity of acute infection as determinants of chronic lung diseases such as asthma and COPD with IL-13-dependent and IL-13/IL-17-independent mechanisms.

Uncleaved NS2-3 is required for production of infectious bovine viral diarrhea virus.

Despite increasing characterization of pestivirus-encoded proteins, functions for nonstructural (NS) proteins NS2, NS2-3, NS4B, and NS5A have not yet been reported. Here we investigated the function of bovine viral diarrhea virus (BVDV) uncleaved NS2-3. To test whether NS2-3 has a discrete function, the uncleaved protein was specifically abolished in two ways: first by inserting a ubiquitin monomer between NS2 and NS3, and second by placing an internal ribosome entry site between the two proteins (a bicistronic genome). In both cases, complete processing of NS2-3 prevented infectious virion formation without affecting RNA replication. We tested the hypothesis that uncleaved NS2-3 was involved in morphogenesis by creating a bicistronic genome in which NS2-3 was restored in the second cistron. With this genome, both uncleaved NS2-3 expression and particle production returned. We then investigated the minimal regions of the polyprotein that could rescue an NS2-3 defect by developing a trans-complementation assay. We determined that the expression of NS4A in cis with NS2-3 markedly increased its activity, while p7 could be supplied in trans. Based on these data, we propose a model for NS2-3 action in virion morphogenesis.