Research

The focus of our laboratory is respiratory viral infections. Our favorite virus is the human rhinovirus. In addition to causing the "common cold," human rhinovirus infections are the most common cause of exacerbations of asthma, COPD and other chronic lung diseases. We also study enterovirus-D68, a respiratory virus which has been associated with a polio-like illness in children. Finally, we are studying human coronavirus-NL63 which, like SARS-CoV2, utilizes the ACE2 receptor and has been associated with severe outbreaks of pneumonia in nursing homes and Kawasaki's disease in children. Our work combines cell culture, animal models and human subjects research. Current projects:

Type innate lymphoid cells (ILC2s) are a major IL-13–producing cell in rhinovirus-infected neonatal mice. Eight days after rhinovirus infection of six day-old mice, Lin−CD25+CD127+ double-positive (DP) and Lin−CD25−CD127− double-negative (DN) ILC2s were characterized for c-kit and Sca-1. Also shown in an image of an ILC2.

EARLY LIFE RHINOVIRUS INFECTION AND CHILDHOOD ASTHMA

Birth cohort studies have found significant associations between early-life wheezing-associated respiratory tract infections and the development of asthma in children. These studies suggest that early life respiratory tract infections have a direct effect on lung and/or immune cell development and the risk of asthma.

To determine possible mechanisms, we established a mouse model of early-life RV infection. Infection of 6 day-old mice, but not mature mice, induces long-lasting mucous metaplasia, eosinophilic inflammation and airways hyperresponsiveness (AHR) which is associated with type 2 innate lymphoid cell (ILC2) expansion and dependent on IL-13, IL-25 and IL-33.

In this proposal, we test the general hypothesis that, following early- life RV infection, development of ILC2-dependent type 2 airway inflammation and mucous metaplasia represents a balance between tuft cell RV-induced IL-25 signaling (promotes the phenotype) and NLRP3- dependent IL-1β signaling (suppresses the phenotype).

Specific Aim 1. Determine the contribution of airway brush (tuft) cells to viral-induced IL-25 production. Specific Aim 2. Determine the role of IL-1β on the development of RV-induced mucous metaplasia and AHR. Specific Aim 3. Determine the effects of early-life RV-C infection.

Completion of the proposed work will provide new insight into the pathogenesis of asthma development, and identify new targets for prevention.

RV-C15 co-localizes with mouse epithelial cell acetyl-α-tubulin and induces expression of pro-inflammatory cytokines. Mouse airway epithelial cells were differentiated at air-liquid interface. A) Cells were infected with sham HeLa cell lysate, RV-C15 or RV-A39 and harvested for immunofluorescence staining with anti-mouse acetyl α-tubulin (red) and anti-VP3 (green). Nuclei are stained with DAPI (blue). Confocal microscopy shows colocalization of cilia and RV-C15 (yellow). The white bar is 50 µ.

MODELS OF RHINOVIRUS-C RESPIRATORY INFECTION AND ASTHMA

Accumulating evidence indicates that infections with a newly-discovered species of rhinovirus, RV-C, are associated with severe respiratory tract infections and asthma exacerbations often requiring hospitalization. In addition, recent data suggest a possible role for early-life RV-C infections in asthma development. Despite increasing recognition of RV-C as a cause of asthmatic disease, virtually nothing is known about the pathogenesis of RV-C infections.

Our preliminary studies show that RV-C15- infected mice show increased type 2 cytokine and mucin gene expression, BAL eosinophils and lineage-negative, CD25+, CD127+ type 2 innate lymphoid cells (ILC2s) compared to RV-A1B-infected mice. In addition, pilot studies from children with natural RV-C infections show increased type 2 cytokine production.

In this application, we test the general hypothesis that, after RV-C infection, airway innate cytokine expression drives ILC2 expansion and development of eosinophilic inflammation and mucous metaplasia.

Specific Aim 1. Determine the contribution of epithelial-derived innate cytokines to RV-C15-induced eosinophilic airway inflammation and hyperresponsiveness (AHR). Specific Aim 2. Determine the contribution of lung ILC2s and macrophages to RV-C-induced airway inflammation and AHR. Specific Aim 3. Determine the effects of early-life RV-C infection on the established asthma phenotype.

HCoV-NL63 infects K18-hACE2 mice. Mouse airway infected with human coronavirus NL63 (green, ACE2; red, nsp3; blue, nsp4). Expression of non-structural proteins nsp3 and nsp4 is evidence of viral replication.

CORONAVIRUS NL63 MOUSE MODEL OF COVID-19 RESPIRATORY DISEASE


SARS-CoV2 is one of a family of seven coronaviruses which cause respiratory infections in humans. In contrast to SARS-CoV2, human coronavirus NL63 (HCoV-NL63) usually causes minor respiratory tract infections. However, NL63 carries many similarities to SARS-CoV2. It shares the same receptor (ACE2). Also, like SARS-CoV2, coronavirus NL63 may cause severe infections in the elderly and has been associated with Kawasaki-like-syndrome.

We are therefore establishing an animal model of NL63 using a transgenic mouse that expresses the human ACE2 receptor. Our general hypothesis is that HCoV-NL63 causes severe respiratory disease in aged mice due to enhanced inflammasome activation.

Specific Aim 1. Establish a mouse model of COVID-19 using coronavirus NL63 and K18-hACE2 transgenic mice. Specific Aim 2. Examine lung NLRP3 inflammasome priming and activation in coronavirus NL63-infected immature, adult and elderly mice. Specific Aim 3. Test the effectiveness of inflammasome inhibition and antivirals on coronavirus NL63-induced lung inflammation.

Establishment of this mouse model of coronavirus infection will provide insight into the cellular and molecular mechanisms underlying COVID-19 and facilitate testing of potential therapeutic interventions.

Confocal Cy3-labeled MyrVP4 (Cy3-MyrVP4) fluorescence resonance energy transfer (FRET) to Cy5-labeled anti-TLR2 (Cy5-anti-TLR2) was assessed by laser photobleaching. A: 293/hTLR2 cells bound with 10 ng of Cy3-MyrVP4 (red) and Cy5-anti-TLR2 (green) (left). DAPI staining of nuclei is an underlay in black. FRET is shown as blue (middle). Colocalization of MyrVP4 and TLR2 is light blue to white (right).

TLR2 AND RHINOVIRUS-INDUCED ASTHMA EXACERBATIONS


Rhinovirus (RV) is the most common cause of asthma exacerbation but underlying mechanisms are not completely known. We have noted colocalization of RV with CD68, a macrophage marker, in the airways of allergen-exposed, RV-infected mice as well as human asthmatics experimentally infected with RV. In mice, depletion of macrophages decreased RV-induced eosinophilic airway inflammation and responsiveness, suggesting that these cells play a direct role in the response. Further, we found that RV-induced cytokine expression is attenuated in macrophages from TLR2 null mice, showing an unexpected role for TLR2 in the response to RV.

In this proposal, we test the novel hypothesis that insertion of myristoylated viral polypeptide (VP)-4 into the macrophage plasma membrane triggers cytokine production by interacting with a TLR2 signaling complex.

Specific Aim 1: Determine the contribution of macrophage TLR2 and its cofactors to RV-induced airway responses. Specific Aim 2: Determine the contribution of myristoylated VP4 to RV-induced airway responses.

Completion of this work could provide new therapeutic targets for RV-induced asthma exacerbations.

Rhinovirus-induced inflammasome activation is Toll-like receptor 2 (TLR2) dependent. Wild-type C57BL/6 (WT) or TLR2−/− mice were inoculated with sham or RV for 24 h. Lung inflammasome activation was assessed by immunoblotting for active IL-1 beta and caspase 1.

RESPIRATORY ENTEROVIRUSES, INFLAMMASOME ACTIVATION AND INNATE IMMUNE CELLS


Respiratory enteroviruses, including rhinoviruses (RVs) and enterovirus D68 (EV-D68), cause acute respiratory tract infections and asthma exacerbations. EV-D68 caused an outbreak of severe respiratory illness in 2014 and is responsible for 14% of acute respiratory illnesses in children biannually. At the same time, RV-C was linked to severe respiratory infections and asthma exacerbations. Our pilot data show that, compared to RV-A1B, inflammasome activation and type 3 innate lymphoid cells (ILC3s) are increased in the lungs of EV-D68-infected mice. In contrast, inflammasome activation is decreased, and type 2 innate lymphoid cells (ILC2s) increased, in RV-C15-infected mice.

We propose that the level of macrophage NLRP3 inflammasome activation and IL-1β produced by different respiratory enteroviruses determines their divergent innate immune cell responses and airway outcomes. EV-D68 triggers intense inflammasome activation and IL-1β-driven ILC3s, leading to IL-17-dependent neutrophilic inflammation. RV-C15 infection elicits minimal inflammasome activation, thus the predominant response is ILC2 expansion and IL-13-dependent eosinophilic inflammation. RV-A1B triggers an intermediate response.

In Specific Aim 1, we will determine the mechanisms by which respiratory enteroviruses EV-D68, RV-A1B and RV-C15 differentially activate the macrophage NLRP3 inflammasome.. In Specific Aim 2, we will determine the role of the NLRP3 inflammasome in the activation of lung innate immune cells.