BackgroundChildren with Down Syndrome (DS) are at high risk for severe respiratory syncytial virus (RSV) infections. DS is associated with impaired cellular responses to oxidative stress and hypoxia; however, these abnormalities have not been explored in trisomy 21 (TS21) airway epithelial cells (AECs) during RSV infection. Understanding these defects is key to identifying factors contributing to severe RSV infections in this high-risk group.MethodsAECs from children with and without DS were analyzed at baseline and after RSV infection to assess NRF2-induced protective genes against oxidative stress and hypoxia, including the enzyme heme oxygenase 1 (HO-1). To investigate DS-specific defects, we focused on miR-155 and BACH1, which regulate NRF2 signaling and HO-1 expression, and are both encoded on chromosome 21. RNA-seq analyses were performed to examine genome-wide hypoxia-related gene responses in control and TS21 AECs at baseline and after RSV infection.ResultsOur findings show that miR-155 inhibits BACH1, leading to increased NRF2-driven HO-1 expression in euploid AECs. In contrast, TS21 AECs from children with DS exhibited impaired HO-1 induction following miR-155 treatment. This was attributed to reduced transcription of the HMOX1 gene, which encodes HO-1, along with global downregulation of hypoxia response genes in DS at baseline and after RSV infection in TS21 AECs.ConclusionsSevere RSV infections in children with DS may be linked to intrinsic defects in AEC responses to hypoxia, including NRF2-driven cytoprotective enzymes like HO-1. These findings offer new mechanistic insights into RSV pathophysiology and potential therapeutic targets in children with DS.

Background Early infancy is marked by high susceptibility to severe viral respiratory infections and reduced protective antibody responses, making rapid development of local airway immunity essential. Despite this, the developmental dynamics of human airway B cells and their interaction with airway epithelial cells (AECs) in early life remain poorly understood. Objective We studied the developmental dynamics of human airway B-cell populations, the variation in AEC-derived B-cell survival and maturation factors with age, and how viral respiratory infections influence their production. Methods Changes in human airway B-cell populations and survival receptors across different pediatric age groups were analyzed by using a single-cell RNA sequencing dataset. The production of B-cell activating factor (BAFF) and other B-cell survival and maturation factors by human AECs was assessed in infants (<12 months) and older children, both at baseline and after viral stimulation in vitro. Additional in vivo validation studies assessed airway BAFF production at baseline and during PCR-confirmed viral respiratory infections across pediatric age groups. Results We observed age-dependent shifts in airway B-cell composition, identifying the BAFF/BAFF-receptor axis as critical for B-cell maturation and survival in early life. Although BAFF production in AECs is initially reduced in infants (<12 months), it can be activated by viral stimuli both in vivo and in vitro. Mechanistic studies showed that BAFF production in human infant AECs is induced by type I and III interferons via JAK/STAT signaling. Conclusion Human AEC JAK/STAT signaling activation regulates the early maturation of airway B-cell responses via local BAFF induction, particularly during viral infections.

Thymic stromal lymphopoietin (TSLP) is a primarily epithelial-derived cytokine that drives type 2 allergic immune responses. Early life viral respiratory infections elicit high TSLP production, which leads to the development of type 2 inflammation and airway hyperreactivity. The goal of this study was to examine in vivo and in vitro the human airway epithelial responses leading to high TSLP production during viral respiratory infections in early infancy. A total of 129 infants (<1-24 m, median age 10 m) with severe viral respiratory infections were enrolled for in vivo (n = 113), and in vitro studies (n = 16). Infants were classified as 'high TSLP' or 'low TSLP' for values above or below the 50th percentile. High versus low TSLP groups were compared in terms of type I-III IFN responses and production of chemokines promoting antiviral (CXCL10), neutrophilic (CXCL1, CXCL5, CXCL8), and type 2 responses (CCL11, CCL17, CCL22). Human infant airway epithelial cell (AEC) cultures were used to define the transcriptomic (RNAseq) profile leading to high versus low TSLP responses in vitro in the absence (baseline) or presence (stimulated) of a viral mimic (poly I:C). Infants in the high TSLP group had greater in vivo type III IFN airway production (median type III IFN in high TSLP 183.2 pg/mL vs. 63.4 pg/mL in low TSLP group, p = 0.007) and increased in vitro type I-III IFN AEC responses after stimulation with a viral mimic (poly I:C). At baseline, our RNAseq data showed that infants in the high TSLP group had significant upregulation of IFN signature genes (e.g., IFIT2, IFI6, MX1) and pro-inflammatory chemokine genes before stimulation. Infants in the high TSLP group also showed a baseline AEC pro-inflammatory state characterized by increased production of all the chemokines assayed (e.g., CXCL10, CXCL8). High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity and increased baseline AEC production of pro-inflammatory chemokines. These findings present a new paradigm underlying the production of TSLP in the human infant airway epithelium following early life viral exposure and shed light on the long-term impact of viral respiratory illnesses during early infancy and beyond childhood.

The coronavirus disease (COVID-19) pandemic has highlighted the critical need to understand the pathobiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human nasal airway epithelial (AE) cells, the point of entry and initial growth of this virus (1–3). ACE2 (angiotensin-converting enzyme 2) is the primary receptor of SARS-CoV-2, and studies in adult human AE cells have found that IFN enhances ACE2 expression (4, 5). This shifts the paradigm for SARS-CoV-2 pathogenesis and therapy by identifying that this virus may enhance its spread by exploiting host IFN antiviral responses. However, IFN-induced expression of ACE2 has not been demonstrated in nasal AE cells of infants, an age group linked to milder COVID-19 (6) and reduced ACE2 airway expression (7). Examining this is needed to address the possibility that the immaturity of pediatric AE cells protects them from SARS-CoV-2 owing to lower IFN-induced expression of ACE2 or by mechanisms independent of IFN-stimulated ACE2 expression. Accordingly, in this study, we examine the ability of the human infant AE cells to upregulate ACE2 in response to IFN-g, IFN-l, and synthetic viral mimic and IFN inducer (poly (I:C)).

We need a better risk stratification system for the increasing number of survivors of extreme prematurity suffering the most severe forms of bronchopulmonary dysplasia (BPD). However, there is still a paucity of studies providing scientific evidence to guide future updates of BPD severity definitions. Our goal was to validate a new predictive model for BPD severity that incorporates respiratory assessments beyond 36 weeks postmenstrual age (PMA). We hypothesized that this approach improves BPD risk assessment, particularly in extremely premature infants. This is a longitudinal cohort of premature infants (≤32 weeks PMA, n = 188; Washington D.C). We performed receiver operating characteristic analysis to define optimal BPD severity levels using the duration of supplementary O2 as predictor and respiratory hospitalization after discharge as outcome. Internal validation included lung X-ray imaging and phenotypical characterization of BPD severity levels. External validation was conducted in an independent longitudinal cohort of premature infants (≤36 weeks PMA, n = 130; Bogota). We found that incorporating the total number of days requiring O2 (without restricting at 36 weeks PMA) improved the prediction of respiratory outcomes according to BPD severity. In addition, we defined a new severity category (level IV) with prolonged exposure to supplemental O2 (≥120 days) that has the highest risk of respiratory hospitalizations after discharge. We confirmed these findings in our validation cohort using ambulatory determination of O2 requirements. In conclusion, a new predictive model for BPD severity that incorporates respiratory assessments beyond 36 weeks improves risk stratification and should be considered when updating current BPD severity definitions.

Studies using human airway epithelial cells (AECs) derived from adults with asthma have confirmed that type 2 airway epithelial cytokines, such as TSLP (thymic stromal lymphopoietin), are key mediators in the pathogenesis of the disease (1), and this fundamental knowledge has led to the discovery of novel asthma therapies (2). In contrast, the airway epithelium of human infants has been remarkably understudied despite compelling evidence that asthma often begins in early life (3). In addition, prior studies have established that airway secretion of TSLP occurs during viral respiratory infections in young children (4, 5). Thus, our goal in this human-based study was to characterize the production of TSLP in vitro in primary infant AECs and in vivo during natural viral respiratory infections in human infants. This study was approved by the Institutional Review Board of Children’s National Health System and included parental informed consent.

Introduction: Viral bronchiolitis is a term often used to group all infants with the first episode of severe viral respiratory infection. However, this term encompasses a collection of different clinical and biological processes. We hypothesized that the first episode of severe viral respiratory infection in infants can be subset into clinical phenotypes with distinct outcomes and underlying airway disease patterns. Methods: We included children (≤2 years old) hospitalized for the first time due to PCR-confirmed viral respiratory infection. All cases were categorized based on primary manifestations (wheezing, sub-costal retractions and hypoxemia) into mild, hypoxemia or wheezing phenotypes. We characterized these phenotypes using lung-X-rays, respiratory outcomes and nasal protein levels of antiviral and type 2 cytokines (IFNγ, IL-10, IL-4, IL-13, IL-1β, and TNFα). Results: A total of 50 young children comprising viral respiratory infection cases (n = 41) and uninfected controls (n = 9) were included. We found that 22% of viral respiratory infection cases were classified as mild (n = 9), 39% as hypoxemia phenotype (n = 16) and 39% as wheezing phenotype (n = 16). Individuals in the hypoxemia phenotype had more lung opacities, higher probability of PICU admission and prolonged hospitalizations. Subjects in the wheezing phenotype had higher probability of recurrent sick visits. Nasal cytokine profiles showed that individuals with recurrent sick visits in the wheezing phenotype had increased nasal airway levels of type 2 cytokines (IL-13/IL-4). Conclusion: Clinically-based classification of the first episode of severe viral respiratory infection into mild, hypoxemia or wheezing phenotypes provides critical information about respiratory outcomes, lung disease patterns and underlying airway immunobiology.

Infants requiring hospitalization due to a viral lower respiratory tract infection (LRTI) have a high risk of developing recurrent respiratory illnesses in early life and asthma beyond childhood. Notably, all validated clinical scales for viral LRTI have focused on predicting acute severity instead of recurrence. We present a novel clinical approach combining individual risk factors with bedside clinical parameters to predict recurrence after viral LRTI hospitalization in young children. A retrospective longitudinal cohort of young children (≤3 years) designed to define clinical predictive factors of recurrent respiratory illnesses within 12 months after hospitalization due to PCR-confirmed viral LRTI. Data collection was through electronic medical record. We included 138 children hospitalized with viral LRTI. Using automatic stepwise logistic model selection, we found that the strongest predictors of recurrence in infants hospitalized for the first time were severe prematurity (≤32 weeks’ gestational age, OR=5.19; 95% CI 1.76 to 15.32; p=0.002) and a clinical score that weighted hypoxemia, subcostal retractions and wheezing (OR=3.33; 95% CI 1.59 to 6.98; p<0.001). After the first hospitalization, the strongest predictors of subsequent episodes were wheezing (OR=5.62; 95% CI 1.03 to 30.62; p=0.04) and family history of asthma (OR=5.39; 95% CI 1.04 to 27.96; p=0.04). We found that integrating individual risk factors (eg, prematurity or family history of asthma) with bedside clinical assessment (eg, wheezing, subcostal retractions or hypoxemia) can predict the risk of recurrence after viral LRTI hospitalization in infants. This strategy may enable clinically oriented subsetting of infants with viral LRTI based on individual predictors for recurrent respiratory illnesses during early life.

INTRODUCTION:IFN lambda (type III-IFN-λ1) is a molecule primarily produced by epithelial cells that provides an important first-line defence against viral respiratory infections and has been linked to the pathogenesis of viral-induced wheezing in early life. The goal of this study was to better understand the regulation of innate IFN-lambda responses in vitro in primary human infant airway epithelial cells (AECs) and in vivo using nasal aspirates during viral respiratory infections. METHODS:IFN-lambda protein levels were quantified: (a) in human infant AECs exposed to (poly(I:C) dsRNA) under different experimental conditions (n = 8 donors); and (b) in nasal aspirates of young children (≤3 years) hospitalized with viral respiratory infection (n = 138) and in uninfected controls (n = 74). In vivo IFN-lambda airway levels during viral infections were correlated with individual characteristics and respiratory disease parameters. RESULTS:Our in vitro experiments showed that the poly(I:C)-induced innate production of IFN lambda in human infant AECs is regulated by (a) p38-MAPK/NF-kB dependent mechanism; and (b) exposure to pro-inflammatory signals such as IL1β. Our in vivo studies demonstrated that (a) infants (<18 months) had higher virus-induced IFN-lambda airway secretion; (b) subjects with RSV infection showed the highest IFN-lambda airway levels; and (c) individuals with the highest virus-induced IFN-lambda levels (>90th percentile) had higher viral loads and were more likely to have respiratory sick visits within 12 months of discharge (OR = 5.8). CONCLUSION:IFN-lambda responses to dsRNA in the human infant airway epithelium are regulated by p38-MAPK and NF-kB signalling. High in vivo IFN-lambda production is influenced by virus type and associated with recurrent respiratory sick visits in young children.

BACKGROUND:MicroRNAs (miRs) control gene expression and the development of the immune system and antiviral responses. MiR-155 is an evolutionarily-conserved molecule consistently induced during viral infections in different cell systems. Notably, there is still an unresolved paradox for the role of miR-155 during viral respiratory infections. Despite being essential for host antiviral TH1 immunity, miR-155 may also contribute to respiratory disease by enhancing allergic TH2 responses and NFkB-mediated inflammation. The central goal of this study was to define how airway miR-155 production is related to TH1, TH2, and pro-inflammatory cytokine responses during naturally occurring viral respiratory infections in young children.METHODS:Normalized nasal airway levels of miR-155 and nasal protein levels of IFN-γ, TNF-α, IL-1β, IL-13, IL-4 were quantified in young children (≤2 years) hospitalized with viral respiratory infections and uninfected controls. These data were linked to individual characteristics and respiratory disease parameters.RESULTS:A total of 151 subjects were included. Increased miR-155 levels were observed in nasal samples from patients with rhinovirus, RSV and all respiratory viruses analyzed. High miR-155 levels were strongly associated with high IFN-γ production, increased airway TH1 cytokine polarization (IFN-γ/IL-4 ratios) and increased pro-inflammatory responses. High airway miR-155 levels were linked to decreased respiratory disease severity in individuals with high airway TH1 antiviral responses.CONCLUSIONS:The airway secretion of miR-155 during viral respiratory infections in young children is associated with enhanced antiviral immunity (TH1 polarization). Further studies are needed to define additional physiological roles of miR-155 in the respiratory tract of human infants and young children during health and disease.

Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins (Mfn1/2), and dynamin related protein 1 (Drp1), we find these conventional pathways for ER to the mitochondria trafficking are dispensable for vMIA trafficking to OMM. Instead, mutations in vMIA that change its hydrophobicity alter its trafficking to mitochondria. Superresolution imaging showed that PACS-2- and Mfn-mediated membrane apposition or hydrophobic interactions alter vMIA's ability to organize in nanoscale clusters at the OMM. This shows that signal-anchored MAM proteins can make use of hydrophobic interactions independently of conventional ER-mitochondria pathways to traffic from the ER to mitochondria. Further, vMIA hydrophobic interactions and ER-mitochondria contacts facilitate proper organization of vMIA on the OMM.

The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses.

The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100–150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization.