In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions.

How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.

CXCR6 orchestrates brain CD8+ T cell residency and limits mouse Alzheimer's disease pathology.

Neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by innate immune-mediated inflammation, but functional and mechanistic effects of the adaptive immune system remain unclear. Here we identify brain-resident CD8+ T cells that coexpress CXCR6 and PD-1 and are in proximity to plaque-associated microglia in human and mouse AD brains. We also establish that CD8+ T cells restrict AD pathologies, including ß-amyloid deposition and cognitive decline. Ligand-receptor interaction analysis identifies CXCL16-CXCR6 intercellular communication between microglia and CD8+ T cells. Further, Cxcr6 deficiency impairs accumulation, tissue residency programming and clonal expansion of brain PD-1+CD8+ T cells. Ablation of Cxcr6 or CD8+ T cells ultimately increases proinflammatory cytokine production from microglia, with CXCR6 orchestrating brain CD8+ T cell-microglia colocalization. Collectively, our study reveals protective roles for brain CD8+ T cells and CXCR6 in mouse AD pathogenesis and highlights that microenvironment-specific, intercellular communication orchestrates tissue homeostasis and protection from neuroinflammation.

Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer.

CD8+ cytotoxic T cells (CTLs) orchestrate antitumour immunity and exhibit inherent heterogeneity1,2, with precursor exhausted T (Tpex) cells but not terminally exhausted T (Tex) cells capable of responding to existing immunotherapies3-7. The gene regulatory network that underlies CTL differentiation and whether Tex cell responses can be functionally reinvigorated are incompletely understood. Here we systematically mapped causal gene regulatory networks using single-cell CRISPR screens in vivo and discovered checkpoints for CTL differentiation. First, the exit from quiescence of Tpex cells initiated successive differentiation into intermediate Tex cells. This process is differentially regulated by IKAROS and ETS1, the deficiencies of which dampened and increased mTORC1-associated metabolic activities, respectively. IKAROS-deficient cells accumulated as a metabolically quiescent Tpex cell population with limited differentiation potential following immune checkpoint blockade (ICB). Conversely, targeting ETS1 improved antitumour immunity and ICB efficacy by boosting differentiation of Tpex to intermediate Tex cells and metabolic rewiring. Mechanistically, TCF-1 and BATF are the targets for IKAROS and ETS1, respectively. Second, the RBPJ-IRF1 axis promoted differentiation of intermediate Tex to terminal Tex cells. Accordingly, targeting RBPJ enhanced functional and epigenetic reprogramming of Tex cells towards the proliferative state and improved therapeutic effects and ICB efficacy. Collectively, our study reveals that promoting the exit from quiescence of Tpex cells and enriching the proliferative Tex cell state act as key modalities for antitumour effects and provides a systemic framework to integrate cell fate regulomes and reprogrammable functional determinants for cancer immunity.

SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity.

Cancer cells evade T cell-mediated killing through tumour-immune interactions whose mechanisms are not well understood1,2. Dendritic cells (DCs), especially type-1 conventional DCs (cDC1s), mediate T cell priming and therapeutic efficacy against tumours3. DC functions are orchestrated by pattern recognition receptors3-5, although other signals involved remain incompletely defined. Nutrients are emerging mediators of adaptive immunity6-8, but whether nutrients affect DC function or communication between innate and adaptive immune cells is largely unresolved. Here we establish glutamine as an intercellular metabolic checkpoint that dictates tumour-cDC1 crosstalk and licenses cDC1 function in activating cytotoxic T cells. Intratumoral glutamine supplementation inhibits tumour growth by augmenting cDC1-mediated CD8+ T cell immunity, and overcomes therapeutic resistance to checkpoint blockade and T cell-mediated immunotherapies. Mechanistically, tumour cells and cDC1s compete for glutamine uptake via the transporter SLC38A2 to tune anti-tumour immunity. Nutrient screening and integrative analyses show that glutamine is the dominant amino acid in promoting cDC1 function. Further, glutamine signalling via FLCN impinges on TFEB function. Loss of FLCN in DCs selectively impairs cDC1 function in vivo in a TFEB-dependent manner and phenocopies SLC38A2 deficiency by eliminating the anti-tumour therapeutic effect of glutamine supplementation. Our findings establish glutamine-mediated intercellular metabolic crosstalk between tumour cells and cDC1s that underpins tumour immune evasion, and reveal glutamine acquisition and signalling in cDC1s as limiting events for DC activation and putative targets for cancer treatment.

Lipid signalling enforces functional specialization of Treg cells in tumours.

Regulatory T cells (Treg cells) are essential for immune tolerance1, but also drive immunosuppression in the tumour microenvironment2. Therapeutic targeting of Treg cells in cancer will therefore require the identification of context-specific mechanisms that affect their function. Here we show that inhibiting lipid synthesis and metabolic signalling that are dependent on sterol-regulatory-element-binding proteins (SREBPs) in Treg cells unleashes effective antitumour immune responses without autoimmune toxicity. We find that the activity of SREBPs is upregulated in intratumoral Treg cells. Moreover, deletion of SREBP-cleavage-activating protein (SCAP)-a factor required for SREBP activity-in these cells inhibits tumour growth and boosts immunotherapy that is triggered by targeting the immune-checkpoint protein PD-1. These effects of SCAP deletion are associated with uncontrolled production of interferon-γ and impaired function of intratumoral Treg cells. Mechanistically, signalling through SCAP and SREBPs coordinates cellular programs for lipid synthesis and inhibitory receptor signalling in these cells. First, de novo fatty-acid synthesis mediated by fatty-acid synthase (FASN) contributes to functional maturation of Treg cells, and loss of FASN from Treg cells inhibits tumour growth. Second, Treg cells in tumours show enhanced expression of the PD-1 gene, through a process that depends on SREBP activity and signals via mevalonate metabolism to protein geranylgeranylation. Blocking PD-1 or SREBP signalling results in dysregulated activation of phosphatidylinositol-3-kinase in intratumoral Treg cells. Our findings show that metabolic reprogramming enforces the functional specialization of Treg cells in tumours, pointing to new ways of targeting these cells for cancer therapy.

CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.

Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2-4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.

Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.

Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells1. Here we use an in vivo pooled CRISPR-Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8+ T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8+ T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia. By using a secondary genome-scale CRISPR-Cas9 screening, we identify BATF as the key target of REGNASE-1 and as a rheostat that shapes antitumour responses. Loss of BATF suppresses the increased accumulation and mitochondrial fitness of REGNASE-1-deficient CD8+ T cells. By contrast, the targeting of additional signalling factors-including PTPN2 and SOCS1-improves the therapeutic efficacy of REGNASE-1-deficient CD8+ T cells. Our findings suggest that T cell persistence and effector function can be coordinated in tumour immunity and point to avenues for improving the efficacy of adoptive cell therapy for cancer.

Deficiency in ST2 signaling ameliorates RSV-associated pulmonary hypertension.

Pulmonary hypertension (PH) observed during respiratory syncytial virus (RSV) bronchiolitis is associated with morbidity and mortality, especially in children with congenital heart disease. Yet, the pathophysiological mechanisms of RSV-associated PH remain unclear. Therefore, this study aimed to investigate the pathophysiological mechanism of RSV-associated PH. We used a translational mouse model of RSV-associated PH, in which wild-type (WT) and suppression of tumorigenicity 2 (ST2) knockout neonatal mice were infected with RSV at 5 days old and reinfected 4 wk later. The development of PH in WT mice following RSV reinfection was evidenced by elevated right ventricle systolic pressure, shortened pulmonary artery acceleration time (PAT), and decreased PAT/ejection time (ET) ratio. It coincided with the augmentation of periostin and IL-13 expression and increased arginase bioactivity by both arginase 1 and 2 as well as induction of nitric oxide synthase (NOS) uncoupling. Absence of ST2 signaling prevented RSV-reinfected mice from developing PH by suppressing NOS uncoupling. In summary, ST2 signaling was involved in the development of RSV-associated PH. ST2 signaling inhibition may be a novel therapeutic target for RSV-associated PH.NEW & NOTEWORTHY We report that the pathogenic role of ST2-mediated type 2 immunity and mechanisms contribute to RSV-associated pulmonary hypertension. Inhibiting ST2 signaling may be a novel therapeutic target for this condition.

Gfi1-Foxo1 axis controls the fidelity of effector gene expression and developmental maturation of thymocytes.

Double-positive (DP) thymocytes respond to intrathymic T-cell receptor (TCR) signals by undergoing positive selection and lineage differentiation into single-positive (SP) mature cells. Concomitant with these well-characterized events is the acquisition of a mature T-cell gene expression program characterized by the induction of the effector molecules IL-7Rα, S1P1, and CCR7, but the underlying mechanism remains elusive. We report here that transcription repressor Growth factor independent 1 (Gfi1) orchestrates the fidelity of the DP gene expression program and developmental maturation into SP cells. Loss of Gfi1 resulted in premature induction of effector genes and the transcription factors forkhead box protein O1 (Foxo1) and Klf2 in DP thymocytes and the accumulation of postselection intermediate populations and accelerated transition into SP cells. Strikingly, partial loss of Foxo1 function, but not restored survival fitness, rectified the dysregulated gene expression and thymocyte maturation in Gfi1-deficient mice. Our results establish the Gfi1-Foxo1 axis and the transcriptional circuitry that actively maintain DP identity and shape the proper generation of mature T cells.

New mouse model of pulmonary hypertension induced by respiratory syncytial virus bronchiolitis.

Pulmonary hypertension (PH) has been observed in up to 75% of infants with moderate to severe respiratory syncytial virus (RSV) bronchiolitis and is associated with significant morbidity and mortality in infants with congenital heart disease. The purpose of the present study was to establish a mouse model of PH secondary to RSV bronchiolitis that mimics the disease etiology as it occurs in infants. Neonatal mice were infected with RSV at 5 days of age and then reinfected 4 wk later. Serum-free medium was administered to age-matched mice as a control. Echocardiography and right ventricular systolic pressure (RVSP) measurements via right jugular vein catheterization were conducted 5 and 6 days after the second infection, respectively. Peripheral capillary oxygen saturation monitoring did not indicate hypoxia at 2-4 days post-RSV infection, before reinfection, and at 2-7 days after reinfection. RSV-infected mice had significantly higher RVSP than control mice. Pulsed-wave Doppler recording of the pulmonary blood flow by echocardiogram demonstrated a significantly shortened pulmonary artery acceleration time and decreased pulmonary artery acceleration time-to-ejection time ratio in RSV-infected mice. Morphometry showed that RSV-infected mice exhibited a significantly higher pulmonary artery medial wall thickness and had an increased number of muscularized pulmonary arteries compared with control mice. These findings, confirmed by RVSP measurements, demonstrate the development of PH in the lungs of mice infected with RSV as neonates. This animal model can be used to study the pathogenesis of PH secondary to RSV bronchiolitis and to assess the effect of treatment interventions. NEW & NOTEWORTHY This is the first mouse model of respiratory syncytial virus-induced pulmonary hypertension, to our knowledge. This model will allow us to decipher molecular mechanisms responsible for the pathogenesis of pulmonary hypertension secondary to respiratory syncytial virus bronchiolitis with the use of knockout and/or transgenic animals and to monitor therapeutic effects with echocardiography.

Crawling with Virus: Translational Insights from a Neonatal Mouse Model on the Pathogenesis of Respiratory Syncytial Virus in Infants.

The infant immune response to respiratory syncytial virus (RSV) remains incompletely understood. Here we review the use of a neonatal mouse model of RSV infection to mimic severe infection in human infants. We describe numerous age-specific responses, organized by cell type, observed in RSV-infected neonatal mice and draw comparisons (when possible) to human infants.

Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.

Regulatory T cells and IL10 suppress pulmonary host defense during early-life exposure to radical containing combustion derived ultrafine particulate matter.

BACKGROUND: Exposure to elevated levels of particulate matter (PM) is associated with increased risk of morbidity and mortality due to respiratory tract viral infections in infants. Recent identification of environmentally persistent free radicals (EPFRs) in the PM from a variety of combustion sources suggests its role in the enhancement of disease severity of lower respiratory tract infections (LRTI). Our previous studies demonstrated that acute exposure to EPFRs induces pulmonary immunosuppression allowing for enhanced influenza disease severity. Here, we determine the mechanism of EPFR-induced immunosuppression and its impact on the immune response towards influenza infection. METHODS: Neonatal mice (3 days old) were acutely exposed to DCB (combustion derived PM with chemisorbed EPFR) for seven consecutive days. Four days post-exposure (dpe), mice were infected with influenza virus. Pulmonary T cell phenotypes including regulatory T cells (Tregs) were analyzed by flow cytometry. The role of IL10 in EPFR-induced exacerbation of influenza disease severity was determined by administering recombinant IL10 (rIL10) to wild type mice or by using IL10 deficient (IL10-/-) neonatal mice. Mice were assessed for morbidity by measuring percent weight change and pulmonary viral load. RESULTS: Neonatal mice exposed to EPFRs had a significant increase in pulmonary Tregs and the immunosuppressive cytokine IL10 following influenza infection, which coincided with decreased protective T cell responses to influenza infection at 6 dpi. Depletion of Tregs in EPFR-exposed neonatal mice resulted in increased protective, adaptive T cell responses, whereas adoptive transfer of Tregs from EPFR-exposed neonates to air-exposed neonatal mice suppressed adaptive T cell responses towards influenza infection. Further, treatment with rIL10 could recapitulate EPFR-induced exacerbation of morbidity and pulmonary viral load compared to air exposed and influenza infected mice, whereas, EPFR-exposed IL10-/- neonates exhibited significant reductions in morbidity, pulmonary viral load and adaptive T cell responses following influenza infection. CONCLUSIONS: Neonatal exposure to EPFRs induced Tregs and IL10 resulting in suppressed adaptive T cell responses and enhanced influenza disease severity in neonatal mice. Depletion of Tregs increased adaptive T cell responses and deficiency of IL10 reduced morbidity and conferred enhanced protection against influenza virus.

Exposure to environmentally persistent free radicals during gestation lowers energy expenditure and impairs skeletal muscle mitochondrial function in adult mice.

We have investigated the effects of in utero exposure to environmentally persistent free radicals (EPFRs) on growth, metabolism, energy utilization, and skeletal muscle mitochondria in a mouse model of diet-induced obesity. Pregnant mice were treated with laboratory-generated, combustion-derived particular matter (MCP230). The adult offspring were placed on a high-fat diet for 12 wk, after which we observed a 9.8% increase in their body weight. The increase in body size observed in the MCP230-exposed mice was not associated with increases in food intake but was associated with a reduction in physical activity and lower energy expenditure. The reduced energy expenditure in mice indirectly exposed to MCP230 was associated with reductions in skeletal muscle mitochondrial DNA copy number, lower mRNA levels of electron transport genes, and reduced citrate synthase activity. Upregulation of key genes involved in ameliorating oxidative stress was also observed in the muscle of MCP230-exposed mice. These findings suggest that gestational exposure to MCP230 leads to a reduction in energy expenditure at least in part through alterations to mitochondrial metabolism in the skeletal muscle.

Regulation of inflammatory biomarkers by intravenous methylprednisolone in pediatric ARDS patients: Results from a double-blind, placebo-controlled randomized pilot trial.

OBJECTIVE: A double-blind, randomized controlled trial showed that low-dose glucocorticoid therapy in pediatric ARDS patients is feasible and may improve both ventilation and oxygenation indices in these patients. However, the molecular mechanisms underlying potential changes in outcomes remain unclear. Based on these clinical findings, this study was designed to examine the effects of intravenous methylprednisolone on circulating inflammatory biomarkers in pediatric ARDS patients. DESIGN: Double-blind, placebo-controlled randomized trial with blood collection on study entry and day 7. SETTING: Tertiary care children's hospital. PATIENTS: Children (0-18years) with ARDS undergoing mechanical ventilation. INTERVENTIONS: 35 children were randomized within 72h of mechanical ventilation. The glucocorticoid group received methylprednisolone 2mg/kg loading dose followed by 1mg/kg/day continuous infusion from days 1 to 7. Both groups were ventilated following the ARDSnet recommendations. WBC and differential cell counts, plasma cytokines and CRP levels, and coagulation parameters were analyzed on days 0 and 7. RESULTS: At study entry, the placebo group had higher IL-15 and basophil levels. On day 7, in comparison to study entry, the placebo group had lower IL-1α, IFN-γ and IL-10 levels. The glucocorticoid group had lower INF-α, IL-6, IL-10, MCP-1, G-CSF and GM-CSF levels, and higher IL-17α levels on day 7 in comparison to study entry. Total and differential cell counts remained unchanged within the placebo group between days 0 and 7, whereas in the glucocorticoid group total WBC and platelets counts were increased on day 7. Pearson's correlation studies within the placebo and glucocorticoid groups revealed positive and negative correlations between cytokine levels, cell counts, coagulation parameters and relevant clinical parameters of disease severity identified in our previous study. Multiple regression models identified several cytokines as predictors for alterations in clinical parameters of disease severity. CONCLUSION: This pilot study shows the feasibility of simultaneously measuring multiple inflammatory cytokines, cell counts and coagulation parameters in pediatric ARDS patients. We report statistical models that may be useful for future, larger trials to predict ARDS severity and outcomes.

Serotonin 5-HT2 receptor activation prevents allergic asthma in a mouse model.

Asthma is an inflammatory disease of the lung characterized by airways hyper-responsiveness (AHR), inflammation, and mucus hyperproduction. Current mainstream therapies include bronchodilators that relieve bronchoconstriction and inhaled glucocorticoids to reduce inflammation. The small molecule hormone and neurotransmitter serotonin has long been known to be involved in inflammatory processes; however, its precise role in asthma is unknown. We have previously established that activation of serotonin 5-hydroxytryptamine (5-HT)(2A) receptors has potent anti-inflammatory activity in primary cultures of vascular tissues and in the whole animal in vasculature and gut tissues. The 5-HT(2A) receptor agonist, (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI] is especially potent. In this work, we have examined the effect of (R)-DOI in an established mouse model of allergic asthma. In the ovalbumin mouse model of allergic inflammation, we demonstrate that inhalation of (R)-DOI prevents the development of many key features of allergic asthma, including AHR, mucus hyperproduction, airways inflammation, and pulmonary eosinophil recruitment. Our results highlight a likely role of the 5-HT2 receptors in allergic airways disease and suggest that 5-HT2 receptor agonists may represent an effective and novel small molecule-based therapy for asthma.

Impaired gamma delta T cell-derived IL-17A and inflammasome activation during early respiratory syncytial virus infection in infants.

Respiratory syncytial virus (RSV) infection remains a significant global health burden disproportionately affecting infants and leading to long-term lung disease. Interleukin (IL)-17A has been shown to be involved in regulating viral and allergic lung inflammatory responses, which has led to a more recent interest in its role in RSV infection. Using a neonatal mouse model of RSV, we demonstrate that neonates fail to develop IL-17A responses compared with adult mice; the main immediate IL-17A contributor in adults were γδ T cells. Antibody neutralization of IL-17A in adult mice caused increased lung inflammation and airway mucus from RSV, whereas exogenous IL-17A administration to RSV-infected neonates caused decreased inflammation but no change in airway mucus. We also observed a lack of pro-inflammatory cytokine production (IL-1ß, IL-6) from infected neonates. Using human cord blood mononuclear cells (CBMCs) and adult peripheral blood mononuclear cells (PBMCs), we compared inflammasome activation by direct retinoic acid-inducible gene I agonism; CBMCs failed to induce pro-inflammatory cytokines or IL-17A(+) γδ T cells compared with PBMCs. Our results indicate that RSV disease severity is in part mediated by a lack of inflammasome activation and IL-17A production in neonates.

Limited type I interferons and plasmacytoid dendritic cells during neonatal respiratory syncytial virus infection permit immunopathogenesis upon reinfection.

UNLABELLED: Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action. IMPORTANCE: Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-α treatment or adoptive transfer of adult pDCs capable of producing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-α is a promising target for future RSV vaccine design.

Building a better neonatal mouse model to understand infant respiratory syncytial virus disease.

BACKGROUND: Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infection in infants; and severe RSV infection in infants is associated with asthma development. Today, there are still no vaccines or specific antiviral therapies against RSV. The mechanisms of RSV pathogenesis in infants remain elusive. This is partly due to the fact that the largely-used mouse model is semi-permissive for RSV. The present study sought to determine if a better neonatal mouse model of RSV infection could be obtained using a chimeric virus in which the F protein of A2 strain was replaced with the F protein from the line 19 clinical isolate (rA2-19F). METHODS: Five-day-old pups were infected with the standard laboratory strain A2 or rA2-19F and various immunological and pathophysiological parameters were measured at different time points post infection, including lung histology, bronchoalveolar lavage fluid (BALF) cellularity and cytokines, pulmonary T cell profile, and lung viral load. A cohort of infected neonates were allowed to mature to adulthood and reinfected. Pulmonary function, BALF cellularity and cytokines, and T cell profiles were measured at 6 days post reinfection. RESULTS: The rA2-19F strain in neonatal mice caused substantial lung pathology including interstitial inflammation and airway mucus production, while A2 caused minimal inflammation and mucus production. Pulmonary inflammation was characterized by enhanced Th2 and reduced Th1 and effector CD8(+) T cells compared to A2. As with primary infection, reinfection with rA2-19F induced similar but exaggerated Th2 and reduced Th1 and effector CD8(+) T cell responses. These immune responses were associated with increased airway hyperreactivity, mucus hyperproduction and eosinophilia that was greater than that observed with A2 reinfection. Pulmonary viral load during primary infection was higher with rA2-19F than A2. CONCLUSIONS: Therefore, rA2-19F caused enhanced lung pathology and Th2 and reduced effector CD8(+) T cell responses compared to A2 during initial infection in neonatal mice and these responses were exacerbated upon reinfection. The exact mechanism is unknown but appears to be associated with increased pulmonary viral load in rA2-19F vs. A2 infected neonatal lungs. The rA2-19F strain represents a better neonatal mouse model of RSV infection.