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Adenosine deaminase-1 (ADA-1) plays both enzymatic and non-enzymatic roles in regulating immune cell function. Mutations in the ADA1 gene account for 15% of heritable severe-combined immunodeficiencies. We determined previously that ADA1 expression defines and is instrumental for the germinal center follicular helper T cell (T(FH)) phenotype using in vitro human assays. Herein, we tested whether ADA-1 can be used as an adjuvant to improve vaccine efficacy in vivo. In vitro, ADA-1 induced myeloid dendritic cell (mDC) maturation as measured by increased frequencies of CD40-, CD83-, CD86-, and HLA-DR-positive mDCs. ADA-1 treatment also promoted the secretion of the T(FH)-polarizing cytokine IL-6 from mDCs. In the context of an HIV-1 envelope (env) DNA vaccine, co-immunization with plasmid-encoded ADA-1 (pADA) enhanced humoral immunity. Animals co-immunized with env DNA and pADA had significantly increased frequencies of T(FH) cells in their draining lymph nodes and increased HIV-binding IgG in serum. Next, mice were co-immunized with subtype C env gp160 DNA and pADA along with simultaneous immunization with matched gp140 trimeric protein. Mice that received env gp160 DNA, pADA, and gp140 glycoprotein had significantly more heterologous HIV-specific binding IgG in their serum. Furthermore, only mice that received the combination ADA-1 adjuvanted vaccine had detectable neutralizing antibody responses. These studies support the use of ADA-1 as a vaccine adjuvant to qualitatively enhance germinal center responses and represent a novel application of an existing therapeutic agent that can be quickly translated for clinical use.

Neonates exhibit increased susceptibility to respiratory viral infections, attributed to inflammation at the developing pulmonary air-blood interface. IFN I are antiviral cytokines critical to control viral replication, but also promote inflammation. Previously, we established a neonatal murine influenza virus (IV) model, which demonstrates increased mortality. Here, we sought to determine the role of IFN I in this increased mortality. We found that three-day-old IFNAR-deficient mice are highly protected from IV-induced mortality. In addition, exposure to IFNβ 24 h post IV infection accelerated death in WT neonatal animals but did not impact adult mortality. In contrast, IFN IIIs are protective to neonatal mice. IFNβ induced an oxidative stress imbalance specifically in primary neonatal IV-infected pulmonary type II epithelial cells (TIIEC), not in adult TIIECs. Moreover, neonates did not have an infection-induced increase in antioxidants, including a key antioxidant, superoxide dismutase 3, as compared to adults. Importantly, antioxidant treatment rescued IV-infected neonatal mice, but had no impact on adult morbidity. We propose that IFN I exacerbate an oxidative stress imbalance in the neonate because of IFN I-induced pulmonary TIIEC ROS production coupled with developmentally regulated, defective antioxidant production in response to IV infection. This age-specific imbalance contributes to mortality after respiratory infections in this vulnerable population.

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity and a key contributor to the large health care burden associated with prematurity, longer hospital stays, higher hospital costs, and frequent re-hospitalizations of affected patients through the first year of life and increased resource utilization throughout childhood. This disease is associated with abnormal pulmonary function that may lead to BPD-associated pulmonary hypertension (PH), a major contributor to neonatal mortality and morbidity. In the absence of any definitive treatment options, this life-threatening disease is associated with high resource utilization during and after neonatal intensive care unit (NICU) stay. The goal of this study was to test the safety and efficacy of a small molecule derivative of chitin, AVR-48, as prophylactic therapy for preventing experimental BPD in a mouse model. Two doses of AVR-48 were delivered either intranasally (0.11 mg/kg), intraperitoneally (10 mg/kg), or intravenously (IV) (10 mg/kg) to newborn mouse pups on postnatal day (P)2 and P4. The outcomes were assessed by measuring total inflammatory cells in the broncho-alveolar lavage fluid (BALF), chord length, septal thickness, and radial alveolar counts of the alveoli, Fulton’s Index (for PH), cell proliferation and cell death by immunostaining, and markers of inflammation by Western blotting and ELISA. The bioavailability and safety of the drug were assessed by pharmacokinetic and toxicity studies in both neonatal mice and rat pups (P3-P5). Following AVR-48 treatment, alveolar simplification was improved, as evident from chord length, septal thickness, and radial alveolar counts, total inflammatory cells were decreased in the BALF, Fulton’s Index was decreased and lung inflammation and cell death were decreased, while angiogenesis and cell proliferation were increased. AVR-48 was found to be safe and the no-observed-adverse-effect level (NOAEL) in rat pups was determined to be 100 mg/kg when delivered via IV dosing with a 20-fold safety margin. With no reported toxicity and with a shorter half-life, AVR-48 is able to reverse the worsening cardiopulmonary phenotype of experimental BPD and BPD-PH, compared to controls, thus positioning it as a future drug candidate.

The neonatal period presents a complex scenario where the threshold of reactivity toward colonizing microbiota, maternal antigens, autoantigens, and pathogens must be carefully moderated and balanced. CD8+ T cells are critical for the response against intracellular bacteria and viruses, but this immune compartment maintains altered function relative to adult counterparts because of the unique challenges which infants face. Here, we review our current understanding of the factors which may promote the attenuation and altered function of the neonatal CD8+ T-cell response and potential avenues for future study. Specifically, we have focused on the neonatal CD8+ T-cell ontogeny, memory formation, TCR structure and repertoire, TCR inhibitory receptors, and the clinical implications of altered neonatal CD8+ T-cell function. Special emphasis has been placed on examining the response of preterm neonates relative to term neonates and adults.

Background Endogenous pulmonary stem cells (PSCs) play an important role in lung development and repair; however, little is known about their role in bronchopulmonary dysplasia (BPD). We hypothesize that an endogenous PSC marker stage-specific embryonic antigen-1 (SSEA-1) and its enzyme, α1,3-fucosyltransferase IX (FUT9) play an important role in decreasing inflammation and restoring lung structure in experimental BPD. Methods We studied the expression of SSEA-1, and its enzyme FUT9, in wild-type (WT) C57BL/6 mice, in room air and hyperoxia. Effects of intraperitoneal administration of recombinant human FUT9 (rhFUT9) on lung airway and parenchymal inflammation, alveolarization, and apoptosis were evaluated. Results On hyperoxia exposure, SSEA-1 significantly decreased at postnatal day 14 in hyperoxia-exposed BPD mice, accompanied by a decrease in FUT9. BPD and respiratory distress syndrome (RDS) in human lungs showed decreased expression of SSEA-1 as compared to their term controls. Importantly, intraperitoneal administration of FUT9 in the neonatal BPD mouse model resulted in significant decrease in pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leakage, alveolar simplification, and cell death in the hyperoxia-exposed BPD mice. Conclusions An important role of endogenous PSC marker SSEA-1 and its enzyme FUT9 is demonstrated, indicating early systemic intervention with FUT9 as a potential therapeutic option for BPD. Impact Administration of rhFUT9, an enzyme of endogenous stem cell marker SSEA-1, reduces pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leak and cell death in the BPD mouse model.SSEA-1 is reported for the first time in experimental BPD models, and in human RDS and BPD.rhFUT9 treatment ameliorates hyperoxia-induced lung injury in a developmentally appropriate BPD mouse model.Our results have translational potential as a therapeutic modality for BPD in the developing lung.

The neonatal period presents a complex scenario where the threshold of reactivity toward colonizing microbiota, maternal antigens, autoantigens, and pathogens must be carefully moderated and balanced. CD8(+) T cells are critical for the response against intracellular bacteria and viruses, but this immune compartment maintains altered function relative to adult counterparts because of the unique challenges which face infants. Here, we review our current understanding of the factors that may promote the attenuation and altered function of the neonatal CD8(+) T cell response and potential avenues for future study. Specifically, we have focused on the neonatal CD8(+) T cell ontogeny, memory formation, T-cell receptor (TCR) structure and repertoire, TCR inhibitory receptors, and the clinical implications of altered neonatal CD8(+) T cell function. Special emphasis has been placed on examining the response of preterm neonates relative to term neonates and adults.

Respiratory viral infections contribute substantially to global infant losses and disproportionately affect preterm neonates. Using our previously established neonatal murine model of influenza infection, we demonstrate that three-day old mice are exceptionally sensitive to influenza virus infection and exhibit high mortality and viral load. Intranasal pre- and post-treatment of neonatal mice with Lactobacillus rhamnosus GG (LGG), an immune modulator in respiratory viral infection of adult mice and human preterm neonates, considerably improves neonatal mice survival after influenza virus infection. We determine that both live and heat-killed intranasal LGG are equally efficacious in protection of neonates. Early in influenza infection, neonatal transcriptional responses in the lung are delayed compared to adults. These responses increase by 24 hours post-infection, demonstrating a delay in the kinetics of the neonatal anti-viral response. LGG pretreatment improves immune gene transcriptional responses during early infection and specifically upregulates type I IFN pathways. This is critical for protection, as neonatal mice intranasally pre-treated with IFNβ before influenza virus infection are also protected. Using transgenic mice, we demonstrate that the protective effect of LGG is mediated through a MyD88-dependent mechanism, specifically via TLR4. LGG can improve both early control of virus and transcriptional responsiveness and could serve as a simple and safe intervention to protect neonates., Author summary Viral lung infections are the number one reason for hospitalization of children under the age of 5 and are a major public health concern. Premature babies, or those born before 37 weeks gestation, are particularly susceptible to viral infections, but exact mechanisms for this susceptibility have not been determined. Here, using a pre-clinical infant mouse model of influenza virus infection, we have found increased neonatal susceptibility to respiratory viral infection compared to adults, and we demonstrate that a probiotic administered intranasally prior to infection provides dramatic protection to infant mice by inducing production of a key anti-viral cytokine, type I interferons.

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8+ T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366–374)–specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366–374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257–264)-specific (OT-I) CD8+ T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8+ T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

Infants less than six months of age are particularly susceptible to respiratory viral infection-associated morbidity and mortality. Type I Interferons, IFN I, a potent anti-viral cytokine which controls viral replication, can also result in the production of pathogenic reactive oxygen species, ROS. Infants are particularly susceptible to ROS because of immature antioxidant production, such as superoxide dismutase 3, and demonstrate increased pathology under oxidative stress conditions. Using our previously established neonatal mouse influenza virus model, we sought to determine if IFN I was protective or deleterious in neonatal influenza virus infection. Three-day old neonatal IFN I receptor, IFNAR-deficient and C57Bl/6 mice were infected with PR8, H1N1 influenza virus and tracked for survival. IFNAR deficient neonatal mice had improved survival, 80% survival versus 20% survival in C57Bl/6 pups. To determine the mechanism of IFN I toxicity, pulmonary type II epithelial cells were isolated 48 hours post-infection and IFNb was applied to the epithelial cells ex vivo. A flow cytometric CellROX assay was performed to determine production of ROS. When exposed to IFNb, neonatal lung epithelial cells produce more ROS than adult lung cells. To determine if an antioxidant could mitigate oxidative stress during in vivo neonatal influenza virus infection, 3 day old murine neonates were treated with an antioxidant, N-acetyl cysteine, NAC, after influenza virus infection. Survival improved from 10% in sham-treated pups to 60% in NAC-treated pups. We speculate that IFN I-induced ROS, coupled with the neonatal inability to neutralize ROS, increases mortality in this vulnerable population.

The underlying mechanisms that regulate neonatal immune suppression are poorly characterized. CD31 (PECAM1) is highly expressed on neonatal lymphocytes and is a known modulator of TCR signaling. To further characterize the role of CD31 in the neonatal CTL response, 3-d and 7-d-old murine neonates were infected with influenza virus and compared to adults. The majority of the pulmonary viral-specific CTLs in the 3-d-old murine neonate retain CD31 expression, whereas adult CTLs have decreased CD31 expression. In addition, CD31+ neonatal viral-specific CTLs demonstrate decreased IFN-γ production, decreased proliferative capacity, and increased likelihood of death. At the peak of infection, sorted neonatal effector CTLs continue to transcribe CD31, indicating a developmental regulation of expression. To explore potential mechanisms for this reduced function, we compared the expression of the transcription factors Eomesodermin (Eomes) and T-bet; there was a significant increase in Eomes paired with a reduction in T-bet in CD31+ neonatal effector CTLs in the lung. Furthermore, in vitro stimulated neonatal CTLs significantly reduce IFN-γ production upon CD31 signaling. Altogether, these data indicate that neonatal CTLs may retain elevated levels of CD31 to maintain peripheral T cell suppression during the bridge to ex utero life.

CD31, expressed by the majority of the neonatal T cell pool, is involved in modulation of T-cell receptor (TCR) signalling by increasing the threshold for T cell activation. Therefore, CD31 could impact neonatal tolerance or immune responses during infection. Cells were harvested from murine neonates at various days post-birth, human late preterm and term cord blood and adult peripheral blood. The number of CD31 expressing CD4+ and CD8a+ T cells increase in the neonatal mouse over the first week of life, which is consistent with previous work demonstrating an increase in CD31 expression across human gestation. Human samples were activated in vitro over a five-day period to simulate acute inflammation similar to chorioamnionitis or early onset sepsis. The frequency of human CD31+ CD4+ and CD8a+ T cells decrease upon activation, with infants having a lower frequency of CD31+ cells compared to adults. In order to further evaluate the regulation of CD31 during acute infection, murine neonates at 3 and 7 days of age were infected with influenza. There was an increased number of naïve and activated CD31+ T lymphocytes at the site of infection at 6 and 9 days post-infection in the 3-day old neonates (p

Neonates have an immunological immaturity which persists from fetal life into the newborn period and can result in a poorly protective response to intracellular pathogens. We have previously shown a less complex TCR repertoire may be responsible for reduced CD8+ T cell immunity in a neonatal murine model of influenza virus infection. Therefore, we sought to determine if there were similar differences in the TCR repertoire across the human life span. High-throughput sequencing of the TCRβ CDR3 regions was performed in sorted, naïve CD8+ T cells from extremely preterm (23–27 weeks gestation), term (39–40 weeks gestation), children aged 16 months to 4 years old, and adults aged 25–50 years (n=5 in each group). There were differences in the CDR3 length across the life span, with preterm infants having a shorter CDR3 length, which was due to fewer N1 and N2 additions (p

Infants less than six months of age are particularly susceptible to influenza infection due to their impaired cytotoxic T cell response. The neonate relies on innate immunity to contain infection without excessive inflammatory response, by using pattern recognition molecules, like collectins and TLRs. TLR2 has been identified as a bacterial recognition receptor. However, little is known about its role in viral recognition and associated pathogenesis, particularly in the neonate. To address this, 3-day old TLR-2−/− and control C57Bl/6 mice were infected with influenza type A virus strain PR/8/34 (H1N1) and tracked for survival. Bronchoalveolar lavage fluid (BALF) was collected and lungs were harvested at post infection day (PID) 1, 3, and 6. BALF was analyzed by flow cytometry for cellular infiltration in the airways. RNA was isolated and analyzed via real-time PCR for viral loads and various cytokines. There was a striking difference in survival between the TLR-2−/− (61%) and control neonates (25%) (p< 0.01). Surprisingly, TLR-2−/− mice had higher viral loads than controls (p < 0.05) on PID 1, which was coupled with lower neutrophil numbers in the BALF (p < 0.02). No significant differences were noted on PID 3 or 6 for neutrophil counts or viral loads. On PID 3, TLR-2−/− mice had more than a 2-fold lower transcription of CXCL-5, a key chemoattractant for neutrophils. Therefore, the resistance of neonatal TLR-2−/− mice to influenza infection was not due to heightened influenza clearance. Rather, there was impaired neutrophil recruitment, which potentially reduced pulmonary inflammation. Future studies using our neonatal murine model could elucidate the mechanism of TLR-2 and CXCL-5 mediated inflammation during neonatal viral infection.