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The Development of Methods to Account for Physiologic Dynamic Changes and Their Effects on the Pharmacokinetics of Therapeutic Monoclonal Antibodies and other Therapeutics
Get PDFPhysiologic changes in the body can drastically affect the clearance of a medication, and therefore increase the variability in exposure to the medication. Physiologic changes that can have a profound effect on the exposure of a medication can stem from changes CYP enzymes, transport proteins, binding protein expression, organ function, immune reactivity, and health status to name a few; with the focus of this dissertation on the dynamic changes in the ontogeny of MRP2 (an apical liver transport protein) and the dynamic changes caused by an immune response to a therapeutic monoclonal antibody (mAb). Several approaches can be used to limit or capture the changes in the pharmacokinetics of a medication caused by ontogeny and immune reactivity related dynamic changes. Three approaches were investigated in this dissertation: 1) preventing/limiting immunogenicity’s effect on a therapeutic mAb, hence eliminating the increase in clearance and variability, 2) using a pharmacometric PK-ADA modeling approach to model immunology-related dynamic and variable effects on a therapeutic mAb and 3) using a systems pharmacology strategy to model the ontogeny changes in a transport protein (MRP2) and the dynamic effects on its drug substrates. In the preclinical and clinical setting, anti-drug antibodies (ADA) that develop against therapeutic mAbs can influence patient safety and interfere with product efficacy. Thus, my first focus in this dissertation investigates methods to limit/prevent immunogenicity and therefore help to eliminate a source of variability and clearance that can be seen in preclinical and clinical studies. My first study investigates the use of immune suppressants in mitigating ADA responses to a fully-humanized mAb in preclinical animal studies. Three groups of Sprague Dawley rats (n=18) were treated with low (0.01 mg/kg), moderate (50 mg/kg), or high (300 mg/kg) doses of a mAb. Experimental groups also received either methotrexate or tacrolimus/sirolimus immune suppression. Methotrexate significantly lowered the incidence of anti-variable region antibodies at moderate mAb dose (P\u3c0.05), while tacrolimus/sirolimus did likewise at moderate and high doses (P\u3c0.01) of mAb. With the exception of low dose mAb plus methotrexate, all immunosuppressed groups displayed more than a 70-fold decrease in ADA magnitude (P\u3c0.05). This abrogation in ADA response correlated with higher mAb exposure in the circulation by week 4 for the moderate and high dosed mAb groups. This method provides an approach to mitigate preclinical immunogenicity by the use of immunosuppressant modalities. Such preconditioning can support preclinical drug development of human therapeutics that are antigenic to animals but not necessarily to humans. Similar approaches to reduce immunogenicity will likely play an essential role with advances in novel therapeutics like fully human mAbs, recombinant proteins, fusion proteins as well as bispecific- and drug-conjugated antibodies. In some cases there may not be a method to reduce/eliminate immunogenicity and the dynamic changes in the elimination of a therapeutic mAb that result. In a preclinical setting, ADA typically influences both multiple dose toxicity studies, as well as preliminary pharmacokinetic (PK) analysis by leading to an increase in clearance of the therapeutic mAb. This increase in clearance caused by ADA can be highly variable due to each animal’s polyclonal immune response to a therapeutic mAb. My second focus aims to account for ADA and its variable effect on a fully human therapeutic mAb. I used data acquired from our previous study that investigated the use of immunosuppressant therapy in mitigating ADA responses to a mAb in a preclinical Sprague Dawley rat study and incorporated much of the data from that study, which included three mAb dosing groups and three immunomodulation therapies. A pharmacometric PK-ADA modeling approach was used to analyze the data. Our model was able to simultaneously capture the pharmacokinetics of the mAb in the presence and absence of ADA, accounting for an immune reaction’s highly variable effect on a therapeutic mAb concentration-time profile. The pharmacometric PK-ADA methodology used in this study demonstrates a modeling strategy that can be applied to other therapeutic mAbs to assess the immunogenicity of a therapeutic mAb and the dynamic effect immunogenicity has on the pharmacokinetics. This modeling methodology can further be applied to the simulation of therapeutic mAbs in the presence of varying rates, magnitudes and affinities of ADA reactions, aiding in the development of appropriately powered toxicology studies and an accurate pharmacokinetic evaluation of a human therapeutic mAb in a preclinical setting. Transport proteins play an important role in determining the disposition of medications in the human body. The expression of transport proteins in the body is not constant throughout childhood development, which affects the pharmacokinetics of a medication that is a substrate of the transport protein. Multidrug resistance protein 2 (MRP2) represents a major hepatic transporter whose expression is dynamic throughout development. MRP2 plays a vital role in the biliary excretion of various organic anions and cations along with glutathione-, glucuronate-, or sulfate-conjugates of several drug substrates. Our third aim is to evaluate the effect the ontogeny of MRP2 has on the pharmacokinetics of ceftriaxone to better understand how a transport protein contributes to the disposition of its substrates throughout childhood development. In order to accomplish our aim, a systems pharmacology modeling approach was used to understand MRP2’s contribution to the elimination of ceftriaxone and the effect of ontogeny changes on the pharmacokinetics of ceftriaxone in pediatric patients. Data from ex vivo studies, preclinical in vivo studies and clinical studies were used to inform our model. Results from the study demonstrate the contribution of MRP2 to the pharmacokinetics of ceftriaxone. Our model was able to capture ceftriaxone’s pharmacokinetics, and MRP2’s contribution to its clearance, allowing for the prediction of pediatric ceftriaxone concentrations. This modeling strategy can also be used to evaluate ontogeny changes in other biochemical transposition proteins, and the subsequent effect on the pharmacokinetics of other therapeutically used compounds. In summary, our work has successfully provided approaches to limit/prevent dynamic changes caused by immune reactions to a therapeutic mAb, demonstrate a pharmacometric PK-ADA approach that can capture the PK changes and variability caused by ADA formation on a therapeutic mAb and demonstrate a systems pharmacology model approach which accounts for the ontogeny of a transport protein and the resultant PK effects on its substrate through childhood development. The following chapters describe and discuss these novel approaches
Metabolic Regulation of Cellular Signaling
Get PDFUsing the biochemically tractable Xenopus oocyte model system, we have previously characterized a novel metabolic regulation of cell death. We found that glucose-6-phosphate (G6P) via the pentose phosphate pathway leads to increased nicotinamide adenine dinucleotide phosphate (NADPH) levels, a subsequent increase in cytosolic acetyl-coenzyme A and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). We recently identified coenzyme A (CoA), derived from the breakdown of acetyl-CoA, as the key metabolic signal that mediates a novel mechanism of calmodulindependent activation of CaMKII. CoA binds directly to the calmodulin (CaM) binding domain (CaMBD) of CaMKII resulting in its activation and downstream inhibitory phosphorylation of caspase-2, suppressing apoptosis. In this dissertation, we questioned whether there are other CaMBD containing proteins metabolically regulated by CoA. In an unbiased approach, CaM binding proteins were first isolated from Xenopus extract using a CaM-Sepharose column. Purified CaM binding proteins were then incubated with CoA-Sepharose in a second purification step and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results indicate the presence of numerous CaM-binding proteins that also bind CoA and are thus potentially metabolically regulated. In a targeted approach, we tested the ability of aberrant glucose signaling to regulate the CaM-binding protein PI3K. We found that addition of G6P, mimicking aberrant glucose metabolism, or CoA to X. laevis egg extracts activated Akt in a phosphatidylinositol 3-kinase (PI3K), phosphoinositidedependent protein kinase 1(PDPK1)-dependent manner. Additionally, we show that CoA binds directly to and activates PI3K. These findings uncover a novel mechanism of PI3K activation by aberrant glucose metabolism and suggest a potentially unknown constitutive activation pathway of PI3K/Akt by aberrant glucose signaling
Psychometric Evaluation of the Nursing Child Assessment Teaching (NCAT) Scale in Two Samples of Mothers and Children
Get PDFThe mother-child relationship (MCR) has received an increasing amount of attention over the last several decades regarding its effect on long-term child development. Because the quality and quantity of interactions in the MCR have been established as important predictors of the child’s development, early identification of areas in the relationship requiring support and intervention is essential for promoting positive child outcomes. Observational assessment of the mother and child is considered best practice in evaluating maternal-child interaction (MCI). The Nursing Child Assessment Teaching (NCAT) scale is an instrument that has been used to quantify the quality of interaction in the MCR during the first 36-months of a child’s life. While studies have shown the NCAT scale as both a reliable and valid instrument, limited evidence exists confirming theoretical congruence between the scale and the Barnard Model it is based on. These analyses were conducted using data from two large, demographically different samples, the Nursing Child Assessment Satellite Training (NCAST) database and the Conditions Affecting Neurocognitive Development and Learning in Early Childhood database. Item response theory, confirmatory factor analysis, and multiple indicators multiple causes modeling were used to examine the psychometric properties of the NCAT scale and describe the interactions between mothers and children from these samples. Results of analyses offer support for the Barnard Model and the potential for instrument abbreviation, which may provide researchers and practitioners a more concise, reliable way of measuring MCI. In addition, the predictive validity of both the full NCAT scale and resulting abbreviated NCAT scale (NCAT-AB) was established by obtaining Pearson correlations and associated probabilities for MCI at 12-months, measured by the NCAT and NCAT-AB, and child cognitive and language development at 36-months, as measured by the Bayley Scales of Infant and Toddler Development, Third Edition. This study provided critical appraisal of the NCAT scale, supported the continued use of the NCAT scale in evaluating MCI, and contributed to the growing body of literature surrounding the importance of the MCR on early child development
Opening a Student-Run Pro Bono Occupational Therapy Pediatric Clinic
Get PDFThis presentation described the opening of a student-run pro bono pediatric therapy clinic that provides services to children/families who are economically disadvantaged. The benefits of participation for students and clients were reviewed, and fund-raising and the development of a mission statement were discussed
Using S. pombe to Study the Biological Roles of the Histone Deacetylases Sir2 and Clr3 and the DEAD-Box RNA Helicase Ded1
Get PDFThe fission yeast Schizosaccharomyces pombe provides a good model system to quickly study basic mechanisms underlying biological pathways conserved in higher eukaryotes. Here we utilized fission yeast to study the roles of the histone deacetylases (HDACs) Sir2 and Clr3 in heterochromatin formation and cancer associated mutations of the DEAD-box RNA helicase DDX3X, homolog of fission yeast Ded1, in translational control. Heterochromatin in fission yeast is characterized by hypoacetylation as well as methylation of histone H3 on lysine 9 (H3K9me). Heterochromatin assembly can now be separated into three distinct steps: heterochromatin establishment, spreading, and maintenance. These steps involve the actions of the histone H3K9 methyltransferase Clr4 along with the RNAi pathway. HDACs are also required for heterochromatin assembly, with the H3K9 HDAC Sir2 and H3K14 HDAC Clr3 participating in the processes of heterochromatin establishment and maintenance. Here, we show that both a serine rich patch within the N-terminal domain of Sir2 and the HDAC activity of Sir2 are required for proper recruitment of Sir2 to chromatin and for heterochromatin establishment. We also report that Sir2 shares similarity in global transcriptional control with Mit1 and Chp2, components of the Snf2/HDAC-containing repressor complex (SHREC), suggesting a connection between Sir2 and SHREC in transcriptional regulation. We also identified additional sites that are deacetylated by the SHREC HDAC Clr3 in addition to H3K14, specifically H2B K5, 6, 10, and 15. Using fission yeast, this study also examined the biological consequence of medulloblastoma associated mutations of the DEAD-box ATP-dependent RNA helicase DDX3X. In fission yeast, Ded1 is an essential protein with connections to both the RNAi pathway and translation of transcripts with complex UTRs. Here we show that human DDX3X can functionally complement for growth defects observed in thermosensitive ded1 mutants while specific cancer-associated DDX3X mutants (A222P, G302V, G325E, and P568L) cannot complement this growth defect. Fission yeast bearing these specific DDX3X mutants exhibit defects in the expression of specific proteins, suggesting the mutant proteins impair translational control
Genome-scale Precision Proteomics Identifies Cancer Signaling Networks and Therapeutic Vulnerabilities
Get PDFMass spectrometry (MS) based-proteomics technology has been emerging as an indispensable tool for biomedical research. But the highly diverse physical and chemical properties of the protein building blocks and the dramatic human proteome complexity largely limited proteomic profiling depth. Moreover, there was a lack of high-throughput quantitative strategies that were both precise and parallel to in-depth proteomic techniques. To solve these grand challenges, a high resolution liquid chromatography (LC) system that coupled with an advanced mass spectrometer was developed to allow genome-scale human proteome identification. Using the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction, we enabled precise proteome quantification with isobaric labeling. We then applied these advanced proteomics tools for cancer proteome analyses on high grade gliomas (HGG) and rhabdomyosarcomas (RMS). Using systems biology approaches, we demonstrated that these newly developed proteomic analysis pipelines are able to (i) define human proteotypes that link oncogenotypes to cancer phenotypes in HGG and to (ii) identify therapeutic vulnerabilities in RMS. Development of high resolution liquid chromatography is essential for improving the sensitivity and throughput of mass spectrometry-based proteomics to genome-scale. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse phase long column (100 µm x 150 cm, 5 µm C18 beads) coupled with Q Exactive MS. The column was capable of achieving a peak capacity of approximately 700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading amount was about 6 micrograms of peptides, although the column allowed loading as many as 20 micrograms. Gas phase fractionation of peptide ions further increased the number of peptides identified by ~10%. Moreover, the combination of basic pH LC pre-fractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a postmortem brain sample of Alzheimer’s disease. As deep RNA sequencing of the same specimen suggested that ~16,000 genes were expressed, current analysis covered more than 60% of the expressed proteome. Isobaric labeling quantification by mass spectrometry has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from co-isolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT) labeled E. coli peptides at 1 : 3 : 10 ratios, and added in ~20-fold more rat peptides as background, followed by the analysis of two dimensional liquid chromatography-MS/MS. Systematic investigation indicated that the quantitative interference was impacted by LC fractionation depth, MS isolation window and peptide loading amount. Exhaustive fractionation (320 x 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicated that intermediate fractionation (40 x 2 h) and y1 ion-based correction allowed accurate and deep TMT protein profiling, which represents a straightforward and affordable strategy in isobaric labeling proteomics High throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma mouse models driven by mutated receptor tyrosine kinase (RTK) oncogenes, platelet-derived growth factor receptor alpha (PDGFRA) and neurotrophic receptor tyrosine kinase 1 (NTRK1), analyzing 13,860 proteins (11,941 genes) and 30,431 phosphosites by mass spectrometry. Systems biology approaches identified numerous functional modules and master regulators, including 41 kinases and 26 transcription factors. Pathway activity computation and mouse survival curves indicate the NTRK1 mutation induces a higher activation of AKT targets, drives a positive feedback loop to up-regulate multiple other RTKs, and shows higher oncogenic potency than the PDGFRA mutation. Further integration of the mouse data with human HGG transcriptome data determines shared regulators of invasion and stemness. Thus, multi-omics integrative profiling is a powerful avenue to characterize oncogenic activity. There is growing emphasis on personalizing cancer therapy based on somatic mutations identified in patient’s tumors. Among pediatric solid tumors, RAS pathway mutations in rhabdomyosarcoma are the most common potentially actionable lesions. Recent success targeting CDK4/6 and MEK in RAS mutant adult cancers led our collaborator Dr. Dyer’s group to test this approach for rhabdomyosarcoma. They achieved synergistic killing of RAS mutant rhabdomyosarcoma tumor cells by combining MEK and CDK4/6 inhibitors in culture but failed to achieve efficacy in vivo using orthotopic patient derived xenografts (O-PDXs). To determine how rhabdomyosarcomas evade targeting of CDK4/6 and MEK, we collaborated to perform large-scale deep proteomic, phosphoproteomic, and epigenomic profiling of RMS tumors. Integrative analysis of these omics data detected that RMS tumor cells rapidly compensate and overcome CDK4/6 and MEK combination therapy through 6 myogenic signal transduction pathways including WNT, HH, BMP, Adenyl Cyclase, P38/MAPK and PI3K. While it is not feasible to target each of these signal transduction pathways simultaneously in RMS, we discovered that they require the HSP90 chaperone to sustain the complex developmental signal transduction milieu. We achieved specific and synergistic killing of RMS cells using sub-therapeutic concentrations of an HSP90 inhibitor (ganetespib) in combination with conventional chemotherapy used for recurrent RMS. These effects were seen in the most aggressive recurrent RMS orthotopic patient derived xenografts irrespective of RAS pathway perturbations, histologic or molecular classification. Thus, multi-omics integrative cancer profiling using our newly developed tools is powerful to identify core signaling transduction networks, tumor vulnerability (master regulators) for novel cancer therapy
Quantifying Joint Coordination Variability in Anterior Cruciate Ligament-Reconstructed Individuals During Walking
Get PDFThe knee is the second most common joint to sustain injury. An estimated 200,000 anterior cruciate ligament (ACL) ruptures occur each year in the United States alone, and about 100,000 ACL reconstruction (ACLR) surgeries are performed annually. There is a significant risk of developing osteoarthritis of the knee after incurring an ACL injury, and the incidence of ipsilateral or contralateral injury is six times greater in individuals who have a surgically repaired ACL. Past studies have analyzed kinetic and kinematic characteristics of individual lower extremity joints to reveal differences between subjects with and without ACLR. Despite reports of altered kinematic performance in individuals with ACLR compared to healthy controls, most of the analyses did not evaluate coordinative function, and thus neglected to consider how the lower limb acts as a linked chain. Therefore, the present study used a method based on dynamical systems theory to quantify coordination and account for the interaction between joints in the lower extremity. The purpose of the study was to quantify and compare joint coordination variability and joint coordination patterns between individuals with ACLR and matched controls. Institutional Review Board (IRB) approval was obtained prior to data collection, and all subjects signed an informed consent form. Twenty subjects (nine females, eleven males; body mass index (BMI) 25±3.5 kg/m2) who had undergone unilateral ACLR (thirteen right, seven left) and been cleared to return to full activity were compared to twenty control subjects matched by gender and BMI (nine females, eleven males; BMI 22.4±2.4 km/m2). Kinematic and kinetic data during walking were collected in the UTHSC Motion Analysis Laboratory. A vector coding technique was used to calculate coupling angles for six joint couplings involving the hip, knee, and ankle across four periods within the stance phase. Joint coordination variability was defined as the standard deviation of the coupling angle between trials within a subject, and joint coordination patterns were based on coupling angle magnitude. Individuals with ACLR exhibited increased joint coordination variability and altered joint coordination patterns compared to the matched controls during the stance phase of walking. These results suggested that coordinative function may not be fully restored in individuals with ACLR following rehabilitation. Increased coordination variability from a normal, or optimal amount as well as altered coordination patterns may result from a deficit in sensorimotor control, and represent risk of re-injury. Further investigation that is prospective, focuses primarily on hip-knee coupled motion in frontal and transverse planes, and includes assessment of EMG in addition to kinematics may contribute relevant information for improving ACL injury prevention and rehabilitation
Assessing Neuronal Synchrony and Brain Function Through Local Field Potential and Spike Analysis
Get PDFStudies of neuronal network oscillations and rhythmic neuronal synchronization have led to a number of important insights in recent years, giving us a better understanding of the temporal organization of neuronal activity related to essential brain functions like sensory processing and cognition. Important principles and theories have emerged from these findings, including the communication through coherence hypothesis, which proposes that synchronous oscillations render neuronal communication effective, selective, and precise. The implications of such a theory may be universal for brain function, as the determinants of neuronal communication inextricably shape the neuronal representation of information in the brain. However, the study of communication through coherence is still relatively young. Since its articulation in 2005, the theory has predominantly been applied to assess cortical function and its communication with downstream targets in different sensory and behavioral conditions. The results herein are intended to bolster this hypothesis and explore new ways in which oscillations coordinate neuronal communication in distributed regions. This includes the development of new analytic tools for interpreting electrophysiological patterns, inspired by phase synchronization and spike train analysis. These tools aim to offer fast results with clear statistical and physiological interpretation
Design, Synthesis, and Evaluation of Novel Positron Emission Tomography Radiotracers
Get PDFNeuroblastoma (NB) is the most common extracranial tumor in patients under 1 year of age and it constitutes about 8-10% of all childhood cancer. It originates from neural crest cells that normally differentiate to form the sympathetic ganglia, adrenal medulla and other paraspinal sites where sympathetic nervous system tissue is present. Even with an extensive treatment regimen that typically includes surgery, chemotherapy, total body irradiation and autologous stem cell transplantation, the 5-year event-free survival is \u3c50% for high risk patients, and there are numerous long-term side effects associated with treatment. This body of work investigated two projects for improving patient outcomes through the development of positron emission tomography (PET) radiotracers that could be used for therapy planning. The goal of the first project was to design, synthesize, and evaluate PET radiotracers that could measure the enzymatic activation of Irinotecan (CPT-11), a potent chemotherapeutic used in the treatment of colon cancer and several pediatric solid tumors. CPT-11 itself is a prodrug which is converted in vivo to SN-38, via metabolism by carboxylesterase (CE) enzymes. St. Jude Children’s Research Hospital researchers have designed a two-pronged protocol of tumor-targeted CPT-11 chemotherapy combining the complementary approaches of a) specific modulation of human CE in normal tissues to improve drug delivery, and b) tumor-targeted activation of prodrug using neural progenitor cells (NPC) transfected with a mutant human CE cDNA. The tumor-selective trafficking of NPC allows over-expression of CE within the tumor. This prodrug/activating enzyme therapeutic approach has shown extremely encouraging preclinical results in the treatment of NB (90% 1-year survival in mice). However, successful translation of this novel therapeutic approach into general clinical practice requires a better understanding of progenitor cell trafficking, duration and intensity of enzymatic activity and the ultimate biological fate of the therapeutic construct. Toward this end, PET radiotracers were developed based on extensive structure-activity relationship (SAR) studies of CE binding. The goal of the second project was to design, synthesize, and evaluate PET radiotracers that could identify the presence of the tropomyosin receptor kinase B (TrkB). TrkB is not normally found in sympathetic nervous tissue, which is the tissue NB develops from, and thus is a potential target for imaging and therapy. The presence of TrkB and its neurotrophin, brain derived neurotrophic factor (BDNF), have been reported to protect neuroblastoma tumor cells from chemotherapy-induced apoptosis via a phosphatidylinositol 3’-kinase pathway. Radiotracers were synthesized and evaluated for their ability to identify TrkB both in vitro and in vivo. PET radiosynthetic procedures were optimized to synthesize novel radiotracers for imaging targets that could help clinicians monitor therapy or identify markers that would aid in therapy planning for NB patients. The method development could be applied to future compounds that show improved chemical characteristics for synthesis and selectivity
Role of Dendritic Cells in Pathology of Respiratory Syncytial Virus Infection in Neonates
Get PDFRespiratory syncytial virus (RSV) is one of the leading causes of bronchiolitis in children. We have shown that neonatal mice respond to primary RSV infection with T helper type 2 (Th2) biased immune responses, which are enhanced following reinfection. Dendritic cells (DCs) including myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) play important roles in driving host responses to RSV infection. mDCs present antigens to help Th cells differentiate, and pDCs protect against viral infection through type I interferons (IFNs). Despite data demonstrating importance of mDCs and pDCs in protection against RSV, it has not been studied in an age appropriate model. Using a neonatal mouse model, we have shown that downregulation of IL-4 receptor alpha (IL-4Rα) on pulmonary myeloid dendritic cells (mDCs) via antisense oligonucleotides protected against RSV induced Th2 immunopathology. Thus, we examined the role of IL-4Rα on mDCs in RSV infection. Here, we discovered that IL-4Rα is developmentally regulated such that neonates have higher levels of IL-4Rα on mDCs. To determine if this elevated expression of IL-4Rα on mDCs was responsible for RSV pathogenesis in neonatal mice, we specifically deleted it from neonatal mDCs (or overexpressed it on adult mDCs) and studied RSV pathogenesis using our neonatal mouse model of RSV infection. Deletion of IL-4Rα from mDCs in our neonatal RSV infection model resulted in reduced disease as evidenced by reduction in Th2 biased inflammation and mucus cell hyperplasia and production. This was accompanied by improved lung function and enhanced mDC maturation status after infection. Furthermore, overexpression of IL-4Rα on adult mDCs was able to induce RSV disease similar to that observed in our neonatal model of RSV infection (i.e., Th2 biased responses including mucus hyperproduction). In vitro CD4+ T cell differentiation assays using mDCs from neonatal littermate control or IL-4Rα-/- mice were also performed to determine the specificity of the in vivo response. Low levels of type I IFNs have been reported in the nasal aspirates of RSV-infected infants. Since pDCs are responsible for a significant proportion of type I IFN production, we characterized the role of type I IFNs and pDC responses in the immunopathogenesis during RSV reinfection in our neonatal mouse model. We found that neonatal pDCs, while recruited to the airways, are recruited in low numbers in the response to neonatal RSV infection. Further, those pDCs that are recruited produce insufficient quantities of type I IFNs. Supplying IFN-α or adult pDCs locally to the neonatal mouse prior to RSV infection in neonates abrogated RSV induced immunopathophysiologies and this protection remained even after reinfection. Specifically, it reduced Th2 responses and lung inflammation and improved lung function. This improvement was due to a decrease in viral load and IL-4Rα expression on Th2 cells after IFN-α treatment. The severity of RSV pathogenesis in infants stems partly from immature immune responses. Our data demonstrate that developmentally regulated IL-4Rα expression on mDCs and inefficient type I IFN production from neonatal pDCs are critical for protection against RSV induced immunopathophysiologies. This study highlights critical differences between neonatal and adult DCs in RSV infection