Born in France, Dr. Gaudilliere studied Engineering at Ecole Polytechnique before completing an MD-PhD degree from the Harvard-MIT Health Sciences and Technology program. During his postdoctoral fellowship in Dr. Garry Nolan’s laboratory (Stanford University), Dr. Gaudilliere developed and standardized a pipeline to implement high dimensional mass cytometry (a.k.a flow cytometry by time of flight mass spectrometry, or CyTOF) in clinical studies.
Dr. Gaudilliere's research group combines high parameter mass cytometry (suspension and imaging mass cytometry) with other proteomics approaches to study how the human immune system responds and adapts to physiological or pathological stressors. Ongoing studies in the Gaudilliere lab focus on several clinical scenarios including, 1) surgical recovery and traumatic injury, 2) pregnancy and preterm birth (in collaboration with the Bill and Melinda Gates and the March of Dimes foundation, 3) neurocognitive recovery after stroke.
Dr. Gaudilliere is also a Board-Certified Anesthesiologist and works clinically in the operating room 25% of his time.
Honors & Awards
Promise in biomedical research, Doris Duke Charitable Foundation (2017)
Kozaka research award, International Anesthesia Research Society (2013)
Stanford Society for Physician Scholar's award, SSPS (2011, 2012)
Albert J. Ryan Fellowship, Harvard Medical School (2004)
Carnot Foundation Scholarship, Ecole Polytechnique, Paris, France (2000)
George Lurcy's Fellowship, Harvard University (2000)
BS, Ecole Polytechnique, Engineering (2000)
PhD, Harvard Medical School, Neurobiology (2005)
Medical Education: Harvard Medical School (2009) MA
Residency: Stanford University Anesthesiology Residency (2013) CA
Board Certification: American Board of Anesthesiology, Anesthesia (2014)
Brice Gaudilliere, Gabriela Fragiadakis, Martin Angst, Garry Nolan. "United States Patent S13-373 Flow cytometry methods for the diagnosis of prolonged convalescence after traumatic injury", Leland Stanford Junior University
Azad Bonni, Aryaman Shalizi, Brice Gaudilliere. "United StatesMethods and compositions for modulating synapse formation", Harvard University
Current Research and Scholarly Interests
The advent of high dimensional flow cytometry has revolutionized our ability to study and visualize the human immune system. Our group combines high parameter mass cytometry (a.k.a Cytometry by Time of Flight Mass Spectrometry, CyTOF), with advanced bio-computational methods to study how the human immune system responds and adapts to acute physiological perturbations. The laboratory currently focuses on two clinical scenarios: surgical trauma and pregnancy.
Deep immune profiling of patients undergoing and recovering from surgery: Using high dimensional mass cytometry, we have recently shown that the signaling behavior of specific innate immune cells measured before surgery in patients blood was strongly associated with surgical recovery. Prospective validation of reported immune correlates of surgical recovery are underway. Ongoing work in humans and animal models focuses on the mechanisms by which pre-operative habilitation interventions may alter a patient’s immune state to improve recovery after surgery.
Deep Immune profiling of normal and preterm pregnancy: Our group is an integral component of a multi-disciplinary effort aiming at understanding the mechanisms of preterm birth, and identifying predictive factors of premature delivery. We have now developed a pipeline and the analytical framework to integrate the single-cell analysis of immune signaling networks by mass cytometry and proteomic profiling of secreted serum factors with the precise phenotyping of pregnancy-related clinical outcomes. In a pilot cross-sectional study of non-pregnant women, we identified candidate immune signatures that differentiated women with a history of preterm or term pregnancies. Longitudinal studies in pregnant patients are ongoing to validate these findings.
Detection of Immune Changes as a Result of Surgical Trauma in Human Subject
Surgical trauma triggers a massive inflammatory response. Over time, both the innate and adaptive branches of the immune system are affected by surgical trauma. The purpose of this study to characterize the cellular and molecular mechanisms immune response to surgical trauma. Additionally, detailed information about patients' recovery profile will be recorded over a period of 6 weeks, with the eventual goal of linking immune responses to recovery profiles.
Immune Modulation by Enhanced vs Standard Prehabilitation Program Before Major Surgery
Over 30 million surgeries are performed annually in the US. Up to 30% of surgical patients experience delayed surgical recovery, marked by prolonged post-surgical pain, opioid consumption, and functional impairment, which contributes $8 billion annually to US health care costs. Novel interventions that improve the resolution of pain, minimize opioid exposure, and accelerate functional recovery after surgery are urgently needed. Multi-modal pre-operative optimization programs (or "prehab") integrating exercise, nutrition, and stress reduction have been shown to safely and effectively improve outcomes after surgery. However, no objective biological markers assess prehab effectiveness and are able to tailor prehab programs to individual patients. Surgery is a profound immunological perturbation, during which a complex network of innate and adaptive immune cells is mobilized to organize the recovery process of wound healing, tissue repair, and pain resolution. As such, the in-depth assessment of a patient's immune system before surgery is a promising approach to tailor prehab programs to modifiable biological markers associated with surgical recovery. The primary goal of this clinical trial is to determine the effect of a personalized prehab program on patients immunological status before surgery.
Immune-modulation Effects of an Arginine Rich Nutritional Supplement in Surgical Patients
The primary objective of this study is to characterize the immune-modulatory effects of arginine-rich nutritional supplements in patients undergoing surgery. Numerical and functional changes of all circulating immune cells will be assessed with mass cytometry.
Stanford is currently not accepting patients for this trial. For more information, please contact Julian Silva, MA, 650-724-9341.
- Integration of mechanistic immunological knowledge into a machine learning pipeline improves predictions NATURE MACHINE INTELLIGENCE 2020
Preferential inhibition of adaptive immune system dynamics by glucocorticoids in patients after acute surgical trauma.
2020; 11 (1): 3737
Glucocorticoids (GC) are a controversial yet commonly used intervention in the clinical management of acute inflammatory conditions, including sepsis or traumatic injury. In the context of major trauma such as surgery, concerns have been raised regarding adverse effects from GC, thereby necessitating a better understanding of how GCs modulate the immune response. Here we report the results of a randomized controlled trial (NCT02542592) in which we employ a high-dimensional mass cytometry approach to characterize innate and adaptive cell signaling dynamics after a major surgery (primary outcome) in patients treated with placebo or methylprednisolone (MP). A robust, unsupervised bootstrap clustering of immune cell subsets coupled with random forest analysis shows profound (AUC = 0.92, p-value = 3.16E-8) MP-induced alterations of immune cell signaling trajectories, particularly in the adaptive compartments. By contrast, key innate signaling responses previously associated with pain and functional recovery after surgery, including STAT3 and CREB phosphorylation, are not affected by MP. These results imply cell-specific and pathway-specific effects of GCs, and also prompt future studies to examine GCs' effects on clinical outcomes likely dependent on functional adaptive immune responses.
View details for DOI 10.1038/s41467-020-17565-y
View details for PubMedID 32719355
An immune clock of human pregnancy.
2017; 2 (15)
The maintenance of pregnancy relies on finely tuned immune adaptations. We demonstrate that these adaptations are precisely timed, reflecting an immune clock of pregnancy in women delivering at term. Using mass cytometry, the abundance and functional responses of all major immune cell subsets were quantified in serial blood samples collected throughout pregnancy. Cell signaling-based Elastic Net, a regularized regression method adapted from the elastic net algorithm, was developed to infer and prospectively validate a predictive model of interrelated immune events that accurately captures the chronology of pregnancy. Model components highlighted existing knowledge and revealed previously unreported biology, including a critical role for the interleukin-2-dependent STAT5ab signaling pathway in modulating T cell function during pregnancy. These findings unravel the precise timing of immunological events occurring during a term pregnancy and provide the analytical framework to identify immunological deviations implicated in pregnancy-related pathologies.
View details for PubMedID 28864494
Clinical recovery from surgery correlates with single-cell immune signatures.
Science translational medicine
2014; 6 (255): 255ra131-?
Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.
View details for DOI 10.1126/scitranslmed.3009701
View details for PubMedID 25253674
Proteomic signatures predict preeclampsia in individual cohorts but not across cohorts - implications for clinical biomarker studies.
The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians
Early identification of pregnant women at risk for preeclampsia (PE) is important, as it will enable targeted interventions ahead of clinical manifestations. The quantitative analyses of plasma proteins feature prominently among molecular approaches used for risk prediction. However, derivation of protein signatures of sufficient predictive power has been challenging. The recent availability of platforms simultaneously assessing over 1000 plasma proteins offers broad examinations of the plasma proteome, which may enable the extraction of proteomic signatures with improved prognostic performance in prenatal care.The primary aim of this study was to examine the generalizability of proteomic signatures predictive of PE in two cohorts of pregnant women whose plasma proteome was interrogated with the same highly multiplexed platform. Establishing generalizability, or lack thereof, is critical to devise strategies facilitating the development of clinically useful predictive tests. A second aim was to examine the generalizability of protein signatures predictive of gestational age (GA) in uncomplicated pregnancies in the same cohorts to contrast physiological and pathological pregnancy outcomes.Serial blood samples were collected during the first, second, and third trimesters in 18 women who developed PE and 18 women with uncomplicated pregnancies (Stanford cohort). The second cohort (Detroit), used for comparative analysis, consisted of 76 women with PE and 90 women with uncomplicated pregnancies. Multivariate analyses were applied to infer predictive and cohort-specific proteomic models, which were then tested in the alternate cohort. Gene ontology (GO) analysis was performed to identify biological processes that were over-represented among top-ranked proteins associated with PE.The model derived in the Stanford cohort was highly significant (p = 3.9E-15) and predictive (AUC = 0.96), but failed validation in the Detroit cohort (p = 9.7E-01, AUC = 0.50). Similarly, the model derived in the Detroit cohort was highly significant (p = 1.0E-21, AUC = 0.73), but failed validation in the Stanford cohort (p = 7.3E-02, AUC = 0.60). By contrast, proteomic models predicting GA were readily validated across the Stanford (p = 1.1E-454, R = 0.92) and Detroit cohorts (p = 1.1.E-92, R = 0.92) indicating that the proteomic assay performed well enough to infer a generalizable model across studied cohorts, which makes it less likely that technical aspects of the assay, including batch effects, accounted for observed differences.Results point to a broader issue relevant for proteomic and other omic discovery studies in patient cohorts suffering from a clinical syndrome, such as PE, driven by heterogeneous pathophysiologies. While novel technologies including highly multiplex proteomic arrays and adapted computational algorithms allow for novel discoveries for a particular study cohort, they may not readily generalize across cohorts. A likely reason is that the prevalence of pathophysiologic processes leading up to the "same" clinical syndrome can be distributed differently in different and smaller-sized cohorts. Signatures derived in individual cohorts may simply capture different facets of the spectrum of pathophysiologic processes driving a syndrome. Our findings have important implications for the design of omic studies of a syndrome like PE. They highlight the need for performing such studies in diverse and well-phenotyped patient populations that are large enough to characterize subsets of patients with shared pathophysiologies to then derive subset-specific signatures of sufficient predictive power.
View details for DOI 10.1080/14767058.2021.1888915
View details for PubMedID 33653202
Data-Driven Modeling of Pregnancy-Related Complications.
Trends in molecular medicine
A healthy pregnancy depends on complex interrelated biological adaptations involving placentation, maternal immune responses, and hormonal homeostasis. Recent advances in high-throughput technologies have provided access to multiomics biological data that, combined with clinical and social data, can provide a deeper understanding of normal and abnormal pregnancies. Integration of these heterogeneous datasets using state-of-the-art machine-learning methods can enable the prediction of short- and long-term health trajectories for a mother and offspring and the development of treatments to prevent or minimize complications. We review advanced machine-learning methods that could: provide deeper biological insights into a pregnancy not yet unveiled by current methodologies; clarify the etiologies and heterogeneity of pathologies that affect a pregnancy; and suggest the best approaches to address disparities in outcomes affecting vulnerable populations.
View details for DOI 10.1016/j.molmed.2021.01.007
View details for PubMedID 33573911
Single-cell peripheral immunoprofiling of Alzheimer's and Parkinson's diseases.
2020; 6 (48)
Peripheral blood mononuclear cells (PBMCs) may provide insight into the pathogenesis of Alzheimer's disease (AD) or Parkinson's disease (PD). We investigated PBMC samples from 132 well-characterized research participants using seven canonical immune stimulants, mass cytometric identification of 35 PBMC subsets, and single-cell quantification of 15 intracellular signaling markers, followed by machine learning model development to increase predictive power. From these, three main intracellular signaling pathways were identified specifically in PBMC subsets from people with AD versus controls: reduced activation of PLCgamma2 across many cell types and stimulations and selectively variable activation of STAT1 and STAT5, depending on stimulant and cell type. Our findings functionally buttress the now multiply-validated observation that a rare coding variant in PLCG2 is associated with a decreased risk of AD. Together, these data suggest enhanced PLCgamma2 activity as a potential new therapeutic target for AD with a readily accessible pharmacodynamic biomarker.
View details for DOI 10.1126/sciadv.abd5575
View details for PubMedID 33239300
- Towards personalized medicine in maternal and child health: integrating biologic and social determinants. Pediatric research 2020
Multiomic immune clockworks of pregnancy.
Seminars in immunopathology
Preterm birth is the leading cause of mortality in children under the age of five worldwide. Despite major efforts, we still lack the ability to accurately predict and effectively prevent preterm birth. While multiple factors contribute to preterm labor, dysregulations of immunological adaptations required for the maintenance of a healthy pregnancy is at its pathophysiological core. Consequently, a precise understanding of these chronologically paced immune adaptations and of the biological pacemakers that synchronize the pregnancy "immune clock" is a critical first step towards identifying deviations that are hallmarks of peterm birth. Here, we will review key elements of the fetal, placental, and maternal pacemakers that program the immune clock of pregnancy. We will then emphasize multiomic studies that enable a more integrated view of pregnancy-related immune adaptations. Such multiomic assessments can strengthen the biological plausibility of immunological findings and increase the power of biological signatures predictive of preterm birth.
View details for DOI 10.1007/s00281-019-00772-1
View details for PubMedID 32020337
Discovery and validation of biomarkers to aid the development of safe and effective pain therapeutics: challenges and opportunities.
Nature reviews. Neurology
Pain medication plays an important role in the treatment of acute and chronic pain conditions, but some drugs, opioids in particular, have been overprescribed or prescribed without adequate safeguards, leading to an alarming rise in medication-related overdose deaths. The NIH Helping to End Addiction Long-term (HEAL) Initiative is a trans-agency effort to provide scientific solutions to stem the opioid crisis. One component of the initiative is to support biomarker discovery and rigorous validation in collaboration with industry leaders to accelerate high-quality clinical research into neurotherapeutics and pain. The use of objective biomarkers and clinical trial end points throughout the drug discovery and development process is crucial to help define pathophysiological subsets of pain, evaluate target engagement of new drugs and predict the analgesic efficacy of new drugs. In 2018, the NIH-led Discovery and Validation of Biomarkers to Develop Non-Addictive Therapeutics for Pain workshop convened scientific leaders from academia, industry, government and patient advocacy groups to discuss progress, challenges, gaps and ideas to facilitate the development of biomarkers and end points for pain. The outcomes of this workshop are outlined in this Consensus Statement.
View details for DOI 10.1038/s41582-020-0362-2
View details for PubMedID 32541893
Systematic Immunophenotyping Reveals Sex-Specific Responses After Painful Injury in Mice.
Frontiers in immunology
2020; 11: 1652
Many diseases display unequal prevalence between sexes. The sex-specific immune response to both injury and persistent pain remains underexplored and would inform treatment paradigms. We utilized high-dimensional mass cytometry to perform a comprehensive analysis of phenotypic and functional immune system differences between male and female mice after orthopedic injury. Multivariate modeling of innate and adaptive immune cell responses after injury using an elastic net algorithm, a regularized regression method, revealed sex-specific divergence at 12 h and 7 days after injury with a stronger immune response to injury in females. At 12 h, females upregulated STAT3 signaling in neutrophils but downregulated STAT1 and STAT6 signals in T regulatory cells, suggesting a lack of engagement of immune suppression pathways by females. Furthermore, at 7 days females upregulated MAPK pathways (p38, ERK, NFkB) in CD4T memory cells, setting up a possible heightened immune memory of painful injury. Taken together, our findings provide the first comprehensive and functional analysis of sex-differences in the immune response to painful injury.
View details for DOI 10.3389/fimmu.2020.01652
View details for PubMedID 32849569
View details for PubMedCentralID PMC7403191
Hypothalamic circuitry underlying stress-induced insomnia and peripheral immunosuppression
2020; 6 (37)
View details for DOI 10.1126/sciadv.abc2590
Multiomics Characterization of Preterm Birth in Low- and Middle-Income Countries.
JAMA network open
2020; 3 (12): e2029655
Worldwide, preterm birth (PTB) is the single largest cause of deaths in the perinatal and neonatal period and is associated with increased morbidity in young children. The cause of PTB is multifactorial, and the development of generalizable biological models may enable early detection and guide therapeutic studies.To investigate the ability of transcriptomics and proteomics profiling of plasma and metabolomics analysis of urine to identify early biological measurements associated with PTB.This diagnostic/prognostic study analyzed plasma and urine samples collected from May 2014 to June 2017 from pregnant women in 5 biorepository cohorts in low- and middle-income countries (LMICs; ie, Matlab, Bangladesh; Lusaka, Zambia; Sylhet, Bangladesh; Karachi, Pakistan; and Pemba, Tanzania). These cohorts were established to study maternal and fetal outcomes and were supported by the Alliance for Maternal and Newborn Health Improvement and the Global Alliance to Prevent Prematurity and Stillbirth biorepositories. Data were analyzed from December 2018 to July 2019.Blood and urine specimens that were collected early during pregnancy (median sampling time of 13.6 weeks of gestation, according to ultrasonography) were processed, stored, and shipped to the laboratories under uniform protocols. Plasma samples were assayed for targeted measurement of proteins and untargeted cell-free ribonucleic acid profiling; urine samples were assayed for metabolites.The PTB phenotype was defined as the delivery of a live infant before completing 37 weeks of gestation.Of the 81 pregnant women included in this study, 39 had PTBs (48.1%) and 42 had term pregnancies (51.9%) (mean [SD] age of 24.8 [5.3] years). Univariate analysis demonstrated functional biological differences across the 5 cohorts. A cohort-adjusted machine learning algorithm was applied to each biological data set, and then a higher-level machine learning modeling combined the results into a final integrative model. The integrated model was more accurate, with an area under the receiver operating characteristic curve (AUROC) of 0.83 (95% CI, 0.72-0.91) compared with the models derived for each independent biological modality (transcriptomics AUROC, 0.73 [95% CI, 0.61-0.83]; metabolomics AUROC, 0.59 [95% CI, 0.47-0.72]; and proteomics AUROC, 0.75 [95% CI, 0.64-0.85]). Primary features associated with PTB included an inflammatory module as well as a metabolomic module measured in urine associated with the glutamine and glutamate metabolism and valine, leucine, and isoleucine biosynthesis pathways.This study found that, in LMICs and high PTB settings, major biological adaptations during term pregnancy follow a generalizable model and the predictive accuracy for PTB was augmented by combining various omics data sets, suggesting that PTB is a condition that manifests within multiple biological systems. These data sets, with machine learning partnerships, may be a key step in developing valuable predictive tests and intervention candidates for preventing PTB.
View details for DOI 10.1001/jamanetworkopen.2020.29655
View details for PubMedID 33337494
Landscape of coordinated immune responses to H1N1 challenge in humans.
The Journal of clinical investigation
Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.
View details for DOI 10.1172/JCI137265
View details for PubMedID 33044226
Changes in pregnancy-related serum biomarkers early in gestation are associated with later development of preeclampsia.
2020; 15 (3): e0230000
Placental protein expression plays a crucial role during pregnancy. We hypothesized that: (1) circulating levels of pregnancy-associated, placenta-related proteins throughout gestation reflect the temporal progression of the uncomplicated, full-term pregnancy, and can effectively estimate gestational ages (GAs); and (2) preeclampsia (PE) is associated with disruptions in these protein levels early in gestation; and can identify impending PE. We also compared gestational profiles of proteins in the human and mouse, using pregnant heme oxygenase-1 (HO-1) heterozygote (Het) mice, a mouse model reflecting PE-like symptoms.Serum levels of placenta-related proteins-leptin (LEP), chorionic somatomammotropin hormone like 1 (CSHL1), elabela (ELA), activin A, soluble fms-like tyrosine kinase 1 (sFlt-1), and placental growth factor (PlGF)-were quantified by ELISA in blood serially collected throughout human pregnancies (20 normal subjects with 66 samples, and 20 subjects who developed PE with 61 samples). Multivariate analysis was performed to estimate the GA in normal pregnancy. Mean-squared errors of GA estimations were used to identify impending PE. The human protein profiles were then compared with those in the pregnant HO-1 Het mice.An elastic net-based gestational dating model was developed (R2 = 0.76) and validated (R2 = 0.61) using serum levels of the 6 proteins measured at various GAs from women with normal uncomplicated pregnancies. In women who developed PE, the model was not (R2 = -0.17) associated with GA. Deviations from the model estimations were observed in women who developed PE (P = 0.01). The model developed with 5 proteins (ELA excluded) performed similarly from sera from normal human (R2 = 0.68) and WT mouse (R2 = 0.85) pregnancies. Disruptions of this model were observed in both human PE-associated (R2 = 0.27) and mouse HO-1 Het (R2 = 0.30) pregnancies. LEP outperformed sFlt-1 and PlGF in differentiating impending PE at early human and late mouse GAs.Serum placenta-related protein profiles are temporally regulated throughout normal pregnancies and significantly disrupted in women who develop PE. LEP changes earlier than the well-established biomarkers (sFlt-1 and PlGF). There may be evidence of a causative action of HO-1 deficiency in LEP upregulation in a PE-like murine model.
View details for DOI 10.1371/journal.pone.0230000
View details for PubMedID 32126118
VoPo leverages cellular heterogeneity for predictive modeling of single-cell data.
2020; 11 (1): 3738
High-throughput single-cell analysis technologies produce an abundance of data that is critical for profiling the heterogeneity of cellular systems. We introduce VoPo (https://github.com/stanleyn/VoPo), a machine learning algorithm for predictive modeling and comprehensive visualization of the heterogeneity captured in large single-cell datasets. In three mass cytometry datasets, with the largest measuring hundreds of millions of cells over hundreds of samples, VoPo defines phenotypically and functionally homogeneous cell populations. VoPo further outperforms state-of-the-art machine learning algorithms in classification tasks, and identified immune-correlates of clinically-relevant parameters.
View details for DOI 10.1038/s41467-020-17569-8
View details for PubMedID 32719375
Integration of mechanistic immunological knowledge into a machine learning pipeline improves predictions.
Nature machine intelligence
2020; 2 (10): 619–28
The dense network of interconnected cellular signalling responses that are quantifiable in peripheral immune cells provides a wealth of actionable immunological insights. Although high-throughput single-cell profiling techniques, including polychromatic flow and mass cytometry, have matured to a point that enables detailed immune profiling of patients in numerous clinical settings, the limited cohort size and high dimensionality of data increase the possibility of false-positive discoveries and model overfitting. We introduce a generalizable machine learning platform, the immunological Elastic-Net (iEN), which incorporates immunological knowledge directly into the predictive models. Importantly, the algorithm maintains the exploratory nature of the high-dimensional dataset, allowing for the inclusion of immune features with strong predictive capabilities even if not consistent with prior knowledge. In three independent studies our method demonstrates improved predictions for clinically relevant outcomes from mass cytometry data generated from whole blood, as well as a large simulated dataset. The iEN is available under an open-source licence.
View details for DOI 10.1038/s42256-020-00232-8
View details for PubMedID 33294774
View details for PubMedCentralID PMC7720904
- Cyt-Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2019 Conference Workshops. Cytometry. Part A : the journal of the International Society for Analytical Cytology 2019; 95 (12): 1236–74
CytoNorm: A Normalization Algorithm for Cytometry Data.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
High-dimensional flow cytometry has matured to a level that enables deep phenotyping of cellular systems at a clinical scale. The resulting high-content data sets allow characterizing the human immune system at unprecedented single cell resolution. However, the results are highly dependent on sample preparation and measurements might drift over time. While various controls exist for assessment and improvement of data quality in a single sample, the challenges of cross-sample normalization attempts have been limited to aligning marker distributions across subjects. These approaches, inspired by bulk genomics and proteomics assays, ignore the single-cell nature of the data and risk the removal of biologically relevant signals. This work proposes CytoNorm, a normalization algorithm to ensure internal consistency between clinical samples based on shared controls across various study batches. Data from the shared controls is used to learn the appropriate transformations for each batch (e.g., each analysis day). Importantly, some sources of technical variation are strongly influenced by the amount of protein expressed on specific cell types, requiring several population-specific transformations to normalize cells from a heterogeneous sample. To address this, our approach first identifies the overall cellular distribution using a clustering step, and calculates subset-specific transformations on the control samples by computing their quantile distributions and aligning them with splines. These transformations are then applied to all other clinical samples in the batch to remove the batch-specific variations. We evaluated the algorithm on a customized data set with two shared controls across batches. One control sample was used for calculation of the normalization transformations and the second control was used as a blinded test set and evaluated with Earth Mover's distance. Additional results are provided using two real-world clinical data sets. Overall, our method compared favorably to standard normalization procedures. The algorithm is implemented in the R package "CytoNorm" and available via the following link: www.github.com/saeyslab/CytoNorm © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
View details for DOI 10.1002/cyto.a.23904
View details for PubMedID 31633883
Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).
European journal of immunology
2019; 49 (10): 1457–1973
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
View details for DOI 10.1002/eji.201970107
View details for PubMedID 31633216
- Differential Dynamics of the Maternal Immune System in Healthy Pregnancy and Preeclampsia FRONTIERS IN IMMUNOLOGY 2019; 10
- A pilot study showing a stronger H1N1 influenza vaccination response during pregnancy in women who subsequently deliver preterm JOURNAL OF REPRODUCTIVE IMMUNOLOGY 2019; 132: 16–20
- How Clinical Flow Cytometry Rebooted Sepsis Immunology CYTOMETRY PART A 2019; 95A (4): 431–41
A year-long immune profile of the systemic response in acute stroke survivors.
Brain : a journal of neurology
Stroke is a leading cause of cognitive impairment and dementia, but the mechanisms that underlie post-stroke cognitive decline are not well understood. Stroke produces profound local and systemic immune responses that engage all major innate and adaptive immune compartments. However, whether the systemic immune response to stroke contributes to long-term disability remains ill-defined. We used a single-cell mass cytometry approach to comprehensively and functionally characterize the systemic immune response to stroke in longitudinal blood samples from 24 patients over the course of 1 year and correlated the immune response with changes in cognitive functioning between 90 and 365 days post-stroke. Using elastic net regularized regression modelling, we identified key elements of a robust and prolonged systemic immune response to ischaemic stroke that occurs in three phases: an acute phase (Day 2) characterized by increased signal transducer and activator of transcription 3 (STAT3) signalling responses in innate immune cell types, an intermediate phase (Day 5) characterized by increased cAMP response element-binding protein (CREB) signalling responses in adaptive immune cell types, and a late phase (Day 90) by persistent elevation of neutrophils, and immunoglobulin M+ (IgM+) B cells. By Day 365 there was no detectable difference between these samples and those from an age- and gender-matched patient cohort without stroke. When regressed against the change in the Montreal Cognitive Assessment scores between Days 90 and 365 after stroke, the acute inflammatory phase Elastic Net model correlated with post-stroke cognitive trajectories (r = -0.692, Bonferroni-corrected P = 0.039). The results demonstrate the utility of a deep immune profiling approach with mass cytometry for the identification of clinically relevant immune correlates of long-term cognitive trajectories.
View details for DOI 10.1093/brain/awz022
View details for PubMedID 30860258
Differential Dynamics of the Maternal Immune System in Healthy Pregnancy and Preeclampsia.
SAGE PUBLICATIONS INC. 2019: 271A
View details for Web of Science ID 000459610400616
- Multiomics modeling of the immunome, transcriptome, microbiome, proteome and metabolome adaptations during human pregnancy BIOINFORMATICS 2019; 35 (1): 95–103
Predicting Acute Pain After Surgery: A Multivariate Analysis.
Annals of surgery
To identify perioperative practice patterns that predictably impact postoperative pain.Despite significant advances in perioperative medicine, a significant portion of patients still experience severe pain after major surgery. Postoperative pain is associated with serious adverse outcomes that are costly to patients and society.The presented analysis took advantage of a unique observational data set providing unprecedented detailed pharmacological information. The data were collected by PAIN OUT, a multinational registry project established by the European Commission to improve postoperative pain outcomes. A multivariate approach was used to derive and validate a model predictive of pain on postoperative day 1 (POD1) in 1008 patients undergoing back surgery.The predictive and validated model was highly significant (P = 8.9E-15) and identified modifiable practice patterns. Importantly, the number of nonopioid analgesic drug classes administered during surgery predicted decreased pain on POD1. At least 2 different nonopioid analgesic drug classes (cyclooxygenase inhibitors, acetaminophen, nefopam, or metamizol) were required to provide meaningful pain relief (>30%). However, only a quarter of patients received at least 2 nonanalgesic drug classes during surgery. In addition, the use of very short-acting opioids predicted increased pain on POD1, suggesting room for improvement in the perioperative management of these patients. Although the model was highly significant, it only accounted for a relatively small fraction of the observed variance.The presented analysis offers detailed insight into current practice patterns and reveals modifications that can be implemented in today's clinical practice. Our results also suggest that parameters other than those currently studied are relevant for postoperative pain including biological and psychological variables.
View details for DOI 10.1097/SLA.0000000000003400
View details for PubMedID 31188202
Understanding health disparities.
Journal of perinatology : official journal of the California Perinatal Association
Based upon our recent insights into the determinants of preterm birth, which is the leading cause of death in children under five years of age worldwide, we describe potential analytic frameworks that provides both a common understanding and, ultimately the basis for effective, ameliorative action. Our research on preterm birth serves as an example that the framing of any human health condition is a result of complex interactions between the genome and the exposome. New discoveries of the basic biology of pregnancy, such as the complex immunological and signaling processes that dictate the health and length of gestation, have revealed a complexity in the interactions (current and ancestral) between genetic and environmental forces. Understanding of these relationships may help reduce disparities in preterm birth and guide productive research endeavors and ultimately, effective clinical and public health interventions.
View details for PubMedID 30560947
GateFinder: Projection-based Gating Strategy Optimization for Flow and Mass Cytometry.
Bioinformatics (Oxford, England)
High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging.Here we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers, and clarify underlying biology.The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://firstname.lastname@example.org or email@example.com.Supplementary data are available at Bioinformatics online.
View details for PubMedID 29850785
Multicenter Systems Analysis of Human Blood Reveals Immature Neutrophils in Males and During Pregnancy.
Journal of immunology
2017; 198 (6): 2479-2488
Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. In this study, we identified a gene signature of immature-like neutrophils, characterized by the overexpression of genes encoding for several granule-containing proteins, which was found at higher levels (up to 3-fold) in young (20-30 y old) but not older (60 to >89 y old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap formation at baseline and upon microbial or sterile autoimmune stimuli. The expression levels of the immature-like neutrophil signature increased linearly with pregnancy, an immune state of increased susceptibility to certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women during late pregnancy. Thus, our findings show novel sex differences in innate immunity and identify a common neutrophil signature in males and in pregnant women.
View details for DOI 10.4049/jimmunol.1601855
View details for PubMedID 28179497
Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states
2017; 23 (2): 174-184
Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N(4)-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.
View details for DOI 10.1038/nm.4267
View details for Web of Science ID 000393729000009
View details for PubMedID 28092664
Deep Immune Profiling of an Arginine-Enriched Nutritional Intervention in Patients Undergoing Surgery.
Journal of immunology (Baltimore, Md. : 1950)
Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific, as they reduce infection rates in elective surgery, but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably, AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery, consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly, AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells, which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms.
View details for PubMedID 28794234
Mass cytometry: The time to settle down.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2017; 91 (1): 12–13
View details for PubMedID 28106953
Freehand Versus Guided Surgery: Factors Influencing Accuracy of Dental Implant Placement.
2017; 26 (4): 500–509
Patient anatomy, practitioner experience, and surgical approach are all factors that influence implant accuracy. However, the relative importance of each factor is poorly understood. The present study aimed to identify which factors most critically determine implant accuracy to aid the practitioner in case selection for guided versus freehand surgery.One practitioner's ideal implant angulation and position was compared with his achieved position radiographically for 450 implants placed using a conventional freehand method. The relative contribution of 11 demographic, anatomical, and surgical factors to the accuracy of implant placement was systematically quantified.The most important predictors of angulation and position accuracy were the number of adjacent implants placed and the tooth-borne status of the site. Immediate placement also significantly increased position accuracy, whereas cases with narrow sites were significantly more accurate in angulation. Accuracy also improved with the practitioner's experience.These results suggest tooth-borne, single-implant cases performed later in the practitioner's experience are most appropriate for freehand placement, whereas guided surgery should be considered to improve accuracy for multiple-implant cases in edentulous or partially edentulous sites.
View details for PubMedID 28731896
The road ahead: Implementing mass cytometry in clinical studies, one cell at a time.
Cytometry. Part B, Clinical cytometry
2017; 92 (1): 10–11
View details for PubMedID 27874247
A Proteomic Clock of Human Pregnancy.
American journal of obstetrics and gynecology
Early detection of maladaptive processes underlying pregnancy-related pathologies is desirable, as it will enable targeted interventions ahead of clinical manifestations. The quantitative analysis of plasma proteins features prominently among molecular approaches used to detect deviations from normal pregnancy. However, derivation of proteomic signatures sufficiently predictive of pregnancy-related outcomes has been challenging. An important obstacle hindering such efforts were limitations in assay technology, which prevented the broad examination of the plasma proteome.The recent availability of a highly-multiplexed platform affording the simultaneous measurement of 1,310 plasma proteins opens the door for a more explorative approach. The major aim of this study was to examine whether analysis of plasma collected during gestation of term pregnancy would allow identifying a set of proteins that tightly track gestational age. Establishing precisely-timed plasma proteomic changes during term pregnancy is a critical step in identifying deviations from regular patterns due to fetal and maternal maladaptations. A second aim was to gain insight into functional attributes of identified proteins, and link such attributes to relevant immunological changes.Pregnant women participated in this longitudinal study. In two subsequent subsets of 21 (training cohort) and 10 (validation cohort) women, specific blood specimens were collected during the first (7-14 wks), second (15-20 wks), and third (24-32 wks) trimesters, and 6 wks post-partum for analysis with a highly-multiplexed aptamer-based platform. An elastic net algorithm was applied to infer a proteomic model predicting gestational age. A bootstrapping procedure and piece-wise regression analysis was used to extract the minimum number of proteins required for predicting gestational age without compromising predictive power. Gene ontology analysis was applied to infer enrichment of molecular functions among proteins included in the proteomic model. Changes in abundance of proteins with such functions were linked to immune features predictive of gestational age at the time of sampling in pregnancies delivering at term.An independently validated model consisting of 74 proteins strongly predicted gestational age (p = 3.8x10-14, R = 0.97). The model could be reduced to eight proteins without losing its predictive power (p = 1.7x10-3, R = 0.91). The three top ranked proteins were glypican 3, chorionic somatomammotropin hormone, and granulins. Proteins activating the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway were enriched in the proteomic model, chorionic somatomammotropin hormone being the top ranked protein. Abundance of chorionic somatomammotropin hormone strongly correlated with STAT5 signaling activity in CD4 T cells, the endogenous cell-signaling event most predictive of gestational age.Results indicate that precisely timed changes in the plasma proteome during term pregnancy mirror a "proteomic clock". Importantly, the combined use of several plasma proteins was required for accurate prediction. The exciting promise of such a "clock" is that deviations from its regular chronological profile may assist in the early diagnoses of pregnancy-relate pathologies and point to underlying pathophysiology. Functional analysis of the proteomic model generated the novel hypothesis that somatomammotropin hormone may critically regulate T-cell function during pregnancy.
View details for PubMedID 29277631
Deep Immune Profiling in Trauma and Sepsis: Flow Is the Way to Go!
Critical care medicine
2017; 45 (9): 1577–78
View details for PubMedID 28816846
Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy.
Journal of immunology
Preterm labor and infections are the leading causes of neonatal deaths worldwide. During pregnancy, immunological cross talk between the mother and her fetus is critical for the maintenance of pregnancy and the delivery of an immunocompetent neonate. A precise understanding of healthy fetomaternal immunity is the important first step to identifying dysregulated immune mechanisms driving adverse maternal or neonatal outcomes. This study combined single-cell mass cytometry of paired peripheral and umbilical cord blood samples from mothers and their neonates with a graphical approach developed for the visualization of high-dimensional data to provide a high-resolution reference map of the cellular composition and functional organization of the healthy fetal and maternal immune systems at birth. The approach enabled mapping of known phenotypical and functional characteristics of fetal immunity (including the functional hyperresponsiveness of CD4(+) and CD8(+) T cells and the global blunting of innate immune responses). It also allowed discovery of new properties that distinguish the fetal and maternal immune systems. For example, examination of paired samples revealed differences in endogenous signaling tone that are unique to a mother and her offspring, including increased ERK1/2, MAPK-activated protein kinase 2, rpS6, and CREB phosphorylation in fetal Tbet(+)CD4(+) T cells, CD8(+) T cells, B cells, and CD56(lo)CD16(+) NK cells and decreased ERK1/2, MAPK-activated protein kinase 2, and STAT1 phosphorylation in fetal intermediate and nonclassical monocytes. This highly interactive functional map of healthy fetomaternal immunity builds the core reference for a growing data repository that will allow inferring deviations from normal associated with adverse maternal and neonatal outcomes.
View details for PubMedID 27793998
- In Reply. Anesthesiology 2016; 124 (6): 1414-1415
Patient-specific Immune States before Surgery Are Strong Correlates of Surgical Recovery
2015; 123 (6): 1241-1255
Recovery after surgery is highly variable. Risk-stratifying patients based on their predicted recovery profile will afford individualized perioperative management strategies. Recently, application of mass cytometry in patients undergoing hip arthroplasty revealed strong immune correlates of surgical recovery in blood samples collected shortly after surgery. However, the ability to interrogate a patient's immune state before surgery and predict recovery is highly desirable in perioperative medicine.To evaluate a patient's presurgical immune state, cell-type-specific intracellular signaling responses to ex vivo ligands (lipopolysaccharide, interleukin [IL]-6, IL-10, and IL-2/granulocyte macrophage colony-stimulating factor) were quantified by mass cytometry in presurgical blood samples. Selected ligands modulate signaling processes perturbed by surgery. Twenty-three cell surface and 11 intracellular markers were used for the phenotypic and functional characterization of major immune cell subsets. Evoked immune responses were regressed against patient-centered outcomes, contributing to protracted recovery including functional impairment, postoperative pain, and fatigue.Evoked signaling responses varied significantly and defined patient-specific presurgical immune states. Eighteen signaling responses correlated significantly with surgical recovery parameters (|R| = 0.37 to 0.70; false discovery rate < 0.01). Signaling responses downstream of the toll-like receptor 4 in cluster of differentiation (CD) 14 monocytes were particularly strong correlates, accounting for 50% of observed variance. Immune correlates identified in presurgical blood samples mirrored correlates identified in postsurgical blood samples.Convergent findings in pre- and postsurgical analyses provide validation of reported immune correlates and suggest a critical role of the toll-like receptor 4 signaling pathway in monocytes for the clinical recovery process. The comprehensive assessment of patients' preoperative immune state is promising for predicting important recovery parameters and may lead to clinical tests using standard flow cytometry.
View details for DOI 10.1097/ALN.0000000000000887
View details for PubMedID 26655308
Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2015; 87 (9): 817-829
Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1) ) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort. © 2015 International Society for Advancement of Cytometry.
View details for DOI 10.1002/cyto.a.22720
View details for PubMedID 26190063
Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2014; 85 (12): 1011-1019
Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. © 2014 International Society for Advancement of Cytometry.
View details for DOI 10.1002/cyto.a.22573
View details for PubMedID 25274027
A FOXO-Pak1 transcriptional pathway controls neuronal polarity
GENES & DEVELOPMENT
2010; 24 (8): 799-813
Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.
View details for DOI 10.1101/gad.1880510
View details for Web of Science ID 000276730300008
View details for PubMedID 20395366
View details for PubMedCentralID PMC2854394
PIASx is a MEF2 SUMO E3 ligase that promotes postsynaptic dendritic morphogenesis
JOURNAL OF NEUROSCIENCE
2007; 27 (37): 10037-10046
Postsynaptic morphogenesis of dendrites is essential for the establishment of neural connectivity in the brain, but the mechanisms that govern postsynaptic dendritic differentiation remain poorly understood. Sumoylation of the transcription factor myocyte enhancer factor 2A (MEF2A) promotes the differentiation of postsynaptic granule neuron dendritic claws in the cerebellar cortex. Here, we identify the protein PIASx as a MEF2 SUMO E3 ligase that represses MEF2-dependent transcription in neurons. Gain-of-function and genetic knockdown experiments in rat cerebellar slices and in the postnatal cerebellum in vivo reveal that PIASx drives the differentiation of granule neuron dendritic claws in the cerebellar cortex. MEF2A knockdown suppresses PIASx-induced dendritic claw differentiation, and expression of sumoylated MEF2A reverses PIASx knockdown-induced loss of dendritic claws. These findings define the PIASx-MEF2 sumoylation signaling link as a key mechanism that orchestrates postsynaptic dendritic claw morphogenesis in the cerebellar cortex and suggest novel functions for SUMO E3 ligases in brain development and plasticity.
View details for DOI 10.1523/JNEUROSCI.0361-07.2007
View details for Web of Science ID 000249415000024
View details for PubMedID 17855618
Transcription factor Sp4 regulates dendritic patterning during cerebellar maturation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (23): 9882-9887
Integration of inputs by a neuron depends on dendritic arborization patterns. In mammals, the genetic programs that regulate dynamic remodeling of dendrites during development and in response to activity are incompletely understood. Here we report that knockdown of the transcription factor Sp4 led to an increased number of highly branched dendrites during maturation of cerebellar granule neurons in dissociated cultures and in cerebellar cortex. Time-course analysis revealed that depletion of Sp4 led to persistent generation of dendritic branches and a failure in resorption of transient dendrites. Depolarization induced a reduction in the number of dendrites, and knockdown of Sp4 blocked depolarization-induced remodeling. Furthermore, overexpression of Sp4 wild type, but not a mutant lacking the DNA-binding domain, was sufficient to promote dendritic pruning in nondepolarizing conditions. These findings indicate that the transcription factor Sp4 controls dendritic patterning during cerebellar development by limiting branch formation and promoting activity-dependent pruning.
View details for DOI 10.1073/pnas.0701946104
View details for Web of Science ID 000247114100061
View details for PubMedID 17535924
View details for PubMedCentralID PMC1887555
A calcium-regulated MEF2 surnoylation switch controls postsynaptic differentiation
2006; 311 (5763): 1012-1017
Postsynaptic differentiation of dendrites is an essential step in synapse formation. We report here a requirement for the transcription factor myocyte enhancer factor 2A (MEF2A) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. A transcriptional repressor form of MEF2A that is sumoylated at lysine-403 promoted dendritic claw differentiation. Activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of MEF2A at serine-408 and, thereby, promoted a switch from sumoylation to acetylation at lysine-403, which led to inhibition of dendritic claw differentiation. Our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain.
View details for DOI 10.1126/science.1122513
View details for Web of Science ID 000235456900049
A CaMKII-NeuroD signaling pathway specifies dendritic morphogenesis
2004; 41 (2): 229-241
The elaboration of dendrites is fundamental to the establishment of neuronal polarity and connectivity, but the mechanisms that underlie dendritic morphogenesis are poorly understood. We found that the genetic knockdown of the transcription factor NeuroD in primary granule neurons including in organotypic cerebellar slices profoundly impaired the generation and maintenance of dendrites while sparing the development of axons. We also found that NeuroD mediated neuronal activity-dependent dendritogenesis. The activity-induced protein kinase CaMKII catalyzed the phosphorylation of NeuroD at distinct sites, including endogenous NeuroD at Ser336 in primary neurons, and thereby stimulated dendritic growth. These findings uncover an essential function for NeuroD in granule neuron dendritic morphogenesis. Our study also defines the CaMKII-NeuroD signaling pathway as a novel mechanism underlying activity-regulated dendritic growth that may play important roles in the developing and mature brain.
View details for Web of Science ID 000221457800009
View details for PubMedID 14741104
Characterization of a neurotrophin signaling mechanism that mediates neuron survival in a temporally specific pattern
JOURNAL OF NEUROSCIENCE
2003; 23 (19): 7326-7336
The temporally specific nature of neurotrophic factor-induced responses is a general feature of mammalian nervous system development, the mechanisms of which remain to be elucidated. We characterized a mechanism underlying the temporal specificity by which BDNF selectively promotes the survival of newly generated, but not mature, granule neurons of the mammalian cerebellum. We found that BDNF specifically induces the extracellular signal-regulated kinase 5 (ERK5)-myocyte enhancer factor (MEF2) signaling pathway in newly generated granule neurons and thereby induces transcription of neurotrophin-3 (NT-3), a novel gene target of MEF2. Inhibition of endogenous ERK5, MEF2, or NT-3 in neurons by several approaches including disruption of the NT-3 gene in mice revealed a requirement for the ERK5-MEF2-NT-3 signaling pathway in BDNF-induced survival of newly generated granule neurons. These findings define a novel mechanism that underlies the antiapoptotic effect of neurotrophins in a temporally defined pattern in the developing mammalian brain.
View details for Web of Science ID 000184817700011
View details for PubMedID 12917366
RNA interference reveals a requirement for myocyte enhancer factor 2A in activity-dependent neuronal survival
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (48): 46442-46446
RNA interference (RNAi) provides a powerful method of gene silencing in eukaryotic cells, including proliferating mammalian cells. However, the utility of RNAi as a method of gene knock-down in primary postmitotic mammalian neurons remained unknown. Here, we asked if RNAi might be utilized to allow the assessment of the biological function of a specific gene in the nervous system. We employed a U6 promoter-driven DNA template approach to induce hairpin RNA-triggered RNAi to characterize the role of the transcription factor myocyte enhancer factor 2A (MEF2A) in the neuronal activity-dependent survival of granule neurons of the developing rat cerebellum. We found that the expression of MEF2A hairpin RNAs leads to the efficient and specific inhibition of endogenous MEF2A protein expression in primary cerebellar granule neurons. We also found that RNAi of MEF2A reduces significantly MEF2 response element-mediated transcription in granule neurons and inhibits activity-dependent granule neuron survival. Taken together, our RNAi experiments have revealed that MEF2A plays a critical role in activity-dependent neuronal survival. In addition, our findings indicate that RNAi does operate in postmitotic mammalian neurons and thus offers a rapid genetic method of studying gene function in the development and function of the mammalian nervous system.
View details for DOI 10.1074/jbc.M206653200
View details for Web of Science ID 000179529300096
View details for PubMedID 12235147