Doctor of Medicine and Doctor of Philosophy (MDPhD)
Discovery and characterization of novel primary immunodeficiency diseases in humans
Stuart Turvey, MBBS, DPhil, FRCPC is a Professor of Pediatrics at the University of British Columbia where he holds both the Tier 1 Canada Research Chair in Pediatric Precision Health and the Aubrey J. Tingle Professorship in Pediatric Immunology. He is a Pediatric Immunologist based at BC Children’s Hospital, and Director of Clinical Research at the Child & Family Research Institute. Prior to coming to Vancouver, Dr Turvey completed both his Pediatric Residency and Allergy/Immunology Fellowship at Children’s Hospital, Harvard Medical School, Boston. He holds a medical degree (MB BS) from the University of Sydney, Australia and a doctorate (DPhil) in Immunology from Oxford University where he was a Rhodes Scholar. Dr Turvey is a Fellow of the Royal College of Physicians and Surgeons of Canada and a Diplomate of the American Board of Pediatrics.
Dr Turvey provides clinical care in the specialty of Clinical Immunology, while his research program focuses on pediatric infectious and inflammatory diseases.
• Innate immunity
• Host defense
• Primary immunodeficiency diseases (PIDs)
• Role of Toll-like receptors in human disease
• Airway inflammation in cystic fibrosis
• Asthma and allergic disease
• Microbiome in health and disease
Despite much effort, we know little about how the healthy child is protected from infectious disease, and even less about why some children develop inflammatory disorders. Why do some healthy children succumb to overwhelming bacterial infection, while others either survive infection or do not become infected at all? Why do some children suffer crippling juvenile arthritis or life-threatening asthma?
My research program is translational, interdisciplinary and unique in its focus on understanding the role of innate immunity in infectious and inflammatory diseases of childhood. Starting with a population of children with a defined infectious or inflammatory disease phenotype (e.g., undue susceptibility to infection, juvenile idiopathic arthritis, asthma), I aim to determine the underlying cellular, molecular and genetic abnormalities responsible for the disease through detailed immunological, genomic and proteomic analysis. The new knowledge generated by this approach will aid diagnosis, elucidate mechanisms of disease pathogenesis and, ultimately, identify novel targets for anti-inflammatory and anti-infectious therapeutic agents.
Genetics of Susceptibility to Childhood Infection Until very recently, clinical immunologists have focused most attention on patients with a ‘noisy clinical phenotype’—multiple, severe and recurrent infections. Indeed, the origin of primary immunodeficiencies is generally attributed to Bruton’s 1952 description of X-linked agammaglobulinemia in a boy whose repeated pneumococcal infections demanded clinical attention. Today, enhanced understanding of human immunity combined with ever more sophisticated tools to dissect the immune response have allowed clinical immunologists to look beyond these "noisy", severely immunocompromised patients to individuals with less obvious immune defects. We have entered the era of "subtle" primary immunodeficiencies that will begin to precipitate a fundamental change and expansion of the focus of clinical immunologists. My lab is involved in searching for subtle genetic immune defects in apparently healthy children who have serious immunological disorders. This journey towards subtlety is anticipated to translate into better care for our patients through improved diagnosis, combined with tailored treatment and targeted prophylaxis. Canadian Healthy Infant Longitudinal Development (CHILD) Study Over the past 30 years there has been an increasing concern about the effects of environment on health. In particular, since infants spend the majority of their time indoors, there is intense interest in the impact indoor pollution has on the health of our children. Our indoor environment has become a public health priority as growing evidence suggests that unseen environmental contaminants in our living spaces may have important effects on children's health and development. The Canadian Healthy Infant Longitudinal Development (CHILD) Study is a multicentre, multidisciplinary, longitudinal, population-based birth-cohort study of 5,000 children enrolled "pre-birth" and followed for five years (www.canadianchildstudy.ca). The main purpose of this study is to determine what aspects of the environment interact with genetic factors to affect children’s health and development. I am a co-principal investigator for the CHILD study and I lead the Vancouver study site. Innate Immunity and Lung Inflammation in Cystic Fibrosis Cystic fibrosis (CF) is the most common, deadly genetic disease affecting young Canadians. Even today, only half of the people living with CF will survive beyond their mid-thirties. New treatments for CF are critically needed. Lung disease is the major life-limiting factor for people living with CF. Lung injury in CF occurs through a vicious cycle of airway blockage, infection and inflammation. Current CF treatments rely upon physiotherapy to reduce airway blockage and antibiotics to treat the infections, but these treatments do not specifically deal with inflammation. New treatments to simultaneously target airway inflammation are likely to provide substantial additional benefits in improving the quality and length of life for those with CF. Through synergistic studies harnessing the power of cell biology, chemistry and functional genomics, we are working to identify optimal "druggable" targets responsible for CF airway inflammation and to discover novel anti-inflammatory drugs. Ultimately, these experiments are designed to develop new therapies for safely reducing lung inflammation and improving the quality and length of life of people with CF.
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Primary immunodeficiency diseases or inborn errors of immunity are a group of rare genetic disorders in which key elements of the immune system are absent or dysfunctional. This typically manifests as greatly enhanced susceptibility to infection, autoimmunity, autoinflammation, malignancy, and/or atopy. Studying these patients can be very informative as they provide a rare opportunity to uncover the fundamental role of single genes in human immunity and disease, which can inform the development of targeted precision therapeutics and patient treatment.Germline mutations affecting caspase recruitment domain (CARD)—B cell CLL/lymphoma 10 (BCL10)—MALT1 paracaspase (MALT1) [CBM] complexes or ‘CBM-opathies’ have recently been linked to a broad spectrum of diseases, including invasive fungal disease, lymphoproliferation, severe atopic disease, psoriatic skin diseases, and profound combined immunodeficiency. These disorders can be difficult to diagnose as their (i) pathogenesis is incompletely understood; (ii) phenotypes are heterogeneous and can be similar to other PIDs; and (iii) incidence is low. With increased use of diagnostic next generation sequencing (NGS) in the clinic, a large number of variants of uncertain significance have been identified in these genes, further complicating diagnosis. The research presented in this thesis was focused on advancing the diagnosis, understanding, and treatment of germline CBM-opathies. We accomplished this by collecting and reporting all known clinical parameters from patients with germline mutations affecting CBM complexes and classified/unified these under the new term ‘CBM-opathies.’ Furthermore, we performed NGS on various patients with combined immunodeficiency, designed assays to functionally validate the pathogenicity of identified variants, and translated our findings into clinical care. Through this work, we were able to (i) classify the pathogenicity of four novel variations of CARD11; (ii) define the role of CARD11 in human B cell development, signalling, and function; (iii) and uncover the role of MALT1 paracaspase activity in mediating the pathogenesis of B cell Expansion with NF-кB and T cell Anergy (BENTA) disease.
Asthma is a chronic inflammatory atopic disease of the airways affecting one in ten children in Westernized countries. The microflora hypothesis of allergic disease proposes the intestinal microbiota as a potential mechanistic link connecting environmental exposures to changes in the developing immune system. Further, animal model studies allude to an early life critical window, during which the immune system is most vulnerable to compositional and functional changes in the intestinal microbiota.We conducted an epidemiological assessment of early life environmental factors associated with atopy and wheezing at age 1 year and preschool-age asthma in children enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) Study (n = 2,695). Here, we identified early life microflora hypothesis related variables (e.g. pre- and post-natal antibiotic exposure, gestational age, and birth mode) as risk and protective factors for asthma and atopic disease. Informed by this epidemiological assessment, we used 16S ribosomal RNA sequencing and quantitative polymerase chain reaction to analyze the 3-month and 1-year fecal microbiota of one-year-old CHILD Study subjects positive or negative for atopy and wheezing (n = 319) and among this same cohort, those who were diagnosed with preschool-age asthma or non-atopic non-wheezing controls (n = 76). The fecal microbiota of atopic wheezing subjects compared to controls showed decreases in the abundances of four gut bacterial genera, Faecalibacterium, Lachnospira, Rothia, and Veillonella, combined with a reduction in fecal acetate at the 3-month time point only. Further, we found shifts in the relative abundances of two bacterial taxa in the 3-month fecal microbiota of preschool-age asthmatic children compared to controls; Lachnospira remained decreased among asthmatic children and Clostridium neonatale was increased in asthmatics. Quartile analysis at 3-months revealed a negative association between the ratio of these two bacteria (Lachnospira/Clostridium neonatale) and asthma risk. Altogether, this research highlights environmental factors that may be contributing to gut bacterial alterations in subjects with asthma or atopic disease. Additionally, these microbial alterations were no longer present by 1-year of age, suggesting the first 100 days of life as the critical window during which taxa-specific gut bacterial dysbiosis is associated with asthma and atopic disease in humans.
Cystic fibrosis (CF) is characterized by a progressive decline in lung function due to airway obstruction, infection, and inflammation. CF patients are particularly susceptible to respiratory infection by a variety of pathogens, and the inflammatory response in CF is dysregulated and prolonged. This thesis identifies and characterizes BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 as putative anti-inflammatory molecules in CF, and explores the CF inflammatory response to rhinovirus infection. BPIFA1 and BPIFB1 are proposed innate immune molecules expressed in the upper airways. We interrogated BPIFA1/BPIFB1 single-nucleotide polymorphisms in data from the North American genome-wide association study (GWAS) for lung disease severity in CF and discovered that the G allele of rs1078761 was associated with reduced lung function in CF patients. Microarray and qPCR gene expression analysis implicated rs1078761 G as being associated with reduced BPIFA1 and BPIFB1 gene expression, suggesting that decreased levels of these genes are detrimental in CF.Functional assays to characterize the role of BPIFA1 and BPIFB1 in CF indicated that these molecules do not have an anti-bacterial role against P. aeruginosa, but do have an immunomodulatory function in CF airway epithelial cells. To further investigate the mechanism of action of BPIFA1 and BPIFB1 during bacterial infection, gene expression was profiled using RNA-Seq in airway epithelial cells stimulated with P. aeruginosa and treated with recombinant BPIFA1 and BPIFB1.Viral infections are now recognized to play an important role in the short and long term health of CF patients. Rhinovirus is emerging as a lead viral pathogen although little is known about the inflammatory response triggered by rhinovirus in the CF lung. To investigate whether CF patients have a dysregulated response to rhinovirus infection, primary airway epithelial cells from CF and healthy control children were infected with rhinovirus and gene expression profiles were assessed by RNA-Seq. Although rhinovirus stimulation resulted significantly altered gene expression, the response to infection was not different in CF patients compared to healthy controls. However, CF cells had significantly higher rhinovirus levels than controls, indicating that CF patients may have a deficient antiviral response allowing for increased rhinovirus replication.
An increasing number of infectious and inflammatory conditions have identified ER stress and IL-1β as central driving forces in inflammation. In the following studies, we have identified several important pathways that promote the cycle of pulmonary inflammation in cystic fibrosis (CF), including the IL-1β regulation by NF-κB, IL-1β stimulation of neutrophil chemokine production by airway epithelial cells, and inhibition of neutrophil chemokine secretion through ER stress signaling. Based on our observations, we conclude that CF environmental factors such as increased NF-κB priming by chronic infection and the presence of ER stressors can significantly augment inflammatory responses by controlling the maturation of IL-1β. These effects appear to be cell type and cytokine-specific, as downstream induction of CXCL1 and IL-8, key neutrophil chemokines that we found were associated with lung disease severity in CF patients, can also be suppressed by ER stress-mediated inhibition of STAT3 signaling in airway epithelial cells.Interestingly, we also found that by varying the time of TLR stimulation relative to the induction of ER stress (particularly by proteasome inhibition), we could obtain differential effects on IL-1β production and maturation. We reasoned that these effects could be important in haematological malignancies where proteasome inhibition has recently become a successful first-line treatment. Not only was tumour cell cytotoxicity significantly increased by addition of a TLR adjuvant, IL-1β production could be controlled depending on when the adjuvant was added relative to the proteasome inhibitor. This could be a useful method in which IL-1β-mediated responses can be manipulated while also increasing tumour cell death.
No abstract available.
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Human inborn errors of immunity (IEIs) are a group of genetic disorders in which specific components of the human immune system are missing, dysfunctional, or poorly regulated. IEIs negatively impact the protective functions of the immune system, increasing susceptibility to infections, autoimmunity, inflammation, and malignancy. The investigation of monogenic IEIs enables the diagnosis and treatment of the affected individuals and provides unique opportunities to understand the role of the immune system and the functional roles of its constituents.Toll-like receptor (TLR) signaling is a key aspect of the innate immune response, and interleukin-1 receptor-associated kinase 4 (IRAK4) plays a vital role in the TLR signaling cascade which enables the production of protective inflammatory cytokines. Human IRAK4-deficiency is an autosomal recessive IEI which presents with a blunted inflammatory response to infection and susceptibility to certain bacteria. We demonstrate that the novel IRAK4 (c.1049delG, p.(Gly350Glufs*15)) variant abrogates IRAK4 protein expression and abolishes TLR signaling, expanding the knowledge of pathogenic variants causing human IRAK4-deficiency.We have also identified 3 unrelated individuals with a phenotype of combined immunodeficiency (agammaglobulinemia and infectious susceptibility) who carry a novel heterozygous de novo missense variant in Interferon Regulatory Factor 4 (IRF4) gene (c.284C>G, p.T95R). IRF4 is a transcription factor expressed in immune cells which plays a critical role in the differentiation of various immune cells. We demonstrate adaptive immune cell differentiation defects in proband primary cells, as well as decreased effector cytokine production in T cells. We also demonstrate that the IRF4 T95R variant is able to bind the canonical GAAA binding site as well as a novel GATA binding site. Therefore, we propose that the IRF4 T95R variant is defined as a neomorphic variant, as it has gained novel DNA-binding functions. This increase in DNA-binding sequence promiscuity may result in a dominant negative redistribution of the wild-type IRF4 and the combined immunodeficiency phenotype in the proband. We have characterized a novel neomorphic variant in the IRF4 gene and defined a novel human IEI which highlights the potential of IRF4 as an immunomodulatory therapeutic target.
Monogenic immune disorders are a group of conditions caused by single gene mutations affecting how the immune system develops, functions, or both. It is now appreciated that certain monogenic immune disorders can present with severe allergic disease, such as asthma, food allergy, elevated eosinophil counts and/or atopic dermatitis. Studying single-gene defects that lead to atopy has identified critical molecules in the pathogenesis of allergic inflammation. These discoveries have created new therapeutic targets for allergic diseases, which carry an immense health and economic burden in Canada. Germline gain-of-function mutations in JAK1 are a newly described monogenic cause of severe atopy, with affected patients demonstrating profound eosinophilia and allergic inflammation. The initial report identified a dramatic clinical response to the combined JAK1/2 inhibitor ruxolitinib. We sought to determine the long-term clinical outcomes and response to ruxolitinib of patients carrying a germline JAK1 gain-of-function mutation. We further aimed to characterize the mechanisms behind JAK1-mediated atopic immune dysregulation, including its effect on T lymphocytes–key regulators in allergic inflammation–and hematopoiesis. We demonstrate that long-term ruxolitinib use is associated with improvements in growth, eosinophilia, and allergic inflammation, however, anemia represents a dose-limiting adverse effect. T cell immunophenotypic analysis revealed severe skewing towards a TH2 phenotype pre-ruxolitinib treatment, in keeping with the allergic clinical manifestations. RNA sequencing data from patients and a transgenic zebrafish model both containing the same JAK1 gain-of-function mutation identified increased expression of genes involved in cytokine signaling, type 1 interferon and antiviral immune responses, and alternative macrophage activation, as well as genes whose role in allergic immune dysregulation warrants further investigation. In conclusion, this work demonstrates a critical role for JAK1 in atopic immune dysregulation, specifically driving lymphocyte responses towards a TH2 phenotype. Combined JAK1/2 inhibition can reverse much of the allergic inflammation, with dramatic clinical effects. This has important implications for our understanding of the pathogenesis and potential therapeutic targets for early life allergic immune dysregulation.
Introduction:Atopic dermatitis (AD) is a prevalent, heterogeneous allergic disease of the skin, impacting quality of life and often a prelude to other incurable allergic diseases. Microbes residing in the gut in early-life play an important role in shaping the function of the immune system and may be critical in the prevention or treatment of AD. Characterizing the early-life gut microbiota in children with AD may lead to new therapeutic and preventative measures involving alteration of the gut microbiota.Hypotheses: The specific hypotheses tested in this study are: 1) there is dysbiosis (or imbalance) in the gut microbiota of infants with clinically relevant AD phenotypes; and 2) there are differences in gut microbiota composition between extrinsic and intrinsic AD endotypes.Methods:Leveraging stool samples and data from the Canadian Healthy Infant Longitudinal Development (CHILD) study, early-life gut microbiota of deeply phenotyped children with AD was assessed. AD at five years of age, moderate/severe AD, and persistent AD phenotypes and intrinsic and extrinsic AD endotypes were formalized. 16S rRNA gene sequencing was used to analyze stool samples to assess gut microbial diversity, maturation and taxa abundance between phenotypes and healthy controls.Results:Gut microbial diversity and maturation was significantly decreased in children with AD at five years of age compared to healthy controls. Epulopiscium, Aggregatibacter, Turicibater, Phascolarctobacterium, Citrobacter and Prevotella were detected at significantly different levels in the early-life gut microbiota of infants with clinically-relevant AD and healthy controls. Microbial diversity and gut maturation indices in children with extrinsic AD were significantly decreased compared to healthy controls, whereas children with intrinsic AD had no differences in diversity or gut maturation compared to healthy controls. Taxa abundance were different between intrinsic and extrinsic AD endotypes.Conclusion:There are differences in the early-life gut microbiota of children with various clinically-relevant AD phenotypes compared to children without AD. Thoroughly phenotyping AD is important for proper treatment or prevention, particularly for considering gut microbiota alteration as a therapeutic method. This knowledge contributes to the process of profiling the gut microbiota of children with AD with the goal of creating live biotherapeutics for prevention of AD.
Asthma and allergic diseases are rapidly becoming the most common chronicdiseases in the developed world. Nearly 1 in 3 Canadians suffer from some form of allergyand more than 300 million individuals in the developed world suffer from asthma. Thesecomplex disorders are caused by the interaction of various genetic and environmental factors.Genome-wide association studies have been widely used to identify genes associated withasthma susceptibility. The gene, ORMDL3, was shown to be associated with early-onsetasthma. Asthmatic patients have elevated expression levels of this gene. The gene encodes atransmembrane protein localized in the endoplasmic reticulum (ER) that may be involved inER stress and inflammation. Its functional role in asthma pathogenesis, however, has yet tobe elucidated. In this research, we investigated the functional role of ORMDL3 in innateimmunity. Experimentally, ORMDL3 expression levels were manipulated in vitro in airwaycells using overexpression plasmid and siRNA technologies. The effects of ORMDL3expression levels on inflammatory responses were then explored. After manipulation ofORMDL3 expression levels, cells were stimulated with various immune response-inducingfactors. Supernatants collected after stimulation were analyzed and no differences in proinflammatorycytokine production were observed. These results suggest that variation inORMDL3 expression levels does not affect innate immune production of IL-6 and IL-8 inairway cells. ORMDL3 knockdown also did not affect expression of other immune-relatedgenes.
IkBα is an important regulator of inflammation. Single nucleotide polymorphisms (SNPs) rs3138053, rs2233406 and rs2233409 in the promoter of the gene NFKBIA, which encodes for IkBα, have been shown to be associated with a variety of infectious and inflammatory conditions. In this study, we investigated the functional impact of the promoter variants of NFKBIA on human immune responsiveness. Using a coding SNP that was in strong linkage disequilibrium (LD) with NFKBIA SNPs rs3138053/rs2233406/rs2233409, we designed and validated an allele-specific PCR assay that could detect subtle differences in allele ratios between the major (ACC) and minor (GTT) promoter variants of SNPs rs3138053/rs2233406/rs2233409. Peripheral blood mononuclear cells (PBMCs) of homozygous (ACC/ACC) and heterozygous (ACC/GTT) individuals were stimulated with 100ng/ml LPS and live cultures of Streptococcus pneumoniae (moi 7.8-30) serotype 14 for 3 and 4 hours. PBMCs of neonatal NFKBIA homozygotes and heterozygotes were stimulated with various Toll-like-receptor (TLR) ligands of the innate immunity cascade to assay for differences in the innate immune response.NFKBIA heterozygotes of European descent displayed 1.21 (1.14-1.27 95% CI)-1.26 (1.18-1.34 95% CI) fold higher expression of the major allele transcript (ACC) relative to the minor allele transcript (GTT). For the same ethnicity, at 3 hours stimulation, NFKBIA homozygotes (ACC/ACC) produced higher levels of NFKBIA mRNA than heterozygotes following stimulation with LPS (1.4 fold. p=0.0095) and S. pneumoniae (1.51 fold, p=0.024). Higher TNFα secretion was seen from PBMCs of heterozygotes as compared to homozygotes (of European descent) in the presence of LPS (1.57 fold , p