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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