Publications by authors named "Janet Markle"

22 Publications

  • Page 1 of 1

Human T-bet Governs Innate and Innate-like Adaptive IFN-γ Immunity against Mycobacteria.

Cell 2020 Dec 8;183(7):1826-1847.e31. Epub 2020 Dec 8.

St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY 10065, USA; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Necker Hospital for Sick Children, 75015 Paris, France; University of Paris, Imagine Institute, 75015 Paris, France; Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France; Howard Hughes Medical Institute, New York, NY, USA. Electronic address:

Inborn errors of human interferon gamma (IFN-γ) immunity underlie mycobacterial disease. We report a patient with mycobacterial disease due to inherited deficiency of the transcription factor T-bet. The patient has extremely low counts of circulating Mycobacterium-reactive natural killer (NK), invariant NKT (iNKT), mucosal-associated invariant T (MAIT), and Vδ2 γδ T lymphocytes, and of Mycobacterium-non reactive classic T1 lymphocytes, with the residual populations of these cells also producing abnormally small amounts of IFN-γ. Other lymphocyte subsets develop normally but produce low levels of IFN-γ, with the exception of CD8 αβ T and non-classic CD4 αβ T1 lymphocytes, which produce IFN-γ normally in response to mycobacterial antigens. Human T-bet deficiency thus underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2 γδ T cells) and IFN-γ production by them, with mycobacterium-specific, IFN-γ-producing, purely adaptive CD8 αβ T, and CD4 αβ T1 cells unable to compensate for this deficit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.10.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770098PMC
December 2020

Do not let them slip through the net: Catching a case of leaky severe combined immunodeficiency.

J Paediatr Child Health 2020 05 13;56(5):809-811. Epub 2019 Nov 13.

Department of Paediatrics, Christchurch Hospital, Christchurch, New Zealand.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jpc.14690DOI Listing
May 2020

Tuberculosis and impaired IL-23-dependent IFN-γ immunity in humans homozygous for a common missense variant.

Sci Immunol 2018 12;3(30)

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA.

Inherited IL-12Rβ1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ immunity and are rare monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for the common P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 individuals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic areas than in ethnicity-adjusted controls ( = 8.37 × 10; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular responses to IL-23, but not to IFN-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ via activation of TYK2 and JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12, responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12 and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated, rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive P1104A missense variant of selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciimmunol.aau8714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341984PMC
December 2018

Human IFN-γ immunity to mycobacteria is governed by both IL-12 and IL-23.

Sci Immunol 2018 12;3(30)

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA.

Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rβ1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rβ2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αβ T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4 T cells, including, in particular, mycobacterium-specific T1* cells (CD45RACCR6), is dependent on both IL-12 and IL-23. Last, we show that , , and have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rβ2 or IL-23R deficiency, relative to IL-12Rβ1 deficiency, is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R- and IL-12Rβ2-deficient than IL-12Rβ1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ-dependent immunity to mycobacteria, both individually and much more so cooperatively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciimmunol.aau6759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380365PMC
December 2018

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

Ann Neurol 2018 11 8;84(5):766-780. Epub 2018 Nov 8.

Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD.

Objective: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function.

Methods: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples.

Results: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology.

Interpretation: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ana.25351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295419PMC
November 2018

Mendelian susceptibility to mycobacterial disease: 2014-2018 update.

Immunol Cell Biol 2019 04 25;97(4):360-367. Epub 2018 Oct 25.

Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM, UMR 1163, Necker Hospital for Sick Children, Paris, France.

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. Since 1996, disease-causing mutations have been found in 11 genes, which, through allelic heterogeneity, underlie 21 different genetic disorders. We briefly review here progress in the study of molecular, cellular and clinical aspects of MSMD since the last comprehensive review published in 2014. Highlights include the discoveries of (1) a new genetic etiology, autosomal recessive signal peptide peptidase-like 2 A deficiency, (2) TYK2-deficient patients with a clinical phenotype of MSMD, (3) an allelic form of partial recessive IFN-γR2 deficiency, and (4) two forms of syndromic MSMD: RORγ/RORγT and JAK1 deficiencies. These recent findings illustrate how genetic and immunological studies of MSMD can shed a unique light onto the mechanisms of protective immunity to mycobacteria in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/imcb.12210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438774PMC
April 2019

Disruption of an antimycobacterial circuit between dendritic and helper T cells in human SPPL2a deficiency.

Nat Immunol 2018 09 20;19(9):973-985. Epub 2018 Aug 20.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York , NY, USA.

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory T1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a mice lack cDC2s, have CD4 T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory T1* cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41590-018-0178-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130844PMC
September 2018

The human CIB1-EVER1-EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses.

J Exp Med 2018 09 1;215(9):2289-2310. Epub 2018 Aug 1.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY

Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of (encoding EVER1) or (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human β-papillomaviruses (β-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to β-HPVs of EV patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20170308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122964PMC
September 2018

IRF4 haploinsufficiency in a family with Whipple's disease.

Elife 2018 03 14;7. Epub 2018 Mar 14.

Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Paris, France.

Most humans are exposed to (Tw). Whipple's disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance. We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.32340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915175PMC
March 2018

Visceral leishmaniasis in two patients with IL-12p40 and IL-12Rβ1 deficiencies.

Pediatr Blood Cancer 2017 06 22;64(6). Epub 2016 Nov 22.

Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Institut National de la Santé et de la Recherche Médicale, Paris, France.

Mutations of the IL12B and IL12RB1 genes underlie the development of IL-12 p40 and IL-12Rβ1 deficiencies, respectively, both of which cause predisposition to infection with weakly virulent mycobacteria and Salmonella. Infections with other intramacrophagic organisms have only been rarely observed. We identified two patients with visceral leishmaniasis who had autosomal recessive IL-12 p40 and IL-12Rβ1 deficiencies, respectively. This finding demonstrates the importance of IFN-γ immunity in the control of leishmaniasis. We also searched the literature for similar reports in patients with these and other primary immunodeficiencies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pbc.26362DOI Listing
June 2017

Transduction of Herpesvirus saimiri-Transformed T Cells with Exogenous Genes of Interest.

Curr Protoc Immunol 2016 11 1;115:7.21C.1-7.21C.12. Epub 2016 Nov 1.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York.

Human T cells can be transformed and expanded with herpesvirus saimiri (HVS). HVS-transformed T cells from patients have facilitated the study of a broad range of primary immunodeficiencies (PID) in which T-cell development or function is altered. However, the utility of HVS-transformed T cells for genetic studies has been limited by technical challenges in the expression of exogenous genes, including wild-type or mutant alleles. A novel, gamma retrovirus-based method for the simple and reliable transduction, purification, and study of HVS-transformed T cells is described. © 2016 by John Wiley & Sons, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cpim.15DOI Listing
November 2016

The mutation significance cutoff: gene-level thresholds for variant predictions.

Nat Methods 2016 Feb;13(2):109-10

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.3739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980758PMC
February 2016

The human gene damage index as a gene-level approach to prioritizing exome variants.

Proc Natl Acad Sci U S A 2015 Nov 19;112(44):13615-20. Epub 2015 Oct 19.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U.1163, Necker Hospital for Sick Children, 75015 Paris, France; Paris Descartes University, Imagine Institute, 75015 Paris, France; Howard Hughes Medical Institute, New York, NY 10065; Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, 75015 Paris, France

The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing). The GDI server, data, and software are freely available to noncommercial users from lab.rockefeller.edu/casanova/GDI.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1518646112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640721PMC
November 2015

IMMUNODEFICIENCIES. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations.

Science 2015 Aug 9;349(6248):606-613. Epub 2015 Jul 9.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065, USA.

Human inborn errors of immunity mediated by the cytokines interleukin-17A and interleukin-17F (IL-17A/F) underlie mucocutaneous candidiasis, whereas inborn errors of interferon-γ (IFN-γ) immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F-producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4(+)CCR6(+)CXCR3(+) αβ T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, RORγT, or both.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaa4282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668938PMC
August 2015

Identification of the inflammasome Nlrp1b as the candidate gene conferring diabetes risk at the Idd4.1 locus in the nonobese diabetic mouse.

J Immunol 2015 Jun 11;194(12):5663-73. Epub 2015 May 11.

Genetics and Genome Biology, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada; Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada

Type 1 diabetes in the NOD mouse model has been linked to >30 insulin-dependent diabetes (Idd) susceptibility loci. Idd4 on chromosome 11 consists of two subloci, Idd4.1 and Idd4.2. Using congenic analysis of alleles in NOD and NOD-resistant (NOR) mice, we previously defined Idd4.1 as an interval containing >50 genes that controlled expression of genes in the type 1 IFN pathway. In this study, we report refined mapping of Idd4.1 to a 1.1-Mb chromosomal region and provide genomic sequence analysis and mechanistic evidence supporting its role in innate immune regulation of islet-directed autoimmunity. Genetic variation at Idd4.1 was mediated by radiation-sensitive hematopoietic cells, and type 1 diabetes protection conferred by the NOR allele was abrogated in mice treated with exogenous type 1 IFN-β. Next generation sequence analysis of the full Idd4.1 genomic interval in NOD and NOR strains supported Nlrp1b as a strong candidate gene for Idd4.1. Nlrp1b belongs to the Nod-like receptor (NLR) gene family and contributes to inflammasome assembly, caspase-1 recruitment, and release of IL-1β. The Nlrp1b of NOR was expressed as an alternative spliced isoform that skips exon 9, resulting in a premature stop codon predicted to encode a truncated protein. Functional analysis of the truncated NOR Nlrp1b protein demonstrated that it was unable to recruit caspase-1 and process IL-1β. Our data suggest that Idd4.1-dependent protection from islet autoimmunity is mediated by differences in type 1 IFN- and IL-1β-dependent immune responses resulting from genetic variation in Nlrp1b.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1400913DOI Listing
June 2015

Comparison of assembly algorithms for improving rate of metatranscriptomic functional annotation.

Microbiome 2014 28;2:39. Epub 2014 Oct 28.

Molecular Structure and Function, Hospital for Sick Children, Peter Gilgan Center for Research and Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada ; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada ; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Background: Microbiome-wide gene expression profiling through high-throughput RNA sequencing ('metatranscriptomics') offers a powerful means to functionally interrogate complex microbial communities. Key to successful exploitation of these datasets is the ability to confidently match relatively short sequence reads to known bacterial transcripts. In the absence of reference genomes, such annotation efforts may be enhanced by assembling reads into longer contiguous sequences ('contigs'), prior to database search strategies. Since reads from homologous transcripts may derive from several species, represented at different abundance levels, it is not clear how well current assembly pipelines perform for metatranscriptomic datasets. Here we evaluate the performance of four currently employed assemblers including de novo transcriptome assemblers - Trinity and Oases; the metagenomic assembler - Metavelvet; and the recently developed metatranscriptomic assembler IDBA-MT.

Results: We evaluated the performance of the assemblers on a previously published dataset of single-end RNA sequence reads derived from the large intestine of an inbred non-obese diabetic mouse model of type 1 diabetes. We found that Trinity performed best as judged by contigs assembled, reads assigned to contigs, and number of reads that could be annotated to a known bacterial transcript. Only 15.5% of RNA sequence reads could be annotated to a known transcript in contrast to 50.3% with Trinity assembly. Paired-end reads generated from the same mouse samples resulted in modest performance gains. A database search estimated that the assemblies are unlikely to erroneously merge multiple unrelated genes sharing a region of similarity (<2% of contigs). A simulated dataset based on ten species confirmed these findings. A more complex simulated dataset based on 72 species found that greater assembly errors were introduced than is expected by sequencing quality. Through the detailed evaluation of assembly performance, the insights provided by this study will help drive the design of future metatranscriptomic analyses.

Conclusion: Assembly of metatranscriptome datasets greatly improved read annotation. Of the four assemblers evaluated, Trinity provided the best performance. For more complex datasets, reads generated from transcripts sharing considerable sequence similarity can be a source of significant assembly error, suggesting a need to collate reads on the basis of common taxonomic origin prior to assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/2049-2618-2-39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4236897PMC
November 2014

Microbiome manipulation modifies sex-specific risk for autoimmunity.

Gut Microbes 2014 Jul 9;5(4):485-93. Epub 2014 Jul 9.

Department of Immunology; University of Toronto; Toronto, ON Canada; Program in Genetics and Genomic Biology; The Hospital for Sick Children; Toronto, ON Canada; Department of Medical Biophysics; University of Toronto; Toronto, ON Canada.

Despite growing evidence for a causal role of environmental factors in autoimmune diseases including the rise in disease frequencies over the past several decades we lack an understanding of how particular environmental exposures modify disease risk. In addition, many autoimmune diseases display sex-biased incidence, with females being disproportionately affected but the mechanisms underlying this sex bias remain elusive. Emerging evidence suggests that both host metabolism and immune function is crucially regulated by the intestinal microbiome. Recently, we showed that in the non-obese diabetic (NOD) mouse model of Type 1 Diabetes (T1D), the gut commensal microbial community strongly impacts the pronounced sex bias in T1D risk by controlling serum testosterone and metabolic phenotypes (1). Here we present new data in the NOD model that explores the correlations between microbial phylogeny, testosterone levels, and metabolic phenotypes, and discuss the future of microbiome-centered analysis and microbe-based therapeutic approaches in autoimmune diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/gmic.29795DOI Listing
July 2014

γδ T cells are essential effectors of type 1 diabetes in the nonobese diabetic mouse model.

J Immunol 2013 Jun 26;190(11):5392-401. Epub 2013 Apr 26.

Programme in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario MG5 1L7, Canada.

γδ T cells, a lineage of innate-like lymphocytes, are distinguished from conventional αβ T cells in their Ag recognition, cell activation requirements, and effector functions. γδ T cells have been implicated in the pathology of several human autoimmune and inflammatory diseases and their corresponding mouse models, but their specific roles in these diseases have not been elucidated. We report that γδ TCR(+) cells, including both the CD27(-)CD44(hi) and CD27(+)CD44(lo) subsets, infiltrate islets of prediabetic NOD mice. Moreover, NOD CD27(-)CD44(hi) and CD27(+)CD44(lo) γδ T cells were preprogrammed to secrete IL-17, or IFN-γ upon activation. Adoptive transfer of type 1 diabetes (T1D) to T and B lymphocyte-deficient NOD recipients was greatly potentiated when γδ T cells, and specifically the CD27(-) γδ T cell subset, were included compared with transfer of αβ T cells alone. Ab-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection, supporting a nonredundant, pathogenic role of γδ T cells in this model. The potent contributions of CD27(-) γδ T cells and IL-17 to islet inflammation and diabetes reported in this study suggest that these mechanisms may also underlie human T1D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1203502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836168PMC
June 2013

Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity.

Science 2013 Mar 17;339(6123):1084-8. Epub 2013 Jan 17.

Program in Genetics and Genome Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipient's microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1233521DOI Listing
March 2013

Generation and analysis of a mouse intestinal metatranscriptome through Illumina based RNA-sequencing.

PLoS One 2012 27;7(4):e36009. Epub 2012 Apr 27.

Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Canada.

With the advent of high through-put sequencing (HTS), the emerging science of metagenomics is transforming our understanding of the relationships of microbial communities with their environments. While metagenomics aims to catalogue the genes present in a sample through assessing which genes are actively expressed, metatranscriptomics can provide a mechanistic understanding of community inter-relationships. To achieve these goals, several challenges need to be addressed from sample preparation to sequence processing, statistical analysis and functional annotation. Here we use an inbred non-obese diabetic (NOD) mouse model in which germ-free animals were colonized with a defined mixture of eight commensal bacteria, to explore methods of RNA extraction and to develop a pipeline for the generation and analysis of metatranscriptomic data. Applying the Illumina HTS platform, we sequenced 12 NOD cecal samples prepared using multiple RNA-extraction protocols. The absence of a complete set of reference genomes necessitated a peptide-based search strategy. Up to 16% of sequence reads could be matched to a known bacterial gene. Phylogenetic analysis of the mapped ORFs revealed a distribution consistent with ribosomal RNA, the majority from Bacteroides or Clostridium species. To place these HTS data within a systems context, we mapped the relative abundance of corresponding Escherichia coli homologs onto metabolic and protein-protein interaction networks. These maps identified bacterial processes with components that were well-represented in the datasets. In summary this study highlights the potential of exploiting the economy of HTS platforms for metatranscriptomics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036009PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338770PMC
September 2012

Naringenin prevents dyslipidemia, apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin resistance.

Diabetes 2009 Oct 10;58(10):2198-210. Epub 2009 Jul 10.

Department of Vascular Biology, Robarts Research Institute, Ontario, Canada.

Objective: The global epidemic of metabolic syndrome and its complications demands rapid evaluation of new and accessible interventions. Insulin resistance is the central biochemical disturbance in the metabolic syndrome. The citrus-derived flavonoid, naringenin, has lipid-lowering properties and inhibits VLDL secretion from cultured hepatocytes in a manner resembling insulin. We evaluated whether naringenin regulates lipoprotein production and insulin sensitivity in the context of insulin resistance in vivo.

Research Design And Methods: LDL receptor-null (Ldlr(-/-)) mice fed a high-fat (Western) diet (42% calories from fat and 0.05% cholesterol) become dyslipidemic, insulin and glucose intolerant, and obese. Four groups of mice (standard diet, Western, and Western plus 1% or 3% wt/wt naringenin) were fed ad libitum for 4 weeks. VLDL production and parameters of insulin and glucose tolerance were determined.

Results: We report that naringenin treatment of Ldlr(-/-) mice fed a Western diet corrected VLDL overproduction, ameliorated hepatic steatosis, and attenuated dyslipidemia without affecting caloric intake or fat absorption. Naringenin 1) increased hepatic fatty acid oxidation through a peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1alpha/PPARalpha-mediated transcription program; 2) prevented sterol regulatory element-binding protein 1c-mediated lipogenesis in both liver and muscle by reducing fasting hyperinsulinemia; 3) decreased hepatic cholesterol and cholesterol ester synthesis; 4) reduced both VLDL-derived and endogenously synthesized fatty acids, preventing muscle triglyceride accumulation; and 5) improved overall insulin sensitivity and glucose tolerance.

Conclusions: Thus, naringenin, through its correction of many of the metabolic disturbances linked to insulin resistance, represents a promising therapeutic approach for metabolic syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db09-0634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2750228PMC
October 2009

Selective up-regulation of LXR-regulated genes ABCA1, ABCG1, and APOE in macrophages through increased endogenous synthesis of 24(S),25-epoxycholesterol.

J Biol Chem 2007 Feb 23;282(8):5207-16. Epub 2006 Dec 23.

Robarts Research Institute Vascular Biology Group, Department of Biochemistry, University of Western, London, Ontario, Canada.

Liver X receptor (LXR) activation represents a mechanism to prevent macrophage foam cell formation. Previously, we demonstrated that partial inhibition of oxidosqualene:lanosterol cyclase (OSC) stimulated synthesis of the LXR agonist 24(S),25-epoxycholesterol (24(S),25-epoxy) and enhanced ABCA1-mediated cholesterol efflux. In contrast to a synthetic, nonsteroidal LXR activator, TO-901317, triglyceride accumulation was not observed. In the present study, we determined whether endogenous 24(S),25-epoxy synthesis selectively enhanced expression of macrophage LXR-regulated cholesterol efflux genes but not genes that regulate fatty acid metabolism. THP-1 human macrophages incubated with the OSC inhibitor (OSCi) RO0714565 (15 nM) significantly reduced cholesterol synthesis and maximized synthesis of 24(S),25-epoxy. Endogenous 24(S),25-epoxy increased ABCA1, ABCG1, and APOE mRNA abundance and consequently increased cholesterol efflux to apoAI. In contrast, OSCi had no effect on LXR-regulated genes LPL (lipoprotein lipase) and FAS (fatty acid synthase). TO-901317 (>or=10 nM) significantly enhanced expression of all genes examined. OSCi and TO-901317 increased the mRNA and precursor form of SREBP-1c, a major regulator of fatty acid and triglyceride synthesis. However, conversion of the precursor to the active form (nSREBP-1c) was blocked by OSCi-induced 24(S),25-epoxy but not by TO-901317 (>or=10 nm), which instead markedly increased nSREBP-1c. Disruption of nSREBP-1c formation by 24(S),25-epoxy accounted for diminished FAS and LPL expression. In summary, endogenous synthesis of 24(S),25-epoxy selectively up-regulates expression of macrophage LXR-regulated cholesterol efflux genes without stimulating genes linked to fatty acid and triglyceride synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M611063200DOI Listing
February 2007