Publications by authors named "Colby Zaph"

56 Publications

Context-dependent roles of B cells during intestinal helminth infection.

PLoS Negl Trop Dis 2021 May 13;15(5):e0009340. Epub 2021 May 13.

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

The current approaches to reduce the burden of chronic helminth infections in endemic areas are adequate sanitation and periodic administration of deworming drugs. Yet, resistance against some deworming drugs and reinfection can still rapidly occur even after treatment. A vaccine against helminths would be an effective solution at preventing reinfection. However, vaccines against helminth parasites have yet to be successfully developed. While T helper cells and innate lymphoid cells have been established as important components of the protective type 2 response, the roles of B cells and antibodies remain the most controversial. Here, we review the roles of B cells during intestinal helminth infection. We discuss the potential factors that contribute to the context-specific roles for B cells in protection against diverse intestinal helminth parasite species, using evidence from well-defined murine model systems. Understanding the precise roles of B cells during resistance and susceptibility to helminth infection may offer a new perspective of type 2 protective immunity.
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http://dx.doi.org/10.1371/journal.pntd.0009340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118336PMC
May 2021

c-Rel employs multiple mechanisms to promote the thymic development and peripheral function of regulatory T cells in mice.

Eur J Immunol 2021 May 7. Epub 2021 May 7.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.
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http://dx.doi.org/10.1002/eji.202048900DOI Listing
May 2021

Intestinal-epithelial LSD1 controls goblet cell maturation and effector responses required for gut immunity to bacterial and helminth infection.

PLoS Pathog 2021 Mar 31;17(3):e1009476. Epub 2021 Mar 31.

CEMIR-Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.
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http://dx.doi.org/10.1371/journal.ppat.1009476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041206PMC
March 2021

The Histone Methyltransferase DOT1L Is Essential for Humoral Immune Responses.

Cell Rep 2020 12;33(11):108504

Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia; Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Histone modifiers are essential for the ability of immune cells to reprogram their gene expression during differentiation. The recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like) induces oncogenic gene expression in a subset of B cell leukemias. Despite its importance, its role in the humoral immune system is unclear. Here, we demonstrate that DOT1L is a critical regulator of B cell biology. B cell development is defective in Dot1lMb1 mice, culminating in a reduction of peripheral mature B cells. Upon immunization or influenza infection of Dot1lCd23 mice, class-switched antibody-secreting cells are significantly attenuated and germinal centers fail to form. Consequently, DOT1L is essential for B cell memory formation. Transcriptome, pathway, and histological analyses identified a role for DOT1L in reprogramming gene expression for appropriate localization of B cells during the initial stage of the response. Together, these results demonstrate an essential role for DOT1L in generating an effective humoral immune response.
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http://dx.doi.org/10.1016/j.celrep.2020.108504DOI Listing
December 2020

The Methyltransferase DOT1L Controls Activation and Lineage Integrity in CD4 T Cells during Infection and Inflammation.

Cell Rep 2020 12;33(11):108505

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia. Electronic address:

CD4 T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T-cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and are resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th2 cell lineage commitment and stability and suggest that inhibition of DOT1L may provide a therapeutic strategy to limit type 2 immune responses.
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http://dx.doi.org/10.1016/j.celrep.2020.108505DOI Listing
December 2020

A chemical biology toolbox to study protein methyltransferases and epigenetic signaling.

Nat Commun 2019 01 3;10(1):19. Epub 2019 Jan 3.

Structural Genomics Consortium, University of Toronto, Toronto, ON, M5G 1L7, Canada.

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4 T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond.
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http://dx.doi.org/10.1038/s41467-018-07905-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318333PMC
January 2019

Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses.

PLoS One 2018 6;13(12):e0208343. Epub 2018 Dec 6.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

Successful T-dependent humoral responses require the production of antibody-secreting plasmablasts, as well as the formation of germinal centers which eventually form high-affinity B cell memory. The ability of B cells to differentiate into germinal center and plasma cells, as well as the ability to tailor responses to different pathogens, is driven by transcription factors. In T cells, the T-box transcription factors T-bet and Eomesodermin (Eomes) regulate effector and memory T cell differentiation, respectively. While T-bet has a critical role in regulating anti-viral B cell responses, a role for Eomes in B cells has yet to be described. We therefore investigated whether Eomes was required for B cell differentiation during either Th1 or Th2 cell-biased immune responses. Here, we demonstrate that deletion of Eomes specifically in B cells did not affect B cell differentiation in response to vaccination, as well as following viral or helminth infection. In contrast to its established role in CD8+ T cells, Eomes did not influence memory B cell differentiation. Finally, the use of an Eomes reporter mouse confirmed the lack of Eomes expression during immune responses. Thus, germinal center and plasma cell differentiation and the formation of isotype-switched memory B cells in response to infection are independent of Eomes expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208343PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283461PMC
May 2019

HIC1 links retinoic acid signalling to group 3 innate lymphoid cell-dependent regulation of intestinal immunity and homeostasis.

PLoS Pathog 2018 02 22;14(2):e1006869. Epub 2018 Feb 22.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity.
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http://dx.doi.org/10.1371/journal.ppat.1006869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823476PMC
February 2018

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential.

Cell Stem Cell 2018 02 25;22(2):177-190.e7. Epub 2018 Jan 25.

Biomedical Research Centre, Department of Medical Genetics, 2222 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada. Electronic address:

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of β-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease.
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http://dx.doi.org/10.1016/j.stem.2017.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031334PMC
February 2018

The orphan nuclear receptor RORα and group 3 innate lymphoid cells drive fibrosis in a mouse model of Crohn's disease.

Sci Immunol 2016 Sep 2;1(3):eaaf8864. Epub 2016 Sep 2.

Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

Fibrosis is the result of dysregulated tissue regeneration and is characterized by excessive accumulation of matrix proteins that become detrimental to tissue function. In Crohn's disease, this manifests itself as recurrent gastrointestinal strictures for which there is no effective therapy beyond surgical intervention. Using a model of infection-induced chronic gut inflammation, we show that -deficient mice are protected from fibrosis; infected intestinal tissues display diminished pathology, attenuated collagen deposition, and reduced fibroblast accumulation. Although is best known for its role in group 2 innate lymphoid cell (ILC2) development, we find that -induced fibrosis is independent of eosinophils, signal transducer and activator of transcription 6 signaling, and T helper 2 cytokine production, arguing that this process is largely ILC2-independent. Instead, we observe reduced levels of ILC3- and T cell-derived interleukin-17A (IL-17A) and IL-22 in infected gut tissues. Furthermore, using / bone marrow chimeric mice, we show that restoring ILC function is sufficient to reestablish IL-17A and IL-22 production and a profibrotic phenotype. Our results show that RORα (retinoic acid receptor-related orphan receptor α)-dependent ILC3 functions are pivotal in mediating gut fibrosis, and they offer an avenue for therapeutic intervention in Crohn's-like diseases.
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http://dx.doi.org/10.1126/sciimmunol.aaf8864DOI Listing
September 2016

Loss of Vascular CD34 Results in Increased Sensitivity to Lung Injury.

Am J Respir Cell Mol Biol 2017 12;57(6):651-661

1 The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular, and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild-type (WT) and Cd34 mice by bleomycin administration. We found that Cd34 mice displayed severe weight loss and early mortality compared with WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34 mice reconstituted with WT hematopoietic cells exhibited early mortality compared with WT mice reconstituted with Cd34 cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together, our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced tissue damage.
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http://dx.doi.org/10.1165/rcmb.2016-0386OCDOI Listing
December 2017

The orphan nuclear receptor ROR alpha and group 3 innate lymphoid cells drive fibrosis in a mouse model of Crohn's disease.

Sci Immunol 2016 Sep;1(3)

The Biomedical Research Centre, University of British Columbia, V6T 1Z3.

Fibrosis is the result of dysregulated tissue regeneration and is characterized by excessive accumulation of matrix proteins that become detrimental to tissue function. In Crohn's disease, this manifests itself as recurrent gastrointestinal strictures for which there is no effective therapy beyond surgical intervention. Using a model of infection-induced chronic gut inflammation, we show that -deficient mice are protected from fibrosis; infected intestinal tissues display diminished pathology, attenuated collagen deposition and reduced fibroblast accumulation. Although is best known for its role in ILC2 development, we find that -induced fibrosis is independent of eosinophils, STAT6 signaling and Th2 cytokine production arguing that this process is largely ILC2-independent. Instead, we observe reduced levels of ILC3- and T cell-derived IL-17A and IL-22 in infected gut tissues. Furthermore, using / bone marrow chimeric mice, we show that restoring ILC function is sufficient to re-establish IL-17A and IL-22 production and a profibrotic phenotype. Our results show that RORα-dependent ILC3 functions are pivotal in mediating gut fibrosis and they offer an avenue for therapeutic intervention in Crohn's-like diseases.
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http://dx.doi.org/10.1126/sciimmunol.aaf8864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489332PMC
September 2016

The Lysine Methyltransferase G9a in Immune Cell Differentiation and Function.

Front Immunol 2017 11;8:429. Epub 2017 Apr 11.

Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

G9a (KMT1C, EHMT2) is a lysine methyltransferase (KMT) whose primary function is to di-methylate lysine 9 of histone H3 (H3K9me2). G9a-dependent H3K9me2 is associated with gene silencing and acts primarily through the recruitment of H3K9me2-binding proteins that prevent transcriptional activation. Gene repression via G9a-dependent H3K9me2 is critically required in embryonic stem (ES) cells for the development of cellular lineages by repressing expression of pluripotency factors. In the immune system, lymphoid cells such as T cells and innate lymphoid cells (ILCs) can differentiate from a naïve state into one of several effector lineages that require both activating and repressive mechanisms to maintain the correct gene expression program. Furthermore, the long-term immunity to re-infection is mediated by memory T cells, which also require specific gene expression and repression to maintain a quiescent state. In this review, we examine the molecular machinery of G9a-dependent functions, address the role of G9a in lymphoid cell differentiation and function, and identify potential functions of T cells and ILCs that may be controlled by G9a. Together, this review will highlight the dynamic nature of G9a-dependent H3K9me2 in the immune system and shed light on the nature of repressive epigenetic modifications in cellular lineage choice.
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http://dx.doi.org/10.3389/fimmu.2017.00429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387087PMC
April 2017

Mastering gut permeability: New roles for old friends.

Eur J Immunol 2017 02;47(2):236-239

Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

Mast cells are innate immune cells that respond rapidly to infection in barrier tissues such as the skin and intestinal mucosa. Expulsion of parasitic worms in the gut involves a robust type 2 host response, and an acute mastocytosis is often generated at the site of infection. However, the role of mast cells in resistance to worm infections appears to be parasite specific. Mast cells are also involved in tissue repair, but the long-term contribution of mast cell activation after worm expulsion has not been definitively studied. In this issue of European Journal of Immunology, Sorobetea et al. [Eur. J. Immunol. 2017. 47: 257-268] demonstrate that activated mast cells persist in the large intestinal lamina propria and intraepithelial compartment long after worm expulsion, resulting in continued local and systemic presence of the mast cell protease mast cell protease 1 (MCPt-1) and enhanced intestinal permeability. In this commentary, we discuss these findings in the wider context of mast cell function in health and disease.
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http://dx.doi.org/10.1002/eji.201646842DOI Listing
February 2017

Early-life antibiotic treatment enhances the pathogenicity of CD4 T cells during intestinal inflammation.

J Leukoc Biol 2017 04 29;101(4):893-900. Epub 2016 Dec 29.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada;

The incidence of inflammatory bowel diseases (IBDs) has steadily increased in recent decades-a phenomenon that cannot be explained by genetic mutations alone. Other factors, including the composition of the intestinal microbiome, are potentially important contributors to the increased occurrence of this group of diseases. Previous reports have shown a correlation between early-life antibiotic (Abx) treatment and an increased incidence of IBD. In this report, we investigated the effects of early-life Abx treatments on the pathogenicity of CD4 T cells using an experimental T cell transfer model of IBD. Our results show that CD4 T cells isolated from adult mice that had been treated with Abx during gestation and in early life induced a faster onset of IBD in -deficient mice compared with CD4 T cells of untreated mice. Ex vivo functional analyses of IBD-inducing CD4 T cells did not show significant differences in their immunologic potential ex vivo, despite their in vivo phenotype. However, genome-wide gene-expression analysis revealed that these cells displayed dysregulated expression of genes associated with cell-cycle regulation, metabolism, and cellular stress. Analysis of Abx-treated CD4 T cell donors showed systemically elevated levels of the stress hormone corticosterone throughout life compared with untreated donors. The cohousing of Abx-treated mice with untreated mice decreased serum corticosterone, and a consequent transfer of the cells from cohoused mice into -deficient mice restored the onset and severity of disease to that of untreated animals. Thus, our results suggest that early-life Abx treatment results in a stress response with high levels of corticosterone that influences CD4 T cell function.
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http://dx.doi.org/10.1189/jlb.3MA0716-334RRDOI Listing
April 2017

Chronic Trichuris muris infection alters hematopoiesis and causes IFN-γ-expressing T-cell accumulation in the mouse bone marrow.

Eur J Immunol 2016 11 26;46(11):2587-2596. Epub 2016 Sep 26.

The Biomedical Research Centre, University of British Columbia, Vancouver, Canada.

Proinflammatory cytokines produced during immune responses to infectious stimuli are well-characterized to have secondary effects on the function of hematopoietic progenitor cells in the BM. However, these effects on the BM are poorly characterized during chronic infection with intestinal helminth parasites. In this study, we use the Trichuris muris model of infection and show that Th1 cell-associated, but not acute Th2 cell-associated, responses to chronic T. muris infection cause a major, transient expansion of CD48 CD150 multipotent progenitor cells in the BM that is dependent on the presence of adaptive immune cells and IFN-γ signaling. Chronic T. muris infection also broadly stimulated proliferation of BM progenitor cells including CD48 CD150 hematopoietic stem cells. This shift in progenitor activity during chronic T. muris infection correlated with a functional increase in myeloid colony formation in vitro as well as neutrophilia in the BM and peripheral blood. In parallel, we observed an accumulation of CD4 , CD8 , and CD4 CD8 (double negative) T cells that expressed IFN-γ, displaying activated and central memory-type phenotypes in the bone marrow during chronic infection. Thus, these results demonstrate that Th1 cell-driven responses in the intestine during chronic helminth infection potently influence upstream hematopoietic processes in the BM via IFN-γ.
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http://dx.doi.org/10.1002/eji.201646326DOI Listing
November 2016

Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection.

PLoS Pathog 2016 09 6;12(9):e1005876. Epub 2016 Sep 6.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7-/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection.
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http://dx.doi.org/10.1371/journal.ppat.1005876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012677PMC
September 2016

G9a regulates group 2 innate lymphoid cell development by repressing the group 3 innate lymphoid cell program.

J Exp Med 2016 06 13;213(7):1153-62. Epub 2016 Jun 13.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia

Innate lymphoid cells (ILCs) are emerging as important regulators of homeostatic and disease-associated immune processes. Despite recent advances in defining the molecular pathways that control development and function of ILCs, the epigenetic mechanisms that regulate ILC biology are unknown. Here, we identify a role for the lysine methyltransferase G9a in regulating ILC2 development and function. Mice with a hematopoietic cell-specific deletion of G9a (Vav.G9a(-/-) mice) have a severe reduction in ILC2s in peripheral sites, associated with impaired development of immature ILC2s in the bone marrow. Accordingly, Vav.G9a(-/-) mice are resistant to the development of allergic lung inflammation. G9a-dependent dimethylation of histone 3 lysine 9 (H3K9me2) is a repressive histone mark that is associated with gene silencing. Genome-wide expression analysis demonstrated that the absence of G9a led to increased expression of ILC3-associated genes in developing ILC2 populations. Further, we found high levels of G9a-dependent H3K9me2 at ILC3-specific genetic loci, demonstrating that G9a-mediated repression of ILC3-associated genes is critical for the optimal development of ILC2s. Together, these results provide the first identification of an epigenetic regulatory mechanism in ILC development and function.
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http://dx.doi.org/10.1084/jem.20151646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4925019PMC
June 2016

SETD7 Controls Intestinal Regeneration and Tumorigenesis by Regulating Wnt/β-Catenin and Hippo/YAP Signaling.

Dev Cell 2016 Apr;37(1):47-57

The Biomedical Research Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis.
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http://dx.doi.org/10.1016/j.devcel.2016.03.002DOI Listing
April 2016

Low-Dose Intestinal Trichuris muris Infection Alters the Lung Immune Microenvironment and Can Suppress Allergic Airway Inflammation.

Infect Immun 2016 02 7;84(2):491-501. Epub 2015 Dec 7.

The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia

Immunological cross talk between mucosal tissues such as the intestine and the lung is poorly defined during homeostasis and disease. Here, we show that a low-dose infection with the intestinally restricted helminth parasite Trichuris muris results in the production of Th1 cell-dependent gamma interferon (IFN-γ) and myeloid cell-derived interleukin-10 (IL-10) in the lung without causing overt airway pathology. This cross-mucosal immune response in the lung inhibits the development of papain-induced allergic airway inflammation, an innate cell-mediated type 2 airway inflammatory disease. Thus, we identify convergent and nonredundant roles of adaptive and innate immunity in mediating cross-mucosal suppression of type 2 airway inflammation during low-dose helminth-induced intestinal inflammation. These results provide further insight in identifying novel intersecting immune pathways elicited by gut-to-lung mucosal cross talk.
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http://dx.doi.org/10.1128/IAI.01240-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730564PMC
February 2016

Dendritic-cell expression of Ship1 regulates Th2 immunity to helminth infection in mice.

Eur J Immunol 2016 Jan 30;46(1):122-30. Epub 2015 Nov 30.

Biomedical Research Centre, University of British Columbia, Vancouver, Canada.

In mouse models of infection with the gastrointestinal parasite Trichuris muris, appropriate dendritic-cell (DC) Ag sampling, migration, and presentation to T cells are necessary to mount a protective Th2-polarized adaptive immune response, which is needed to clear infection. SH2-containing inositol 5'-phosphatase 1 (SHIP-1) has been shown to be an important regulator of DC function in vitro through the negative regulation of the phosphoinositide 3-kinase (PI3K) pathway, but its role in vivo is relatively unexplored. In the current work, mice with a specific deletion of SHIP-1 in DCs (Ship1(ΔDC) ) were infected with the parasite T. muris. Ship1(ΔDC) mice were susceptible to infection due to ineffective priming of Th2-polarized responses. This is likely due to an increased production of interleukin (IL) 12p40 by SHIP-1-deficient DCs, as in vivo antibody blockade of IL-12p40 was able to facilitate the clearing of infection in Ship1(ΔDC) mice. Our results describe a critical role for SHIP-1 in regulating the ability of DCs to efficiently prime Th2-type responses.
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http://dx.doi.org/10.1002/eji.201545628DOI Listing
January 2016

Goblet Cell Derived RELM-β Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation.

PLoS Pathog 2015 Aug 18;11(8):e1005108. Epub 2015 Aug 18.

Division of Gastroenterology, Department of Pediatrics, Child and Family Research Institute, Vancouver, Canada.

Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-β is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-β's role in host defense is unclear. Thus, wildtype and RELM-β gene deficient mice (Retnlb-/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb-/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb-/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-β did not directly increase IEC proliferation, rather RELM-β acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb-/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-β to Retnlb-/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-β and goblet cells play an unexpected, yet critical role in recruiting CD4+ T cells to the colon to protect against an enteric pathogen, in part via the induction of increased IEC proliferation.
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http://dx.doi.org/10.1371/journal.ppat.1005108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540480PMC
August 2015

Lineage-specific regulation of allergic airway inflammation by the lipid phosphatase Src homology 2 domain-containing inositol 5-phosphatase (SHIP-1).

J Allergy Clin Immunol 2015 Sep 5;136(3):725-736.e2. Epub 2015 Mar 5.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:

Background: Inpp5d (Src homology 2 domain-containing inositol-5-phosphatase [Ship1])-deficient mice experience spontaneous airway inflammation and have enhanced sensitivity to allergen-induced airway inflammation.

Objective: We hypothesized that lineage-specific deletion of Ship1 expression in cells known to be crucial for adaptive TH2 responses would uncover distinct roles that could either positively or negatively regulate susceptibility to allergic airway inflammation (AAI).

Methods: Ship1 expression was deleted in B cells, T cells, or dendritic cells (DCs), and the resulting Ship1(ΔB cell), Ship1(ΔT cell), Ship1(ΔDC), or Ship1(F/F) (wild-type) control mice were evaluated in a model of house dust mite (HDM)-induced AAI.

Results: Unlike germline panhematopoietic Ship1 deletion, deletion of Ship1 selectively in either the B-cell, T-cell, or DC lineages did not result in spontaneous airway inflammation. Strikingly, although loss of Ship1 in the B-cell lineage did not affect HDM-induced AAI, loss of Ship1 in either of the T-cell or DC lineages protected mice from AAI by skewing the typical TH2 immune response toward a TH1 response.

Conclusions: Although panhematopoietic deletion of Ship1 leads to spontaneous lung inflammation, selective deletion of Ship1 in T cells or DCs impairs the formation of an adaptive TH2 response and protects animals from HDM-induced AAI.
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http://dx.doi.org/10.1016/j.jaci.2015.01.019DOI Listing
September 2015

Regulation of CD4 T-cell differentiation and inflammation by repressive histone methylation.

Immunol Cell Biol 2015 Mar 13;93(3):245-52. Epub 2015 Jan 13.

The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada.

Repressive epigenetic modifications such as dimethylation and trimethylation histone H3 at lysine 9 (H3K9me2 and H3K9me3) and H3K27me3 have been shown to be critical for embryonic stem (ES) cell differentiation by silencing cell lineage-promiscuous genes. CD4(+) T helper (T(H)) cell differentiation is a powerful model to study the molecular mechanisms associated with cellular lineage choice in adult cells. Naïve T(H) cells have the capacity to differentiate into one of the several phenotypically and functionally distinct and stable lineages. Although some repressive epigenetic mechanisms have a critical role in T(H) cell differentiation in a similar manner to that in ES cells, it is clear that there are disparate functions for certain modifications between ES cells and T(H) cells. Here we review the role of repressive histone modifications in the differentiation and function of T(H) cells in health and disease.
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http://dx.doi.org/10.1038/icb.2014.115DOI Listing
March 2015

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells.

Proc Natl Acad Sci U S A 2014 Sep 18;111(35):12853-8. Epub 2014 Aug 18.

Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge, MA 02139;

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.
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http://dx.doi.org/10.1073/pnas.1407358111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156762PMC
September 2014

Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2-inducing allergen exposures.

J Allergy Clin Immunol 2014 Apr;133(4):1142-8

The Biomedical Research Centre, University of British Columbia, Vancouver. Electronic address:

Background: Allergic inflammation involves the sensitization of naive CD4(+) T cells to allergens, resulting in a TH2-skewed inflammatory response. Although antigen presentation by dendritic cells to T cells in the lymph node is crucial for TH2 cell development, the innate signals that initiate adaptive type 2 inflammation and the role of group 2 innate lymphoid cells (ILC2s) are poorly understood.

Objective: We sought to investigate the influence of ILC2s and the route of priming on the development of an adaptive type 2 immune response to lung allergens.

Methods: Wild-type and ILC2-deficient mice were exposed intranasally or systemically to the TH2-inducing antigens house dust mite or ovalbumin in a model of allergic airway inflammation or the TH17-inducing bacterial antigen Saccharopolyspora rectivirgula in a model of hypersensitivity pneumonitis. The formation of an adaptive immune response was evaluated based on serum antibody titers and production of T cell-derived cytokines (IL-4, IL-5, IL-13 and IL-17A).

Results: We find that lung ILC2s play a critical role in priming the adaptive type 2 immune response to inhaled allergens, including the recruitment of eosinophils, TH2 cytokine production and serum IgE levels. Surprisingly, systemic priming with ovalbumin, with or without adjuvants, circumvents the requirement for ILC2s in inducing TH2-driven lung inflammation. ILC2s were also found to be dispensable for the sensitization to TH1- or TH17-inducing antigens.

Conclusion: These data highlight a critical role for ILC2s in the development of adaptive type 2 responses to local, but not systemic, antigen exposure.
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http://dx.doi.org/10.1016/j.jaci.2014.02.033DOI Listing
April 2014

Methyltransferase G9A regulates T cell differentiation during murine intestinal inflammation.

J Clin Invest 2014 May 25;124(5):1945-55. Epub 2014 Mar 25.

Inflammatory bowel disease (IBD) pathogenesis is associated with dysregulated CD4⁺ Th cell responses, with intestinal homeostasis depending on the balance between IL-17-producing Th17 and Foxp3⁺ Tregs. Differentiation of naive T cells into Th17 and Treg subsets is associated with specific gene expression profiles; however, the contribution of epigenetic mechanisms to controlling Th17 and Treg differentiation remains unclear. Using a murine T cell transfer model of colitis, we found that T cell-intrinsic expression of the histone lysine methyltransferase G9A was required for development of pathogenic T cells and intestinal inflammation. G9A-mediated dimethylation of histone H3 lysine 9 (H3K9me2) restricted Th17 and Treg differentiation in vitro and in vivo. H3K9me2 was found at high levels in naive Th cells and was lost following Th cell activation. Loss of G9A in naive T cells was associated with increased chromatin accessibility and heightened sensitivity to TGF-β1. Pharmacological inhibition of G9A methyltransferase activity in WT T cells promoted Th17 and Treg differentiation. Our data indicate that G9A-dependent H3K9me2 is a homeostatic epigenetic checkpoint that regulates Th17 and Treg responses by limiting chromatin accessibility and TGF-β1 responsiveness, suggesting G9A as a therapeutic target for treating intestinal inflammation.
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http://dx.doi.org/10.1172/JCI69592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001530PMC
May 2014

The retinoic acid-metabolizing enzyme Cyp26b1 regulates CD4 T cell differentiation and function.

PLoS One 2013 22;8(8):e72308. Epub 2013 Aug 22.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of Cyp26b1, the T cell-specific family member, in CD4(+) T cells. Mice with a conditional knockout of Cyp26b1 in T cells (Cyp26b1 (-/-) mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iTreg) cell- and TH17 cell-polarizing conditions in vitro. Further, Cyp26b1 expression was differentially regulated in iTreg and TH17 cells. Transfer of naïve Cyp26b1 (-/-) CD4(+) T cells into Rag1 (-/-) mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of Cyp26b1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target Cyp26b1 for the treatment of inflammatory bowel disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072308PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750006PMC
June 2014

Control of the hippo pathway by Set7-dependent methylation of Yap.

Dev Cell 2013 Jul 11;26(2):188-94. Epub 2013 Jul 11.

The Biomedical Research Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

Methylation of nonhistone proteins is emerging as a regulatory mechanism to control protein function. Set7 (Setd7) is a SET-domain-containing lysine methyltransferase that methylates and alters function of a variety of proteins in vitro, but the in vivo relevance has not been established. We found that Set7 is a modifier of the Hippo pathway. Mice that lack Set7 have a larger progenitor compartment in the intestine, coinciding with increased expression of Yes-associated protein (Yap) target genes. Mechanistically, monomethylation of lysine 494 of Yap is critical for cytoplasmic retention. These results identify a methylation-dependent checkpoint in the Hippo pathway.
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http://dx.doi.org/10.1016/j.devcel.2013.05.025DOI Listing
July 2013

Requirement for core 2 O-glycans for optimal resistance to helminth infection.

PLoS One 2013 29;8(3):e60124. Epub 2013 Mar 29.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

The migration of lymphocytes to the small intestine is controlled by expression of the integrin α4β7 and the chemokine receptor CCR9. However, the molecules that specifically regulate migration to the large intestine remain unclear. Immunity to infection with the large intestinal helminth parasite Trichuris muris is dependent upon CD4(+) T cells that migrate to the large intestine. We examine the role of specific chemokine receptors, adhesion molecules and glycosyltransferases in the development of protective immunity to Trichuris. Mice deficient in expression of the chemokine receptors CCR2 or CCR6 were resistant to infection with Trichuris. Similarly, loss of CD34, CD43, CD44 or PSGL-1 had no effect on resistance to infection. In contrast, simultaneous deletion of the Core2 β1,6-N-acetylglucosaminyltransferase (C2GnT) enzymes C2GnT1 and C2Gnt2 resulted in delayed expulsion of worms. These results suggest that C2GnT-dependent modifications may play a role in migration of protective immune cells to the large intestine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060124PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612062PMC
September 2013