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Www.ias2013.org Kuala Lumpur, Malaysia, 30 June - 3 July 2013 The impact of SIV infection on gut innate lymphocyte populations and gene expression Ronald.

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Presentation on theme: "Www.ias2013.org Kuala Lumpur, Malaysia, 30 June - 3 July 2013 The impact of SIV infection on gut innate lymphocyte populations and gene expression Ronald."— Presentation transcript:

1 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 The impact of SIV infection on gut innate lymphocyte populations and gene expression Ronald S. Veazey, DVM, PhD Division of Comparative Pathology Tulane National Primate Research Center Covington, Louisiana Tulane University School of Medicine New Orleans, Louisiana

2 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Differences between innate and adaptive immunity? Adaptive immunity previously defined as having ”immunologic memory” but lines are increasingly blurred – innate lymphoid cells (ILC) do not have antigen specific receptors (CD3) Emerging evidence shows innate lymphoid cells develop independently in GALT, and develop immunologic memory Murine studies indicate GALT develops through immune responses to bacteria mediated through specialized ”Lymphoid tissue inducer” (Lti), possible stem cells, and other innate lymphoid cell (ILC) subsets

3 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Innate lymphoid cells Newly proposed nomenclature: Group 1: ILC1: ILCs that produce IFNγ. Prototypical member is the NK cell. NK cells display both cytotoxic activity, and produce IFNγ following activation. Group 2: ILC2: ILCs that produce type 2 cytokines (including IL-5 and IL-13) and are dependent on GATA-binding protein 3 (GATA3) and retinoic acid receptor- related orphan receptor-α (RORα) for development and function. Group 3: ILC3: ILCs that produce IL-17 and/or IL-22 and depend on the transcription factor RORγt for development and function. Lymphoid tissue inducer cells (LTi) are prototype. Spits, H., D. Artis, et al. (2013). "Innate lymphoid cells--a proposal for uniform nomenclature." Nat Rev Immunol 13(2): “ILCs should be categorized into three groups based on the cytokines that they can produce and the transcription factors that regulate their development and function”

4 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Innate lymphoid cells Proinflammatory retinoic-acid-receptor-related orphan receptor- gammat-positive (RORgammat+) innate lymphoid cells (ILCs) differentiate from distinct fetal liver RORgammat(+) precursors and are crucial for immune homeostasis (ILC17, 22, etc.). Sawa, Eberl et al., Lineage relationship analysis of RORgammat+ innate lymphoid cells. Science 330: Mucosal (RORgammat(+)innate lymphoid cells (ILCs) are an important innate lymphocyte population required for immunity to intestinal infections Klose, Diefenbach, et al., A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells. Nature 494: ILC regulate CD4+ T-cell responses to intestinal bacteria. Hepworth, Sonnenberg et al, Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria. Nature 498:

5 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 PLoS Pathog September; 8(9) Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection. Namita Rout,…Amitinder Kaur PLoS Pathog ;8(8) ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIV(mac)251 challenge. Alpert MD, ….Evans DT. Blood Jun 3;115(22): Epub 2010 Mar 25. CD16- natural killer cells: enrichment in mucosal and secondary lymphoid tissues and altered function during chronic SIV infection. Reeves RK,……Johnson RP. Blood Sep 22;118(12): Epub 2011 Jul 26. Gut inflammation and indoleamine deoxygenase inhibit IL-17 production and promote cytotoxic potential in NKp44+ mucosal NK cells during SIV infection. Reeves RK, …Johnson RP. Recent studies of NK / ILC in SIV-infected macaques

6 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Phenotyping innate lymphoid cells (ILC) in normal macaque blood (Xu, Veazey et al, Mucosal Immunol 2012) Gating strategy: Lineage negative, CD3negCD8++ C3negCD8a neg (DC, B cells, etc.) C3negCD8a HIGH (ILC’s)

7 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 SIV/IgG immune complexes in SIV-infected macaques block detection of CD16 but not cytolytic activity of natural killer cells. Wei and Fultz, et al, Clin and Vacc Immunol 2006 Comparison of anti-human CD16 mAb clones on SIV- infected macaque cells before and after “washing” CD16 detection is artificially masked by some anti-SIV immune complexes CD16 clone DJ130 (Dako) best results

8 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Lymphocytes IL-17+ IL-17 CD3 CD3+CD4+ (Th17) CD3+CD8+ (Tc17) CD3-CD8 high (ILC) IL-17 PBMCJej LPLColon LPLSpleen Tonsil Duodenum ILC that secrete IL-17 are restricted to mucosal tissues in macaques

9 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 IL-17 secreting cells in jejunum of normal rhesus macaques Blood Oct 1;112(7): Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Brenchley…Douek, et al. *

10 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Intestinal ILC17 cells are depleted in SIV infection

11 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Summary: Some ILC17 (ILC3) cells secrete IL-22, TNF- , but not IFN-  nor granzyme B (not cytolytic) SIV infection results in significant loss of ILC17 cells, especially in the jejunum, which persists throughout SIV infection. Loss of ILC17 cells (and IL-17 in general) may contribute to loss of intestinal mucosal integrity and disease progression in human immunodeficiency virus (HIV)/SIV infection.

12 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Small Intestine (Jejunum) Epithelial cells (Enterocytes) Intraepithelial Lymphocytes Lamina propria Cells (LPC’s) Fibrovascular stroma Approach to reduce tissue complexity Separation by Percoll gradients: SIV infection pre infection (6 weeks) Resection biopsy 6-8 cm 21 d post infection Resection biopsy 6-8 cm 90d post infection Resection biopsy 6-8 cm N=5 Changes in intestinal gene expression in SIV: Experimental Design

13 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Genome wide changes in the jejunum lamina propria in SIV infection (measured by Affymetrix rhesus arrays - 54,675 capture probes) 1) Intestinal lamina propria

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16 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Upregulation of multiple genes associated with immune activation

17 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Downregulated genes associated with oxidative phosphorylation, IFN , IL-17, B cell “help”

18 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Gene changes in lamina propria in chronic (90 days) SIV infection

19 Kuala Lumpur, Malaysia, 30 June - 3 July 2013  LBP (lipopolysaccharide binding protein)  Expressed by macrophages and Paneth cells in response to LPS  LBP-LPS complex together with CD14 activates TLR4 pathway  CD70  Cytokine that belongs to the TNF family of ligands  Expressed only on activated T cells (CD4 & CD8) and binds to CD27 (receptor)  Induces proliferation of co-stimulated T cells  May bind CD27 on memory B cells and induce plasma cell differentiation (hypergammaglobulinemia)  CD38  Ectoenzyme and an activation marker for CD4, CD8 and B cells  JNK3 (Jun-N-terminal kinase 3)  Activated by LPS and proinflammatory cytokines Important Up-regulated genes at 90 d PI

20 Kuala Lumpur, Malaysia, 30 June - 3 July 2013  CXCL18  Chemotactic for naïve T cells and non activated lymphocytes  May be a protective response (moderation of inflammation)  TLR8  B inds ssRNA (HIV/SIV)  Signaling induces IFN  production leading to immune activation  Downregulation may be a protective response or cellular dysfunction (DC, mac)  IL-8  Produced mainly by macrophages, dendritic cells  Can Inhibit HIV replication in PBMCs and ectocervical tissue explants  AICD  Required for somatic hypermutation and class switch recombination Important Down-regulated genes at 90 d PI

21 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Intestinal Epithelium PLoS One. 2013; Intestinal Epithelium Reveals Transcriptional Signatures Consistent with Disturbances in Enterocyte Maturation and Differentiation during the Course of SIV Infection. Mohan, Veazey, Lackner et al.

22 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Gene changes in intestinal epithelium in acute (21 days) SIV infection

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25 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Transcription factors and signaling pathways known to regulate intestinal epithelial gene expression 1.Wnt-TCF7L2 /TCF4 signaling:  Crypt cell proliferation  Paneth cell differentiation  Directs epithelial cell migration along the Villi 2.NOTCH signaling:  Crypt cell proliferation and cell fate decisions 3.Sonic and Indian Hedgehog signaling:  Crypt formation, spacing and villus development 4. EPH/Ephrins: Regulated by Wnt signaling:  Progenitor cell migration up the crypt 5. PTEN/PI3K :  Crypt stem cell renewal 6. BMP signaling:  Crypt stem cell proliferation independent of Wnt signaling 7.LKB1 signaling:  Regulates epithelial cell polarity

26 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 WNT Dkk CK1 GSK3  Dsh/ Dvl CK1 GBP P CK1 LRP Axin  -catenin TCF4/LEF CBP  -catenin BCL9 Pygo GSK3  Dsh/ Dvl  -catenin APC Ub  -TrCP Target Genes Myc, Cyclin D1 TCF1, PPAR-  MMP-7, Axin-2 CD44 Axin Frizzled Wnt-  catenin-TCF7L2 signaling pathway Groucho Downregulated d21: Wnt 10A Frizzled TCF7/TCF4

27 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Gene changes in intestinal epithelium in chronic (90 days) SIV infection

28 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Upregulated genes in the intestinal epithelium at 90 d PI (most involved with epithelial cell proliferation)  NOTCH and NOTCH target genes (HES4, HES7) and EZH2 (histone methyltransferase)  ETS homologous factor: Regulate epithelial cell proliferation and differentiation  FGF4 and 12: Fibroblast growth factor 4 and 12  Stimulates intestinal epithelial cell proliferation  Kruppel like factor 12: Induces cell proliferation (up in invasive gastric cancers)  PI3K regulatory subunit 2 (beta), Inositol polyphosphate-4-phosphatase, type 1  Increases cell survival, growth and proliferation (altered in 40% of colorectal cancers)  Defensin Beta 119  Antimicrobial peptide  Mucin 5B, BMPR1A  Upregulated in H. pylori induced gastric disease (anti-microbial ?)  Lubricates intestinal contents

29 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Downregulated genes in the intestinal epithelium at 90 d PI (most involved in epithelial differentiation and defense)  Paraxonase: Epithelial Defense response to pathogens  PDX1: Pancreatic and duodenal homeobox 1  Regulates gene expression in Enteroendocrine cells  Focal Adhesion Kinase: Important for epithelial cell motility, survival and healing  Kruppel like factor 6 and 10  A ubiquitous transcription factor that induces cell differentiation  FOXP2 and P4: Epithelial cell specification and differentiation  TGF  3: Induces cell differentiation and possess anti-inflammatory properties  Mucin 13: Cell signaling in epithelial cells  Solute carrier family 5 and 15: Uptake and digestion of tripeptides and monocarboxylates

30 Kuala Lumpur, Malaysia, 30 June - 3 July 2013  Tight Junctions  Claudin 22  Angiomotin like-1  Adherens Junctions  Cadherin 5, 11, 23  Catenin (Cadherin associated protein) alpha 1 (Anchor protein links Cadherins to actin filament)  Desmosomes  Desmoglein 2: Component of Desomosomes  Desmocollin: Component of Desomosomes  Junctional Plakoglobin: Component of Desomosomes  Hemidesmosomes  Laminin beta 3, 4, gamma 1, gamma 2  Required for hemidesmosome assembly  Binding, attaching and migration of cells (epithelial healing)  Integrin alpha 1, 3 and 6  Receptor for laminins  Plays a critical structural role in hemidesmosomes Cell adhesion Molecules downregulated 90 d PI:

31 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Summary: Early SIV infection results in upregulation of genes associated with intestinal epithelial apoptosis, proliferation, and repair Upregulation of numerous genes associated with lymphocyte activation, LPS pathways Downregulation of most epithelial adhesion / tight junction molecules indicating early barrier dysfunction Downregulation of anti-microbial genes

32 Kuala Lumpur, Malaysia, 30 June - 3 July 2013 Acknowledgements Tulane National Primate Research Center Huanbin Xu Mahesh Mohan Terri Rasmussen Andrew Lackner Northwestern University, Chicago Tom Hope Queens University Belfast Karl Malcolm Washington University, MO Herbert “Skip” Virgin Scott Handley Case Western Reserve University Michael Lederman Eric Arts Cornell University John Moore Pj Klasse St George’s Hospital, London Robin Shattock NCI-Frederick Jeff Lifson Mike Piatak NIH / NIAID Nancy Miller Susan Plaeger Opendra Sharma Jim Turpin


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