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The impact of SIV infection on gut innate lymphocyte populations and gene expression

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.

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The impact of SIV infection on gut innate lymphocyte populations and gene expression

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  1. 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. Differences between innate and adaptive immunity? • Adaptive immunity previously defined as having ”immunologicmemory” but lines areincreasinglyblurred – innatelymphoidcells (ILC) do not have antigen specific receptors (CD3) • Emergingevidence shows innatelymphoidcellsdevelopindependentlyin GALT, and developimmunologicmemory • Murine studies indicate GALT developsthroughimmune responses to bacteriamediatedthroughspecialized ”Lymphoidtissue inducer” (Lti), possible stem cells, and otherinnatelymphoidcell (ILC) subsets

  3. Innate lymphoid cellsNewly proposed nomenclature: Spits, H., D. Artis, et al. (2013). "Innate lymphoid cells--a proposal for uniform nomenclature." Nat Rev Immunol 13(2): 145-149. “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” • Group 1: ILC1: ILCs that produce IFNγ. Prototypical member is the NK cell. NK cells display both cytotoxic activity, and produce IFNγfollowing 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 RORγtfor development and function. Lymphoid tissue inducer cells (LTi) are prototype.

  4. Innate lymphoid cells • Proinflammatoryretinoic-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., 2010. Lineage relationship analysis of RORgammat+ innate lymphoid cells. Science 330:665-669. • Mucosal (RORgammat(+)innate lymphoid cells (ILCs) are an important innate lymphocyte population required for immunity to intestinal infectionsKlose, Diefenbach, et al., 2013. A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells. Nature 494:261-265. • ILC regulate CD4+ T-cell responses to intestinal bacteria. Hepworth, Sonnenberg et al, 2013. Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria. Nature 498:113-117.

  5. Recent studies of NK / ILC in SIV-infected macaques Blood. 2010Jun 3;115(22):4439-46. Epub 2010 Mar 25. CD16- natural killer cells: enrichment in mucosalandsecondarylymphoid tissues andalteredfunctionduringchronic SIV infection. ReevesRK,……Johnson RP. Blood. 2011 Sep 22;118(12):3321-30. Epub 2011 Jul 26. Gut inflammationandindoleaminedeoxygenaseinhibit IL-17 productionandpromotecytotoxicpotential in NKp44+ mucosal NK cellsduring SIV infection. Reeves RK, …Johnson RP. PLoS Pathog. 2012 September; 8(9) Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection. Namita Rout,…AmitinderKaur PLoSPathog. 2012;8(8) ADCC develops over time during persistent infection with live-attenuated SIV and isassociatedwithcomplete protection against SIV(mac)251 challenge. Alpert MD, ….Evans DT.

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

  7. CD16 detection is artificially masked by some anti-SIV immune complexes Comparison of anti-human CD16 mAb clones on SIV-infected macaque cells before and after “washing” 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 VaccImmunol 2006 CD16 clone DJ130 (Dako) best results

  8. ILC that secrete IL-17 are restricted to mucosal tissues in macaques IL-17+ Lymphocytes IL-17 CD3 Duodenum PBMC Jej LPL Colon LPL Spleen Tonsil CD3+CD4+ (Th17) CD3+CD8+ (Tc17) CD3-CD8high (ILC) IL-17

  9. IL-17secreting cells in jejunum of normal rhesus macaques * Blood. 2008 Oct 1;112(7):2826-35. Differential Th17 CD4 T-celldepletion in pathogenicandnonpathogeniclentiviralinfections. Brenchley…Douek, et al.

  10. Intestinal ILC17 cells are depleted in SIV infection

  11. Summary: Some ILC17 (ILC3) cells secrete IL-22, TNF-a, but not IFN-g 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. Changes in intestinal gene expression in SIV: Experimental Design Small Intestine (Jejunum) Approach to reduce tissue complexity Separation by Percoll gradients: Intraepithelial Lymphocytes Lamina propria Cells (LPC’s) Fibrovascular stroma Epithelial cells (Enterocytes) N=5 SIV infection 90d post infection Resection biopsy 6-8 cm pre infection (6 weeks) Resection biopsy 6-8 cm 21 d post infection Resection biopsy 6-8 cm

  13. Genome wide changes in the jejunum lamina propria in SIV infection (measured by Affymetrix rhesus arrays - 54,675 capture probes) 1) Intestinal lamina propria

  14. Upregulation of multiple genes associated with immune activation

  15. Downregulated genes associated with oxidative phosphorylation, IFNg, IL-17, B cell “help”

  16. Gene changes in lamina propria in chronic (90 days) SIV infection

  17. Important Up-regulated genes at 90 d PI • 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

  18. Important Down-regulated genes at 90 d PI • CXCL18 • Chemotactic for naïve T cells and non activated lymphocytes • May be a protective response (moderation of inflammation) • TLR8 • Binds 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

  19. 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.

  20. Gene changes in intestinal epithelium in acute (21 days) SIV infection

  21. Transcription factors and signaling pathways known to regulate intestinal epithelial gene expression • Wnt-TCF7L2 /TCF4 signaling: • Crypt cell proliferation • Paneth cell differentiation • Directs epithelial cell migration along the Villi • NOTCH signaling: • Crypt cell proliferation and cell fate decisions • 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 • LKB1 signaling: • Regulates epithelial cell polarity

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

  23. Gene changes in intestinal epithelium in chronic (90 days) SIV infection

  24. 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

  25. 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

  26. Cell adhesion Molecules downregulated 90 d PI: • 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 • JunctionalPlakoglobin: 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

  27. 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

  28. Tulane National Primate Research Center HuanbinXu Mahesh Mohan Terri Rasmussen Andrew Lackner Northwestern University, Chicago Tom Hope Queens University Belfast Karl Malcolm Washington University, MO Herbert “Skip” Virgin Scott Handley Acknowledgements • Case Western Reserve University • Michael Lederman • Eric Arts • Cornell University • John Moore • PjKlasse • 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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