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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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
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Innate lymphoid cells
Newly 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 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.
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Innate lymphoid cells
• Proinflammatory retinoic-acid-receptor-related orphan receptorgammat-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 infections Klose, 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.
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Recent studies of NK / ILC in SIV-infected macaques
Blood. 2010 Jun 3;115(22):4439-46. 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. 2011 Sep 22;118(12):3321-30. 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.
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,…Amitinder Kaur
PLoS Pathog. 2012;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.
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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.)
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CD16 detection is artificially masked by some anti-SIV immune complexes
Comparison of anti-human CD16 mAb clones on SIVinfected 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 Vacc Immunol
2006
CD16 clone DJ130
(Dako) best results
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ILC that secrete IL-17 are restricted to mucosal tissues in macaques
Lymphocytes
IL-17+
9.35
26.1
IL-17
PBMC
CD3+CD4+
(Th17)
CD3+CD8+
(Tc17)
CD3-CD8high
(ILC)
Jej LPL
1.72
19.2
0.289
29.3
0.0385
25.7
73.5
CD3
Colon LPL
3.51
3.67
11.1
Spleen
0.36
Tonsil
0.468
Duodenum
1.78
0.502
0.284
2.96
0.181
2.74
13.9
IL-17
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IL-17 secreting cells in jejunum of normal rhesus macaques
*
Blood. 2008 Oct 1;112(7):2826-35.
Differential Th17 CD4 T-cell depletion in pathogenic and
nonpathogenic lentiviral infections. Brenchley…Douek, et al.
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Intestinal ILC17 cells are depleted in SIV infection
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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.
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Changes in intestinal gene expression in SIV: Experimental Design
N=5
SIV infection
pre infection (6 weeks)
Resection biopsy 6-8 cm
90d post infection
Resection biopsy 6-8 cm
21 d post infection
Resection biopsy 6-8 cm
Approach to reduce tissue complexity
Separation by Percoll gradients:
Small Intestine (Jejunum)
Intraepithelial
Lymphocytes
Epithelial cells
(Enterocytes)
Lamina propria
Cells (LPC’s)
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Fibrovascular
stroma
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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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Upregulation of multiple genes
associated with immune activation
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Downregulated genes associated with
oxidative phosphorylation, IFNg, IL-17,
B cell “help”
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Gene changes in lamina propria in chronic (90 days) SIV infection
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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
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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 IFNa 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
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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.
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Gene changes in intestinal epithelium in acute (21 days) SIV infection
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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
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Wnt-catenin-TCF7L2 signaling pathway
WNT
LRP
Dkk
CK1
Axin
Frizzled
Dsh/
Dvl
Downregulated d21:
GSK3
Wnt 10A
Frizzled
TCF7/TCF4
Dsh/
Dvl
-catenin
CK1
GBP
P
GSK3
Axin
-catenin
APC
Ub
CK1
-TrCP
Groucho
Pygo
BCL9
CBP
-catenin
TCF4/LEF
Target Genes
Myc, Cyclin D1
TCF1, PPAR-
MMP-7, Axin-2
CD44
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Gene changes in intestinal epithelium in chronic (90 days) SIV infection
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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
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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
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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
 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
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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
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Acknowledgements
Tulane National Primate
Research Center
Huanbin Xu
Mahesh Mohan
Terri Rasmussen
Andrew Lackner
Case Western Reserve University
Michael Lederman
Eric Arts
Cornell University
John Moore
Pj Klasse
Northwestern University, Chicago
Tom Hope
St George’s Hospital, London
Robin Shattock
Queens University Belfast
Karl Malcolm
NCI-Frederick
Jeff Lifson
Mike Piatak
Washington University, MO
Herbert “Skip” Virgin
Scott Handley
NIH / NIAID
Nancy Miller
Susan Plaeger
Opendra Sharma
Jim Turpin
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