Multi-functional plasmacytoid dendritic cells redistribute to gut

tissues during simian immunodeficiency virus infection

Haiying Li, Jacqueline Gillis,

R. Paul Johnson and R. Keith

Reeves

Division of Immunology, New England Pri-

mate Research Center, Harvard Medical

School, Southborough, MA, USA

doi:10.1111/imm.12132

Received 17 March 2013; revised 31 May

2013; accepted 03 June 2013.

Correspondence: R. Keith Reeves, Division

of Immunology, New England Primate

Research Center, Harvard Medical School,

One Pine Hill Drive, Southborough, MA

01772-9102, USA.

E-mail: roger_reeves@hms.harvard.edu

Senior author: R. Keith Reeves

Summary

The objective of this study was to determine the systemic effects of

chronic simian immunodeficiency virus (SIV) infection on plasmacytoid

dendritic cells (pDCs). pDCs play a critical role in antiviral immunity, but

current data are conflicting on whether pDCs inhibit HIV/SIV replication,

or, alternatively, contribute to chronic immune activation and disease.

Furthermore, previous pDC studies have been complicated by incomplete

descriptions of generalized depletion during HIV/SIV infection, and the

effects of infection on pDCs outside peripheral blood remain unclear. In

scheduled-sacrifice studies of naive and chronically SIV-infected rhesus

macaques we evaluated the distribution and functionality of pDCs in mul-

tiple tissues using surface and intracellular polychromatic flow cytometry.

As previously observed, pDCs were reduced in peripheral blood and

spleens, but were also depleted in non-lymphoid organs such as the liver.

Interestingly, pDCs accumulated up to fourfold in jejunum, colon and

gut-draining lymph nodes, but not in peripheral lymph nodes. Most unex-

pectedly, SIV infection induced a multi-functional interferon-a, tumour

necrosis factor-a, and macrophage inflammatory protein-1b cytokine

secretion phenotype, whereas in normal animals these were generally dis-

tinct and separate functions. Herein we show a systemic redistribution of

pDCs to gut tissues and gut-draining lymph nodes during chronic SIV

infection, coupled to a novel multi-functional cytokine-producing pheno-

type. While pDC accumulation in the mucosa could aid in virus control,

over-production of cytokines from these cells could also contribute to the

increased immune activation in the gut mucosa commonly associated with

progressive lentivirus infections.

Keywords: interferon-a; plasmacytoid dendritic cells; simian immunodefi-

ciency virus.

Introduction

Plasmacytoid dendritic cells (pDCs), also referred to as

natural-interferon-producing cells, respond to microbial

pathogens by rapidly producing interferon-a (IFN-a), aswell as other pro-inflammatory cytokines such as tumour

necrosis factor-a (TNF-a) and macrophage inflammatory

protein-1b (MIP-1b) initiating a cascade of both innate

and adaptive immune responses. In normal infections,

the pDC response rapidly resolves, but recent evidence

suggests that during pathogenic HIV/SIV infections pDCs

are continually stimulated,1,2 resulting in over-production

of cytokines. However, despite their potent antimicrobial

role, pDCs are also directly implicated in suppression

of T helper type 17 cells and innate lymphoid cells, and

promoting generalized apoptosis and chronic inflam-

mation.3–9

As first reported over a decade ago, multiple groups have

verified that pDC numbers are severely reduced in blood

and lymph nodes during HIV infection.2,10–12 Further-

more, loss of pDCs begins during primary infection, is

associated with increasing viral loads, and is only partially

reversible by highly active anti-retroviral therapy. A com-

parable loss of pDCs has been demonstrated during acute

ª 2013 John Wiley & Sons Ltd, Immunology, 140, 244–249244

IMMUNOLOGY OR IG INAL ART ICLE

and chronic simian immunodeficiency virus (SIV) infec-

tion of rhesus, pig-tailed and cynomolgus macaques,13–15

but not non-pathogenic host species of SIV such as sooty

mangabeys and African green monkeys.7,16,17 Studies of

acute pathogenic SIV infection in macaques have demon-

strated a transient increase of pDCs in peripheral blood

because of rapid egress from the bone marrow, followed by

rapid depletion of circulating pDCs and accumulation of

apoptotic pDCs in lymph nodes.13,18 These data, combined

with evidence that pDCs are targets of infection for SIV

and HIV,13,19–22 led to a model where pDCs were generally

depleted during progressive lentivirus infection. However,

recently we entertained an alternative hypothesis – that

pDCs are not depleted but are trafficking elsewhere. Indeed

we and others have now shown that in fact pDCs are not

depleted by HIV/SIV infection, but rather accumulate in

the colorectum through an a4b7-dependent homing mech-

anism.6,17 This finding has redefined the view of pDCs in

lentivirus disease, but was incomplete, only focusing on

pDCs in blood and colorectal biopsies. Furthermore, the

functionality of pDCs in the mucosae is still unclear.

Herein, we address this deficit by investigating the systemic

effects of SIV infection on pDC redistribution.

Materials and methods

Animals

Twenty-nine Indian rhesus macaques (Macaca mulatta)

were analysed; 19 SIV-naive macaques and 10 macaques

infected chronically with SIVmac239. All animals were

free of simian retrovirus type D, simian T-lymphotrophic

virus type 1 and herpes B virus, were housed at the New

England Primate Research Center and were maintained in

accordance with the guidelines of the Committee on Ani-

mals of the Harvard Medical School and the Guide for

the Care and Use of Laboratory Animals.

Cell processing

Macaque peripheral blood mononuclear cells were

isolated from EDTA-treated blood by density gradient

centrifugation over lymphocyte separation medium (MP

Biomedicals, Solon, OH) and contaminating red

blood cells were lysed using a hypotonic ammonium

chloride solution. Mononuclear cells were isolated

from various tissue sections by both enzymatic and

mechanical disruption as described previously for our

laboratory.5,23,24

Antibodies and flow cytometric analyses

Flow cytometry staining of mucosal dendritic cells was

performed as previously described.6 LIVE/DEAD Aqua

dye (Invitrogen, Carlsbad, CA) and isotype-matched

controls and/or fluorescence-minus-one controls were

included for all assays. Except where noted, all antibodies

were obtained from BD Biosciences (La Jolla, CA) and

included fluorochrome-conjugated monoclonal antibodies

to the following molecules: Caspase-3 (Alexa647 conju-

gate, clone C92-605), CD3 [allophycocyanin (APC)-Cy7

conjugate, clone SP34.2], CD4 [Alexa700 and peridinin

chlorophyll protein (PerCp)-Cy5.5 conjugates, clone

L-200], CD8 (APC-H7 and PerCp-Cy5.5 conjugates, clone

SK1), CD11c (APC conjugate, clone S-HCL3), CD14

[Alexa700 and phycoerythrin (PE)-Cy7 conjugates, clone

M5E2], CD20 (PerCp-Cy5.5 conjugate, clone L27), CD45

(Pacific Blue conjugate, clone D058-1283), CD123 (PE

and PE-Cy7 conjugates, clone 7G3), HLA-DR (PE-Texas

Red conjugate, clone Immu-357, Beckman-Coulter), and

Ki67 (FITC conjugate, clone B56). Acquisitions were

made on an LSR II (BD Biosciences) and analysed using

FLOWJO (version 9.5) software (Tree Star Inc., Ashland,

OR).

Intracellular cytokine staining

After isolation, fresh mucosal mononuclear cells were

resuspended in RPMI-1640 (Sigma-Aldrich, St Louis,

MO) containing 10% fetal bovine serum alone (R10) or

with imiquimod (Sigma-Aldrich) at a final concentration

of 10 lM. Golgiplug (brefeldin A) and Golgistop (monen-

sin) were added to all samples at final concentrations of

6 lg/ml and cells were then cultured for 12 hr at 37° in

5% CO2. After culture, pDCs were surface-stained using

markers as shown in Fig. 1, and then cells were permeabi-

lized using Caltag Fix & Perm. Intracellular cytokine

staining was performed for MIP-1b (FITC conjugate,

clone 24006, R&D Systems, Minneapolis, MN), IFN-a(PE conjugate, clone 225.C, Chromaprobe, Maryland

Heights, MO) and TNF-a (Alexa 700 conjugate, Mab11).

Raw data analysis was performed using FLOWJO software

and multi-parametric analyses were done using SPICE

(version 5.22).25

Plasma virus load quantification

RNA copy equivalents were determined in EDTA-treated

plasma using a quantitative real-time RT-PCR assay based

on amplification of conserved sequences in gag.26 The

limit of detection for this assay was 30 viral RNA copy

equivalents/ml plasma.

Statistical analyses

All statistical analyses were performed using GRAPHPAD PRISM

6.0 software (GraphPad Software, Inc., La Jolla, CA). Non-

parametric Mann–Whitney U-tests and Spearman correla-

tion tests were used where indicated and values of P < 0�05were assumed to be significant in all analyses.

ª 2013 John Wiley & Sons Ltd, Immunology, 140, 244–249 245

SIV-induced redistribution of pDCs

Results

Chronic SIV infection induces redistribution of pDCsfrom lymphoid organs to gut mucosae and associateddraining lymph nodes

In a planned serial-sacrifice study we sought to comprehen-

sively evaluate pDC distribution in both naive and chroni-

cally SIV-infected macaques. We first defined pDCs as

CD45+ live mononuclear cells expressing HLA-DR but neg-

ative for lineage markers (CD3, CD14, CD20). Plasmacy-

toid DCs were also CD123bright, but negative for CD11c, a

common myeloid dendritic cell marker (Fig. 1a). Similar

to previous reports in HIV-infected persons and SIV-

infected macaques,10,11,13,18 we observed a three-fold reduc-

tion of pDCs in peripheral blood (Fig. 1b). pDCs were also

FS

C

Aqu

a dy

e

CD

20

CD

123

HLA

-DR

(a)

CD45 FSC CD3 CD14 CD11c

1

10Naive

Chronic

*** *

*

*** *

* *

(b)

0·010

0·100

% p

DC

s of

mon

onuc

lear

cel

ls

PBMC Bone marrow Spleen Liver Colon Jejunum PaLN MLN PLN0·001

1

10(c)

*

0·010

0·100

1

% m

DC

s of

mon

onuc

lear

cel

ls

PBMC Bone marrow Spleen Liver Colon Jejunum PaLN MLN PLN0·001

ND

250 K

200 K

150 K

100 K

50 K

00

0

102

102

103

103

104

104

105 0 103 104 105 0 103 104 105 0 103 104 105

105

0

102

103

104

105

0

103

104

105

0

103

104

105

250 K200 K150 K100 K50 K0

Figure 1. Distribution of plasmacytoid dendritic cells (pDCs) in lymphoid and mucosal tissues from naive and simian immunodeficiency virus

(SIV) -infected macaques. (a) Representative gating strategy for CD123+ pDCs and CD11c+ myeloid DCs (mDCs) among live tissue mononuclear

cells. Frequencies of (b) pDCs and (c) mDCs in multiple tissue sites. Box and whisker plots show medians and ranges of between 6 and 19 ani-

mals per tissue group. Mann–Whitney U-tests were used for naive versus SIV comparisons; *P < 0�05; **P < 0�01; ***P < 0�001. Only statisti-

cally significant differences are shown. PBMC, peripheral blood mononuclear cells; PaLN, MLN and PLN, pararectal/paracolonic, mesenteric and

peripheral (axillary, inguinal) lymph nodes; ND, not done.

ª 2013 John Wiley & Sons Ltd, Immunology, 140, 244–249246

H. Li et al.

reduced in bone marrow of SIV-infected macaques, albeit

not significantly, and previous reports have suggested that

this reflects an efflux of precursor cells into the circula-

tion.18 Interestingly, we also found pDCs to be reduced in

spleen and liver, suggesting a potential loss of pDCs from

both lymphoid and non-lymphoid organs. Finally, we

sought to enumerate pDCs in mucosae and associated

lymph nodes. We and others recently reported that pDC

loss in blood is actually reflective of migration to the gut

mucosa,6,17 but these initial studies only demonstrated this

phenomenon in colorectal biopsies. In this current study

we found that pDCs are not only increased in colorectal

biopsies, but in total colonic and jejunum tissues with four-

fold and threefold increases, respectively. Furthermore, we

found, perhaps unexpectedly, that pDCs also accumulated

in pararectal/paracolonic and mesenteric lymph nodes, but

not in inguinal and axillary lymph nodes. These data indi-

cate that pDCs not only migrate and/or expand in the gut

mucosa but also in the gut-associated draining lymph

nodes. Interestingly, tissue numbers of pDCs did not corre-

late with viral loads in these animals (data not shown). By

comparison, myeloid DC frequencies in each of these tis-

sues were largely unchanged (Fig. 1c), suggesting that the

redistribution of pDCs is cell-specific.

Gut pDCs from SIV-infected macaques develop anovel multi-functional phenotype

Some studies have suggested that during chronic HIV/

SIV infection pDCs are dysfunctional2,11,27,28 whereas it

has also been argued that pDCs are continually activated

during infection and contribute to chronic immune acti-

vation.7 However, previous studies have focused on pDCs

in peripheral blood and the results have been inconclu-

sive. In a smaller animal cohort we previously found that

pDCs in the gastrointestinal tract produced IFN-a, MIP-

1b and TNF-a.6 Now using a simultaneous three-function

assay in a large cohort of normal and SIV-infected maca-

ques, we verified that colonic pDCs produce high levels

of IFN-a and MIP-1b but very little TNF-a in normal

animals (Fig. 2a). Consistent with previous findings in

SIV-infected macaques pDC production of IFN-a was

slightly, but not significantly, reduced while TNF-aproduction was up-regulated. Interestingly, IFN-a mean

fluorescence intensity was significantly increased in

SIV-infected macaques (Fig. 2b), suggesting a bulk

increase in IFN-a due to numerical expansion of pDCs.

However, the most unexpected finding was that although

changes in overall cytokine production during infection

were minimal, the per cell cytokine production was sig-

nificantly altered during infection. In normal animals

IFN-a, MIP-1b and TNF-a were generally produced by

discrete subpopulations of pDCs, whereas in infected ani-

mals there was a significant increase in both bi-functional

and tri-functional cells (Fig. 2c).

Gut pDC functions are associated with changes inT-cell activation and turnover

As increased cytokine production and alterations in the

gut mucosa are considered to be prime mechanisms of

inducing chronic immune activation we next evaluated

relationships between ex vivo gut pDC cytokine produc-

tion (as measured in Fig. 2a) and systemic markers of T-

cell activation and turnover. To do so we quantified

intracellular Ki67 as a marker of proliferation/activation

and intracellular caspase-3 as a marker of apoptosis/turn-

over in bulk circulating CD4+ CD3+ and CD8+ CD3+

T cells. Despite its role as an inflammatory cytokine, we

found no correlations between MIP-1b production and

either of these T-cell markers (data not shown). However,

TNF-a production by gut pDCs was positively associated

with caspase-3 expression in both CD4+ (R = 0�886,P = 0�033) and CD8+ T cells (R = 0�600, P = 0�042), aswell as with Ki67 expression in CD8 T cells (R = 0�429,P = 0�042). Interferon-a production also significantly cor-

related with caspase-3 expression in CD4+ T cells

(R = 0�371, P = 0�049). Collectively, these data indicate

that increased gut-homing and cytokine production by

pDCs during SIV infection could contribute to the net

increase in systemic immune activation observed in pro-

gressive disease.

Discussion

Until recently, our understanding of the role of pDCs in

lentivirus disease has been somewhat incomplete and

complicated by the perception of generalized pDC deple-

tion. This deficit has made addressing the question of

whether or not pDCs are ‘helpful’ or ‘hurtful’ to control-

ling disease difficult. To help better refine the model of

lentivirus-induced modulation of pDC dynamics we pres-

ent two new bodies of data: (i) pDCs are reduced in

blood and non-lymphoid organs, but accumulate in the

mucosae and associated lymph nodes; and (ii) chronic

SIV infection alters mucosal pDC cytokine secretion by

inducing a multi-functional phenotype.

In the post-highly active anti-retroviral therapy era one

of the greatest causes of ongoing disease in HIV patients

is chronic immune activation, contributing to cardiovas-

cular diseases, non-AIDS-related cancers, and liver and

kidney failure.29–31 Although the breakdown of the gut

microenvironment leads to activation via microbial trans-

location,8,9,32 it is unclear why activation persists in the

absence of obvious HIV replication. As pDCs are the pri-

mary source of IFN-a in the SIV-infected gut, our new

data demonstrating the overall redistribution to gastroin-

testinal tissues raises the question – could pDCs be con-

tributing to apoptosis in the gut, so fuelling microbial

translocation and activation? Although the frequency of

TNF-a+ and IFN-a+ pDCs did not significantly increase,

ª 2013 John Wiley & Sons Ltd, Immunology, 140, 244–249 247

SIV-induced redistribution of pDCs

the relative increase in pDCs in mucosal sites may con-

tribute to a net increase in cytokines in the gut, which we

have observed previously.5 Indeed a significant shift in

mean fluorescence intensity of IFN-a in pDCs from

SIV-infected macaques (Fig. 2) supports this notion. Fur-

thermore, HIV stimulates pDCs to secrete IFN-a and

indoleamine 2,3-dioxygenase in vitro that inhibits T-cell

proliferation and induces apoptosis.33,34 This observation

is in line with our observed correlation between pDC-

produced cytokines and T-cell activation and apoptosis in

the circulation.

The alteration in the cytokine production profile of

gut pDCs during SIV infection is puzzling. pDC cyto-

kine production has long been shown to occur in

sequential waves35 – IFN-a followed by pro-inflamma-

tory cytokines. Our data suggest that through some

unknown mechanism SIV may induce these functions

simultaneously in the gut. Indeed, seminal work from

O’Brien et al.1 also indicated that HIV induces a persis-

tent IFN-a-producing phenotype in pDCs that may per-

sist with production of other cytokines, perhaps as a

result of a change in transcriptional regulation. Hence,

what we are demonstrating as multi-functional pDCs

may be the first in vivo observation of pDCs that have

become static in cytokine production and partial matu-

rity. Although pDCs are highly responsive to HIV RNA

and DNA, this functional phenotype could also be a

result of alternative stimuli such as CpG-DNA or micro-

particles.32,36,37 Regardless, regulation of gut-trafficking

and cytokine production in pDCs is probably highly

complex, but delineation of such mechanisms will

undoubtedly lead to a better understanding of how

80

(a) (b)

(c)

IFN-α TNF-αMIP-1β IFN-α TNF-αMIP-1β

IFN-α

TNF-αMIP-1β

IFN-α

TNF-α

MIP-1β

% C

ytok

ine+

pD

Cs

% C

ytok

ine+

pD

Cs

60

40

20

0

40

30

20

10

80

60

40

20

0

80 300 *

**

*

*

Naive

SIV

200

MF

I

100

0

300

200

100

00

500

1000

1500

2000

Naive

SIV60

40

20

0

+

+

+

+

+ +

+

+

+

+

+

+

–

––

–

–

–

–

–

–

Figure 2. Plasmacytoid dendritic cell (pDC) function in colonic tissue. (a) Percentages and (b) mean fluorescence intensities (MFIs) of colonic

pDCs from naive and simian immunodeficiency virus (SIV)-infected macaques expressing intracellular interferon-a (IFN-a), macrophage inflam-

matory protein-1b (MIP-1b) and tumour necrosis factor-a (TNF-a) following imiquimod stimulation. Bars represent means � SEM of six to

eight animals per group. (c) Multi-parametric SPICE (version 5.22) analyses of the data shown in (a). Bars represent poly-functional subpopula-

tions of stimulated pDCs and correspond to pie slices as shown. Arcs summarize overlap of each of the three functions. For multi-parametric

analyses, non-responsive cells are not included in the analyses. Mann–Whitney U-tests were used for naive versus SIV comparisons; *P < 0�05.Only statistically significant differences are shown.

ª 2013 John Wiley & Sons Ltd, Immunology, 140, 244–249248

H. Li et al.

pDCs balance antimicrobial properties and activation/

apoptosis in the gut.

Acknowledgements

The authors thank Angela Carville, Elaine Roberts and

Joshua Kramer for animal care, and Tristan Evans and

Michelle Connole for expert technical assistance. This

work was supported by a CHAVI/HVTN Early Career

Investigator award, grant number U19 AI067854, a Har-

vard University CFAR grant, number P30 AI060354 (both

to RKR), and NIH NEPRC base grant P51 OD011103.

Disclosures

All authors report no conflicts of interest.

References

1 O’Brien M, Manches O, Sabado RL et al. Spatiotemporal trafficking of HIV in human

plasmacytoid dendritic cells defines a persistently IFN-a-producing and partially

matured phenotype. J Clin Invest 2011; 121:1088–101.

2 Sabado RL, O’Brien M, Subedi A et al. Evidence of dysregulation of dendritic cells in

primary HIV infection. Blood 2010; 116:3839–52.

3 Favre D, Lederer S, Kanwar B et al. Critical loss of the balance between Th17 and T

regulatory cell populations in pathogenic SIV infection. PLoS Pathog 2009; 5:

e1000295.

4 Favre D, Mold J, Hunt PW et al. Tryptophan catabolism by indoleamine 2,3-dioxygen-

ase 1 alters the balance of TH17 to regulatory T cells in HIV disease. Sci Transl Med

2010; 2:32ra36.

5 Reeves RK, Rajakumar PA, Evans TI et al. Gut inflammation and indoleamine deoxy-

genase inhibit IL-17 production and promote cytotoxic potential in NKp44+ mucosal

NK cells during SIV infection. Blood 2011; 118:3321–30.

6 Reeves RK, Evans TI, Gillis J, Wong FE, Kang G, Li Q, Johnson RP. SIV infection

induces accumulation of plasmacytoid dendritic cells in the gut mucosa. J Infect Dis

2012; 206:1462–8.

7 Bosinger SE, Sodora DL, Silvestri G. Generalized immune activation and innate

immune responses in simian immunodeficiency virus infection. Curr Opin HIV AIDS

2011; 6:411–8.

8 Brenchley JM, Price DA, Schacker TW et al. Microbial translocation is a cause of sys-

temic immune activation in chronic HIV infection. Nat Med 2006; 12:1365–71.

9 Estes JD, Harris LD, Klatt NR et al. Damaged intestinal epithelial integrity linked to

microbial translocation in pathogenic simian immunodeficiency virus infections. PLoS

Pathog 2010; 6:e1001052.

10 Fitzgerald-Bocarsly P, Jacobs ES. Plasmacytoid dendritic cells in HIV infection: striking

a delicate balance. J Leukoc Biol 2010; 87:609–20.

11 Chehimi J, Campbell DE, Azzoni L et al. Persistent decreases in blood plasmacytoid

dendritic cell number and function despite effective highly active antiretroviral therapy

and increased blood myeloid dendritic cells in HIV-infected individuals. J Immunol

2002; 168:4796–801.

12 Lepelley A, Louis S, Sourisseau M et al. Innate sensing of HIV-infected cells. PLoS

Pathog 2011; 7:e1001284.

13 Reeves RK, Fultz PN. Disparate effects of acute and chronic infection with

SIVmac239 or SHIV-89.6P on macaque plasmacytoid dendritic cells. Virology 2007;

365:356–68.

14 Brown KN, Trichel A, Barratt-Boyes SM. Parallel loss of myeloid and plasmacytoid den-

dritic cells from blood and lymphoid tissue in simian AIDS. J Immunol 2007;

178:6958–67.

15 Malleret B, Karisson I, Maneglier B et al. Effect of SIVmac infection on plasmacytoid

and CD1c+ myeloid dendritic cells in cynomolgus macaques. Immunology 2008;

124:223–33.

16 Diop OM, Ploquin MJ, Mortara L et al. Plasmacytoid dendritic cell dynamics and a

interferon production during simian immunodeficiency virus infection with a non-

pathogenic outcome. J Virol 2008; 82:5145–52.

17 Kwa S, Kannanganat S, Nigam P et al. Plasmacytoid dendritic cells are recruited to the

colorectum and contribute to immune activation during pathogenic SIV infection in

rhesus macaques. Blood 2011; 118:2763–73.

18 Brown KN, Wijewardana V, Liu X, Barratt-Boyes SM. Rapid influx and death of plas-

macytoid dendritic cells in lymph nodes mediate depletion in acute simian immunode-

ficiency virus infection. PLoS Pathog 2009; 5:e1000413.

19 Donaghy H, Gazzard B, Gotch F, Patterson S. Dysfunction and infection of freshly iso-

lated blood myeloid and plasmacytoid dendritic cells in patients infected with HIV-1.

Blood 2003; 101:4505–11.

20 Patterson S, Rae A, Hockey N, Gilmour J, Gotch F. Plasmacytoid dendritic cells are

highly susceptible to human immunodeficiency virus type 1 infection and release infec-

tious virus. J Virol 2001; 75:6710–3.

21 Schmidt B, Scott I, Whitmore RG, Foster H, Fujimura S, Schmitz J, Levy JA. Low-level

HIV infection of plasmacytoid dendritic cells: onset of cytopathic effects and cell death

after PDC maturation. Virology 2004; 329:280–8.

22 Smed-Sorensen A, Lore K, Vasudevan J, Louder MK, Andersson J, Mascola JR, Spetz

AL, Koup RA. Differential susceptibility to human immunodeficiency virus type 1

infection of myeloid and plasmacytoid dendritic cells. J Virol 2005; 79:8861–9.

23 Reeves RK, Evans TI, Gillis J, Wong FE, Connole M, Carville A, Johnson RP Quantifi-

cation of mucosal mononuclear cells in tissues with a fluorescent bead-based polychro-

matic flow cytometry assay. J Immunol Methods 2011; 367:95–8.

24 Reeves RK, Gillis J, Wong FE, Yu Y, Connole M, Johnson RP et al. CD16– natural killer

cells: enrichment in mucosal and secondary lymphoid tissues and altered function dur-

ing chronic SIV infection. Blood 2010; 115:4439–46.

25 Roederer M, Nozzi JL, Nason MC. SPICE: exploration and analysis of post-cytometric

complex multivariate datasets. Cytometry A 2011; 79A:167–74.

26 Cline AN, Bess JW, Piatak M Jr, Lifson JD. Highly sensitive SIV plasma viral load assay:

practical considerations, realistic performance expectations, and application to reverse

engineering of vaccines for AIDS. J Med Primatol 2005; 34:303–12.

27 Tilton JC, Manion MM, Luskin MR et al. Human immunodeficiency virus viremia

induces plasmacytoid dendritic cell activation in vivo and diminished a interferon pro-

duction in vitro. J Virol 2008; 82:3997–4006.

28 Conry SJ, Milkovich KA, Yonkers NL et al. Impaired plasmacytoid dendritic cell

(PDC)-NK cell activity in viremic human immunodeficiency virus infection attributable

to impairments in both PDC and NK cell function. J Virol 2009; 83:11175–87.

29 Klatt NR, Funderburg NT, Brenchley JM. Microbial translocation, immune activation,

and HIV disease. Trends Microbiol 2013; 21:6–13.

30 Byrnes M, Travers K, Burns M, Sapra S. A systematic literature review examining solu-

ble and cellular biomarkers in HIV patients receiving antiretroviral therapy. J Int AIDS

Soc 2012; 15:18172.

31 Reiss P. HIV, co-morbidity and ageing. J Int AIDS Soc 2012; 15(Suppl. 4):18073.

32 Jiang W, Ledermann M, Hunt P et al. Plasma levels of bacterial DNA correlate with

immune activation and the magnitude of immune restoration in persons with antiretro-

viral-treated HIV infection. J Infect Dis 2009; 199:1177–85.

33 Boasso A, Herbeuval JP, Hardy AW, Anderson SA, Dolan MJ, Fuchs D, Shearer GM.

HIV inhibits CD4+ T-cell proliferation by inducing indoleamine 2,3-dioxygenase in

plasmacytoid dendritic cells. Blood 2007; 109:3351–9.

34 Chang JJ, Altfeld M. Innate immune activation in primary HIV-1 infection. J Infect Dis

2010; 202(Suppl. 2):S297–301.

35 Piqueras B, Connolly J, Freitas H, Palucka AK, Banchereau J. Upon viral exposure,

myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to

recruit immune effectors. Blood 2006; 107:2613–8.

36 Gasper-Smith N, Crossman DM, Whitesides JF et al. Induction of plasma (TRAIL),

TNFR-2, Fas ligand, and plasma microparticles after human immunodeficiency virus type

1 (HIV-1) transmission: implications for HIV-1 vaccine design. J Virol 2008; 82:7700–10.

37 Schiller M, Parcina M, Heyder P et al. Induction of type I IFN is a physiological

immune reaction to apoptotic cell-derived membrane microparticles. J Immunol 2012;

189:1747–56.

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SIV-induced redistribution of pDCs