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Modified interferon-α subtypes production and chemokine networks in the thymus during acute simian immunodeficiency virus infection, impact on thymopoiesis

Dutrieux, Jacquesa,b,c,d,*; Fabre-Mersseman, Véroniquea,b,c,*; Charmeteau-De Muylder, Bénédictea,b,c; Rancez, Magalia,b,c; Ponte, Rosaliea,b,c; Rozlan, Sandraa,b,c; Figueiredo-Morgado, Suzannea,b,c; Bernard, Amandinea,b,c; Beq, Stéphaniea,b,c; Couëdel-Courteille, Annea,b,c,d; Cheynier, Rémia,b,c

doi: 10.1097/QAD.0000000000000249
BASIC SCIENCE
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SDC

Objectives: Thymus dysfunction characterizes human/simian immunodeficiency virus (SIV) infections and contributes to physiopathology. However, both the mechanisms involved in thymic dysfunction and its precise timing remain unknown. We here analyzed thymic function during acute SIV infection in rhesus macaques.

Design and methods: Rhesus macaques were intravenously infected with SIVmac251 and bled every 2/3 days or necropsied at different early time points postinfection. Naive T-cell counts were followed by flow cytometry and their T-cell receptor excision circle content evaluated by qPCR. Thymic chemokines were quantified by reverse transcription-qPCR and localized by in-situ hybridization in thymuses collected at necropsy. Thymic interferon alpha (IFN-α) subtype production was quantified by reverse transcription-qPCR combined to heteroduplex tracking assay. The effect of thymic IFN-α subtypes was tested on sorted triple negative thymocytes cultured on OP9-hDL1 cells.

Results: A reduced intrathymic proliferation history characterizes T cells produced during the first weeks of infection. Moreover, we evidenced a profound alteration of both chemokines and IFN-α subtypes transcriptional patterns in SIV-infected thymuses. Finally, we showed that IFN-α subtypes produced in the infected thymuses inhibit thymocyte proliferation, still preserving their differentiation capacity.

Conclusion: Thymopoiesis is deeply impacted from the first days of SIV infection. Reduced thymocyte proliferation – a time-consuming process – together with modified chemokine networks is consistent with thymocyte differentiation speed-up. This may transiently enhance thymic output, thus increasing naive T-cell counts and diversity and the immune competence of the host. Nonetheless, long-lasting modification of thymic physiology may lead to thymic exhaustion, as observed in late primary HIV infection.

Supplemental Digital Content is available in the text

aINSERM, U1016, Institut Cochin

bCNRS, UMR8104

cUniversité Paris Descartes, Sorbonne Paris Cité

dUniversité Paris Diderot, Paris, France.

*Jacques Dutrieux and Véronique Fabre-Mersseman contributed equally to the writing of the article.

Correspondence to Rémi Cheynier, Département Immunité Infection Inflammation, Institut Cochin, 27 rue du Faubourg Saint Jacques, 75014 Paris, France. Tel: +33 1 40 51 65 41; fax: +33 1 40 51 65 10; e-mail: remi.cheynier@inserm.fr

Received 21 October, 2013

Revised 4 February, 2014

Accepted 4 February, 2014

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).

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Introduction

T-cell maturation in the thymus allows maintaining a broadly diversified population of naive T cells in the periphery that permits the immune system to react against most invading pathogens. Part of this diversity originates from proliferation that occurs between T-cell receptor beta (TCRB) and T-cell receptor alpha (TCRA) rearrangements at both the intermediate single-positive (ISP) and double-positive differentiation steps [1]. During primary HIV infection, premature thymus aging, characterized by reduced intrathymic precursor T-cell proliferation and thymic export, participates in naive T-cell exhaustion and immunodeficiency [2–4]. This could be a consequence of either the cytokine storm accompanying viral replication or infection of the thymus itself, or both. Indeed, the thymus is infected in patients with acquired immune deficiency syndrome [5], in HIV-infected severe combined immunodeficiency-hu mice [6,7], and in acutely simian immunodeficiency virus (SIV)-infected newborn macaques [8]. Moreover, the cytokine storm triggered by HIV/SIV infection contributes to the initial control of viral replication, but also contributes to the immunopathology of the infection [9]. Among the cytokines whose production is strongly enhanced during acute infection, interferon alpha (IFN-α) was suggested as contributing to HIV-induced thymic dysfunction [2,10]. Indeed, apart from its antiviral effect principally triggered by stimulation of PKR, 2′5′OAS, or Mx proteins, IFN-α also limits viral replication by inhibition of cell cycling through stimulation of p21 and p27 (Kip1) expression, thus limiting cyclin-D2 and cyclin-E expression [11,12].

In both HIV-infected and SIV-infected individuals, plasma IFN-α concentration peaks around day 10 of acute infection and persists, in organs, during chronic pathogenic infections [13–15]. In contrast, in nonpathogenic SIV infection models, rapidly controlled IFN-α response is accompanied by preserved CD4+ T-cell compartment, suggesting a role in pathogenesis [16–18]. As in humans, rhesus macaque IFN-α family is composed of 13 closely related proteins that certainly unequally contribute to plasma IFN-α levels. The relative expression of these subtypes depends either upon the activating stimulus or the producing cell type [19,20]. At the cell surface, these molecules interact with the same membrane-associated receptor, although with different affinity, leading to variable antiproliferative activities [21].

Thymocyte differentiation also involves chemokine-induced migrations of the differentiating cells within thymic cortex and medulla [22]. The chemokines implicated in T-cell differentiation in primates remain barely studied. Nevertheless, similar to mice, one could expect CCL19, CCL21, CCL25, and CXCL12 to be the major factors implicated in intrathymic cell migrations [22,23].

In the present study, we evaluated the consequences of acute SIV infection on thymopoiesis in rhesus macaques. We measured the impact of SIV infection on the production of recent thymic emigrants (RTEs). We analyzed IFN-α subtype transcription in the thymus of acutely SIV-infected monkeys and tested their effect on thymocyte proliferation. On the same thymuses, we evaluated the expression of chemokines implicated in thymocyte migrations.

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Methods

Animals and simian immunodeficiency virus infection

Eighteen Chinese rhesus macaques were intravenously inoculated with 50 AID50 of the pathogenic SIVmac251 isolate (provided by Dr Anne-Marie Aubertin, INSERM U544, Strasbourg, France). Blood was longitudinally sampled every 3–4 days from nine animals for 3 weeks [10 ml on ethylenediaminetetraacetic acid (EDTA)]. The other nine macaques were euthanatized (intravenous injection of 10 ml of pentobarbital) at day 3 (n = 3), 7 (n = 2), 10 (n = 2), and 14 (n = 2) postinfection. The four animals sacrificed at day 10 and 14 postinfection were included in the longitudinal blood sample analysis. Healthy animals (n = 3) served as controls. Thymuses were collected at necropsy and immediately treated or conserved. The animal experimentation ethical committee Paris 1 approved the experiments (n°2011–0001).

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Quantification of plasma interferon-α

Plasma IFN-α was quantified using VeriKine Human Interferon-Alpha ELISA Kit according to the manufacturer's instructions (PBL InterferonSource, Piscataway, New Jersey, USA).

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T-cell receptor excision circle and simian immunodeficiency virus-DNA quantifications

Simian sj T-cell receptor excision circle (TREC), DJβ1TRECs (DJβ 1.1–1.6) and DJβ2TRECs (DJβ 2.1–2.7) were quantified by multiplex nested qPCR using LightCycler480 technology (Roche Diagnostics, Meylan, France), together with the CD3γ chain as housekeeping gene, as described [2,24,25]. TREC contents were adjusted to circulating naive (CD95CD28+) CD4+ and CD8+ T-cell counts (sjTREC/105 naive T cells). Intrathymic precursor T-cell proliferation was estimated by the sj/βTREC ratio as described in [2,4].

SIV DNA was quantified as described in [26]. Plasmid harboring a single copy of both CD3γ and SIV-Gag amplicons was used to generate standard curves (see Table, Supplemental Digital Content 1, http://links.lww.com/QAD/A501 which shows Macaque-adapted primers and probes).

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Chemokine mRNA quantification

CCL19, CCL21, CCL25, and CXCL12 mRNA quantifications were performed in triplicate as described [27]. Total mRNA was extracted using an RNeasy kit (Qiagen, Courtaboeuf, France) according to manufacturer's instructions and reverse transcribed using the QuantiTect Rev. Transcription Kit (Qiagen). Specific cDNAs were quantified by duplex nested qPCR together with hypoxanthine phosphoribosyltransferase (HPRT) as housekeeping gene, using similar conditions as for TREC quantifications (see Table, Supplemental Digital Content 2, http://links.lww.com/QAD/A501 which shows macaques adapted primers). qPCR was performed on 1/280th of the PCR products in LightCycler480 SYBR Green I Master (Roche), using ‘inner’ primers (10 min at 95°C, 40 cycles of 1 s at 95°C, 10 s at 64°C, 15 s at 72°C). To generate standard curves, plasmids containing a single copy of HPRT amplicon and a single copy of one chemokine amplicon were used.

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Interferon-α subtypes mRNA quantification

Total mRNA was purified using an RNeasy kit (Qiagen) according to manufacturer's instructions, including two steps of DNase treatment (RNase-Free DNase Set; Qiagen). Reverse transcription was performed using the QuantiTect Rev. Transcription Kit (Qiagen). Total IFN-α coding cDNAs were quantified using the same protocol as for chemokines (see Table, Supplemental Digital Content 2, http://links.lww.com/QAD/A501). Plasmid harboring a single copy of both HPRT and IFN-α amplicons was used to generate standard curves.

IFN-α subtypes were quantified by heteroduplex tracking assay (HTA), adapted from [28]. Briefly, total IFN-α cDNA was amplified using inner primers (15 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 60°C, 5 min at 72°C). PCR products were then mixed with human IFN-αB2 or G probes synthesized using inner primers containing fluorescein or Rox dyes (Sigma Aldrich, Saint-Quentin-Fallavier, France) and EDTA (2 mmol/l), denatured at 95°C for 5 min, and rapidly chilled on ice to allow random probe-to-target heteroduplex formation. Heteroduplexes were resolved on polyacrylamide gels (acrylamide/bisacrylamide ratio 19 : 1, 4% stacking gel and 12% running gel). Fluorescence was detected using a Typhoon 9400 (GE Healthcare, Aulnay-sous-Bois, France). IFN-α subtypes were identified and quantified using TotalLab Quant 11.3 (TotalLab, Newcastle Upon Tyne, UK). Absolute mRNA copy number of each IFN-α subtype was calculated using relative quantification of each subtype and absolute quantification of total IFN-α mRNA.

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Production and function of interferon-α subtypes

Simian IFN-α subtype genes (numbered as they appear on the chromosome, starting by the closest to the centromere) were synthesized, cloned in pM+Ppgk.EP expression vector (Cytheris S.A., Issy Les Moulineaux, France) and transiently transfected using FreeStyle Max CHO Expression System (Invitrogen, Saint-Aubin, France), according to manufacturer's instructions. Supernatants collected at day 7 posttransfection were concentrated 15-fold (Amicon Ultra-15 Centrifugal Filter Device; Merck Millipore, Molsheim, France) and treated using acidic purification method adapted from [29,30] (pH 2.0 for 6 days at 4°C). After centrifugation and dialysis against phosphate-buffered saline (PBS), the IFN-α subtypes were quantified on polyacrylamide gel using SYPRO-Orange (Sigma Aldrich).

The effect of the different IFN-α subtypes on thymocyte development was evaluated on fluorescence-activated cell sorting (FACS)-purified healthy simian triple-negative thymocytes (see flow cytometry sorting and analysis section) cultured during 14 days on OP9-hDL1 cells (provided by Dr N. Taylor, CNRS-UMR5535, Montpellier, France) with recombinant simian glycosylated IL-7 (5 ng/ml, Cytheris S.A.), Flt3L (5 ng/ml) (Miltenyi Biotec, Paris, France), and simian IFN-α subtypes added every other day (100 ng/ml). Thymocyte differentiation was evaluated by FACS using anti-CD4-peridinin-chlorophyll protein-cyanin5.5 (PerCP-Cy5.5) (clone L200; BD Biosciences, Le Pont de Claix, France) and anti-CD8-pacific blue (clone RPA-T8, BD Biosciences).

In order to evaluate the impact of IFN-α subtypes on thymocyte proliferation, triple-negative thymocytes (CD3CD4CD8) were FACS-purified, labeled with carboxyfluorescein succinimidyl ester (CFSE; 2 μmol/l; Invitrogen), and cultured for 12 days on OP9-hDL1 in the same conditions. At day 5, 9, and 12, cells were sampled and FACS-analyzed using anti-CD4-PerCP-Cy5.5 and anti-CD8-pacific blue. Thymocyte proliferation was evaluated by measurement of CFSE dilution at day 5, 9, and 12 on differentiated cells (double-positive, CD4+CD8+; SP4, CD4+CD8, and SP8, CD4CD8+). Analyses were performed using FlowJo 9.5.2 software (Treestar, Ashland, Oregon, USA).

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Thymocytes flow cytometry sorting

Freshly isolated thymic cells from healthy macaques were stained with anti-CD3-fluorescein isothiocyanate (clone SP34-2; BD Biosciences), anti-CD4-PerCP-Cy5.5, anti-CD8-pacific blue, and anti-sphingosine-1-phosphate receptor 1 (S1PR1)-allophycocyanin (clone 218713; R&D Systems, Minneapolis, Minnesota, USA). Triple-negative cells, (CD3-CD4CD8) were analyzed and sorted using a BD FACS ARIA III (BD Biosciences).

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Peripheral blood mononuclear cell flow cytometry analysis

Peripheral blood mononuclear cells were Ficoll-purified and stained using anti-CD3-fluorescein isothiocyanate, anti-CD4-PerC-Cy5.5, anti-CD8-pacific blue, anti-CD28-phycoerythrin (clone CD28.2; BD Biosciences), anti-CD95-allophycocyanin (clone DX2; BD Biosciences), anti-CD31-Biotin (clone WM59; AbD Serotec, Colmar, France), and Streptavidin-phycoerythrin-TexasRed (BD Biosciences). Naive T cells (CD3+CD95CD28+ CD4+ and CD8+ cells), RTEs (CD3+CD95CD28+CD31+CD4+ cells), central memory T cells (TCM, CD3+CD95+CD28+ CD4+ and CD8+ cells), and effector memory T cells (TEM, CD3+CD95+CD28 CD4+ and CD8+ cells) were analyzed using FlowJo 9.5.2 software.

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Immunohistochemistry-combined in-situ hybridization

Digoxigenin-labeled RNA probes were generated using digoxigenin (DIG) RNA Labeling Kit Sp6/T7 (Roche) according to manufacturer's recommendations from CCL19, CCL25, and CCL21-containing pCRII-Topo plasmids (Invitrogen).

Thymus cryosections (12 μm) were fixed for 20 min in cold formalin buffer (Labonord, Fontenay-sous-Bois, France) and acetylated twice in 0.1 mol/l triethanolamine solution containing 0.25% acetic anhydride (Sigma Aldrich). Prehybridization (1 h at 58°C) and hybridization (3 days at 58°C) were performed in hybridization buffer [50% formamide, 2X saline-sodium citrate (SSC), 1X Denhardt's solution, 0.05 mol/l EDTA, 0.5 mg/ml salmon sperm DNA (Sigma Aldrich)] using 2 ng/μl of probes.

After washing (15 min in 5X SSC and twice 30 min in 0.5X SSC), DIG was detected with alkaline phosphatase-labeled sheep anti-DIG antibody (Roche) in 0.1% bovine serum albumin (BSA) for 2 h and revealed with NBT/BCIP (Roche) overnight. Sections were washed and fixed in 95% ethanol.

After saturation with PBS containing 0.5% BSA and incubation with anti-HLA-DR antibody (clone TAL.1B5; Abcam, Paris, France) for 1 h at room temperature (RT), specific immunostaining was revealed using ImmPRESS antimouse Ig (peroxidase) Polymer Detection Kit (Vector Laboratories, Nanterre, France), dehydrated and mounted in VectaMount medium (Vector Laboratories), according to the manufacturer's instructions.

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Immunohistological staining

Thymus cryosections (10 μm) were fixed for 20 min at 4°C in 2% paraformaldehyde/PBS, rinsed in PBS, permeabilized with 0.2% Triton X-100/PBS at RT for 8 min, rinsed in PBS, and blocked with 5% BSA/2% normal goat serum in PBS at RT for 30 min. After blocking endogenous biotin with the Streptavidin/Biotin Blocking Kit (Vector Laboratories), sections were successively incubated overnight at 4°C with primary antibodies, rinsed in 0.5% Tween20/PBS, incubated at RT in the dark for 30 min with secondary antibodies, rinsed in 0.5% Tween20/PBS, and incubated at RT in the dark for 15 min with Alexa Fluor 488 streptavidin (Molecular Probes, Cergy Pontoise, France). Sections were washed in 0.5% Tween20/PBS and then in PBS alone, counterstained with 4,6-diamidino-2-phenylindol (Molecular Probes), and mounted in Fluoromount-G medium (Southern Biotechnology, Birmingham, Alabama, USA).

Primary antibodies were polyclonal rabbit antihuman IFN-α (PBL InterferonSource) and mouse monoclonal antibodies to HLA-DR (clone TAL.1B5, IgG1; Abcam) or mouse monoclonal antibodies to CXCL12 (clone 79018, IgG1; R&D systems) and HLA-DR (clone TÜ36, IgG2b; BD Biosciences). Secondary antibodies were biotin-conjugated goat antirabbit antibody, Alexa Fluor 488-conjugated goat antimouse IgG1, Alexa Fluor 546-conjugated goat antimouse IgG1, and Alexa Fluor 546-conjugated goat antimouse IgG2b (Molecular Probes).

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Results

Acute simian immunodeficiency virus infection modifies thymic physiology in vivo

To evaluate the consequences of SIV infection on thymic function, we analyzed naive T-cell subsets and TREC content in the blood of 12 macaques followed over the first 21 days of SIV infection (Fig. 1 and see Figure, Supplemental Digital content 3, http://links.lww.com/QAD/A501 which shows T-cell subsets in SIV-infected macaques). Circulating RTEs (CD31+ naive CD4+ T cells [31]) counts gradually declined over the first 10 days, but rebounded to baseline values between day 10 and day 14 and subsequently plateaued (Fig. 1a). This was also the case for naive CD4+ and CD8+ T-cell counts as well as TCM CD8+ and TEM CD4+ (see Figure, Supplemental Digital Content 3, http://links.lww.com/QAD/A501). In contrast, sjTREC frequencies remained constant over the first 10 days and then significantly declined at day 14 and subsequently plateaued (Fig. 1b). Finally, DJβTREC frequencies increased up to day 10, dropped at day 14, and raised again by day 21 (Fig. 1c). As a consequence, the sj/βTREC ratio, a marker for thymocyte proliferation [2], gradually declined over the first days of infection, with a nadir between day 7 and day 10 postinfection (median sj/βTREC ratio: 10 and 4.6 at day 0 and nadir, respectively; P = 0.014) and significantly rebounded by day 14 (P = 0.017; Fig. 1d and see Figure, Supplemental Digital Content 4, http://links.lww.com/QAD/A501 which shows sj/βTREC ratio and plasma IFN-α concentration in SIV-infected macaques).

Fig. 1

Fig. 1

Interestingly, the nadir of the sj/βTREC ratio coincided with the peak of plasma IFN-α positivity between day 7 and day 10 (see Figure, Supplemental Digital Content 4, http://links.lww.com/QAD/A501). Moreover, the amplitude of plasma IFN-α response directly correlated with the decline of sj/βTREC ratio (r = −0.52, P = 0.034; Fig. 1e), suggesting a role for IFN-α in the impairment of thymocyte proliferation.

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A subset of interferon-α subtypes is expressed in simian immunodeficiency virus-infected thymuses

In order to evaluate the role of IFN-α in the modifications of thymic function during acute SIV infection, we analyzed IFN-α transcription in the thymus of acutely SIV-infected macaques. Surprisingly, whereas thymic infection gradually increased by day 3 (see Figure, Supplemental Digital Content 5, http://links.lww.com/QAD/A501 which shows SIV DNA in thymuses from SIV-infected macaques), total IFN-α transcription – already detectable in the thymus of uninfected macaques – was not significantly changed following infection (Fig. 2a). However, the transcription pattern of the 12 IFN-α subtypes was profoundly modified during this period. Indeed, whereas only a few IFN-α genes were transcribed in uninfected thymuses (Fig. 2b and c), thymic IFN-α expression rapidly diversified, by day 3 of infection (Fig. 2b). IFN-α1, 2, and 3 were coexpressed in seven of the nine infected macaques, IFN-α7 and 14 in six monkeys, and IFN-α8 and 13 in six and five animals, respectively (Fig. 2d). Other subtypes were sporadically observed (Fig. 2d).

Fig. 2

Fig. 2

Fig. 2

Fig. 2

Immunohistochemical labeling confirmed IFN-α expression in all the thymuses (Fig. 2e). Compatible with being IL-3R+ plasmacytoid dendritic cells (pDCs) [32,33], most IFN-α+ cells were Mamu-DR+, located in the medulla and predominantly concentrated in Hassall's corpuscles (Fig. 2e).

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Interferon-α subtypes expressed in infected thymuses inhibit thymocyte proliferation

The biological activity of the 12 simian IFN-α subtypes was evaluated on simian triple-negative thymocytes cultured on human Notch receptor ligand Delta-like 1-expressing murine stromal cells (OP9-hDL1) [34]. Addition of the different IFN-α subtypes led to reduced frequency of differentiated (double-positive, SP4 and SP8) cells (Fig. 3a). Only modest for IFN-α1 and 6 (two-fold reduction as compared to cultures without IFN-α; P < 0.05), the reduction of differentiated cell numbers was high in the presence of IFN-α3, 4, 13, and 14 (80–90% reduction; P < 0.05 as compared to IFN-α1 and 6) and quite complete (>90%) in cultures containing IFN-α2, 5, 7, 8, 9, and 12/15 (P < 0.05 as compared to IFN-α1 and 6; Fig. 3a). Longitudinal analysis showed that differentiated cell frequencies strongly increased between day 9 and 12 in cultures without IFN-α, but reached a plateau by day 5 in the presence of IFN-α5, 6, and 13, suggesting antiproliferative effect of IFN-α subtypes (Fig. 3b).

Fig. 3

Fig. 3

We next evaluated thymocyte proliferation through longitudinal measurement of CFSE dilution in thymocytes differentiating on OP9-hDL1 cells (Fig. 3c and Fig. 4). In cultures without IFN-α, differentiated thymocytes (double-positive, SP4 and SP8) demonstrated higher proliferation than triple-negatives with more than 90% of differentiated cells having achieved at least nine divisions by day 9 (Fig. 4a and b). At any time point, triple-negative cell proliferation was delayed in the presence of IFN-α (Fig. 4a). However, IFN-α2, 3, 4, 7, 8, and 12/15 more efficiently retarded cell proliferation than IFN-α1, 6, 9, and 14, IFN-α5 and 13 having an intermediate activity.

Fig. 4

Fig. 4

In contrast, in the presence of IFN-α, thymocyte differentiation occurred with very limited proliferation. Indeed, cells that differentiated in cultures containing IFN-α2, 3, 4, 7, 8, and 12/15 barely proliferated (>85% inhibition as compared to medium alone; P < 0.05; Fig. 3c and 4b). Thus, three IFN-α subtypes transcribed in the thymus during acute SIV infection (IFN-α2, 3, and 7) strongly impacted on thymocyte proliferation, still allowing their differentiation.

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Simian immunodeficiency virus infection modifies thymic chemokine network

Both CCL25 (Fig. 5a, top panel) and CCL19 (Fig. 5a, central panel) mRNA levels were significantly increased by day 3 postinfection. In contrast, CXCL12 mRNA transcription was significantly lower in infected macaques than in controls (Fig. 5a, bottom panel). CCL25 mRNA was almost exclusively localized in the cortex (Fig. 5b, top panel), CCL19 mRNA in the medulla (Fig. 5b, bottom panel), and CXCL12 mostly in thymic subcapsular region (Fig. 5c). Finally, CCL21 transcription, mostly found in the medulla, remained stable following infection (see Figure, Supplemental Digital Content 6, http://links.lww.com/QAD/A501 which shows thymic expression of CCL21 and S1PR1 during acute SIV infection). Of note, S1PR1 expression by SP4 and SP8 mature thymocytes was not modified following infection (see Figure, Supplemental Digital Content 6, http://links.lww.com/QAD/A501).

Fig. 5

Fig. 5

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Discussion

As expected, circulating T-cell numbers dropped during the first 10 days of SIV infection. However, whereas memory CD4+ T-cell numbers remained low after acute infection, all naive T-cell subsets rapidly rebounded by day 14 (Fig. 1 and see Figure, Supplemental Digital Content 3, http://links.lww.com/QAD/A501). Analysis of TREC content demonstrates that thymic export contributes to this rebound. Indeed, despite stable sjTREC frequency, we observed a significant increase in DJβTREC frequency, leading to reduced sj/βTREC ratio in the infected macaques (Fig. 1). Considering that modified sj/βTREC ratio exclusively signs changes in thymocyte proliferation [2], this demonstrates that, on average, circulating RTEs at day 7/10 of SIV infection had a shorter intrathymic proliferative history than those sampled before infection. The only hypothesis that explains a global reduction of the sj/βTREC ratio is the release in the RTE pool of new cells characterized by a reduced intrathymic proliferation (low sj/βTREC ratio). Moreover, in order to impact on the global sj/βTREC ratio, newly exported cells should be numerous enough to outnumber preexisting TREC-containing cells, suggesting substantial thymic export.

Surprisingly, total level of IFN-α mRNA levels remained stable in the thymus of SIV-infected macaques. However, in human thymus, thymocytes and medullary pDCs constitutively express IFN-α [35]. During acute SIV infection, pDCs accumulate in lymphoid organs and represent the major source of IFN-α production ([14,36] and [37]). In contrast, IFN-α production is barely sustained by this cell type in chronic HIV/SIV infection [14,15]. In the thymus of acutely infected monkeys, IFN-α-producing cells were mostly localized within Hassall's bodies (Fig. 2e), suggesting a role of Hassall's corpuscle-associated DC-Lamp+ dendritic cells in IFN-α production [38]. Moreover, at later time points, a punctuated labeling suggested diffusion of the cytokine into the tissue (Fig. 2e).

IFN-α is a complex family of proteins produced by most cell types in response to a large array of danger signals. However, the expression of IFN-α subtypes depends on the stimuli and the cell type involved [19,20,39]. Moreover, after intraperitoneal Poly (I:C) treatment in mice, following the waves of IFN-α subtypes expression during the first 6 h, only a few IFN-α remained expressed in the longer term [28]. This is concordant with our observation of the expression of a group of IFN-α subtypes in the infected thymus and the stability of the expressed subtypes between day 3 and day 14 in the thymuses of SIV-infected macaques. However, thymic expressed IFN-α subtypes could differ from those expressed at the other sites sustaining acute viral replication as well as during chronic infection. A comprehensive analysis of the IFN-α subtypes produced in these various organs could help in a better understanding of this particularly important response to viral infections.

Longitudinal analysis of differentiated (CD4+CD8+, CD4+CD8 and CD4CD8+) thymocytes in culture (Figs. 3 and 4) showed that whereas differentiated thymocytes had extensively proliferated in IFN-α-free cultures, in the presence of IFN-α, thymocyte differentiation occurred with reduced proliferation, leading to a smaller number of differentiated cells at day 14. However, in IFN-α2, 3, 4, 7, 8, and 12/15-containing cultures, the vast majority of both triple-negative and differentiated cells barely cycled by day 5 (Fig. 4) while the frequency of differentiated cells reached a maximum (Fig. 3b). This strongly suggests that in normal conditions cells ready to differentiate at the beginning of the cultures proliferate and then eventually differentiate whereas, in the presence of specific IFN-α subtypes, they immediately differentiate. Accordingly, considering that cell cycling is a time-consuming process, IFN-α-induced inhibition of proliferation should accelerate thymopoiesis, provided that thymocyte trafficking is not also impaired during acute infection.

In mice and humans, a few chemokines are strongly involved in thymocyte trafficking [40]. Cortical CCL25 expression drives CD34+ cells entry into and their migration through the cortex while they differentiate into DN3 cells [41]. CXCL12 plays a role in the retention of double-negatives and early double-positives in the outer cortex [42,43], where they proliferate between TCRB and TCRA rearrangements, before their migration to the medulla [44,45]. Finally, CCL19 is implicated in the migration of positively selected thymocytes from the cortex to the medulla, as well as to the transport of differentiated cells through the medulla [46], allowing sphingosine-1-phosphate-driven export of mature T cells through cortico-medullary blood vessels [47]. Accordingly, the increased transcription of both CCL19 and CCL25, together with decreased expression of CXCL12, is compatible with faster migration processes within the thymus. Together with the inhibition of thymocyte proliferation, such modifications of thymic chemokine networks most probably accelerate thymocyte migrations within the thymus and result in increased thymic export. Indeed, if thymopoiesis is accelerated during the first days after infection, thymic output should be composed of already mature cells close to leave the thymus at the time of infection caught up by cells that rapidly differentiated from double-negative cells in the presence of thymic IFN-α production. Enhanced thymic export should translate into an increase of the RTE compartment in SIV-infected macaques. Such an increased thymic output was observed in acutely HIV-infected patients [48]. However, massive cell homing from the blood to both lymphoid (spleen and lymph nodes) and nonlymphoid (gut, lungs, sexual mucosae) organs characterizes acute HIV/SIV infection. RTE migration into organ, which strongly diminishes circulating RTE numbers, certainly masks the increased thymic export. Whether newly produced RTEs also home in organs remains to be established, but preferential migration of ‘old’ RTEs could help visualizing the change in sj/βTREC ratio. Normalization of RTE counts and sj/βTREC ratio after day 10 may suggest that preinfection RTEs return to circulation and dilute the RTEs produced during acute infection. However, in the long term, inhibition of thymocyte proliferation, reducing total number of lymphoid cells in the thymus, could affect thymic epithelial cells/thymocytes cross-talk and stromal cell survival [49], and, thus, may contribute to thymic involution and naive T-cell depletion that characterizes HIV infection [2,24].

Our data provide original evidence that deep modifications of thymic physiology occur during acute SIV infection. Local production of specific IFN-α subtypes that directly restrict immature thymocyte proliferation, together with altered thymic chemokine networks, may lead to accelerated thymocyte maturation, most probably resulting in transient increase of thymic output. Whether modification of IFN-α and chemokine productions within the thymus is a more general feature of acute viral infections remains to be established. However, an increased thymic output characterizes acute measles infection in both children and rhesus macaques [50], suggesting a common response to viral infections. Accordingly, identification of human IFN-α subtypes that, similarly to simian IFN-α2, 3, 4, 7, and 12/15, impact on thymopoiesis could be of major importance to improve both natural and vaccine-elicited immune responses.

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Acknowledgements

The authors would like to thank Dr Céline Gommet and the staff of the Institut Pasteur primate center. The authors greatly acknowledge M. Andrieu, M. Favier, and F. Letourneur of the Cochin Institute Cytometry and Immunobiology, Morphology and Histology and Genomics Facilities. They also thank Drs Rafick-Pierre Sékaly, Anne Hosmalin, and Bruno Lucas for critically reading this article.

J.D., V.F.M., and B.C.dM. performed most of the experiments. M.R., A.C.C., and R.P. performed immunohistochemistry and in-situ hybridization experiments. S.F.M. and A.B. contributed to chemokine quantifications. S.R. and S.B. cloned simian IFN-α subtypes. J.D., M.R., A.C.C., and R.C. analyzed the data and wrote the article. All the authors discussed the results and commented on the article.

This work was carried out in partial fulfillment of J.D.'s PhD thesis at Université Paris Diderot, Paris, France. This work was supported by ANRS (Agence Nationale de Recherches sur le SIDA et les Hépatites Virales), SIDACTION, INSERM, CNRS and Univeristé Paris Descartes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.

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Conflicts of interest

The authors report no conflicts of interest.

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Keywords:

acute viral infection; chemokines; HIV; interferon-α subtypes; physiopathology; simian immunodeficiency virus; sj/bTREC ratio; TREC; thymopoiesis; thymus

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