Rapid Involution of the Thymus Differentiation-Associated Gene 5

Activation of Melanoma Differentiation-Associated Gene 5 Causes Rapid Involution of the Thymus

This information is current as David Anz, Raffael Thaler, Nicolas Stephan, Zoe Waibler, of October 1, 2021. Michael J. Trauscheid, Christoph Scholz, Ulrich Kalinke, Winfried Barchet, Stefan Endres and Carole Bourquin J Immunol 2009; 182:6044-6050; ; doi: 10.4049/jimmunol.0803809 http://www.jimmunol.org/content/182/10/6044 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activation of Melanoma Differentiation-Associated Gene 5 Causes Rapid Involution of the Thymus1

David Anz,2* Raffael Thaler,2* Nicolas Stephan,* Zoe Waibler,† Michael J. Trauscheid,‡ Christoph Scholz,*§ Ulrich Kalinke,¶ Winfried Barchet,‡ Stefan Endres,3* and Carole Bourquin*

In the course of infection, the detection of pathogen-associated molecular patterns by specialized pattern recognition receptors in the host leads to activation of the innate immune system. Whereas the subsequent induction of adaptive immune responses in secondary lymphoid organs is well described, little is known about the effects of pathogen-associated molecular pattern-induced activation on primary lymphoid organs. Here we show that activation of innate immunity through the virus-sensing melanoma differentiation-associated gene 5 (MDA-5) receptor causes a rapid involution of the thymus. We observed a strong decrease in

thymic cellularity associated with characteristic alterations in thymic subpopulations and microanatomy. In contrast, immune Downloaded from stimulation with potent TLR agonists did not lead to thymic involution or induce changes in thymic subpopulations, demonstrating that thymic pathology is not a general consequence of innate immune activation. We determined that suppression of thymocyte proliferation and enhanced apoptosis are the essential cellular mechanisms involved in the decrease in thymic size upon MDA-5 activation. Further, thymic involution critically depended on type I IFN. Strikingly however, no direct action of type I IFN on thymocytes was required, given that the decrease in thymic size was still observed in mice with a selective deletion of the type I

IFN receptor on T cells. All changes observed were self-limiting, given that cessation of MDA-5 activation led to a rapid recovery http://www.jimmunol.org/ of thymic size. We show for the ﬁrst time that the in vivo activation of the virus-sensing MDA-5 receptor leads to a rapid and reversible involution of the thymus. The Journal of Immunology, 2009, 182: 6044–6050.

he innate immune system represents the first line of de- RIG-I recognizes viral ssRNA with a 5Ј-triphosphate motif and fense against viral infections. Initiation of antiviral im- short dsRNA, MDA-5 is activated by long dsRNA (3–5). Vi- T mune responses is critically dependent on the activation rally encoded RNA can also be detected by another family of of innate pattern recognition receptors that recognize evolutionar- pattern recognition receptors, the TLRs; long dsRNA and ss- ily conserved structures, termed pathogen-associated molecular RNA sequences can activate innate immunity through the en- patterns (1). In particular, the cytoplasmic helicases melanoma dif- dosomally located TLR3 and TLR7, respectively (6–10). Stim- by guest on October 1, 2021 ferentiation-associated gene 5 (MDA-5)4 and retinoic acid induc- ulation of pattern recognition receptors leads to the initiation of ible gene I (RIG-I) play an essential role in sensing viral RNA and an innate immune response characterized by the production of in generating immune responses to RNA viruses (2–4). Whereas a large panel of proinflammatory cytokines (7, 11). Among these, the type I IFNs IFN-␣ and IFN-␤ play an essential role in preventing viral spread through the induction of apoptosis and *Division of Clinical Pharmacology, University of Munich, Munich, Germany; †Jun- ior Research Group NG2, Paul-Ehrlich-Institut, Langen, Germany; ‡Institute of Clin- the suppression of cell proliferation (12, 13). In secondary lym- ical Chemistry and Pharmacology, University Hospital, University of Bonn, Bonn, phoid organs such as the spleen, the lymph nodes, or the GALT, Germany; §Department of Obstetrics and Gynecology Maistrasse, University of Mu- nich, Munich, Germany; and ¶Department of Experimental Infection Research, Twin- stimulation by pathogen-associated molecular patterns leads to core–Center for Experimental and Clinical Infection Research, Hannover Medical the generation of adaptive T and B cell responses against School, Hannover, Germany pathogens. Received for publication November 13, 2008. Accepted for publication March In contrast to the well-characterized effects of pattern recog- 11, 2009. nition receptor activation in secondary lymphoid organs, the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance consequences of innate immune activation on the primary lym- with 18 U.S.C. Section 1734 solely to indicate this fact. phoid organs, the bone marrow and thymus, are still unclear. 1 This study was supported by grants from the Ludwig-Maximilians-Universita¨t Within these organs, continuous proliferation of pluripotent Mu¨nchen Excellent Research Professorship (to S.E.), from the Else-Kro¨ner Fresenius progenitors is necessary to supply the organism with immune Foundation (to S.E. and C.B.), and from German Research Foundation Grants DFG En 169/7-2 and Graduiertenkolleg 1202 (to S.E. and C.B.), Excellence Cluster CIPSM cells and to maintain organ integrity. In the bone marrow, pro- 114 (to S.E.), BA3544/1-1 (to W.B.), and SFB-TR 36 (to S.E.). This work is part of liferation is suppressed during viral infections, an effect that is the doctoral thesis of R.T. and N.S. supported by Graduiertenkolleg 1202. in part mediated by the antiproliferative action of type I IFN 2 D.A. and R.T. contributed equally to this study. (14). Indeed, neutropenia, a hallmark of bone marrow suppres- 3 Address correspondence and reprint requests to Dr. Stefan Endres, Division of Clin- sion, is one of the most common side effects of IFN-␣ treatment ical Pharmacology, Ludwig-Maximilians Universita¨t, Ziemssenstrasse 1, 80336 Mu- nich. E-mail address: [email protected] in hepatitis C-infected patients (15, 16). 4 Abbreviations used in this paper: MDA-5, melanoma differentiation-associated gene Little is known about the impact of innate immune activation on 5; RIG-I, retinoic acid-inducible gene I; IFNAR, type I IFN receptor; poly(I:C), poly- structure and function of the thymus in vivo. The development of inosinic-polycytidylic acid; DN, double negative; DP, double positive; TEC, thymic T cells from bone marrow progenitors takes place exclusively in epithelial cells. the thymus, and an efficient thymic output of lymphocytes is there- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 fore crucial for the maintenance of the naive T cell pool in the www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803809 The Journal of Immunology 6045

periphery (17–19). An important fraction of self-reactive T cells is sions of thymus and spleen were washed twice with PBS before resuspen- deleted in the thymus by apoptosis, and thymic cellularity is main- sion in the provided buffer and incubation with annexin V and propidium tained by vigorous proliferation of immature thymocytes. Viral iodide. Cells were subsequently analyzed by flow cytometry. infections are in some cases associated with in vivo alterations of Statistical analysis thymic function; thymic atrophy and a reduced T cell output are All data are presented as mean Ϯ SEM and were analyzed as appropriate seen in HIV-infected patients (20) and have also been described in by the unpaired Student t test or the ANOVA test. Statistical analysis was a mouse model of reovirus infection (21). The mechanisms in- performed using SPSS software. volved remain, however, unclear. We describe here for the first time that in vivo activation of the virus-sensing MDA-5 receptor Results causes involution of the thymus. Stimulation of innate immunity through MDA-5 causes involution of the thymus Materials and Methods To investigate whether in vivo activation of innate immunity af- Mice fects the thymus, we treated mice with the dsRNA molecule Female C57BL/6 and BALB/c mice were purchased from Harlan-Winkel- poly(I:C). Poly(I:C) stimulates the immune system through two mann. Experiments were done on C57BL/6 mice unless indicated other- different pathways mediated by activation of either the endosomal Ϫ/Ϫ wise. Type I IFN receptor-deficient mice (IFNAR ) were backcrossed TLR3 or the cytoplasmic helicases RIG-I and MDA-5 (4–6, 24). 20 times on the C57BL/6 background (22). CD4creϩ/ϪIFNARflox/flox mice, ϩ Ϫ Ϫ Ϫ Adult mice were injected twice with poly(I:C) at 3-day intervals, CD19cre / IFNARflox/flox, and MDA-5 / mice have been previously described (23, 24). Mice were at least 8 wk of age at the onset of experiments. and organs were examined 24 h after the second injection. We Animal studies were approved by the local regulatory agency (Regierung observed a substantial decrease in the volume of the thymus (Fig. Downloaded from von Oberbayern, Munich, Germany). 1A) reflected by a Ͼ3-fold reduction in thymic weight (Fig. 1B). In Ligands for MDA-5 and TLRs contrast, no decrease in weight was observed for the spleen (Fig. 1B) and the peripheral lymph nodes (data not shown), demonstrat- Polyinosinic acid-polycytidylic acid (poly(I:C); InvivoGen) was applied i.p. (250 ␮gin250␮l of distilled water). The fully phosphothioate-mod- ing that this effect was selective for the thymus. The rapid invo- ified CpG oligonucleotide 1826 (5Ј-TCCATGACGTTCCTGACGTT-3Ј; lution was due to a strong reduction in cellularity, whereby the Coley Pharmaceutical Group), LPS (Sigma-Aldrich), and R848 (Alexis average number of thymocytes dropped from Ͼ130 ϫ 106 cells to Biochemicals) were injected s.c. into the flank in 200 ␮l of PBS (100, 5, Ͻ3 ϫ 106 cells (Fig. 1C). To determine which pattern recognition http://www.jimmunol.org/ ␮ and 20 g, respectively). receptor was involved in poly(I:C)-induced thymic involution, we Flow cytometry examined thymic pathology in mice deficient for MDA-5. This Single-cell suspensions were stained with anti-CD3-Pacific Blue or PerCP, cytoplasmic helicase plays a crucial role in the recognition of anti-CD4-PE-Cy7, anti-CD8-allophycocyanin-Alexa750, anti-CD25-PE many common viruses by the innate immune system (10). In strik- Ϫ Ϫ and anti-CD44-allophycocyanin (all BD Biosciences). Events were mea- ing contrast to wild-type mice, poly(I:C) treatment of MDA-5 / sured on a FACSCanto II flow cytometer (BD Biosciences) and analyzed mice did not affect the macroscopic aspect of the thymus and only with FlowJo software (TreeStar). slightly decreased thymic weight (Fig. 1D), indicating an essential

Histology role for this receptor in thymic pathology. To investigate whether by guest on October 1, 2021 Specimens were fixed in formalin before embedding in paraffin blocks. The decrease of thymic size is a general consequence of innate immune resulting tissue sections were stained with H&E. For immunofluorescence activation, we treated mice with ligands for different TLRs. Al- analysis, 5-␮m frozen cryosections were fixed in acetone before blocking though application of the TLR7 ligand R848 led to a moderate with 10% goat serum. Rat anti-mouse CD4 (Biolegend) and rat anti-mouse decrease in thymic weight, neither stimulation with the TLR4- CD8 (BD Biosciences) were used as primary Abs. Because both Abs are activating LPS nor stimulation with the TLR9 ligand CpG DNA derived from the same species, we used a protocol based on sequential application of primary Abs and detection with Fab fragments. First, we resulted in a significant decrease of thymic size (Fig. 1E). All three applied the anti-CD8 Ab followed by detection with biotinylated goat-anti- TLR ligands are potent stimulators of innate immunity that effi- rat IgG Fab fragments; these Fab fragments fully saturate the first primary ciently induce production of proinflammatory cytokines and lym- Ab and thus prevent binding of subsequently applied anti-rat secondary phocyte activation in vivo (7). Thus, in vivo immune activation Abs (25). After detection with a Cy2-conjugated streptavidin, the second primary Ab (anti-CD4) was applied followed by detection with rhodamine through the cytoplasmic helicase MDA-5, but not through TLRs in red X-conjugated mouse anti-rat IgG. To prevent cross-reactions of the general, rapidly leads to involution of the thymus. goat anti-rat Fab fragments to endogenous mouse IgG, tissues were gen- erally blocked with (Fab) goat anti-mouse IgG before staining. Images MDA-5-induced thymic suppression is characterized by a ϩ ϩ were obtained using a fluorescence microscope (Axiovert 2000; Carl Zeiss) decrease in CD4 CD8 double-positive (DP) thymocytes and processed using Adobe Photoshop for adjustment of contrast and size. The stages of T cell maturation within the thymus are character- Intrathymic FITC injection ized by the differential expression of surface markers. Immature Intrathymic injections of FITC were performed as described (26). Poly(I:C) progenitor cells are double-negative (DN) for both CD4 and CD8 was applied at days 1 and 3 after FITC injection, and organs were isolated upon immigration and can be further differentiated into four stages at day 4 to assess thymocyte emigration to the periphery. of maturation: the progenitor cells first express CD44 (DN1 cells), ϩ ϩ Ϫ ϩ In vivo BrdU proliferation assay then become consecutively CD44 CD25 (DN2), CD44 CD25 (DN3), and finally CD44ϪCD25Ϫ (DN4). DN cells subsequently Mice received three i.p. injections of 2 mg of BrdU in PBS (Sigma-Al- drich) at 6-h intervals. Thymus and spleen were isolated 6 h after the last acquire double positivity for CD4 and CD8 in the thymic cortex injection of BrdU. After surface staining, single-cell suspensions were and finally lose the expression of either CD4 or CD8 to leave the fixed and permeabilized using a ready-mixed kit from Ebioscience. BrdU thymus as mature, single-positive T lymphocytes (17, 27). After incorporation was detected using FastImmune Anti-BrdU FITC with treatment with poly(I:C), we observed a significant drop in the cell DNAse according to the manufacturer’s instructions (BD Biosciences), and count for all major thymic populations (Fig. 2A). In contrast, the FITCϩ cells were quantified by flow cytometry. loss in cellularity was prevented to a large extent in MDA-5-de- Detection of apoptotic cells ficient mice after treatment with poly(I:C) in all thymic subpopu- An Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) was used lations including the DN subsets (Fig. 2B). Proportional analysis for detection of apoptotic cells. After surface staining, single-cell suspen- demonstrated a strong decrease within the fraction of DP cells, 6046 INVOLUTION OF THE THYMUS BY MDA-5 ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 2. Decrease in thymocyte subpopulations upon MDA-5 activation. Mice were treated with poly(I:C) as described in Fig. 1. Thymo- cytes were counted and analyzed by flow cytometry. A, Absolute cell numbers for thymocyte subpopulations, means of 5 mice Ϯ SEM. B, Poly(I:C) (pI:C)-induced change in cell numbers (ratio of untreated to poly(I:C) treated) in the indicated thymocyte subpopulations of wild-type (WT; n ϭ 4) and MDA-5-deficient (n ϭ 5) mice. C, Proportions of thymocyte subpopulations in wild-type (n ϭ 4) and MDA-5-deficient (n ϭ 5) mice;

means Ϯ SEM are indicated as bars. D, Means of the proportions within by guest on October 1, 2021 FIGURE 1. MDA-5-induced decrease in size, weight, and cellularity of ϩ Ϫ ϩ ϩ the thymus. A, Mice were treated twice (days 0 and 3) with poly(I:C) DN thymocyte subsets (DN1, CD44 CD25 ; DN2, CD44 CD25 ; DN3, Ϫ ϩ Ϫ Ϫ ϭ Ϯ (pI:C), and organs were examined 24 h after the second injection. Macro- CD44 CD25 and DN4, CD44 CD25 ); n 5 mice for each group scopic aspect of the thymus of five untreated (top row) and four poly(I: SEM. E, Fraction of DP thymocytes of BALB/c mice 8 h after the last of C)-treated (bottom row) mice; B, weight of the thymus and spleen: each two injections (days 0 and 3) of poly(I:C), R848 (ligand for TLR7), LPS ϭ data point represents one individual mouse, and the means are depicted as (ligand for TLR4), and CpG (ligand for TLR9) for n 5 mice per group. Ͻ ءءء Ͻ ءء Ͻ ء a bar; C, mean number of thymocytes for n ϭ 5 (control) and n ϭ 4 , p 0.05; , p 0.01; , p 0.001; comparison with untreated (poly(I:C)) mice per group. Error bars indicate SEM. D, Macroscopic as- unless indicated by brackets. pect and means of thymic weight from n ϭ 5 WT and MDA-5-deficient mice Ϯ SEM. The means of untreated mice were set to 100%. E, Thymus weight of BALB/c mice 8 h after the last of two injections (days 0 and 3) mice treated with LPS, R848, or CpG. In striking contrast to of poly(I:C), R848 (ligand for TLR7), LPS (ligand for TLR4) and CpG mice treated with poly(I:C), the fraction of DP cells was not ϭ (ligand for TLR9) for n 5 mice per group. Results are representative of decreased in any of the three groups treated with TLR ligands ,ء .(at least three independent experiments. ø ϭ untreated; pI:C ϭ poly(I:C (Fig. 2E). Thus, stimulation of MDA-5, but not TLR activation, p Ͻ 0.001; n.s., not significant; comparison ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 with untreated unless indicated by brackets; WT, wild type. leads to a reduction of the DP thymocyte fraction. MDA-5-induced immune activation leads to a reduction of the dropping from Ͼ80% to Ͻ40% of all thymocytes, whereas a rel- thymic cortex and altered histomorphology ative increase was seen within the single-positive cells (Fig. 2C). Histologically, the thymus is divided into a highly cellular cortex These changes were nearly entirely absent in MDA-5-deficient with strong proliferative activity and a medulla defined by a coarse mice stimulated with poly(I:C), confirming the essential role of reticulum with lower lymphocyte density (27). To assess changes the MDA-5 activation pathway for the suppressive effect of in thymic microanatomy upon in vivo stimulation of innate im- poly(I:C) on the thymus (Fig. 2C). Stimulation with poly(I:C) munity, organs from poly(I:C)-treated mice were examined by his- further led to a relative increase in the DN1 compartment that tology. Although both thymic cortex and medulla were markedly was accompanied by a drop in the DN3 fraction (Fig. 2D). decreased in volume, the reduction of the cortex was clearly more Thus, the overall decrease in thymic cellularity by stimulation pronounced, resulting in a decreased ratio of cortical to medullary of innate immunity is characterized by a strong reduction of the space (Fig. 3A). Furthermore, the corticomedullary border was DP cell fraction with specific changes within the DN cell sub- blurred, resulting in a disorganized aspect of thymic microanat- set. To examine whether the potent immune activation induced omy. Fluorescent double staining for CD4 (red) and CD8 (green) by synthetic TLR ligands could also modify the distribution of demonstrated a decrease in the DP-cell fraction in the cortex de- thymocyte subpopulations, we analyzed thymocyte fractions in tected by a reduced merged (yellow) color signal (Fig. 3B). To The Journal of Immunology 6047

2 Untreated Poly (I:C) 1.6

1.2

0.8

0.4 FITC-positive cells (%) FITC-positive

0 CD4+ CD8+ CD4+ CD8+ Spleen LN FIGURE 4. Recent thymic emigrants in secondary lymphoid organs after treatment with poly(I:C). All mice received intrathymic injections of FITC followed by two poly(I:C) applications (at days 1 and 3 after injection of FITC). The spleen and the peripheral lymph nodes (LN) were isolated 24 h after the last application of poly(I:C). The number of FITCϩ- emigrated lymphocytes was measured by ﬂow cytometry. Bars indicate Downloaded from means of 5 control and 6 poly(I:C)-treated mice Ϯ SEM. The decrease of FITCϩ CD4ϩ and CD8ϩ cells upon poly(I:C) treatment is not signiﬁcant.

ptosis of developing T lymphocytes. In addition, poly(I:C)-trig- gered antiproliferative effects on thymocytes could contribute to http://www.jimmunol.org/ thymic involution. To explore the mechanisms involved, we first measured T cell emigration out of the thymus after treatment with poly(I:C). FITC was injected directly into the thymus (26), and peripheral lymphoid organs were analyzed 4 days later for the presence of FITC-positive recent thymic emigrants. Two applications of poly(I:C) did not increase the fraction of emigrated lymphocytes detected in the peripheral lymphoid organs (Fig. 4). In- stead, lymphocyte emigration was slightly reduced, albeit not by guest on October 1, 2021 significantly, after treatment with poly(I:C). To investigate whether innate immune activation affects T cell proliferation in the thymus, we quantified proliferating cells by measuring the in vivo incorporation of BrdU. DP thymocytes that represent the most abundant population in the thymus showed the highest proliferative activity in untreated mice (Fig. 5A). Upon immunostimulation with poly(I:C), we observed a marked suppression of proliferation for DP cells and for single-positive CD8 cells (Fig. 5A). The DN and CD4 subpopulations showed low baseline proliferation that was not significantly affected by poly(I:C) (data not shown). The level of proliferation in DP and FIGURE 3. MDA-5-mediated histomorphological changes of the thy- CD8 cells tended to recover as early as 48 h after the last appli- mus. A, H&E-stained sections of the thymus 48 h after the second of two injections of poly(I:C). B, Cryosections of the thymus were double-stained cation of poly(I:C). In the spleen, we observed conversely an in- with anti-CD4 and anti-CD8 mAbs. Images show the cortical areas of the crease in the number of proliferating CD8 T cells and B cells upon thymus: CD4 (red); CD8 (green); DP cells (yellow merged color signal). C, poly(I:C) treatment, indicating that suppression of cellular prolif- Thymic histology from untreated and poly(IC)-treated wild-type or MDA- eration is specific for the thymus (Fig. 5A). 5-deficient mice. C, cortex; M, medulla. To evaluate the role of apoptosis in thymic involution, we as- sessed the percentage of apoptotic cells within the thymocyte subpopulations upon treatment with poly(I:C). Early and late apopto- assess the importance of MDA-5-mediated signaling in histomor- sis was significantly enhanced in DP and DN thymocytes, phological changes, thymi from MDA-5-deficient mice treated respectively (Fig. 5B). No changes in apoptosis were observed for with poly(I:C) were examined. In contrast to wild-type mice, no T cells in the spleen (data not shown). Taken together, our results histological alterations were observed in MDA-5-deficient mice show that thymic involution following MDA-5 stimulation results (Fig. 3C), confirming the essential role of MDA-5 for poly(I:C)- from a marked decrease in thymocyte proliferation associated with induced thymic pathology. increased rates of apoptosis.

Involution is caused by suppression of proliferation and MDA-5-mediated thymic suppression depends on the type I IFN enhanced apoptosis of thymocytes receptor The rapid thymocyte loss upon MDA-5 activation could be due Antiproliferative and proapoptotic effects are characteristic fea- either to enhanced emigration of thymocytes or to increased apo- tures of type I IFN activity. Recent studies in fetal thymic organ 6048 INVOLUTION OF THE THYMUS BY MDA-5 ACTIVATION Downloaded from

FIGURE 5. Decreased in vivo proliferation and enhanced apoptosis of thymocytes upon treatment with poly(I:C). Mice were treated twice (days 0 and 3) with poly(IC). A, BrdU was applied 18 h before the organs were taken. The percentage of BrdUϩ cells was determined 24 or 48 h after the second injection of poly(I:C). Indicated thymocyte and splenocyte subsets http://www.jimmunol.org/ were analyzed by flow cytometry. Bars indicate means of 4 to 5 mice Ϯ SEM. B, Organs were examined 24 h after the second injection of poly(I:C) for the percentage of early apoptotic (annexin Vϩ, propidium iodideϪ; top) and late apoptotic (annexin Vϩ, propidium iodideϩ; bottom) thymocytes gated on the indicated subpopulations. Bars indicate means of 5 mice Ϯ .p Ͻ 0.01; comparison with untreated ,ءء ;p Ͻ 0.05 ,ء .SEM cultures suggested that type I IFN inhibits thymocyte development in vitro (28). To examine a possible involvement of type I IFN in by guest on October 1, 2021 MDA-5-mediated thymic reduction, we treated mice deficient for IFNAR with poly(I:C). In contrast to the decrease in thymic weight FIGURE 7. Recovery of thymus weight and structure after treatment seen in wild-type animals, we observed no significant decrease in with poly(I:C) (p(I:C)). A, Time course of thymus weight in BALB/c mice after a single injection of poly(I:C) on day 0; mean of n ϭ 4 mice for each time point. B, Development of thymus weight after two injections of poly(I:C); means of n ϭ 4 for each group. C, Histology of the thymus 10 days after the last of two injections of poly(I:C). C, cortex; M, medulla. .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء

IFNAR-deficient mice (Fig. 6, top). Further, the characteristic loss of DP thymocytes was completely absent in these mice. Because the receptor for type I IFN is expressed by a broad spectrum of cells in the organism (29), we investigated whether thymic involution is mediated through direct action of type I IFN on developing T cells. We used CD4-creϩ/ϪIFNARflox/flox-transgenic mice in which the IFNAR is selectively deleted on all T cells. Loss of thymic weight and cellularity in these mice were comparable with those of wild-type animals (Fig. 6, middle). To examine whether B cells, representing an important fraction of immune cells in the mouse, may be involved in type I IFN-mediated thymic involution, CD19-creϩ/ϪIFNARflox/flox mice that lack IFNAR expression on B cells, were injected with poly(I:C). Here again, as in wild-type mice, thymic weight and the fraction of DP cells were strongly reduced (Fig. 6, bottom). These data demonstrate that thymic involution is not mediated by the direct action of FIGURE 6. IFNAR-dependent involution of the thymus upon treatment type I IFN on T cells or B cells. with poly(I:C). Mice were treated twice (days 0 and 3) with poly(I:C), and organs were examined 48 h after the last injection. The thymic weight and Suppression of thymic cellularity occurs rapidly and is ϭ Ϫ/Ϫ ϭ the fraction of DP thymocytes of wild-type (WT; n 5), IFNAR (n self-limiting 5), CD4creϩ/ϪIFNARflox/flox (n ϭ 7) and CD19creϩ/ϪIFNARflox/flox (n ϭ 4) mice are shown as means Ϯ SEM; means of untreated mice were set to Typically, the secretion of type I IFN is an early and short-lasting p Ͻ 0.01; comparison to untreated. event (2–48 h) upon viral infection or stimulation by synthetic ,ءء ;p Ͻ 0.05 ,ء .100% The Journal of Immunology 6049 ligands (30). Because thymic suppression is dependent on type I the thymus. Isolated thymic plasmacytoid dendritic cells produce IFN, we examined whether poly(I:C)-mediated involution is re- large amounts of type I IFNs upon viral stimulation and suppress versible upon cessation of treatment by determining thymus the development of CD34ϩCD1aϪ thymic progenitor cells in co- weight at different times after a single application of poly(I:C). A culture assays (38, 39). Inhibition of thymic development by significant reduction was observed as early as 24 h after stimula- IFN-␣ has also been shown in newborn mice, where treatment with tion (Fig. 7A). Suppression was less pronounced than after two an active IFN-␣2/␣1 hybrid molecule was associated with de- injections of poly(I:C) (Fig. 7, A and B). After 3 days, an increase creased cellularity of bone marrow and thymus (40). Additionally, in thymus weight was detectable, and weight returned to initial type I IFN induced by poly(I:C) is reported to suppress fetal thy- levels 1 wk after stimulation (Fig. 7A). Recovery was delayed after mic organ cultures in vitro (28) and to block output of thymocytes two consecutive applications of poly(I:C) and did not reach initial in vivo (31). Here we demonstrate that type I IFN does not act levels 10 days after the second injection (Fig. 7B). All histological directly on T cells, given that mice in which the IFNAR deficiency alterations observed during thymic involution were reversible, and is restricted to T cells (CD4-Creϩ/ϪIFNARflox/flox) show a decrease the ratio of cortical to medullary tissue was restored 10 days after in thymic cellularity similar to that of wild-type mice upon stim- the last injection (Fig. 7C). These results demonstrate that MDA- ulation with poly(I:C). Similarly, we observed no direct effects of 5-induced suppression of the thymus is self-limiting and organ type I IFN on B cells. This suggests that the site of action of type integrity is restored within 10 days after cessation of immune I IFN is on nonlymphoid cells that, once stimulated, suppress T stimulation. cell development in the thymus. Among non-immune cells resid- ing in the thymus, thymic epithelial cells (TEC) represent a pop- Discussion ulation that plays an essential role in thymocyte development and Our results demonstrate that stimulation of innate immunity by in proliferation (41, 42). It has been reported that type I IFN, by Downloaded from vivo activation of MDA-5 leads to involution of the thymus. Fur- directly acting on TECs, induces phenotypical and functional al- thermore, we show that immune activation by a panel of potent terations of these cells in vitro that may impair thymocyte prolif- TLR agonists neither caused involution of the thymus nor induced eration (43). It has further been shown that TECs themselves have changes within the relative fractions of thymic subpopulations. In- the ability to produce type I IFN in response to transfection with deed, the only significant TLR-mediated effect observed was a poly(I:C) in vitro (43). It is thus possible that TECs can respond to mild decrease in thymic weight induced by two applications of the in vivo stimulation by poly(I:C) and produce type I IFN that could http://www.jimmunol.org/ potent TLR7 ligand R848. These novel findings expand on recent essentially contribute to the MDA-5-induced thymic involution. results describing blockade of thymic output by stimulation with Because MDA-5 is expressed ubiquitously in all lymphoid and the dsRNA molecule poly(I:C) (31). In addition, our data support various nonlymphoid tissues (44), systemic production of type I the concept that MDA-5, rather than TLR3 or RIG-I, is involved IFN upon poly(I:C) treatment may additionally affect TEC function during the in vivo recognition of synthetic long dsRNA. Previous and, consequently, thymocyte development. Finally, we cannot ex- studies examining the relative importance of these receptors and clude that the effects seen in DN thymocyte subset may result from their downstream signaling pathways for immune stimulation by a direct action by type I IFN: in CD4-Creϩ/ϪIFNARflox/flox mice, poly(I:C) have focused on the induction of proinflammatory cyto- the IFNAR is irreversibly deleted upon first expression of the CD4 by guest on October 1, 2021 kines (4, 5, 24). Here we describe thymic pathology as a novel Ag during T cell maturation, so that T lymphocyte precursors in functional readout that reaffirms the importance of MDA-5 as an in the DN stage remain susceptible to type I IFN-mediated effects. vivo mediator of poly(I:C) activity. Taken together, our results Activation of RIG-I-like helicases and TLRs has evolved as a show that in vivo activation of innate immunity through the cyto- promising strategy to activate the immune system for therapeutic plasmic helicase MDA-5, but not through TLRs in general, leads purposes. We and others have shown that the ligand for TLR9, to involution of the thymus. CpG, can be successfully used as an adjuvant for vaccination and Stimulation of pattern recognition receptors such as MDA-5, CpG oligonucleotides are in clinical trials for the therapy of in- RIG-I, TLR3, or TLR7 with synthetic ligands mirrors the immune fectious and malignant diseases (45–48). Further, TLR7 agonists activation induced by infections with RNA viruses (2, 4). Infection support Ag-specific T and B cell activation (49) and are effectively of mice with reovirus, an RNA virus that activates innate immunity used against cutaneous malignant or premalignant lesions (50). via RIG-I and MDA-5 (32), has been associated with atrophy of Stimulation of the RIG-I receptor using triphosphate RNA or of the thymus (21). In humans, a massive involution of the thymus MDA-5 with poly(I:C) mediates potent antitumoral effects in mice was observed in children who died from acute infection with mea- (51, 52). Finally, type I IFN itself is used for the treatment of sles virus, a ssRNA virus that induces type I IFN via the MDA-5 cancer and chronic viral infections as hepatitis (16, 53). All of receptor (33–35). Infection with HIV also leads to thymic damage these molecules could affect thymic cellularity in humans, in par- and results in a reduced overall emigration of thymocytes that is ticular potent inducers of type I IFN such as ligands for MDA-5 or reversible upon antiviral treatment (20, 36). Furthermore, a de- RIG-I. Thus, the impact on thymic function should be considered crease in thymic cellularity has also been described in a model of during therapeutic immune activation, specifically in the case of T cell-restricted overexpression of lymphotoxins, proteins known chronic treatment regimens. to be induced upon viral infection (37). Taken together, these ob- servations and our results suggest that viral infections may repre- Acknowledgments sent a natural trigger for rapid and reversible involution of the We thank Nadja Sandholzer, Stefanie Bauer, and the entire immunology thymus. Furthermore, our results suggest that thymic involution group at the Paul-Ehrlich-Institut for expert technical assistance. may occur selectively upon infection with viruses known to activate innate immunity via MDA-5. Disclosures In our study, the synthetic dsRNA poly(I:C) causes thymic in- The authors have no financial conflict of interest. volution. This molecule is known as potent inducer of type I IFN (7), and we delineate here a key role for this cytokine in thymic References suppression. It has been shown that plasmacytoid dendritic cells, 1. Medzhitov, R., and C. A. Janeway, Jr. 1997. Innate immunity: the virtues of a the professional type I IFN producers, are abundantly present in nonclonal system of recognition. Cell 91: 295–298. 6050 INVOLUTION OF THE THYMUS BY MDA-5 ACTIVATION

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