L’immunoprotéasome : régulateur de transcription et promoteur de survie cellulaire

Le protéasome (CP) contrôle la majorité des fonctions cellulaires par la dégradation des protéines intracellulaires. En plus d’exprimer le CP, les vertébrés expriment également l’immunoprotéasome (IP), caractérisé par des préférences de dégradation distinctes. Le rôle le mieux caractérisé pour l’IP est la génération d’antigènes adaptés pour la liaison au complexe majeur d’histocomptabilité de classe I (CMH-I). Cependant, les nombreux phénotypes observés au niveau de cellules déficientes en IP ou avec une mutation révèlent que l’IP influence des fonctions immunitaires indépendamment de la génération d’antigènes et peut atténuer le stress présent au niveau de cellules non-immunitaires. L’objectif de cette thèse était de caractériser les rôles de l’IP qui ne sont pas reliés à la génération d’antigènes associés au CMH-I. L’analyse du transcriptome de cellules dendritiques IP-déficientes en cours de maturation révèle que l’IP affecte l’expression de plus de 8 000 transcrits. L’IP affecte l’expression génique principalement au niveau transcriptionnel en contrôlant l’abondance de régulateurs de transcriptions tels que NF-κB et les membres des familles IRF et STAT. Les cellules dendritiques IP-déficientes sont également moins efficaces pour activer des lymphocytes T CD8+, même chargées artificiellement avec des quantités optimales d’antigènes associés au CMH-I. En outre, nos études montrent que l’IP est fortement exprimé au niveau de cellules de patients atteints de leucémie myéloïde aigue. L’expression de l’IP est intrinsèque aux leucémies, puisque qu’elle n’est pas corrélée à la présence de lymphocytes sécréteurs d’IFN-γ. De plus, l’expression d’IP est particulièrement élevée au niveau de leucémies monocytaires et/ou possédant un réarrangement MLL. Notamment, des analyses de corrélation montrent que l’IP est connecté à des gènes impliqués dans le métabolisme, l’activité mitochondriale et la réponse au stress. En effet l’inhibition de la sous-unité PSMB8 de l’IP mène à l’accumulation de protéines ubiquitinées et la mort de cellules leucémiques monocytaires. Globalement, nos travaux montrent que le rôle de l’IP n’est pas limité à la génération d’antigènes, mais qu’il peut contrôler l’expression génique et la survie des leucémies.By regulating protein degradation, constitutive proteasomes (CP) control practically all cellular functions. In addition to CP, vertebrates express immunoproteasomes (IP), which display distinct substrate preferences. The first non-redundant role ascribed to IP is its enhanced ability to generate MHC I-associated antigens. However, deletion or inhibition of IP subunits can affect several immune cell functions independently of MHC-I antigen generation. Moreover, recent work has shown that IP can be expressed in non-immune cells to deal with cell stress. Thus, we wished to investigate the roles of IP that are not related to antigen generation and that are not redundant with the CP. Based on profiling of WT and IP-deficient maturing mouse dendritic cells (DCs), we report that IP regulate the expression of more than 8,000 transcripts. The broad impact of IP on gene expression is cell-autonomous, mediated mainly at the transcriptional level, and involves major signaling pathways including IRFs, NF-kB and STATs. Moreover, even when engineered to present optimal amounts of antigenic peptides, IP-deficient DCs are inefficient for in vivo T-cell priming. In addition, consistent with the fact that cancer cells endure proteotoxic stress, we report that acute myeloid leukemia (AML) cells from patients express high levels of IP genes. Expression of IP genes in AML is a cell-autonomous and IFN-independent feature that correlates with the methylation status of IP genes, and is particularly high in AML with a monocytic phenotype and/or MLL rearrangement. Notably, IP inhibition leads to accumulation of polyubiquitinated proteins and cell death in IPhigh but not IPlow AML cells. Co-clustering analysis reveals that genes correlated with IP subunits in monocytic AMLs are primarily implicated in cell metabolism and proliferation, mitochondrial activity and stress responses. Overall, our studies show that the role of IP is not limited to antigen processing and reveals major non-redundant roles for IP in transcription regulation and resistance to cell stress in AML

Interleukin-21 Accelerates Thymic Recovery from Glucocorticoïd-Induced Atrophy

<div><p>Both physiological and psychological stress cause thymic atrophy via glucocorticoïd (GC)-dependent apoptosis of double-positive (DP) thymocytes. Given the pervasiveness of stress, GC-induced thymic atrophy is arguably the most common type of acquired immunodeficiency. We recently reported that interleukin-21 (IL-21) has a unique ability to expand the small subset of DP thymocytes (CD69⁺) which are ongoing positive selection, and that administration of IL-21 increases thymic output in aged mice. The goal of this study was to evaluate whether IL-21 could mitigate GC-induced thymic atrophy. In contrast to double-negative (DN) and single-positive (SP) thymocytes, most DP thymocytes (CD69⁻) do not constitutively express the IL-21 receptor (IL-21R). Accordingly, CD69⁻ DP thymocytes from PBS-treated mice were unresponsive to IL-21 administration. However, following GC injection, surviving CD69⁻ DP thymocytes up-regulated IL-21R and responded to IL-21 treatment as evidenced by enhancement of Bcl6 expression and phosphorylation of STAT1, STAT3 and STAT5. Consequently, IL-21 administration to GC-treated mice accelerated thymic recovery by expanding considerably DP thymocytes and, to a lesser extent, DN thymocytes. However, IL-21-induced expansion of DN/DP thymocytes did not alter the diversity of the intrathymic or peripheral T-cell receptor (TCR) repertoire. We conclude that IL-21 dramatically accelerates recovery from GC-induced thymic atrophy.</p></div

Administration of rIL-21 to DEX-injected animals induces Bcl-6 expression in DP thymocytes.

<p>A–B) Representative flow-cytometry analysis of Bcl2 expression in DN and DP thymocytes derived from PBS-, DEX/PBS- or DEX/rIL-21-treated mice. C) Compiled mean fluorescent intensity for Bcl2 analysis. D–E) Representative flow-cytometry analysis for Bcl2l1 in DN and DP thymocytes from the same experimental groups. F) Compiled mean fluorescent intensity for Bcl2l1 analysis. G–H) Representative flow-cytometry analysis for Bcl6 in DN and DP thymocytes using same experimental groups. I) Compiled mean fluorescent intensity for Bcl6 analysis. For all experiments performed, we tested 3 mice per group, *<i>P</i><0.05. Data shown are representative of 3 separate experiments.</p

Analysis of TCR diversity.

<p>A–B) Flow-cytometry analysis of 15 TCRVβ-chains using intrathymic CD4 (A) or CD8 (B) SP T cells. C–D) Similar flow-cytometry analyses were performed using spleen CD4 (A) or CD8 (B) SP T cells. For both analyses, T cells were derived from PBS- (dark blue), DEX/PBS- (blue) or DEX/rIL-21-treated mice (light blue). We tested 3 mice per group. Data shown are representative of 3 separate experiments.</p

Proposed model for rIL-21- accelerated recovery following induction of acute thymic atrophy.

<p>Based on data presented herein, we propose the following model: under normal circumstances, only a small fraction of DP thymocytes (CD69⁺ post-selection DP3s which represent 6% of DPs) express the IL-21R (step 1). Upon DEX administration, 90–95% of DP thymocytes are depleted by apoptosis within 48 hrs (step 2). At that stage, the thymic cortico-medullary demarcation is blurred. The surviving DPs (CD69⁻ DP1s) upregulate IL-21R on their cell surface and become responsive to rIL-21. Upon rIL-21 administration, pSTAT1, pSTAT3 and pSTAT5 are activated while post-transcriptional mechanisms lead to accumulation of Bcl6. These signaling events accelerate thymic recovery from DEX-induced atrophy.</p

STAT phosphorylation in thymocytes.

<p>A–B) Representative flow-cytometry analysis of pSTATs in fractionated thymocytes derived from PBS- or DEX-treated animals supplemented with 10ng/ml rIL-21 (red histograms) or no cytokines (black histograms). We tested 3–6 mice per group in separate experiments.</p