Laure Dumoutier & Jean-Christophe Renauld

Sommige ziekten, zoals astma (longziekte), psoriasis (huidziekte), de ziekte van Crohn (darmziekte) of kanker, zijn gekoppeld aan een ontregeling van de reactie op ontstekingen. In ons laboratorium proberen we meer inzicht te krijgen in de rol van cytokines, kleine eiwitten die een cruciale signaleringsrol spelen in het ontstekingsproces. Ons onderzoek richt zich in het bijzonder op de cytokines IL9 en IL-22.

Cytokines worden geproduceerd door cellen zoals de lymfocyten als reactie op een prikkel. Deze proteïnen veroorzaken in verschillende cellen de uitdrukking van specifieke receptoren. Ze spelen een essentiële rol in de reacties van de gastheer tegen de infecties. Een overmatige reactie kan echter aan de basis liggen van bepaalde ziekten.

Ons laboratorium kijkt onder andere naar huidziekten, zoals psoriasis, en naar ziekten die te maken hebben met het spijsverteringsstelsel, zoals colitis. De twee cytokines die we bestuderen, IL-9 en IL-22, hebben we ontdekt in ons laboratorium.

IL-9

We hebben aangetoond dat het cytokine IL-9 goede en slechte kanten heeft, afhankelijk van de ziekte.

Zo is IL-9 betrokken bij de bescherming tegen worminfecties, terwijl het een schadelijke rol speelt bij astma en bij de ontwikkeling van T-lymfocyten.

We hebben een muismodel ontwikkeld met een worminfectie die lijkt op Trichuris trichuria, een worm die infecties in de darmen van de mens veroorzaakt. Hiermee hebben we aangetoond dat muizen bij aanwezigheid van IL-9 volledig beschermd zijn tegen infectie. Als we de activiteit van IL-9 echter blokkeren, overleven de wormen en blijft de infectie duren.

IL-9 heeft ook een slechte kant. Als er, na blootstelling aan verschillende allergenen, teveel van wordt gevormd, kan het bronchiale hyperactiviteit veroorzaken. Onze observaties toonden aan dat IL-9 astma bevordert door de productie van een ander cytokine te stimuleren, namelijk IL-13.

Astma is een chronische ontstekingsziekte van het ademhalingssysteem en wordt gekenmerkt door de omkeerbare obstructie van de luchtwegen en door bronchiale spasmen. De symptomen zijn hoest, een piepend geluid en druk op de borst en een moeilijke ademhaling. De veronderstelde rol van IL-9 bij astmapatiënten wordt bevestigd door genetische studies van andere groepen, die een verband aantonen tussen IL-9, de IL-9 receptor genen en de ziekte. Bovendien hebben we een toename van IL-9 cytokines kunnen aantonen bij astmapatiënten. We werken samen met farmaceutische bedrijven om moleculen te maken die de activiteit van IL-9 kunnen blokkeren, om zo de levenskwaliteit van astma patiënten te verbeteren.

IL-9 speelt een schadelijke rol bij de vorming van tumoren. Wij hebben in vivo aangetoond dat een overexpressie van de cytokine niet de ontwikkeling van T-lymfocyten of het aantal ervan wijzigt, maar wel leidt tot een laag-frequente spontane lymfoomvorming.

Onze bevindingen tonen aan dat IL-9 geen conventioneel oncogeen is, maar dat het eerder de ontwikkeling van tumoren bevordert als reactie op externe prikkels. Een ontregeling van de IL-9 signalering is ook betrokken bij tumortransformatie. Dit proces is bestudeerd in een in-vitromodel van tumorgenese. Daarmee hebben we oncogene mutaties geidentificeerd in het gen JAK1, een intracellulair proteïne dat een sleutelrol speelt in het opwekken van reacties op verschillende cytokines. Activatie van JAK1 wordt vaak waargenomen bij kankers en draagt bij tot oncogenese.

IL-22

Recent onderzochten we de rol van IL-22 in huidpathologieën, zoals psoriasis. Psoriasis is een chronische huidziekte die 2% van de wereldbevolking treft en die gekenmerkt wordt door het loskomen van dikke huidschilfers. De schilfers zijn het resultaat van een abnormale woekering van de keratinocyten (cellen van de epidermis), in gang gezet door immuuncellen en cytokines. Psoriasis evolueert in cycli met periodes van verbetering en verslechtering. Het is niet besmettelijk en bevat een genetische component. Door gebruik te maken van experimentele psoriasismodellen hebben we overexpressie van IL-22 kunnen aantonen in de huidletsels. Bovendien leidt een afremming van de activiteit van IL-22 tot een verbetering van de symptomen. De vorming van schilfers vermindert en de roodheid door de ontsteking verbetert. Dit wijst erop dat de cytokine een nadelige rol speelt in proriasis.

We zijn ook van start gegaan met translationele studies (link tussen fundamenteel en klinisch onderzoek) naar contactallergie. Dit is een ontstekingspathologie van de huid, veroorzaakt door contact met externe substanties. Momenteel gaat onze aandacht uit naar contactallergieën die veroorzaakt worden door bestanddelen in parfums en cosmetica, zoals methylisothiazolinon, een bewaarmiddel dat in bepaalde shampoos zit. We willen het immunologisch proces typeren dat gepaard gaat met de allergische reactie. We hebben ook een experimenteel contactallergiemodel ontwikkeld om de rol van verschillende factoren in de ziekte te kunnen begrijpen. Dit onderzoek wordt gevoerd in samenwerking met de dienst dermatologie van het Ziekenhuis Sint-Lucas.

In tegenstelling tot de huid, hebben we een beschermende rol van IL-22 kunnen aantonen in ontstekingsziekten die het maag-darm kanaal treffen. We hebben aangetoond dat de cytokine noodzakelijk is om de integriteit van de intestinale slijmvliezen te beschermen en zo een te ernstige ontsteking te verhinderen. De ziekte van Crohn en ulceratieve colitis zijn de meest courante chronische ontstekingsziekten van het maag-darmstelsel. De ziekte van Crohn kan elk deel van het maagdarmkanaal treffen, colitis enkel de dikke darm en het rectum. De ziekte van Crohn wordt veroorzaakt door een chronische ontsteking waarbij het eigen immuunsysteem het maagdarmkanaal aanvalt. Op dit moment kunnen patiënten nog niet genezen van deze ziekte. Ze worden behandeld om de symptomen onder controle te houden.

In de toekomst gaan we ons onderzoek uitbreiden tot andere cytokines die lijken op IL-22 en die ook een overexpressie vertonen in onze experimentele psoriasis- of colitismodellen. We hopen dat dit onderzoek ons zal helpen om een beter inzicht te krijgen in de ontstekingsreactie bij deze twee ziekten dat we de behandeling van de patiënten misschien kunnen verbeteren.

Our team is deciphering the biological activities of cytokines in inflammatory and tumoral processes using various preclinical models. In addition, we are investigating the signal transduction pathways underlying these activities at the molecular level.

Our work focuses on Interleukin-9 (IL-9) and IL-22, two cytokines discovered in our laboratory. IL-9 is produced by a particular T lymphocyte population, called TH9, and plays a role in immune responses against intestinal parasites and asthma. Dysregulation of IL-9 signaling is also implicated in tumoral transformation, and this process has been studied in an in vitro tumorigenesis model, leading to the identification of oncogenic mutations of the JAK1 gene. IL-22, originally identified as a gene induced by IL-9 in T lymphocytes, upregulates the production of acute phase reagents and antibacterial proteins in the liver, the lung and intestinal mucosae, and in the skin. IL-22 appears to play a key role in wound healing and skin inflammation processes such as psoriasis. The role of these cytokines in inflammation is being investigated using transgenic and gene-targeted mice for these cytokines and their receptors, and by using an original strategy of anti-cytokine vaccination.

Interleukin-9

Interleukin-9 (IL-9) was discovered in our group through its ability to sustain antigen-independent growth of certain murine T helper clones. Although IL-9 did not turn out to be a T cell growth factor for freshly isolated T cells, it was found particularly potent on T cell lymphomas as an anti-apoptotic agent. To determine the biological activities of this factor, we generated transgenic mice overexpressing this cytokine. Analysis of these animals disclosed two essential properties of IL-9: its activity on mast cells and eosinophils with consecutive implications in asthma, and its tumorigenic potential in T lymphocytes.

IL-9 tg mice : Parasite infections and asthma

Although IL-9 overproduction does not affect mouse viability and IL-9 transgenic mice did not show any major abnormality at first look, they were found to harbor increased numbers of mast cells in the intestinal and respiratory epithelia, and were characterized by a general hypereosinophilia. This phenotypic characteristic was found to increase the capacity of these animals to expel nematodes such as Trichinella spiralis or Trichuris muris, suggesting that IL-9 administration could protect susceptible hosts against these parasites. Conversely, blocking IL-9 activity resulted in a failure to expel T. muris parasites and a decreased eosinophilic response against the parasite. The other side of the coin was the discovery that IL-9 overexpression, such as that characterizing the IL-9 transgenic animals, resulted in bronchial hyperresponsiveness upon exposure to various allergens. Our observations showed that IL-9 promotes asthma through both IL-13-dependent and IL-13-independent pathways. The potential aggravating role of IL-9 in asthma was confirmed by genetic analyses, performed by others, and pointing to both IL-9 and the IL-9 receptor genes as major candidate genes for human asthma. In addition, we found that asthma patients produce increased amounts of IL-9.

IL-9 tg mice : T cell lymphomas

IL-9 transgenic animals showed normal T cell development and T cell numbers but spontaneously developed thymic lymphomas at low frequency. Two lines of evidence indicate that IL-9 is not a conventional oncogene, but rather favors tumor development in response to exogenous stimuli. Firstly, the tumor incidence was significantly lower when mice were maintained under pathogen-free conditions. Secondly, all IL-9 transgenic mice, when exposed to subliminal doses of a chemical carcinogen or to irradiation, developed T cell lymphomas that were innocuous in wild type mice. The anti-apoptotic activity of IL-9 provides an attractive explanation for these observations, namely that IL-9 could lead to increased survival of abnormal cells generated by exposure to minimal doses of oncogenic stimuli. The potential implication of IL-9 in oncology was also confirmed in human systems by its constitutive expression in Hodgkin lymphomas.

IL-9R and signal transduction

Analysis of the mode of action of IL-9 at the molecular level was initiated by cloning the murine and human IL-9 receptor (IL-9R) cDNAs. By further dissecting the signal transduction cascade triggered by IL-9, we showed that, upon IL-9 binding, the IL-9R associates with a co-receptor protein called γc. This induces the phosphorylation of the JAK1 and JAK3 tyrosine kinases, which are associated with IL-9R and γc, respectively. A single tyrosine residue of the IL-9R is then phosphorylated and acts as a docking site for 3 transcription factors of the STAT family, STAT-1, -3 and -5, which become phosphorylated and migrate to the nucleus, where they activate the transcription of a number of genes. This pathway is common to many cytokines but is often dispensable for their biological activities. For IL-9, our group demonstrated that activation of the STAT transcription factors is crucial for all the effects of IL-9 studied on various cell lines, including positive and negative regulation of cell proliferation, as well as inhibition of corticoid-induced apoptosis in T cell lymphomas. Further analysis demonstrated that STAT-1, -3 and -5 play specific, redundant and synergistic roles in the different activities of IL-9 in vitro. The pathways responsible for IL-9-induced proliferation were studied in detail, and this process was found to depend mainly on the activation of STAT-5, on the recruitment of the IRS-1 adaptor, and on the activation of the Erk MAP-Kinase pathway.

Role of JAK1 overexpression and mutation in tumor cell transformation

Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Our observations indicate that JAK overexpression plays a role in such processes. Using a murine proB cell line that strictly depends on IL-3 for growth in vitro, cytokine-independent and tumorigenic clones were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquired IL-9 responsiveness during a first step of selection, and progressed after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but, more importantly, allowed a second selection step that resulted in cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway. Recently, we elucidated the mechanism responsible for the second step of this tumoral transformation process, as we found that the majority of the cytokine-independent tumorigenic clones acquired an activating mutation in the kinase or in the pseudokinase domain of JAK1.

In parallel to these observations, in collaboration with Professor Marco Tartaglia at the University of Rome, we identified activating mutations in JAK1 in 20% of T-cell acute lymphoblastic leukemia (T-ALL) and in 3% of B-ALL patients, confirming the relevance of our in vitro model-derived JAK1 mutations for human malignancies. Further analysis of human ALL samples showed that JAK1-mutated leukemias share a type I IFN transcriptional signature, suggesting that these mutants not only activate growth-promoting pathways, but also antiviral pathways. Expression of these activating JAK1 mutants in murine hematopoietic cell lines recapitulated this signature in the absence of IFN, but also strongly potentiated the in vitro response to IFN. Finally, we also showed in an in vivo leukemia model that cells expressing mutants such as JAK1(A634D) are hypersensitive to the anti-proliferative and anti-tumorigenic effect of type I IFN, suggesting that type I IFNs should be considered as a potential therapy for ALL with JAK1 activating mutations.

While most JAK1 mutants were sensitive to ATP-competitive JAK inhibitors, mutations targeting Phe958 and Pro960 in the hinge region of the kinase domain rendered JAK1 not only constitutively active, but also resistant to all tested JAK inhibitors. Furthermore, mutation of the homologous Tyr931 in JAK2 wild-type or JAK2 V617F mutant found in myeloproliferative neoplasms also conferred resistance to JAK inhibitors, including the clinically used INCB018424. These observations indicate that in JAK mutation positive patients, treatment with JAK inhibitors is likely to contribute to the selection of these mutations that combine increased oncogenicity and drug resistance.

IL-22

Searching for genes specifically regulated by IL-9 in lymphomas, we identified a new gene that turned out to encode a 179 amino acid long protein, including a potential signal peptide and showing a weak but significant sequence homology with IL-10. This protein, originally designated IL-TIF for IL-10-related T-cell derived Inducible Factor, was later renamed IL-22. Despite its structural homology with IL-10, IL-22 fails to recapitulate any of IL-10 biological activities. Biological activities of IL- 22 include the induction of acute phase proteins in liver and protection against experimental hepatitis and colitis. Among the different T cell subsets, IL-22 was found to be preferentially produced by TH17 cells, which are associated with several autoimmune and inflammatory processes, and the aryl hydrocarbon receptor AhR turned out to be a major regulator of the expression of this cytokine.

IL-22 and psoriasis

We assessed the role of IL-22 in a mouse model of psoriasis, where skin inflammation is triggered by topical application of the TLR7/8 agonist, imiquimod. At the macroscopic level, scaly skin lesions induced by daily application of imiquimod in wild-type mice were almost completely absent in IL-22–deficient mice and in mice treated with a blocking anti–IL-22 Ab. At the microscopic level, IL-22–deficient mice showed a dramatic decrease in the development of pustules and neutrophil infiltration and a partial decrease in acanthosis. At the molecular level, the absence or inhibition of IL-22 strongly decreased the expression of chemotactic factors such as CCL3 and CXCL3 and of biomarkers such as S100A8, S100A7, and keratin 14, which reflect the antimicrobial and hyperproliferative responses of keratinocytes.

IL-22 and asthma

In contrast with this pro-inflammatory effect of IL-22 in skin inflammation, asthma models show that IL-22 can have a protective anti-inflammatory activity in lungs. This protective effect of IL-22 has been attributed to an inhibition of IL-13 activity on lung epithelial cells either for CCL17/TARC induction or for IL-25 production. Inhibiting IL-22 in vivo, through antibody treatment or by gene targeting, increased expression of these inflammatory mediators, infiltration by eosinophils and broncho-hyperrersponsiveness.

IL-22 production

Both the psoriasis and asthma models have challenged the dogma that IL-22 is mainly produced by TH17 lymphocytes, and gamma-delta T cells as well as innate lymphoid cells turned out to be the major producers of this cytokine. We characterized the cells responsible for IL-22 production in response to TLR agonists such as LPS or flagelin. We identified a new innate lymphoid spleen cell population expressing CD25, CCR6 and IL-7R representing 1% of spleen cells from recombination activating gene (Rag2)-deficient mice. This population comprises 60% to 70% CD4+ cells, which produce IL-22, and are still present in common g chain-deficient mice; the CD4- subset coexpresses IL-22 and IL-17, and is common g chain-dependent. These cells share a transcriptional program with NKp46+ RORgt+ cells found in intestinal mucosae and involved in antibacterial responses. The importance of IL-22 production for the LPS-triggered response is highlighted by the fact that IL-22-deficient mice are more resistant to LPS-induced mortality, pointing to the pro-inflammatory activity of this cytokine.

IL-22 receptors and signal transduction

Although IL-22 does not share any biological activity with IL-10, these 2 cytokines share a common component of their respective receptor complex, IL-10Rß. Anti-IL-10Rß antibodies indeed block the IL-22-induced acute phase response in HepG2 cells. All receptor complexes for IL-10-related cytokines include a long chain and a short chain, based on the length of the cytoplasmic domain of these transmembrane proteins. IL-10Rß is a typical short chain component, with only 76 amino acids in the cytoplasmic domain, whose main function seems to consist of recruiting the Tyk2 tyrosine kinase. In addition to IL-10R ß, IL-22 signaling requires the expression of a long chain protein, called IL-22R, which comprises a 319 amino acid long cytoplasmic domain. This chain associates with JAK1 and is responsible for the activation of cytoplasmic signaling cascades such as the JAK/STAT, ERK, JNK and p38 MAP kinase pathways. An unexpected feature of the IL-22R chain is the fact that the C-terminal domain of this receptor is constitutively associated with STAT3, and that STAT3 activation by this receptor does not require the phosphorylation of the receptor, in contrast to the mechanism of STAT activation by most other cytokine receptors.

In addition to the membrane IL-22 receptor complex, we identified a protein of 231 amino acids, that presents 33% amino acid identity with the extracellular domains of IL-22R but is without any cytoplasmic or transmembrane domain. This soluble receptor has been named IL-22 binding protein (IL-22BP), because it binds IL-22 and blocks its activities in vitro, demonstrating that this protein can act as an IL-22 antagonist.

The crystal structure of IL-22, alone and bound to its cellular receptor IL-22R or to its soluble receptor IL-22BP, has been characterized in collaboration with Professor Igor Polikarpov of the University of Sao Paulo and is illustrated below.

We have showed that the IL-22R chain also forms a functional heterodimeric receptor complex by associating with IL-20Rß, the second short chain member of the IL-10R-related receptor family. This complex mediates STAT-1 and –3 activation by IL-20 and IL-24, but not by IL-22. Further more, IL-20 and IL-24 can also bind to other complexes consisting of IL-20Rα and IL-20Rß.

Anti-cytokine vaccination

In addition to conventional gene targeting strategies that were used in our lab to generate mice deficient in the IL-9R, in IL-22 or in IL-22R, we developed a new strategy of anti-cytokine vaccination. Vaccinated mice produce anti-cytokine autoantibodies that block the biological activities of endogenous cytokines. Neutralizing auto-antibodies against cytokines such as IL-9, IL-12 and IL-17 have been induced upon vaccination with the autologous cytokines chemically coupled with OVA (IL-9, IL-17) or with the Pan DR T helper epitope PA-DRE (IL-12). This strategy contributed to the demonstration of IL-9’s role in an intestinal helminth infection, IL-12’s role in atherosclerosis and of IL-17’s role in experimental autoimmune encephalomyelitis. More recently, we developed another procedure for anti-cytokine vaccination, by taking advantage of tumor cells expressing peptides, fused with a human transmembrane proteins, as vaccines. These vaccination methods represent simple and convenient approaches to knock down the in vivo activity of soluble regulatory proteins, including cytokines and their receptors, and are validated with additional targets in inflammatory models.

Complete list on PubMed