METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF TH2 MEDIATED DISEASES

Abstract

The present invention relates to methods and pharmaceutical composition for the treatment of T-helper type 2 (Th2)-mediated diseases. More particularly, the present invention relates to an inhibitor of the Suv39h1-HP1a silencing pathway for use in the treatment of a T-helper type 2 (Th2)-mediated disease, in particular allergic asthma.

Claims

1. A method of using an inhibitor of the Suv39h1-HP1α silencing pathway to treat a T-helper type 2 (Th2)-mediated disease.

2. The method of claim 1 wherein the inhibitor of the Suv39h1-HP1α silencing pathway selected from the group consisting of inhibitors of H3K9-histone methyltransferase Suv39h1; inhibitors of H3K9-histone methyltransferase Suv39h1 gene expression, inhibitors of HP1α gene expression and inhibitors of the binding of H3K9me3 to HP1α.

3. The method of claim 1 wherein the inhibitor of the Suv39h1-HP1α silencing pathway is an epipolythiodioxopiperazine.

4. The method of claim 1 wherein said T-helper type 2 (Th2)-mediated disease is selected from the group consisting of graft immune diseases (chronic GVHD), autoimmune diseases (especially organ non-specific autoimmune diseases), type-Th2 allergic diseases, ulcerative colitis, systemic lupus erythematodes, myasthenia gravis, systemic progressive scleroderma, rheumatoid arthritis, interstitial cystitis, Hashimoto's diseases, Basedow's diseases, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, atrophic gastritis, pernicious anemia, Addison diseases, pemphigus, pemphigoid, lenticular uveitis, sympathetic ophthalmia, primary biliary cirrhosis, active chronic hepatitis, Sjogren's syndrome, multiple myositis, dermatomyositis, polyarteritis nodosa, rheumatic fever, glomerular nephritis (lupus nephritis, IgA nephtopathy, and the like), allergic encephalitis, atopic allergic diseases (for example, bronchial asthma, allergic rhinitis, allergic dermatitis, allergic conjunctivitis, pollinosis, urticaria, food allergy and the like), Omenn's syndrome, vernal conjunctivitis and hypereosinophilic syndrome.

5. The method of claim 1 wherein the T-helper type 2 (Th2)-mediated disease is asthma or allergic asthma.

6. A method for screening a drug for the treatment of a Th2-mediated disease comprising the steps of testing a plurality of test substances for their ability to inhibit the Suv39h1-HP1α silencing pathway and selecting the substance capable of inhibiting said pathway.

7. A method for screening a drug for the treatment of a Th2-mediated disease with an agent selective for SUV39H1 inhibition identified by: (a) contacting a SUV39H1 with a test substance, and measuring the level of methylation of an amino acid of histone H3, (b) contacting a second Histone H3 lysine 9 (H3K9) histone methyl transferase with the test substance, and measuring the level of methylation of an amino acid of history H3, and selecting as said drug a test substance that preferentially inhibits SUV39H1 compared to the second H3K9 histone methyl transferase.

Description

FIGURES

[0063] FIGS. 1A-C. Suv39h1 regulates Th2 stability in vivo and influences allergic lung inflammation: Wild type or Suv39h1-deficient mice were injected intraperitoneally on days 0 and 7 with OVA/Alum. On days 55 thru 60 challenged with OVA intranasally and sacrificed on day 61 for analysis. (a) IL4 or IFNγ producing OVA-specific splenocytes were enumerated using dual color ELISpot (left and middle panels). From the same animals the total number of CD4.sup.+ splenocytes expressing T1/ST2 and producing IFNγ were enumerated by flow cytometry (right panel). (b) The serum levels of OVA-specific IgG1 (left panel) or IgG2c (right panel) were quantified using ELISA. PBS immunized control values were subtracted as background from (a) and (b). (c) Periodic acid Schiff staining was used to quantify the number of mucus positive cells in the lung of OVA challenged animals. All data shown is a representative of two independent experiments with 6 mice per group and is represented as mean±s.e.m.

[0064] FIG. 2. Chaetocin treatment results in less allergen-induced lung pathology. Mice were injected intraperitoneally on days 0 and 7 with OVA/Alum. On day 17 mice were challenged intranasally with OVA and DMSO (OVA+Veh.) or Chaetocin (OVA+Chaetocin) and sacrificed on day 22 for analysis. Periodic acid Schiff staining was used to quantify the number of mucus positive cells (pink staining) in the lung of OVA challenged animals. Images are 200× magnification. All data shown is a pool of two independent experiments with 6 mice per group and is represented as mean±s.e.m.

EXAMPLE 1

Suv39H1 and HP1A Control the Fidelity of the TH2 Cell Lineage

[0065] Material & Methods

[0066] Mice: C57BL/6 were obtained from Charles River (Les Oncins, France) and housed in the animal facility of Institut Curie. We maintained Suv39h1 knockout mice, a kind gift from T. Jenuwein,.sup.12 on a mixed 129SVxC57BL/6 background. The HP1α and HP1γ mutant mouse lines were established at the MCI/ICS (Mouse Clinical Institute—Institut Clinique de la Souris-, Illkirch, France; http://www-mci.u-strasbg.fr) and maintained on a mixed 129SVxC57BL/6 background. The details of the strategy are available upon request (project IR00001073/K316). The HP1α targeting vector comprises 1) 3.9 kb of 5 homology arm in intron 3, 2) a floxed fragment of 1.3 kb comprising a LoxP site, 156 bp of intron 3, exon 3 and 1030 bp of intron 4 and a floxed neo-resistance cassette also surrounded by FRT sites and 3) a 3.4 kb of 3′ homology arm of intron 4. This construct was electroporated in ES cells (MCI-129sv/Pas) and 733 G-418 resistant clones were screened by PCR with 5′ and 3′ external primers and a LoxP specific pair of primers. One positive clone for homologous recombination and with only one insertion was isolated and confirmed by Southern blot analysis with a 5′ external probe and two Neo specific probes. The karyotype of this ES clone was verified to be normal and this clone was injected in wild-type mice (C57B1/6J). Offsprings were screened by PCR for germ line transmission. Positive mice were then crossed with CMV-Cre transgenic mice to excise the floxed sequence and backcrossed to wild type to obtain HP1α−/− mice. All control wild type mice used were sex-matched littermate controls.

[0067] CD4.sup.+ T cell purification: Single cells suspensions of spleens and lymph nodes (mesenteric, inguinal, axillary and brachial) were pooled and after red blood cell lysis CD4.sup.+ T cells purified by negative selection using Miltenyi CD4.sup.+ T cell isolation kit (Miltenyi Biotec). To obtain naïve CD4.sup.+ T cells, these cells were further purified either by sorting CD44.sup.low CD.sup.neg cells using a FACSAria (BD) or FacsVantage (BD) or by positive selection using CD62Lmicrobeads (Miltenyi Biotec). The isolation procedure was performed using 1×PBS (Gibco) containing 0.5% BSA and 2 mM EDTA. Cultures obtained from each sorting procedure yielded similar results.

[0068] CD4.sup.+ T cell cultures: tissue culture-treated 96 well flat bottom plates (Techno Plastic Products) were coated with 1 mg/ml of anti-CD3e (clone 145-2C11, BD) and anti-CD28 (clone 37.51, BD) in 1×PBS (Gibco) for 1-3 hours at 37° C. 5% CO.sub.2. Wells were then washed twice with 1×PBS. For non-biased culture conditions wells were seeded with 1×10.sup.6/ml CD4.sup.+ T cells in RPMI+Glutamax (Gibco) containing 10% FCS (Gibco), 0.1 mM 2-mercaptoethanol and penicillin and streptomycin. For Th1 culture conditions the medium contained 5 ng/ml of recombinant mouse IL12 (R&D systems) and 10 mg/ml of anti-IL4 (clone 11B.11, BD or eBiosciences). For Th2 culture conditions the medium contained 50 ng/ml of recombinant mouse IL4 (R&D systems) and 10 mg/ml of anti-IL12 (C17.8, BD or eBiosciences) and anti-IFNg (clone XMG1.2, BD or eBiosciences). Cells were cultured at 37° C. in 5% CO.sub.2. After three days the cells were transferred to fresh plates and the medium was supplemented with a 2× cocktail of the above reagents with the addition of 30 U/ml of recombinant human IL-2 (Chiron). If necessary cell populations were expanded every 3 days in medium plus cytokines as described above For secondary cultures, primary cultures were counted and 1×10.sup.6/ml were seeded into 96 well plates in the conditions described above.

[0069] Cell staining for flow cytometric analysis: All antibodies used were purchased from BD with the exception of anti-T-bet (4B10) and anti-Gata3 (HG3-31) which were purchased from Santa Cruz Biotechnology. All staining was done in round-bottom 96 well plates (Corning Life Sciences). Cell Surface staining was performed for 25 minutes at 4° C. in 1×PBS 0.5% BSA and 2 mM EDTA. For intracellular cytokine staining cells were restimulated at 37° C. with PMA (25 ng/ml, Sigma) and ionomycin (1 mM, Sigma) for 4 hours with the addition of Brefeldin A (5 mg/ml) for the last 2 hours. Cell surface staining was performed and then cells were fixed with 2% formaldehyde for 10 minutes at room temperature. The cells were then washed in 1×PBS and then permeabilized by resuspension in Perm/Wash buffer (BD). Cells were centrifuged and intracellular cytokine staining was performed with anti-IFNg and anti-IL4 in permwash buffer for 25 minutes at 4° C. For intracellular transcription factor staining, cells were fixed and permeabilized with 1×PBS containing 0.2% Triton X-100 for 5 minutes at room temperature. Cells were then washed and staining performed in 1×PBS containing 5% BSA. Secondary anti-mouse alexa 488 or 647 (Molecular Probes) were used to detect T-bet and GATA3 primary antibodies. A mouse IgG1 isotype was used as a control. For some experiments fluorophore conjugated T-bet and GATA3-specific antibodies were used (ebioscience). For CFSE experiments, purified T cells were incubated for 10 min at 37° C. in PBS with 2.5 μM CFSE (Molecular Probes). All FACS acquisition was performed on a FACScalibur II (BD) using Cell Quest software. Analysis was performed using FlowJo software (Treestar).

[0070] Western blotting: Cell pellets were lysed in 1× NuPAGE LDS sample buffer (Invitrogen) and 1× NuPAGE reducing agent (Invitrogen). After 30 min treatment with 25 U benzonase nuclease (Novagen), lysate from 2×10.sup.5 cells were loaded on NuPAGE Bis/Tris 4-12% gradient gels (Invitrogen), using a 1×MOPS migration buffer (Invitrogen). After transfer, the membranes were blocked with 5% milk in PBS 0.05% Tween and then incubated with primary antibodies and peroxidase-conjugated secondary antibodies. Bound antibodies were revealed using the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) according to the manufacturers' directions. The intensity of the bands was quantified by densitometry and was expressed as arbitrary units. Anti-HP1α (Euromedex, 2HP-2G9), anti-HP1γ (Euromedex,2MOD-1G6) and anti-HP1β (Euromedex, 1MOD-1A9).

[0071] Chromatin immunoprecipitation: For ChIP of wild type cells, CD4.sup.+ T cells from 4-5 week old female C57BL/6 mice were purified by negative selection as described above. For ChIP of Suv39h1-deficient and littermate cells CD4.sup.+CD44.sup.low cells were prepared as described above. ChIP analysis was carried out essentially as described elsewhere.sup.37. Briefly, cells were fixed for 10 min in 1% formaldehyde (wt/vol) at room temperature. Formaldehyde was quenched by 0.125 M glycine for 5 min. Cells were then washed with cold PBS and lysed. The suspension of nuclei was sonicated to achieve an average 200-500 bp length of genomic DNA fragments. Specific antibodies (H3K9me3 (ab 8898 Abcam), H3K9ac (17-657, Upstate), Histone-H3 (ab1791 Abcam), HP1α (05-689, Upstate) and rabbit or mouse IgG (Upstate) were coupled to Dynal protein A beads (Invitrogen). 5 μg of chromatin from each sample was taken ChIP with anti-H3K9me3, 10 μg and for ChIP with anti-H3K9ac and 15 pg for ChIP with anti-HP1α. DNA was purified after crosslink reversal. Real time PCR was performed in triplicate using the Sybr green detection system (Qiagen). Primer sequences are listed in Supplementary table 1. Chromatin immunoprecipitations were performed at least three times with independently cultured cells, results were expressed as a percentage of input DNA and averaged.

[0072] Allergen-Induced Asthma: Suv39h1-deficient and wild type littermate controls were injected intraperitoneally on days 0 and 7 with 10 μg OVA (Sigma) in PBS mixed with 50 μl Imject Alum (Thermo Scientific), Imject Alum alone or PBS alone. On days 55 thru 60 anaesthetized mice were challenged with 50 μg OVA in 30 μl of PBS or PBS alone intranasally. On day 61 mice were sacrificed for analysis.

[0073] Isolation of Total Lung Cells: Right lung lobes were excised and cut into small pieces (˜1-2 mm.sup.2). Pieces were transferred into 5 mLs of a freshly prepared collagenase digestion solution containing 1,500 U Collagenase Type 2 (Worthington) and 75 ul of a 10 mg/mL DNase I (Roche) stock solution. Lungs were digested for 1 hour at 37 C under constant horizontal shaking. 5 mL lung digest was transferred into a 40 m sieve and pushed through with a sterile syringe top. Following centrifugation red blood cells were lysed with ACK lysis buffer for 1-2 minutes at room temperature. Total lung cells were washed 2 additional times and counted.

[0074] Lung Fixation and Histology: Left lung lobe was carefully excised and fixed in 3.7% PFA in PBS overnight. Lobes were moved into 70% ETOH the next day. Lung samples were embedded, cut and stained with haematoxylin and eosin (H&E) for cellular infiltration analysis and Periodic acid-Schiff (PAS) for goblet cell hyperplasia analysis. Images were acquired on an Eclipse 90i Upright microscope at the Nikon Imaging Center at the Institut Curie. Mucus index was calculated using ImageJ software (NIH) on entire lung sections.

[0075] Serum Ig: Flat bottom 96-well Nunc-Immuno Plates (Thermo Scientific) were coated with 10 μg/mL OVA or 1/1000 goat anti-mouse Ig (H+L) in PBS (Southern Biotechnologies, cat#1010-01) overnight at 4 C. Coating liquid was discarded and wells were blocked with 150 μl PBS+1% BSA for 2 hours at 37 C. After discarding blocking buffer, sample and standards were added in 50 L per well overnight at 4 C. Standards were purchased from Southern Biotechnologies (Mouse IgG1, clone 15H6, starting dilution 10 ng/mL in PBS) and (Mouse IgG2c, clone 6.3, starting dilution long/mL). Serum samples were diluted in PBS 1/100 for IgG2c and 1/1,000 for IgG1. Wells were washed 3× with PBS+0.01% Tween20. Detection IgG1 (goat anti-mouse IgG1-biotin, 0.5 mg/mL) and IgG2c (goat anti-mouse IgG2c-biotin, 0.5 mg/mL) both from Southern Biotechnologies and used at 1/5000 in 50 μl of PBS for 2 hours at 37 C. Wells were washed 3× as before and Strepavidin-HRP was added in 50 L of PBS (R&D, 1/200 dilution) for 30 minutes at 37 C. After 3 washes, 150 L of TMB substrate was added to each well (Sigma). The reaction was stopped by adding 50 μl/well of 1N H.sub.2SO.sub.4. Plates were read at 450 nm and 570 nm, the latter for background wavelength correction.

[0076] ELISpot: Splenocytes were seeded at 1×10.sup.6 and 0.5×10.sup.6 cells per well with and without 50 μg/mL OVA for 2 days. Dual IFNg/IL4 ELISpot was performed according to manufacture's protocol (R&D Systems, cat# ELD5217).

[0077] Results

[0078] After antigen encounter, CD4.sup.+ helper T (Th) cell differentiation follows distinct developmental programs that generate different types of effector T lymphocytes, including Th1, Th2, Th17 or Treg. Each subtype is characterized by specific expression of lineage-instructive transcription factors and signature cytokines.sup.1,2. Recently, the stability and plasticity of Th phenotypes has been recognized as critical to immune responses.sup.3,4.

[0079] Differentiation of Th1 and Th2 cells from a common precursor occurs by cross-antagonism between the master-regulators T-bet and GATA-3, respectively, and leads to a mutually exclusive expression of cytokines genes.sup.1,2,3. Th1 cells produce IFNγ, which is important for clearance of intracellular pathogens, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13 and are critical for humoral immunity and clearance of extracellular pathogens.sup.1.

[0080] As Th1 and Th2 phenotypes are heritable through cellular divisions.sup.5,6,7, it has been proposed that epigenetic modifications regulate T cell differentiation.sup.1,3,4,8. Interestingly, correlations were observed between levels of several histone-H3 and H4 modifications with activity or silencing of cytokine genes in committed Th1 and Th2 cells.sup.9. Moreover, the combination of active (H3K4me3) and inactive (H3K27me3) marks in the loci of transcription factors was proposed to shape their transcriptional potential.sup.10. However, the actual pathways underlying the establishment of epigenetic marks have not been directly manipulated to demonstrate their implication in T cell differentiation.sup.3.

[0081] Intriguingly, the role of one important epigenetic mark predominantly associated with transcriptionally inert heterochromatin, trimethylation of the lysine 9 residue of Histone-H3 (H3K9me3), has not been investigated thoroughly during Th cell differentiation. A 2-member family of histone methyltransferases (HMTases), Suv39h1 and Suv39h2.sup.11,12,13 (also known as KMT1A and KMT1B, respectively.sup.14) are responsible for trimethylation of H3K9 in pericentric heterochromatin. H3K9me3 in turn, can serve as a binding site for a histone binding adaptor family, heterochromatin protein 1 (HP1α, β and γ).sup.15,16,17. HP1 was originally identified in Drosophila and defined based on its property to act as a dominant suppressor of position effect variegation on gene silencing.sup.18. Since then, a number of groups have provided evidence that a HP1 repressive loop can silence transgene expression in mammals.sup.15,17,19,20. Current views propose that Suv39h1 trimethylates H3K9 in pericentromeric regions leading to the recruitment of HP1, which either sterically inhibits the binding of transcriptional machinery or attracts diverse chromatin modifiers, allowing maintenance and propagation of heterochromatin.sup.21. Suv39h1 can also associate with histone deacetylases (HDACs) and other HMTases, suggesting a complex mechanism of Suv39h1-mediated silencing, including maintenance of a hypoacetylated state of H3K9.sup.22,23.

[0082] In order to investigate a role for this silencing system in Th cell differentiation, we examined the post-translational modifications to H3K9 within the promoters of Th lineage determining genes by chromatin immunoprecipitation (ChIP), using antibodies specific for H3K9me3 (a mark usually associated with silencing) and H3K9ac (a typical mark of active genes). Importantly, we screened for batches of commercial antibodies that did not cross-react with other histone marks and identified one batch of anti-H3K9me3 with low cross reactivity to all other marks tested. Anti-H3K9me3 effectively precipitated major satellite sequences from pericentric heterochromatin, where this mark is enriched, and did not precipitate the promoter region of active Gapdh gene. On the contrary, H3K9ac was associated with the promoter of the active Gapdh gene, but not with major satellites.

[0083] During in vitro CD4.sup.+ Th differentiation we observed by day 7 an increase of repressive H3K9me3 in the promoters of lineage-inappropriate cytokine genes (I14 in Th1 and Ifng in Th2) and an increase of H3K9ac at the promoters of lineage-specifying cytokine genes. The promoter of Ifng initially contained higher levels of H3K9me3 in naïve CD4.sup.+ T cells than that of IL4. During lineage commitment, we observed a 2.5 fold increase in repressive H3K9me3 in the promoter of silent Ifng (in Th2 cells) and a four-fold increase in the promoter of silent IL4 (in Th1 cells). Similarly, a five-fold enrichment of H3K9ac was observed in the promoter of active Ifng and up to 30-fold in the active I14 promoter.

[0084] We then examined H3K9 modifications at the promoters of the genes coding Th1 and Th2 lineage-instructive transcription factors. There was a high level of the repressive H3K9me3 mark at the promoter of Tbx21 (coding for T-bet) in naïve cells, yet during Th1 differentiation, H3K9me3 decreased, concomitant with a five-fold increase in H3K9ac. In Th2 cells, the hypermethylated and hypoacetylated status of H3K9 at the Tbx21 promoter was maintained. The promoter of Th2-specifying Gata3 displayed an approximate six-fold increase in H3K9me3 in Th1 cells and two-fold increase in H3K9ac in Th2 cells. Our results show that the balance between trimethylation and acetylation of H3K9 in Th1 and Th2 gene promoters is regulated in a lineage-specific manner, and correlates with gene silencing or activation, respectively.

[0085] The observed modifications in H3K9 could, of course, be either a cause or consequence of the differences in gene expression between Th1 and Th2 cells. To address this, we used mice lacking the H3K9 tri-methyltransferase Suv39h1 (in adult tissues Suv39h2 expression if restricted to testes.sup.11). As previously reported.sup.12,24, these mice developed normally and had the predicted ratio and phenotype of hematopoietic cells in the lymphoid organs. Next we explored a possible role for Suv39h1 in Th cell polarization. There was little difference between wild type (age and sex-matched littermate controls) and Suv39h1-deficient cells in up-regulation of surface activation markers or cell proliferation in Th 1 or Th2 culture conditions. We then examined the differentiation of CD4.sup.+ T cells for 7 days in non-biased (NB), Th1 and Th2 conditions by assessing intracellular cytokine accumulation and expression of Th1 and Th2 master regulators, Tbet and Gata3, respectively. We did not find any major differences between the wild type littermate cells and Suv39h1-deficient cells. Thus, Suv39h1-deficient cells polarize normally in vitro, suggesting that H3K9 trimethylation is not involved in the silencing of the opposite loci during differentiation. However, these cells were polarized in highly biased conditions where they were incubated with the optimal concentrations of the polarizing cytokines and antibodies neutralizing the opposite cytokines. We therefore reasoned that incubating the already differentiated Th1 or Th2 cells under the opposite polarizing conditions may reveal potential defects in silencing of the opposite loci.

[0086] To test this possibility, differentiated Th1 or Th2 cells (day 7 of the primary cultures) were re-stimulated for 2-3 days under the opposite polarizing conditions (Th1 conditions for Th2 cells, and Th2 conditions for Th1). In wild type Th2 cells, we observed only low induction of the silenced Th1 or Th2 genes by Th1 cells in the secondary cultures, indicating that the silencing of the opposite lineage genes during Th1/Th2 differentiation is irreversible under these conditions. Similarly, Suv39h1-deficient Th1 cells did not express IL4 or GATA3 in secondary Th2 cultures. In contrast, upon exposure of Suv39h1-deficient Th2 cells to secondary Th1 conditions, the silenced IFNγ and T-bet were both induced in a significant proportion (˜30%) of the cells.

[0087] The increase in plasticity observed in the Suv39h1-deficient Th2 cells when cultured in Th1 conditions could be due to a silencing defect in fully differentiated Th2 cells, or to uncommitted cells (Gata-3/IL4-negative) that persisted in the Th2 cultures and induced expression of IFNγ and T-bet under Th1 conditions. To address this question, we gated on Th2 cells that maintained expression of IL4 or GATA3 after secondary Th1 re-stimulation and examined their levels of IFNγ or T-bet. The proportion of IL4 and GATA3-positive Th2 cells that co-express IFNγ and T-bet in the secondary Th1 cultures was increased in the Suv39h1-deficient cells. Therefore, in the absence of Suv39h1, increased levels IFNγ and T-bet are due to a true plasticity of differentiated IL4.sup.+/GATA3.sup.+ Th2 cells, and not to undifferentiated T cells in the Th2 cultures. We conclude that Suv39h1 restricts Th2 plasticity by locking out the Th1 lineage genes in differentiated Th2 cells.

[0088] In order to address the mechanisms involved in the stable silencing of the Th1 lineage genes IFNγ and T-bet, we analyzed wild type and Suv39h1-deficient Th2 cells by ChIP, using antibodies against both H3K9me3 and H3K9ac. Importantly, the Suv39h1-deficient Th2 cell populations were indistinguishable from wild type Th2 cells in terms of IL4/GATA3 and IFNγ/T-bet expression. Both populations were also homogenous, as GATA3 was expressed in virtually 100% of the cells. As expected, ChIP analysis for major satellites showed a three-fold decrease of H3K9me3, in comparison to wild type littermates, while no H3K9ac was detected. Opposite results were obtained with ChIP for the active Gadph gene: no H3K9m3 signal and a strong H3K9ac signal, which was unchanged in Suv39h1-deficient cells.

[0089] As expected from our analysis, the patterns of methylation and acetylation reflected Th2 polarization (i.e. higher levels of H3K9me3 in silenced genes and of H3K9ac in active genes). In Suv39h1-deficient Th2 cells, H3K9me3 levels in the promoter of Tbx21 persisted, while a reproducible increase in acetylation was also observed here. In wild type Th2 cells, repressive H3K9me3 is non-uniformly spread across regulatory elements (so-called conserved non-coding regions, CNS) encompassing 5′ and 3′ gene flanking sequences, which are indispensable for proper expression of IFNγ.sup.1. Suv39h1-deficient Th2 cells showed specific decreases in H3K9me3 levels in several, but not all regulatory elements. Significant decrease in densities of H3K9me3 was observed in the promoter of Ifng and proximal CNS (+54 kb), and stronger decrease at CNS (−53 kb) and proximal CNS (−6 kb). At the same time, we observed hyperacetylation of H3K9 within the promoter of Ifng and CNS regions from which H3K9me3 was decreased. Of note, ChIP with the antibodies for histone H3 did not show any significant changes in nucleosomal density at the regions of interest in Suv39h1-deficient cells. The imbalance between repressive and active H3K9 modifications in the Suv39h1-deficient Th2 cells was restricted to the Ifng and Tbx21 gene promoters, while other Th1-related transcription factors, such as Stat1 and Stat4, as well as Th2-related Stat6 did not show major alterations. In Suv39h1-deficient cells there was no increase in H3K9ac at promoters of Th2-specifying IL4 and Gata3. Therefore, in Th2 cells, Suv39h1 deficiency causes an imbalance between H3K9me3 and H3K9ac at the Th1 gene loci, resulting in incomplete silencing and increased Th2-to-Th1 plasticity. As Suv39h1 is known to associate with HDACs.sup.22, the observed increase of H3K9ac in Suv39h1 gene targets in Th2 cells might reflect the loss of this silencing component. These results suggest that the incomplete silencing of the Th1 genes (Ifng and Tbx21) in Suv39h1-deficient Th2 cells is the consequence of the observed imbalance of the H3K9me3/H3K9ac ratio in the corresponding gene promoters.

[0090] Given the known connection between Suv39h1 and HP1 in heterochromatin.sup.21, we investigated the potential involvement of HP1 in Th1 gene silencing in Th2 cells by generating mice deficient in HP1α by targeted deletion. These mice developed normally, and had the predicted ratio and phenotype of hematopoietic cells in the lymphoid organs. When stimulated, CD4.sup.+ T cells from HP1α-deficient mice displayed comparable up-regulation of cell surface activation markers and cell division profiles to wild type. After seven days of differentiation in polarizing in Th1 or Th2 conditions, we detected no difference in intracellular cytokine profiles or T-bet and GATA3 expression levels between the HP1α-deficient and wild type cells. We conclude that, similar to Suv39h1-deficient cells, CD4.sup.+ T cells deficient for HP1α have no defects in activation, proliferation or Th1/Th2 polarization.

[0091] We then investigated the putative role for HP1α in Th2-plasticity by re-culturing HP1α-deficient Th1 or Th2 cells under the opposite polarization conditions, as before. We observed a marked increase in IFNγ production by HP1α-deficient Th2 cells compared to wild type cells, similar to that observed in cells lacking Suv39h1. There were also a similar proportion of IL4.sup.+ cells from HP1α-deficient mice that expressed IFNγ in these secondary cultures (˜22%). HP1α-deficient Th1 cells behaved as their wild type counterparts in the corresponding opposite experiment. We also observed greater induction of T-bet in the secondary Th1 cultures of HP1α-deficient Th2 cells, as compared to wild type. Thus, HP1α is involved in the maintenance of Th1 gene silencing in Th2 cells.

[0092] Due to the high degree of homology between the three HP1-isoforms, we tested if the effects observed were specific to HP1α. To do this, we used cells from HP1γ deficient mice (HP1β-deficiency is lethal in mice.sup.25). We did not see any difference in the production of IFNγ between HP1γ-deficient and wild type Th2 cells, showing that reversibility of silencing of Th1 gene expression in Th2 cells is specific to HP1α. Therefore, HP1α is required for the effective silencing of the Th1 gene loci in Th2 cells and the restriction of Th2-to-Th1 plasticity, possibly via Suv39h1.

[0093] We tested this possibility directly by measuring the recruitment of HP1α to the Th1 promoter in both wild type and Suv39h1-deficient Th2 cells. HP1α was indeed bound to the silenced Ifng and Tbx21 promoters in wild type Th2 cells. However, in Suv39h1-deficient Th2 cells this binding was markedly reduced, showing that the recruitment of HP1α to Th1 gene promoters in Th2 cells is Suv39h1-dependent. We conclude that during Th2 lineage commitment, silencing by the Suv39h1/HP1 pathway lock out the Th1-specifying genes, thereby restricting Th2-to-Th1 plasticity.

[0094] Finally, we reasoned that if the defective silencing of the Ifng and Tbx21 loci in the Suv39h1-deficient Th2 cells compromised lineage commitment and increased plasticity, we would observe a shift toward a Th1 responses in vivo. We investigated this possibility in a model of ovalbumin (OVA)-induced allergic asthma that promotes a strict Th2-type response resulting in allergen-induced lung pathology.sup.26. As expected, immunological sensitization of wild type mice to OVA resulted in the generation of an antigen-specific Th2 response characterized by OVA-specific production of IL4 and low levels of IFNγ in the spleen (FIG. 1a). In addition to the generation of IL4.sup.+ T cells, in Suv39h1-deficient mice we also observed abundant allergen-specific IFNγ.sup.+ cells and IL4/IFNγ double producers, indicative of Th2 instability (FIG. 1a). Suv39h1-deficient mice also had increased numbers of CD4.sup.+ T cells that expressed the Th2 marker T1/ST2 and produced IFNγ (FIG. 1a; right panel). Evidence of an increased Th1 response in the Suv39h1-deficient mice was further supported by the different IgG isotypes in the serum. In wild type mice, as expected, we found high levels of OVA-specific IgG1 (a surrogate marker for the induction of Th2 responses), and barely detectable levels of the Th1-induced OVA-specific IgG2c.sup.27 in the serum (FIG. 1b). In line with a response skewed toward Th1, serum from the Suv39h1 KO mice contained increased levels of IgG2c when compared to their wild type counterparts (FIG. 1b). Therefore, consistent with our in vitro results, Suv39h1-deficient mice show defective Th2-lineage stability in vivo.

[0095] To determine whether the increase in Th2 plasticity had a pathophysiological consequence, we evaluated lung inflammation in the immunized mice. Indicative of strong Th2-mediated lung disease.sup.28, OVA-immunization of wild type mice resulted in an intense eosinophil infiltration and mucus production in the lungs (FIG. 1c). In Suv39h1-deficient mice, we observed a marked reduction in both eosinophil infiltration and mucus production. Similar results were also obtained in bone-marrow irradiation chimeras, in which the lung epithelium expressed Suv39h1, indicating that the differences between wild type and Suv39h1-deficient mice are restricted to the hematopoietic compartmen. We found that increased Th2 plasticity caused skewed Th1 responses in a strict Th2 model, resulting in reduced allergen-specific inflammatory response (as suggested before by others.sup.29,30,31). Taken together, these results indicate that Suv39h1-silencing controls the stability of Th2 cell identity in vivo.

[0096] In conclusion, we have identified an epigenetic pathway responsible for maintaining the phenotypic stability of Th2 cells by silencing Th1 genes. The Suv39h1-HP1α loop has been long associated with constitutive pericentric heterochromatin where it maintains a stable, silent environment.sup.17,32. However, whether this is restricted to pericentric heterochromatin has remained unexplored. Here we show that these factors could contribute to the regulation of haematopoietic genes, such as the Ifng and Tbx21. We suggest a model in which the loss of Suv39h1 in Th2 cells leads to the perturbation of the homeostasis in H3K9 post-translational modifications in silenced Th1-loci. This decrease of repressive H3K9me3 concomitant with the acquisition of the active H3K9ac mark has no consequences in normal polarizing culture conditions. However, under in vivo conditions, this imbalance leads to a loss of Th2 stability and an acquisition of a chimeric Th2+Th1 phenotype. This is potentially due to both the increased permissiveness of Ifng locus for Tbet and a repulsion of HP1α repressive element by increased levels of H3K9ac as has been observed previously.sup.33. Given that we observed a similar phenotype with HP1α-deficient cells, we propose that HP1α serves as a Suv39h1/H3K9me3-dependant molecular lock. Suv39h-mediated silencing mechanisms are known to involve, in addition to HP1α, diverse co-repressors such as KAP1 and DNA methyltransferases (DNMT) and HDACs, which can directly modify chromatin.sup.19,22,34,35,36. Indeed in the increase in H3K9ac in Suv39h1 target regions strongly suggests a loss of HDACs. In the case of the Th2 cell lineage, the exact mechanism of the multimeric Suv39h1/HP1a “locking” module will be the subject of further investigations to elucidate the control of stability and plasticity of Th phenotypes.

EXAMPLE 2

Chaetocin Treatment Results in Less Allergen-Induced Lung Pathology

[0097] Methods:

[0098] 6-8 week old female C57B1/6 mice were injected intraperitoneally on days 0 and 7 with 10 mg OVA (Sigma) in PBS mixed with 50 μl Imject Alum (Thermo Scientific). On days 17 to 22 anaesthetized mice were sensitized intranasally with 50 mg OVA in 30 ul of PBS mixed with 0.25 mg/kg of chaetocin (Sigma) or with vehicle (DMSO). On day 22 mice were sacrificed for analysis.

[0099] Results:

[0100] To assess whether allergic asthma pathology could be reduced by the inhibition of Suv39h1 we used a model of OVA induced allergic asthma. Mice were treated as shown in the experimental design above. In this model mice develop allergic responses that results in the production of mucus in the airways. FIG. 2 shows that the production of mucus in the airways was significantly greater in mice treated with the allergen (OVA) and the vehicle control that when OVA was administered in conjunction with chaetocin. These results show that inhibition of Suv39h1 in the lung reduces allergic asthma pathology. We conclude that chaeotocin has a potential usage for asthma treatment.

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