METHODS AND COMPOSITIONS FOR MODULATING MACROPHAGES POLARIZATION

Abstract

Inventors have surprisingly found that Emricasan is a much more potent inhibitor of monocyte differentiation compared to q-VD-OH by its ability to efficiently inhibit caspase-8, which is instrumental to this process. In addition, they have demonstrated that Emricasan alleviates the IL4-mediated M2-like polarization of human macrophages. Moreover, Emricasan also hampers bleomycin-induced pulmonary fibrosis in mice, a disease associated with an infiltration of M2-macrophages. Finally, caspase-8 deficient mice were found to be resistant to bleomycin-induced pulmonary fibrosis. As a whole, their findings indicate that the beneficial effect of Emricasan relies on its ability to inhibit caspase-8, and its capacity to prevent monocyte differentiation and M2 polarization of macrophages. Accordingly, the invention relates to a caspase 8 inhibitor for use in the polarization of macrophages.

Claims

1. A method of polarizing a macrophages, comprising contacting the macrophage with a caspase 8 inhibitor.

2. The method according to claim 1 wherein the macrophage is a type 2 macrophage.

3. The method according to claim 1 wherein the macrophage is a type 1 macrophage.

4. The method according to claim 1, wherein said caspase 8 inhibitor is Emricasan.

5. A method of treating a macrophage related diseases in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a caspase 8 inhibitor.

6. The method according to claim 5, wherein the macrophage related disease is selected from the group consisting: a solid cancer, a fibrotic diseases, hepatic fibrosis, systemic sclerosis, allergy, asthma, atherosclerosis and Alzheimer's disease.

7. The method according to claim 6, wherein the fibrotic disease is lung fibrosis.

8. The method according to claim 6 wherein the solid cancer is selected from the group consisting of: adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

9. The method according to claim 5, wherein the caspase 8 inhibitor is administered in combination with a classical treatment.

10. (canceled)

11. The method according to claim 9, wherein the classical treatment is administration of a natural or synthetic compound, immunotherapy, chemotherapy or radiotherapy.

12. A pharmaceutical composition comprising a caspase 8 inhibitor.

13. The pharmaceutical composition according to claim 12, wherein the caspase 8 inhibitor is Emricasan.

14. (canceled)

15. The method of claim 8, wherein the bile duct cancer is peripheral cancer, distal bile duct cancer, or intrahepatic bile duct cancer; the bone cancer is osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, or multiple myeloma; the brain and central nervous system cancer is meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, ganglioglioma, Schwannoma, germinoma or craniopharyngioma; the breast cancer is ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ or gynecomastia; the endometrial cancer is endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, or clear cellcarcinoma; the gallbladder cancer is mucinous adenocarcinoma or small cell carcinoma; the gastrointestinal carcinoid tumor is a choriocarcinoma or a chorioadenoma destruens carcinoma; the kidney cancer is renal cell cancer; the liver cancer is hemangioma, hepatic adenoma, focal nodular hyperplasia, or hepatocellular carcinoma; the lung cancer is small cell lung cancer or non-small cell lung cancer; the paranasal sinus cancer is esthesioneuroblastoma or midline granuloma; the rhabdomyosarcoma is embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma or pleomorphic rhabdomyosarcoma; the skin cancer is melanoma or nonmelanoma skin cancer; the testicular cancer is seminoma or nonseminoma germ cell cancer; the thyroid cancer is follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma or medullary thyroid carcinoma; and/or the uterine cancer is uterine leiomyosarcoma.

Description

FIGURES

[0099] FIG. 1. Emricasan inhibits CSF-1-induced monocyte differentiation at the micromolar level. Human peripheral blood monocytes from healthy donors were exposed for 2 days to 100 ng/mL CSF-1 alone or in combination with indicated concentrations (μM) of Emricasan which was added 30 min before CSF-1 treatment. (A) Macrophagic differentiation of monocytes from 3 different healthy donors was examined by 3-color flow cytometric analysis. The results are expressed as percentage of CD71/CD163 or CD16/CD163 double positive cells and represent the mean±SD of 3 independent experiments performed in duplicate. (B) Cell death from 3 different healthy donors was examined by flow cytometry analysis. The results are expressed as percentage of AnnexinV/DAPI double positive cells and represent the mean±SD of 3 independent experiments performed in duplicate. n.s. denotes not statistically significant according to a paired student t test. *P<0.05, **P<0.01, ***P<0.001 according to a paired student t test (versus d2).

[0100] FIG. 2. Emricasan is a more effective inhibitor of CSF-1-induced monocyte differentiation compared to Q-VD-OPh. Human blood monocytes were exposed for 2 days to 100 ng/mL CSF-1 alone or in combination with indicated concentrations of Emricasan or Q-VD-OPh (qVD) which were added 30 min before CSF-1 treatment. Macrophagic differentiation of monocytes from 3 different healthy donors was examined by 3-color flow cytometric analysis. The results are expressed as percentage of CD71/CD163 or CD16/CD163 double positive cells and represent the mean±SD of 3 independent experiments performed in duplicate. n.s. denotes not statistically significant according to a paired student t test. *P<0.05, **P<0.01, ***P<0.001 according to a paired student t test (versus d2).

[0101] FIG. 3. Both Q-VD-Oph and Emricasan hamper in the same way apoptosis in untreated monocytes. Human blood monocytes were exposed for 1 day to indicated concentrations of Q-VD-OPh (qVD) or Emricasan. Measures of caspase-3 (DEVD-AMC) and caspase-8 (IETD-AMC) activities. The results are expressed as A.U./mg/min and represent the mean the mean±SD of 3 independent experiments performed in triplicate.

[0102] FIG. 4. Emricasan is an effective inhibitor of caspase-8 and caspase-3. The ability of Emricasan or Q-VD-OPh (qVD) to inhibit caspases activities were assessed using active recombinant proteins of caspase-8 and caspase-3. (A) Measures of caspase-8 activity (IETD-AMC). IETD-CHO treatment is used as positive control in the in vitro assay. The results are expressed as A.U./min and represent the mean of 3 independent experiments realized in duplicate (B) Measures of caspase-3 activity (DEVD-AMC). DEVD-CHO is used as positive control in the in vitro assay. The results are expressed as A.U./min and represent the mean of 3 independent experiments realized in duplicate.

[0103] FIG. 5. Emricasan is a potent inhibitor of CSF-1-induced caspases activation. Human blood monocytes were exposed for 2 days or 3 days to 100 ng/mL CSF-1 alone or in combination with indicated concentrations of Emricasan or Q-VD-OPh (qVD) which were added 30 min before CSF-1 treatment. Caspases activities from 3 different healthy donors was examined by flow cytometry analysis. The results are expressed as percentage of IETD or DEVD positive cells and represent the mean±SD of 3 independent experiments performed in duplicate. n.s. denotes not statistically significant according to a paired student t test. *P<0.05, **P<0.01, ***P<0.001 according to a paired student t test (versus d2). Asterisks indicate cleavage fragments. Each panel is representative of at least 3 independent experiments.

[0104] FIG. 6. Emricasan blocks the M2-polarization of CSF-1-derived macrophages. Human monocytes were differentiated during 7 days with 100 ng/mL CSF-1. Emricasan was added 60h before the end of CSF-1 treatment. (A) Functional assay of CSF-1-derived macrophages exposed for 7 days to 100 ng/mL CSF- with or without Emricasan. The results are expressed as MFI and represent the mean of 3 independent experiments performed in duplicate. **P<0.01 according to a paired student t test (B) Macrophage polarization was evaluated by 3-color flow cytometric analysis. The percentage indicates cells that express both CD206/CD200R or CD163/CD200R. The results represent the mean±SD of 3 independent experiments performed in duplicate. **P<0.01, ***P<0.001 according to a paired student t test. Human monocytes were differentiated during 5 days with 100 ng/mL CSF-1 and then polarized into M2-macrophages (IL-4) for 2 days. Emricasan was added 16 h before the IL-4 treatment. The results are expressed as percentage of CD200R/CD206 or CD200R/CD163 double positive cells and represent the mean±SD of 3 independent experiments performed in duplicate.

[0105] FIG. 7. Pharmacologic and genetic inhibition of caspase-8 prevents bleomycin-induced pulmonary fibrosis. (A) Quantification of Sirius Red labeling intensity. Results are expressed as fold change in Sirius Red staining in treated compared to control mice (bleomycin was compared to untreated, bleomycin+Emricsan to Emricasan alone). Each dot or square is an individual mouse. *P<0.05 according to Mann-Whitney test. (B) Quantification of airspace number/mm2 of parenchymal tissue. Results expressed as fold change in treated compared to control mice as in B. *P<0.05 according to Mann-Whitney test. (C) Quantification of Sirius Red labeling intensity. Results expressed as fold change in treated compared to control mice, C8 KO+bleomycin compared to C8 KO as in B. *P<0.05 according to Mann-Whitney test. (D) Quantification of airspace number/mm2 of parenchymal tissue. Results are expressed as fold change in treated compared to untreated wild-type mice, as in panel E. *P<0.05 according to Mann-Whitney test. (E) Cytokines were measured in broncho-alveolar lavage fluid collected from bleomycin-treated wild-type (wt) and LysM-Cre/Caspase-8 flox/flox (C8 KO) mice treated with bleomycin. Results are expressed as fold-changes compared to untreated mice. *P<0.05, **P<0.01 according to Mann-Whitney test.

[0106] FIG. 8: Emricasan dampens the M2-polarization of CSF-1-derived macrophages. Human monocytes were differentiated during 5 days with 50 ng/mL CSF-1 and then polarized into MO-macrophages (CSF-1) or M2-macrophages (IL-4) for 24 (mRNA) or 48 hours. Emricasan (3 μM) was added 16h before the polarization. The expression of the indicated mRNA is analyzed by qPCR (mean ±SEM of 6 independent experiments). *P<0.05, **P<0.01 according to a paired student t test (versus M2-macrophages).

EXAMPLE

[0107] Material and Methods

[0108] Reagents and Antibodies

[0109] Human CSF-1 was purchased from Miltenyi (130-096-493). Emricasan (IDN-6556) was purchased from Euromedex (S7775-5mg). Q-VD-OPh was from Clinisciences (A1901-5 mg). Caspase-8, Caspase-3, Caspase-7 and HSP60 antibodies were purchased from Cell Signaling Technology (catalog numbers were 9746, 9662, 9492 and 12165 respectively). Mouse caspase-8 was from R&D Systems (AF705). HRP-conjugated rabbit anti-goat was purchased from Dako (P0449) and HRP-conjugated goat anti-rabbit was from Cell Signaling (5127). Active recombinant caspase-8 (ALX-201-062) and −3 (ALX-201-059) were from Enzo life sciences.

[0110] Human Monocyte Culture and Differentiation

[0111] Human peripheral volunteers were obtained from healthy donors with informed consent following the Declaration of Helsinki according to recommendations of an independent scientific review board. The project has been validated by The Etablissement Francais du Sang, the French national agency for blood collection (protocol N°ALM/PLER/FO/001). Blood samples were collected using ethylene diamine tetraacetic acid-containing tubes. Mononucleated cells were first isolated using Ficoll Hypaque (Eurobio, CMSMSL0101). Then, we used the autoMACS® Pro Separator (Miltenyi, France) to perform cell enrichment. An initial positive selection, which included antibody targeting CD14, was used for monocyte enrichment (Miltenyi, 130-050-201). Purified monocytes from human were grown in RPMI 1640 medium with glutamax-I (Life Technologies, 61870044) supplemented with 10% (vol/vol) foetal bovine serum (Life Technologies). Macrophage differentiation was induced by adding into the culture medium 100 ng/mL CSF-1 and was visualized using standard optics (20x/0.35 Ph1) equipped with an AxioCam ERc camera (Zeiss, France). Phase images of the cultures were recorded with the Zen 2 software (Zeiss).

[0112] Flow Cytometry

[0113] To analyze the macrophagic differentiation of monocytes, the cells were washed with ice-cold phosphate buffered saline (PBS, Life Technologies, 14190169), incubated at 4° C. for 10 min in PBS/bovine serum albumin (BSA 0.5%, Dutscher, 871002) with anti-CD16, anti-CD71 and anti-CD163 or isotype controls (Miltenyi and BD Biosciences, catalog numbers were 130-113-396, 130-097-628 and 551374). Finally, the cells were washed and fixed in 2% paraformaldehyde (EMS, 15710). To perform macrophage polarization, purified monocytes were plated at 0.3×106 per mL in RPMI 1640 medium with glutamax-I supplemented with 10% (vol/vol) fetal bovine serum plus CSF-1 for 5+2 days to differentiate into M0 macrophages. 20 ng/mL IL-4 (Miltenyi, 130-094-117) was added after 5 days of differentiation for two days to polarize into M2-macrophages. To analyze the macrophage polarization, cells were detached using PBS/EDTA/BSA, washed with PBS, and incubated at 4° C. for 10 min in PBS/bovine serum albumin with anti-CD200R (Biolegend, 329308), anti-CD206 (Miltenyi, 130-100-034) and anti-CD163 (Miltenyi, 130-097-628) or isotype controls. Finally, the cells were washed and fixed in 2% paraformaldehyde (EMS, 15710). Fluorescence was measured with a MACSQuant® Analyzer (Miltenyi, Paris, France). To analyze the cell death, cells were washed with ice-cold PBS and incubated at 4° C. for 15 min in a specific buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2) with AnnexinV-FITC (Miltenyi, 130-097-928) and DAPI (Sigma-Aldrich, D9542). Fluorescence was measured with a MACSQuant® Analyzer (Miltenyi, Paris, France). To analyze the ability of macrophages to phagocyte bacteria, we used Vybrant® Phagocytosis Assay Kit according manufacture's instruction (ThermoFisher, V-6694). Briefly, macrophages were detached and incubated with fluorescein-labeled E. coli (K-12 strain) for 30 min. Next, cells were washed twice with PBS and resuspended in PBS. Fluorescence, that indicate the internalization of particles, was measured with a MACSQuant® Analyzers (Miltenyi, France). Trypan blue solution was used to quench the fluorescence from particles that were not internalized. To detect caspase activity, we used FITC-DEVD-FMK or FITC-IETD-FMK according to the manufacturer's instruction (Promocell, green caspase-3 or caspase-8 staining kits, PK-CA577-K183 or PK-CA57-188).

[0114] Caspase Activity Measurement Assay

[0115] After stimulation, cells were lysed for 30 min at 4° C. in lysis buffer (50 mM HEPES pH 8, 150 mM NaCl, 20 mM EDTA, 1 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin and 0.2% Triton X-100) and lysates were cleared at 16 000 g for 15 min at 4° C. Each assay (in triplicate) was performed with 10 μg of protein prepared from control or stimulated cells. Briefly, cellular extracts were then incubated in a 96-well plate with 0.2 mM of DEVD-AMC (Caspase-3) or IETD-AMC (Caspase-8) as substrates for various times at 37° C. Caspase activity was measured either following emission at 460 nm (excitation at 390 nm) in the presence or not of 10 μM of DEVD-CHO or IETD-CHO. Enzyme activities were expressed in arbitrary units (A.U.) per min and per mg of proteins. The same protocol was used with 0.25 units of active recombinant caspase-8 (Enzo, ALX-201-062) or -3 (Enzo, ALX-201-059) in each triplicate.

[0116] Immunoblot Assays

[0117] Cells were lysed for 30 min at 4° C. in lysis buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 20 mM EDTA, PhosphoSTOP (Sigma, 04906837001), complete protease inhibitor mixture (Sigma, 11836153001), 1% Triton X-100 (Sigma, T9284)]. Lysates were centrifuged at 20,000 g (15 min, 4° C.) and supernatants were supplemented with concentrated loading buffer (4× Laemmli buffer). Fifty micrograms of proteins were separated and transferred following standard protocols before analysis with the chemiluminescence detection kit (GE Healthcare, RPN2105).

[0118] Whole-Transcriptome RNA-seq

[0119] The RNA integrity (RNA Integrity Score≥7.0) was checked on the Agilent 2100 Bioanalyzer (Agilent) and quantity was determined using Qubit (Invitrogen). SureSelect Automated Strand Specific RNA Library Preparation Kit was used according to manufacturer's instructions with the Bravo Platform. Briefly, 50 to 200 ng of total RNA sample was used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. The fragmented mRNA samples were subjected to cDNA synthesis and were further converted into double stranded DNA using the reagents supplied in the kit, and the resulting dsDNA was used for library preparation. The final libraries were bar-coded, purified, pooled together in equal concentrations and subjected to paired-end sequencing on Novaseq-6000 sequencer (Illumina) at Gustave Roussy.

[0120] RNA-Sequencing Analysis

[0121] Quantification. Quality of raw FastQ files was assessed with Fastqc v0.11.8 and Fastq-screen v0.13.0. Quality report was gathered with MultiQC v1.8. Abundance estimation was performed with Salmon v0.14.1 using following parameters: —libType A —validateMappings —numBootstraps 60. Salmon index was created using Human Gencode reference annotation release 33 and using following parameters —gencode —keepDuplicates. Differential analysis. Statistical analysis was performed using R 3.6.1. Transcript expression levels were aggregated in gene expression levels using tximport v1.14.0 Bioconductor package. At this step only protein coding genes were considered. We also decided to keep only high quality annotations therefore genes annotated as “automated annotation” in GENCODE were discarded. DESeq2 v1.26.0 method was used to identify differentially expressed genes between groups with an adjusted p-value threshold of 0.05.

[0122] Reverse-Transcription and Real-Time Polymerase Chain Reaction

[0123] RNA was prepared from 5×106 cells using the RNeasy Mini Kit according to manufacturer's protocol (Qiagen, 74104). Each cDNA sample was prepared using AMV RT and random primers (Promega, M510F and C1181). Real-time polymerase chain reaction (PCR) was performed using the SyBR Green detection protocol (Life Technologies, 4367659). Briefly, 5 ng of total cDNA, 500 nM (each) primers, and 5 μL SyBR Green mixture were used in a total volume of 10 μL. Detection of multiple endogenous controls (ACTB, L32 and UBIQUITIN) were used to normalize the results. Specific forward and reverse primers are accessible upon request.

[0124] Animal Models

[0125] C57/BL6 female mice (8 weeks-old) were purchased from Charles River Laboratories (L'arbresle, France). Caspase-8 flox/flox mice were kindly provided by Hedrick's laboratory (UCSD) (PMID:16148088) and crossed with LysMCre transgenic mice (PMID: 10621974). Animal genotyping was done by PCR using primers indicated in Table 1, and by immunoblotting.

[0126] Lung Fibrosis Model

[0127] Procedures were approved by our Institutional Ethical Committee (CEEA 26) and the French Ministry of Research (#9861). Animals were injected intraperitoneally with bleomycin sulfate (0.1 mg/g body weight) once a week during three weeks with or without subcutaneous injection of Emricasan (18 μg/g body weight) (MedChemtronica) twice a day. To quantify the extent of collagen fibers, left lungs were fixed in 4% formaldehyde, paraffin embedded, cut into 4 μm sections, stained with Sirius Red, scanned using a microscopy virtual slide system (Olympus VS120), and analyzed using ImageJ 1.50b software. To quantify airspace number, tissue sections 4-μm stained with Sirius Red were scanned using a NanoZoomer-SQ (Hamamatsu Corporation, Japan). Images of entire lung sections were recorded by means of NDP.view.2 software (Hamamatsu Corporation) and analyzed at ×20 magnification with a pixel size of 0.452 μm. To quantify fibrosis, we used a numerical software program that allows a fully automatic selection of airspaces (alveoli and ducts) from the entire lung sections, without the large bronchi and vessels. Fibrosis severity was indicated by the ratio between the number of airspaces and the total area of parenchymal tissue.

[0128] Macrophage Collection and Analysis

[0129] Right lungs were digested with the Lung Dissociation kit (Miltenyi Biotec, Somerville, Mass., USA) and filtered before eliminating erythrocytes with ACK to collect nucleated cells. These cells were washed with ice-cold PBS, incubated with Fc block (Murine TruStain FcX, Biolegend, London, UK, 1/50 dilution) for 15 min, incubated with antibodies (Table 1) for 20 minutes at 4° C., washed, and analyzed with a BD LSRFortessa X-20 flow cytometer and FlowJo software v. 10.0.00003. Interstitial macrophages were selected according to their larger size (FSC) and granularity (SSC) as CD45+, GR1−, CD11b high, SiglecF−, IAIE+, CD24− cells, alveolar macrophages as CD45+, GR1−, CD11b low, SiglecF high cells and inflammatory monocytes were selected as CD45+ positive, CD11b high, SiglecF−, IA-IE− cells.

[0130] Broncho-Alveolar Lavage Fluid

[0131] We collected the broncho-alveolar fluid (BALF) of sacrificed animals by cannulating their trachea and ligating their right lung before slowly delivering 300 μl PBS in the left lung and retrieving the liquid through the cannula, which was repeated twice. BALF was centrifuged at 600 g for 10 minutes at 4° C. before collecting the acellular fraction that was kept at −80° C., up to cytokine analysis. Interleukin-2 (IL-2), IL-5, IL-6, chemokine (C-X-C motif) ligand 1 (CXCL1 or KC), were quantified using Mouse Pro-Inflammatory Panel 1 V-Plex according to the manufacturer's guidelines (MSD), the chemiluminescence signal being measured on a Sector Imager 2400 (MSD). A Milliplex TGFβ1, Single Plex magnetic bead kit (Merck Millipore) and the Bio-Plex200 system (Bio-Rad) were used to measured TGFβ1.

[0132] Statistical Analysis

[0133] Statistical analysis was performed using a paired Student t test and significance was considered when P values were lower than 0.05. The results are expressed as the mean±SEM. For mouse experiment analyses, investigators were blinded. Data are presented as means±SE. Statistical significance was determined by Mann-Whitney test. All the tests were two-tailed.

[0134] Results

[0135] Low Concentrations of Emricasan Inhibit CSF-1-Induced Monocyte Differentiation

[0136] Caspase inhibition using pancaspase inhibitors such as z-VAD-fmk or Q-VD-OPh inhibits CSF-1-induced monocyte differentiation (Jacquel et al., Blood 2009, Sci Reports 2018). We investigated here the effect of increasing concentrations of Emricasan, a pancaspase inhibitor that has recently achieved phase 2 clinical trials in patients suffering liver failure, on human primary monocyte differentiation induced by CSF-1 (FIG. 1). Human primary monocytes treated with CSF-1 for 2 days exhibited a robust increase in the expression of CD71/CD163 and CD16/CD163 antigens, a hallmark of macrophagic differentiation, generating 98% and 92% of double positive cells, respectively, as assessed by flow cytometry (data not shown). Emricasan added at day 0 triggered a dose-dependent inhibition of macrophagic differentiation in the low micromolar range. Quantification of the Emricasan effect in three different donors confirmed a strong inhibitory effect of this pancaspase inhibitor at low micromolar concentrations (1-2 μM) (FIG. 1A).

[0137] Induction of differentiation by CSF-1 is known to inhibit the spontaneous apoptosis of monocytes that occurs rapidly in culture in the absence of this cytokine. We thus analyzed the effect of various concentrations of Emricasan on apoptosis induction in differentiating monocytes. CSF-1 reduced apoptotic cell rate three times as shown by annexin V staining at 48 h compared to untreated monocytes (FIG. 1B). Emricasan failed to induced significant loss of Annexin V staining at low concentrations, but slightly increased DAPI staining at higher concentrations (3 μM). In conclusion, Emricasan did not induce apoptosis in undifferentiated monocytes and low concentrations of Emricasan (up to 2 μM) were not toxic for primary human monocytes, indicating that Emricasan can be used beneficially in place of other pancaspase inhibitors.

[0138] We therefore investigated in comparison with Emricasan the inhibitory effect of Q-VD-OPh, a pancaspase inhibitor widely used in the literature to block apoptotic caspases, on CSF-1-induced monocyte differentiation. Monocytes treated with CSF-1 for 2 days in the absence of Q-VD-OPh, exhibited an increased expression of CD71/CD163 and CD16/CD163 antigens generating 97% and 78% of double positive cells, respectively, as assessed by flow cytometry (data not shown). Q-VD-OPh added at day 0 triggered a dose-dependent inhibition of macrophagic differentiation in the 75-125 μM range that was however weak compared to the effect of Emricasan (data not shown). When Q-VD-OPh was added twice, i.e at day 0 and day 1 a much robust inhibition of monocyte differentiation was achieved, that was however weaker than the one obtained with Emricasan used at a single and lower dose (data not shown). Quantification of the Q-VD-OPh effect on several different donors confirmed a significant inhibitory effect of this pancaspase inhibitor at 100-125 μM, but only when added twice (data not shown). We next directly analyzed on the same experiment the ability of Q-VD-OPh added twice (at days 0 and 1) and Emricasan added only one time to impair CSF-1-mediated monocyte differentiation (data not shown). As expected, monocytes treated with CSF-1 for 2 days exhibited a robust increase in the expression of CD71/CD163 and CD16/CD163 antigens, generating 95% and 74% of double positive cells, respectively, as assessed by flow cytometry. Emricasan in the 1.5-2.5 μM range efficiently inhibited monocyte differentiation while 2 successive treatments with100 μM Q-VD-OPh were necessary to achieve an identical inhibition, further demonstrating the superiority of Emricasan towards Q-VD-OPh. Quantification of these results on 3 different donors confirmed the higher potency of Emricasan versus Q-VD-OPh to inhibit CSF-1-induced monocyte differentiation into macrophages (FIG. 2). All together these data indicate that Emricasan is a highly potent inhibitor of caspases during differentiation of human monocytes into macrophages and can be used beneficially instead of the less active and specific compounds Q-VD-OPh or z-VAD-fmk.

[0139] Both Q-VD-OPh and Emricasan are Potent Inhibitors of Apoptosis in Untreated Monocytes

[0140] When cultured in the absence of CSF-1, human monocytes rapidly underwent apoptosis as assessed by Annexin V/DAPI staining (FIG. 1B). Human freshly isolated monocytes were left untreated or treated with different concentrations of Q-VD-OPh or Emricasan for 24 h. In the absence of pancaspase inhibitors, 51% of monocytes exhibited increased Annexin V staining at 24 h, indicative of apoptotic cell death induction. Concentrations of Q-VD-OPh as low as 5 μM were sufficient to abrogate Annexin V staining after 24 h in culture without CSF-1 indicating that Q-VD-OPh is much more efficient to block caspase activation induced during apoptosis than during CSF-1-mediated differentiation. A single concentration of Emricasan (2 μM) was sufficient to obtain the same effect (data not shown). We confirmed by Western Blot experiments that both inhibitors abrogated the cleavage of the zymogens of caspases 8, 3 and 7 in their active 17-20 kDa fragments (data not shown). Finally, we also verified that all the concentrations of Q-VD-OPh and Emricasan efficiently inhibited caspase 3 activity in untreated monocytes, using Ac-DEVD-AMC as substrate (FIG. 3). Therefore it appears that Q-VD-OPh is a potent inhibitor of apoptotic caspases but conversely to Emricasan, a weaker inhibitor of the activation of caspases that specifically occurred and are essential for proper monocyte differentiation. As a whole these findings show that Emricasan is more active on those caspase activities that trigger differentiation of monocytes.

[0141] Effect of Caspase Inhibitors on Recombinant Caspases-8 and -3

[0142] To investigate further the differential effect of Q-VD-OPh and Emricasan on caspase activities and monocyte differentiation, we performed dose-response curves for both inhibitors on recombinant caspase-8 and -3 activities in vitro. Caspase-8 was assessed using Ac-IETD-AMC as substrate. Q-VD-OPh abrogated caspase-8 activity at 50 μM, with an IC50 around 1 μM, whereas Emricasan fully inhibited caspase-8 activity at 0.2 μM and exhibited an IC50 of only 0.012 μM, that was in the range of Ac-IETD-CHO, a highly potent caspase-8 inhibitor (IC50=0.015 μM) (FIG. 4A). The same experiment was reproduced using recombinant caspase-3 and Ac-DEVD-AMC as substrate (FIG. 4B). Importantly, the dose-response curve for Q-VD-OPh and Emricasan inhibition of caspase-3 were perfectly stackable (maximal inhibition at 10 μM and IC50 in the 0.5 μM range), indicating that both inhibitors were equally efficient to inhibit recombinant caspase-3 in vitro. In conclusion, the better efficiency of Emricasan to inhibit CSF-1-induced human monocyte differentiation in vitro and ex vivo likely relies on its ability to abrogate caspase-8 activity which is crucial for this process.

[0143] Emricasan Efficiently Inhibits Caspase 8 Activity in Cellulo in Differentiating Monocytes

[0144] We have shown previously that the superiority of Emricasan compared with Q-VD-OPh relies on its better efficiency towards caspase-8 in vitro using a recombinant caspase. To assess caspase-8 activity in cellulo, human primary monocytes were incubated with or without CSF-1 in either the presence of different concentrations of Emricasan or a maximal concentration of Q-VD-OPh (100 μM, 2 times). After 2 days, caspase-8 activity was assessed by flow cytometry using Ac-IETD-FITC as substrate (data not shown). 88% of differentiated monocytes exhibited high caspase-8 staining, indicative of caspase-8 activation (data not shown). Q-VD-OPh added twice at 100 μM induced a strong inhibition of caspase-8 activity, whereas Emricasan abrogated caspase-8 activity at the single dose of 2 μM, in agreement with its effect on monocyte differentiation (FIGS. 1A and 1B and FIG. 2). Quantification of caspase-8 activity in several experiments confirmed a very strong inhibition of the percentage of cells expressing active caspase-8 (FIG. 5). A potent inhibition of caspase -3 activity in cellulo was observed in identical conditions (FIG. 5).

[0145] We also checked in parallel that the different caspase inhibitors blocked CSF-1-mediated monocyte differentiation (data not shown). Finally, we verified using western blot experiments the cleavage of caspases-3 and -7 in their differentiation-like characteristic fragments of 26 and 30 kDa in monocytes treated 2 days with CSF-1 (data not shown). Importantly, we established that Q-VD-OPh and Emricasan impaired the cleavage of effector caspases-3 and -7 at their specific differentiation cleavage site.

[0146] Emricasan Impedes Macrophage Polarization Ex Vivo

[0147] Although caspases are necessary for the differentiation of monocytes into macrophages induced by CSF-1, their role in the polarization of macrophages into the M1 or M2 phenotype has not been explored so far. To investigate a possible implication of caspases during these processes, primary human monocytes were firstly incubated 5 days with CSF-1 to induce macrophagic differentiation, and next treated with IL-4 during 2 days to induce polarization towards M2 phenotype. Emricasan dampened M2-like polarization of M0 macrophages induced by IL4 (FIGS. 6A and 6B) and at the same time induced M1-like markers.

[0148] Emricasan Impedes Monocyte-Derived Cell Changes Induced by IL-4

[0149] In IL4-polarized macrophages, Emricasan inhibited the generation of CD200R+/CD206+ and CD200R+/CD163+ double-positive cells (FIG. 6B lower panel). To confirm the inhibitory effect of Emricasan on IL4-mediated M2 polarization, we performed RNAseq analysis on macrophages from three different donors. We found that IL-4 both induces anti-inflammatory and represses pro-inflammatory markers, an effect that was counteracted by Emricasan (data not shown). This mirror effect was confirmed by analyzing the mRNA level of CD200R and CCL18, two well-known anti-inflammatory molecules, and CXCL8, a proinflammatory one. Indeed, CD200R and CCL18 expressions were increased by IL-4, an effect counteracted by Emricasan, while CXCL8 level was diminished by IL-4 but upregulated when IL-4 was combined with Emricasan (FIG. 8). In conclusion, caspase inhibition by Emricasan prevented the up-regulation of anti-inflammatory actors to the benefit of pro-inflammatory molecules, thus orienting polarization of human macrophages towards a pro-inflammatory phenotype.

[0150] Caspase-8 Inhibition Prevents Bleomycin-Induced Lung Fibrosis in Mice

[0151] Emricasan has been shown to improve liver functions in patients suffering liver diseases in several recent studies (Frenette C T, Clin Gastroenterol Hepatol. 2019 Mar; 17(4):774-783 ; Barreyro F J, Liver Int. 2015 Mar; 35(3):953-66 ; Baskin-Bey E S, Am J Transplant. 2007 Jan; 7(1):218-25). We investigate whether this could be also the case in bleomycin-induced pulmonary fibrosis in mice, a disease associated with M2-macrophage infiltration. Weekly intraperitoneal injection of bleomycin sulfate (0.1 mg/g body weight) to 2-month old mice generates a lung fibrosis that, after three weeks, can be visualized by Sirius Red staining of collagen fibers (data not shown) and quantified using ImageJ 1.50b software (FIG. 7A). Complete obliteration of alveoli, which is a key feature of pulmonary fibrosis, provokes a decrease in airspace number that can also be quantified (FIG. 7B). Subcutaneous injection of Emricasan twice a day (18 μg/g body weight) for three weeks dramatically decreases lung fibrosis intensity (data not shown), as verified by quantifying Sirius Red staining intensity (FIG. 7A) and air space (FIG. 7B). To further explore the role of caspases in bleomycin-induced lung fibrosis, we generated mice with LysM promoter guided, Cre recombinase-induced deletion of caspase-8 (Caspase-8-/−). Genotyping of the models validated both the presence of the foxed alleles in mouse tail DNA and the appropriate deletion of targeted alleles in macrophages (data not shown). LysM-Cre driven caspase-8 gene deletion protected the animals from bleomycin induced lung fibrosis (data not shown), as indicated by a decreased network of collagen fibers (FIG. 7C) and a lower restriction of airspace (FIG. 7D). Analysis of cytokines in the broncho-alveolar fluid (BALF) collected from bleomycin treated animals revealed a decreased level of TGFβ1, IL-2, IL-5, IL-6 and KC in the BALF of Caspase-8-/− mice (FIG. 7E).

[0152] Conclusion:

[0153] In conclusion, we first established that compared to Q-VD-OPh, a classically and widely used pancaspase inhibitor, Emricasan durably impairs CSF-1-induced monocyte differentiation and this at very low micromolar range and therefore represents an excellent alternative to other pancaspase inhibitors. Moreover, we confirmed in vitro using recombinant caspases the much more greater efficiency of Emricasan on caspase-8. By contrast, we found no difference in the ability of Q-VD-OPh and Emricasan to inhibit caspase-3 activity in vitro and to impair apoptosis of human monocytes ex vivo suggesting an equivalent effect of both inhibitors on caspase-3. More precisely, we found that Emricasan was 100 times more potent on caspase-8 activity and monocyte differentiation than Q-VD-OPh. These original results demonstrate that Emricasan can be used to block the specific activation of caspases observed during CSF-1-mediated differentiation of monocytes into macrophages. Indeed, in response to CSF-1, a caspases activation cascade is initiated by the cleavage and the activation of caspase-8 in an original multimolecular complex composed of FADD, FLIP, RIP1 and caspase-8. Importantly, and that's what makes it so original, there is no cell death receptor within this platform. Caspase-8 activation secondly triggers the cleavage and activation of caspase-3 that ultimately cleaves several protein substrates, among which some such as NPM1 (nucleophosmin 1) are thought to play a role in the differentiation process. Interestingly, the cleavage site of effector caspases, i.e. caspase-3 and caspase-7, during monocyte differentiation are completely different from the one are cleaved when monocytes underwent spontaneous apoptosis in the absence of CSF-1 (data not shown). This distinct mode of caspases cleavage, and accordingly their specific activation, may explain the differences observed in the sensitivity to various caspase inhibitors observed during differentiation of monocytes. Once differentiated, macrophages play an important role in tissue development, inflammation, anti-pathogenic defense, homeostasis and cancer and their major functions are phagocytosis, antigen presenting and cytokine production. In activated immune responses, macrophages are a heterogenous population, exerting a combination of pro-inflammatory (M1-macrophages) and anti-inflammatory (M2-macrophages) functions. Deciphering the process of macrophage polarization, recruitment, and functions may provide insights for the development of new therapies to manipulate the balance of M1/M2 phenotype, number, and distribution of macrophage, in order to enhance anti-microbial defense or dampen detrimental inflammation. In this study, we demonstrated that caspases targeting using Emricasan, a clinically available pan-caspase inhibitor, may be a promising approach to evaluate the ability of Emricasan to modify the M2 polarization of CSF-1-induced macrophages.

REFERENCES

[0154] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.