Patent Application
20220087950
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 24, 2021, is named MTV-19201_SL.txt and is 9,328 bytes in size.
Macrophages play an essential role in development, tissue homeostasis and repair, and immunity. Most macrophages exhibit multi-dimensional spectrum of phenotypes in response to various physiological and pathological signals. Because of their critical function in maintaining tissue homeostasis and repair, dysregulation of macrophage polarization has been implicated in contributing to many human diseases including cancer, fibrosis, obesity, diabetes, and infectious, cardiovascular, inflammatory and neurodegenerative diseases. Accordingly, there is a great need to identify modulators of macrophage activation for disease intervention.
In one aspect, described herein is a method of identifying a modulator of macrophage activation. The method comprises contacting a primary macrophage cell with a candidate agent; monitoring or photographing the morphology of the cell contacted with the candidate agent; and optionally comparing the cell's morphology in the presence of the candidate agent with the cell's morphology in the absence of the candidate agent; wherein a change in morphology in the presence of the candidate agent is indicative of modulation of macrophage activation. Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the primary macrophage cell is a bone marrow-derived macrophage or a monocyte-derived macrophage. In some embodiments, the morphology of the cell is monitored or photographed by a microscope, such as a fluorescence microscope. In some embodiments, the morphology of the cell is monitored or photographed by Opera Phenix high content screening system or CellProfiler. In some embodiments, the morphology of the cell is changed from elongated shape to round shape. In some embodiments, the modulator activates a M1-like macrophage, deactivates a M2-like macrophage, changes a tumor-associated macrophage (TAM) to M1-like macrophage, changes a M2-like macrophage to a M1-like macrophage, changes a M-CSF macrophage to a M1-like macrophage, changes a GM-CSF macrophage to a M1-like macrophage, changes a primary macrophage to a M1-like macrophage, induces LPS, IFNγ or TNFα, or activates a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin. In some embodiments, the modulator is a M1-activating compound. In some embodiments, the modulator is cytochalasin-B, fenbendazole, parbendazole, methiazole, alprostadil, FTY720, penfluridol, taxol, smer-3, cantharidin, SCH79797, mitoxantrone, niclosamide, MS275, HMN-214, DPI, thiostrepton, evodiamine, cucurbitacin-I, NVP 231, Chlorhexidine, Diphenyleneiodonium, LE135, Fluvoxamine, Mocetinostat, Pimozide, NP-010176, Celastrol, FTY720, WP1130, Prulifloxacin, dihydrocelastryl diacetate, or Quinolinium. In some embodiments, the M1-like macrophage mediates a pro-inflammatory response, an anti-microbial response, and/or an anti-tumor response. In some embodiments, the modulator treats cancer, fibrosis, and/or an infectious disease. In some embodiments, the cancer is hematological malignancy, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, or superficial spreading melanoma. In some embodiments, the infectious disease is a viral infection, or a bacterial infection. The infection may be associated with COVID-19 (SARS-CoV-2), SARS-CoV, MERS-CoV, Ebola virus, influenza, cytomegalovirus, variola and group A streptococcus, or sepsis.
In some embodiments, the morphology of the cell is changed from round shape to elongated shape. In some embodiments, the modulator activates a M2-like macrophage, deactivates a M1-like macrophage, changes a M1-like macrophage to a M2-like macrophage, changes a M-CSF macrophage to a M2-like macrophage, changes a GM-CSF macrophage to a M2-like macrophage, changes a primary macrophage to a M2-like macrophage, modulator induces a M2-activating stimuli selected from IL4, IL13 and IL10, or inhibits a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin. In some embodiments, the modulator is a M2-activating compound. In some embodiments, the modulator is Bostunib, Su11274, Alsterpaullone, Alrestatin, Bisantrene, triptolide, lovastatin, QS 11, Regorafenib, Sorafenib, MLN2238, GW-843682X, KW 2449, Axitinib, JTE 013, Purmorphamine, Arcyriaflavin A, Dasatinib, NVP-LDE225, 1-Naphthyl PP1, Selamectin, MGCD-265, podofilox, colchicine, or vinblastine sulfate. In some embodiments, the M2-like macrophage mediates an anti-inflammatory or a tissue repair response. In some embodiments, the modulator treats an inflammatory disease, a metabolic disease, an autoimmune disease, or a neurodegenerative disease. In some embodiments, the inflammatory disease, the metabolic disease, or the autoimmune disease is diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), hepatic steatosis, non-alcoholic steatohepatitis, cirrhosis, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), cardiovascular disease, remote tissue injury after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and resultant glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, Chronic open-angle glaucoma, acute closed angle glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, Anti-Neutrophilic Cytoplasmic Autoantibody vasculitis, Purtscher retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, hyperacute rejection, hemodialysis, chronic occlusive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barre syndrome, Myasthenia Gravis, Bullous Pemphigoid, or myositis. In some embodiments, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barre{acute over ( )} syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, or Huntington's disease.
In one aspect, described herein is a method of treating cancer, fibrosis, or an infectious disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator changes the morphology of a macrophage cell from elongated shape to round shape. Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the modulator activates a M1-like macrophage, deactivates a M2-like macrophage, changes a tumor-associated macrophage (TAM) to M1-like macrophage, changes a M2-like macrophage to a M1-like macrophage, changes a M-CSF macrophage to a M1-like macrophage, changes a GM-CSF macrophage to a M1-like macrophage, changes a primary macrophage to a M1-like macrophage, induces a M1-activating stimuli selected from LPS, IFNγ and TNFα, or activates a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin. In some embodiments, the modulator is a M1-activating compound. In some embodiments, the modulator is cytochalasin-B, fenbendazole, parbendazole, methiazole, alprostadil, FTY720, penfluridol, taxol, smer-3, cantharidin, SCH79797, mitoxantrone, niclosamide, MS275, HMN-214, DPI, thiostrepton, evodiamine, cucurbitacin-I, NVP 231, Chlorhexidine, Diphenyleneiodonium, LE135, Fluvoxamine, Mocetinostat, Pimozide, NP-010176, Celastrol, FTY720, WP1130, Prulifloxacin, dihydrocelastryl diacetate, or Quinolinium. In some embodiments, the M1-like macrophage mediates a pro-inflammatory response, an anti-microbial response, and/or an anti-tumor response. In some embodiments, the cancer is hematological malignancy, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, or superficial spreading melanoma. In some embodiments, the method further comprises administering to the subject an effective amount of a second cancer therapy. In some embodiments, the second cancer therapy comprises cancer immunotherapy. In some embodiments, the cancer immunotherapy comprises administering an immune checkpoint inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to an immune checkpoint protein. The immune checkpoint protein may be CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAGS, TIM-3 or VISTA. The immune checkpoint inhibitor may be atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, BGB-A317, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 or STI-A1010. In some embodiments, the second cancer therapy comprises the administration of a chemotherapy agent, such as rituxumab, thiotepa, cyclosphosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, dynemicin, clodronate, esperamicin; neocarzinostatin chromophore, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triaziquone; 2,2′,2″-trichlorotriethylamine, trichothecene, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, oxaliplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, RFS 2000, difluoromethylomithine, retinoic acid or capecitabine. In some embodiments, the infectious disease is a viral infection, or a bacterial infection. In some embodiments, the infection is associated with COVID-19 (SARS-CoV-2), SARS-CoV, MERS-CoV, Ebola virus, influenza, cytomegalovirus, variola and group A streptococcus, or sepsis.
In one aspect, described herein is a method of treating an inflammatory disease, a metabolic disease, an autoimmune disease, or a neurodegenerative disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator changes the morphology of a macrophage cell from round shape to elongated shape. Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the modulator activates a M2-like macrophage, deactivates a M1-like macrophage, changes a M1-like macrophage to a M2-like macrophage, changes a M-CSF macrophage to a M2-like macrophage, changes a GM-CSF macrophage to a M2-like macrophage, changes a primary macrophage to a M2-like macrophage, induces a M2-activating stimuli selected from IL4, IL13 and IL10, or inhibits a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin. In some embodiments, the modulator is a M2-activating compound. In some embodiments, the modulator is Bostunib, Su11274, Alsterpaullone, Alrestatin, Bisantrene, triptolide, lovastatin, QS 11, Regorafenib, Sorafenib, MLN2238, GW-843682X, KW 2449, Axitinib, JTE 013, Purmorphamine, Arcyriaflavin A, Dasatinib, NVP-LDE225, 1-Naphthyl PP1, Selamectin, MGCD-265, podofilox, colchicine, or vinblastine sulfate. In some embodiments, the M2-like macrophage mediates an anti-inflammatory or a tissue repair response. In some embodiments, the inflammatory disease, the metabolic disease, or the autoimmune disease is diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), hepatic steatosis, non-alcoholic steatohepatitis, cirrhosis, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), cardiovascular disease, remote tissue injury after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and resultant glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, Chronic open-angle glaucoma, acute closed angle glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, Anti-Neutrophilic Cytoplasmic Autoantibody vasculitis, Purtscher retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, hyperacute rejection, hemodialysis, chronic occlusive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barre syndrome, Myasthenia Gravis, Bullous Pemphigoid, or myositis. In some embodiments, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barre{acute over ( )} syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, or Huntington's disease.
In one aspect, described herein is a method of treating cancer, fibrosis, or an infectious disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator activates a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin. Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the modulator is cytochalasin-B, fenbendazole, parbendazole, methiazole, alprostadil, FTY720, penfluridol, taxol, smer-3, cantharidin, SCH79797, mitoxantrone, niclosamide, MS275, HMN-214, DPI, thiostrepton, evodiamine, cucurbitacin-I, NVP 231, Chlorhexidine, Diphenyleneiodonium, LE135, Fluvoxamine, Mocetinostat, Pimozide, NP-010176, Celastrol, FTY720, WP1130, Prulifloxacin, dihydrocelastryl diacetate, or Quinolinium. In some embodiments, the cancer is hematological malignancy, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, or superficial spreading melanoma. In some embodiments, the method further comprises administering to the subject an effective amount of a second cancer therapy. In some embodiments, the second cancer therapy is cancer immunotherapy, such as an immune checkpoint inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAGS, TIM-3 or VISTA. In some embodiments, the immune checkpoint inhibitor is atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, BGB-A317, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 or STI-A1010. In some embodiments, the second cancer therapy is a chemotherapy agent, such as rituxumab, thiotepa, cyclosphosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, dynemicin, clodronate, esperamicin; neocarzinostatin chromophore, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triaziquone; 2,2′,2″-trichlorotriethylamine, trichothecene, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, oxaliplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, RFS 2000, difluoromethylomithine, retinoic acid or capecitabine. In some embodiments, the infectious disease is a viral infection, or a bacterial infection. In some embodiments, the infection is associated with COVID-19 (SARS-CoV-2), SARS-CoV, MERS-CoV, Ebola virus, influenza, cytomegalovirus, variola and group A streptococcus, or sepsis.
In one aspect, described herein is a method of treating an inflammatory disease, a metabolic disease, an autoimmune disease, or a neurodegenerative disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator inhibits a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin. Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the modulator is Bostunib, Su11274, Alsterpaullone, Alrestatin, Bisantrene, triptolide, lovastatin, QS 11, Regorafenib, Sorafenib, MLN2238, GW-843682X, KW 2449, Axitinib, JTE 013, Purmorphamine, Arcyriaflavin A, Dasatinib, NVP-LDE225, 1-Naphthyl PP1, Selamectin, MGCD-265, podofilox, colchicine, or vinblastine sulfate. In some embodiments, the inflammatory disease, the metabolic disease, or the autoimmune disease is diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), hepatic steatosis, non-alcoholic steatohepatitis, cirrhosis, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), cardiovascular disease, remote tissue injury after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and resultant glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, Chronic open-angle glaucoma, acute closed angle glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, Anti-Neutrophilic Cytoplasmic Autoantibody vasculitis, Purtscher retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, hyperacute rejection, hemodialysis, chronic occlusive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barre syndrome, Myasthenia Gravis, Bullous Pemphigoid, or myositis. In some embodiments, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barre{acute over ( )} syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, or Huntington's disease.
In one aspect, described herein is a method of treating an inflammatory disease, a metabolic disease, an autoimmune disease, or a neurodegenerative disease. The method comprises administering to a subject in need thereof an effective amount of diphenyleneiodonium (DPI). Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the inflammatory disease, the metabolic disease, or the autoimmune disease is diabetes, obesity, Non-alcoholic fatty liver disease (NAFLD), hepatic steatosis, non-alcoholic steatohepatitis, cirrhosis, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), cardiovascular disease, remote tissue injury after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and resultant glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, Chronic open-angle glaucoma, acute closed angle glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, Anti-Neutrophilic Cytoplasmic Autoantibody vasculitis, Purtscher retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, hyperacute rejection, hemodialysis, chronic occlusive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barre syndrome, Myasthenia Gravis, Bullous Pemphigoid, or myositis. In some embodiments, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barre{acute over ( )} syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, or Huntington's disease.
Macrophages are remarkably plastic and in response to different local stimuli can polarize toward multi-dimensional spectrum of phenotypes, including the pro-inflammatory M1-like and the anti-inflammatory M2-like states. Using a high throughput phenotypic screen, ˜300 compounds that potently activated primary human macrophages toward either pro-inflammatory (M1-like) or anti-inflammatory (M2-like) state were identified from a library of ˜4000 FDA-approved drugs, bioactive compounds and natural products. Among the hits, ˜30 were capable of reprogramming M1-like macrophages toward M2-like state and another ˜20 were capable of reprogramming M2-like macrophages toward M1-like state. Transcriptional analysis of 34 non-redundant hits on macrophage reprogramming by RNA-seq identified shared pathways through which the selected hits modulate macrophage activation, as well as new unique targets and pathways by which individual compound stimulates macrophage activation. One M1-activating compound, thiostrepton, was further shown to reprogram tumor-associated macrophages toward M1-like state in mice and exhibit potent anti-tumor activity either alone or in combination with an antibody therapeutic. Described herein are new compounds, targets and pathways involved in macrophage activation. The methods described herein provide a valuable resource not only for studying the macrophage biology but also for developing novel therapeutics or repositioning known drugs for treating diseases through modulating macrophage activation.
Macrophages are a key class of phagocytic cells that readily engulf and degrade dying/dead cells and invading bacteria and viruses. As such, macrophages play an essential role in development, tissue homeostasis and repair, and immunity. Consistently, macrophages are generated during early ontogeny and throughout the adult life. In mammals, the first wave of macrophages is generated from the yolk sac and gives rise to macrophages in the central nervous system, i.e., microglia, for example. The second wave of macrophages is generated from fetal liver and give rise to alveolar macrophages in the lung and Kupffer cells in the liver among others. After birth, macrophages are generated from the bone marrow where hematopoietic stem cells give rise to monocytes, which differentiate into tissue resident macrophages upon migration from blood into specific tissues.
A remarkable feature of macrophages is their plasticity: the ability to respond to local stimuli to acquire different phenotypes and functions so as to respond to changing physiological needs. Macrophages from different tissues exhibit different phenotypes and functions. For example, Kupffer cells in the liver function in the degradation of toxic and waste products as well as in the maintenance of metabolic homeostasis, whereas alveolar macrophages in the lung function in removal of dust, microorganisms, and surfactants from the respiratory surfaces despite their common origin from fetal liver. Within the same tissue, macrophages are heterogeneous and can change phenotypes and functions in response to changing local tissue environment. For example, macrophages can eliminate antibody-bound tumor cells through Fc receptor-mediated phagocytosis (antibody-dependent cellular phagocytosis or ADCP). However, once adapted to the tumor microenvironment, the tumor-associated macrophages (TAM) suppress anti-tumor immune responses and promote tumor growth and metastasis.
Macrophage plasticity underlies their ability to be activated toward a spectrum of phenotypes and acquire diverse functions. One extreme is the classically activated pro-inflammatory M1 macrophages and the other extreme is the alternatively activated anti-inflammatory M2 macrophages. By expressing inflammatory cytokines, such as IFNγ and TNFα, and reactive oxygen species, M1 macrophages mediate anti-microbial and anti-tumor responses, but can also cause inflammation and tissue damage if hyper-activated. In contrast, by expressing anti-inflammatory cytokines, such as IL-10, TGFβ and arginase, M2 macrophages mediate tissue repair, but can also mediate fibrosis if dysregulated. While M1 and M2 serves to define the opposite activating states of macrophages in simplistic manner, most macrophages exhibit multi-dimensional spectrum of phenotypes in response to various physiological and pathological signals. By transcriptional profiling of human monocyte-derived macrophages (hMDMs) in response to 29 different stimuli, such as pro- and anti-inflammatory cytokines, 49 gene expression modules that are associated with macrophage activation were identified. Many aspects of macrophage activation/plasticity remain poorly defined. In particular, how small molecules modulate macrophage activation remains to be elucidated.
Because of their critical function in maintaining tissue homeostasis and repair, dysregulation of macrophage polarization has been implicated in contributing to many human diseases including cancer, fibrosis, obesity, diabetes, and infectious, cardiovascular, inflammatory and neurodegenerative diseases. For example, TAMs are one of the most abundant immune cells present in solid tumors. Clinical and experimental studies have shown that TAMs produce various membranous and soluble factors that enhance tumor cell growth and invasion as well as suppress anti-tumor immune responses to allow cancer cells to escape immune surveillance. TAMs are derived from circulating monocytes in the tumor microenvironment, which progressively skews macrophages into the immunosuppressive state, phenotypically resembling M2-activated macrophages. Reprogramming M2-like TAMs toward M1-like macrophages is associated with expression of a strong anti-tumor activity. In a remarkable synergy, cyclophosphamide-activated macrophages efficiently eliminate leukemia cells in refractory bone marrow microenvironment in combination with monoclonal antibody therapeutics. Repolarizing TAMs toward a pro-inflammatory, anti-tumorigenic M1-like state proves an efficient approach to cancer immunotherapy either alone or in combination with antibody therapeutics. More broadly, as dysregulation of macrophage activation has emerged as a key determinant in many disease development and progression, modulation of macrophage activation could be a fruitful approach for disease intervention.
Described herein is a high throughput phenotypic screen for small molecules that activate primary human macrophages. By screening a library of 4126 compounds which include FDA-approved drugs, bioactive compounds and natural products, ˜300 potently activated M-CSF cultured macrophages toward pro-inflammatory M1-like or anti-inflammatory M2-like state (or spectrum) were identified. Among the hits, ˜30 were capable of reprogramming M2-like macrophages induced by IL4/IL13 toward pro-inflammatory M1-like macrophages and another ˜20 were capable of reprogramming M1-like macrophages induced by IFNγ/TNFα toward anti-inflammatory M2-like macrophages. By analyzing the effects of the 34 selected hits on macrophage reprogramming through RNA-seq, we identified new cellular pathways that mediate macrophage activation (or reprogramming). M1-activating compounds thiostrepton and cucurbitacin I were further shown to reprogram TAMs toward M1-like macrophages in mice and exhibit potent anti-tumor activity either alone or in combination with monoclonal antibody therapeutics. The examples herein reveal a remarkable plasticity of macrophage polarization and provides a valuable resource not only for studying the macrophage biology but also for developing novel therapeutics or repositioning known drugs for treating diseases through macrophage reprogramming. Furthermore, the phenotypic screen can be extended to much larger compound libraries and in combination with transcriptional profiling is a powerful approach to elucidate the mechanism of action of small molecule compounds in macrophage polarization for precision disease intervention.
The high throughput phenotypic screen described herein is based on macrophage cell shape changes in response to compounds. Cell shape change is a valid phenotypic profiling of macrophage activation based on the following considerations. First, cell shape changes are mediated by changes in cytoskeleton dynamics and are known to associate with different states of cell function in general. More specifically, both mouse and human macrophages exhibit dramatically different cell shapes following activation into different phenotypes in vitro: an elongated shape for M2-like macrophages and round shape for M1-like macrophages. Consistently, we showed that known M1-activating stimuli LPS, IFNγ and TNFα induced round shape of differentiated macrophages whereas known M2-activating stimuli IL4, IL13 and IL10 induced elongated cell shape of differentiated macrophages (
The data herein identifies compounds, targets and pathways that mediate macrophage activation and sheds new light on the underlying molecular mechanisms. In our library, many compounds have known protein targets. Based on functional pathway enrichment analysis of protein targets of M1- or M2-activating compounds, we identified known pathways, such as cytokine, in macrophage activation. More importantly, we identified new pathways, including leptin, VEGF, EGF and neurotransmitter pathways, which mediate macrophage activation. Although studies have shown these pathways in macrophage function, their effects on macrophage activation and underlying mechanisms are unknown. Our transcriptional analysis of macrophages suggests that the ligands of these pathways activate macrophage by regulating gene expression of both typical M1 and M2 modules. For example, in hMDMs, leptin upregulates the expression of typical M1 modules induced by IFNγ while suppresses the expression of chronic inflammation TPP modules (
Macrophages exhibit a multi-dimensional spectrum of phenotypes beyond M1 and M2. Our identification of a diverse panel of macrophage-activating compounds that target GPCRs, enzymes, kinases, nuclear hormone receptors (NHRs), and transporters (
The data described herein also provides a rich resource for exploring compounds/targets/pathways for modulating macrophage activation in disease intervention. Reprogramming macrophage has emerged as a significant approach for treating a variety of diseases. Suppression or reprogramming of M2-like TAMs into M1-like macrophages by small molecule compounds is associated with induction of a strong anti-tumor activity alone or in combination with other therapeutics. Similarly, suppression or reprogramming of M1-like macrophages into M2-like state significantly inhibits the progression of inflammatory and autoimmune diseases. In this study, we confirmed M1-activating compounds thiostrepton and cucurbitacin I potently reprogrammed TAMs toward M1-like macrophages and enhanced anti-tumor activity either alone or in combination with an antibody therapeutic (
Activation of GPR3-β-Arrestin2-PKM2 Pathway in Kupffer Cells Protects Against Obesity and Liver Pathogenesis Through Enhanced Glycolysis
Increasing evidence suggests a critical role of macrophages in regulating body weight and obesity associated pathologies. However, the underlying molecular and cellular mechanisms remain to be elucidated. Here, we show that diphenyleneiodonium (DPI), an agonist of G-protein coupled receptor 3 (GPR3), stimulates both rapid and sustained increase in glycolysis at cellular level and protects mice from high fat diet (HFD) induced obesity and liver pathogenesis. Activation of GPR3 by DPI results in a rapid recruitment of β-arrestin2 to the plasma membrane, formation of β-arrestin2-GAPDH-PKM2 super complex, greatly increased enzymatic activities of GAPDH and PKM2, and therefore a rapid increase in glycolytic activities. DPI stimulation also results in the formation of PKM2 dimers, translocation of PKM2 from the cytosol to the nucleus, transactivation of c-Myc, and transcription of glycolytic genes, leading to a sustained increase in glycolysis. In mice, DPI inhibits HFD-induced obesity and liver pathogenesis by enhancing glycolysis and suppressing inflammatory response of Kupffer cells in a PKM2-dependent manner. In patients with non-alcoholic fatty liver disease (NAFLD), single cell RNA sequencing identifies a population of disease-associated macrophages that exhibit reduced expression of glycolytic genes but increased expression of inflammatory genes. DPI stimulates glycolysis and suppresses inflammatory responses of Kupffer cells from NAFLD patients. These findings identify GPR3-β-arrestin2-PKM2 signaling as a critical pathway for metabolic reprogramming of Kupffer cells and activation of this pathway as a potential approach to inhibit the development of obesity and NAFLD.
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder globally and is induced by fat deposition in the liver. NAFLD progresses through a series of stages: from simple steatosis to non-alcoholic steatohepatitis (NASH) to cirrhosis. Although the disease pathogenesis is not well understood, development of NAFLD is highly correlated with obesity and diabetes, and pathogenetically associated with lipid accumulation, inflammation, injury and fibrosis in the liver. As NFLAD is also a metabolic disorder, mechanisms that link metabolism to inflammation offers insights into the pathogenesis and help to identify targets for therapeutic development.
Kupffer cells (KCs) are the resident macrophages in the liver and the most abundant tissue macrophages in the body. They play a key role in detoxification, pathogen removal and tissue repair and homeostasis, but they can also contribute to the pathogenesis of liver diseases, including NAFLD, as they are involved in the initiation and progression of inflammation and tissue injury. In response to local stimuli, KCs regulate both metabolic and immune functions in the homeostatic liver. Lipids and other metabolites have been shown to not only regulate the expression of genes associated with immune response in human macrophages, but also modulate the activation of KCs in models of fatty liver disease and steatohepatitis. Disease-associated macrophages (DAMs) have been identified by single cell RNA sequencing (scRNAseq) in livers from patients with advanced NAFLD (NASH and cirrhosis) and from mouse models of NASH. DAMs exhibit altered expression of pathways associated with not only inflammation but also metabolism, suggesting that reprogramming dysfunctional macrophages may be a promising strategy to treat NAFLD.
G protein-coupled receptors (GPCRs) play essential roles in metabolic disorders as they serve as receptors for metabolites and fatty acids. In our screen for compounds that can reprogram macrophages, we found that diphenyleneiodonium (DPI), an agonist of GPR3, upregulates expression of genes involved in glycolysis and lipid metabolism. GPR3 is highly expressed in the brain and has been shown to play important roles in neurological processes. GPR3 is considered as a constitutively active orphan receptor that mediates sustained cAMP production in the absence of a ligand. An important mechanism that regulates GPCR signaling is desensitization, involving the receptor kinases (GRKs) and the β-arrestins. GPR3 stimulates the AP production by recruiting the scaffold protein β-arrestin2 to regulate γ-secretase activity. Despite these progresses, little is known about the function and mechanism of GPR3 signaling in other cell types, especially in regulating metabolism.
We have investigated the effect of DPI on metabolic reprogramming of macrophages, the underlying molecular mechanisms, and physiological effect of DPI on high fat diet (HFD)-induced obesity and pathogenesis. We show: i) DPI induces a rapid switch of cellular metabolism from oxidative phosphorylation (OxPhos) to glycolysis in macrophages by stimulating the formation of β-arrestin2-GAPDH-PKM2 super complex with greatly increased enzymatic activities; ii) DPI also induces a prolonged increase in glycolytic activities by stimulating translocation of PKM2 from cytosol to nucleus, transactivation of c-Myc, and transcription of glycolytic genes; iii) DPI inhibits HFD-induced obesity and liver pathogenesis in mice by stimulating glycolysis and suppressing inflammation in KCs and in a manner that requires PKM2 expression in KCs; and iv) DPI also stimulates glycolysis and suppresses inflammation of KCs from patients with NAFLD. These findings identify that GPR3 to β-arrestin2 to PKM2 and to c-Myc signaling is a critical pathway for metabolic reprogramming of macrophages and activation of this pathway in KCs is an approach for therapeutic interventions of obesity and NAFLD.
DPI has been reported as an agonist of GPR3 and an inhibitor of NOX. Consistent with previous observation that NOX-deficiency leads to a lower cellular glycolysis, we found that p47phox−/− BMDMs and inhibition of NOX activity by apocynin in macrophages lead to a significantly reduced basal level of glycolytic activity. However, DPI (50 nM) stimulated a similar level of increase in glycolysis in p47phox−/− BMDMs as in wild-type BMDMs, or with or without inhibitor apocynin, showing that DPI stimulates glycolysis independent of NOX activity. In contrast, although GPR3 knockdown also reduces the basal level of glycolytic activities, DPI (50 nM) failed to stimulate any significant increase in glycolysis, suggesting that DPI stimulates glycolysis through activation of GPR3. Similarly, β-arrestin2 and PKM2 are required for mediating the effect of DPI on glycolysis as knockout of these genes in BMDMs abolishes DPI-induced glycolysis. The difference between β-arrestin2 and PKM2 is that the former is required for maintaining a threshold level of basal glycolytic activity while the latter is not required. These genetic analyses identify a signaling pathway involving GPR3, β-arrestin2 and PKM2 in mediating the effect of DPI on glycolysis as well as NOX, GPR3 and β-arrestin2 in maintaining a threshold level of basal cellular glycolysis. As SIP, a putative endogenous ligand of GPR3, also induces a significant increase in glycolysis in macrophages, the identified pathway likely functions in metabolic reprogramming in response to endogenous ligands.
Consistent with a critical role of β-arrestin2 in GPCR signaling by functioning as a scaffold protein, we show that activation of GPR3 by DPI leads to a rapid recruitment of β-arrestin2 to the plasma membrane (
We found that activation of GPR3 by DPI also promotes the formation of PKM2 dimers in an ERK1/2-dependent manner (
Consistent with the increased glucose consumption through elevated glycolysis, DPI has profound effects on glucose metabolism and on HFD-induced weight gain, lipid deposition and fibrosis in the liver at the organismal level. DPI confers a better glucose tolerance in mice under normal conditions (
Finally, we show the presence of DAMs in the liver of NAFLD patients, which share the same phenotype, including expression of TREM2, CD9, GPNMB, MHCII (HLA-DRB1), C1QA and CLEC10A, as those found in the livers of patients with NASH and cirrhosis. As similar DAMs have been observed in various tissues with diverse pathologies, such as HFD-induced NASH in mice, scar tissues, Alzheimer's disease, and lung fibrosis, DAMs from different diseases may share a common gene expression signature. Our scRNAseq shows that DAMs are inhibited in glycolysis but increased in inflammation as suggested by downregulation of glycolytic genes and upregulation of inflammatory genes (
In one aspect, described herein is a method of identifying a modulator of macrophage activation. The method comprises contacting a primary macrophage cell with a candidate agent; monitoring or photographing the morphology of the cell contacted with the candidate agent; and optionally comparing the cell's morphology in the presence of the candidate agent with the cell's morphology in the absence of the candidate agent; wherein a change in morphology in the presence of the candidate agent is indicative of modulation of macrophage activation.
In another aspect, described herein is a method of treating cancer, fibrosis, or an infectious disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator changes the morphology of a macrophage cell from elongated shape to round shape.
In one aspect, described herein is a method of treating an inflammatory disease, a metabolic disease, an autoimmune disease, or a neurodegenerative disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator changes the morphology of a macrophage cell from round shape to elongated shape.
In another aspect, described herein is a method of treating cancer, fibrosis, or an infectious disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator activates a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin.
In one aspect, described herein is a method of treating an inflammatory disease, a metabolic disease, an autoimmune disease, or a neurodegenerative disease. The method comprises administering to a subject in need thereof an effective amount of a modulator of macrophage activation; wherein the modulator inhibits a serotonin transporter or receptor, a histamine transporter or receptor, a dopamine transporter or receptor, an adrenoceptor, VEGF, EGF and/or leptin.
In another aspect, described herein is a method of treating an inflammatory disease, a metabolic disease, an autoimmune disease, or a neurodegenerative disease. The method comprises administering to a subject in need thereof an effective amount of diphenyleneiodonium (DPI).
Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.
The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, Mass. (2000).
The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.
“Adjuvant” or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject. For example, an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given ligand) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level.
As used herein, the term “antibody” may refer to both an intact antibody and an antigen binding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies and antigen-binding antibody fragments.
The terms “antigen binding fragment” and “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include Fab, Fab′, F(ab′)2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
The terms “increased”, “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold increase, at least about a 1000-fold increase or more as compared to a reference level.
“Immunotherapy” is treatment that uses a subject's immune system to treat cancer and includes, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
“Administering” or “administration of” a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
The present disclosure provides methods of identifying a modulator of macrophage activation, comprising contacting a primary macrophage cell with a candidate agent; monitoring or photographing the morphology of the cell contacted with the candidate agent; and optionally comparing the cell's morphology in the presence of the candidate agent with the cell's morphology in the absence of the candidate agent; wherein a change in morphology in the presence of the candidate agent is indicative of modulation of macrophage activation.
As used herein, the term “test compound” or “candidate agent” refers to an agent or collection of agents (e.g., compounds) that are to be screened for their ability to have an effect on the cell. Test compounds can include a wide variety of different compounds, including chemical compounds, mixtures of chemical compounds, e.g., polysaccharides, small organic or inorganic molecules (e.g., molecules having a molecular weight less than 2000 Daltons, less than 1500 Dalton, less than 1000 Daltons, or less than 500 Daltons), biological macromolecules, e.g., peptides, proteins, peptide analogs, and analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, naturally occurring or synthetic compositions.
Depending upon the particular embodiment being practiced, the test compounds can be provided free in solution, or can be attached to a carrier, or a solid support, e.g., beads. A number of suitable solid supports can be employed for immobilization of the test compounds. Examples of suitable solid supports include agarose, cellulose, dextran (commercially available as, i.e., Sephadex, Sepharose) carboxymethyl cellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins, plastic films, polyaminemethylvinylether maleic acid copolymer, glass beads, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. Additionally, for the methods described herein, test compounds can be screened individually, or in groups. Group screening is particularly useful where hit rates for effective test compounds are expected to be low such that one would not expect more than one positive result for a given group.
A number of small molecule libraries are known in the art and commercially available. These small molecule libraries can be screened using the screening methods described herein. A chemical library or compound library is a collection of stored chemicals that can be used in conjunction with the methods described herein to screen candidate agents for a particular effect. A chemical library comprises information regarding the chemical structure, purity, quantity, and physiochemical characteristics of each compound. Compound libraries can be obtained commercially, for example, from Enzo Life Sciences™, Aurora Fine Chemicals™, Exclusive Chemistry Ltd™, ChemDiv, ChemBridge™, TimTec Inc™, AsisChem™, and Princeton Biomolecular Research™, among others.
Without limitation, the compounds can be tested at any concentration that can exert an effect on the cells relative to a control over an appropriate time period. In some embodiments, compounds are tested at concentrations in the range of about 0.01 nM to about 100 nM, about 0.1 nM to about 500 microM, about 0.1 microM to about 20 microM, about 0.1 microM to about 10 microM, or about 0.1 microM to about 5 microM.
The compound screening assay can be used in a high throughput screen. High throughput screening is a process in which libraries of compounds are tested for a given activity. High throughput screening seeks to screen large numbers of compounds rapidly and in parallel. For example, using microtiter plates and automated assay equipment, a laboratory can perform as many as 100,000 assays per day, or more, in parallel.
The compound screening assays described herein can involve more than one measurement of the cell or reporter function (e.g., measurement of more than one parameter and/or measurement of one or more parameters at multiple points over the course of the assay). Multiple measurements can allow for following the biological activity over incubation time with the test compound. In one embodiment, the reporter function is measured at a plurality of times to allow monitoring of the effects of the test compound at different incubation times.
The screening assay can be followed by a subsequent assay to further identify whether the identified test compound has properties desirable for the intended use. For example, the screening assay can be followed by a second assay selected from the group consisting of measurement of any of: bioavailability, toxicity, or pharmacokinetics, but is not limited to these methods.
The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood (Research Blood Components LLC.) by density gradient centrifugation with Ficoll-Paque Plus (GE healthcare) and LeucoSep™ (Greiner Bio-one). Human monocytes were purified from PBMC using the EasySep™ human monocyte isolation kit (Stemcell Technology) according to the manufacture's protocol. For in vitro differentiation of monocytes into human macrophages (M0, primary macrophage), isolated monocytes were cultured in complete RMPI1640 supplemented with 10% FCS (Gibco), 2 mM L-glutamine (Corning) and 1% PenStrep solution (Corning) in the presence of 50 ng/mL recombinant human M-CSF (Peprotech) for 7 days. Tumor cell line B16F10 were purchased from ATCC and cultured in complete DMEM supplemented with 10% FCS, 1% PenStrep solution and 2 mM L-glutamine. Luciferase-expressing human lymphoma B cell line (GMB) were described in Roghanian et al. Cancer Immunol Res (2019) and cultured in complete RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 0.55 mM 2-mercaptoethanol (Gibco), 1% non-essential amino acids (Lonza), 1 mM sodium pyruvate (Cellgro) and 1% PenStrep solution.
Based on the shape difference of M1 (round) and M2 (elongated) differentiated macrophages, we developed a high throughput method to screen compounds which could modulate macrophage polarization. Human M0 primary macrophages differentiated from monocytes in vitro were seeded using a Multidrop Combi dispenser (Thermo Scientific) at a density of 5,000 cells/well in 50 μL complete RPMI in the presence of 10 ng/mL M-CSF into optical 384-well plates (Cat. 393562, BD Falcon) and cultured for 16 hrs for cell recovery. Around 20% of macrophages in this stage (M0) are elongated. Cells were treated with a library of over 4000 individual compounds or drugs at the final concentration of 20 μM using the CyBi-Well simultaneous pipettor (CyBio). The screening compound library composes of the 2066 bioactive compounds, 320 FDA approved drugs, 440 oncological drugs and 1280 natural compounds from the center for the development of therapeutics in Broad Institute at MIT. After 24 hr incubation, supernatants were removed using the microplate washer (Bioteck) and cells were fixed by adding 50 μL 16% paraformaldehyde (Thermo Scientific) with the dispenser for 20 minutes. Cells were then washed with 50 μL 1×PBS twice and incubated for 20 minutes with NucBlue and AF746 Phalloidin (Invitrogen) to stain nucleus and cytoskeleton. Cells were then washed with 50 μL 1×PBS twice and maintained in PBS for the image acquirement. Plates were read in the Opera Phenix high content screening system (PerkinElmer) to photograph cells using 20× objective in 2 fluorescent channels (Blue and FarRed). A total of 6 different fields in each well and an average of 1,000 cells were imaged per well. CellProfiler was used to identify each cell by overlapping signals from its nucleus and cytoskeleton, and calculate the eccentricity as the parameter to measure the cell morphology. The Z-score was calculated by T-test to measure the difference of cell morphology between each treatment and control. For each row of the 384-well plate, total 4 wells with first and last two columns treated with the same concentration of DMSO were combined as the control for the other 20 treatment wells in that row. In the meantime, classic M1 and M2 stimuli were added to generate the gold-standard Z-score cutoffs with M1 or M2 activation. Classic M1 stimuli include LPS (100 ng/mL), IFNγ (50 ng/mL, Peprotech), TNFα (50 ng/mL, Peprotech), or IFNγ plus TNFα. Classic M2 stimuli include IL-10 (10 ng/mL, Peprotech), IL-4 (10 ng/mL, Peprotech), or IL-13 (5 ng/mL, Peprotech). The gold-standard Z-scores were used as the cutoffs to identify potent compounds to activate macrophage into M1 or M2 state.
To further screen to compounds which could reactivate or reprogram differentiated macrophages, potent 127 M1-activating and 180 M2-activating compounds from the first-round screening were cherry-picked up. Human macrophages were seeded into optical 384-well plates. Sixteen hours later, medium in M1 plates were replaced by M1 differentiating medium (complete RPMI with 50 ng/mL IFNγ and 50 ng/mL TNFα) and medium in M2 plates by M2 differentiating medium (complete RPMI with 5 ng/mL IL-4 and 5 ng/mL IL-13). After 24 hrs cell differentiation, M1 plates (M1 macrophages) and M2 plates (M2 macrophages) were treated with M2-activating compounds and M1-activating compounds respectively for 24 hrs. Two independent experiments were performed with or without replacing differentiating medium right before treatment. Cell imaging and analysis were performed as indicated above.
The identified compounds were classified based on the database from the International Union of Basic and Clinical Pharmacology (IUPHAR)(guidetopharmacology.org). The protein targets of the compounds were text-mined based on the target databases of UPHAR and DrugBank (drugbank.ca). The pathway enrichment analysis of protein targets of compounds was based on the WikiPathways.
B6 mice were purchased from the Jackson Laboratory and maintained in the animal facility at the Massachusetts Institute of Technology (MIT). NSG mice were purchased from the Jackson Laboratory and maintained under specific pathogen-free conditions in the animal facilities at MIT. All animal studies and procedures were approved by the Massachusetts Institute of Technology's Committee for Animal Care. Flow cytometry antibodies specific for mouse CD11b (M1/70), F4/80 (BM8), MHC-II (M5/114.15.2), Ly6C (HK1.4), Ly6G (1A8), Gr-1 (RB6-8C5), CD80 (16-10A1), CD86 (GL-1), CD163 (S150491), CD206 (C068C2), IFNγ (XMG1.2) and TNFα (MP6-XT22) were from Biolegend (USA) and iNOS (CXNFT) as from eBioscience (USA). Flow cytometry antibodies specific for human CD80 (2D10), CD86 (BU63), CD163 (GHI/61) and CD206 (15-2) were frpm Biolegend (USA) and iNOS (4E5) was from Novus Biologicals (USA). Antibody ARG1 (AlexF5) specific for both human and mouse was from eBioscience (USA). B16F10 melanoma specific antibody TA99 for in vivo study was prepared as described. Single cell preparation from different organs, staining of cells with fluorophore-conjugated antibodies and analysis of the stained cells using flow cytometry are as described. Briefly, cells in single cell suspension were incubated with specific antibodies at 4° C. for 20 minutes, washed twice, and resuspended in FACS buffer containing either DAPI. Cells were fixed and permeabilized with Cyto-Fast Fix/Perm buffer set (Biolegend) for intracellular staining according to the manufacture's protocol. Samples were stimulated by the cell stimulation cocktail (eBioscience) for 4 hrs and then fixed/permeabilized for intracellular staining. Cells were run on BD-LSRII, collecting 20,000 to 100,000 live cells per sample. The data were analyzed by FlowJo.
For the melanoma model, an inoculum of 1×106 B16F10 tumor cells was injected subcutaneously on the flank of 8- to 10-week-old male B6 mice in 100 μL sterile PBS. Six days following tumor inoculation, mice were randomized into 4 treatment groups including control (PBS or DMSO), tumor-targeting antibody TA99, compound, compound plus TA99. TA99 was administered at 100 μg per dose intraperitoneally (I.P.). The compound was administrated at the indicated dosage by either I.P. or paratumor injection subcutaneously (S.C.). All mice were dosed at day 6 and day 12 post tumor inoculation for a total of 2 treatments. Tumor size was measured as an area (longest dimension×perpendicular dimension) at day 6, day 12 and day 18 post tumor inoculation. Mice were euthanized for analysis at day 18 post tumor inoculation. For the lymphoma model, 1×107 GMB cells were injected through tail intravenously in 100 μL sterile PBS into 10- to 12-week-old male NSG mice. Mice were treated two weeks post tumor cell engraftment. Tumor-targeting antibody Rituxumab (InvivoGen) was administered at 10 mg/kg intraperitoneally. The compound was administrated I.P. at the indicated dosage. All mice were dosed at week 2 and week 3 post tumor injection for a total of 2 treatments. Tumor growth and spread was visualized using an IVIS Spectrum-bioluminescent imaging system (PerkinElmer) at week 2, week 3 and week 4 post tumor injection. Mice were euthanized for analysis at week 4 post tumor inoculation.
Mice were euthanized and tumor tissues were isolated and fixed with 10% neutral-buffered formalin solution (Sigma-Aldrich) for 24 hours. The tissues were processed with Tissue Processor (Leica Microsystems) and embedded in paraffin. Sections were cut at 5 μm thickness, mounted on polylysine-coated slides (Thermo Fisher Scientific), de-waxed, rehydrated, and processed for hematoxylin and eosin (H&E) staining according to a standard protocol. For immunochemical staining, antigen retrieval was carried out by either microwaving the slides in 0.01 M sodium citric acid buffer (pH 6.0) for 30 min. Sections were then immersed for 1 hour in blocking buffer (3% BSA, 0.2% Triton X-100 in PBS), then incubated in primary antibody in blocking buffer at 4° C. overnight, followed by incubation with secondary antibody conjugated HRP at 4° C. for 1 hour. All lung stained sections were scanned with a high-resolution Leica Aperio Slide Scanner. Images were analyzed by WebScope software.
Mouse bone marrow-derived macrophages (mBMM) were prepared as described previously54. Briefly, fresh bone marrow cells were isolated from B6 mice. Cells were plated into 6-well plate with 1×106/mL in complete RPMI with 2-mercaptoethanol and cultured for 6 days with fresh medium change every 2 days. mBMMs were differentiated to resemble TAMs in the presence of 10 ng/mL mIL-4 and mIL-13 (Peprotech) or 25 mM lactate acid for 24 hrs or tumor conditioned medium (CM). To prepare CM, 70% confluent B16F10 cultured were replaced with fresh medium and the tumor medium was collected and filtered (0.2 μm) 24 hrs later. The mixture of 3 volumes of tumor medium with 1 volume of complete RPMI for mBMM serves as the CM. Expression of Arg, Fizz1 and Vegfa were quantified by qPCR to assess the development of TAMs. Other genes of Tnf, I11b, Nos2, Cxcl 2, Ccl 5, Ym1 and Tgfb serve as macrophage activating markers. To assay the tumor growth inhibition, mBMMs (10,000 cells per well in 96 well plate) were treated with thiostrepton for 24 hrs and then cocultured with equal number of B16 melanoma cells in fresh complete RPMI for 12 hrs. The conditioned medium treated or not treated with thiostrepton were collected and filtered. The numbers of B16 melanoma cells were cultured for 12 hrs with conditioned medium or conditioned medium heated at 95° C. for 5 min. Tumor cells were quantified by flow cytometry to determine the macrophage-dependent killing function.
RNAs were extracted with RNeasy MiniElute kit (Qiagen), converted into cDNA and sequenced using Next-Generation Sequencing (Illumina). RNA-seq data was aligned to the mouse genome (version mm10) and raw counts of each genes of each sample were calculated with bowtie2 2.2.3 and RSEM1.2.15. Differential expression analysis was performed using the program edgeR at P<0.05 with a 2 fold-change. The gene expression level across different samples was normalized and quantified using the function of cpm. Differentially expressed genes were annotated using online functional enrichment analysis tool DAVID (http://david.ncifcrf.gov/). Gene set enrichment analysis were performed with GSEA with FDR q-value<0.05. The heatmap figure was visualized with MeV. To quantify the levels of RNA transcripts, total RNA was extracted from various cells and reverse transcribed by TaqMan® Reverse Transcription Reagents Kit (ABI Catalog No. N8080234), followed by amplification with Sybr Green Master Mix (Roche Catalog No. 04707516001) with specific primers (Table 4) and detected by Roche LightCycler 480. The Ct values were normalized with housekeeping gene GAPDH for comparison.
To determine the central hubs of all stimulation conditions by compounds (refer to
Statistical significance was determined with the two-tailed unpaired or paired Student's t-test. The FDRs were computed with q=p*n/1, (p=P value, n=total number of tests, i=sorted rank of P value).
Raw RNAseq are deposited in the database of Gene Expression Omnibus (GEO) with accession ID: GSE14992 and GSE155551.
Human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and differentiated into macrophages in a 7-day culture in the presence of recombinant human M-CSF. The resulting human monocyte-derived macrophages (hMDMs) were stimulated with different known M1-activating stimuli, including lipopolysaccharide (LPS), IFNγ, TNFα, or IFNγ plus TNFα, or M2-activating cytokines, including IL-10, IL-4 or IL-13, for 24 hours. The M1-activated hMDMs were round with punctate F-actin staining whereas M2-activated hMDMs were elongated with filamentous F-actin staining (
Based on the correlation between cell shape and macrophage activation, we developed a high throughput screen for compounds that activate hMDMs to either M1- or M2-like state (
Table 1 shows pathway analysis of proteins targeted by identified compounds.
Escherichia
coli
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coli
To validate the effect of identified compounds on macrophage activation, we assayed dosage responses of the commercially available top list of compounds to determine their effective concentration (EC) on cell shape change. 20 of 23 selected M1-activating and 4 of 6 M2-activating compounds showed strong dosage effects with an EC below 10 μM (
We also analyzed transcriptional responses of hMDMs to ligands of novel pathways, including serotonin (5HT), dopamine, VEGF, EGF and leptin by RNA-seq. Each ligand induced diverse transcriptional response (
Table 2 shows dosage information of selected compounds on M0 macrophages.
To investigate whether the identified compounds could reprogram or reactivate macrophages after M1- or M2-like differentiation, we rescreened the hits on M1- or M2-activated macrophages. hMDMs were activated into M2-like macrophages by IL-4 plus IL-13 or M1-like macrophages by IFNγ plus TNFα. After removing the differentiating cytokines, M2-like macrophages were treated with each of the 166 M1-activating compounds and M1-like macrophages were treated with each of the 180 M2-activating compounds at a final concentration of either 5 μM and 10 μM. 24 hours later, cell images were taken and cell shapes were quantified. Based on the same Z-score cutoff, 37 M1-activating and 21 M2-activating compounds were identified to induce cell shape changes at the concentration of both 5 μM and 10 μM (
We also rescreened the hits on differentiated macrophages in the presence of differentiating cytokines: either IL-4 plus IL-13 or IFNγ plus TNFα. Surprisingly, more compounds exhibited significant effects on cell shape changes in the presence of these cytokines (67 M1- and 55 M2-activating) than in absence of these cytokines (46 M1- and 25 M2-activating) at the same compound concentration of 5 μM (
Table 3 shows dosage information of selected compounds on differentiated macrophages
To broadly validate the identified compounds on macrophage activation (reprogramming) and to shed light on how the compounds activate macrophages, we selected 17 M1- and 17 M2-activating compounds with ECs below 5 μM and performed transcriptional profiling by RNA-seq. M2-like macrophages induced by IL-4 plus IL-13 were treated with each of the 17 M1-activating compounds at its ECs for 24 hours. Similarly, M1-like macrophages induced by IFNγ plus TNFα were treated with each of the 17 M2-activating compounds at its ECs for 24 hours. Different compounds up-regulated and down-regulated different number of genes (
To investigate the common denominators of macrophage activation, a reverse engineering regulatory network was assembled by ARACNe based on mutual information between each gene pair computed from the compound-perturbing expression profiles. Top 10% central hub genes inferred from the network (n=1255 most interconnected genes) collectively participated in 98,048 interactions. Most of top central hub genes or regulators, such as GBP1, FAM26F, STAT1, have been shown to play essential roles in macrophage activation and function (
To determine if the identified compounds activate macrophages in disease setting in vivo, we selected thiostrepton, a natural cyclic oligopeptide and an approved veterinary antibiotic for treating skin infection, and tested it to activate macrophages to M1-like state. Similar to other thiopeptide antibiotics, thiostrepton inhibits the ribosome function of bacterial protein synthesis. Recently, thiostrepton was shown to exhibit antiproliferative activity in human cancer cells through inhibiting proteasome and/or FOXM1 transcription factor. Following treatment of hMDMs with 2.5 μM thiostrepton for 24 hours, hMDMs were polarized to express proinflammatory cytokines TNFα and IL-1β and down-regulate the M2 chemokine CCL24 (
To determine the effect of thiostrepton on TAM in vitro, mouse bone marrow macrophages (BMMs) were cultured in the conditioned medium (CM) of B16F10 tumor cells in the absence or presence of thiostrepton for 24 hrs. Alternatively, BMMs were cultured in the conditioned medium for 24 hrs first and then treated with thiostrepton for another 24 hrs. The expression of selected genes associated with macrophage polarization was assayed by qPCR. Thiostrepton inhibited the expression of TAM/M2-associated genes Arg1, Fizz1, Vegfa, Ym1 and Tgfb but up-regulated the expression of M1-associated genes Tnf, I11b, Cxcl2 and Nos2 (
To examine whether thiostrepton-activating macrophages or conditioned medium have effects on tumor cell growth, BMMs were treated with thiostrepton for 24 hrs. Equal numbers of primed BMMs and melanoma cells (B16F10) were co-cultured for 12 hrs. Significantly more melanoma cells were lost in the presence of thiostrepton-treated macrophages as compared to the untreated macrophages in a dose-dependent manner (
Next, we examined whether thiostrepon has anti-tumor effect in vivo through activating macrophages. B16F10 melanoma cells were injected subcutaneously into syngeneic C57BL/6 mice. 6 and 12 days later, tumor-bearing mice were treated with either vehicle (DMSO), melanoma specific antibody TA99, thiostrepton, or combination of TA99 and thiostrepton by intraperitoneal injection (I.P.). In a dosage-dependent manner (150 or 300 mg/kg), thiostrepton strongly suppressed the tumor growth alone and additively with TA99 (
To investigate whether tumor-infiltrated macrophages were reprogrammed, we purified TAMs from B16F10 melanoma tumors from mice dosed with thiostrepton or vehicle by I.P. or S.C. at day 18 post tumor engraftment and performed RNA-seq. GSEA and functional enrichment analysis showed that thiostrepton up-regulated the expression of genes associated with inflammatory response and ROS and down-regulated the expression of genes associated with mitotic division in TAMs from mice treated with thiostrepton by both I.P. and S.C. (
To further confirm the anti-tumor effects of thiostrepton in vivo, we injected i.v. luciferase-expressing human B lymphoma cells into NSG mice. Tumor-bearing mice were treated with rituximab (anti-CD20), thiostrepton or both at 2 and 3 weeks post tumor engraftment. Quantification of tumor burden by luciferase imaging showed that thiostrepton alone or together with rituximab significantly reduced the tumor burden in the bone marrow (
C57BL/6 (B6) mice, p47phox−/−, Clec4f-Cre mice were purchased from the Jackson Laboratory and maintained in the animal facility at the Massachusetts Institute of Technology (MIT). PKMflox mice were described in the previous publication. Antibodies specific for CD11b (M1/70), F4/80 (BM8), MHC-II (M5/114.15.2), CD45.2 (104), CD9 (MZ3) for flow cytometry were from Biolegend. Anti-GPR3 (#SC390276) was from Santa Cruz Biotechnology. Anti-β-arrestin2 (#4674), Glycolysis Antibody Sampler Kit (#8337), anti-Myc and anti-FLAG were from Cell Signaling Technology. Anti-PKM2 (#1C11C7) was from Abcam. β-Arrestin2 CRISPR plasmids (sc432139) was from Santa Cruz Biotechnology. pCMV-β-arrestin2-GFP (PS10010), pCMV6-Flag-myc-barrestin2 (PS100001) and Arrb2 mouse siRNA Oligo Duplex (Locus ID 216869) were from Origene. Immortalized Kupffer cell line (ABI-TC192D, AcceGen), human primary KCs (ABC-TC3646, AcceGen), THP-1 (ATCC TIB-202) and 293T (CRL-3216) were cultured following vendor instructions (37° C., 5% CO2). Transfection of ImKCs with siRNAs was accomplished using Lipofectamine™ 2000 (Thermo Fisher Scientific) according to the manufacturer's instruction. Apocynin (PHL83252) was from Sigma.
Mouse BMDMs were prepared. Fresh bone marrow cells were isolated from B6 mice, plated onto a six-well plate with 1×106/mL in complete RPMI with 2-mercaptoethanol and 20% L929 supernatants which were obtained by culturing L-929 cells for 6 days with medium change every 2 days.
293T cells were transfected with FLAG-tagged β-arrestin2, using TransIT®-LT1 Transfection Reagent (Mirus). Thirty-six hours after transfection, the cells were lysed using cold Lysis Buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, 10% glycerol, proteinase inhibitor (Roche Catalog No. 11836153001), and phosphatase inhibitors (Roche Catalog No. 04906845001). The clear supernatants from the lysate were incubated with M2-magnetic beads conjugated with anti-FLAG antibody (Sigma Catalog No. M8823) for 2 hours at 4° C. Then the beads were washed twice and eluted by the 3×FLAG peptides (Sigma Catalog No. F4799) as described in the Sigma manual for Western blotting.
Proteins were extracted from cells with RIPA buffer. Protein concentration was quantified by BCA Protein Assay Kit (Pierce Biotechnology). Samples containing 20 μg total protein were resolved on a 10% SDS-PAGE gel and electro-transferred onto a PVDF membrane (Millipore Corporation). The membrane was blocked in 5% (w/v) fat-free milk in PBST (PBS containing 0.1% Tween-20). The blot was hybridized overnight with primary antibodies: anti-pSRC (D49G4, Cell Signaling Technology, 1:1000) and pSIK1/2/3 (#ab199474, Abcam, 1:1000) according to the recommended dilution in 5% fat-free milk. The blot was washed twice in PBST and then incubated with anti-Rabbit HRP-conjugated secondary antibody (Cell Signaling Technology, 1:2000) in 5% fat-free milk. The membrane was washed twice in PBST and subjected to protein detection by ECL Plus Western Blotting Detection System (GE Healthcare) before being exposed to a Kodak BioMax XAR film. The membrane was stripped and reblotted with the anti-β-tubulin (D49G4, Cell Signaling Technology) for protein loading control.
Protein was extracted from cells in 1× Native PAGE sample buffer (ThermoFisher) containing 1% digitonin followed by 20 min spin at 12,000×g to pellet debris. Protein extracts were analyzed using NativePAGE Novex System (ThermoFisher) and subsequently transferred to PVDF membrane, fixed, and blotted for native proteins.
ImKCs were treated with DPI (#81050, Cayman) at 50 or 500 nM for 6 hrs or 24 hrs. Cells were washed once in ice-cold 0.9% NaCl and lysates were extracted in 80% methanol solution containing internal standards for LC/MS by scraping on dry ice followed by 10-minute mixing with vortex in 4° C. Following lysate extraction, debris were removed by high-speed centrifugation and supernatant was dried using speedvac. Samples were analyzed by LC/MS on QExactive Orbitrap instruments (Thermo Scientific) in Whitehead Institute metabolite profiling core facility. Data analysis was performed using the in-house software described previously (Lewis et al., 2014).
BMDMs or ImKCs were cotransfected with plasmids encoding FLAG-GPR3-GFP or β-arrestin2-RFP. Twenty-four hours after transfection, cells were reseeded into a 24-well glass-bottom plate (Nest, Shanghai, China) and treated with DPI (50 nM), S1P (3 mM), or vehicle control (0.3% DMSO) for the indicated duration. The fluorescent signals of membrane-bound receptor or β-arrestin2 were collected as live images using a total internal reflection fluorescence (TIRF) microscope (Olympus).
OCR and ECAR were measured in isolated tissues or cultured ImKCs using the Seahorse XFe Extracellular Flux Analyzer (Agilent). For tissue respiration assays, 1.0 mg adipose tissue was dissected from inguinal WAT depots by using a surgical biopsy instrument (Integra Miltex Standard Biopsy Punches, Thermo Fisher) and placed into XF96 Islet Capture Microplates and pre-incubated with XF assay medium with pH value at 7.4. XF assay medium supplemented with 1 mM sodium pyruvate, 2 mM GlutaMax™-I, and 25 mM glucose. Isolated MDMs or Kupffer cells were subjected to a mitochondrial stress test by adding oligomycin (2 μM) followed by carbonyl cyanide 4-(trifluoromethoxy), phenylhydrazone (FCCP, 5 μM), and antimycin (1 μM). For glucose stress assay and ECAR measurement, XF assay medium was supplemented only with GlutaMax™-I. Tissue or cells were subjected to a glucose stress test by adding highly concentrated glucose (for tissue, 25 mM; for cells, 10 mM), followed by adding oligomycin (5 μM), FCCP (5 μM), and 2-DG (50 mM). Cells were seeded in culture dishes, and the medium was changed after 6 hours with serum-free DMEM. Cells were incubated for 12-16 hours, and the culture medium was then collected for measurement of glucose and lactate concentrations. Glucose levels were determined using a glucose (GO) assay kit (Sigma). Glucose consumption was the difference in glucose concentration when compared with DMEM. Lactate levels were determined using a lactate assay kit (Eton Bioscience).
BMDMs or Kupffer Cells were fixed and incubated with primary antibodies, and then labeled with Alexa Fluor dye-conjugated secondary antibodies and counterstained with Hoechst 33342 according to standard protocols. Cells were examined using a deconvolution microscope (Zeiss) with a 63-Å oil immersion objective. Axio Vision software from Zeiss was used to deconvolute Z-series images.
The enzymatic activities of PKM and GAPDH were measured using the pyruvate kinase activity assay kit (Biovision, #K709) and GAPDH activity assay kit (Biovision, #K680) according to the manufacturer's protocols, respectively.
The c-Myc activity was assessed using the Myc Reporter kit (BPS Biosciences) and the Dual-Luciferase Reporter System (Promega) according to the manufacturers' instructions. Briefly, 100 μL (1.5×105 cells/mL) control and Kupffer cells were seeded into 96-well plates. After overnight incubation, when cells reached ˜50% confluency, 1 μL of Reporter A (60 ng/μL) in the Myc Reporter kit was transfected into cells using Turbofectin 8.0. After 48 hours, cells were lysed in 25 μL Passive Lysis Buffer (provided in the Dual-Luciferase Reporter kit). 20 μL of cell lysate was transferred to 96-well plates and placed in a 96-well microplate luminometer (GloMax-Multi, Promega). 100 μL Luciferase Assay Reagent II and 100 μL Stop & Glo Reagent (both provided in the Dual-Luciferase Reporter kit) were sequentially injected, and firefly and Renilla luciferase activities were automatically measured. c-Myc activities were determined by the ratios of firefly to Renilla luciferase activities.
C57BL/6 mice at 5 weeks of age (body weight=23-25 g) were randomly assigned to three groups: 5 mice were fed with a normal chow diet for 16 weeks and then injected with saline once every 5 days for 4 weeks; 10 mice were fed with HFD (60 kcal % fat) for 16 weeks to induce obesity and hepatosteatosis and then divided into two groups: HFD+ vehicle (HFD) group (n=5) was injected with the vehicle (PEG3000) and HFD+ DPI group (n=5) was injected with DPI in vehicle (2 mg/kg) i.p. every 5 days for 4 weeks.
Liver samples fixed in 10% buffered formalin were embedded in paraffin, sliced (2 μm sections), and stained with hematoxylin and eosin (H&E). Histological examination for morphological changes was performed in a blinded manner. Liver sections were scored according to the criteria of the NAFLD activity score (NAS).
The GTT were performed in mice 19 weeks after feeding with HFD or NC. For GTT, mice were fasted overnight, followed by an intraperitoneal injection of 1 g/kg glucose. For the ITT, mice were fasted for 6 hours, followed by an intraperitoneal injection of 0.75 units/kg insulin. Blood was obtained from the tail vein before (0 min) and after (15, 30, 60, 90 and 120 min) the injection of glucose or insulin. Glucose levels were measured using an automatic glucometer (Roche Diagnostics, Rotkreuz, Switzerland).
Human liver biopsies were obtained from livers procured from deceased donors deemed unacceptable for liver transplantation. Samples were collected with appropriate institutional ethics approval from The First Affiliated Hospital of Jilin University. All experiments were performed in accordance with the relevant guidelines and regulations. In addition, written informed consent was obtained from each subject. During organ retrieval, donor liver grafts were perfused in situ with cold (HTK) solution (Methapharm) to thoroughly flush out circulating cells, leaving only tissue resident cells that are then used to prepare a single-cell suspension to isolate immune cells. The unused liver caudate lobe post liver transplantation was collected and flushed with HBS+EGTA at 4° C. to remove any non-liver resident cells. Single-cell isolation from the resected caudate lobe was performed with a modified two-step collagenase procedure (MacFarland et al. 2017 ACnano). Single cell suspension was stained with anti-CD45 to sort all immune cells for scRNAseq or anti-CD14 to sort KCs for in vitro treatment by flow cytometry (BD Aria).
Mouse livers were dissected and digested with Collagenase IV (Roche). Single cell suspension was stained with anti-F4/80, anti-CD 11b and anti-Gr-1. F4/80+CD11b+Gr1low macrophages were sorted by flow cytometry (BD Aria). RNAs were extracted with RNeasy MinElute Kit (Qiagen), converted into cDNA and sequenced using Next-Generation Sequencing (Illumina). RNA-seq data were aligned to the human genome (version hg19) and raw counts of each genes of each sample were calculated with bowtie2 2.2.3 (Langmead et al. 2009) and RSEM 1.2.15 (Li et al. 2011). Differential expression analysis was performed using the program edgeR at P<0.05 with a two-fold change (Robinson et al. 2010). The gene expression level across different samples was normalized and quantified using the function of cpm. DEGs were annotated using online functional enrichment analysis tool DAVID (Huang et al. 2007).
Sorted CD45+ cells were resuspended and washed in 0.05% RNase-free BSA in PBS for single-cell library preparation with 10× Chromium Next GEM Single Cell 3′ Kit (10×Genomics according to the manufacturer's instructions. The single-cell cDNA libraries were sequenced by NexSeq500 (IIlumina). Raw sequences were demultiplexed, aligned, filtered, barcode counting, unique molecular identifier (UMI) counting with Cell Ranger software v3.1 (10×Genomics) to digitalize the expression of each gene for each cell. The analysis was performed using the Seurat 3.0 package. We first processed each individual data set separately prior to combining data from multiple samples. The outlier cells with extreme low number (<500) or high number (>5,000) of gene features as doublets, or low total UMI (<1,000) and high mitochondrial ratio (>15%) from each data set were removed. Subsequently, samples were combined based on the identified anchors for the following integrated analysis. We ran principal component analysis (PCA) and used the first 15 principal components (PCs) to perform tSNE clustering. We checked well-defined marker genes for each cluster to identify potential cell populations, such as T cells (CD3E, CD8A, CD4, CD69, IL7R), B and plasma cells (CD19, MS4A1, SDC1), DC (CD11C, CLEC9A), NK cells (CD56, CD16, GZMB). For macrophage analysis, CD14 and CD68 positive clusters were selected for subsequent analyses. We repeated PCA, tSNE clustering on the integrated data sets of macrophages. Differential expression analysis was performed to identify the genes significantly upregulated in each cluster compared with all other cells. For gene sets representing specific cellular functions or pathways, we performed functional enrichment analysis with the biological process of Gene Ontology by the online tool DAVID.
Statistical significance was determined with the two-sided unpaired or paired Student's t test. The FDRs were computed with q=P×n/i, where P=P value, n=total number of tests, and i=sorted rank of P value.
DPI stimulates transcription of many genes in the glycolysis pathway in human primary macrophages (
DPI is an agonist of GPR3 and an inhibitor of GAPDH oxidase (NOX). We first determined the requirement of NOX in DPI-stimulated glycolysis. Bone marrow derived macrophages (BMDMs) were prepared from p47phox−/− mice, which do not have any functional NOX activity as p47phox is the organizer of phagocyte NAPDH oxidase (NOX2). Compared to wild-type (WT) BMDMs, p47phox−/− BMDMs had a significantly lower basal level of glycolysis, glycolytic capacity and glycolytic reserve (
To determine the requirement of GPR3, we knocked down GPR3 by siRNA (siGpr3) in ImKCs. Although GPR3 knockdown was about 70% (
β-arrestin2, encoded by Arrb2, has been reported to bind to GPR3 and is required for GPR3 signaling. To investigate the requirement of β-arrestin2 in DPI-stimulated glycolysis, we constructed Arrb2−/− ImKCs using CRISPR-Cas9 mediated gene editing (
Together, these results show that DPI-stimulated glycolysis is dependent on GPR3 and β-arrestin2 and that activation of GPR3 by DPI leads to rapid trafficking of β-arrestin2 to the plasma membrane.
How does DPI stimulate a rapid increase in glycolytic activity? We investigated the interaction between β-arrestin2 and metabolic enzymes, including PKM2 and GAPDH. To investigate this mechanism, we treated ImKCs with or without DPI for 6 hours and immunoprecipitated β-arrestin2 followed by Western blotting analysis. ERK1/2, enolase, GAPDH and PKM2 co-precipitated with β-arrestin2 (
How does DPI stimulate transcription of genes in the glycolysis pathway? PKM2 is known to be present in monomeric, dimeric and tetrameric forms. While the tetrameric form exhibits glycolytic enzymatic activity, the dimeric form can translocate into the nucleus and function as a transcriptional cofactor to activate expression of c-Myc, which, in turn, can directly activate the transcription of almost all glycolytic genes through binding the classical E-box sequence. To test this mechanism, we first determined if PKM2 is required for DPI-induced transcription of glycolytic genes. BMDMs were prepared from wild-type and Pkm−/− mice, incubated with or without 50 and 500 nM DPI for 24 hours, and the transcript levels of key glycolytic genes were quantified by RT-PCR. In a dose-dependent manner, DPI stimulated the transcription of Pkm, Ldha and Hk2 in the wild-type but not in Pkm−/− BMDMs (
Next, we determined if DPI induces formation of dimeric PKM2 and nuclear translocation. ImKCs were treated with 50 or 500 nM DPI for 6 or 12 hours, lysed and analyzed directly by Native PAGE gel, followed by anti-PKM2 Western blotting. While PKM2 was found in monomeric and tetrameric forms without DPI treatment, dimeric form was induced following DPI treatment in a dose-dependent manner (
We also determined if c-Myc is induced by DPI in a PKM2-dependent manner. As shown in
Taken together, these results show that DPI stimulates sustained increase in glycolytic activity through nuclear translocation of PKM2, transcriptional activation of c-Myc, and transcription of glycolytic genes.
To explore the in vivo consequence of DPI on glycolysis, we examined fast glucose response in DPI pretreated mice. C57BL/6 (B6) mice were injected intraperitoneally (i.p.) with 2 mg/kg DPI and 6 hours later mice were injected i.p. with 1.5 mg/kg glucose. Blood glucose levels were measured before DPI injection, 6 hours after DPI injection and at different time points after glucose injection. As shown in
We also examined the effect of DPI on hepatic steatosis. B6 mice were fed with HFD for 16 weeks. Nine weeks after HFD when mice became obese, DPI (2 mg/kg) was given once every 5 days for a total of 10 doses. DPI also significantly reduced the weight gain without affecting the weekly food intake (
To investigate the cell types in the liver that mediate DPI's effect, we analyzed the expression of PKM2 in different cell types in the livers using known single cell RNAseq data. In both human and mice, PKM2 was highly expressed in Kupffer cells and intermediately expressed in other immune cells, while PKM1 (PKLR) was exclusively expressed in APOC3+ hepatocytes (
To further investigate the effects of DPI on Kupffer cells in vivo, we purified KCs from vehicle- or DPI-treated HFD-fed mice and age-matched mice on the normal diet, and performed RNA-seq. GSEA and functional enrichment analysis showed that upregulation of genes associated with immune and inflammatory responses in KCs from mice fed with HFD or ND (
Single cell RNAseq analysis of liver cells from NASH and cirrhosis patients has identified TREM2+ disease-associated macrophages (DAMs) in the liver that have lower expression of metabolic genes. To determine whether the DAMs are also present in patients with NFALD, we performed scRNAseq of immune cells from liver biopsies of 3 healthy donors and 3 NFALD patients. Fourteen cell clusters were identified, including naïve CD8+ T cells, resident memory CD8+ (TRM) cells, CD4+ T cells, B and plasma cells, CD56low and CD56hi NK cells, macrophages or KCs, neutrophils and proliferating cells (
To directly examine the effect of DPI on human Kupffer cells from NFALD patients, we purified KCs from two NFALD patients and performed the transcriptional analysis by RNA-seq following DPI treatment ex vivo for 24 hours. The same as human MDMs and mouse ImKCs, the expression of glycolytic genes was upregulated by DPI whereas the expression of DAM markers, including APOE, CLEC10A, TREM2 and C1QA, was downregulated (
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 63/080,988, filed Sep. 21, 2020; the contents of which are hereby incorporated herein by reference in their entirety.
This invention was made with Government support under Grant No. R35 CA197605 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63080988 | Sep 2020 | US |
