Patent Application
20210177895
The present invention, in some embodiments thereof, relates to methods of modulating M2 macrophage polarization and use of same in therapy.
Mammalian tissues consist of diverse cell types that include: fibroblasts, epithelial, endothelial and immune lineages. Tissue formation during embryonic development requires the coordinated function and crosstalk between distinct cell types, in specific environmental contexts. Development of the lung into specialized committed cell types is a highly regulated process, characterized by unique pathways and functional properties. In parallel, cells of the immune system migrate from hematopoietic sites to the lung, in order to establish an active immune compartment that interacts with stromal cells, and influences tissue differentiation, growth and function.
The mammalian lung is the central respiratory organ, featuring a diverse set of specialized cell types. Gas exchange in the lung occurs in the alveoli, which are composed of specialized epithelial cells: the alveolar type (AT) 1 cells that mediate gas exchange, and AT2 cells that secrete surfactant and maintain the surface tension of the lungs (Whitsett and Alenghat, 2015). Alveolar epithelial cells branch from their mutual progenitor between the canalicular (E16.5) and saccular (E18.5) stages, resulting in dramatic changes in morphology and gene expression (Treutlein et al., 2014). Another major cell type is the alveolar macrophages (AM), which clear surfactant from the alveolar space, and act as important immune modulators, suppressing unwanted immune responses in the lungs (Hussell and Bell, 2014). AM originate from fetal liver embryonic precursors and are self-maintaining, with no contribution from the adult bone marrow (Epelman et al., 2014; Hashimoto et al., 2013; Murphy et al., 2008; Shibata et al., 2001). The first wave of lung macrophages appears at embryonic day 12.5 (E12.5), followed by a second wave stemming from fetal-liver derived monocytes, which continues its differentiation axis during alveolarization into mature AM (Ginhoux, 2014; Ginhoux and Jung, 2014; Hoeffel and Ginhoux, 2018; Kopf et al., 2015; Tan and Krasnow, 2016).
The immune response in each tissue, and the lung in particular, must be tightly regulated and adapted to its requirements, as aberrant immune activation may cause tissue damage and pathologies including chronic inflammation, fibrosis and autoimmune responses. Hence, each tissue is equipped with a unique signaling environment that interacts with the immune compartment and shapes the gene expression and chromatin landscapes of the cells (Butovsky et al., 2014; Cipolletta et al., 2015; Cohen et al., 2014; Greter et al., 2012; Hussell and Bell, 2014; Lavin et al., 2014; Okabe and Medzhitov, 2014; Panduro et al., 2016; Yu et al., 2017). In the lung context, AM exhibit a tissue specific phenotype, evident by their gene expression and function (Gautier et al., 2012; Guilliams et al., 2013b; Kopf et al., 2015; Lavin et al., 2014). There is a major gap in our understanding of the dynamic signaling during the alveolarization process, as attempts to grow AM ex vivo have not been successful (Fejer et al., 2013). Lung macrophage development and maturation was shown to be dependent on different growth and differentiation cues transmitted from epithelial cells (mainly AT2), innate lymphocytes (ILC) and the AM themselves (de Kleer et al., 2016; Guilliams et al., 2013a; Saluzzo et al., 2017; Yu et al., 2017). The function and crosstalk of other lung resident immune and non-immune cell types in the lung is currently much less understood.
Basophils are thought to be short-lived granulocytic cells, characterized by the presence of lobulated nuclei and secretory granules in the cytoplasm. They complete their maturation in the bone-marrow, before they enter and patrol the bloodstream. Under pathological conditions, such as parasite infection and allergic disorders, basophils are recruited and invade tissue parenchyma (Min et al., 2004; Mukai et al., 2005; Oh et al., 2007), and their major function has been mainly attributed to induction of Th2 responses in allergy, and IL-4 secretion after helminth infection (Mack et al., 2005; Min et al., 2004; Sokol et al., 2009; Sullivan and Locksley, 2009; Tschopp et al., 2006; Tsujimura et al., 2008).
Active modulation of macrophage polarization is therefore an approach in the development for anti-inflammatory and anti-cancer therapies.
Additional related background art:
WO2016185026
EP3072525A1
WO02017097876
Wynn TA, Nat Rev Immunol. 2015 May;15(5):271-82.
According to an aspect of some embodiments of the present invention there is provided a method of treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject in need thereof, the method comprising:
(a) culturing basophils in the presence of IL33 and/or GM-SCF; and
(b) administering to the subject a therapeutically effective amount of the basophils following the culturing,
thereby treating the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in the subject.
According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of basophils having been generated by culturing in the presence of IL33 and/or GM-SCF for use in treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject in need thereof.
According to some embodiments of the invention, the basophils are blood circulating basophils or derived from the bone-marrow.
According to some embodiments of the invention, the method further comprises prior to (a):
(i) isolating the basophils from bone marrow or peripheral blood;
(ii) differentiating the basophils from the bone marrow or peripheral blood in the presence of IL-3 to as to obtain a differentiated culture;
(iii) isolating from the differentiated culture a cKIT- population.
According to some embodiments of the invention, the (ii) is performed for 8-10 days in culture.
According to some embodiments of the invention, the (a) is performed for up to 48 hours.
According to some embodiments of the invention, the culturing is performed so as to achieve a lung basophil phenotype.
According to some embodiments of the invention, the lung basophil phenotype comprises expression of growth factors and cytokines selected from the group consisting of Csf1, Il6, Il13, L1cam, Il4, Ccl3, Ccl4, Ccl6, Ccl9 and Hgf, the expression being higher than in blood circulating basophils.
According to some embodiments of the invention, the lung basophil phenotype comprises an expression signature of Il6, Il13, Cxcl2, Tnf, Osm and Ccl4.
According to some embodiments of the invention, the lung basophil phenotype comprises an expression signature of Fcera1+, Il13ra+ (Cd123), Itga2+ (Cd49b), Cd69+, Cd244+ (2B4), Itgam+ (Cd11b), Cd63+, Cd24a30, Cd200r3+, Il2re, Il18rap+ and C3ar1+.
According to some embodiments of the invention, the basophils are human.
According to some embodiments of the invention, the basophils comprise an expression signature of Fcer1, Il13ra1, Itga2, Cd69, Cd244, Itgam, Cd63, Cd24, Il2ra, Il18rap and C3ar1.
According to some embodiments of the invention, the basophils are autologous to the subject.
According to an aspect of some embodiments of the present invention there is provided a method of treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a signaling molecule selected from the group consisting of IL6, IL13 and HGF, thereby treating the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in the subject.
According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of a signaling molecule selected from the group consisting of IL6, IL13 and HGF for use in treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject
According to some embodiments of the invention, the therapeutically effective amount increases the M1/M2 macrophage ratio.
According to some embodiments of the invention, the subject is a human subject.
According to some embodiments of the invention, the administering is in a local route of administration.
According to some embodiments of the invention, the administering is to the lung.
According to some embodiments of the invention, the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio is an inflammatory disease.
According to some embodiments of the invention, the inflammatory disease is selected from the group consisting of: sepsis, septicemia, pneumonia, septic shock, systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), acute lung injury, aspiration pneumanitis, infection, pancreatitis, bacteremia, peritonitis, abdominal abscess, inflammation due to trauma, inflammation due to surgery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue damage due to chemotherapy or radiotherapy, and reactions to ingested, inhaled, infused, injected, or delivered substances, glomerulonephritis, bowel infection, opportunistic infections, and for subjects undergoing major surgery or dialysis, subjects who are immunocompromised, subjects on immunosuppressive agents, subjects with HIV/AIDS, subjects with suspected endocarditis, subjects with fever, subjects with fever of unknown origin, subjects with cystic fibrosis, subjects with diabetes mellitus, subjects with chronic renal failure, subjects with bronchiectasis, subjects with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, subjects with febrile neutropenia, subjects with meningitis, subjects with septic arthritis, subjects with urinary tract infection, subjects with necrotizing fasciitis, subjects with other suspected Group A streptococcus infection, subjects who have had a splenectomy, subjects with recurrent or suspected enterococcus infection, other medical and surgical conditions associated with increased risk of infection, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, meningococcemia, post-pump syndrome, cardiac stun syndrome, stroke, congestive heart failure, hepatitis, epiglotittis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, pre-eclampsia, eclampsia, HELP syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhagic fever, pelvic inflammatory disease, Legionella, Lyme disease, Influenza A, Epstein-Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, progressive systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, systemic vasculitis, Wegener's granulomatosis, transplants including heart, liver, lung kidney bone marrow, graft-versus-host disease, transplant rejection, sickle cell anemia, nephrotic syndrome, toxicity of agents such as OKT3, cytokine therapy, cryoporin associated periodic syndromes and cirrhosis.
According to some embodiments of the invention, the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio is an autoimmune disease.
According to some embodiments of the invention, the autoimmune disease is selected from the group consisting of Addison's Disease, Allergy, Alopecia Areata, Alzheimer's disease,
Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, Ankylosing Spondylitis, Antiphospholipid Syndrome (Hughes Syndrome), arthritis, Asthma, Atherosclerosis, Atherosclerotic plaque, autoimmune disease (e.g., lupus, RA, MS, Graves' disease, etc.), Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia, Behcet's Disease, Berger's Disease, Bullous Pemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue/Coeliac disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis, Chronic Inflammatory Demyelinating, Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy (Guillain-Barre syndrome), Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), chronic obstructive pulmonary disease (COPD), CREST syndrome, Crohn's disease, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema, Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia, Evan's Syndrome, Exopthalmos, Fibromyalgia, Goodpasture's Syndrome, Hashimoto's Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder (e.g., psoriasis), Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, Insulin Dependent Diabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes, Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki's Disease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, lupus, Lupus Nephritis, Lymphoscytic Lypophisitis, Meniere's Disease, Miller Fish Syndrome/acute disseminated encephalomyeloradiculopathy, Mixed Connective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes (Whitaker's syndrome), Polymyalgia Rheumatica, Polymyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis/ Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis, Raynaud's Phenomenon, Reiter's Syndrome/Reactive arthritis, Restenosis, Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis, Schmidt's syndrome, Scleroderma, Sjorgen's Syndrome, Stiff-Man Syndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2 diabetes, Ulcerative colitis, Uveitis, Vasculitis, Vitiligo, and Wegener's Granulomatosis.
According to some embodiments of the invention, the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio is a pulmonary disease.
According to some embodiments of the invention, the M2/M1 macrophage comprises alveolar macrophages.
According to some embodiments of the invention the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio is a chronic obstructive pulmonary disease (COPD).
According to an aspect of some embodiments of the present invention there is provided a method of treating a disease or disorder that can benefit from increasing an M1/M2 macrophage ratio in a subject in need thereof, wherein the disorder is not associated with basophilia, the method comprising depleting basophils or activity of the basophils in the subject, thereby treating the disease or disorder that can benefit from increasing an M 1/M2 macrophage ratio in the subject.
According to some embodiments of the invention, the depleting is by an agent which depletes the basopohils or the activity of the basophils.
According to an aspect of some embodiments of the present invention there is provided an agent which depletes basopohils or activity of the basophils for use in treating a disease or disorder that can benefit from increasing an M 1/M2 macrophage ratio in a subject in need thereof.
According to some embodiments of the invention, the agent is directed to at least one basophil marker.
According to some embodiments of the invention, the agent targets FceR 1 a, IL33R and/or CSF2Rb.
According to some embodiments of the invention, the agent targets GM-CSF and/or IL33.
According to some embodiments of the invention, the depleting is effected ex-vivo.
According to some embodiments of the invention, the depleting is effected in-vitro.
According to some embodiments of the invention, the basophils are blood circulating basophils.
According to some embodiments of the invention, the basophils are lung resident basophils.
According to some embodiments of the invention, the depleting is effected in a local manner.
According to some embodiments of the invention, the disease or disorder that can benefit from increasing an M1/M2 macrophage ratio is cancer.
According to some embodiments of the invention, the disease or disorder that can benefit from increasing an M1/M2 macrophage ratio is melanoma.
According to some embodiments of the invention, the disease or disorder that can benefit from increasing an M1/M2 macrophage ratio is pulmonary fibrosis.
According to some embodiments of the invention, aid disease or disorder that can benefit from increasing an M 1/M2 macrophage ratio is selected from the group consisting of cancer, fibrotic diseases.
According to an aspect of some embodiments of the present invention there is provided a method of increasing an M1/M2 macrophage ratio, the method comprising depleting basophils having a lung basophil phenotype from a vicinity of macrophages or depleting activity of the basophils, thereby increasing M1/M2 macrophage ratio.
According to an aspect of some embodiments of the present invention there is provided a method of increasing an M2/M1 macrophage ratio, the method comprising enriching for basophils having a lung basophil phenotype in a vicinity of macrophages or an effector of the basophils, thereby increasing M2/M1 macrophage ratio.
According to some embodiments of the invention, the enriching is by GM-CSF and/or IL33.
According to some embodiments of the invention, the effector is selected from the group consisting of IL6, IL13 and HGF.
According to some embodiments of the invention, the method is effected ex-vivo.
According to some embodiments of the invention, the method is effected in-vivo.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
ILC (H) and basophils (I). Colored nodes represent genes up-regulated in the cell type (>2 fold change), and gray nodes represent their interacting partners. Full and empty circles represent ligands and receptors, respectively. *p<0.05, **p<0.01, ***p<0.005.
The present invention, in some embodiments thereof, relates to methods of modulating M2 macrophage polarization and use of same in therapy.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. The various macrophage functions are linked to the type of receptor interaction on the macrophage and the presence of cytokines. Similar to the T helper type 1 and T helper type 2 (TH1-TH2) polarization, two distinct states of polarized activation for macrophages have been defined: the classically activated (M1) macrophage phenotype and the alternatively activated (M2) macrophage phenotype. Similar to T cells, there are some activating macrophages and some suppressive macrophages, therefore, macrophages should be defined based on their specific functional activities. Classically activated (M1) macrophages have the role of effector cells in TH1 cellular immune responses. The alternatively activated (M2) macrophages appear to be involved in immunosuppression and tissue repair. For these reasons, modulating the ratio of Ml/M2 has been considered as a relevant approach for the treatment of inflammation and autoimmunity on the one hand and cancer on the other hand.
Whilst reducing the present invention to practice, the present inventors have identified a lung-resident population of basophils that reside in close proximity to alveoli. These basophils are characterized by a unique gene expression phenotype and cytokine/growth factor secretion. They play an important role in guiding the maturation and function of alveolar macrophages in the lung. It is suggested that a lung resident basophil phenotype is also a hallmark of disease conditions which are not limited to the lung, suggesting that they can be beneficial towards treating medical conditions that can benefit from Ml/M2 modulation.
Specifically, the present inventors report the extensive profiling of immune and non-immune lung cells by single cell RNA-sequencing of 50,770 cells along major time points of lung development. A highly diverse set of cell types and states was observed, and complex dynamics of developmental trajectories were identified, including three waves of macrophage types, from primitive cells to mature AM. Analysis of interacting ligands and receptors revealed a highly connected network of interactions, and highlighted basophils as cells expressing major growth factors and cytokine signaling in the lung. Basophils in the lung reside in close proximity to alveoli, and exhibit a lung specific phenotype, highly diverged from peripheral circulating basophils. Using Il1rl1 (IL-33 receptor) knockout mice and in vitro cultures, the present inventors discovered that lung basophils' education is mediated by the combinatorial imprinting of GM-CSF (Csf2) and IL-33 from the lung environment, and can be recapitulated in vitro by introducing these cytokines. Using antibody depletion strategies, diphtheria toxin-mediated selective depletion of basophils and in-vitro co-culture experiments, the present inventors demonstrate that basophils play an important role in guiding the maturation and function of alveolar macrophages (AM) in the lung. These findings open new clinical strategies to macrophage manipulation and basophil-based therapeutics.
Thus, according to an aspect of the invention, there is provided a method of increasing an M2/M1 macrophage ratio. The method comprises enriching for basophils having a lung basophil phenotype in a vicinity of macrophages or an effector of said basophils, thereby increasing M2/M1 macrophage ratio.
As used herein “M1 macrophages” refer to macrophages characterized by the expression of proinflammatory genes and are typically endowed with an effector function in TH1 cellular immune responses. M1 macrophages according to some embodiments of the present invention can be identified by using FACS, or by their cytokine secretion profile (e.g., TNFa, IL1b), and can be quantified by ELISA for instance or at the RNA level such as by using RT-PCR.
As used herein “M2 macrophages” refer to macrophages that are endowed with an immunosuppression activity and tissue repair. M2 macrophages according to some embodiments of the present invention can be quantified by cell number using specific markers (e.g., MRC1, ARG1) such as by using FACS, or by their cytokine secretion profile (e.g., IL-10, CCL17, CCL22) and can be quantified by ELISA for instance or at the RNA level such as by using RT-PCR.
As used herein “alveolar macrophages” or “AM” refer to a type of macrophages found in the pulmonary alveolus. AM originate from fetal liver embryonic precursors and are self-maintaining, with no contribution from the adult bone marrow.
Mouse AM can be identified using anti-CD45, anti-CD11c, anti-F4/80 and/or anti-SIGLEC-F.
Human AM can be identified using anti-CD45 and/or anti-CD11c
As used herein “increasing” refers to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or even 95%, increase in M2/M1 ratio (M2 polarization) as compared to that in the absence of said enrichment (e.g., GM-CSF, IL33, IL6 and/or IL13), as assayed by methods which are well known in the art (see Examples section which follows).
Increasing an M2/M1 macrophage ratio refers to M2 polarization.
As mentioned, the method of this aspect of the invention is performed by enriching for basophils having a lung basophil phenotype
The present inventors have shown that a lung basophil phenotype can be acquired in vitro (see Examples section which follows).
As used herein “a lung basophil phenotype” refers to a structural and/or functional phenotype.
According to a specific embodiment, the structural phenotype comprises a signature of Fcera1+, Il3ra+ (Cd123), Itga2+ (Cd49b), Cd69+, Cd244+ (2B4), Itgam+ (Cdl11b), Cd63+, Cd24a+, Cd200r3+, Il2ra+, Il18rap+ and C3ar1+; or Fcer1+, Il13ra1+, Itga2+, Cd69+, Cd244+, Itgam+, Cd63+, Cd24+, Il2ra+, Il18rap+ and C3ar1+ in the case of human cells.
According to an additional or alternative embodiment, the structural phenotype comprises expression of key cytokines and growth factors, such as Csf1, Il6, Il13, L1 cam, Il4, Ccl3, Ccl4, Ccl6, Ccl9 and Hgf.
According to an additional or alternative embodiment, the structural phenotype comprises expression of key cytokines and growth factors Il6, Il13, and Hgf.
According to an additional or alternative embodiment, the structural phenotype comprises a distinct gene expression profile of lung basophils from blood-circulating basophils, characterized by a unique gene signature that includes expression of Il6, Il13, Cxcl2, Tnf, Osm and Ccl4
A “functional phenotype” refers to the effect of M2 polarization on macrophages.
According to a specific embodiment, the basophils are mammalian basophils.
According to a specific embodiment, the basophils are human basophils.
According to an embodiment, the enriching is by contacting with GM-CSF and/or IL33.
According to an embodiment, the enriching is by contacting with GM-CSF and IL33.
As used herein “contacting” or methods described herein can be performed, in-vivo, ex-vivo or in-vitro.
According to a specific embodiment, the enriching is effected in vitro or ex vivo.
As used herein “basophils” refer to a specific type of leukocytes called granulocytes, which are characterized by large cytoplasmic granules that can be stained by basic dyes and a bi-lobed nucleus, being similar in appearance to mast cells, another type of granulocyte. Basophils are the least common granulocyte, making only 0.5% of the circulating blood leukocytes, and have a short life span of only 2-3 days (in vivo). Basophils are derived from granulocyte-monocyte progenitors in the bone marrow; where basophil precursors and mast cell precursors arise from an intermediate bipotent basophil-mast cell precursor (Arinobu et al. 2005 and Arinobu et al. 2009). Table 1 shows the markers associated with the different lineage cell types.
Basophils can be identified by the expression of certain markers, which is consistent between humans and mice, refer to Table 2.
According to a specific embodiment, basophils are isolated from the bone-marrow or peripheral blood.
According to a specific embodiment, basophils are produced as follows:
(i) isolating the basophils from bone-marrow.
(ii) differentiating the basophils from the peripheral blood in the presence of IL-3 to as to obtain a differentiated culture;
(iii) isolating from the differentiated culture a cKIT−population.
According to an exemplary protocol, bone marrow (BM) progenitors are harvested and cultured at a predetermined concentration e.g., of 0.1×106-1×106 cells per ml. For BM-derived macrophages (MΦ) differentiation, BM cells are cultured for 6-10 days, e.g., 8 days, in the presence of M-CSF. Then, cells are scraped. For BM-derived basophils differentiation, BM cells are cultured for 7-10 days, in the presence of IL-3 (e.g., 9-10 days). Following, basophils are enriched by magnetic-activated cell sorting for a CD117−population (cKit; Miltenyi Biotec), and re-plated for 16 hours. During differentiation, cultures can be in standard media.
Ex-vivo methods can be done in tissue culture or when possible in a closed system such as by apheresis.
Bone marrow cultures or circulating basophils (peripheral blood) cultures are treated with the differentiation factors. Culturing can be effected while supplementing with IL-3 (5-20 ng/ml, e.g., 10 ng/ml) and M-CSF (5-20 ng/ml, e.g., 10 ng/ml) for cell survival; and/or IL33 (30-70 ng/ml, e.g., 50 ng/ml) and/or GM-CSF (30-70 ng/ml, e.g., 50 ng/ml) for cell activation towards basophils that can regulate M2 polarization of macrophages. Typically, cell activation is performed for 48 hours or less, e.g., 6-48 hours, 12-48 hours, 24-48 hours, 12-36 hours, 18-24 hours, e.g., 24 hours (e.g., IL33+GM-CSF).
As used herein “in a vicinity of macrophages” can refer to a co-culture of basophils and macrophages. Alternatively, “in a vicinity of macrophages” can refer to enriching such that there is an effective amount of basophils having a lung basophil phenotype in vivo, or an effective amount of effectors of said basophils so as to allow polarization to M2 macrophages.
Effectors of basophils having a lung basophil phenotype include, but are not limited to IL6, IL13 and/or HGF (hepatocyte growth factor).
According to another aspect there is provided, a method of increasing an M 1/M2 macrophage ratio, the method comprising depleting basophils having a lung basophil phenotype from a vicinity of macrophages or depleting activity of said basophils, thereby increasing M1/M2 macrophage ratio.
Increasing M1/M2 macrophage ratio also refer to M1 polarization.
As used herein “increasing” refers to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or even 95%, increase in M1/M2 ratio (M1 polarization) as compared to that in the absence of said depletion, as assayed by methods which are well known in the art (see Examples section which follows).
Depleting basophils having a lung basophil phenotype can be effected by any method known in the art, some are described infra.
According to an embodiment, depletion can be effected by an agent targeting a basophil marker.
Such markers are described hereinabove e.g., Fcera1+, Il3ra+ (Cd123), Itaga2+ (Cd49b), Cd69+, Cd244+ (2B4), Itgam+ (Cd11b), Cd63+, Cd24a+, Cd200r3+, Il2ra+, Il18rap+ and C3ar1+; or Fcer1+, Il13ra1+, Itga2+, Cd69+, Cd244+, Itgam+, Cd63+, Cd24+, Il2ra+, Il18rap+ and C3ar 1+or as listed in Table 2.
According to a specific embodiment, the depletion is effected to specifically eliminate basophils having a lung basophil phenotype and not other cell populations (depletion of other cell populations is not affected by more than 20%, 15%, 10%, 5%, 1%, each value is considered a different embodiment).
According to a specific embodiment, such an agent can be an antibody such as an anti Fceral+antibody.
The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.
According to specific embodiments, the antibody comprises an Fc domain.
According to specific embodiments, the antibody is a naked antibody.
As used herein, the term “naked antibody” refers to an antibody which does not comprise a heterologous effector moiety e.g. therapeutic moiety.
According to specific embodiments, the antibody comprises a heterologous effector moiety typically for killing the basophils thereby increasing M1/M2 macrophage ratio. The effector moiety can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner. The effector moiety may be any molecule, including small molecule chemical compounds and polypeptides. Non-limiting examples of effector moieties include but are not limited to cytokines, cytotoxic antibodies, toxins, radioisotopes, chemotherapeutic antibody, tyrosine kinase inhibitors, and other therapeutically active antibodies. Additional description on heterologous therapeutic moieties is further provided hereinbelow.
The antibody may be mono-specific (capable of recognizing one epitope or protein), bi-specific (capable of binding two epitopes or proteins) or multi-specific (capable of recognizing multiple epitopes or proteins).
According to specific embodiments, the antibody is a mono-specific antibody.
According to specific embodiments, the antibody is bi-specific antibody.
Bi-specific antibodies are antibodies that are capable of specifically recognizing and binding at least two different epitopes. The different epitopes can either be within the same molecule or on different molecules such that the bi-specific antibody can specifically recognize and bind two different epitopes on a single RTN4 polypeptide as well as two different polypeptides. Alternatively, a bi-specific antibody can bind e.g. RTN4 and another effector molecule such as, but not limited to e.g. CD2, CD3, CD28, B7, CD64, CD32, CD16. Methods of producing bi-specific antibodies are known in the art and disclosed for examples in U.S. Pat. Nos. 4,474,893, 5,959,084, and 7,235,641, 7,183,076, U.S. Publication Number 20080219980 and International Publication Numbers WO 2010/115589, WO2013150043 and WO2012118903 all incorporated herein by their entirety; and include, for example, chemical cross-linking (Brennan, et al., Science 229,81 (1985); Raso, et al., J. BioI. Chern. 272, 27623 (1997)), disulfide exchange, production of hybrid-hybridomas (quadromas), by transcription and translation to produce a single polypeptide chain embodying a bi-specific antibody, or by transcription and translation to produce more than one polypeptide chain that can associate covalently to produce a bi-specific antibody. The contemplated bi-specific antibody can also be made entirely by chemical synthesis.
Antibodies with more than two valencies are also contemplated.
According to other specific embodiments, the antibody is a multi-specific antibody.
According to specific embodiments, the antibody is a conjugate antibody (i.e. an antibody composed of two covalently joined antibodies).
The antibody may be monoclonal or polyclonal.
According to specific embodiments, the antibody is a monoclonal antibody.
According to specific embodiments, the antibody is a polyclonal antibody.
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
It will be appreciated that for human therapy or diagnostics, humanized antibodies are preferably used.
According to specific embodiments, the antibody is a humanized antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
According to another embodiment, the depletion is effected by depleting activity of the basophils so as to prevent signal communication with the macrophages.
According to a specific embodiment, such an activity is of IL6, IL13 and/or HGF.
Inhibiting the activity of any of these molecules can be done using antibodies for those ligands, or soluble receptors, also referred to as “decoys” that bind to these ligands and prevent their function.
Typically, such soluble receptors comprise the extracellular portion of the receptor molecule and are devoid of the transmembrane domain(s) and the cytoplasmic domain(s).
The receptor of HGF is c-Met receptor.
The receptor for IL6 is Interleukin 6 receptor (IL6R) also known as CD126.
The receptor for IL13 is interleukin-13 receptor.
Small molecule inhibitors of c-MET, IL6R and IL13R are well known in the art and some are already in clinical use. Examples of c-Met inhibitors include, but are not limited to, class I and class II ATP-competitive small molecule c-Met inhibitors, e.g., JNJ-38877605, PF-04217903, XL880, foretinib and AMG458, as well as ATP-non-competitive small molecule c-Met inhibitors such as, Tivantinib (ARQ197). Examples of IL6R inhibitors (e.g, antibodies, Tocilizumab, Sarilumab), small molecules inhibitors of IL6 are taught in WO2013019690, incorporated hereinby reference. An examples of IL13R inhibitor is ASLAN004.
In order to ensure specificity to a specific tissue (when needed), the agent can be accompanied by a specific delivery vehicle e.g., directed to a tissue marker or administered in a local manner e.g., for pulmonary activity e.g., intranasal administration. Modes of administration are described hereinbelow.
As used herein “depletion” refers to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, even total elimination as determined by FACS of the desired cells, be them basophils of a lung phenotype or M2 macrophages.
Methods of Detecting the Expression Level of RNA
The expression level of the RNA in the cells of some embodiments of the invention can be determined using methods known in the arts.
Northern Blot analysis: This method involves the detection of a particular RNA in a mixture of RNAs. An RNA sample is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation. The individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or a nylon-based membrane to which the denatured RNAs adhere. The membrane is then exposed to labeled DNA probes. Probes may be labeled using radio-isotopes or enzyme linked nucleotides. Detection may be using autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of particular RNA molecules and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the gel during electrophoresis.
RT-PCR analysis: This method uses PCR amplification of relatively rare RNAs molecules. First, RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine. Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.
RNA in situ hybridization stain: In this method DNA or RNA probes are attached to the RNA molecules present in the cells. Generally, the cells are first fixed to microscopic slides to preserve the cellular structure and to prevent the RNA molecules from being degraded and then are subjected to hybridization buffer containing the labeled probe. The hybridization buffer includes reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) which enable specific hybridization of the DNA or RNA probes with their target mRNA molecules in situ while avoiding non-specific binding of probe. Those of skills in the art are capable of adjusting the hybridization conditions (i.e., temperature, concentration of salts and formamide and the like) to specific probes and types of cells. Following hybridization, any unbound probe is washed off and the bound probe is detected using known methods. For example, if a radio-labeled probe is used, then the slide is subjected to a photographic emulsion which reveals signals generated using radio-labeled probes; if the probe was labeled with an enzyme then the enzyme-specific substrate is added for the formation of a colorimetric reaction; if the probe is labeled using a fluorescent label, then the bound probe is revealed using a fluorescent microscope; if the probe is labeled using a tag (e.g., digoxigenin, biotin, and the like) then the bound probe can be detected following interaction with a tag-specific antibody which can be detected using known methods.
In situ RT-PCR stain: This method is described in Nuovo GJ, et al. [Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P, et al. [Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction. The reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arcturus Engineering (Mountainview, Calif.).
Methods of Detecting Expression and/or Activity of Proteins
Expression and/or activity level of proteins expressed in the cells of the cultures of some embodiments of the invention can be determined using methods known in the arts.
Enzyme linked immunosorbent assay (ELISA): This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
Western blot: This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
Radio-immunoassay (RIA): In one version, this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I125) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
In an alternate version of the RIA, a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
Fluorescence activated cell sorting (FACS): This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
Immunohistochemical analysis: This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies. The substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.
Ex-vivo or in-vitro cells or cell populations obtainable by any of the methods described herein are also contemplated according to some embodiments of the invention. Cell populations obtained according to some embodiments of the invention are characterized by a level of purity higher than that found in the physiological environment (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the cells are the cells of interest e.g., basophils, or cells differentiated therefrom or macrophages).
As mentioned any of the methods described can be effected ex-vivo or in-vivo.
The ability to modulate the balance between M1 and M2 macrophages, allows harnessing the present teachings towards therapy.
Thus, according to an aspect of the invention there is provided a method of treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject in need thereof, the method comprising:
(a) culturing basophils in the presence of IL33 and/or GM-SCF; and
(b) administering to the subject a therapeutically effective amount of the basophils following the culturing,
thereby treating the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in the subject.
According to another aspect there is provided a therapeutically effective amount of basophils having been generated by culturing in the presence of IL33 and/or GM-SCF for use in treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject in need thereof.
According to another aspect there is provided a method of treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a signaling molecule selected from the group consisting of IL6, IL13 and HGF, thereby treating the disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in the subject.
According to another aspect there is provided a therapeutically effective amount of a signaling molecule selected from the group consisting of IL6, IL13 and HGF for use in treating a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio in a subject.
As used herein “subject” refers to a subject suffering from a disease or disorder that can benefit from increasing an M1/M2 macrophage ratio or from a disease or disorder that can benefit from increasing an M2/M1 macrophage ratio. Alternatively, the subject is at a risk of developing such a disease or disorder.
When administering basophils, the cells can be autologus, non-autologous, allogeneic, syngeneic or xenogeneic (with the proper immune-suppression when needed).
As used herein “disease or disorder that can benefit from increasing M2/M1 macrophage ratio” refers to diseases or disorders (medical conditions in total) that can be ameliorated by suppressing the immune system.
Such typically include, but are not limited to, inflammation, autoimmunity, or injuries.
As used herein, the term “inflammatory disease” as used herein refers to acute or chronic localized or systemic responses to harmful stimuli, such as pathogens, damaged cells, physical injury or irritants, that are mediated in part by the activity of cytokines, chemokines, or inflammatory cells (e.g. macrophages) and is characterized in most instances by pain, redness, swelling, and impairment of tissue function. The inflammatory disease may be selected from the group consisting of: sepsis, septicemia, pneumonia, septic shock, systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), acute lung injury, aspiration pneumonitis, infection, pancreatitis, bacteremia, peritonitis, abdominal abscess, inflammation due to trauma, inflammation due to surgery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue damage due to chemotherapy or radiotherapy, and reactions to ingested, inhaled, infused, injected, or delivered substances, glomerulonephritis, bowel infection, opportunistic infections, and for subjects undergoing major surgery or dialysis, subjects who are immunocompromised, subjects on immunosuppressive agents, subjects with HIV/AIDS, subjects with suspected endocarditis, subjects with fever, subjects with fever of unknown origin, subjects with cystic fibrosis, subjects with diabetes mellitus, subjects with chronic renal failure, subjects with bronchiectasis, subjects with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, subjects with febrile neutropenia, subjects with meningitis, subjects with septic arthritis, subjects with urinary tract infection, subjects with necrotizing fasciitis, subjects with other suspected Group A streptococcus infection, subjects who have had a splenectomy, subjects with recurrent or suspected enterococcus infection, other medical and surgical conditions associated with increased risk of infection, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, meningococcemia, post-pump syndrome, cardiac stun syndrome, stroke, congestive heart failure, hepatitis, epiglotittis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, pre-eclampsia, eclampsia, HELP syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhagic fever, pelvic inflammatory disease, Legionella, Lyme disease, Influenza A, Epstein-Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, progressive systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, systemic vasculitis, Wegener's granulomatosis, transplants including heart, liver, lung kidney bone marrow, graft-versus-host disease, transplant rejection, sickle cell anemia, nephrotic syndrome, toxicity of agents such as OKT3, cytokine therapy, cryoporin associated periodic syndromes and cirrhosis.
As used herein, an “autoimmune disease” is a disease or disorder arising from and directed at an individual's own tissues. Examples of autoimmune diseases include, but are not limited to Addison's Disease, Allergy, Alopecia Areata, Alzheimer's disease, Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, Ankylosing Spondylitis, Antiphospholipid Syndrome (Hughes Syndrome), arthritis, Asthma, Atherosclerosis, Atherosclerotic plaque, autoimmune disease (e.g., lupus, RA, MS, Graves' disease, etc.), Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia, Behcet's Disease, Berger's Disease, Bullous Pemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue/Coeliac disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis, Chronic Inflammatory Demyelinating, Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy (Guillain-Barre syndrome), Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), chronic obstructive pulmonary disease (COPD), CREST syndrome, Crohn's disease, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema, Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia, Evan's Syndrome, Exopthalmos, Fibromyalgia, Goodpasture's Syndrome, Hashimoto's Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder (e.g., psoriasis), Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, Insulin Dependent Diabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes, Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki's Disease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, lupus, Lupus Nephritis, Lymphoscytic Lypophisitis, Meniere's Disease, Miller Fish Syndrome/acute disseminated encephalomyeloradiculopathy, Mixed Connective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes (Whitaker's syndrome), Polymyalgia Rheumatica, Polymyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis/Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis, Raynaud's Phenomenon, Reiter's Syndrome/Reactive arthritis, Restenosis, Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis, Schmidt's syndrome, Scleroderma, Sjorgen's Syndrome, Stiff-Man Syndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2 diabetes, Ulcerative colitis, Uveitis, Vasculitis, Vitiligo, and Wegener's Granulomatosis.
As used herein “disease or disorder that can benefit from increasing an M1/M2 macrophage ratio” refers to diseases or disorders (medical conditions in total) that can be ameliorated by activating the immune system such as evidenced by the secretion of pro-inflammatory cytokines.
Such typically include, but are not limited to, cancer, e.g., metastatic cancer, progressive fibrotic diseases such as for example idiopathic pulmonary fibrosis (IPF), hepatic fibrosis systemic sclerosis, allergy and asthma, atherosclerosis and Alzheimer's disease, pulmonary fibrosis, liver fibrosis. In particularly, the method of the present invention is particularly suitable for the treatment of cancer. As used herein, the term “cancer” has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangio sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In some embodiments, the method of the present invention is particularly suitable for the treatment of metastatic cancer to bone, wherein the metastatic cancer is breast, lung, renal, multiple myeloma, thyroid, prostate, adenocarcinoma, blood cell malignancies, including leukemia and lymphoma; head and neck cancers; gastrointestinal cancers, including esophageal cancer, stomach cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignancies of the female genital tract, including ovarian carcinoma, uterine endometrial cancers, vaginal cancer, and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer, including malignant melanoma or squamous cell cancer.
The cells or agents (e.g., cytokines, growth factors, antibodies) of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term “active ingredient” refers to the cells or agents (e.g., cytokines, growth factors, antibodies) accountable for the biological effect.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. According to a specific embodiment, the localized treatment is to the lung such as by intranasal administration.
Pulmonary administration cells or agents as described herein.
Pulmonary administration may be accomplished by suitable means known to those in the art. Typically, pulmonary administration requires dispensing of the biologically active substance from a delivery device into the oral cavity of a subject during inhalation. For example, compositions comprising cells or agents are administered via inhalation of an aerosol or other suitable preparation that is obtained from an aqueous or nonaqueous solution or suspension form, or a solid or dry powder form of the pharmaceutical composition, depending upon the delivery device used. Such delivery devices are well known in the art and include, but are not limited to, nebulizers, metered dose inhalers, and dry powder inhalers, or any other appropriate delivery mechanisms that allow for dispensing of a pharmaceutical composition as an aqueous or nonaqueous solution or suspension or as a solid or dry powder form. Methods for delivering cells or agents, to a subject via pulmonary administration, including directed delivery to the central and/or peripheral lung region(s), include, but are not limited to, a dry powder inhaler (DPI), a metered dose inhaler (MDI) device, and a nebulizer.
The term “tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (cells or agents (e.g., cytokines, growth factors, antibodies)) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., as described above) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1).
Dosage amount and interval may be adjusted individually to provide effective (e.g., the lung tissue) levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
As used herein the phrase “treatment regimen” refers to a treatment plan that specifies the type of treatment, dosage, schedule and/or duration of a treatment provided to a subject in need thereof (e.g., a subject diagnosed with a pathology). The selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the pathology) or a more moderate one which may relief symptoms of the pathology yet results in incomplete cure of the pathology. It will be appreciated that in certain cases the more aggressive treatment regimen may be associated with some discomfort to the subject or adverse side effects (e.g., a damage to healthy cells or tissue). The type of treatment can include a surgical intervention (e.g., removal of lesion, diseased cells, tissue, or organ), a cell replacement therapy, an administration of a therapeutic drug (e.g., receptor agonists, antagonists, hormones, chemotherapy agents) in a local or a systemic mode, an exposure to radiation therapy using an external source (e.g., external beam) and/or an internal source (e.g., brachytherapy) and/or any combination thereof. The dosage, schedule and duration of treatment can vary, depending on the severity of pathology and the selected type of treatment, and those of skills in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.
As used herein the term “about” refers to ±10%
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells - A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, CA (1990); Marshak et al., “Strategies for Protein Purification and Characterization - A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Mice
Sex- and age-matched Mcpt8-Cre+/−DTAfl/+ and Mcpt8-Cre+/−DTA+/+ littermate controls were used. YFP-expressing Mcpt8-Cre (B6.129-Mcpt8tml(Cre)Lksy/J) (Sullivan et al., 2011) and DTA (B6.129P2-Gt(ROSA) 26Sortml (DTA)Lky/J) (Voehringer et al., 2008) mice were kindly provided by Stephen Galli, Stanford University, and originally obtained from the Jackson Laboratory. Il1rl1−/− (Townsend et al., 2000) mice were kindly provided by Andrew McKenzie, MRC Laboratory of Molecular Biology Cambridge. All these mice were bred and maintained at the animal facility of the Medical University of Vienna under specific pathogen free conditions. All experiments were performed in accordance with Austrian law and approved by the Austrian Federal Ministry of Sciences and Research (BMWFW-66.009/0146-WF/V/3b/2015). C57BL/6 WT pregnant, neonate and adult mice were obtained from Harlan. Mice were housed under specific-pathogen-free conditions at the Animal Breeding Center of the Weizmann Institute of Science. All animals were handled according to the regulations formulated by the Institutional Animal Care and Use Committee.
Tumor Cell Line
B 16F10 murine melanoma cells were maintained in DMEM, supplemented with 10% FCS, 100 U/mL penicillin, 100 mg/mL streptomycin and 1 mM 1-glutamine (Biological Industries). Cells were cultured in a humidified 5% CO2 atmosphere, at 37° C.
Method Details
Lung dissociation and single cell sorting
Single-cell experiments were performed on embryonic mouse lung at E12.5, E16.5, E18.5 and E19.5, on neonate lung at 1, 6, 7, 10, 16, 30h, 2 days, and 7 days PN, and on adult mouse lung (8-12 weeks). In general, embryonic experiments were performed on pooled sibling lungs of one litter (at E12.5 six lungs were pooled, at E16.5, E18.5 and E19.5 three lungs were pooled, at PN time points 2 lungs were pooled, and for adult lungs, samples were not pooled). Embryos were euthanized by laying on a frozen surface, while PN and adult mice were scarified by overdose of anesthesia. For all time points, except E12.5, mice were perfused by injection of cold PBS via the right ventricle prior to lung dissection. Lung tissue was dissected from mice and half tissues were homogenized using lung dissociation kit (Miltenyi Biotec), while enzymatic incubation was adapted to single cell protocol, and therefore was lasted 15 min (for 8 week adult mice, enzymatic digestion was lasted 20 min). The second half of the lung was dissociated as previously documented (Treutlein et al., 2014), briefly cells were supplemented with DMEM/F12 medium (Sigma-Aldrich) containing Elastase (3 U/ml, Worthington) and
DNase (0.33 U/ml, Sigma-Adrich) incubated with frequent agitation at 37° C. for 15 min. Next, an equal volume of DMEM/F12 supplemented with 10%FBS, 1 U/ml penicillin, and 1Uml streptomycin (Biological Industries) was added to single-cell suspensions. Following dissociations, single cell suspension of the same lung was merged and centrifuged at 400 g, 5 min, 4° C. All samples were filtered through a 70 μm nylon mesh filter into ice cold sorting buffer (PBS supplemented with 0.2mM EDTA pH8 and 0.5% BSA).
For calibration of lung dissociation protocol, cells derived from adult mouse lungs were supplemented with 1). DMEM (Biological Industries) containing Liberase (50 μg/ml, Sigma-Aldrich) and DNase (1 μg/ml, Roche); 2). PBS Ca+Mg+(Biological Industries) containing Collagenase IV (1 mg/ml, Worthington) and Dispase (2.4 U/ml, Sigma-Adrich); 3). DMEM/F12 (Sigma-Aldrich) containing Elastase and DNase, as described above; and 4). Enzymes derived from lung dissociation kit (Miltenyi biotec), as described above. Following enzymatic digestion with frequent agitation at 37° C. for 20 min, an equal volume of DMEM supplemented with 10% FBS, 1 U/ml penicillin, and 1Uml streptomycin (Biological Industries), or sorting buffer was added to single-cell suspensions from liberase and collagenase-dispase treatments, respectively. All live cells were sorted, after exclusion of doublets and erythrocytes, for MARS-seq analysis. Single cell analysis of cells extracted by each dissociation technique showed differential distribution of cell types (not shown). Next, we chose dissociation protocol for the study that extracted vast range of cell populations from the immune and the non-immune compartments, without any preference to specific cell type stemming from the dissociation enzymes. Therefore, lung digestions along the study were a combination of elastase digestion, which lead to the extraction of epithelial cells and AM, and miltenyi kit protocol, which led to the extraction of different cell populations from the immune compartment. Importantly, these digestions were not characterized in any cell type preference, like endothelium dominancy that we found following collagenase-dispase and liberase treatments (not shown); however, the percentages of cells observed in the single cell maps are dependent on the different lung dissociation methods (
Isolation of Peripheral Blood Cells
Peripheral blood cells were suspended with 200 of heparin, and washed with PBS supplemented with 0.2mM EDTA pH8 and 0.5% BSA. Cells were suspended with ficoll-Paque™ PLUS (1:1 ratio with PBS, Sigma-Adrich) and centrifuged at 460 g, 20 min, 10° C., with no-break and no-acceleration. The ring-like layer of mononuclear cells was transferred into new tube and washed twice with cold PBS, centrifuged at 400 g, 5 min, 4° C., passed through a 40i.tm mesh filter, and then suspended in ice-cold sorting buffer.
Tumor Microenvironment Dissociation
For purification of basophils from tumor microenvironment, 1×106 cells were suspended in 1000 μl PBS and injected subcutaneous (s.c.) into 8-week mice. Solid tumors were harvested 10 days post injection, cut into small pieces, and suspended with RPMI-1640 supplemented with DNase (12.5 μg/ml, Sigma-Adrich) and collagenase IV (1 mg/ml, Worthington). Tissues were homogenized by GentleMacs tissue homogenizer (Miltenyi Biotec), and incubated at 37° C. for 10 min. Following two times of mechanic and enzymatic dissociation, cells were washed and suspended in red blood lysis buffer (Sigma-Aldrich) and DNase (0.33 U/ml, Sigma-Adrich), incubated for 5 min at room temperature, washed twice with cold PBS, passed through a 40 μm mesh filter, centrifuged at 400 g, 5 min, 4° C. and then resuspended in ice cold sorting buffer.
Spleen Dissociation
Tissue was harvested from 8 week females, suspended with accutase solution (Sigma-Adrich), homogenized by GentleMacs tissue homogenizer (Miltenyi Biotec), and incubated with frequent agitation at 37° C. for 10 min. Cells were washed and suspended in red blood lysis buffer (Sigma-Aldrich) and DNase (0.33 U/ml, Sigma-Adrich), incubated for 3 min at room temperature, washed twice with cold PBS, passed through a 40 μm mesh filter, centrifuged at 400 g, 5 min, 4° C. and then resuspended in ice cold sorting buffer.
Liver Dissociation
Basophils from the liver were isolated by a modification of the two-step collagenase perfusion method of Seglen (Seglen, 1973). Digestion step was performed with Liberase (20μg/ml; Roche Diagnostics) according to the manufacturer's instruction. Liver was minced to small pieces, suspended with PBS and centrifuged at 30 g, 5 min, 4° C. Supernatant was collected in new tube (to remove hepatocytes), suspended with PBS and centrifuged at 30 g, 5 min, 4° C. (this step was repeated twice). Following second wash, supernatant was collected in new tube, centrifuged at 500 g, 5 min, 4° C., and then resuspended in ice-cold sorting buffer.
Flow Cytometry and Sorting
Cell populations were sorted with SORP-aria (BD Biosciences, San Jose, Calif.) or with AriaFusion instrument (BD Biosciences, San Jose, Calif.). Samples were stained using the following antibodies: eF780-conjugated Fixable viability dye, eFluor450-conjugated TER-119, APC-conjugated CD45, FITC-conjugated CD117 (cKit), and PerCPCy5.5-conjugated F4/80 were purchased from eBioscience, PerCP Cy5.5-conjugated FCERal (MARI), APC-Cy7-conjugated Ly6G, FITC-conjugated CD3, PE-Cy7-conjugated CD19, PE-Cy7-conjugated CD31, APC-Cy7-conjugated CD326, APC/Cy7-conjugated TER-119, AF700-conjugated CD45, Pacific blue-conjugated CD49b, PE-conjugated Fcer1 a, PE/Cy7-conjugated CD117, FITC-conjugated Ly6C, PE-conjugated CD11c, BV605-conjugated CD11b and BV605-conjugated Ly-6C were purchased from Biolegend, and FITC-conjugated CD11C was purchased from BD-Pharmingen. Prior to sorting, cells were stained with DAPI or fixable viability dye for evaluation of live/dead cells, and then filtered through a 40 μm mesh. For the sorting of whole immune cell populations, samples were gated for CD45+, for sorting of whole stromal cell samples were gated for CD45−, and for the isolation of basophils, samples were gated for CD45+FCεR1α630 cKit−, after exclusion of doublets, dead cells and erythrocytes. To record marker level of each single cell, the FACS Diva 7 “index sorting” function was activated during single cell sorting. Following the sequencing and analysis of the single cells, each surface marker was linked to the genome-wide expression profile. This methodology was used to optimize the gating strategy. Isolated live cells were single-cell sorted into 384-well cell capture plates containing 2lit of lysis solution and barcoded poly(T) reverse-transcription (RT) primers for single-cell RNA-seq (Jaitin et al., 2014; Paul et al., 2015). Four empty wells were kept in each 384-well plate as a no-cell control during data analysis. Immediately after sorting, each plate was spun down to ensure cell immersion into the lysis solution, and stored at −80° C. until processed.
For evaluation of protein levels of receptors expressed by lung basophils, we performed cell surface staining of PE-conjugated CD131 (CSF2Rb, Miltenyi Biotec), PE/Cy7-conjugated IL-33R (Biolegend), and PacificBlue-conjugated CD49b (Biolegend). For evaluation of intracellular protein levels of ligands expressed by lung basophils, cells were incubated with RPMI-1640 supplemented with 10% FCS, 1mM 1-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries) and GolgiStop (1:1000; for IL-13, BD bioscience, San Jose, Calif.), or Brefeldin A solution (1:1000, for IL-6, Biolegend), for 2h at 37° C., to enable expression of intracellular cytokines, and to prevent their extracellular secretion. Cells were washed, fixed, permeabilized and stained for surface and intracellular proteins using the Cytofix/Cytoperm kit, according to the manufacture's instructions (BD bioscience, San Jose, Calif.). For the intracellular experiments the following antibodies were used: PE-conjugated IL-6 (Biolegend), PE-conjugated IL-13 (eBioscience) and matched Isotype control PE-conjugated Rat IgG1 (Biolegend). Cells were analyzed using BD FACSDIVA software (BD Bioscience) and FlowJo software (FlowJo, LLC).
BM derived cell cultures
BM progenitors were harvested from C57BL/6 8 week old mice and cultured at concentration of 0.5×106 cells/ml. For BM-MΦ differentiation, BM cultures were cultured for 8 days in the presence of M-CSF (50 ng/ml; Peprotech). On day 8, cells were scraped with cold
PBS and replated on 96-well flat bottom tissue culture plates for 16h. For BM-derived basophils differentiation, BM cultures were cultured for 10 days in the presence of IL-3 (30 ng/ml; Peprotech). Basophils were enriched by magnetic-activated cell sorting for CD117−population (cKit; Miltenyi Biotec), and replated on 96-well flat bottom tissue culture plates for 16h. All BM cultures were done in the standard media RPMI-1640 supplemented with 10% FCS, 1mM 1-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries). Every 4 days BM cultures were treated with differentiation factors M-CSF (50 ng/ml) or IL-3 (30 ng/ml). Following replating of BM-derived cells, co-cultured and mono-cultured cells were seeded in concentration of 0.5×106 cells/ml (1:1 ration in co-cultures), and supplemented with IL-3 (10 ng/ml) and M-CSF (lOng/ml) for cell survival, IL33 (50 ng/ml; Peprotech) or GM-CSF (50 ng/ml; Peprotech) for cell activation.
For co-culture of BM-basophils with lung-derived monocytes and undifferentiated macrophages, we sorted CD45+CD115+ myeloid cells from 30h PN lungs and performed the in vitro experiment, as detailed above.
MARS-Seq Library Preparation
Single-cell libraries were prepared as previously described (Jaitin et al., 2014). In brief, mRNA from cell sorted into cell capture plates were barcoded and converted into cDNA and pooled using an automated pipeline. The pooled sample is then linearly amplified by T7 in vitro transcription, and the resulting RNA is fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration is assessed as described earlier (Jaitin et al., 2014).
Lung-Resident Basophil Depletion
For depletion of basophils in neonate lungs, we calibrated a protocol based on previous studies (Denzel et al., 2008; Guilliams et al., 2013). Mice were injected i.n. with 7 μl of 100 μg anti-Fcεr1α (MARI; eBioscience) or IgG isotype control (Armenian hamster, eBioscience) twice, at 10h and 15h following birth. Lungs were purified from injected neonates 30h following birth and CD45+ cells were sorted for RNA-seq analysis.
Phagocytosis Assay
Phagocytosis assays were performed as described earlier (Sharif et al., 2014). AM were isolated by bronchoalveolar lavage (BAL). In brief, the trachea of mice was exposed and cannulated with a sterile 18-gauge venflon (BD Biosciences) and 10m1 of sterile saline were instilled in 0.5m1 steps. Total cell numbers in the retrieved BAL fluid (comprising >95% AM) were counted using a Neubauer chamber. To assess bacterial phagocytosis, 1-2.5×105 AM were plated and allowed to adhere for 3h in RPMI containing 10% fetal calf serum (FCS), 1% penicillin and 1% streptomycin. Next, AM were incubated with FITC-labeled heat-inactivated S. pneumoniae (MOI 100) for 45 min at 37° C. or 4° C. (as a negative control). Cells were washed and incubated with proteinase K (50 m/ml) for 10 min on ice to remove adherent bacteria. Uptake of bacteria was assessed via flow cytometry and the phagocytosis index was calculated as (MFI× % positive cells at 37° C.) minus (MFI× % positive cells at 4° C.).
Single-Molecule Fluorescent in situ Hybridization (smFISH)
Neonates in the age of 7 days were perfused with PBS. Lung tissues harvested and fixed in 4% paraformaldehyde for 3h at 4° C., incubated overnight with 30% sucrose in 2% paraformaldehyde at 4° C. and then embedded in OCT. Cryo-sections (6 μm) were used for hybridization. Probe libraries were designed and constructed as previously described (Itzkovitz et al., 2012, Stellaris Fish Probes # SMF-1082-5, SMF-1063-5, SMF 1065-5). Single molecule FISH probe libraries consisted of 48 probes of length 20 bps. smFISH probe libraries of Il1r11, Il33, and Mcpt8 probes were coupled to Cy3, AF594, and cy5, respectively. Hybridizations were performed overnight in 30° C. DAPI dye for nuclear staining was added during the washes. Images were taken with a Nikon Ti-E inverted fluorescence microscope equipped with a x60 and x100 oil-immersion objective and a Photometrics Pixis 1024 CCD camera using MetaMorph software (Molecular Devices, Downington, Pa.). smFISH molecules were counted only within the DAPI staining of the cell.
Histology and Immunohistochemistry
For histologic examination, paraffin-embedded lung sections were taken at indicated time-points. To stain for proSP-C, endogenous peroxidase activity was quenched and antigen was retrieved with Antigen Unmasking Solution (Vector Laboratories, H-3300). Blocking was done in donkey serum and the slides were then stained with anti-proSP-C (Abcam), followed by secondary goat-anti-rabbit IgG antibody (Vector Laboratories), and signal amplification using the Vectastain ELITE kit (Vector Laboratories). For F4/80 staining, antigen was retrieved using protease type XIV (SIGMA), followed by blocking with rabbit serum and staining with rat-anti-mouse F4/80 mAb (AbD Serotec). A secondary rabbit-anti-rat IgG Ab (Vector Laboratories) was applied and the signal was amplified with Vectastain ELITE kit (Vector Laboratories). For Mcpt8 staining, an anti-GFP Ab (Abcam) was used followed by a secondary biotinylated rabbit-anti-goat IgG Ab (Vector Laboratories). For detection, Peroxidase Substrate kit (Vector) or Vector VIP Peroxidase Kit (Vector Laboratories) was applied. Cell structures were counter-stained with hematoxylin or methylgreen and pictures were taken on an Olympus FSX100 Microscope.
For whole lobe analysis, slides were scanned using a TissueFAXS imaging system (TissueGnostics GmbH) equipped with a Zeiss Axio Imager.Z1 microscope (Carl Zeiss Inc., Jena, Germany). Images were taken using a PCO PixelFly camera (Zeiss).
Tissue Clearing
Tissue clearing protocol was performed as described earlier (Fuzik et al., 2016). In short, lungs at indicated time-points were perfused once with PBS and afterwards with 7.5% formaldehyde in PBS. Lung lobes were fixed in 7.5% formaldehyde in PBS at room temperature overnight. Lung lobes were cleared using CUBIC reagent 1 (25 wt % urea, 25 wt % N,N,N′,N′-tetrakis(2-hydroxypropyl) ethylenediamine and 15 wt % Triton X-100) for 4 days (30h PN, day 8.5) or 7 days (8-weeks) at 37° C. After repeated washes in PBS, lung lobes were incubated in blocking solution (PBS, 2.5% BSA, 0.5% Triton X-100, 3% normal donkey serum) and afterwards placed in primary antibody solution (1:100; goat anti-mouse GFP, abcam) for 4 days (30h PN, day 8.5) or 5 days (8-weeks) at 37° C. After washing the secondary antibody solution (1:500; donkey anti-goat AF555, Invitrogen) was added for 4 days (30h PN, day 8.5) or 5 days (8-weeks) at 37° C. After re-washing with PBS and a fixing step for 2h at room temperature in 7.5% formaldehyde, washing steps were repeated and lung lobes were incubated in CUBIC reagent 2 (50 wt % sucrose, 25 wt % urea, 10 wt % 2,20,20′-nitrilotriethanol and 0.1% v/v % Triton X-100) for another 4 days (30h PN, day 8.5) or 7 days (8-weeks). Cleared lung lobes were imaged in CUBIC reagent 2 with a measured refractive index of 1.45 using a Zeiss Z1 light sheet microscope through 5× detection objective, 5× illumination optics at 561 laser excitation wavelength and 0.56× zoom. Z-stacks were acquired in multi-view tile scan mode by dual side illumination with light sheet thickness of 8.42 μm and 441.9 ms exposure. Stitching, 3D reconstruction, visualization and rendering was performed using Arivis Vision4D Zeiss Edition (v.2.12).
Quantification and Statistical Analysis
Low Level Processing and Filtering
All RNA-Seq libraries (pooled at equimolar concentration) were sequenced using Illumina NextSeq 500 at a median sequencing depth of 58,585 reads per single cell. Sequences were mapped to mouse genome (mm9), demultiplexed, and filtered as previously described (Jaitin et al., 2014), extracting a set of unique molecular identifiers (UMI) that define distinct transcripts in single cells for further processing. We estimated the level of spurious UMIs in the data using statistics on empty MARS-seq wells (median noise 2.7%; not shown). Mapping of reads was done using HISAT (version 0.1.6) (Kim et al., 2015); reads with multiple mapping positions were excluded. Reads were associated with genes if they were mapped to an exon, using the UCSC genome browser for reference. Exons of different genes that shared genomic position on the same strand were considered a single gene with a concatenated gene symbol. Cells with less than 500 UMIs were discarded from the analysis. After filtering, cells contained a median of 2,483 unique molecules per cell. All downstream analysis was performed in R.
Data Processing and Clustering
The Meta-cell pipeline (Giladi et al., 2018) was used to derive informative genes and compute cell-to-cell similarity, to compute K-nn graph covers and derive distribution of RNA in cohesive groups of cells (or meta-cells), and to derive strongly separated clusters using bootstrap analysis and computation of graph covers on resampled data. A full description of the method and downstream analysis is depicted in Figures. Default parameters were used unless otherwise stated.
Clustering of lung development was performed for the immune (CD45+) and non-immune (CD45−) compartments combined. Cells with high (>64) combined expression of hemoglobin genes were discarded (Hba-a2, Alas2, Hba-a1, Hbb-b2, Hba-x, Hbb-b1). We used bootstrapping to derive robust clustering (500 iterations; resampling 70% of the cells in each iteration, and clustering the co-cluster matrix with minimal cluster size set to 20). No further filtering or cluster splitting was performed on the meta-cells.
In order to annotate the resulting meta-cells into cell types, we used the metric FP - gene,mc (not shown), which signifies for each gene and meta-cell the fold change between the geometric mean of this gene within the meta-cell and the median geometric mean across all meta-cells. The FP metric highlights for each meta-cell genes which are robustly over-expressed in it compared to the background. We then used this metric to “color” meta-cells for the expression of lineage specific genes such as Clic5 (AT1), Ear2 (macrophages), and Cd79b (B cells), etc. Each gene was given a FP threshold and a priority index—such that coloring for AT1 by Clic5 is favored over coloring for general epithelium by Epcam. The selected genes, priority, and fold change threshold parameters are as follows:
Trajectory Finding
To infer trajectories and align cells along developmental pseudo-time, we used the published package Slingshot (Street et al., 2017). In short, Slingshot is a tool that uses pre-existing clusters to infer lineage hierarchies (based on minimal spanning tree, MST) and align cells in each cluster on a pseudo-time trajectory. Since our data is complex and contains many connected components and time points, we chose to apply Slingshot on subsets of interconnected cells type, namely E16.5 monocytes and macrophage II and III (dataset a), and the fibroblast lineage (dataset b).
For dataset a, we performed Slingshot on all macrophages II-III and on monocyte meta-cells with low relative expression of Ly6c2 (excluding differentiated monocytes and retaining E16.5 monocytes). For each dataset we chose a set of differential genes between the cell types (FDR corrected chi2test, q<10−3, fold change>2). We performed PCA on the log transformed UMI normalized to cell size. We ran Slingshot on the seven top principal components, with monocytes and early fibroblasts as starting clusters.
We first observe strong AT1 and AT2 signatures on day E18.5. This is parallel to disappearance of progenitor epithelium cells. From this we hypothesized that the precise branching point is not sampled with high temporal resolution in our developmental cohort, rendering Slingshot inefficient for this particular case. Instead, we examined whether progenitor epithelial cells on day E16.5 may be already primed toward either AT1 or AT2. To detect AT1 AT2 priming in epithelium progenitors, we used published gene lists of AT1 and AT2 (Treutlein et al., 2014) and computed two scores by the following term: Σgenelog(1+7*UMIgenecell). We then examined score distribution in epithelium progenitors.
Interaction Maps
To visualize all lung interactions, we used a published dataset of ligand and receptor pairs (Ramilowski et al., 2015). We applied a lenient filtering, including all LR with >13 UMI in at least one meta-cell (normalized to meta-cell size). We computed the Spearman correlation between the log transformed UMI (down-sampled to 1000 UMI), and used hierarchical clustering to identify LR modules (cutree with K=15). We built a scaffold of an interaction graph by computing the Spearman correlation between LR modules and connecting edges between modules with ρ>0.4, generating a graph with the Rgraphviz package. We projected single LR on the graph scaffold by computing the mean x,y coordinates across all LR with ρ>0.05 (
To determine enrichment of stroma-stroma and immune-immune interactions we determined for each LR whether it's mainly expressed in the stromal or the immune compartments (log2 fold change >1, not shown). We computed the number of S-S and I-I interactions and compared to 10,000 randomly generated graphs. Importantly, as the interaction graph is not regular, we preserved nodes' degrees for each randomly generated graph. Ligand functional groups were extracted from David GO annotation tool (Huang da et al., 2009), and curated manually.
For projections in
Mapping Cells to the Lung Cluster Model
Given an existing reference single cell dataset and cluster model, and a new set of single cell profiles, we extract for each new cell the K (K=10) reference cells with top Pearson correlation on transformed marker gene UMIs as described above. The distribution of cluster memberships over these K-neighbors was used to define the new cell reference cluster (by majority voting).
Basophil Profiling, Ex Vivo and Co-Culture Analysis
We used the MetaCell pipeline to analyze and filter the following datasets: (a) lung and blood derived basophils (
To compute the combined expression of genes in single cells (
TissueFAXS Quantification
TissueFAXS images were processed by MATLAB (R2014b). Segmentation of alveoli was performed by a custom-made pipeline. Images were converted to grayscale and enhanced, opened and closed with a disk size of 15 pixels. Alveoli were determined by intensity threshold of 200. Areas larger than 300,000 pixels were discarded. Segmentation of nuclei was performed by a similar pipeline (disk size=5 pixels), followed by applying a watershed algorithm, and detection of local minima. Images were converted to L*A*B color-space, and mean values of each nucleus were collected. Nuclei at the edges of the section were discarded. Nuclei with area <Tarea, mean luminance >T1 or high circularity score (>Tcirc) were discarded. Nuclei distances to alveoli (in pixels) were calculated with the bwdist method. Basophils (which are YFP+) are distinguished from other nuclei by their dark brownish hue (
Data and Software Availability All reported data will be uploaded and stored in GEO, accession number GSE119228. Software and custom code will be available by request.
A comprehensive map of the lung cell types during development To understand the contribution of different immune and non-immune cell types and states for lung development and homeostasis, we collected single cell profiles along critical time points of lung development. In order to avoid biases stemming from cell-surface markers or selective tissue dissociation procedures, we combined a broad gating strategy and permissive tissue dissociation protocol, resulting in a comprehensive repertoire of the immune and non-immune cells located in the lung (not shown; Methods). We densely sampled cells from multiple time points of lung embryonic and postnatal development, and performed massively parallel single cell RNA-seq coupled to index sorting (MARS-seq) (Jaitin et al., 2014) (
Lung Compartmentalization is Shaped by Waves of Cellular Dynamics
During embryogenesis and soon after birth, the lung undergoes dramatic environmental changes with its maturation and abrupt exposure to airborne oxygen. Accordingly, our analysis shows that meta-cell composition varies widely at these time points (
In accordance with previous works (Kopf et al., 2015; Tan and Krasnow, 2016), we identified three distinct macrophage subsets, which we term macrophage I-III. These subsets appeared in waves during development, with macrophage I dominating in early pregnancy, macrophage II culminating around birth, and macrophage III steadily increasing since late pregnancy stage, and becoming the majority on day 7 PN (
Lung basophils broadly interact with the immune and non-immune compartments In multicellular organisms, tissue function emerges as heterogeneous cell types form complex communication networks, which are mediated primarily by interactions between ligands and receptors (LR) (Zhou et al., 2018). Examining LR pairs in single cell maps can potentially reveal central cellular components shaping tissue fate (Camp et al., 2017; Zhou et al., 2018). In order to systematically map cellular interactions between cells and reveal potential communication factors controlling development, we characterized LR pairs between all lung cell types (
In the developing lung, modules of LR mainly clustered by cell type (not shown). However, for some LR we could identify significant changes in expression levels in the same cell type during development (not shown). We projected ligands and receptors based on their correlation structure, resulting in a graphical representation of all LR and their interactions, which highlighted their separation into cell type related modules (
(Methods). As expected, cytokines and components of the complement system were found mainly in the immune compartment, as well as the receptors recognizing them (
To identify important cellular communication hubs involved in a large number of interactions between and within compartments, we examined LR expression patterns across different cell types (not shown). From the non-immune compartment, smooth muscle fibroblasts, expressing Tgfb3 and the Wnt ligand Wnt5a (Nabhan et al., 2018), and AT2 cells, characterized by the exclusive expression of interleukin 33 (Il33) and surfactant protein (Sfpta1), were involved in complex NI-NI and NI-I signaling (
Lung basophils are characterized by distinct spatial localization and gene signature In light of the rich interactive profile of basophils (
To molecularly characterize lung basophils, we sought to extensively isolate them by flow cytometry. We gated on basophil specific markers identified in the data (CD45+FceR1α+cKit−), and validated our sorting strategy using MARS-seq analysis (
Notably, the ligands Il6, Hgf and L1cam are exclusively expressed by lung basophils, compared to other lung immune and non-immune cells (
IL33 and GM-CSF Imprint the Lung-Alveolar Basophil Transcriptional Identity
Since lung-resident basophils showed a unique gene expression signature, we analyzed the data for lung specific signals that can serve as differentiation cues for lung basophil receptors (not shown). Csf2 (GM-CSF) is a hematopoietic growth factor, whose role in shaping the lung microenvironment and specifically AM, has long been established (Ginhoux, 2014; Guilliams et al., 2013; Shibata et al., 2001). Interestingly, we found that the major source of Csf2 expression in the lung stemmed from ILC and the basophils themselves, with only a small contribution from AT2 cells. Among all lung cells, basophils expressed the highest RNA and protein levels of Csf2rb, a major receptor for Csf2 (
In order to test whether the lung environmental signals, IL-33 and GM-CSF, are directly responsible for inducing the lung basophil phenotype, we used an in vitro system where we cultured bone marrow (BM)-derived basophils in media supplemented with these cytokines. We differentiated BM-derived cells in IL3 supplemented medium, isolated basophils by negative selection of cKit (BM-basophils), and cultured them in the presence of growth media alone (IL3) or with different combinations of the lung cytokine milieu; GM-CSF and/or IL-33 (
Lung basophils imprint naive macrophages with an alveolar macrophage phenotype The expression of critical lung signaling molecules by basophils prompted us to explore their signaling activity, and contribution in shaping the unique phenotype of other lung resident cells. As lung resident basophils highly express Il6, Il13 and Csfl, three important myeloid growth factors, we hypothesized that they may interact with other myeloid cells, particularly macrophages, via Il6ra, Il13ra and Csf1r (
The effect of lung basophils on AM maturation in vivo, led us to ask whether lung-basophils can promote transition of monocytes or naïve macrophages towards the AM signature directly. For this hypothesis, we performed an in vitro co-culturing assay. Naïve BM-derived macrophages (BM-MΦ) were cultured alone or co-cultured with BM-basophils in growth media supporting both cell types (M-CSF and IL-3, respectively), with or without a combination of
GM-CSF and IL-33, the milieu signaling that primes basophils toward the lung-basophil phenotype (
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.
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This application is a Continuation of PCT Patent Application No. PCT/IL2019/050939 having international filing date of Aug. 21, 2019, which claims the benefit of priority under 35 USC § 119(e) of US Provisional Patent Application No. 62/722,196, filed on Aug. 24, 2018. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62722196 | Aug 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IL2019/050939 | Aug 2019 | US |
| Child | 17183593 | US |
