Patent Application
20080260784
The present invention relates to novel compositions and methods for modulating an immune response in order to prevent or treat disease and/or conditions where T lymphocyte cells have a pathogenic role, such as Th1 or ThIL-17 mediated inflammatory conditions, chronic inflammatory conditions and autoimmune diseases.
Cells of the innate immune system, especially dendritic cells (DCs), direct the differentiation of naïve CD4+ T cells into functionally distinct Th1, Th2, ThIL-17 (also known as Th17) or regulatory T (Tregs) cell subtypes. Activation of immature DCs through binding of conserved microbial molecules to pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs) and integrins, is accompanied by DC maturation and homing to the lymph nodes, where the mature DCs present antigen (Ag) to naïve T cells. Activation of dendritic cells by pathogen derived molecules plays a critical role in regulating the differentiation of naïve CD4+ T cells into distinct T cell subtypes. Th1 cells confer protection against intracellular infection but are also associated with inflammatory responses and autoimmune disease, whereas Th2 cells are involved in allergic responses. IL-17 producing T cells (ThIL-17 or Th17) are a pathogenic subset of T cells which are characterised by the production of IL-6, TNF-alpha and, in particular, IL-17 and IL-17F, important pro-inflammatory cytokines which contribute to autoimmune disease. Treg cells are capable of suppressing Th1, Th2 and Th17 responses.
Immune Mediated Conditions
Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system. Individuals with this disease have autoreactive T cells (T cells that recognize self antigens), which together with interleukin (IL)-1beta and tumour necrosis factor (TNF) alpha, participate in the formation of inflammatory lesions along the myelin sheath of nerve fibres. The cerebrospinal fluid (CSF) of patients with MS contains activated T cells, which infiltrate the brain tissue and cause the characteristic inflammatory lesions, destroying the myelin. Experimental autoimmune encephalomyelitis (EAE) is an animal model for MS. It is induced in mice or rats by injection of mylein basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG) or peptides thereof with complete Freund's adjuvant. The disease can also be induced by transfer of MBP or MOG-specific T cells that secrete IL-17 (called ThIL-17 cells or Th17 cells). The animals develop cellular infiltration of the myelin sheaths of the central nervous system, resulting in demyelination and eventually paralysis. The clinical signs and pathological changes resemble MS.
Crohn's disease and ulcerative colitis are inflammatory bowel diseases in humans. These autoimmune diseases are inflammatory conditions of the intestine mediated by CD4+ T cells.
Many autoimmune and chronic inflammatory diseases have no satisfactory treatment and in most cases steroids and non-steroidal anti-inflammatory drugs are employed. However, these are non-specific and have side effects. More recently, drugs that inhibit key inflammatory cytokines, in particular tumour necrosis factor-alpha (TNF-alpha), have been developed. These include antibodies or soluble TNF receptors that are effective against certain autoimmune diseases, but are associated with side effects (including recurrent tuberculosis and cancers) and are limited to diseases where TNF-alpha is the key mediator of pathology. Another therapeutic approach is the direct administration of anti-inflammatory cytokines (e.g. IL-10), but this is compromised by the short half-life of cytokines in vivo. Alternative strategies employ agents that induce anti-inflammatory cytokines, such as IL-10, which will have a direct immunosuppressive effect in vivo. Molecules that promote the induction of suppressor or regulatory T cells have the potential to limit inflammatory Th1-mediated immune responses and also the T cells which secrete IL-17 (ThIL-17) which also mediate inflammatory responses in T cell mediated autoimmune responses.
Helminth Infection
Infection with Helminth parasites has been associated with immunosuppression and compromised T-cell responses. Infection with the liver fluke Fasciola hepatica induces highly polarized Th2 responses in mice, with high levels of the cytokines IL-4, IL-5 and IL-10 and also IgG1 antibodies. Infection with F. hepatica suppressed IFN-gamma secreting T cell (Th1 cell) responses and protection against the bacterial pathogen, Bordetella pertussis. Infection with F. hepatica also inhibited Th1 responses and protection against B. pertussis induced with a whole cell pertussis vaccine.
F. hepatica excretory/secretory (ES) components have also been shown to suppress Th1 responses induced by a whole cell B. pertussis vaccine. This suppression was shown to be reversed by an inhibitor of cathepsin L proteinase, this suggesting that cathepsin L proteinase was responsible for mediating the suppressive effect.
Further, cathepsin L proteinase purified from F. hepatica ES component suppressed Th1 response induced with Pw vaccine. These studies also demonstrated that the suppression induced by F. hepatica or the cathepsin L proteinase were mediated by IL-4 as suppression of the immune response was seen to be reversed in IL-4 defective mice.
The conclusion of the above studies was therefore that cathepsin L proteinase was responsible for, and mediated the suppressive effect of, F. hepatica and F. hepatica ES component and that the suppression was mediated through IL-4 induction.
A method of preventing the onset and progression of autoimmune diseases, inflammatory conditions or immune mediated disorders through the modulation of the T cell responses which are causative of these conditions would be highly advantageous in the prevention and treatment of these conditions.
The inventors of the present invention have surprisingly identified that the excretory/secretory (ES) component of Fasciola hepatica comprises compounds other than cathepsin L proteinase which mediate suppression of the immune response. While it was considered that suppression of the immune response by cathepsin L proteinase was mediated by IL-4 cytokine production, the present inventors have identified that the ES component mediates immune suppression through a number of alternative mechanisms which serve to modulate the immune response. In particular, the inventors have identified that the excretory/secretory (ES) component of Fasciola hepatica interacts with the cells of the innate immune system to modulate the immune response through the modulation of cytokine expression, in particular the upregulation of IL-10 cytokine levels, through the activation of dendritic cells into a phenotype which promotes a T regulatory phenotype (which produces IL-10) and which skews the immune response away from the production of T cells having a Th1 and/or ThIL-17 phenotype.
In particular, the inventors have identified that the ES component of F. hepatica stimulates the production of anti-inflammatory cytokines, such as IL-10, which can serve as potent modulators of the immune response.
Further, the inventors have also surprisingly shown that the ES fraction from F. hepatica can activate dendritic cells into a phenotype that promotes the production of regulatory T cells (Tregs), these Tregs in turn modulating the immune response, through the suppression of Th1 and ThIL-17 type responses. In particular, such dendritic cells have been shown to exhibit low expression levels of cell surface markers CD80, CD86, CD40 and MHC class II, while higher expression levels of CCR5 are observed over the expression levels of these cell surface markers on naïve dendritic cells. The IL-17 producing T cell subset secretes a cytokine profile including IL-17, IL-17F, IL-17H, IL-17F, IL-17A, IL-6 and TNF. IL-17 producing T (Th17 (ThIL-17)) cells, driven by IL-1 and IL-23 or IL-6 and TGF-beta, are a distinct subtype of inflammatory T cells from Th1 cells and are pathogenic in many autoimmune diseases and chronic inflammatory conditions.
These effects are independent to the immune modulatory effects which have been hitherto observed in relation to cathepsin L proteinase. Importantly, the immunomodulatory effects observed by the present inventors are not suppressed by cathepsin L proteinase inhibitors and are not IL-4 dependent. Without wishing to be bound by theory, the inventors therefore predict that the immunomodulatory effects mediated by the ES component are mediated, at least in part, by components or products derived from the ES component other than a cathepsin L proteinase.
Modulation of the response and cytokine profile expressed by a specific cell type of the immune system can lead, in turn, to a wider modulation of the overall immune response. The inventors have further surprisingly shown that the ES fraction from F. hepatica inhibits the induction of IFN-gamma, IL-17 producing Th1 cells (Th17 (ThIL-17) cells) and Th1 cells, through either the inhibition of IL-12 and IL-23, which promotes expansion of the Th1 and ThIL-17 cells, or by inhibiting the activation of Th1 and ThIL-17 cells, or by suppressing the function of Th1 and ThIL-17 cells.
According to a first aspect of the present invention there is provided a composition for the treatment or prophylaxis of a T cell mediated inflammatory immune response, said composition comprising the excretory/secretory (ES) component of Fasciola hepatica or a fraction thereof, or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom.
Where the composition comprises a fraction, derivative product, mutant, fragment or variant of a protein or peptide isolated from the ES component of Fasciola hepatica, then said fraction or molecule exhibits the same immune modulatory activity as identified by the inventors herein in relation to the ES component, namely, the suppression of the production and/or activity of IFN-gamma, IL-17 producing Th1 cells (Th17 (ThIL-17) cells) and Th1 cells, through either the inhibition of IL-12 and IL-23, which promotes expansion of the Th1 and ThIL-17 cells, or by inhibiting the activation of Th1 and ThIL-17 cells, or by suppressing the function of Th1 and ThIL-17 cells.
Suitably the protein isolated from the ES component is not Cathepsin L. Suitably the T cell mediated inflammatory immune response treated by the composition is a pro-inflammatory immune response. Typically the pro-inflammatory immune response is a Th1 mediated immune response, the development of which is suppressed following the administration of the composition of this aspect of the invention. In particular, the composition suppresses or inhibits the induction and function of Th1 cells. Suitably, said suppression or inhibition of the production of Th1 cells is caused by the production of anti-inflammatory cytokines, in particular IL-10. Suitably, IL-10 production is induced following dendritic cell maturation which is induced by administration of the ES component or a fraction or product thereof. Said suppression may further be mediated by the enhancement of the production or function of the cytokine TGF-beta. Further, said suppression of the pro-inflammatory immune response may result from the ES component or components thereof stimulating Tregs (regulatory T cells) to mediate suppression of a pro-inflammatory immune response by cell to cell contact.
Suitably the ES component comprises the excretory/secretory product released by the Fasciola hepatica parasites. In one embodiment the ES component may be provided in an isolated or purified form. The ES component may be obtained by collection of the ES component from live tissues.
In an alternative embodiment the ES component may be fractionated into a plurality of fractions, with at least one of said resulting fractions being used in the composition of this aspect of the invention. In a further embodiment, specific molecules may be isolated or purified from the ES component. In a further embodiment, the specific molecule may be produced by recombinant means.
In one embodiment, the ES component lacks at least one cathepsin L proteinase (FhCatL) protein.
In further embodiments, fractions or peptides isolated or identified as being present within the ES component are produced by recombinant means. Such a product may contain more than one peptide component as identified from the ES component.
A second aspect of the present invention provides a pharmaceutical composition for the prevention and/or treatment of a T cell mediated pro-inflammatory condition, wherein the composition comprises the excretory/secretory (ES) component of Fasciola hepatica or a fraction or a derivative, mutant, fragment or variant of a protein or peptide isolated therefrom along with a pharmaceutically acceptable excipient or carrier.
By T cell mediated pro-inflammatory condition, we mean an immune response which is pro-inflammatory in nature, which is mediate by Th1 cells and CTLs (cytotoxic T lymphocytes). The pro-inflammatory response may be driven by pro-inflammatory cytokines such as IL-12 and IFN-gamma.
According to a further aspect of the present invention there is provided a method for modulating a T cell mediated immune response in a subject in need of such treatment, the method comprising the steps of;
Suitably the ES component of Fasciola hepatica, or the fraction or derivative product, mutant, fragment or protein derived therefrom, serves to mediate at least one of:
Suitably the T cell mediated immune response is a T cell mediated pro-inflammatory immune response.
Suitably the subject is in need of treatment of, or protection against, an autoimmune disease.
In one embodiment of this aspect of the invention the T cell mediated pro-inflammatory immune response is suppressed. Without wishing to be bound by theory, this suppression may result from the step of contacting an immune cell with an agent comprising the excretory/secretory (ES) component from Fasciola hepatica, in accordance with the method of this aspect of the invention.
As herein defined, the term “upregulation” when used in relation to the increase in the production of a cytokine means that the activity or expression of that cytokine is greater than that observed in resting cells.
As herein defined, the term “suppression” when used in relation to the decrease in the production of a cytokine means that the activity or expression of the cytokine is lower than that observed in resting cells.
As herein defined, the term “inhibition” when used in relation to the inhibition in the production of a cytokine means that the activity or expression of the cytokine is inhibited or substantially inhibited when compared to the activity or expression level observed in resting cells.
Suitably the administration of the ES component results in the suppression of a pro-inflammatory cytokine such as IL-12 and/or IFN-gamma and the upregulation of an anti-inflammatory cytokine such as IL-10 and /or TGF-beta.
A further embodiment of the invention provides for the effective amount of the agent comprising the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom to couple, bind or otherwise associate with at least one cell surface activation molecule of at least one type of immune cell, this resulting in the suppression, inhibition or down-regulation of one or more functional activities of that cell.
In one embodiment of the invention the immune cell whose function is modulated is at least one cell of the innate immune system. Suitably, the cell is a cell type with antigen processing and presenting function, such as an antigen presenting cell (APC), for example a dendritic cell, or a macrophage, or a B cell.
Where the APC is a dendritic cell, it may be an immature dendritic cell, a semi-mature dendritic cell, or it may be a mature dendritic cell.
In a further embodiment the cell of the innate immune system is a cell which does not function as an antigen presenting cell, for example a mast cell. Mast cells secrete cytokines such as IL-4 and are accordingly known to have a role in facilitating the immune response. However, they do not have an associated antigen processing function.
In one embodiment, the subject is a mammal. In a further embodiment, the mammal is a human.
Without wishing to be bound by theory, the inventors of the present invention believe that the down-regulation of the T cell response which results following the administration of the excretory/secretory (ES) component from Fasciola hepatica is caused by modulating the activity of antigen presenting cells (APC), and in particular dendritic cells (DC), in inducing the production of regulatory T cells (Tregs) which express a profile of cytokines, such as IL-10 and/or TGF-beta, which suppress an immune response.
Without wishing to be bound by theory, the inventors believe that at least one specific protein molecule is present within the excretory/secretory (ES) component which is responsible for the modulation of immune function, said at least one protein being responsible for the observed immune modulation. In addition or in the alternative to the presence of a protein, a lipid, lipopetide or peptide molecule may be present in the ES component and responsible for downregulation.
In addition to the promotion of the expression of IL-10, the ES component fraction may also induce expression of other anti-inflammatory cytokines from the cells of the innate immune system.
Further, although the inventors have identified that the dendritic cell function is modulated following exposure to the ES component, other cells of the innate immune system, such as macrophages, may also have their function modulated, with this modulation promoting an anti-inflammatory response.
Further, the ES component may further comprise at least one Toll-like receptor (TLR) ligand. However, in an alternative embodiment of this aspect of the invention, a TLR ligand (agonist) may be administered exogenously along with the composition of the invention.
Accordingly, in one embodiment the method may further include the step of administering at least one TLR agonist along with said composition.
The TLR agonist may have binding specificity for at least one of TLR-2, TLR-3, TLR-4, TLR-5, TLR-7, TLR-8and TLR-9. Specific examples of suitable TLR agonists include, but are not limited to, Pam3CSK4, Zymosan, PolyIC, dsRNA, LPS (lipopolysaccharide), monophosphoryl lipid A (MPL), Flagellin, CpG-ODN (CPG-oligodeoxynucledtides), Imiquimod, R838 and R837. Further, whole bacteria such as Bordetella pertussis and Mycobacterium tuberculosis may also act as TLR agonists (Toll agonists). Further, suitable analogues to the TLR agonists listed above may also be used, wherein said analogues function to activate at least one Toll-like receptor.
A further aspect of the present invention provides a method for treating an autoimmune disease, comprising the steps of:
Suitably the autoimmune disease is selected from the group consisting of: rheumatoid arthritis or Multiple Sclerosis. Further autoimmune diseases are detailed hereinafter.
Suitably the ES component or fraction thereof or protein isolated therefrom is capable of suppressing an immune response selected from the group consisting of at least one of:
Further, the ES component or fraction thereof or protein isolated therefrom may further suppress the development of the immune response by:
As herein defined, the term “inhibition” when used in relation to the inhibition of the production of a cytokine means that the activity or expression of the cytokine is inhibited or substantially inhibited when compared to the activity or expression level observed in resting cells.
The term “administering an ES component protein” includes both the administration of the ES component protein as well as the administration of a nucleic acid sequence encoding an ES component protein. In the later case, the ES component protein is produced in vivo in the animal.
In order to obtain fractions or peptide derived from the ES component, the ES component may be separated by gel filtration chromatography, such as Sepharyl S-3000, and then analysed by polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western.
By “effective amount” it is meant a concentration of ES component or the fraction thereof or protein isolated therefrom which, when administered to a patient suffering from an autoimmune disease, produces a therapeutically beneficial effect. The effective amount of ES component or the fraction thereof or protein isolated therefrom of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
In another embodiment, the present invention provides a method of inducing immune tolerance to a transplanted organ or tissue in a recipient animal comprising administering an effective amount of the ES component from Fasciola hepatica or a fraction thereof or a protein isolated therefrom to the recipient animal prior to the transplantation of the organ or tissue.
The invention includes a use of an effective amount of the ES component from Fasciola hepatica or a fraction thereof or a protein isolated therefrom to induce immune tolerance to a transplanted organ or tissue.
The term “inducing immune tolerance” means rendering the immune system unresponsive to a particular antigen without inducing a prolonged generalized immune deficiency. The term “antigen” means a substance that is capable of inducing an immune response. In the case of autoimmune disease, immune tolerance means rendering the immune system unresponsive to an auto-antigen that the host is recognizing as foreign, thus causing an autoimmune response. In the case of allergy, immune tolerance means rendering the immune system unresponsive to an allergen that generally causes an immune response in the host. In the case of transplantation, immune tolerance means rendering the immune system unresponsive to the antigens on the transplant. An alloantigen refers to an antigen found only in some members of a species, such as blood group antigens. A xenoantigen refers to an antigen that is present in members of one species but not members of another. Correspondingly, an allograft is a graft between members of the same species and a xenograft is a graft between members of a different species.
The recipient can be any member of the animal kingdom including rodents, pigs, cats, dogs, ruminants, non-human primates and preferably humans. The organ or tissue to be transplanted can be from the same species as the recipient (allograft) or can be from another species (xenograft). The tissues or organs can be any tissue or organ including heart, liver, kidney, lung, pancreas, pancreatic islets, brain tissue, cornea, bone, intestine, skin and heamatopoietic cells.
The method of the invention may be used to prevent graft versus host disease wherein the immune cells in the transplant mount an immune attack on the recipient's immune system. This can occur when the tissue to be transplanted contains immune cells, such as when bone marrow or lymphoid tissue is transplanted when treating leukaemias, aplastic anaemias and enzyme or immune deficiencies, for example.
Accordingly, in another embodiment, the present invention provides a method of preventing or inhibiting graft versus host disease in a recipient animal receiving an organ or tissue transplant comprising administering an effective amount of the ES component from Fasciola hepatica or a fraction thereof or a protein isolated therefrom to the organ or tissue prior to the transplantation in the recipient animal.
The invention includes a use of an effective amount of the ES component from Fasciola hepatica or a fraction thereof or a protein isolated therefrom to prevent or inhibit graft versus host disease.
The methods of the present invention may also be used to treat or prevent an allergic reaction. In an allergic reaction, the immune system mounts an attack against a generally harmless, innocuous antigen or allergen. Allergies that may be prevented or treated using the methods of the invention include, but are not limited to, hay fever, asthma, atopic eczema as well as allergies to poison oak and ivy, house dust mites, bee pollen, nuts, shellfish, penicillin and numerous others.
Accordingly, in a further embodiment, the present invention provides a method of preventing or treating an allergy comprising administering an effective amount of the ES component from Fasciola hepatica or a fraction thereof or a protein isolated therefrom to an animal having, or suspected of having, an allergy. The invention includes a use of an effective amount of the ES component from Fasciola hepatica or a fraction thereof or a protein isolated therefrom to prevent or treat an allergy.
In another aspect, the present invention provides a method of preventing immune suppression mediated in a host suffering from infection with Fasciola hepatica comprising administering an effective amount of the ES component from Fasciola hepatica or a fraction thereof to an animal in need thereof.
A further aspect of the present invention relates to a method for the treatment and/or prophylaxis of a disease or a condition that would benefit from the down-regulation of an immune response, the method comprising the step of:
Suitably the immune response is an inflammatory immune response, such as the immune response mediated by Th1 cells or ThIL-17 cells.
The subject may be a mammal, in particular a human. Suitably, the subject has a condition which is an immune mediated condition, such as an allergy or an autoimmune disorder. In one embodiment the condition is an autoimmune disease, in particular a Th1-mediated and/or ThIL-17-mediated autoimmune disease.
In specific embodiments, the immune mediated disease may include, but not be limited to autoimmune conditions and diseases such as, but not limited to; multiple sclerosis, Crohn's disease, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, stroke, athlerosclerosis, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, lupus erythematosus, systemic lupus erythematosus, Hashimotos thyroiditis, myasthenia gravis, autoimmune anaemia, thrombocytopenia, asthma or an atopic disease.
The definition of “immune-mediated disease” also includes other immune-mediated disorders such as one or more of dermatitis (including atopic dermatitis and eczematous dermatitis), Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves ophthalmopathy, sarcoidosis, colitis, primary biliary cirrhosis, uveitis posterior, interstitial lung fibrosis, and coeliac disease.
In further embodiments, the disease or condition may include neurodegenerative conditions where the expression of specific cytokine profiles has been shown to contribute to the onset or progression of disease. Such conditions and diseases may include Alzheimer's disease (AD), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.
The neurodegenerative condition may also include acute inflammation conditions of the brain which result following bacterial and viral infections.
In addition to the above indicated autoimmune conditions, the present invention may be extended to any immune mediated disorder where an undesirable or unwanted immune response is triggered by the presentation of antigen.
Accordingly, a yet further aspect of the present invention provides a method for the prophylaxis and/or therapeutic treatment of a condition characterised by an aberrant, unwanted or otherwise inappropriate immune-mediated response in a subject, the method comprising administering to the subject an effective amount of an agent comprising the excretory/secretory (ES) component from Fasciola hepatica or a fraction, derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom, which agent couples, binds or otherwise associates with at least one cell type belonging to the innate immune system, for a time and under conditions sufficient to suppress, inhibit or otherwise down-regulate the immune activity of said cell type.
Typically the at least one cell type which belongs to the innate immune system comprises an antigen presenting cell (APC) such as a dendritic cell (DC), or a follicular DC, a macrophage or a B cell. Alternatively the at least one cells type which belongs to the innate immune system may be at least one cell which does not exhibit specific antigen presenting cell function such as mast cells.
Where the APC is a dendritic cell, it may be an immature, semi-mature, or a mature dendritic cell.
A further aspect of the present invention provides for the use of the excretory/secretory (ES) component from Fasciola hepatica or a fragment thereof or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom for the treatment of an immune mediated disease.
A further aspect of the present invention provides for the use of the excretory/secretory (ES) component from Fasciola hepatica or a fragment, derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of an immune mediated disease.
A yet further aspect of the present invention provides for the use of the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of an autoimmune disease.
In one embodiment, the ES component lacks the cathepsin L proteinases.
In a further embodiment, the composition further comprises a cathepsin L proteinase inhibitor.
A still further aspect of the present invention provides for the use of the excretory/secretory (ES) component from Fasciola hepatica or a fraction, derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of an inflammatory condition.
A still further aspect of the present invention provides for the use of the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of a neurodegenerative disease.
A still further aspect of the present invention provides for the use of the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of a cardiac disease or condition.
Dendritic Cell Modulation
The inventors have further identified that the excretory/secretory (ES) component from Fasciola hepatica can be used to modulate the cytokine expression profile and maturation state of dendritic cells.
Dendritic cells are key modulators of the immune response. The cytokine profiles which they express have an important influence on the development of the immune response. In particular, cytokines expressed by dendritic cells can be important in skewing an immune response down the Th1 or Th2 pathway, or in inducing the production of ThIL-17 or T cells with suppressor activity.
Dendritic cells isolated from the peritoneal cavity of F. hepatica infected mice secreted high concentrations of IL-10 and had significantly lower cell surface expression of CD80, CD86, CD40 and MHC Class II, but higher levels of CCR5 than dendritic cells from naive mice. F. hepatica is seen to suppress T cell responses by inducing dendritic cell production of cytokines, such as IL-10 or TGF-β, which induce suppressor T cell activity.
Accordingly, a further aspect of the present invention provides a method for suppressing an immune response suitable for the treatment of an immune-mediated disease in a subject in need of such treatment, the method comprising the steps of:
In one embodiment, the dendritic cells are activated to express high levels of the cytokine IL-10, or other anti-inflammatory cytokines such as TGF-beta, or other cytokines which promote the induction of Tregs.
In one embodiment of this aspect of the invention the dendritic cells are autologous to the subject.
In one embodiment the dendritic cells are immature dendritic cells. Alternatively the dendritic cells are mature dendritic cells.
In a further embodiment the dendritic cells may be administered along with a Toll-Like Receptor (TLR) ligand, an example of which is Pam3Cys.
Further, a self-antigen may be optionally co-administered along with the dendritic cells. An example of a self antigen is MOG or myelin basic protein.
A yet further aspect of the present invention provides for the use of the excretory/secretory (ES) component of Fasciola hepatica for the treatment of dendritic cells in the preparation of a medicament for the treatment of an autoimmune disease.
Inhibition of IL-17 Activity
The present inventors have further surprisingly shown that the excretory/secretory (ES) component from Fasciola hepatica suppresses the induction of T cells which express IL-17 (so called ThIL-17 cells). IL-17 has been identified as a key modulator of the onset and progression of autoimmune diseases and chronic inflammatory conditions, in particular multiple sclerosis, colitis and rheumatoid arthritis. Accordingly the modulation of IL-17 levels by an agent comprising the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom provides a novel therapy applicable to the prophylaxis and/or treatment of autoimmune diseases and chronic inflammatory conditions.
According to a yet further aspect of the present invention there is provided a method of prophylaxis and/or treatment of an immune-mediated condition, the method comprising the step of:
According to a still further aspect of the present invention there is provided a method of prophylaxis and/or treatment of an immune-mediated condition, the method comprising the step of:
Typically, the inhibition of IL-23 will further result in the inhibition of the production of IL-17.
A still further aspect of the present invention provides the use of the excretory/secretory (ES) component from Fasciola hepatica or a derivative, mutant, fragment or variant of a protein isolated therefrom in the preparation of a medicament to inhibit the production of IL-17 for the treatment of an immune-mediated disease.
IL-12 Suppression
The inventors have further identified that the production of IL-12 is suppressed as a result of ES administration. IL-12 has a role in effecting the promotion of immune responses, and in particular those mediated by Th1 and ThIL-17 cells. This suppression may further be extended to members of the IL-12 cytokine family, such as IL-23 and IL-27.
Accordingly, a further aspect of the invention provides a composition for the suppression of IL-12 production or the production of an IL-12 family member, said composition comprising the excretory/secretory (ES) component of Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom.
A further still aspect provides a pharmaceutical composition for use in the suppression of IL-12 production or the production of an IL-12 family member, wherein the composition comprises the excretory/secretory (ES) component of Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom. A yet further aspect of the present invention provides a method for suppressing the production of IL-12 or an IL-12 family member, the method comprising the step of;
In one embodiment, the IL-12 family member is IL-23 and/or IL-27.
Liver Fluke Homogenate (LFH)
In addition to the administration of the ES (excretory/secretory) component of Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom, the compositions, uses of said compositions and methods of the present invention can also be achieved through the use of liver fluke homogenate (LFH) instead of the ES component.
The inventors have surprisingly identified that the administration of LFH may modulate the expression of certain cytokines which have importance in immune mediated inflammatory conditions. For example, liver fluke homogenate (LFH) or a fraction, derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom may modulate the expression levels of the cytokines IL-4 and IL-10.
Accordingly a further aspect of the invention provides a composition for the prevention and/or treatment of an inflammatory condition, said composition comprising liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom.
In one embodiment the inflammatory condition is a T cell mediated inflammatory immune response.
A yet further aspect of the present invention provides a pharmaceutical composition for the prevention and/or treatment of an inflammatory condition, wherein the composition comprises liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom.
In one embodiment the inflammatory condition is a T cell mediated inflammatory immune response.
A yet further aspect of the present invention provides a method for modulating an inflammatory condition, the method comprising the steps of;
In one embodiment the inflammatory condition is a T cell mediated inflammatory immune response.
In one embodiment, the inflammatory condition is an immune mediated condition, such as an allergy or an autoimmune disorder. In further embodiments, the condition is an autoimmune disease, in particular a Th1-mediated and/or ThIL-17-mediated autoimmune disease. In specific embodiments, the immune mediated disease may include, but not be limited to multiple sclerosis, Crohn's disease, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, stroke, athlerosclerosis, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, lupus erythematosus, systemic lupus erythematosus, Hashimotos thyroiditis, myasthenia gravis, autoimmune anaemia, thrombocytopenia, asthma or an atopic disease.
In addition to identifying a novel and unexpected use for LFH in the modulation of immune-mediated conditions, such as those listed above, the present inventors have also surprisingly identified that LFH is particularly effective in modulating IL-4 levels.
According to a further still aspect of the present invention there is provided a method for the prophylaxis and/or treatment of cognitive dysfunction, the method comprising the step of administering an agent comprising liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom to an individual in need of such therapy.
A further aspect of the invention extends to the use of an agent comprising liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the treatment of cognitive dysfunction.
A further still aspect relates to the use of liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of cognitive dysfunction.
In a preferred embodiment, the administration of the LFH results in the up-regulation of anti-inflammatory cytokines, for example IL-4 and the down-regulation of pro-inflammatory cytokines such as IL-1. Preferably the upregulation will be in the hippocampus, most preferably this upregulation of the anti-inflammatory cytokine profile will be present in the microglial cells. It is preferred that LFH or ES be administered to the brain or directly to another area of the central nervous system (CNS).
Typically, the anti-inflammatory cytokines which are upregulated are IL-4, IL-10 and TGF-beta. The pro-inflammatory cytokines which are down-regulated are typically IL-1 beta and TNF-alpha. Preferably the modulation of pro-inflammatory and anti-inflammatory cytokine levels is in the hippocampus, most preferably this modulation of the cytokine profile is in the microglial cells.
A yet further aspect of the present invention provides a method of treating cognitive dysfunction through direct administration of liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom to the brain or CNS.
A further still aspect relates to the use of liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of cognitive dysfunction wherein the medicament is administered directly to the brain or CNS.
A further still aspect relates to the use of liver fluke homogenate (LFH) or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom in the preparation of a medicament for the treatment of an immune mediated condition.
Toll-Like Receptor (TLR) Agonist
The present inventors have surprisingly identified that a TLR agonist is present in the ES (excretory/secretory) component of Fasciola hepatica. This TLR agonist may have important utility as an adjuvant in the induction of an immune response.
The TLR agonist could, for example, be administered along with a therapeutic, such as a vaccine for the treatment of a cancer or a malignant condition, or along with a vaccine directed to an infectious disease.
In further aspects, the present invention extends to the use of the TLR agonist identified within the ES (excretory/secretory) component of Fasciola hepatica in a method for inducing the maturation of dendritic cells.
Accordingly a yet further aspect of the present invention provides a method for inducing a Th1 response in a subject suitable for the treatment of cancer or an infectious disease, the method comprising the steps of:
Definitions
As used herein, the term “immune cell” includes cells that are of haematopoietic origin and play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells, natural killer cells, myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
As used herein, the term “T cell” includes CD4+ T cells and CD8+ T cells. The term “T cell” also includes both T helper 1 type T cells and T helper 2 type T cells and also Th-IL17 cells.
As used herein, the term “antigen-presenting cell” or “antigen-presenting cells” or its abbreviation “APC” or “APCs” refers to a cell or cells capable of endocytotic adsorption, processing and presenting of an antigen. The term includes professional antigen presenting cells, for example, B lymphocytes, monocytes, dendritic cells (DCs) and Langerhans cells, as well as other antigen presenting cells such as keratinocytes, endothelial cells, glial cells, fibroblasts and oligodendrocytes. The term “antigen presenting” means the display of antigens as peptide fragments bound to MHC molecules on the cell surface. Many different kinds of cells may function as APCs including, for example, macrophages, B cells, follicular dendritic cells and dendritic cells.
As used herein, the term “immune response” includes T cell mediated and/or B cell mediated immune responses that are influenced by modulation of T cell co-stimulation. The term immune response further includes immune responses that are indirectly effected by T cell activation such as antibody production (humoral responses) and the activation of cytokine responsive cells such as macrophages.
As used herein, the term “dendritic cell” or “dendritic cells” (DC) refers to a dendritic cell or cells in its broadest context and includes any DC that is capable of antigen presentation. The term includes all DC that initiate an immune response and/or present an antigen to T lymphocytes and/or provide T-cells with any other activation signal required for stimulation of an immune response. Reference herein to “DC” should be read as including reference to cells exhibiting dendritic cell morphology, phenotype or functional activity and to mutants or variants thereof. The morphological features of dendritic cells may include, but are not limited to, long cytoplasmic processes or large cells with multiple fine dendrites. Phenotypic characteristics may include, but are not limited to, expression of one or more of MHC class I molecules, MHC class II molecules, CD1 or CD4.
As used herein, the term “antigen” is any organic or inorganic molecule capable of stimulating an immune response. The term “antigen” as used herein extends to any molecule such as, but not limited to, a peptide, polypeptide, protein, nucleic acid molecule, carbohydrate molecule, organic or inorganic molecule capable of stimulating an immune response.
A “subject” in the context of the present invention includes and encompasses mammals such as humans, primates and livestock animals (e. g. sheep, pigs, cattle, horses and donkeys), laboratory test animals such as mice, rabbits, rats and guinea pigs, and companion animals such as dogs and cats. It is preferred for the purposes of the present invention that the mammal is a human.
Treatment
The term ‘treatment’ is used herein to refer to any regimen that can benefit a human or non-human mammal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects.
More specifically, reference herein to “therapeutic” and “prophylactic” treatment is to be considered in its broadest context. The term “therapeutic” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition.
Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term “prophylactic” may be considered as reducing the severity or the onset of a particular condition. “Therapeutic” may also reduce the severity of an existing condition.
Administration
The excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom for use in the present invention may be administered alone but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected depending on the intended route of administration.
The excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom for use in the present invention may be administered to a patient in need of treatment via any suitable route. The precise dose will depend upon a number of factors, including the precise nature of the form of the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom to be administered.
Although the preferred route of administration is parenterally (including subcutaneous, intramuscular, intravenous), some further suitable routes of administration include (but are not limited to) oral, rectal, nasal, topical (including buccal and sublingual), infusion, vaginal, intradermal, intraperitoneally, intracranially, intrathecal and epidural administration or administration via oral or nasal inhalation, by means of, for example, a nebuliser or inhaler, or by an implant.
For intravenous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection or Lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules. Implantable or microcapsular sustained release matrices include polylactides (U.S. Pat. No. 3,773,919; EP-A-0058481) copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981, and Langer, Chem. Tech. 12:98-105,1982).
Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A. R., Lippincott Williams & Wilkins; 20th edition (Dec. 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H. C. et al. 7th Edition ISBN 0-683305-72-7, the entire disclosures of which are herein incorporated by reference.
Pharmaceutical Compositions
As described above, the present invention extends to a pharmaceutical composition for the modulation of an immune response and in particular the down regulation of a T cell response wherein the composition comprises at least the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom, or a derivative, fragment, variant, mutant or recombinant version thereof.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient (i.e. the excretory/secretory (ES) component from Fasciola hepatica or a derivative product, or a mutant, fragment or variant of a protein or peptide isolated therefrom), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art.
Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, intranasal or via oral or nasal inhalation.
The formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze dried powder.
Dose
The composition is preferably administered to an individual in a therapeutically “effective amount” or a “desired amount”, this being sufficient to show benefit to the individual.
As defined herein, the term an “effective amount” means an amount necessary to at least partly obtain the desired response, or to delay the onset or inhibit progression or halt altogether the onset or progression of a particular condition being treated.
The amount varies depending upon the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation and other relevant factors. It is expected that the amount will fall in a relatively broad range, which may be determined through routine trials.
Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners, physicians or other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or at other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.
Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
As used herein, the term “inhibits” when used in relation to the modulation of the T cell response means the partial or complete down-regulation of proliferation and/or activity of the T cells.
Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise.
The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention, and further, with reference to the figures.
Material and Methods
Animals and Immunisation
Female BALB/c mice were purchased from Harlan Olac (Bicester, United Kingdom) and used at 6 to 8 weeks old, with four mice per group. Mice were housed in individually ventilated cages and all experiments were performed according to regulations of the Irish Department of Health, the European Union and the Ethics Committee of Trinity College Dublin. Mice were immunized subcutaneously (s.c.) in the flank with depyrogenated keyhole limpet hemocyanin (KLH) (5 μg; Calbiochem, La Jolla, Calif.); KLH (5 μg) with ES (20 or 50 μg; or with Dulbecco's PBS (Sigma, Poole, United Kingdom) in a final volume of 200 μl. Seven days after immunization, mice were sacrificed by cervical dislocation, and serum and inguinal lymph nodes were collected
Preparation of Excretory-Secretory Products from F. hepatica
Live, adult flukes were collected from bovine livers at a local abattoir. Flukes were washed in several changes of Ca2+- and Mg2+-free Dulbeccos PBS, and then incubated overnight in PBS, at 37° C. in 5% CO2 in air. Supernatant fluid containing excretory-secretory (ES) products was harvested, clarified by centrifugation at 13,000 g for 15 mins at 4° C. and passed through a 0.22 μm filter. The protein concentration of the ES preparation was determined using a bicinchoninic acid protein kit (Pierce). Antigen preparations were stored at −80° C. until required.
Infection of Mice with F. hepatica
Metacercariae were obtained from Compton Paddock Laboratories (Berkshire, UK). Viability was examined using a stereomicroscope before orally infecting mice with 10 metacercariae of F. hepatica. This resulted in infection in 100% of animals. Mice were sacrificed by cervical dislocation 3 weeks after infection, unless otherwise stated.
Isolation and Culture of Bone Marrow-Derived DC (BMDC)
Bone marrow-derived immature DC were prepared by culturing bone marrow cells obtained from the femurs of mice in RPMI 1640 medium with 10% FCS supplemented with supernatant (10%) from a GM-CSF-expressing cell line (J558-GM-CSF). On day 3, fresh medium with 10% GM-CSF cell supernatant was added to adherent cells. On day 7, cells were collected, washed, and used for assays. BMDC were cultured at 1×106 cells/ml.
Purification of peritoneal macrophages, CD11c+ DC and CD4+ T-cells
For isolation of peritoneal macrophages, peritoneal exudate cells (PEC) were harvested from mice by lavage. PEC were incubated in 6-well tissue culture plates for 2 hrs to allow plastic adherence of macrophages. Non-adherent cells were removed by washing and adherent macrophages were harvested from the plates for stimulation. CD4+ T-cells or CD11c+ dendritic cells were isolated from spleen and PEC using positive selection with MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and an autoMACS cell sorting instrument. Following lysis of red blood cells, single cell suspensions of either PEC or spleen cells were incubated with either MACS CD4 or CD11c immunomagnetic beads (Miltenyi Biotec), and allowed to pass through the autoMACS using positive selection. The purity of CD4+ T-cells and CD11c+ dendritic cells after autoMACS separation were routinely 90-95% as estimated by FACScan analysis using FITC-conjugated CD4 or CD11c respectively.
J774 Cell Culture
J774.A1 cells (ATCC number TIB 67), were grown in tissue culture flasks at 37° C. in 5% CO2 in air with RPMI 1640 medium containing 10% foetal calf serum (FCS). When cells reached confluence, they were removed from culture, centrifuged at 1200 rpm for 5 mins to pellet cells, and reseeded in fresh flasks at a concentration of 1×106/ml. When cells reached sufficient numbers for experiments, cells were then collected by vigorous pipetting and were centrifuged at 1200 rpm for 5 mins. The cells were washed once with 10% RPMI and then seeded into 24-well culture plates at 1×106/ml at a volume of 1 ml/well for stimulations.
Induction or Inhibition of Cytokine Production
J774 and peritoneal macrophages, bone marrow-derived DC, MACS-isolated CD11c+ DC and peritoneal exudate cells were cultured 24-well tissue culture plates at a cell density of 1×106/ml. In some experiments, peritoneal exudate cells and J774 macrophages were also stimulated with ES in the presence of the cathepsin inhibitor E-64 (Sigma) at a concentration of 10 mM, or with heat-inactivated ES, which was heated to 100° C. for 20 mins. Following incubation for 24 hrs at 37° C. in 5% CO2 in air, supernatants were removed and stored until analysis of IL-10, IL-12p40 concentrations using commercially available immunoassay kits (R&D systems), or pairs of monoclonal antibodies (Pharmingen) for IL-4 and IL-5, IL-12p70 and TGF-β. The effect of ES on IL-12 production was determined by pre-stimulating BMDC or J774 macrophages (1×106/ml) for 2 hrs with ES (4 and 20 μg/ml), followed by addition of LPS (10 ng/ml) and IFN-γ (20 ng/ml), or LPS only, and incubation for a further 22 hrs. Supernatants were removed and concentrations of IL-10, IL-12 p40 and/or IL-12 p70 determined by immunoassay. The effect of ES on Con A-induced cytokine production and proliferation was determined by stimulating spleen cells (2×106/ml) from naïve mice with ES (4 and 20 μg/ml) in the presence or absence of Con A (5 μg/ml). Supernatants were removed following incubation for 72 hrs, at 37° C. in 5% CO2 in air, and T-cell cytokine concentrations determined by ELISA. Proliferation of T-cells was measured by [3H] thymidine incorporation over the last 4 hrs of a 48 hr culture. To assess cytokine induction by ES in vivo, BALB/c mice were injected s.c. in the flank with 50 μg ES in a total volume of 200 μl/mouse. Six hours later, inguinal nodes were removed and homogenized in 1 ml ice cold PBS. After removal of cells by centrifugation at 1200 rpm for 5 mins at 4° C., supernatants were removed and analyzed for the presence of IL-4 and IL-10 using commercially available cytokine DuoSets (R&D). In a separate experiment, groups of 4 BALB/c mice were injected with 300 μl of either PBS or ES (50 μg). After 2 hours, peritoneal exudate cells (PEC) from PBS- and ES-injected mice were harvested by peritoneal lavage in ice-cold PBS. Cells were blocked with 50% PBS/FCS (v/v) and surface labelled with anti-CD11c, before fixing and permeabilising for intracellular cytokine labelling by adding PE-labelled anti-IL-10, and FITC-labelled anti-IL-4 and anti-IFN-γ monoclonal antibodies, or rat control Ig. After 30 mins on ice, cells were then washed with PBS/BSA/azide and acquired immediately. A total of 20,000 cells were acquired per sample. Cells were gated on the CD11c+ population, and the expression of intracellular proteins was analyzed on a FACSCalibur flow cytometer using CellQuest software.
Suppression Assay
CD4+ T cells were purified from OVA-specific TCR transgenic (TCR-Tg) mice using MACS microbeads and the AutoMacs system (described above). OVA-specific T-cells (1×105/ml) were cultured with OVA323-339 peptide (2 μg/ml), and irradiated spleen cells (2×106/ml) as antigen presenting cells (APC). This OVA-specific response served as the control. F. hepatica-specific T-cells (FH T-cells) were isolated from peritoneal lavage fluid of infected mice 3 weeks after infection using CD4+ MACS microbeads (as above). FH T-cells were then cultured either without any antigen or APC, or with liver fluke homogenate (LFH) at 20 μg/ml, or ES (20 μg/ml), together with irradiated spleen cells from naïve BALB/c mice as APC. OVA-specific and FH T-cells were then cultured together in a 1:1 ratio, either in the same well or separated by a semi-permeable membrane (transwell). After 72 hrs incubation at 37° C. in 5% CO2 in air, supernatants were removed from above and below the transwell, and from the co-cultured cells for determination of IL-4, IL-5, IL-10 and IFN-γ levels by ELISA. Proliferation was determined by 3H-thymidine incorporation after 96 hrs culture.
Analysis of Innate Cell Activation by FACS
BMDC or J774 macrophages (1×106/ml) were cultured in medium only, ES (20 μg/ml) or E. coli LPS (10 ng/ml). Cells were recovered, and surface marker expression was assessed by flow cytometry using fluorescently labeled antibodies (BD Pharmingen). After blocking with 50% FCS/PBS (w/v), cells were incubated for 30 mins on ice in the dark with antibodies specific for mouse CD80, CD86, CD11c (for BMDC only), MHC Class II, CD40 and CCR5. Cells labeled with appropriate isotype matched antibodies with irrelevant specificity acted as controls. A total of 20,000 cells per sample were analyzed on a FACScalibur flow cytometer. Analysis was performed using CellQuest software (version 3.3; Becton Dickinson Immunocytometry Systems, San Jose, Calif.).
Modulation of J774 Surface Marker Expression
The effect of ES on LPS-induced cell surface marker expression was determined by pre-stimulating J774 macrophages (1×106/ml) for 2 hrs with ES (20 μg/ml), followed by addition of LPS (10 ng/ml), and incubation for a further 22 hrs. After 24 h cells were recovered, washed and blocked, and expression of surface markers were assessed using CD86, CD80, CD40, CCR5 and MHC Class II. Cells incubated with an isotype-matched directly conjugated Ab with irrelevant specificity acted as a control. After incubation for 30 mins on ice in the dark, cells were washed and immunofluorescence analysis was performed on a FACScan (Becton Dickinson) and analyzed using CellQuest software; 20,000 cells were analyzed per sample.
Flow Cytometric Analysis of Intracellular Cytokine Synthesis
To analyse cytokine production in DC and T-cells from F. hepatica-infected mice, peritoneal exudate cells (PEC) were first incubated with 50% PBS/FCS (v/v) for 20 mins at room temperature. DC and T-cell were then stained with anti-CD11c and anti-CD4 respectively. For detection of intracellular IL-10, IL-4 and IFN-γ, cells were fixed and permeabilised using a commercially available intracellular cytokine staining kit (Caltag). PE-conjugated anti-IL-10, and FITC-conjugated IL-4, IFN-γ mAb or rat control Ig were added at a predetermined saturating concentration for 30 min. Cells were then washed with PBS/BSA/azide and acquired immediately. A total of 50,000 cells were acquired per sample. Cells were gated on CD11c or CD4 for DC and T-cells respectively, and the expression of intracellular proteins was analyzed on a FACSCalibur flow cytometer using CellQuest software.
Antigen-Specific Cytokine Production
Spleen (2×106/ml) or lymph node cells (1×106/ml) from immunized mice or infected mice were cultured at 37° C. and 5% CO2 in RPMI medium with either MOG peptide (10 and 100 μg/ml) or KLH (2 to 50 μg/ml) or phorbol 12-myristate 13-acetate (PMA) (25 ng/ml; Sigma) and anti-CD3 (0.5 μg/ml; BD Pharmingen, San Diego, Calif.) or medium only. After 72 hrs, supernatants were collected for cytokine detection and the medium was replaced. Proliferation was assessed by [3H]thymidine incorporation. Concentrations of IL-4, IL-5, IL-10 and IFN-γ were determined by immunoassay.
Induction of EAE and ES Treatment
Mice were each injected s.c. with 150 μg of MOG35-55 peptide in CFA containing 5 mg/ml Mycobacterium tuberculosis H37Ra (Difco) in the base of the tail. All mice received 500 ng of pertussis toxin (Sigma) by i.p. injection on days 0 and +2 post-immunization. For treatment with ES, 50 μg of ES in PBS was injected i.p. every second day. Mice were assessed daily for clinical signs of EAE, and scored as follows: 1=tail paralysis, 2=wobbly gait, 3=hind limb weakness, 4=hind limb paralysis, 5=complete paralysis of hind and fore limbs, 6=death. The disease index was calculated by adding all daily average disease scores, dividing the average day of onset, and multiplying by 100. Experiments were terminated when control EAE mice displayed clinical scores of 3-4. Mice were sacrificed by cervical dislocation, and serum and spleens were harvested.
EAE was induced in C57BU6 mice by immunization with MOG35-55 peptide in CFA on day 0 and injection of pertussis toxin days 0 and 2. One group of mice was left untreated. A second group was infected with 10 metacercariae of F. hepatica one day before induction of EAE. A third was injected i.p. with 50 μg ES, one day before induction of EAE and every second day thereafter. 20 days after induction of EAE, mice were sacrificed and spleen removed. Spleen cells (2×106/ml) were stimulated with medium only, MOG35-55 peptide (10 and 100 μg/ml), or anti-CD3 and PMA. Supernatants were removed after 3 days and IL-17, IFN-γ, IL-4 and IL-10 concentrations determined by ELISA. Results are mean values for groups of 4-5 mice tested in triplicate.
Results
Infection with F. hepatica Inhibits Dendritic Cell Maturation
Following oral infection with F. hepatica metacercariae, the parasites migrate from the gut through the peritoneal cavity to the liver. Therefore the peritoneal cavity is exposed to products of the liver fluke. Here we examined the influence of a F. hepatica infection on cells of the innate immune response in the peritoneal cavity. Mice were infected with F. hepatica and 3 weeks after infection, cells were recovered from the peritoneal cavity. Cells from naïve mice served as controls. Dendritic cells (DC) were examined for expression of cells surface markers indicative of maturation. In comparison with cells from control mice, DC from F. hepatica had significantly lower expression of MHC class II and the co-stimulatory molecules, CD80, CD86 and CD40 and higher expression of CCR5 (
A High Frequency of Dendritic Cells from the Peritoneal Cavity of F. hepatica Infected Mice Secrete IL-10 and a Lower Frequency Secrete IL-4
Mice were infected with F. hepatica and 3 weeks after infection, cells were recovered from the peritoneal cavity. Cells from naïve mice served as controls. CD11c+ DC were examined for intracellular IL-4, IL-10 and IFN-γ by immunofluoresence analysis (
A High Frequency of IL-10 Secreting T Cells with a Regulatory Phenotype are Detected in the Peritoneal Cavity of F. hepatica Infected Mice
Since IL-10-producing DC have been shown to promote the induction of IL-10-secreting regulatory T (Treg) cells, we examined the possibility that infection with F. hepatica was also associated with the induction of Treg cells. BALB/c mice were infected with F. hepatica and 3 weeks after infection, cells were recovered from the peritoneal cavity. Cells from naïve mice served as controls. CD4+ T cells were examined for intracellular IL-4, IL-10 and IFN-γ by immunofluoresence analysis (
T cells from the peritoneal cavity were also assessed for surface expressions of surface markers know to be associated with Treg cells. Immunofluorescence analysis with specific antibodies revealed that expression of CTLA-4, IL-10R, CD28, T1/ST2, and CCR5 were enhanced on cells from F. hepatica infected mice (
Fasciola hepatica Induces FOXP3-Expressing T-Cells
T Cells from the Peritoneal Cavity of F. hepatica Infected Mice Have Suppressor Activity
Having demonstrated that T cells in the peritoneal cavity of F. hepatica infected mice secrete IL-10 and a have a regulatory phenotype, we examined their suppressor activity in co-culture experiments with T cells from ovalbumin (OVA) T cell receptor (TCR) transgenic (Tg) mice. A high frequency of DO11.10 TCR Tg mice are specific for OVA and are a useful readout of antigen-specific responses. T cells from DO11.10 TCR Tg mice proliferate and secrete IFN-γ, IL-4, IL-5 and IL-10 following stimulation with antigen (OVA) and antigen presenting cells (APC) in vitro. Co-cultivation with T cells from the peritoneal cavity of F. hepatica infected mice had a marked suppressive effect on proliferation and IFN-γ production. This was observed with and without stimulation with antigen (liver fluke homogenate; LFH or excretory/secretory fraction from F. hepatica; ES). IL-4 production was also reduced especially with un-stimulated T cells from F. hepatica infected mice. In contrast, IL-5 and IL-10 production was enhanced, especially with antigen-stimulated cells from F. hepatica infected mice (
We next assessed the mechanisms of suppression, specifically the role of cell contact versus soluble factors. Here the T cells from DO11.10 TCR Tg mice were separated from the T cells from the F. hepatica infected mice by a semi-permeable membrane. Stimulation of the T cells from the peritoneal cavity of F. hepatica infected mice with LFH or ES resulted in the production of high concentrations of IL-5 and IL-10 and low concentrations of IFN-γ. IL-4 was also detected following stimulation with LFH, but not with ES (
These findings demonstrate that T cells from F. hepatica infected mice suppress T cell responses to an unrelated antigen in a bystander fashion. Furthermore this suppression is in part mediated by a soluble factor, possibly IL-10, which was secreted in high concentrations by T cells from F. hepatica infected mice.
IL-10 Production During F. hepatica Infection Regulates IL-5 and IFN-γ Production in Vivo
In order to examine the role of IL-10 in the suppression of immune responses in vivo, C57BU6 and IL-10 knockout mice were infected with F. hepatica and cytokine production was examined in the peritoneal fluid and from mesenteric lymph node cells re-stimulated with antigen in vitro.
IL-10 and IL-5, but not IL-4 and IFN-γ were detected in the peritoneal fluid of C57BL/6 mice infected with F. hepatica (
Having demonstrated that F. hepatica infection induces IL-10 production from innate cells and T cells, we examined the influence of F. hepatica ES on IL-10 production by cells of the innate immune system. Peritoneal exudate cells, peritoneal macrophages, J774 macrophages, bone marrow-derived DC and CD11c+ DC from the peritoneum and the spleen all secreted IL-10 in responses to ES (
ES Stimulates Innate IL-10 and IL-4 Production in Vivo
Having demonstrated the induction of IL-10 by innate immune cells in vitro, we examined the ability of ES to stimulate innate cytokine production in vivo. Mice were injected s.c. in the flank and the draining lymph nodes were removed 6 hours later and homogenized and IL-4 and IL-10 concentrations determined by ELISA. Alternatively mice were injected i.p. and peritoneal DC were assessed for cytokine production after 2 hours. The results revealed that s.c injection of ES stimulated the production of significant concentrations of IL-10 and lower concentrations of IL-4 in the draining lymph node (
ES Inhibits TLR Agonist-Induced IL-12p70 Production
IL-10 production by innate cells is associated with the induction of Treg cells, whereas IL-12 promotes the expansion of IFN-γ-secreting Th1 cells. Therefore we examined the influence of ES on IL-12p40 and IL-12p70 production by macrophages and DC. ES alone did not stimulate IL-12p70, but did stimulate IL-12p40 and IL-10 production from J774 macrophages (
The Immunomodulatory Effect of ES is Mediated by a Heat-Sensitive Molecule Other Than a Cathepsin
It has previously been reported that cathepsin L proteinase from Fasciola hepatica has immunomodulatory properties. Here we assessed the role of cathepsins in the induction of IL-10 production. We first assessed the effect of heat treatment on the activity of ES. Heat-treatment of ES significantly reduced its ability to stimulate IL-10 production by peritoneal exudate cells (
ES Modulates Surface Marker Expression on DC and Macrophages
DC and macrophages are potent antigen presenting cells, but must first be activated to enhance expression of MHC class II and co-stimulatory molecules. Fully mature DC promote expansion of effector T cells, especially Th1 cells, whereas immature or partially mature DC promote expansion of anergic or regulatory T cells. Here we found that ES enhanced surface expression of CD86, CD80, CD40 and MHC class II in DC from C57BL/6 mice. The level of activation was similar to that observed with LPS. However, unlike LPS which suppressed CCR5 expression (CCR5 expression is normally reduced following DC maturation), ES enhanced CCR5 expression. In IL-10-defective mice this enhancement was not observed. Furthermore, CD80, CD86, CD40 and MHC class II was enhanced in ES-stimulated DC from IL-10-defective mice (
ES also enhanced surface expression of CD80, CD86, CD40, CCR5 and MHC class II on J774 macrophages (
ES Induced IL-10 Producing Treg Cells and Suppresses Proliferation and IFN-γ Production by Effector T Cells.
We examined the influence of ES on the induction of T cell responses to a model antigen, KLH. Mice were injected with ES only, KLH only or KLH and ES and draining lymph nodes were removed after 7 days and cells re-stimulated with antigen (KLH) in vitro. In this experiment, KLH alone did not induce and antigen-specific immune responses, although responses were detected against the positive control stimulus, anti-CD3 and PMA. In contrast, co-injection with ES (50 μg) generated T cells that secreted high concentrations of antigen-specific IL-10 and lower concentrations of IL-5, but no IL-4 or IFN-γ (
We also examined the influence of ES on T cell response to the mitogen, Concanavlin A (Con A) in vitro. Spleen cells stimulated with Con A proliferated and secreted IFN-γ and low concentrations of IL-10 (
F. hepatica Infection or Parenteral Administration of ES Protects Against Experimental Autoimmune Encephalomyelitis (EAE).
Self-antigen-specific T cells that secrete IFN-γ and IL-17 mediated pathology in a number of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. T cell mediated autoimmune diseases are controlled in normal individuals by natural and inducible Treg cells that suppress pathogenic T cells. Having shown that F. hepatica infection and ES induces IL-10-secreting Treg cells, we examined the influence of the infection or parenteral administration of EAE on the development of EAE in chronic disease model, involving administration of myelin oligodendrocyte glycoprotein (MOG) peptide in complete Freund's adjuvant (CFA) with pertussis toxin (PT). Untreated mice developed clinical symptoms of EAE after 12 days and reached a clinical score of grade 3 or above after 20 days and had to be sacrificed at this stage. In contrast, F. hepatica infected mice did not develop symptoms until day 14 and ES treated until day 16 (
F. hepatica Infection or Parenteral Administration of ES Suppresses Induction of Pathogenic T Cells During Development of EAE.
T cells mediate many autoimmune diseases and it has traditionally been considered that IFN-γ-secreting Th1 cells are responsible for the inflammatory pathology. However recent evidence suggests that IL-17-secreting T cells mediate much of the damage in many chronic inflammatory and autoimmune diseases, including rheumatoid arthritis, colitis, multiple sclerosis and EAE, the murine model for multiple sclerosis. Here that development of EAE was associated with the induction of MOG-specific IL-17 and IFN-γ-secreting T cells in untreated mice (
Fasciola hepatica Infection Slows the Onset and Attenuates the Clinical Signs of EAE in C57BU6 Wildtype and IL-10-Defective Mice.
F. hepatica Infection Inhibits Pathogenic IFN-γ and IL-17 Secreting Cells During EAE by an IL-10-Independent Mechanism.
Fasciola hepatica Infection Attenuates Clinical Signs of DSS-Induced Colitis in BALB/c Mice.
Fasciola hepatica Infection Suppresses Pro-Inflammatory Cytokines Production in the Colon During DSS-Induced Colitis
Therapy with Fasciola hepatica ES Products Attenuates Clinical Signs of DSS-Induced Colitis in BALB/c Mice
Therapy with Fasciola hepatica ES Products Suppresses Pro-Inflammatory Cytokine Production During DSS-Induced Colitis in BALB/c Mice
ES Inhibits Antigen-Specific IL-17 and IFN-γ Production to Co-administered Antigen.
The present inventors have discovered that infection with F. hepatica or administration of F. hepatica ES to mice induces a high frequency of IL-10secreting regulatory T (Treg) cells and that ES products have the capacity to inhibit autoimmune diseases. We demonstrated that F. hepatica infected mice have a very high frequency of CD4+ T-cells in the peritoneal cavity that secreted very high concentrations of IL-10, in the absence of IL-4. These Treg cells induced by infection inhibited proliferation and IFN-production by OVA-specific T-cells in a co-culture system or when separated by a semi-permeable membrane, indicating soluble factor mediated suppression. Furthermore, dendritic cells (DC) from the peritoneal cavity of F. hepatica infected mice secreted high concentrations of IL-10 and had significantly lower cell surface expression of CD80, CD86, CD40 and MHC class II, but higher CCR5, than DC from naïve mice, indicating an immature status. When used as antigen presenting cells for OVA-specific T-cells, DC from F-hepatica infected mice induced significantly lower cytokine production, when compared with DC from naïve mice. These findings demonstrate that F. hepatica suppresses T cell responses by modulating DC activation and Treg cell induction.
It has been also unexpectedly shown that F. hepatica ES modulated DC maturation and enhanced IL-10 production in vitro. Addition of a cathepsin L proteinase inhibitor did not reverse the modulatory effect of ES. Furthermore, injection of mice with ES prevented the development of experimental autoimmune encephalitis (EAE), a murine model for multiple sclerosis. T cells that secrete IL-17, termed ThIL17 cells, are pathogenic in EAE and our data suggests that ES suppresses the induction of ThIL-17 cells, either by inhibiting IL-23, which promotes the expansion of ThIL-17 cells or by inhibiting the activation or function of ThIL-17 cells. The findings suggest that the ES fraction from F. hepatica includes components, other than cathepsin L proteinase, which interact with cells of the innate immune system, stimulates the induction of anti-inflammatory cytokines, and activates dendritic cells into a phenotype that promotes the induction of Treg cells, while inhibiting ThIL-17 cells and thereby prevents the development of autoimmune diseases.
All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005/0811 | Dec 2005 | IE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IE2006/000136 | 12/5/2006 | WO | 00 | 6/5/2008 |
