WO2010136766A2 - Methods and compositions for treating nf-kappa b mediated disorders - Google Patents

Abstract

A method for preventing or treating an NF-kappaB mediated disorder, such as an inflammatory disorder, an autoimmune disorder, or cancer, in a subject suffering from or at risk of suffering from an NF-kappaB mediated disorder is disclosed, wherein the method comprising administering to the subject an effective amount of a protein comprising an amino acid sequence comprising SEQ ID NO:1, or a fragment or variant thereof.

Description

METHODS AND COMPOSITIONS FOR TREATING NF-KAPPA B MEDIATED

DISORDERS

The present application relates to methods and compositions for treating NF-kappaB mediated disorders, especially disorders associated with high levels of NF-kappaB regulated cytokines.

NF-KappaB is known to participate in a number of diseases, in particular via its regulation of cytokines. Whilst treatments are available for many of these diseases, there is a need for additional and improved treatments.

Example diseases which are mediated by NF-kappaB include inflammatory disorders, autoimmune diseases and cancer. A common theme amongst many of these diseases is inflammation and its associated biological pathways.

Inflammation is a complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue.

In the absence of inflammation, wounds and infections would never heal and progressive destruction of the tissue would compromise the survival of the organism. However, an inflammation that runs unchecked can also lead to a host of diseases. It is for this reason that inflammation is normally closely regulated by the body.

Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells which are present at the site of inflammation and is characterised by simultaneous destruction and healing of the tissue from the inflammatory process. The effectiveness of current therapies for NF-kappaB mediated disorders, for example inflammatory disorders, can vary and their use is often accompanied by adverse side effects. Thus, improved therapeutic agents and methods for the treatment of NF-kappaB mediated disorders are needed.

SUMMARY OF THE INVENTION

Remarkably, it has now been found that the protein NIeE is a potent inhibitor of NFKappaB and may be used to treat NF-kappaB mediated disorders, especially disorders associated with high levels of NF-kappaB regulated cytokines.

According to one aspect of the present invention, there is provided a method for preventing or treating an NF-kappaB mediated disorder in a subject suffering from or at risk of suffering from an NF-kappaB mediated disorder, the method comprising administering to the subject an effective amount of a protein comprising an amino acid sequence comprising SEQ ID NO: 1 , or a fragment or variant thereof.

In this respect, SEQ ID NO:1 corresponds to the NIeE protein (T3SS secreted effector NIeE homolog [Escherichic coli O127:H6 str. E2348/69]).

Surprisingly, it has been found that the NIeE protein is able to downregulate NF- kappaB activation and proinflammatory cytokine production in mammalian cells. As a result of this activity, it has been found that the NIeE protein can be used in the treatment of NF- kappaB mediated disorders such as inflammatory disorders, cancer and autoimmune diseases.

According to another aspect of the present invention, there is provided use of a protein comprising an amino acid sequence comprising SEQ ID NO:1, or a fragment or variant thereof in the manufacture of a medicament for the treatment or prophylaxis of an NF-kappaB mediated disorder in a subject suffering from or at risk of suffering from an NF-kappaB mediated disorder.

According to a further aspect of the present invention, there is provided a protein comprising an amino acid sequence comprising SEQ ID NO:1, or a fragment or variant thereof for use in the treatment or prophylaxis of an NF-kappaB mediated disorder in a subject suffering from or at risk of suffering from an NF-kappaB mediated disorder.

Preferably, the fragments or variants thereof comprise an amino acid sequence that has at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 1.

Preferably, the fragments thereof comprise at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight consecutive amino acids from SEQ ID NO:1. Longer fragments are also preferred, for example at least about 10, 15, 20, 25, 30, 50, 75, 100, 150 and up to at least about 200 amino acids of SEQ ID NO:1. Fragments may also include truncated peptides that have x amino acids deleted from the N- terminus and/or C-terminus. In such truncations, x may be 1 or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more), but preferably less than 150 amino acids of SEQ ID NO:l.

Preferably, the fragments or variants thereof are functional fragments or variants thereof.

Preferably, the NF-kappaB mediated disorder is selected from an inflammatory disorder, an autoimmune disorder, or cancer.

Preferably, the NF-kappaB mediated disorder is associated with high levels of NF- kappaB regulated cytokines.

Preferably, the inflammatory disorder is selected from arthritis, inflammatory bowel disease, Crohn's Disease and ulceritive colitis.

Preferably, the autoimmune disorder is selected from an allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, autoimmune vasculitis, nephrotic syndrome and rheumatoid arthritis. Preferably, the cancer is selected from breast cancer, ovarian cancer, bladder cancer, lung cancer, thyroid cancer, glioblastoma, stomach cancer, endometrial cancer, kidney cancer, colon and colorectal cancer, pancreatic cancer, prostate cancer, leukemia, myeloma, B lymphoma and non-hodgkins lymphoma.

In some embodiments, the NF-kappaB mediated disorder is selected from sepsis, infectious diseases, transplant rejection, malignancy, pulmonary disorder, intestinal disorder, cardiac disorder and inflammatory bone disorders.

Preferably, the NF-kappaB mediated disorder is selected from psoriatic arthropathy, ankylosing spondylitis, juvenile rheumatoid arthritis, Still's disease, systemic lupus erythematosis, Sjogren's disease, mixed connective tissue disorder, polymyalgia rheumatica, giant cell arteritis, Wegener's granulomatosis, Kawasaki's disease, Bechet's disease, psoriasis, Graves disease, Hashimoto's thyroiditis, asthma, Type 1 diabetes, Type 2 diabetes, ischemic heart disease, peripheral vascular disease, stroke, pyoderma gangrenosum, sarcoidosis, Dercum's disease, toxic epidermal necrolysis, idiopathic uveitis or scleritis, birdshot retinochoroiditis, uveitic and diabetic cystoid macular edema, age-related macular degeneration, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), depression, schizophrenia, Alzheimer's disease, vascular dementia, glomerulonephritis, atherosclerosis, restenosis, graft v. host reactions, septic shock, cachexia, anorexia, multiple sclerosis, gram negative sepsis, endotoxic shock, neoplastic diseases, uveitis (e.g., childhood and seronegative), lupus and other diseases mediated by immune complexes such as pemphigus and glomerulonephritis, congential hyperthyroidism (CH), delayed type hypersensitivity (DTH) such as contact hypersensitivity, sarcoidosis, chronic arthritis, adult still disease, scleroderma, giant cell arteritis, SAPHO syndrome, primary biliary cirrhosis (PBC), myelodysplastic syndromes, vasculitis, hematologic malignancies, cochleovestibular disorders, macrophage activation syndrome, interstitial lung disease, hepatitis C, ovulation induction, and myelodysplastic syndromes.

Example embodiments of the present invention will now be described with reference to the accompanying figures in which: Figure 1 shows that EPEC inject the effectors Tir (translocated intimin receptor) and NIeD into monocyte-derived dendritic cells, (a) Monocyte-derived dendritic cells were either cultured with EPEC-GFP, EPEC ΔescN-GFP or left untreated. Cells were left to adhere on coverslips and fluorescence stained for Tir. Images were taken by confocal microscopy. Shown is a representative image of 3 independent experiments. Scale bar indicates 10 μm. (b) Monocyte-derived dendritic cells were either cultured with EPEC-GFP or EPEC ΔescN- GFP. Cells were left to adhere on coverslips and fluorescence stained for CDl Ic. Shown is a representative image of 3 independent experiments. Scale bar indicates 10 μm. (c) Monocyte- derived dendritic cells were cultured in medium containing 1 mM IPTG for 3 h with either EPEC NIeD-TEM, EPEC ΔescN- NIeD-TEM or left untreated. Cells were afterwards loaded with CCF2-AM for 1 h and then analyzed by flow cytometry. Cells shown in the plot labeled "uninfected, - CCF2" were untreated and not loaded with CCF2-AM for control purposes. Dot plots are representative of 3 independent experiments, (d) T84 cells and mdDCs were cultured in a transwell system with T84 cells on the apical side and mdDCs on the basal side of the membrane. Either EPEC NIeD-TEM-I or EPEC ΔescN- NIeD-TEM-I was added to the apical compartment for 2 h under the addition of ImM IPTG. MdDC were detached by gentle centrifugation, loaded with CCF2-AM for 1 h at RT and cells were analyzed for blue and green fluorescence by confocal microscopy. Images are representative of 3 experiments. Scale bar indicates 50 μm;

Figure 2 shows that the effector NIeD is translocated into Peyer's patch dendritic cells (a) Human Peyer's patch mononuclear cells (PPMCs) in a single cell suspension were cultured in medium containing 1 mM IPTG for 3 h with either EPEC NIeD-TEM-I, EPEC ΔescN- NIeD-TEM-I or left untreated. As a control sample, PPMCs were cultured with EPEC NIeD-TEM-I without IPTG. After culture, cells were loaded with 1 μM CCF2-AM for 1 h and then further stained for HLA-DR and lineage markers to identify DCs. After the initial gating on DCs, cells were analyzed for blue and green fluorescence. Dot plots are representative of 3 independent experiments, (b-g) Whole human Peyer's patch biopsies were cultured for 4 h in medium containing 1 mM IPTG with either EPEC NIeD-TEM-I, EPEC ΔescN- NIeD-TEM-I or left untreated. A single cell suspension was prepared by tissue digestion using collagenase under the addition of gentamicin. Cells were loaded with 1 μM CCF2/AM and further stained for HLA-DR and lineage markers. Cells were then analyzed for blue and green fluorescence by flow cytometry. Figs. 2 b,c,d, show the analysis of the whole PP population after indicated treatments, and Figs. 2 e,f,g show the analysis of only the PP dendritic cells;

Figure 3 shows that EPEC impairs proinflammatory cytokine production in mdDCs and Peyer's patch cells in a type 3 secretion-dependent way. (a) MdDCs were cultured with EPEC wt or EPEC ΔescN for 4 h in a MOI of 1:100. The concentration of IL-8, TNF and IL-6 in culture supernatants was determined by ELISA. Data show the means + SD and are representative for 4 independent experiments, (bl) MdDCs were left untreated, treated with LPS 10 ng/ml or cultured with EPEC wt or EPEC ΔescN for 4 h in a MOI of 1:100 at start of culture. After that, gentamicin was added and cells were further cultured with 2 μM monensin for 4 h to block secretion of cytokines. CDl Ic was stained on the cell surface followed by intracellular TNF staining. Cells were analyzed by flow cytometry. Plots are representative for 3 experiments. Figure 3b2 illustrates that EPEC reduces the expression of co-stimulatory molecules on mdDCs. MdDCs were left untreated, treated with LPS lOng/ml or cultured with EPEC ΔescN for 3h. Gentamicin was added and cells cultured for 24h. Expression of the DC marker CDl Ic as well as the costimulatory and maturation markers, CD80, CD83, and CD86 was analyzed by flow cytometry. The plots show one of 3 experiments with similar results, (c) MdDCs were cultured in a MOI of 1:100 with EPEC wt, EPEC ΔescN, LPS 10 ng/ml or left untreated for indicated times. Cells were lysed and cytosolic and nuclear fractions prepared which were subjected to SDS-PAGE and immunoblotting. Levels of IkappaB alpha, phospho- IkappaB alpha and beta-actin were detected in the cytosolic fraction. p65 and histone Hl were detected in the nuclear fraction. Shown is a representative blot of 3 independent experiments, (d) Isolated PPMCs were cultured in a MOI of 1:100 with EPEC wt, EPEC ΔescN, LPS 10 ng/ml or left untreated for 4 h. After that, gentamicin was added and cells further cultured over night. The concentrations of IL-8, TNF and IL-6 in supernatants were determined by ELISA. Cultures were set up in duplicates. Data are means + SD. Analysis was done by students t-test, p< 0.5. The shown graph is representative for 3 independent experiments;

Figure 4 shows that overexpression of the effector NIeE impairs the proinflammatory response of mdDCs and HeLa cells, (a) MdDCs were either left untreated or cultured in a MOI of 1 :100 with EPEC wt, EPEC ΔescN, EPEC Δlsland 1, EPEC Δlsland 4, EPEC Δlsland 5, EPEC Δlsland 1,4, EPEC Δlsland 4,5 for 3 h. IL-8, TNF and IL-6 were determined in supernatants by ELISA. Results are representative of three independent experiments, (b) MdDCs were either left untreated or cultured in a MOI of 1 : 100 with EPEC wt, EPEC ΔescN, EPEC ΔNleE, EPEC ΔNleE complemented with NIeE for 3 h. IL-8, TNF and IL-6 were determined in supernatants by ELISA. Results are representative of four independent experiments, (c) HeLa cells were either left untransfected, transfected with the empty myc- vector, NleE-myc or EspB-myc. After 30 min of IL-I treatment, cells were stained for myc and NF-kappaB-p65 and afterwards analysed for nuclear p65 staining by confocal microscopy. Statistical analysis of the microscopic pictures is shown in (d). Counting was done in 12 randomly chosen pictures, taken at 400fold magnification. "NIeE myc transfected" and "NIeE myc untransfected" show the values of the either NleE-myc positive cells or untransfected cells, respectively in the same sample. "EspB myc transfected" and "EspB myc untransfected" accordingly. Values are means + SD. Analysis was done by students t-test, p< 0.01. (Number of analysed cells per condition n= >113). Results are representative for 3 independent experiments, (e) The luminescence readouts of 57A HeLa cells that were transfected with the myc- vector or different amounts of NleEO-myc plasmid, cultured for 36 hours and then treated with TNF-α for 6h before adding luciferase substrate and analysing luminescence are shown. Increasing amounts of transfected NIeE reduce nuclear factor kappa B-p65 activation in a dose-dependent manner. The graph shows mean values + SD from 3 experiments with n between 6 and 12. (f) Immunoblots of nuclear factor kappa B-p65 in nuclear extracts of MdDCs that were cultured with EPEC strains and stimulated with IL- lβ for indicated times are shown. The lower blot shows histone Hl as a loading control. The figure shows one of 2 similar experiments.

Figure 5 shows Z-stack of the T84 cell and DC transwell system. Confocal Z-stack images of T84 cells (apical side) and mdDCs (basal side) on a transwell membrane with EPEC-GFP in the apical compartment. Occludin (tight junction protein) is stained with Alexa fluor 647 and CDl Ic (DC marker) is labeled with Alexa fiuor 555. Nuclear counterstaining was achieved with DAPI. Cells were cultured for 2 h with bacteria before fixation and immunofluorescence staining;

Figure 6 shows that EPEC does not reduce viability of mdDCs. MdDCs were either cultured with EPEC wt or EPEC ΔescN for 4 h in a MOI of 1 : 100 or left untreated. Cells were then resuspended in annexin binding buffer and stained with anti-annexin V and PI. Cells were analyzed by flow cytometry to determine percentages of apoptotic and necrotic cells in the population. Results are representative for 3 independent experiments;

Figure 7 shows that EPEC impairs NF-kappaB signaling in HeLa cells. HeLa cells were transfected with pDsRed-p65. For live cell imaging, EPEC wt or EPEC ΔescN were added to the culture medium and HeLa cells imaged by confocal microscopy for 80 min under constant temperature (37°C) and CO₂ (5%) conditions;

Figure 8 shows that EPEC does not reduce viability of PPMCs. PPMCs were cultured for 4 h either with EPEC wt, EPEC ΔescN in a MOI of 1:100, treated with LPS 10 ng/ml or left untreated. Cells were then resuspended in annexin binding buffer and stained with anti- annexin V and PI. Cells were analysed by flow cytometry to determine percentages of apoptotic and necrotic cells in the population. Results are representative for 3 independent experiments;

Figure 9 shows the amino acid sequence of NIeE (SEQ ID NO:1); and

Figure 10 shows the nucleic acid sequence (SEQ ID NO:2) which encodes the amino acid sequence of NIeE (SEQ ID NO:1)

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods and compositions for treating NF-kappaB mediated disorders, for example inflammatory disorders, autoimmune disorders and cancer.

The methods used in the invention and detailed examples of the invention are set out below.

Within this specification embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the invention. Within this specification, the terms "comprises" and "comprising" are interpreted to mean "includes, among other things". These terms are not intended to be construed as "consists of only".

Within this specification, the term "about" means plus or minus 20%, more preferably plus or minus 10%, even more preferably plus or minus 5%, most preferably plus or minus 2%.

As used herein, the term "effective amount" means the amount of a composition which is required to treat or prevent the specified indication.

As used herein, the term "subject" refers to an animal, preferably a mammal and in particular a human. In a particular embodiment, the subject is a mammal, in particular a human, who is suffering from or is at risk of suffering from an NF-kappB mediated disorder. The term "subject" is interchangeable with the term "patient" as used herein.

Also encompassed by the present invention are pharmaceutical compositions comprising the claimed protein for the claimed uses.

For clinical use, a compound (eg. a protein) according to the present invention or prodrug form thereof is formulated into a pharmaceutical formulation which is formulated to be compatible with its intended route of administration, for example for oral, rectal, parenteral or other modes of administration. Pharmaceutical formulations are usually prepared by mixing the active substance with a conventional pharmaceutically acceptable diluent or carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Examples of pharmaceutically acceptable diluents or carrier are water, gelatin, gum arabicum, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal silicon dioxide, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Such formulations may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifϊers, flavouring agents, buffers, and the like.

The formulations can be further prepared by known methods such as granulation, compression, microencapsulation, spray coating, etc. The formulations may be prepared by conventional methods in the dosage form of tablets, capsules, granules, powders, syrups, suspensions, suppositories or injections. Liquid formulations may be prepared by dissolving or suspending the active substance in water or other suitable vehicles. Tablets and granules may be coated in a conventional manner.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, 'chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum mono stearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a compound according to an embodiment of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Within this specification, "identity," as it is known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Percentage identity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), all of which are incorporated herein by reference in their entirety. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Preferred computer program methods to determine percentage identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984), which is incorporated herein by reference in its entirety), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990), which is incorporated herein by reference in its entirety). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. MoI. Biol. 215: 403-410 (1990), which is incorporated herein by reference in its entirety). As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of "SEQ ID NO: A" it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of "SEQ ID NO: A." In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of "SEQ ID NO:B" is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of "SEQ ID NO: B." In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Within this specification, the term "treatment" means treatment of an existing disease and/or prophylactic treatment in order to prevent incidence of a disease. As such, the methods of the invention can be used for the treatment, prevention, inhibition of progression or delay in the onset of disease. The term "treatment" may also mean treatment of the symptoms of a disease rather than complete cure of the source of the disease.

EXAMPLES

Intestinal dendritic cells (DCs) send processes into the gut lumen to sample pathogens. Non-invasive enteropathogenic Escherichia coli (EPEC) colonizes the surface of gut epithelial cells using a type three secretion system (T3SS) to inject effector proteins that modify epithelial cell function, and is typical of the types of pathogens that gut DCs need to sense to initiate protective immunity. We investigated whether EPEC might also inject proteins into DCs to prevent immune recognition. Using a FRET based system linked to T3SS we clearly showed that EPEC can inject effector proteins into in vitro grown myeloid DCs (mDCs), human Peyer's patch DCs and DCs sending processes across model gut epithelium. Myeloid DCs cultured with a mutant EPEC which cannot translocate effector proteins, were found to secrete large amounts of pro-inflammatory cytokines and increase expression of CD80, CD83 and CD86. In marked contrast, wild type EPEC barely elicited proinflammatory cytokine production and shut off nuclear translocation of NF-kappaB p65. By sequentially deleting effector protein genes in the known pathogenicity islands, we identified NIeE on pathogenicity Island 4 as being critical for this effect. Over-expression of NIeE in HeLa cells completely prevented p65 accumulation in response to IL- lβ activation, and luciferase production in an NFKappaB reporter cell line. Our results show that EPEC translocates effector proteins into human dendritic cells to dampen the danger signal elicited by its own pathogen associated molecular patterns.

Abbreviations

DCs, dendritic cells; EPEC, enteropathogenic Escherichia coli; FBS, fetal bovine serum; GFP, green fluorescent protein; IKB, inhibitory factor kappa B; mdDCs, monocyte- derived dendritic cells; NF-κB, nuclear factor kappa B; pAb, polyclonal antibody; PAMPS, pathogen associated molecular patterns; PI, propidium iodide; PPMCs, Peyer's patch mononuclear cells; T3SS, type three secretion system; RLU, relating light unit.

Dendritic cells (DCs) directly sense commensals and pathogens in the gut by sending processes between gut epithelial cells into the lumen (1, 2, 3). The DCs recognize pathogen associated molecular patterns (PAMPS) on bacteria and microbial products via engagement of Toll-like receptors and NOD molecules, leading to protective immunity to pathogens or tolerance to commensals (4).

Enteropathogenic Escherichia coli (EPEC) is one of the main causes of infant diarrhea in developing countries. EPEC belongs to the group of pathogens which are non-invasive but colonize the intestinal mucosa using the type three secretion system (T3SS) to form attaching and effacing (A/E) lesions (5). EPEC as well as enterohaemorrhagic E. coli (EHEC) shows an unexplained tropism for the follicle-associated epithelium (FAE) overlying organized gut associated lymphoid tissue in the early stage of infection (6).

A number of studies have shown that EPEC elicits a proinflammatory response in gut epithelial cells (7, 8). However, other work suggests that the situation is more complex and that EPEC can reduce inflammatory cytokine production dependent on a functional T3SS (9, 10, 11). There are however no studies on the interactions between EPEC and gut immune cells. Since EPEC colonizes the surface of lymphoid structures in the gut, the site at which protective mucosal immune responses are generated, we investigated whether EPEC might also inject effectors into DCs as they reach into the lumen. Here we show that this is indeed the case and have identified an EPEC protein of previously unknown function, NIeE, which down regulates NF-kappaB activation and proinflammatory cytokine production in mammalian cells.

EPEC injects the translocated intimin receptor (Tir) and the effector NIeD into monocyte- derived dendritic cells

Tir is a receptor molecule that has been shown to be inserted by EPEC into the plasma membrane of epithelial cells by T3S (12, 13). It acts as the receptor for intimin, a bacterial outer-membrane protein that mediates EPECs intimate attachment to the host cell. Bacterial attachment is followed by injection of effector proteins via T3SS, subverting several host cell functions (5). To determine if EPEC can also translocate Tir into human dendritic cells, we co-cultured human blood monocyte-derived dendritic cells (mdDCs) for 4 h with GFP- expressing EPEC (EPEC-GFP) and EPEC ΔescN-GFP, a GFP-expressing mutant which lacks the ATPase escN and therefore cannot translocate proteins by T3SS. Confocal imaging revealed that Tir was detectable in the plasma membrane of mdDCs cultured with EPEC-GFP (Fig Ia). Dendritic cells that had been cultured with the ΔescN mutant were also closely surrounded by bacteria, but Tir was not detectable in the membrane of the DCs. EPEC-GFP and EPEC ΔescN-GFP surrounded mdDCs equally well (Fig Ib). To directly visualize EPEC effectors in mdDCs, we made use of an established reporter system which is based on the fusion of the bacterial effector protein NIeD and the beta-lactamase TEM-I. This fusion results in expression and translocation of TEM-I together with NIeD. The presence of TEM-I can be detected in cells by its ability to cleave the green fluorescent substrate CCF2/AM to yield blue fluorescence. Approximately 20% of mdDCs co-cultured with EPEC NIeD-TEM-I and subsequently loaded with CCF2/AM emitted blue fluorescence, indicating injection of the fusion protein (Fig Ic). In contrast, mdDCs cultured with EPEC ΔescN NIeD-TEM-I, showed very little blue fluorescence (Fig Ic).

We next seeded mdDCs on the basal side of membrane filters that had a confluent layer of T84 cells with very high transepithelial resistance (TER) on the apical side, and added either EPEC NIeD-TEM-I or EPEC ΔescN NIeD-TEM-I to the apical side of the epithelial cells (as shown in a z-stack image in the supplementary Fig 5). After 2 h incubation, mdDCs were detached from the membrane and loaded with CCF2/AM. Confocal microscopy revealed that among mostly green cells, some mdDCs from the transwell with EPEC NIeD-TEM-I emitted blue fluorescence, indicating intracellular beta-lactamase activity. Blue cells were completely undetectable in mdDCs detached from membranes of transwells cultured with EPEC ΔescN NIeD-TEM-I or uninfected (Fig Id). Infection with EPEC NIeD-TEM-I led to only a small fall in TER and no bacteria were cultivatable from the basal side of the transwell (data not shown). These results show that EPEC injects effector proteins into DCs, including those DCs reaching through an epithelial monolayer.

EPEC translocates the effector NIeD into dendritic cells of human Peyer 's patches

EPEC colonizes the FAE of Peyer' s patches (PP) of the human ileum. So we next explored the possibility that EPEC could inject effector proteins into PP DCs. We firstly co- cultured single cell suspensions of PP mononuclear cells (PPMCs) with EPEC NIeD-TEM-I and the escN mutant and subsequent loading with CCF2/AM. The PPMCs identified as DCs (lineage negative / HLA-DR positive) were then analyzed for blue and green fluorescence. Forty seven percent of the DCs cultured with EPEC NIeD-TEM-I (+IPTG) emitted blue fluorescence and 12% a double positive green/blue signal (indicating that not all CCF2/AM had been cleaved) compared to 1.4% of the cells cultured with the ΔescN mutant (Fig 2a). Next, we determined if EPEC can inject effector proteins into DCs of intact PP biopsies. PP biopsies were cultured with either EPEC NIeD-TEM-I or EPEC ΔescN NIeD-TEM-I for 4 h before single cell suspension were made in culture medium containing gentamicin to kill all bacteria. Cells were then loaded with CCF2/AM and stained for DC markers. In PPMCs from biopsies cultured with EPEC NIeD-TEM-I, we detected a blue signal (1%) and double positive green/blue signal (66%, Fig 2b). The DCs in this sample emitted a blue signal (11%) and double positive green/blue signal (82%, Fig 2e). We could hardly detect any blue or blue/green cells from biopsies cultured with the escN mutant (Fig 2c, f) or from uninfected biopsies (Fig 2d, g). These results suggest that EPEC injects effector proteins into PP DCs. We are aware that EPEC could inject effector proteins into cells at the edges of the cut biopsies; however the vast majority of DCs in human PP are present in the subepithelial dome, distant from the cut edge. EPEC reduces the expression of co-stimulatory molecules on dendritic cells.

Dendritic cells are known to up-regulate co-stimulatory molecules upon contact with bacterial components. To investigate if EPEC can influence the expression of these cell surface markers, we analyzed the co-stimulatory molecules CD80 and CD86 as well as the maturation marker CD83 on mdDCs after culture with EPEC. After culture with the escN mutant or LPS, mdDCs strongly unregulated expression of CD80, CD83, and CD86 (Fig 3 b2). Cells cultured with wild type EPEC showed only moderate increase in accessory molecules. The difference was unlikely to be due to differences in the ability of the two strains to come into contact with the DCs (Fig Ib).

Dendritic cells and Peyer 's patch cells co-cultured with EPEC show a markedly reduced proinflammatory response

To look at the inflammatory response, we cultured mdDCs with EPEC wild type (wt) and EPEC ΔescN for up to 4h and collected the culture supernatants. DCs cultured with the ΔescN mutant produced very high levels of the proinflammatory cytokines IL-8, TNF and IL- 6, while cells cultured with the wild type strain secreted small amounts of these cytokines (Fig 3 a). TNF production by mdDCs was further investigated by intracellular cytokine staining. Forty five percent of CDl Ic+ mdDCs cultured with EPEC ΔescN were positive for TNF compared to 4.7% of mdDCs cultured with EPEC wt and 17% of cells cultured with LPS (Fig 3bl).

To exclude that the low cytokine concentrations were due to cell death, we stained the mdDCs for Annexin V as indicator of apoptosis and propidium iodide (PI) as a viability dye. Fig 6 shows the dot plots of mdDCs analyzed for Annexin V and PI staining. Viability of mdDCs cultured with EPEC wt and EPEC ΔescN was not decreased compared to uninfected mdDCs.

Stimulation of DCs with bacteria leads to marked increase in surface expression of the co- stimulatory molecules CD80, CD86 and of the maturation marker CD83. EPEC ΔescN elicited a marked increase in the expression of all of these molecules, but wt EPEC had only little effect (Fig 3b2.

Since the the expression of IL-8 and TNF is mainly regulated by the transcription factor NF-kappaB, we addressed the question if EPEC affects NF-kappaB signaling in DCs. Immunoblotting revealed a lower content of NF-kappaBp65 in the nuclear extracts after co- culture with the wild type EPEC for 3 h compared to extracts from mdDCs cultured with the ΔescN mutant (Fig 3 c). The level of phosphorylated IkappaB alpha decreased in cytosolic fractions of mdDCs cultured with EPEC wt but was almost constant for the time of the experiment in the cytosol of cells cultured with the ΔescN mutant. Accordingly, total IkappaB alpha increased after 2 h in cells cultured with the wild type but remained low in cells cultured with the mutant (Fig 3c). For live cell imaging, we transfected HeLa cells with pdsRedl-p65 and added either EPEC wt or EPEC ΔescN. For the latter there was rapid nuclear p65 accumulation which persisted for 40 min. For the wt EPEC, we could barely detect accumulation of p65 in the nucleus of any cells (Fig 7).

To study if EPEC also reduces proinflammatory cytokine production in PPMCs, we cultured PPMCs with EPEC for 4 h after which gentamicin was added to avoid bacterial overgrowth. IL- 8, TNF and IL-6 concentrations were determined in culture supernatants and revealed significantly lower concentrations for cells cultured with EPEC wt compared to cells in culture with ΔescN (Fig 3d). We performed Annexin V and PI staining on these cells at the end of culture to investigate if there were differences in cell viability. Neither of the bacterial strains increased cell death in our experimental procedures (Fig 8).

Identification of the effector protein NIeE as the T3SS effector which is involved in inhibiting NF-kappaB activation and pro-inflammatory cytokine production

By employing site directed mutagenesis of the different pathogenicity islands in EPEC, we identified Island 4 (encoding effectors EspL, NIeE and NIeB) as a possible locus for the effector that might be responsible for the modulation of NF-kappaB signaling in mdDCs. (For a list of the mutants which had no effect, see supplementary Table I). Cells cultured with Island 4 mutants showed markedly higher production of IL-8, TNF and IL-6 than cells cultured with the wild type (Fig 4a). By selective mutagenesis of the effectors encoded by Island 4, we identified NIeE, which is an effector protein translocated by T3SS, as the most likely protein that targets the NF-kappaB pathway. MdDCs cultured with EPEC ΔnleE showed high secretion of IL-8, TNF and IL-6 and this was markedly reduced in mdDCs cultured with EPEC ΔnleE complemented with a plasmid encoding NIeE (Fig 4b). NIeE was cloned into the myc-tagged vector pRK5 and transfected into HeLa cells. As controls, cells were either transfected with the empty vector or a plasmid encoding for the effector EspB-myc. After treatment with IL- lβ, cells were analyzed for the presence of p65 in the nucleus by confocal microscopy. No NleE-myc transfected cells showed translocation of p65 into the nucleus (Fig 4c, d), whereas 39% of EspB-myc transfected cells (Fig 4c,d) showed nuclear p65. A similar range of nuclear p65 was seen in untransfected and vector transfected cells. In the transfected samples, we also analyzed the untransfected cells ("NIeE myc untransfected" and "EspB myc untransfected", respectively) which showed nuclear p65 in 37% and 38% of cells (Fig 4c, d). We also used an NFkappaB reporter cell line to quantify the effect of NIeE. TNF elicited a large increase in luciferase activity; however transfection with graded amounts of plasmid expressing NIeE reduced luciferase activity in a dose- dependent fashion (Fig 4e). The importance of NIeE for the down-regulation of the inflammatory response in DCs was confirmed by immunoblotting for p65. DCs cultured with EPEC AnIeE showed strong nuclear translocation of p65 after 30 min and Ih of infection, whereas cells cultured with the complemented strain had little nuclear p65 after Ih (Fig 4f). We attribute the more rapid kinetics compared to wt bacteria to the over-expression of NIeE. We also sought to determine if NIeE could inhibit NFkappaB activation in DCs in response to another signal. When we added IL- lβ after Ih of infection with EPEC AnIeE, there was still high induction of nuclear p65 after 30 minutes, however cells pre-treated with NIeE complemented bacteria were unresponsive to IL-I β (Fig 4f).

We show here for the first time that EPEC use the T3SS to inject effector proteins into human gut DCs. We have attempted to mimic the physiological context in which this will occur in the human gut, namely by focusing on human PP cells wherever feasible, and particularly on DCs, which send processes into the gut lumen to sample pathogens. Taken together, our observations that EPEC injects effector proteins into mdDCs, mdDCs reaching through a model epithelium, PPMCs, and DCs in whole PP biopsies suggest that the process of injection into DCs is indeed a phenomenon with in vivo significance.

The second and critically important part of the study is the functional consequences of this interaction. When DCs were cultured with an EPEC ΔescN mutant there was massive NFkappaB activation and the secretion of large amounts of IL-8, TNF and IL-6. EscN is an ATPase which is essential for the formation of the T3SS molecular syringe and for the secretion of effector molecules. The massive response elicited by these bacteria in DCs is presumably in response to recognition of PAMPS on the bacteria by DCs. There is not a priori reason to assume that the wt EPEC has any deficiency in PAMPS, yet when it was co- cultured with DCs (and HeLa cells), after a brief burst of NF-kappaB activation and cytokine secretion, the inflammatory response was shut off. This strongly suggested that the inhibition was due to an effector protein and so we sequentially knocked out the major pathogenicity islands in wt EPEC to attempt to abolish the effect. Deletion of pathogenicity Island 4 abolished the inhibitory effect and when we focused down on the 3 proteins encoded on this locus, deletion of nleE, also abolished the inhibitory effect. Island 4 corresponds to the locus encoding the 3 effectors, NIeE, NIeB and EspL on the recently described integrative element IE6 of EPEC E2348/69 genome (14). This island is conserved in A/E pathogens, known as O- Island 122 in EHEC O157:H7, and is significantly associated with the most pathogenic strains (15, 16).

A copy of Island 4, Island 5, is present on the integrative element IE2 in EPEC E2348/69 but the genes for nleB and espL have frameshift mutations and are theoretically non- functional. The copy of nleE on Island 5 has an internal deletion of 118 nucleotides but the open reading frame is conserved suggesting that the protein is produced. However to avoid the possible complementation by these effectors we also deleted Island 5 together with Island 4. NIeB, EspL, and NleE produced by A/E mouse pathogen Citrobacter rodentium are required for full colonization and full induction of disease in mice (16-18). Mutation of nleB leads to the most important attenuation of virulence with a strong reduction of colonization and no mortality in infected mice (16, 17). Mutation of espL (entL) or nleE induce a lower but significant reduction in colonization and a delayed mortality (16, 18). EspL (EspL2) produced by EHEC was recently shown to modify the host cell cytoskeleton via binding to annexin-2, thought to be potentially involved in adherence (19).

We were able to verify the anti-inflammatory effects of NleE itself by direct transfection into HeLa cells and showed that it completely inhibited NF-kappaB activation. This study clearly shows that EPEC can reduce the proinflammatory response of host cells by injecting NleE.

Material and methods 2

Reagents and cytokines

Recombinant human Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) and interleukin-4 (IL-4) were obtained from PeproTech (London, UK). Lipopolysaccharide from Escherichia coli 055:B5, IPTG, gentamicin, BSA, EDTA and cell culture reagents were purchased from Sigma (Gillingham, UK). Interleukin-lβ (IL- lβ) was obtained from R&D Systems (Abingdon, UK).

Antibodies

Antibodies for immunofluorescence staining: dendritic cells were labeled with a mouse pAb against CDl Ic (1:40, abeam, Cambridge, UK). The EPEC translocated intimin receptor (Tir) was decorated with a rabbit serum containing pAb against Tir (1:500; kindly provided by Gad Frankel). Anti-myc clone 4A6 was purchased from Cambridge Bioscience (Cambridge, UK) and used 1:500. NF-kappaBp65 was stained with rabbit anti-p65 (1 :500, Santa Cruz, Heidelberg, Germany). Anti-occludin (1:200) was purchased from Santa Cruz. Secondary antibodies were Cy3 and Cy5-conjugated or Alexa 555 and Alexa 488-conjugated (Invitrogen, Paisley, UK).

Antibodies for flow cytometry: monoclonal antibodies CDl Ic-APC, TNF-PE-Cy7 and HLA-DR-PE-Cy7 as well as appropriate isotype controls were purchased from Becton Dickinson (Oxford, UK). The dendritic cell exclusion cocktail (= lineage: antibodies against CD3, CD 14, CD 16, CD 19, CD34) was PE-Cy5-conjugated and purchased from AbD Serotec (Oxford, UK).

Antibodies for immunoblotting: Rabbit anti IkappaB alpha and rabbit anti-NF- kappaBp65 were bought from Santa Cruz. Mouse anti-phospho-IkappaB alpha was obtained from Cell Signaling (Danvers, MA, USA). Rabbit anti-beta-actin (abeam) served as a cytosolic loading control. Mouse anti-histone Hl was used as nuclear loading control (AbD Serotec).

Patients Biopsies of Peyer's patches were taken in the terminal ileum from patients undergoing routine colonoscopy who had no signs of intestinal inflammation and no history of inflammatory bowel diseases. The reason for colonoscopy in these patients was mostly a change in bowel habits, screening for colon cancer and polypectomies. If necessary, the dye indigocarmin was sprayed onto the ileal mucosa to facilitate identification of Peyer's patches in adult patients. All patients who took part in this study did so after informed consent. Pediatric tissue was obtained with fully informed parental consent. This study was approved by the local ethics committee.

Processing of Peyer's patch biopsies

Biopsies were carefully washed in sterile HBSS (without calcium and magnesium, containing 100 U/ml penicillin and 100 μg/ml streptomycin). Epithelial cells were removed with 1 mM EDTA in HBSS containing antibiotics under agitation for 30 min at 37°C. A single cell suspension was prepared in RPMI/ 10% FBS/ penicillin & streptomycin / gentamicin with collagenase D (1 mg/ml; Roche, Burgess Hill, UK) and DNase (10 U/ml; Roche) under agitation for Ih at 37°C, then passed through a cell strainer. Cells were washed twice with RPMI medium containing 10% fetal bovine serum (FBS) without antibiotics and kept on ice until further use.

Differentiation of dendritic cells from human blood monocytes

Peripheral blood was obtained from buffy coats (National Blood Service, Brentwood, UK). Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation with Ficoll-Paque (GE Healthcare, Buckinghamshire, UK) for 30 min at room temperature followed by three washing steps with PBS containing 0.5% BSA and 2 mM EDTA. For magnetic labeling of blood monocytes, PBMCs were incubated with MACS CD 14 micro beads (Miltenyi Biotech, Surrey, UK) for 15 min at 4°C. The labeled cells were separated with MS columns according to manufacturer's instructions. Isolated monocytes were cultured in RPMI medium supplemented with 10% FBS, 2 mM L-Glutamine, 1% non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin. GM-CSF and IL-4 were added to the medium at final concentrations of 800 U/ml each. Cells were cultured over a period of six days with cytokines being replenished after 3 days by removing half of the culture medium and adding back the same volume of medium containing fresh GM-CSF and IL-4 resulting in a final concentration of 800 U/ml and 500 U/ml, respectively. Before infection experiments, cells were cultured over night without antibiotics.

Bacterial strains, constructions of mutant strains and expression vectors

All EPEC strains were grown on Luria-Bertani (LB) plates with the required antibiotics and single colonies were picked for culture in LB broth. See Table 1 for details about the strains used in this study. For expression of green fluorescent protein (GFP), strains were electroporated with the plasmid pFVP25.1 encoding for the gfpmut3a gene. To obtain strains expressing the NIeD-TEM-I fusion protein, bacteria were electroporated with the plasmid pCX-NleD encoding for the effector NIeD fused to the beta-lactamase TEM-I under the control of the IPTG-inducible promoter Ptrc.

Primers and plasmids used in this study are listed in Table 2 and 3, respectively. Mutants of the EPEC O127:H6 strain E2348/69 constructed for this study were obtained using the PCR one-step λ Red recombinase method (42). Briefly, each mutation was obtained using a PCR product containing an antibiotic resistance gene flanked by the 50 bases from the 5' and 3' ends of the target locus. Plasmids pKD3 (mutations of Island 1 and Island 5) and pSB315 (mutations of Island 4 and nleEl) were used as PCR templates. The PCR products were electroporated into the recipient strains carrying the Red system expression plasmid pKD46 and mutants were selected on LB plates with kanamycin or chloramphenicol. Recombinant clones were cured of pKD46 plasmid by growth at the non permissive temperature (42⁰C) and mutation confirmed by different PCR reactions using primers flanking the targeted region and primers into the antibiotic resistance gene. Mutation of Island 1 was obtained with primers Nlel-EPEC-FRT-for and NleD-EPEC-FRT-rev inducing a deletion of a 5923bp fragment encompassing the gene for nlel, nleB2, nleC and nleD. Island 5 mutant was obtained with primers NleB3-EPEC-FRT-for and NleE2-EPEC-FRT-rev inducing a deletion of a 5680 locus containing the gene for nleE2 and the pseudogenes for nleB3 and espL2 but not the efal/lifA-like gene. Island 4 mutant was obtained with primers NleBl-EPEC-Kn315- for and NleEl-EPEC-Kn315-rev and has a deletion of a 5822bp locus containing the effectors genes nleEl, nleBl and EspLl but not the efal/lifA gene. The mutation of nleEl was obtained using the primers pair NleE-EPEC-Kn315-for and NleEl-EPEC-Kn315-rev. The 2

genome sequence of the EPEC O127:H6 strain E2348/69 was recently published and the pathogenicity islands encoding the effectors deleted in this study have been annotated (14). Island 1 in this study refers to the locus containing the genes nleG, nleB, nleC and nleD located on the lambda-like prophage which is called PP4 (14). Island 4 and Island 5 refers to the loci containing the genes or pseudo-genes nleE, nleB and espL and are located on the integrative elements called IE6 and IE2 (14).

The gene encoding the effectors proteins were amplified by PCR using Deep Vent DNA polymerase (New England BioLabs, Ipswich, Massachusetts, USA) and genomic DNA from EPEC E2348/69 strain. PCR products obtained with primer pairs EspL-EcoRV-for/EspL- Pstl-rev and EspL-EcoRV-for/EspL2HA-Pstl-rev were digested by EcoRV and Pstl and cloned into pSAlO digested with Smaland Pstl giving plasmids pSA-espL and pSA- espL2HA respectively. PCR products obtained with primer pairs NIeBl -EcoRl -for/NleBl- Pstl-rev, NleEl-EcoRl-for-NleEl-Pstl-rev and NleEl-EcoRl-for/NleE2HA-Pstl-rev were digested with EcoRl and Pstl and cloned into the corresponding sites of pSAlO giving plasmids pSA-nleBl, pSA-nleEl and pSA-nle2HA respectively. PCR products amplified with primer pairs NleE-E69-BamHl-for/ NleE-E69-EcoRl-rev and EspB-BamHl-for/EspB- EcoRl-rev were digested by BamHland EcoRl and cloned into pRK5 giving plasmids pRK- nleE and pRK-espB respectively. All plasmids were verified by DNA sequencing.

Co-culture of dendritic cells and EPEC strains

The evening before infection, mdDCs were washed twice with HBSS, counted and resuspended in fresh RPMI medium/10% FBS and seeded in 24-well plates. Bacteria were cultured for 8h at 37⁰C in LB broth, then sub cultured 1:500 in RPMI medium/10% FBS at 37°C over night. After determination of the OD600, bacteria were washed twice with HBSS and resuspended in fresh cell culture medium. Bacterial cells were added to the dendritic cells and incubated at 37°C for the desired time of the experiment. Culture supernatants were collected and the cells further processed for lysate preparation.

Translocation assay using NIeD-TEM-I beta-lactamase fusion protein For the investigation of type three secretion (T3S) into dendritic cells by EPEC, we employed a previously published reporter system that utilizes a translational fusion between the effector protein NIeD and the beta-lactamase TEM-I (43, 44). Injection of TEM-I into host cells only occurs when NIeD is translocated. The presence of TEM-I in the host cell can be detected with the green fluorescent substrate CCF2/AM (520 nm emission) which easily enters the cells and emits blue fluorescence (447 nm emission) after cleavage by TEM-I. Dendritic cells were co-cultured with EPEC E69 pcX-NleD-TEM-1 in RPMI/10% FBS/lmM IPTG for indicated periods of time. After washing, cells were resuspended in HBSS and loaded with 1 μM CCF2/AM for 2 h at room temperature (CCF2/AM loading kit from Invitrogen). The cells were washed and further analyzed for green and blue fluorescence by confocal microscopy or flow cytometry.

Flow cytometry

For surface stainings, cells were resuspended in FACS blocking buffer containing 20% human serum (Sigma) in PBS and left on ice for 30 min before adding fluorochrome- conjugated antibodies. Staining was performed for 30 min on ice followed by two washing steps with FACS buffer (PBS containing 0.02% sodium azide, 2% FBS and 2 mM EDTA). For intracellular cytokine staining, the cells were initially cultured with 2 μM monensin for 4 h to block secretion. Cells were then fixed in Leucoperm (AbD Serotec) solution A for 15 min at RT, followed by permeabilization in solution B and staining with the fluorochrome- conjugated antibody for 30 min. Flow cytometry was performed using the LSR II analyzer (Becton Dickinson) and data were analyzed with either FACS Diva or WinList software.

Cell Viability assay

Annexin V and propidium iodide (PI) staining was employed to identify apoptotic and necrotic cells. 105 cells were resuspended in 100 μl binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaC12) and stained with 5 μl Annexin V-FITC and 5 μg /ml PI (both BD Bioscience) for 15 min. 400 μl binding buffer were added and cells analyzed immediately by flow cytometry.

Co-culture ofT84 cells and human dendritic cells on transwell membranes T84 cells (epithelial colonic cancer cell line) of passage numbers 7-15 were cultured in Dulbecco's Modified Eagle Medium (DMEM) / Hams Nutrient Fl 2 Mix, supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1% non-essential amino acids and 2 mM L-glutamine at 37°C with 5% CO2. For transwell experiments, cells were seeded on the upper side of collagen-treated membranes (6.5 mm diameter, 3 μm pore size; Corning, New York, USA) and cultured for 13 to 15 days. Medium was changed every other day and cells were used for experiments when the transepithelial resistance reached > 1000 Ωcm2. Prior to the infection, T84 and dendritic cells were cultured without antibiotics over night. The transwell inserts were positioned upside down in a 6- well plate and a drop containing 4x10⁵ dendritic cells was placed onto the membrane. DCs were allowed to adhere for 4 h at 37°C. Afterwards, the transwell inserts were turned around in a new 24-well plate with fresh medium and bacteria were added from the apical side of the T84 cells (upper transwell compartment). During incubation, multiple medium samples were taken from the lower transwell compartments and plated on LB agar plates. The plates showed no bacterial colonies after over night culture and ensured that bacteria were only present in the upper compartment and could not interact with the mdDCs from the basal side. After 2 h of infection, the supernatants of the upper compartment were collected and the plate gently centrifuged to detach the DCs from the membrane. Supernatants in the lower transwell compartment were collected and the DCs further processed for the beta-lactamase translocation assay. In parallel experiments, the membranes was processed for immunofluorescence staining, cut out of the plastic insert and mounted on glass slides.

Culture and transfection of HeLa cells

HeLa cells were cultured in DMEM with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and seeded on poly-L-lysine-coated cover glasses placed in 24 well plates. The following day, cells were transfected with plasmids encoding for NleE-myc, EspB-myc or the empty vector with Fugene HD (Roche) in a ratio of 5:2 (Fugene : DNA). Twenty- four hours after transfection, cells were treated with 20 ng/ml recombinant IL- lβ (R&D) for 30 min. Cells were further processed for immunofluorescence staining. 9

For live cell imaging, HeLa cells were seeded on glass chamber slides and transfected with pDsRed-p65 (45). Imaging was performed by confocal microscopy (LSM 510 META, Carl Zeiss Jena, Germany) with the cells kept in an environmental chamber at 37°C and 5% CO2.

Luciferase reporter assay

57A HeLa cells (53) which are stably transfected with the luciferase gene under the control of NFkappaB regulatory elements, were transfected with the plasmid encoding for NleE-myc or the empty vector. To ensure an equal transfection efficiency of different amounts of DNA, transfection was monitored by co-transfection of a GFP-encoding plasmid (Lonza, Basel, Switzerland). After 36h, cells were seeded in duplicates and treated with 10 ng/ml TNF for 6h. Luciferase substrate (Promega, Southampton, UK) was added to the cells and the luminescence measured.

Immunofluorescence staining and confocal microscopy

After infection experiments, dendritic cells were washed twice with PBS, seeded on poly-L-lysine coated coverslips and allowed to adhere for 1 h at room temperature. Cells were fixed with 3% paraformaldehyde/PBS for 15 min and permeabilised with 0.1% Triton X in PBS for 5 min. Non-specific binding sites were blocked with 10% horse serum (Sigma). Primary and secondary antibodies were applied in a humidifying chamber for 45 min each at 37°C. Nuclear counterstaining was achieved by adding DAPI (1 μg/ml; Sigma) during incubation with the secondary antibody. After washing in PBS, coverslips were mounted in Immu-Mount (Thermo Scientific, Waltham, MA, USA). Specificity of antibodies was controlled by using isotype control immunoglobulins (Sigma). The specificity of secondary antibodies was confirmed by omitting primary antibodies. Images were acquired by confocal laser scanning microscopy with the LSM 510 Meta, Plan-Neofiuar 4Ox oil/0.50 objective (Carl-Zeiss, Jena, Germany) and processed by LSM 5 Image Examiner and Adobe Photoshop software.

Determination of cytokine concentration in culture supernatants Cytokine concentrations in culture supernatants were determined by enzyme linked immunosorbent assay (ELISA) using either kits from R&D Systems or ImmunoTools (Friesoythe, Germany) and according to the protocols of the manufacturers.

Preparation oflysates and extraction ofcytosolic and nuclear fractions

Biopsies were lysed in RIPA buffer containing protease and phosphatase inhibitors (protease inhibitor cocktail and phosphatase inhibitor cocktail 2, both obtained from Sigma). Dendritic cells were washed and lysed immediately after infection experiments and the cytosolic and nuclear extracts were prepared according to the manufacturer's protocol (Nuclear/Cytosol Fractionation Kit, Bio Vision, Cambridge BioScience, Cambridge, UK).

Immunoblotting

Samples were mixed with LDS-sample buffer (Invitrogen) containing 10% mercaptoethanol and heated for 10 min at 70°C. SDS-PAGE was performed with the NuPage system (Invitrogen) using BisTris Gels. The proteins were transferred to nitrocellulose membranes and blocked with either non-fat milk or bovine serum albumin for 1 h at room temperature in 0.1% Tween/PBS before incubation with primary antibodies. The secondary antibodies were conjugated to horseradish peroxidase (DAKO, Cambridgeshire, UK) and incubated on the membranes for 1 h at room temperature. ECL Plus detection system (GE Healthcare) was applied before the membranes were exposed to X-ray film.

Statistical analysis

The software GraphPad Instat was used to perform statistical tests. A p< 0.05 was considered significant.

Table 1. Bacterial strains used in this stud

Table 2: Primers used in this study

Table 3: Plasmids used in this study

Plasmids Description Reference

Table 4. Bacterial strains tested on mdDCs for their otential to affect IL-8 production

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications are covered by the appended claims.

References (the content of which are incorporated herein by reference in their entirety)

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Claims

3CLAIMS

1. A method for preventing or treating an NF-kappaB mediated disorder in a subject suffering from or at risk of suffering from an NF-kappaB mediated disorder, the method comprising administering to the subject an effective amount of a protein comprising an amino acid sequence comprising SEQ ID NO:1, or a fragment or variant thereof.

2. A method according to claim 1, wherein the fragments or variants thereof comprise an amino acid sequence that has at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:1.

3. A method according to claim 1 or 2, wherein the fragments thereof comprise (i) at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight consecutive amino acids from SEQ ID NO:1.

4. A method according to any preceding claim, wherein the fragments or variants thereof are functional fragments or variants thereof.

5. A method according to any preceding claim, wherein the NF-kappaB mediated disorder is associated with high levels of NF-kappaB regulated cytokines.

6. A method according to any preceding claim, wherein the NF-kappaB mediated disorder is selected from an inflammatory disorder, an autoimmune disorder, or cancer.

7. A method according to claim 6, wherein the inflammatory disorder is selected from arthritis, inflammatory bowel disease, Crohn's Disease, ulceritive colitis and Irritable Bowel Syndrome.

8. A method according to claim 6, wherein the autoimmune disorder is selected from an allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, autoimmune vasculitis, nephrotic syndrome and rheumatoid arthritis.

9. A method according to claim 6 wherein the cancer is selected from breast cancer, ovarian cancer, bladder cancer, lung cancer, thyroid cancer, glioblastoma, stomach cancer, endometrial cancer, kidney cancer, colon and colorectal cancer, pancreatic cancer, prostate cancer, leukemia, myeloma, B lymphoma and non-hodgkins lymphoma.

10. A method according to any of claims 1 to 5, wherein the NF-kappaB mediated disorder is selected from sepsis, infectious diseases, transplant rejection, malignancy, pulmonary disorder, intestinal disorder, cardiac disorder, inflammatory bone disorders, psoriatic arthropathy, ankylosing spondylitis, juvenile rheumatoid arthritis, Still's disease, systemic lupus erythematosis, Sjogren's disease, mixed connective tissue disorder, polymyalgia rheumatica, giant cell arteritis, Wegener's granulomatosis, Kawasaki's disease, Bechet's disease, psoriasis, Graves disease, Hashimoto's thyroiditis, asthma, Type 1 diabetes, Type 2 diabetes, ischemic heart disease, peripheral vascular disease, stroke, pyoderma gangrenosum, sarcoidosis, Dercum's disease, toxic epidermal necrolysis, idiopathic uveitis or scleritis, birdshot retinochoroiditis, uveitic and diabetic cystoid macular edema, age-related macular degeneration, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), depression, schizophrenia, Alzheimer's disease, vascular dementia, glomerulonephritis, atherosclerosis, restenosis, graft v. host reactions, septic shock, cachexia, anorexia, multiple sclerosis, gram negative sepsis, endotoxic shock, neoplastic diseases, uveitis (e.g., childhood and seronegative), lupus and other diseases mediated by immune complexes such as pemphigus and glomerulonephritis, congential hyperthyroidism (CH), delayed type hypersensitivity (DTH) such as contact hypersensitivity, sarcoidosis, chronic arthritis, adult still disease, scleroderma, giant cell arteritis, SAPHO syndrome, primary biliary cirrhosis (PBC), myelodysplasia syndromes, vasculitis, hematologic malignancies, cochleovestibular disorders, macrophage activation syndrome, interstitial lung disease, hepatitis C, ovulation induction, and myelodysplastic syndromes.

11. Use of a protein comprising an amino acid sequence comprising SEQ ID NO:1, or a fragment or variant thereof in the manufacture of a medicament for the treatment or prophylaxis of an NF-kappaB mediated disorder in a subject suffering from or at risk of suffering from an NF-kappaB mediated disorder.

12. A protein comprising an amino acid sequence comprising SEQ ID NO:1, or a fragment or variant thereof for use in the treatment or prophylaxis of an NF-kappaB mediated disorder in a subject suffering from or at risk of suffering from an NF-kappaB mediated disorder.