WO2013039872A1

WO2013039872A1 - Compositions and methods for treating inflammatory bowel diseases

Info

Publication number: WO2013039872A1
Application number: PCT/US2012/054592
Authority: WO
Inventors: Joel V. Weinstock
Original assignee: Tufts Medical Center
Priority date: 2011-09-12
Filing date: 2012-09-11
Publication date: 2013-03-21

Abstract

The present invention relates to compositions and methods for treating inflammatory bowel diseases. In particular, the present invention relates to helminth-based therapeutic compositions and methods for treating inflammatory bowel diseases.

Description

COMPOSITIONS AND METHODS FOR TREATING INFLAMMATORY BOWEL

DISEASES

This application claims priority to provisional patent application 61/533,472, filed September 12, 2011, which is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under DK38327 and DK058755 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Inflammatory bowel diseases (IBD) are defined by chronic, relapsing intestinal inflammation of obscure origin. IBD refers to two distinct disorders, Crohn's disease and ulcerative colitis (UC). Both diseases appear to involve either a dysregulated immune response to GI tract antigens, a mucosal barrier breach, and/or an adverse inflammatory reaction to a persistent intestinal infection. The GI tract luminal contents and bacteria constantly stimulate the mucosal immune system, and a delicate balance of proinflammatory and anti-inflammatory cells and molecules maintains the integrity of the GI tract, without eliciting severe and damaging inflammation. It is unknown how the IBD inflammatory cascade begins, but constant GI antigen-dependent stimulation of the mucosal and systemic immune systems perpetuates the inflammatory cascade and drives lesion formation.

There is no known cure for IBD, which afflicts 2 million Americans. Current methods of managing IBD symptoms cost an estimated $1.2 billion annually in the United States alone.

In patients with IBD, ulcers and inflammation of the inner lining of the intestines lead to symptoms of abdominal pain, diarrhea, and rectal bleeding. Ulcerative colitis occurs in the large intestine, while in Crohn's, the disease can involve the entire GI tract including the small and large intestines. For most patients, IBD is a chronic condition with symptoms lasting for months to years. It is most common in young adults, but can occur at any age. It is found worldwide, but is most common in industrialized countries such as the United States, England, and northern Europe. It is especially common in people of Jewish descent and has racial differences in incidence as well. The clinical symptoms of IBD are intermittent rectal bleeding, crampy abdominal pain, weight loss and diarrhea. Diagnosis of IBD is based on the clinical symptoms, the use of a barium enema, but direct visualization (sigmoidoscopy or colonoscopy) is the most accurate test. Protracted IBD is a risk factor for colon cancer. The risk for cancer begins to rise significantly after eight to ten years of IBD.

Some patients with UC only have disease in the rectum (proctitis). Others with UC have disease limited to the rectum and the adjacent left colon (proctosigmoiditis). Yet others have UC of the entire colon (universal IBD). Symptoms of UC are generally more severe with more extensive disease (larger portion of the colon involved with disease).

The prognosis for patients with disease limited to the rectum (proctitis) or UC limited to the end of the left colon (proctosigmoiditis) is better than that of full colon UC. Brief periodic treatments using oral medications or enemas may be sufficient. In those with more extensive disease, blood loss from the inflamed intestines can lead to anemia, and may require treatment with iron supplements or even blood transfusions. Rarely, the colon can acutely dilate to a large size when the inflammation becomes very severe. This condition is called toxic megacolon. Patients with toxic megacolon are extremely ill with fever, abdominal pain and distention, dehydration, and malnutrition. Unless the patient improves rapidly with medication, surgery is usually necessary to prevent colon rupture.

Crohn's disease can occur in all regions of the gastrointestinal tract. With this disease intestinal obstruction due to inflammation and fibrosis occurs in a large number of patients. Granulomas and fistula formation are frequent complications of Crohn's disease. Disease progression consequences include intravenous feeding, surgery and colostomy.

The most commonly used medications to treat IBD are anti-inflammatory drugs such as the aminosalicylates. The salicylate preparations have been effective in treating mild to moderate disease. They can also decrease the frequency of disease flares when the medications are taken on a prolonged basis. Examples of aminosalicylates include sulfasalazine, olsalazine, and mesalamine. All of these medications are given orally in high doses for maximal therapeutic benefit. These medicines are not without side effects. Azulfidine and other such preparations can cause nausea, allergic reaction, hair loss, headache, and in rare cases they can cause kidney or pulomonary inflammation.

Corticosteroids are more potent and faster-acting than aminosalicylates in the treatment of IBD, but potentially serious side effects limit the use of corticosteroids to patients with more severe disease. Side effects of corticosteroids usually occur with long term use. They include thinning of the bone and skin, infections, diabetes, muscle wasting, rounding of faces, psychiatric disturbances, and, on rare occasions, destruction of hip joints. Corticosteroids are not effective for maintenance of disease remission.

In IBD patients who do not respond to aminosalicylates or corticosteroids, medications that suppress the immune system are used. Examples of immunosuppressants include azathioprine, 6-mercaptopurine and methotrexate. Immunosuppressants used in this situation help to control IBD and allow gradual reduction or elimination of corticosteroids. However, immunosuppressants cause increased risk of infection, cancer, excessive immune suppression, liver and/or pancreatic disease, and the need for hospitalization.

Clearly there is a great need for identification of the pathophysiological basis of IBD, which include Crohn's disease and ulcerative colitis so as to develop more rational and safer approaches to disease treatment.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for treating inflammatory bowel diseases. In particular, the present invention relates to helminth-based therapeutic compositions and methods for treating autoimmune disease (e.g., inflammatory bowel diseases).

For example, in some embodiments, the present invention provides a method of treating an inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), comprising: a) contacting an isolated immune system cell (e.g., dendritic cell, macrophage cell, or regulatory T cell (Treg)) from a subject diagnosed with an IBD with a helminth, or a non-viable extract or molecule from a helminth, to generate a treated immune system cell (e.g., in vitro); and b) administering the treated immune system cell to the subject. In some embodiments, the administering reduces or eliminates symptoms of IBD. In some embodiments, the helminth is Heligmosomoides polygyrus. In other embodiments, the helminth parasite is a nematode, and may be selected from the group such as Ascaris lumbricoides, Enterobius vermicularis, Trichuris trichiura, Ancylostoma duodenale and Necator americanus, Strongyloides stercoralis and Trichinella spiralis. In other embodiments of the invention, the helminthic parasite is a platyhelminth, and may be selected from, for example, trematodes and cestodes, such as Fasciolopsis, Echinostoma and Heterophyes species, Clonorchis sinensis, Opisthorchis viverrini, Opisthorchis felineus, Fasciola hepatica, Schistosoma species, Diphyllobothrium species, Taenia saginata, Taenia solium and

Hymenolepsis nana. In still other embodiments, the helminthic parasite is selected from filarial parasites or lung flukes. In additional embodiments, the helminthic parasites are selected from, for example, Trichuris muris, Trichinella spiralis, Nippostrongylus prasiliensis, Heligrnonsomoides polygyrus, Hymenolepsis nanan, Angiostrongylus species, Trichuris suis, Ascaris suum, Trichuris vulpis, Toxocara species, Gnathostoma species, Ancylostoma species, Anisakis species and Pseudoterranova species. In some embodiments, the isolated immune system cell is in peripheral blood or is purified from peripheral blood, intestines or other tissue. In some embodiments, the Treg is cell is a Foxp3+/IL10- and/or a Foxp3+/IL10+ Treg. In some embodiments, the administering is intravenous, subcutaneous, intramuscular or intraperitoneal administration.

In other embodiments, the present invention provides a method of treating an inflammatory bowel disease (IBD), comprising: administering a helminth extract,

fractionated extract or purified extract to a subject, wherein the administering reduces or eliminates symptoms of IBD. In some embodiments, the extract is administered orally. In some embodiments, the extract is in a capsule that dissolves in the intestine.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

Figure 1 shows that Hp DC inhibit cytokine production in Rag mice reconstituted with 10⁶ IL 10 KO T cells, given i.p., and exposed to piroxicam (NSAID) to induce colitis.

Figure 2 shows that DC regulation requires cell contact.

Figure 3 shows that Hp induces regulatory DC in colitis.

Figure 4 shows experimental design (top panel); the severity of inflammation in colons of mice receiving A) no DC, B) DC from mice with no prior Hp infection (DC No Hp), or C) DC from mice after Hp infection {Hp DC) (middle panel) and the effect of cell transfer on mucosal cytokine production (lower panel).

Figure 5 shows that DC-transfer does not diminish the relative number of OT2 cells that appear in the gut or MLN. Figure 6 shows that exposure to Hp before T cell reconstitution does not significantly increase the relative number of Foxp3+ T cells in the colon of the mice. Panel A: The capacity of Hp to induce Treg in the colon was analyzed using two experimental designs. Panel B shows that in the no colitis model, Hp exposure decreased the relative number of CD4+ T cells that expressed Foxp3 in the TI.

Figure 7 shows that exposure to Hp before T cell reconstitution decreases the relative number CD4+ Tcells expressing Foxp3 in the colon or MLN of non-colitic mice and has little effect on Foxp3+ T cells in colitic animals.

Figure 8 shows that Hp macrophages inhibit cytokine production.

Figure 9 shows that Hp macrophage transfer A) protects mice from colitis and B) blocks the mucosal cytokine response to OVA.

Figure 10 shows that macrophage transfer diminishes the relative number of OT2 T cells that appear in the gut.

Figure 11 shows that Hpb infection increased the number of Foxp3+ Tregs in the colon of Foxp3/IL10 reporter mice.

Figure 12 shows that colonic Foxp3+ T cells from Hpb infected mice blocked the development of colitis in a CD4+CD25- T cell transfer model of IBD. A) Cells from either Hpb infected (Hpb) or uninfected animals (no Hpb) were adoptively transferred via i.p.

injection into Rag mice along with CD4+ CD25- WT and OT2 splenic T cells. B) Colitis was scored for severity on a 4-point scale in stained histological sections. C) Dispersed colonic LPMC were cultured 48h in vitro without or with OVA (10 ug/ml) to stimulate cytokine release.

Figure 13 shows that colonic LPMC Foxp3+ T cells from Hpb-infected mice readily accumulated in Rag intestine after adoptive transfer.

Figure 14 shows that both colonic Foxp3+ IL10- and Foxp3+ IL10+ T cells from Hpb infected mice blocked colitis. A) Foxp3+/IL10- and Foxp3/IL10+ T cells were adoptively transferred via i.p. injection into Rag mice along with CD4+ CD25- WT and OT2 splenic T cells. B) Colitis was scored for severity on a 4-point scale in stained histological sections. C) Dispersed colonic LPMC were cultured 48h in vitro without or with OVA (10 ug/ml) to stimulate cytokine release.

Figure 15 shows that transfer of colonic Foxp3+/IL10- T cells from Hpb-infected mice into the Rag colitis model caused substantial numbers of Foxp3+/IL10- and

Foxp3+/IL10+ CD4+ T cells to accumulate in the Rag colon. Figure 16 shows that Foxp3+ T cells from the colon of IL10 KO mice infected with Hpb could not prevent colitis. A) Colitis was scored for severity on a 4-point scale in stained histological sections. B) Dispersed colonic LPMC were cultured 48h in vitro without or with OVA (10 ug/ml) to stimulate cytokine release. Culture supernatants were assayed for IFNy and IL 17 using ELISA.

Figure 17 shows an exemplary model for Treg subset differential function in the colon.

DEFINITIONS

As used herein, the term "immune system cell" refers to any cell that functions in the immune system to induce innate and/or adaptive immunity. In some embodiments, immune system cells are white blood cells. Examples include, but are not limited to, dendritic cell, macrophages, T cells (e.g., regulatory T cell), monocytes, lymphocytes, basophil, neutrophil, and eosinophil.

As used herein, the term "subject" refers to any organisms that is treated using the methods described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.

The term "diagnosed," as used herein, refers to the recognition of a disease by its signs and symptoms, or genetic analysis, pathological analysis, histological analysis, and the like.

As used herein, the term "purified" or "to purify" refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non- immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Such examples are not however to be construed as limiting the sample types applicable to the present invention. DETAILED DESCRIPTION OF THE INVENTION

Immunological diseases like IBD are infrequent in less developed countries, possibly because helminthic infections provide protection by modulating host immunity. In IBD murine models, the helminth Heligmosomoides polygyrus bakeri (Hp) prevents colitis. Hp protects mice from IBD through interaction with innate immunity. Experiments conducted during the course of development of embodiments of the present invention utilized a Rag IBD model where animals were reconstituted with IL10-/- T cells to make them susceptible to IBD and with OVA antigen-responsive, transgenic OT2 T cells to allow study of a gut antigenic response. It was determined if altered DC function was a mechanism underlying IBD protection by Hp. Intestinal DC from Hp-infected Rag mice added to lamina propria mononuclear cells (LPMC) isolated from colitic animals blocked OVA IFNg and IL17 responses in vitro through direct contact with the inflammatory LPMC. Transfer of DC from Hp-infected mice into Rag mice reconstituted with IL10-/- T cells protected the animals from IBD, and LPMC from these mice lost OVA responsiveness. After DC transfer, OT2 T cells populated the intestines normally. The OT2 T cells were rendered antigen-nonresponsive through regulatory action of LPMC on non-T cell elements. The DC did not function through altering Foxp3 T cell frequency. Thus, Hp modulates intestinal DC function, rendering them regulatory.

Experiments conducted during the course of development of embodiments of the present invention determined that exposure of Rag mice to Hp induces regulatory-type DC in the intestines. This indicates that Hp does not require direct interaction with T or B cells to render intestinal DC regulatory. These regulatory DC, in Hp-induce protection from colitis, mediate protection since DC transfer is sufficient to render animals resistant to IBD even if the recipient mice never experienced Hp infection. Since inappropriate T cell activation to luminal antigens is believed to underlie the etiology of human IBD, the capacity of these DC to block an antigen-specific T cell response in the gut leads to suppression of colitis.

DC are present in all tissues. DC in MLN, Peyer's patches and the LP are

continuously exposed to a multitude of luminal antigens. In different regions of the intestines, various DC subsets have been characterized based on the expression of cell surface molecules like CD103, CX3Crl and CDl lb (Ng et al, 2010 Inflamm. Bowel Dis. 16: 1787-1807).

Further experiments conducted during the course of development of embodiments of the present invention demonstrated that Hpb infection could protect mice from colitis through activation of colonic Treg. Hpb infection increased the number of T cells expressing Foxp3 in the colon. Foxp3+/IL10- and Foxp3+/IL10+ T cell subsets isolated from the colon of Hpb infected mice prevented colitis when adoptively transferred into a murine model of inflammatory bowel disease, while Tregs from uninfected mice could not provide protection. Only the transferred colonic Foxp3+/IL10- T cells from Hpb infected mice readily

accumulated in the colon and MLN of recipient mice, and they reconstituted both the

Foxp3+/IL10- and Foxp3+/IL10+ T cell subsets. However, transferred Foxp3+/IL10+ T cells disappeared. IL10 expression by Foxp3+ T cells was necessary for colitis prevention. Thus, Hpb infection activates colonic Foxp3+ T cells making them highly regulatory.

Various animal models of IBD describe that regulatory-type T cells are important for maintaining mucosal immune homeostasis and for controlling colitis (Boden et al, 2008 Current Opinion in Gastroenterology. 24(6):733-741). Hpb infection stimulates Foxp3 mRNA expression in T cells (Elliott et al, 2004 Eur. J. Immunol. 34:2690-2698) and expands the number of Foxp3+ T cells in the mesenteric lymph nodes (Grainger et al., 2010. J. Exp. Med. 207:2331-2341). Secretions from Hpb can induce T cells to express Foxp3 (Grainger et al, 2010. J. Exp. Med. 207:2331-2341). T cells from the MLN of Hpb-infected, IL10 deficient mice transferred into helminth-na^'ive animals will arrest colitis attesting to the importance of T cells in the control of IBD (Elliott et al., supra).

IL10 is a regulatory cytokine important for immune homeostasis in the gut. Mice deficient in IL10 (Berg et al, 1996. J. Clin. Invest. 98: 1010-1020) or IL10R (Spencer et al, 1998. J. Exp. Med. 187:571-578) develop spontaneous colitis. Humans with a mutation in the ILIOR are prone to IBD further highlighting the importance of IL10 in the protection from this disease process (Glocker et al, 2009. N. Engl. J. Med. 361 :2033-2045). IL10 comes from several sources. However, recent evidence describes that IL10 from Treg has importance in protecting the mucosa from inflammation (Rubtsov et al., 2008. Immunity 28:546-558).

Accordingly, embodiments of the present invention provide compositions and methods for treating IBD using ex vivo expose to helminthes or fractioned helminth components.

I. Helminths

As described herein, embodiments of the present invention utilize helminths or one or more components derived from helminths. The present invention is not limited to a particular helminth. Exemplary helminths are described herein and in U.S. Patent Applications

7,250,173, 7,833,537, 6,764,838, each of which is herein incorporated by reference in its entirety.

Parasites are living entities that dwell on or in other creatures during some part of their life cycles, drawing nourishment from the host. Parasites that inhabit the intestines have a complex interplay with the mucosal immune system. They must establish a tranquil relationship with host mucosal defenses to survive.

Helminths are elaborate multicellular worms with complex life cycles and

development. The nematodes (non-segmented roundworms) and the platyhelminths

(flatworns) are the two groups of helminths that colonize the human intestines. Perhaps more than a third of the population of the world currently shelter one or more of these organisms. The life-time exposure rate, however, is actually much more. The prevalence of helminths is highest in warm climates and in populations subject to crowding, poor sanitation and impure food supply. Inflammatory bowel disease (IBD), rheumatoid arthritis and autoimmune diseases are rare in these same regions.

Nematodes that frequently inhabit the human gut are Ascaris lumbricoides,

Enterobius vermicularis (pin worm), Trichuris trichiura (whipworm), Ancylostoma duodenale and Necator americanus (hookworms), and Strongyloides stercoralis. Trichinella spiralis infests the small intestine briefly.

The platyhelminths include the trematodes and cestodes. The most common adult trematodes that reside in the human intestines are Fasciolopsis, Echinostoma and

Heterophyes species. Those that live in the biliary system include Clonorchis sinensis, Opisthorchis viverrini. The platyhelminths include the trematodes and cestodes. The most common adult trematodes that reside in the human intestines are Fasciolopsis, Echinostoma and Heterophyes species. Those that live in the biliary system include Clonorchis sinensis, Opisthorchis viverrini and felineus, and Fasciola hepatica. Schistosoma dwell in the venous system, but several species chronically affect the gut by the passage of eggs through the intestinal wall. Adult cestodes commonly infecting humans are Diphyllobothrium species (fish tapeworm), Taenia saginata (beef tapeworm), Taenia solium (pork tapeworm) and Hymenolepsis nana (dwarf tapeworm).

The second general group of helminthic parasites that can be utilized in the present invention include helminths that colonize animals. These include, but are not limited to, Trichuris muris (mouse whipworm), Trichinella spiralis, Nippostrongylus brasiliensis,

Heligmonsomoides polygyrus and Hymenolepsis nana, all of which are intestinal helminths that infect mice. Additionally, Angiostrongylus is a rat helminth. Trichuris suis and Ascaris suum are pig helminths that can infect humans. Trichuris vulpis, Toxocara species,

Gnathostoma, and Ancylostoma are dog or cat helminths that also can infect humans.

Anisakis and Pseudoterranova are nematodes of marine mammals that can transmit to humans. Bird schistosomes can transiently infect humans. Such schistosomes include S. douthitti, Trichobilharzia ocellata, T. stagnicolae, T. physellae, and Gigantobilharzia huronensis.

The host acquires various helminthic species through contact with soil, food or water contaminated with the infective form of the parasite. Children most frequently harbor helminthic infections because of their close contact with soil and suboptimal hygienic practices. Helminths incite an intestinal Th2 response, which can cause worm expulsion or limit the magnitude of infection. Most children living in non-industrialized countries have these parasites. Many helminthic species survive for years within the gut, biliary tree or mesenteric veins making thousands of eggs daily. Thus, beginning in childhood, these worms and/or their ova release molecules that bathe the intestinal mucosal surface for years inciting Th2-type inflammation.

Dysregulation of the immune system leading to an excessive Thl response may be the cause of several human diseases. Some diseases due to dominant Thl responses include IBD, rheumatoid arthritis, sarcoidosis, multiple sclerosis, and insulin-dependent diabetes melitis.

IBD is more common in temperate climates. A major environmental factor predisposing to IBD, CD and UC is underexposure during childhood to intestinal helminths. People in industrialized countries are living in increasingly hygienic environments and are acquiring helminths much less frequently. The decreasing frequency of helminthic infections correlates with the increasing prevalence of CD. A case in point is the marked increase in the frequency of CD in young Asians and Africans after residing in Israel for greater than 10 years. Also, the frequency of helminthic infestation differs between the Jewish Israelis and Arabs. In 1969, stool examinations of hospitalized patients in Arab- predominant East Jerusalem contained helminthic ova over 60% of the time. The frequency in Israeli-predominant East Jerusalem was 10% or less.

For use in embodiments of the present invention, helminths are maintained in laboratory conditions. In some embodiments, helminths are cycled through intermediate and preparatory animals grown in SPF conditions. Samples of helminth populations are tested to ensure phenotypic stability such as colonization rates, fecundity, and susceptibility to antihelminthics. II. Therapeutic Methods

In some embodiments, the present invention provides compositions and method for treating IBD (e.g., CD and UC). In some embodiments, the present invention provides compositions and methods for treating autoimmune diseases. Examples of autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis (underactive thyroid), autoimmune hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Wegener's granulomatosis, autoimmune hepatitis, Beliefs disease, primary bilary cirrhosis, scleroderma, ulcerative colitis, Sjogren's syndrome, type 1 diabetes mellitus, uveitis, Graves' disease, myocarditis, rheumatic fever, ankylosing spondylitis, rheumatoid arthritis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, multiple sclerosis, polymyositis, Guillain-Barre syndrome, alopecia areata, pemphigus/pemphigoid, psoriasis, vitiligo, food allergy, allergic rhinitis, asthma, chronic hepatitis, sclerosing and cholangitis.

Epidemiological data provide a genetic susceptibility to the development of Crohn's disease (CD) and ulcerative colitis (UC). The incidence of CD in industrialized societies has increased from the 1950s until the mid 1980s, and now is from 1 to 8 per 100,000 persons per year. This indicates that unknown changes in our environment have affected the frequency of CD. In some embodiments, the present invention provides ex vivo methods of treating colitis. In some embodiments, the methods comprise exposing immune system cells (e.g., dendritic cells, Treg, or macrophage) of an individual to a helminth or helminths product and then returning the cells to the subject.

Macrophages, dendritic cells, and/or Tregs may be obtained from peripheral blood, either purified or unpurified. For example, in some embodiments, whole blood is exposed to the helminth or helminth product. In other embodiments, macrophages, Tregs and/or dendritic cells are purified from blood using any number of acceptable methods (e.g., flow cytometry).

In other embodiments, helminth extracts or purified components are administered as a therapeutic. Extracts can be obtained using known methods. In some embodiments, extract fractions or purified components are obtained. The activity of an extract, fractionated extract, or purified extract component can be determined using any suitable method, including but not limited to, those disclosed in example 1 below.

Any number of different species of helminths are suitable for use in the compositions and methods described herein (e.g., including but not limited to, those described above). Any number of life forms of helminths are suitable for use in embodiments of the present invention (e.g., egg, larval, cercarial or encysted larval forms). In some embodiments, a preparatory animal is utilized to obtain an appropriate life cycle of helminth.

Eggs: Helminths are maintained in SPF preparatory animals, for example, SPF pigs.

To harvest eggs, the animals are given a special diet low in coarse fiber. Animals are given an oral purgative to induce defecation. Stool is collected and enzymatically digested to free eggs. Eggs are isolated from liquefied stool by flotation on density gradients, screen filtration, Visser filtration, or centrifugal elutriation. Preservation of eggs varies with the helminth used. Eggs from helminths that are resistant to dessication are dried, compounded with inert material, incorporated into an enteric capsule, and refrigerated. Eggs from helminths that are susceptible to dessication are preserved by refrigeration in liquid medium or by adding cryoprotectant and freezing in liquid nitrogen. Viable eggs are washed, mixed with chilled lactose-free pudding or other vehicle at the location for use

Larvae: Some helminths (i.e. hookworms) require a soil maturation phase before they can colonize humans. Eggs from these agents can be incubated under optimal conditions to mature the embryo, or hatch the egg and provide larval forms. Cercariae: Some helminths have complex life cycles that utilize an intermediate host. The intermediate host sheds the form able to colonize humans. Cercariae are the form for trematode helminths (e.g., flukes) shed by intermediate hosts like snails. Cercariae are isolated from colonized snails grown in SPF conditions. Cercariae are washed. These may be preserved by adding cryoprotectant and freezing in liquid nitrogen. Thawed or fresh cercariae are washed and utilized as needed

Encysted Larvae: Some helminths (e.g., tapeworms) form encysted larvae or cysticerci in intermediate hosts. It is the encysted larval form that initiates human

colonization. Encysted larva are removed from intermediate hosts, for example, cattle or fish or plants grown in SPF conditions. Cysts are washed free of remaining host tissue. Cysts may be preserved by adding cryoprotectant and freezing in liquid nitrogen. Cysts are thawed or used fresh.

In other situations, adult life forms can be obtained from host through administration of strong laxatives or by sacrificing an animal host.

The present invention further provides pharmaceutical compositions (e.g. , comprising helminth extract, fractionated extract, or purified extract component). The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.

Administration may be topical intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.

Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. In some embodiments, capsules that dissolve in the intestines at the site of use are utilized.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

In some embodiments, pharmaceutical compositions further comprise one or more additional agents known to be useful in the treatment of IBD (e.g., immune suppressants such as steroids and the like). Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient or by observations of symptoms of disease. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual extracts or extract components, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state.

In some embodiments, treatments involving ex vivo treatment of macrophages, Tregs or dendritic cells are administered one every day, weeks, every 2 or 3 weeks, monthly or at other intervals.

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1

A. Materials and Methods

Mice and Hp infection:

This study used C57BL/6 Rag2 mice, OT2 and IL10-/- mice (Jackson Laboratory, Bar Harbor, ME). In some experiments, C57BL/6 OT2 CD45.1 mice (a gift from Dr. Fuhlbrigge, BWH, Boston, MA) or IL10 KO Foxp3 eGFP reporter mice (gift from Dr. Cathryn Nagler, University of Chicago, IL) were used. Breeding colonies were maintained in SPF facilities at Tufts University. Animals were housed and handled following national guidelines and as approved by Animal Review Committee. For these experiments, 5- to 6-wk-old mice were colonized with 125 Hp third stage larvae by oral gavage. Infective, ensheathed Hp L3 (U.S. National Helminthological

Collection no. 81930) were obtained from fecal cultures of eggs by the modified Baermann method and stored at 4°C. To de-worm mice, animals were given a single dose of pyrantel pamoate (0.5 mg/mouse, Sigma, St. Louis, MO) via oral gavage. De-worming was confirmed by the absence of adult worms in the duodenum at time of animal sacrifice and by

preliminary experiments that showed the absence of worms in the GI track of mice 1 wk after taking pyrantel pamoate. Colitis model:

Rag mice of similar age (usually 6 to 7 wks old) were reconstituted i.p. with 10⁶ IL10- /- splenic T cells. In some experiments, mice also received 3xl0⁵ OT2 splenic T cells given ip. One wk later, the animals were administered piroxicam mixed into their feed for 2 wks (Piroxicam at 40mg/250g chow wk 1, and 60mg/250 g chow wk 2). Two wks after induction of colitis, the piroxicam (Sigma) was stopped, and the colitis was studied 1 wk later. The colons then were microscopically examined and scored for severity of colitis, and LPMC were isolated for culture (e.g. Figure 1 and 3). In some experiments, Rag mice were infected with Hp (125 larvae) for 2 wks, then treated with pyrantel pamoate to terminate the Hp infection, rested 1 wk, and then reconstituted with T cells as described above. More details regarding the various manipulations of this model are provided in the results section.

Dispersion of splenocytes, and splenic T cell enrichment:

Single cell suspensions of splenocytes were prepared by gentle teasing in RPMI 1640 medium (GIBCO, Grand Island, NY). The cells were washed three times in RPMI. Splenic T cells or CD4+ T cells were isolated by negative selection using the EasySep mouse T cell

Enrichment Kit as outlined by the manufacturer (Stemcell Technologies, #19751, Vancouver, Canada). Viability was determined using exclusion of trypan blue dye.

LPMC isolation and LP cell fractionation:

Gut LPMC were isolated from the terminal ileum or colon as described (Elliott et al.,

2004. Eur. J. Immunol. 34:2690-2698.). Cell viability was 90% as determined by trypan blue exclusion. Dendritic cells (DC)(CD1 lc+) were isolated from dispersed LPMC or MLN cells with mouse CDl lc positive selection Kit #18758 from Stem Cell Technologies that was used according to kit directions. Similar technology was used to isolate macrophages targeting the macrophage surface protein f4/80. The purity of the isolated cells always was greater than 95% as determined by FACS. Cell culture:

LPMC from IL10-/- T cell- and OT2 T cell-reconstituted Rag mice were cultured (2 x 10⁵ cells per well) for 48h in 96-well round-bottomed plates. Cells were cultured alone or with OVA (10 μ^πιΐ) (Sigma). The culture medium was RPMI 1640 containing 10% FCS, 25 mM HEPES buffer, 2 mM L-glutamine, 5 x 10 ⁵ M 2-ME, 1 mM sodium pyruvate, 100 U/ml penicillin, 5 mg/ml gentamicin, and 100 mg/ml streptomycin (complete medium) (all Life Technologies, Gaithersburg, MD). After culture, the supematants were assayed for IFNy and IL17A using ELISA (described below).

In the Rag LPMC/OT2 T cell mix experiments, OT2 Thyl .2 splenic T cells were mixed with LPMC from Rag mice at the ratio of (1 :4). Cells (2xl0⁵) were cultured in RPMI complete medium for 48 h. Some cultured contained OVA at up to 10 μg/ml to stimulate cytokine release. Supematants were assayed for IFNg and IL17 after the incubation using ELISA.

Transwell experiments used the Coming Transwell-96 well permeable support system with a 0.4 um pore size (#3381).

Sandwich ELISAs:

ELISAs were performed using paired antibodies (R & D Systems, Minneapolis, MN) according to manufacturer's instructions. IL17 ELISA was done using primary capture antibody from (R&D Systems, Inc) and biotinylated anti-IL17A antibody (R&D Systems). IL10 was captured with anti-ILlO mAb (R&D Systems) and detected with biotinylated mAb (R&D Systems). To measure IFNy, plates were coated with a mAb to IFNy (HB170, ATCC) and incubated with supernatant. IFNy was detected with polyclonal rabbit anti-IFNy (gift from Dr. Mary Wilson, University of Iowa) followed by biotinylated goat anti-rabbit IgG (AXcell, Westbury, NY). Total TGFb was measured using acid-treated supernatant and mAb240 for capture and biotinylated chicken IgY BAF240 for detection (both R&D

Systems)

Flow cytometry analysis: LPMC were washed twice and adjusted to 10⁷ cells/ml in FACS buffer (LGM) and stained with saturating amounts of conjugated mAb for 30 min at 4°C. Following staining, cells were washed twice and re-suspended in LGM for analysis on a FACSCalibur using Cell Quest software (BD Biosciences, Mountain View, CA). Before adding labeled mAb, each tube received 1 μg of anti-Fc mAb (eBioscience, San Diego, CA) to block nonspecific binding of conjugated Abs to FcRs. The mAbs used for staining or cell sorting were anti- Thyl .2-FITC, or -PECy5, or -APC; anti-CDl lc-FITC; anti-F4/80-PE or Cy5, anti- CD4-PE or -PE-Cy5; anti-CD45.1-APC;anti-CD45.2-APC; anti-CD8-APC or-PE (all from

eBioscience).

Statistical analysis:

Data are means ±SE of multiple determinations. Difference between two groups was compared using Student's i-test. Multiple group comparisons used analysis of variation and Dunnett's t-test. P values <0.05 were considered significant.

B. Results

This study used an IBD model of intestinal inflammation in which Rag mice are reconstituted i.p. with IL10-/- T cells and then the mice are orally feed a NSAID (piroxicam) for 2 wks to induce colitis (Hang et al, 2010. J. Immunol.185:3184-3189). The severe Thl/Thl7-type colitis persists after discontinuing the NSAID. Since the intestinal antigens that drive intestinal immunity are poorly defined, Rag mice are reconstituted with transgenic OT2 T cells bearing MHC Class II dependent TCR that recognize OVA. These cells are given simultaneously with the IL10-/- T cells, and a large number of these OT2 cells subsequently appear in the gut. This permits the study of the regulation of antigen-specific responses in the intestinal LP, since isolated LPMC from these mice will produce large amounts of IFNg and IL17 upon OVA stimulation in vitro.

Rag mice exposed to Hp only before T cell reconstitution are protected from colitis (Hang et al., supra). This indicates that interactions of Hp just with cellular components of innate immunity are sufficient to provide this protection. To extend this observation, these experiments determined that infection with Hp induces regulatory DC in the gut.

DC isolated from Rag mice infected with Hp blocked OVA-induced cytokine secretion in vitro In the initial experiments, it was determined if DC from Hp-infected Rag mice could impair the natural interaction of intestinal pro-inflammatory DC with their effector T cells such as to impede antigenic responses. Rag mice were reconstituted with IL10-/- T cells, and treated with piroxicam to induce colitis. LPMC were isolated from these animals, mixed with splenic OT2 T cells and cultured in vitro with or without OVA antigen to stimulate cytokine production. Some wells also received supplemental DC isolated from the intestines of a separate group of Rag mice that had been infected with Hp for 2 wks before sacrifice {Hp DC). Still other wells were given DC isolated from the gut of age-matched Rag mice that never received Hp infection (DC no Hp). (Fig 1, top panel)

Isolated LPMC from the mice with colitis produced IFNy and IL17 constitutively and secreted substantially more when cultured with OVA (Fig 1). Addition of Hp DC added at a ratio of 1 :5 (DC: LPMC) did not affect constitutive cytokine secretion, but totally blocked OVA-induced stimulation. Addition of the DC no Hp control cells did not block cytokine release.

Supernatants from these cell cultures also were examined for IL10, TGFP and IL4, which are cytokines that can regulate or alter antigenic responses. IL10 or TGFP were secreted in similar amounts under all culture situations (Table 1, number 1). OVA added to the cultures did not increase either IL10 or TGFb release. No IL4 was detected in any cultures.

Other experiments used DC isolated from the MLN of Rag mice with or without Hp infection. DC from the MLN of Hp-infected mice also blocked OVA-induced, cytokine responses equally as well as gut DC when added to the isolated LPMC from the colitic animals (1 :5 cell ratio). The OVA response was not affected by the addition of MLN DC from Rag mice who never received Hp-infection.

DC regulation of the OVA-response requires cell contact

Using Transwell plates, it next was determined if DC regulation of OVA-induced, cytokine production required cell contact. Once more LPMC isolated from colitic animals were mixed with splenic OT2 T cells and cultured in the outer well alone or with gut Hp DC at the usual ratio with or without OVA. As expected, OVA induced strong cytokine responses only in the absence of the DC (Fig 2). However, Hp DC did not alter OVA-induce cytokine secretion when the Hp DC were placed in the inner well separated from the colitic LPMC by a semi-permeable membrane. The intestines of Rag mice protected from colitis through Hp exposure contains regulatory DC

The above experiments indicated that infection of Rag with Hp induces regulatory DC within the gut that can block the intestinal OVA response in vitro. To further test the significance of this observation, it next was determined if animals protected from colitis by Hp infection display regulatory type DC within their intestines.

Rag mice were reconstituted with IL10-/- T cells and OT2 T cells, and then treated with piroxicam to induce colitis. One week after stopping the piroxicam, the mice were sacrificed and the intensity of the colitis was scored by examining histological tissue sections. Also, intestine was dissociated to isolate the LPMC, which were cultured in vitro with or without OVA to stimulate IFNg and IL17 release.

In parallel, a second group of age-matched Rag mice were exposed to Hp for 2 wks and then de-wormed by pharmaceutical means before they received similar T cell

reconstitution and piroxicam exposure. One week after stopping the piroxicam, the mice were sacrificed and the colitis also was scored for intensity of inflammation by examining histological sections. Intestine was dissociated to isolate intestinal DC, which were added to some of the cultures of LPMC from the severely colitic mice to see if these DC would affect the OVA response. (Fig 3, top panel)

T cell-reconstituted, Rag mice developed severe colitis after piroxicam treatment, which was not seen in mice that were infected with Hp before the T cell reconstitution. (No Hp infection vs. Hp infection; inflammatory score 3.7+/-0.4 vs. 1.2+/-0.3, mean+/- SE, N=3 separate experiments) Isolated LPMC from the colitic mice produced IFNg and IL17 whose production was enhanced by OVA stimulation (Fig 3). DC from the Rag mice protected from colitis by Hp infection blocked OVA-induced cytokine secretion when they were added in vitro to the LPMC from the colitic animals.

Hp DC are sufficient to prevent colitis

The next series of experiments ascertained if DC from Hp-infected Rag mice could adoptively transfer protection from colitis. Rag mice received the usual numbers of IL10-/- and OT2 T cells administered i.p. Some mice also received DC isolated from the MLN of Rag mice that had been infected with Hp for 2 wks. An additional group of animals received MLN DC from age -matched Rag mice that never received Hp infection. After NSAID administration, the animals were sacrificed at the appropriate time to assess severity of colitis and the responsiveness of isolated LPMC to OVA stimulation (Fig 4, top panel).

Figure 4 shows, as expected, that severe colitis develops in mice receiving no DC. Mice receiving DC from Hp-infected animals displayed much less colonic inflammation. Transfer of DC from animals never infected with Hp did not affect the intensity of the inflammatory response.

LPMC isolated from mice that received no DC or DC from uninfected mice produced both IFNg and IL17 constitutively when cultured in vitro and even more after OVA stimulation. However, LPMC from Rag mice receiving DC from the infected animals lost their responsiveness to OVA stimulation (Fig 4).

Culture supernatants also contained IL10. OVA stimulation did not increase IL10 secretion. LPMC isolated from the colons of mice that received no DC or DC from uninfected mice produced comparable amounts of this cytokine. However, LPMC from mice receiving DC from the infected animals actually made much less IL10 (Table 1).

Also measured were TGFb and IL4. All cultures produced comparable amounts of

TGFb, and OVA did not stimulate more TGFb release (Table 1). IL4 was not detected.

Additional experiments were conducted using DC isolated from the TI of Rag mice with or without Hp infection. Gut DC from Hp-infected animals adoptively transferred protection from colitis and blocked mucosal responsiveness to OVA comparable to MLN DC. Once again, intestinal DC from uninfected mice had no effect.

OT2 T cell populate the gut and MLN correctly after transfer of DC from Hp-infected mice

The loss of LPMC antigenic responsiveness after transfer of DC from Hp-infected mice could have signified that DC transfer interfered with normal OT2 T cell population of the LP. T cells from C57BL/6 mice express CD45.2. Rag mice were reconstituted with OT2 T cells from C57BL/6 transgenic mice expressing CD45.1 so that OT2 cells within the isolated LPMC could be distinguished from the IL10-/- T cells through differential CD45 display.

One group of Rag mice received CD45.1+ OT2 T cells and CD45.2+ IL10-/- T cells, and then were exposed to piroxicam to induce colitis. Another group of Rag animals were treated as above except they also received DC from the MLN of Rag mice infected with Hp to protect the animals from colitis and to block the intestinal response to OVA. About 1.5% of the LP or MLN CD4+ T cells isolated from the colitic mice displayed CD45.1 (Fig 5). The relative number of LP or MLN CD4+ T cells expressing CD45.1 did not diminish in Rag mice receiving the DC. After Hp DC transfer, intestinal OT2 T cells retain the capacity to produce cytokines after OVA stimulation

Since OT2 T cells readily populated the gut even after regulatory DC transfer, it next was determined if the OT2 T cells could regain OVA responsiveness in a more permissive environment. The experiment as described in the above paragraph was repeated. One group of Rag mice received DC from the MLN of Rag mice infected with Hp. The other group did not receive DC. One wk after stopping the piroxicam, LPMC were isolated from the TI of both groups, and CD45.1+ OT2 T cells were separated from the dispersed LPMC using FACS (Hp OT2 and OT2). Also, the residual LPMC were depleted of T cells (Hp non-T and non-T). OT2 T cells were mixed with either one or the other non-T cell preparation and cultured with or without OVA. All cultures produced comparable amounts of IFNg and IL17 without OVA stimulation (Table 2). OT2 cells from either source did not respond to OVA if the cells were mixed with non-T cells isolated from the intestines of Rag mice that received DC (Table 2). However, OT2 cells responded well to OVA if they were cultured with Non-T cells from mice that did not receive DC.

DC transfer does not increase the relative number of T cells expressing Foxp3 in the intestines or MLN

Regulatory DC can induce the expansion of Foxp3+ T cells in tissues. It was ascertained if the frequency of Foxp3+ T cells would increase if Rag mice were infected with Hp before T cell reconstitution. Rag mice were infected with Hp for 2 wks and then de- wormed by treatment with pyrantel pamoate. One wk later, the mice received Foxp3 eGFP IL10-/- reporter T cells and OT2 T cells. In some experiments, mice received piroxicam to induce colitis whereas others received no such treatment and remained free of colitis (figure 6, panel A shows experimental designs). At the time of sacrifice, LPMC were isolated from the TI and analyzed for Foxp3 T cell expression using flow analysis to detect eGFP. About 98% of the Foxp3+ T cells in the colon, TI and MLN were CD4+. Following Hp infection, mice without colitis displayed a decreased in the relative number of CD4+ T cells in the TI expressing Foxp3 (Figure 6, panel B). In the colitic mice, Hp exposure did not significant increase the proportion of CD4+ T cells in the TI expressing Foxp3 compared to the uninfected control group. This pattern also was seen in dispersed MLN cells and LPMC from the colon (Figure 7). There was no significant shift in the relative number of CD8+ T cells displaying Foxp3 in the gut or MLN either.

Table I: Re ulator DC did not alter either II 1 ΰ or TGFb roduction in the cell cultures

For 1} d 2), superaatants were generated, as described i figure I and figure 4.

respectively. Data are mean pg/ml + SE of three separate experiments.

Table II; OT2 T cells isolated from the TI oi T cell-reconstituted Rag mice that receiv DC from H -infected animals {Hp OT2) respond to OVA when cultured with T celJ- depleted LPMC (Noa-T) isolated from T cell-reconstituted mice receiving no DC

The experimental design is outlined in figure 4, top panel, expect that the recipient mice received CD45,l OT2 cells. Some mice also received DC from the MLN of Rag xnke infected with Hp for 2 ks. Other mice received no DC . At the time of sacrifice. OT2 T cells were isolated from the dispersed colonic LPMC using FACS and anti-CD45, i rnAb (OT2). LPMC deleted of T cells were obtained using FACS and aaiti-Thy 1.2 inA (Non- T). 1x10° OT2 T cells from the TI of either Hp DC recipients or control mice (No DC) were mixed with 2x 1.0" Non-T from the TI of either experimental group (Hp noii-T or non-T, no DC) and cultured without or with O VA. (10 ug ml) for 4Sh. Data are mean pg l+SE of three independent determinations, Presented are data from one of two independent experiments, ""cytokine vs. cytokine + OVA, P<0.01

Example 2

It was determined if macrophages from Tip-infected Rag mice could impair the natural interaction of intestinal pro-inflammatory DC with their effector T cells such as to impede antigenic responses.

Rag mice were reconstituted with 10⁶ IL10 KO T cells, given i.p., and exposed to piroxicam (NSAID) to induce colitis. One wk after stopping the piroxicam, LPMC were isolated from TI and mixed with freshly isolated splenic OT2 T cells (LPMC:OT2; ratio 4: 1). The cells then were cultured in vitro at 2 x l O⁵ cells per well in RPMI complete medium for 48h in 96-well round-bottomed plates with or without OVA (10 μg/ml). Some cultures also received macrophages (4xl0⁵/well) isolated from the TI of Rag mice that had been infected with Hp for 2 wks (Hp Mac), whereas other macrophages came from mice without infection (Mac No Hp). Cell culture supematants were assayed for IFNg and IL17 using ELISAs after the 48h culture period. Data are mean ± SE of 4 independent experiments. *LPMC vs. LPMC+OVA, and LPMC+Mac No Hp vs. LPMC+ Mac No Hp +OVA, p<0.01.

Isolated LPMC from the mice with colitis produced IFNg and IL17 constitutively and secreted substantially more when cultured with OVA (Figure 8). Addition of Hp Mac, given at a ratio of 1 :5 (DC:LPMC), did not affect constitutive cytokine secretion, but totally blocked OVA-induced, cytokine stimulation. Addition of the Mac no Hp control cells did not block cytokine release.

Other experiments used macrophages isolated from the MLN of Rag mice with or without Hp infection. Macrophages from the MLN of Hp-infected mice also blocked OVA- induced, cytokine responses equally as well as gut macrophages when added to the isolated LPMC from the colitic animals (1 :5 cell ratio). The OVA response was not affected by the addition of MLN macrophages from Rag mice who never received Hp-infection.

The next series of experiments ascertained if macrophages from Hp-infected Rag mice could adoptively transfer protection from colitis. Rag mice were infected with Hp for 2 wks and then the Hp were eliminated by treating the animals with a single orally dose of pyrantel pamoate. Age-matched control mice received the same drug treatment, but were not given the infection. One wk after drug treatment, macrophages were isolated from the MLN, and the macrophages from either Hp-infected or control mice were transferred into other groups of Rag mice that received splenic IL10 KO and OT2 T cells by i.p. injection. A third group of Rag mice was reconstituted with IL10 KO and OT2 T cells, but received no macrophages. Animals then were treated with piroxicam to induce colitis.

At the end of the experiment, colonic tissue was examined microscopically to score the severity of the colitis using a 4-point scale. The data in figure 9A represent the severity of inflammation in colons of mice receiving no macrophages (No Mac), macrophages from mice after Hp infection (Hp Mac) or macrophages from mice with no prior Hp infection (Mac No Hp). Data are means + SE from 4 separate experiments each containing 4-5 mice/group. * No Mac or Mac No Hp vs. Hp Mac, p<0.01.

Also studied was the effect of cell transfer on mucosal cytokine production (Figure 9B). LPMC were isolated from the TI of these very same mice. The cells were cultured in vitro at 2¾10⁵ cells per well in RPMI complete medium for 48h with or without OVA (10 μg/ml) to stimulate cytokine release. Cell culture supernatants were assayed for IFNg and IL17 using ELISAs after the 48h culture period. Data are mean ± SE of 4 independent experiments. *LPMC (No Mac) vs. LPMC (No Mac) +OVA and LPMC + Mac No Hp vs. LPMC + Mac No Hp+OVA, p<0.01.

Figure 9 A shows that severe colitis developed in mice receiving no macrophages. Mice receiving macrophages from Hp-infected animals displayed much less colonic inflammation. Transfer of macrophages from animals never infected with Hp did not affect the intensity of the inflammatory response.

LPMC isolated from mice that received no macrophages or macrophages from uninfected mice produced both IFNg and IL17 constitutively when cultured in vitro and even more after OVA stimulation. However, LPMC from Rag mice receiving macrophages from the infected animals lost their responsiveness to OVA stimulation (Fig 9B).

The loss of LPMC antigenic responsiveness after transfer of macrophages from Hp- infected mice signified that macrophage transfer interfered with normal OT2 T cell population of the LP. T cells from C57BL/6 mice express the molecule CD45.2. Rag mice received IL10 KO T cells displaying CD45.2 and OT2 T cells expressing CD45.1. Some mice also received macrophages from the MLN of Rag mice previously infected with Hp (F4/80), while other animals did not receive macrophages (Control). At the end of the experiment, LPMC were isolated from the TI of either the control or macrophage transfer group. Also studied were dispersed MLN cells. Cells were subject to flow analysis to determine the relative number of LP T cells expressing CD45.1. For each group, flow analysis was performed on pooled LPMC or MLN cells isolated from 4 individual mice. Data are representative of 3 independent experiments.

Results are shown in Figure 10. Rag mice were reconstituted with OT2 T cells from transgenic C57BL/6 mice expressing CD45.1 so that OT2 cells within the isolated LPMC could be distinguished from the IL10-/- T cells through differential CD45 display. In this mouse, transgenic TCR expressed on the CD4+ OT2 T cell subset recognize the OVA.

One group of Rag mice received CD45.1 OT2 T cells and CD45.2⁺ IL10-/- T cells, and then were exposed to piroxicam to induce colitis. Another group of Rag animals were treated as above except they also received macrophages from the MLN of Rag mice infected with Hp to protect the animals from colitis and to block the intestinal response to OVA. The relative number of LP CD4+ T cells expressing CD45.1 diminished in Rag mice receiving the macrophage. However, CD45.1 CD4+ T cells populated the MLN normally.

In conclusion, following Hp infection, both DC and macrophage from Hp infected mice acquire the capacity to prevent the interaction of T cells with their antigen presenting cells preventing production of pathogenic cytokines. However, unlike the tolerogenic DC, the regulatory macrophages also impede expansion of potentially pathogenic antigen-specific T cells within the intestines. Example 3

Materials and Methods Mice:

This study used C57BL/6 Rag2 and C57BL/6 wild-type (WT) mice obtained from Jackson Laboratory, Bar Harbor, ME. Also used were C57BL/6 OT2 CD45.1 mice (a gift of Dr. Fuhlbrigge, BWH, Boston, MA) and IL10 KO Foxp3 eGFP reporter mice (gift from Dr. Cathryn Nagler, University of Chicago, IL). Foxp3 mRFP/ILlO eGFP double reporter mice were produced by cross breeding Foxp3 mRFP and IL10 eGFP single reporter mice were obtained from (Richard Flavell, Yale University, CT). Breeding colonies were maintained in SPF facilities at Tufts University. Animals were housed and handled following national guidelines and as approved by our Animal Review Committee. Hpb infection:

For the experiments, 5- to 6-wk-old mice were colonized with 125 Hpb third stage larvae (L3) by oral gavage. And infected mice were used after two weeks. Infective, ensheathed Hpb L3 (U.S. National Helmintho logical Collection no. 81930) were obtained from fecal cultures of eggs by the modified Baermann method and stored at 4°C.

Dispersion of splenic T cells and mesenteric lymph node (MLN), and splenic T cell enrichment:

Single cell suspensions of splenocytes and MLN cells were prepared by gentle teasing in RPMI 1640 medium (GIBCO, Grand Island, NY). The cells were washed three times in RPMI. Splenic CD4+/CD25- T cells were labeled with FITC-CD4 and PE-CD25 mAbs (eBioscience, San Diego, CA) and then isolated by FACS (FACSCalibur using Cell Quest software, BD Biosciences, Mountain View, CA). Viability was determined using exclusion of trypan blue dye.

Lamina propria mononuclear cells (LPMC) isolation and LP cell fractionation:

Gut LPMC were isolated from the colon as described (Elliott et al, Eur. J. Immunol. 34:2690-2698). Foxp3 mRFP+/IL10 eGFP- T cells and Foxp3 mRFP+/IL10 eGFP+ T cells were isolated by FACS. The viability of the isolated cells always was greater than 95% as determined using exclusion of trypan blue dye.

Adoptive cell transfer:

Rag mice of similar age (usually 5 to 6 wks old) were reconstituted i.p. with lx 10⁵

C57BL6 WT CD4+CD25- splenic T cells and 3xl0⁵ OT2 CD45.1 splenic T cells. In some experiments, mice also received lxlO⁵ Foxp3+, Foxp3+/IL10-, Foxp3+/10+ or IL10KO Foxp3+ colon LPMC T cells given by i.p. injection. Colitis model:

Mice received CD4+CD25- splenic T cells from WT and OT2 mice. In some experiments, the animals also received unfractionated colonic Foxp3+ T cells, or colonic Foxp3+/IL10- or Foxp3+IL10+ T cells from Foxp3/IL10 double reporter mice. Some of these reporter mice were infected with Hpb for 2 wks before sacrifice. One wk after T cell transfer, Rag mice were administered piroxicam, a nonsteroid anti-inflammatory drug (NSAID), mixed into their feed for 2 wks (42mg piroxicam /250g chow wk 1, and 62mg

piroxicam/250g chow wk 2). The piroxicam (Sigma) was stopped, and colitis was studied 1 wk later. Thus, it was 4 wks from the day of cell transfer until animal sacrifice. Half of the colons divided longitudinally were fixed, sectioned and stained with H&E for

microscopically examined to score the severity of colitis. The other half was dissociated with collagenase to isolate LPMC, which were analyzed by flow cytometry and cultured in vitro. More details regarding the various manipulations of this model are provided in the results section. Cell culture:

Colonic LPMC from Rag mice reconstituted with C57BL/6 WT CD4+CD25- and OT2 CD4+CD25- T cells were cultured (2.5 ¾10⁵ cells per well) for 48h in 96-well round- bottomed plates. Cells were cultured alone or with OVA (10 μg/ml) (Sigma). The culture medium was RPMI 1640 containing 10% FCS, 25 mM HEPES buffer, 2 mM L-glutamine, 5 x 10 ⁵ M 2-ME, 1 mM sodium pyruvate, 100 U/ml penicillin, 5 mg/ml gentamicin, and 100 mg/ml streptomycin (complete medium) (all Life Technologies, Gaithersburg, MD). After culture, the supernatants were assayed for IFNy, IL17A or IL4 using ELISA (described below). Sandwich ELISAs:

ELISAs were performed using paired antibodies (R & D Systems, Minneapolis, MN) according to manufacturer's instructions. IL17 ELISA was done using primary capture antibody from (R&D Systems, Inc) and biotinylated anti-IL17A antibody (R&D Systems). To measure IFNy, plates were coated with a mAb to IFNy (HB170, ATCC) and incubated with supernatant. IFNy was detected with polyclonal rabbit anti-IFNy (gift from Dr. Mary Wilson, University of Iowa) followed by biotinylated goat anti-rabbit IgG (AXcell,

Westbury, NY).

Flow cytometry analysis:

Isolated LPMC were washed twice and adjusted to 10⁷ cells/ml in FACS buffer (LGM) and stained with saturating amounts of conjugated mAb for 30 min at 4°C. Following staining, cells were washed twice and re-suspended in LGM for analysis on a FACSCalibur using Cell Quest software (BD Biosciences, Mountain View, CA). Before adding labeled mAb, each tube received 1 μg of anti-Fc mAb (eBioscience, San Diego, CA) to block nonspecific binding of conjugated Abs to FcRs. The mAbs used for staining or cell sorting were anti-CD4-FITC, -APC or -PECy5; anti-CD25-PE; anti-CD45.1-APC (all from eBioscience).

Histological evaluation:

A pathologist blinded to the experimental condition graded the severity of the colonic inflammation using a well describe 4-point scale (Berg et al., Gastroenterol. 123: 1527-1542). Statistical analysis:

Data are means +SE of multiple determinations. Difference between two groups was compared using Student's i-test. Multiple group comparisons used analysis of variation and Dunnett's t-test. P values <0.05 were considered significant. Results

Hpb infection induced an increase in the proportion of colonic LPMC CD4+ T cells expressing Foxp3 T cells that express Foxp3 are plentiful in the gut and help to limit mucosal immune responses. Since Hpb can protect mice from colitis (Elliot et al., 2002. Eur. J. Immunol. 34:2690-2698; Elliott et al, 2008 J. Immunol. 181 :2414-2419), it was determined if Hpb infection increased the relative number of Foxp3+ T cells in the colonic mucosa of healthy C57BL/6 Foxp3 reporter transgenic mice. Foxp3mRFP/IL10eGFP double reporter mice were used to allow visualization and then isolation of Treg subsets distinguished by their differential capacity to express IL10.

In the colon of healthy transgenic mice, Foxp3RFP was seen only in T cells, and nearly all the Foxp3+ T cells in the colon were CD4+ (>97%). A small number of CD8+ T cells expressed Foxp3 also (data not shown). In the colonic LP, about 25% of the CD4+ T cells were Foxp3+ and about 60% of them also expressed IL10 (Figure 11 and Table 3). ILlOeGFP also was seen in some CD4+ T cells that were negative for Foxp3 expression.

Reporter mice were infected with Hpb for 2 wks. As compared to age-matched uninfected control animals, there was a modest, but significant increase in the proportion of colonic LP CD4+ T cells that were Foxp3+/IL10- or Foxp3+/IL10+ (Figure 11, Table 3). Also, the proportion of Foxp3- CD4+ T cells expressing IL10 increased slightly as well.

Colonic Foxp3+ T cells from Hpb infected mice prevented colitis and reduced the release of IFNg and IL17 from the colonic LPMC

To study colitis, experiments employed a well established Rag CD4+ CD25- T cell transfer model of IBD (Kjellev et al, 2006. International Immunopharmacology 6: 1341- 1354). To enhance expression of disease, 1 wk after cell transfer, mice were fed a NSAID (piroxicam) for 2 wks. This resulted in all animals developing severe colitis that was evident 1 wk thereafter stopping the NSAID (Four wks after cell transfer). The NSAID disrupts the production of protective arachadonic acid metabolites in the mucosa (Berg eta 1., 2002. Gastroenterol. 123:1527-1542) making the animals more prone to IBD. This is relevant to human IBD, since administration of many types of NSAIDs worsen the disease (Takeuchi et al., 2006. Clinical Gastroenterology & Hepatology 4: 196-202; Chan et al., 2011. Alimentary Pharmacology & Therapeutics 34:649-655). CD4+ CD25- OT2 T cells were adoptively transferred into the Rag mice concomitantly with the other cells so that an antigen specific T cell response could be assayed in the colonic LPMC. OT2 T cells are transgenic T cells that response to OVA. Isolated LPMC from these T cell reconstituted Rag animals respond to OVA with IFNg and IL17 release. These cytokines drive the disease in human IBD and in many animal models of this condition (Abraham and Cho. 2009. Inflam. Bowel Dis. 15: 1090- 1100; Maloy et al, 2011. Nature 474:298-306).

Using this model, it was asked if the Hpb infection affected the functionality of the colonic Foxp3+ T cells isolated from WT mice with regard to their capacity to prevent IBD. Foxp3+ T cells from the colon of Hpb infected or uninfected WT reporter mice were adoptively transferred into Rag mice along with the appropriate splenic CD4+CD25-T cells. Another group of Rag mice received the appropriate CD4+CD25- splenic T cells, but no colonic Foxp3+ T cells. The Rag mice were give piroxicam and sacrificed 4 wks after cell transfer to assess the severity of the colitis. Figure 12 shows that only colonic Foxp3+ T cells from Hpb infected mice protected the mice from IBD.

Colonic LPMC were isolated from the colitis-induced Rag 4 wks after cell transfer and cultured with or without OVA. Colonic LPMC cultured without antigen produced substantial amounts of IFNg and IL17, but much more in the presence of OVA. Only adoptive transfer of colonic Foxp3+ T cells from Hpb infected reporter animals into Rag recipients decreased constitutive and OVA-stimulated cytokine release from the colon LPMC (Figure 12).

Fluorescent colonic Foxp3+ T cells from Hpb infected reporter mice readily accumulated in the colon andMLNofRag recipients

Also studied was if adoptive transfer of colonic reporter Foxp3+ T cells, from Hpb infected mice, into Rag recipients led to accumulation of fluorescent Foxp3+ T cells in their colons. Following such transfers, the colons of Rag mice contained large numbers of

CD4+Foxp3+/IL10- and Foxp3+/IL10+ T cells (Figure 13, Table 4). All tissues were examined at the usual time of sacrifice for this colitis model (4 wks after Foxp3 T cell transfer). Examination of the MLN yielded similar results, although, compared to the colon, the relative number of CD4+ T cells expressing Foxp3 was lower in this tissue (Table 4).

Transfer of colonic Foxp3+ T cells from uninfected reporter mice into Rag recipients resulted in proportionately fewer T cells in the colon that were Foxp3+/IL10- compared to mice receiving cells from infected animals (about 70% less) (Figure 13, Table 4).

Moreover, there were nearly no Foxp3+/IL10+ T cells present. Results were similar for MLN. Also noted was a small, but definite CD4+ T cell subset that was Foxp3-/IL10+. Foxp3+IL10- and Foxp3+IL10+ T cell subsets protected from colitis with comparable efficiency

The Foxp3+ T cells adoptively transferred into Rag mice to prevent colitis were composed of two subsets distinguished by their capacity to express ILIO. Experiments ascertained if the Foxp3+ ILIO- and Foxp3+ IL10+ T cell subsets, obtained from the colon of Hpb infected mice, would provide similar levels of colitis protection. Experiments showed that both subsets afforded comparable protection (Figure 4), and reduced constitutive and OVA-stimulated cytokine release from the colonic LPMC isolated from the colitic mice (Figure 4).

Transfer of colonic Foxp3+IL10- T cells from Hpb infected reporter mice into Rag recipients resulted in accumulation of both Foxp3+/IL10- and Foxp3+/IL10+ T cells in the colon and MLN of the recipient animals

The above observation that Foxp3+/IL10- T cells can protect mice from colitis was unexpected, since previous studies showed that Foxp3+ T cells that make ILIO are important for controlling immune responses in the intestinal mucosa (Rubtsov et al., 2008. Immunity 28:546-558). Thus, further studies determined if transfer of Foxp3+ T cells that cannot produce IL10 result in the accumulation of both Foxp3+ T cell subsets in the colon of Rag recipients.

Rag mice that received colonic Foxp3+IL10- T cells from Hpb infected reporter mice readily acquired large numbers of fluorescent CD4+ Foxp3+IL10- and Foxp3+IL10+ T cells in the colon (Figure 15, Table 5) and MLN (Table 5) at the standard time of sacrifice (4 wks). In these tissues, the relative number of CD4+ T cells expressing Foxp3 with or without IL10 was similar to that observed in Rag mice reconstituted with unfractionated colonic Foxp3+ T cells (Figure 13, Hpb).

It was also ascertained if transfer of Foxp3+IL10+ T cells would yield similar results. A surprising outcome was that Rag recipients of colonic Foxp3+IL10+ T cells, examined 4 wks after transfer, displayed essentially no fluorescent Foxp3+T cells in tissues (Figure 15, Table 5). Examination of colons at earlier time points after cell transfer (wk 1 and 2) revealed only small numbers of such cells. Thus, transferring just colonic Foxp3+/IL10+ T cells failed to stably reconstitute the Foxp3 compartment. Adoptive transfer of colonic Foxp3+ T cells from Hpb infected IL 10-/- mice into Rag recipients resulted in Tregs accumulating within their tissues, but failed to protect them from colitis

To further explore the importance of IL10 in prevention of colitis, IL10-/- Foxp3eGFP reporter mouse were colonized for 2 wks with Hpb. Foxp3+ T cells isolated from the colon of these animals adoptively transferred into Rag recipients did not prevent colitis (Figure 16). Colonic LPMC, isolated from the Rag recipients of IL10-/- Foxp3+ T cells, cultured in vitro produced amounts of IFNg and IL17 without or with OVA stimulation similar to that of control mice.

In the Rag mice that received IL10-/- Foxp3+ T cells, colon and MLN were examined for the presence of Foxp3eGFP+ CD4+ T cells at the 4-wk time of sacrifice. Dispersed colonic LPMC and MLN cells were examined by flow cytometry. Fluorescent IL10-/- Foxp3+ CD4+ T cells were numerous in both tissues. (Mean percentage of CD4+ T cells expressing Foxp3eGFP: Colon, 6.8±1.8 and MLN, 3.4±1.7. Data are means of 3 experiments ±SE.)

All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the invention will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.

Claims

We claim: 1. A method of treating an inflammatory bowel disease (IBD), comprising: a) contacting an isolated immune system cell from a subject diagnosed with an IBD with a helminth to generate a treated immune system cell; and

b) administering said treated immune system cell to said subject, wherein said administering reduces or eliminates symptoms of said IBD.

2. The method of claim 1, wherein said IBD is selected from the group consisting of Crohn's disease and ulcerative colitis.

3. The method of claim 1, wherein said helminth is Heligmosomoides poly gyrus.

4. The method of claim 1, wherein said immune system cell is selected from the group consisting of a macrophage, a dendritic cell, and a regulatory T cell (Treg).

5. The method of claim 1, wherein said isolated immune system cell is in peripheral blood or intestines.

6. The method of claim 1, wherein said isolated immune system cell is purified from peripheral blood or intestines.

7. The method of claim 1, wherein said administering is intravenous,

subcutaneous, intramuscular, or intraperitoneal administration.

8. The method of claim 1, wherein said Treg is a Foxp3+/IL10- and/or

Foxp3+/IL10+ Treg.

9. A method of treating inflammatory bowel disease (IBD), comprising:

administering a helminth extract to a subject, wherein said administering reduces or eliminates symptoms of said IBD.

10. The method of claim 9, wherein said IBD is selected from the group consisting of Crohn's disease and ulcerative colitis.

11. The method of claim 9, wherein said extract is administered orally.

The method of claim 9, wherein said extract is fractionated.

The method of claim 9, wherein said helminth is Heligmosomoides polygyrus.

14. The method of claim 9, wherein said extract is in a capsule that dissolves in the intestine.