Suppression of colitis by adoptive transfer of helminth antigen-treated dendritic cells requires interleukin-4 receptor-α signaling.

Infection with helminth parasites has been explored as a treatment for autoimmune and inflammatory diseases. As helminth antigens have potent immunomodulation properties capable of inducing regulatory programs in a variety of cell types, transferring cells treated with helminth antigens represents a novel extension to helminth therapy. Previous work determined that transfer of bone marrow-derived dendritic cells (DC) pulsed with a crude extract of the tapeworm Hymenolepis diminuta (HD) can suppress colitis in recipient mice. The present study explored the mechanism of disease suppression and the importance of interleukin (IL)-4 signaling. Transfer of HD-DCs suppressed dinitrobenzene sulfonic acid (DNBS)-induced colitis through activation of recipient IL-4 receptor-α. The transferred HD-DCs required IL-4Rα and the capacity to secrete IL-10 to drive IL-4 and IL-10 production and to suppress colitis in recipient mice. Treatment of DCs with IL-4 evokes an alternatively activated phenotype, but adoptive transfer of these cells did not affect the outcome of colitis. Collectively, these studies demonstrate the complexity between IL-4 and IL-10 in donor cells and recipient, and the requirement for parasite- and host-derived factors in this novel form of cell therapy. Thus IL-4Rα signaling is revealed as a pathway that could be exploited for helminth antigen cell-based therapy.

Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer.

Colorectal cancer (CRC) is an important health issue worldwide, accounting for the third place of cancer incidence. Chronic inflammation, as seen in Crohn's disease and ulcerative colitis, is the most important risk factor for developing CRC, as it favours neoplastic transformation by enhancing epithelial cell turnover in the colonic mucosa. Treatments for CRC need to be improved; currently they are not specific and have several secondary effects in patients. The main objective of this work was to evaluate a new therapeutic strategy against a colitis-related colorectal cancer in vivo and in vitro by targeting mTOR-signaling and lactate dehydrogenase A. Together, these mechanisms directly affect tumor energetics. In this study we evaluated a better and more efficient triple therapy against a chronic inflammation-associated CRC in vivo and in vitro. After the development of tumors, mice were treated intraperitoneally during a forty-day period with single drugs or different combinations of Metformin, Sodium Oxamate and Doxorubicin. Targeted inhibition of the mTOR pathway, lactate dehydrogenase A and the concurrent use of Doxorubicin (called in this work as triple therapy), leaded to a notable reduction in the number and size of tumors in mice, and, a significant pro-inflammatory cytokines reduction Besides, we showed that treated cells were induced to early autophagy, and apoptosis cell death. Our results represent a novel and robust therapeutic strategy for overcoming CRC by means of targeting central molecular pathways in cancer by the combination of Metformin, Oxamate, and Doxorubicin leading to a rapid tumor growth inhibition and a dramatic colorectal crypt restoration. Besides, drug combination resulted in a notable reduction of anti-inflammatory cytokines.

Regulation of immunity by Taeniids: lessons from animal models and in vitro studies.

Taeniidae is the largest family of the Cyclophyllidea order of parasites despite being composed of just two genera: Taenia spp and Echinococcus spp. These parasites are flatworms with a terrestrial life cycle, having an immature or larval stage called metacestode, which develops into the mature form within the intestine of the primary host after being consumed in raw or poorly cooked meat. Consumed eggs hatch into oncospheres, penetrate the intestinal walls and are transported via the bloodstream to later develop into metacestodes within the muscles and internal organs of secondary and sometimes primary hosts, thereby initiating the cycle again. Larval stages of both Taenia spp and Echinococcus spp are well known to produce tissue-dwelling, long-lasting infections; in this stage, these parasites can reach centimetres (macroparasites) and both genera may cause life-threatening diseases in humans. Establishing such long-term infections requires an exceptional ability to modulate host immunity for long periods of time. In this review, we analyse the immunoregulatory mechanisms induced by these tapeworms and their products, mainly discussing the importance of taeniid strategies to successfully colonize their hosts, such as antigen-presenting cell phenotype manipulation and the consequent induction of T-cell anergy, among others.

Protection against Naegleria fowleri infection in mice immunized with Cry1Ac plus amoebic lysates is dependent on the STAT6 Th2 response.

We previously reported that intranasal administration of Cry1Ac protoxin alone or in combination with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice. Those results suggested that both antibody responses and innate immune mechanisms may be participating in the protective effects observed. The present study was aimed to investigate whether the STAT6-induced Th2 immune response is essential for the resistance to N. fowleri infection, conferred by immunization with amoebic lysates plus Cry1Ac. STAT6-deficient (STAT6-/-) and wild-type (STAT6+/+) BALB/c mice were immunized by the intranasal route with a combination of N. fowleri lysates plus Cry1Ac, and subsequently challenged with lethal doses of N. fowleri trophozoites. STAT6+/+ mice displayed 100% protection, while no protection was observed in STAT6-/- mice. Significantly higher titres of Th2-associated IgG1 as well as interleukin-4 (IL-4) were found in STAT6+/+ mice, whereas in STAT6-/- mice significantly more IL-12 and IFN-gamma as well as significantly higher titres of Th1-associated IgG2a were detected. Thus, whereas protected STAT6+/+-immunized mice elicited a Th-2 type inclined immune response that produced predominantly humoral immunity, unprotected STAT6-/- mice exhibited a polarized Th1 type cellular response. These findings suggest that the STAT6-signalling pathway is critical for defence against N. fowleri infection.

The divergent roles of alternatively activated macrophages in helminthic infections.

Macrophages play crucial roles in the immune response, as they can initiate, modulate and also be final effector cells during immune responses to infections. Macrophages are derived from myeloid precursor cells in bone marrow and are widely distributed in every tissue of the body. Over the past 10 years, the concepts about macrophage activation have clearly changed; macrophages are not called activated or inactivated as they used to be. These changes in the concept of macrophage response is the result of many in vitro and in vivo studies, but the major support for the current concept of alternatively activated macrophages (AAMphi) comes from parasitic helminth infections. Parasitic helminths have developed complex mechanisms to evade and modulate host immunity. Infections with these parasites induce strong polarized Th2-type immune responses frequently associated with impaired T-cell proliferative responses to parasitic or unrelated antigens. Given the recent advances in understanding the immunoregulatory capabilities of helminthic infections, it has been suggested that macrophages can be a target for immunomodulation. Furthermore, they become altered when a host experiences chronic exposure to helminth parasites or their by-products, which favour the induction of AAMphi. How AAMphi participate in modulating host immunity during helminth infections and what their roles are in clearing or favouring parasite survival remains elusive. Here we review the most recent advances in the literature on AAMphi at the host-parasite interface, including three classes of helminths: nematodes (Brugia, Nippostrongylus, Litomosoides, Heligmosomoides), trematodes (Schistosoma, Fasciola) and cestodes (Taenia, Echinococcus, Hymenolepis).

Intact glycans from cestode antigens are involved in innate activation of myeloid suppressor cells.

During helminthic infections, strong Th2 type-biased responses concomitant with impaired cell-proliferative responses to parasitic and unrelated antigens are major immunological hallmarks. Parasite glycan structures have been proposed to play a role in modulating these responses. To understand early events related to immune modulation during cestode infection, we have examined the role of intact glycans of antigens from Taenia crassiceps in the recruitment of innate cells. Soluble antigens from this cestode contained higher levels of carbohydrates than proteins. Intraperitoneal injection of the antigens rapidly recruited a cell population expressing F4/80(+)/Gr-1(+)surface markers, which adoptively suppressed naïve T-cell proliferation in vitro in response to anti-CD3/CD28 MAb stimulation in a cell-contact dependent manner. Soluble antigens with altered glycans by treatment with sodium periodate significantly reduced the recruitment of F4/80(+)/Gr1(+)cells, concomitantly their suppressive activity was abrogated, indicating that glycans have a role in the early activation of these suppressor cells. Using C3H/HeJ and STAT6-KO mice, we found that expansion and suppressive activity of F4/80(+)Gr1(+)cells induced by T. crassiceps intact antigens was TLR4 and Th2-type cytokine independent. Together with previous studies on nematode and trematode parasites, our data support the hypothesis that glycans can be involved on a similar pathway in the immunoregulation by helminths.

CpG-containing ODN has a limited role in the protection against Toxoplasma gondii.

Bacterial DNA containing immunostimulatory motifs (CpG) induces the development of a T(H1) immune response. Since protection against Toxoplasma gondii is correlated with this type of response, the aim of this work was to determine if a synthetic oligodeoxynucleotide (ODN) containing CpG sequences could be useful as adjuvant for the induction of a long-lasting protective immune response against T. gondii. BALB/c mice immunized with a total soluble antigen of T. gondii (TSA2) mixed with ODN-containing CpG sequences developed a typical TH1 response, as determined by antibody isotypes and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by spleen cells. However, they did not resist a challenge with the virulent RH strain of the parasite. Absence of protection paralleled with lower levels of IFN-gamma, when compared with mice vaccinated with the live tachyzoites of the attenuated ts.4 strain of the parasite, which resisted this challenge. Intraperitoneal injection of ODN alone to mice induced a high degree of resistance to a lethal challenge inoculated by the same route. Nevertheless, this nonspecific protection was transient. Thus, the use of ODN containing CpG motifs as adjuvant is of limited value for the induction of a protective immune response against T. gondii.

The schistosome oligosaccharide lacto-N-neotetraose expands Gr1(+) cells that secrete anti-inflammatory cytokines and inhibit proliferation of naive CD4(+) cells: a potential mechanism for immune polarization in helminth infections.

Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1(+)/CD11b(+)/F4/80(+), as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1(+)/F4/80(+) population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-beta ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1(+) cells adoptively suppressed naive CD4(+) T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4(+) cells involved cell contact and was dependent on IFN-gamma and NO, with a discrete role played by IL-10. Coculture of naive CD4(+)T cells with Gr1(+) suppressor cells did not lead to CD4(+) T cell apoptosis, although it did imprint on naive CD4(+) T cells a response characterized by lower levels of IFN-gamma, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.

Characterization and protective potential of the immune response to Taenia solium paramyosin in a murine model of cysticercosis.

Paramyosin has been proposed as a vaccine candidate in schistosomiasis and filariasis. However, limited information is available about its protective potential against cysticercosis and the immune response it induces. Immunization of mice with recombinant full-length paramyosin of Taenia solium (TPmy) results in about a 52% reduction in parasite burden after a subsequent challenge by intraperitoneal inoculation of Taenia crassiceps cysticerci. Immunization assays using recombinant fragments of TPmy, corresponding approximately to thirds on the amino, central, or carboxyl regions, suggest that protective epitopes are located mostly in the amino-end third. Proliferation assays using T cells obtained from mice immunized with the full-length recombinant TPmy also showed a preferential response to the amino-terminal fragment. In contrast, antibodies in the sera from these mice predominantly recognize epitopes located in the carboxyl-terminal fragment, being the immunoglobulin G1 subclass, the predominant antibody isotype. Characterization of the cellular immune response induced against the protective amino-terminal fragment reveals production of gamma interferon and interleukin-2, but not interleukin-4, suggesting a Th1-like profile.

Intraspleen DNA inoculation elicits protective cellular immune responses.

DNA immunization or inoculation is a recent vaccination method that induces both humoral and cellular immune responses in a range of hosts. Independent of the route or site of vaccination, the transfer of antigen-presenting cells (APC) or antigens into lymphoid organs is necessary. The aim of this investigation was to test whether intraspleen (i.s.) DNA inoculation is capable of inducing a protective immune response. We immunized mice by a single i.s. injection of a DNA construct expressing the immunoglobulin (Ig) heavy-chain variable domain (VH) in which the complementarity-determining regions (CDR) had been replaced by a Taenia crassiceps T-cell epitope. In these mice, immune responses and protective effects elicited by the vaccine were measured. We have shown here for the first time that i.s. DNA inoculation can induce protective cellular immune responses and activate CD8(+) T cells. Also, Ig V(H) appeared to be the minimal delivery unit of "antigenized" Ig capable of inducing T-cell activation in a lymphoid organ. The strategy of introducing T-cell epitopes into the molecular context of the V(H) domain in combination with i.s. DNA immunization could have important implications and applications for human immunotherapy.

Taenia crassiceps cysticercosis: a role for prostaglandin E2 in susceptibility.

Several biological factors are involved in susceptibility and resistance to murine cysticercosis. A substantial body of evidence implies prostaglandins as potent regulators of immune responses during parasitic diseases. Here we evaluated the role played by prostaglandin E2 in cysticercosis. Mice were treated in vivo with prostaglandin E2 or with indomethacin (a prostaglandin E2 synthesis inhibitor) before infection. Parasite growth was enhanced by prostaglandin treatment, which provoked poor Con-A responses, low Th1-type cytokines secretion, and high levels of IL-6 and IL-10. In contrast, mice receiving indomethacin showed a reduction in parasite load parallel to a strong Con-A response and high levels of IL-2 and IFN-gamma, concomitantly with a decrease in IL-4, IL-6 and IL-10 production. Indirect in vitro studies suggest that an important source of prostaglandin E2 production could be related to host's adherent cells. However, prostaglandin E2 from parasite origin cannot be discarded.

DNA pulsed macrophage-mediated cDNA expression library immunization in vaccine development.

cDNA expression library immunization (cDELI), based on the use of a large number of pathogen's cDNA clones, has been previously used in our laboratory in an experimental model of murine Taenia crassiceps cysticercosis. In this study we show that ex vivo immunization of mice with macrophages pulsed in vitro with plasmid DNA containing cDNA expression library (2x10(4) clones) leads to significant reduction of parasite load. Additionally, pathogen-specific proliferation of splenocytes from immunized mice is demonstrated indicating the induction of cellular protective immune response and the efficacy of the proposed strategy to identify vaccine candidate antigens. Our method may represent an attractive tool in vaccine discovery applicable to any host-pathogen system and may be useful especially for the pathogens with large genomes and complicated life cycles.

Phage-displayed T-cell epitope grafted into immunoglobulin heavy-chain complementarity-determining regions: an effective vaccine design tested in murine cysticercosis.

A new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (V(H)) domain with the Taenia crassiceps epitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4(+) and CD8(+) T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig V(H) domains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction.

Susceptibility to Trypanosoma cruzi is modified by a previous non-related infection.

Helminth infections are frequently massive, chronic and strong inductors of Th2-type cytokines. This implies that infection by such parasites could alter the susceptibility to subsequent infections by other pathogens, particularly intracellular parasites. We therefore explored whether a persistent infection, caused by Taenia crassiceps cysticerci, in BALB/c mice could affect susceptibility to a later infection by Trypanosoma cruzi. We found that the presence of the cysticerci indeed modified the immune response and the susceptibility to T. cruzi, and that these modifications depended on the time-course evolution of the initial infection. Coinfection with the protozoan in the early stages of the helminth infection, induced a delay on the onset of parasitaemia, early specific production of IFN-gamma and high specific production of IL-4. A significant increase in susceptibility to T. cruzi was observed only when mice were coinfected in late stages when the helminth load is greater and a Th2 type response against it is predominant. The in vitro specific response to T. cruzi antigens was then characterized by low levels of both IFN-gamma and IL-4. These findings suggest that chronic helminth infections could potentially have a significant influence over the immune response and hence susceptibility to other pathogens.

Th1-type cytokines improve resistance to murine cysticercosis caused by Taenia crassiceps.

Resistance and susceptibility to different parasitic diseases have been associated with the predominance of Th1- or Th2-type immune responses. In experimental murine cysticercosis a Th1 response seems to be involved in resistance, whereas Th2 activity is associated with heavy parasite intensities. To test this notion the roles of Th1- and Th2-type cytokines in infected mice were studied after treatment with anticytokine monoclonal antibodies or with recombinant murine cytokines during early stages of infection. Mice receiving anti-interleukin 10 (IL-10) carried lower parasite intensities than did control mice and developed a strong Th1-type response, whereas mice receiving anti-interferon gamma (IFN-gamma) showed a dramatic increase in susceptibility. Treatment with recombinant cytokines confirmed these results; mice receiving IFN-gamma and IL-2 showed low parasite numbers, whereas IL-10 induced a significant increase in parasite loads. Thus, the Th1-type immune response plays a fundamental role in protection against Taenia crassiceps cysticercosis, whereas Th2, at least through IL-10, favors parasite establishment.

Protection against murine cysticercosis using cDNA expression library immunization.

Genetic or DNA-based immunization, including genomic immunization, has shown to be a viable alternative approach to induce protective immunity against a number of pathogens in several disease models. Here we describe a new method, cDNA expression library immunization (cDELI), based on the use of a large number of cDNA clones. This immunization strategy was tested in experimental murine Taenia crassiceps cysticercosis model. A partial cDNA expression library of 2 x 10(4) members was constructed in eukaryotic expression vector pcDNA3 and used to immunize BALB/c female mice subcutaneously (s.c.) and intramuscularly (i.m.). In both cases significant reduction of parasite load (up to 65%) was obtained. We were unable to directly measure T. crassiceps-specific humoral immune response in any of the immunized mice, although the expression of pathogen proteins in vitro in macrophages transfected with cDNA expressing plasmids was demonstrated. Also, in three out of five randomly selected immunized mice detectable levels of interferon-gamma (IFN-gamma) were obtained. cDELI has additional advantages compared with recently developed single gene or genomic immunization approaches because a cDNA population represents only those genes that are being expressed in the pathogen cells and the selection of stage-specific antigens is possible. The use of cDELI could be particularly attractive for the pathogens with complicated life cycles and large genomes.

Shift from an early protective Th1-type immune response to a late permissive Th2-type response in murine cysticercosis (Taenia crassiceps).

In early stages of experimental murine cysticercosis caused by Taenia crassiceps, there is a clear but transient Th1-type immune response (characterized by high levels of interleukin [IL]-2, interferon-gamma, concanavalin A, and antigen specific response, delayed-type hypersensitivity, and immunoglobulin [Ig]G2a antibodies) that associates with a low rate of parasite reproduction. As time of infection progresses an energic and more permanent Th2-type response follows (characterized by high levels of IL-4, IL-6, IL-10, IgG2b, and IgG1 antibodies) that in turn associates with an increment in the rate of parasite reproduction. The sequential activation of Th1-type and Th2-type responses in murine cysticercosis would appear to favor progressively parasite reproduction, explaining the long time residence and the massive parasite intensity reached in chronic infections.

Stressful stimulation of newborn mice increases the in vitro proliferative response in T cells from thymus.

The effects of prolonged stressful stimulation on the in vitro proliferative response of thymic T cells and the thymic zinc concentration were investigated in newborn Balb/c mice. Animals were stressed by intraperitoneal injections with aliquots from a heat-killed staphyloccocal suspension over one month. The splenic T lymphocytes from the stressed animals showed a significant reduction in the in vitro response to Concanavalin A (Con-A) stimulation. However, an unexpected and significant increase in proliferative response was observed when thymic lymphocytes from stressed animals were stimulated with the same mitogen. The intrathymic zinc levels were regularly elevated in stressed mice, in contrast to those values obtained in the thymus from healthy control mice. These results suggest that neonatal stress can disrupt the intrathymic maturation and the selection of pre-T lymphocytes. The increment of the in vitro proliferative response of T cells from the thymus of stressed mice may be caused by proportionally higher amounts of intrathymic lymphoid subpopulations expressing a mature phenotype and functionality.

A role for 17-beta-estradiol in immunoendocrine regulation of murine cysticercosis (Taenia crassiceps).

In experimental murine cysticercosis caused by Taenia crassiceps, parasite reproduction is favored by thymectomy or by orchidectomy, and restricted by ovariectomy. Hormonal reconstitution experiments showed that 17-beta-estradiol increases parasite numbers whereas 5-alpha-dihydrotestosterone was ineffective. Parasite numbers decreased with increments in cellular immunity but were insensitive to antibody levels. A possible immunoendocrinological interaction involving estrogen as a depressor of cellular immunity is envisaged in the control of cysticercosis.

Thymus-related cellular immune mechanisms in sex-associated resistance to experimental murine cysticercosis (Taenia crassiceps).

The role of sex, thymus, and cellular immune mechanisms in mouse resistance to experimental cysticercosis with Taenia crassiceps was studied in male and female susceptible mice treated with cyclophosphamide, as well as in mice neonatally thymectomized and passively transferred with T-enriched lymphoid cells. High doses of cyclophosphamide increased delayed hypersensitivity and resistance of mice of both sexes without affecting antibody production. Neonatal thymectomy diminished resistance in both sexes but depressed delayed hypersensitivity in females only, without significantly affecting antibody response in either sex. Passive transfer of T-enriched lymphoid cells to thymectomized mice restored resistance to control levels without greatly affecting delayed hypersensitivity. Thus, our results indicate that cell-associated immune mechanisms are implicated in resistance to murine cysticercosis with T. crassiceps. Because neonatal thymectomy nearly equalized the intensity of infection of female and male mice, it is argued that the thymus is importantly involved in the interaction between gonads and the immune system in the control of this cysticercosis.