Muscle Larvae Excretory-secretory Products Research Articles

Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity.

The immunomodulatory potential of Trichinella spiralis muscle larvae excretory-secretory products (ES L1) has been well documented in vitro on dendritic cells (DCs) and in animal models of autoimmune diseases. ES L1 products possess the potential to induce tolerogenic DCs and consequently trigger regulatory mechanisms that maintain immune homeostasis. The use of ES L1 as a potential treatment for various inflammatory disorders proved to be beneficial in animal models, although the precise immunomodulatory factors have not yet been identified. This study aimed at the isolation and characterization of ES L1 components that possess galectin family member properties. Galectin-1-like proteins (TsGal-1-like) were isolated from ES L1 based on the assumption of the existence of a lactose-specific carbohydrate-recognition domain and were recognized by anti-galectin-1 antibodies in Western blot. This TsGal-1-like isolate, similar to galectin-1, induced DCs with tolerogenic properties and hence, the capacity to polarize T cell response towards a regulatory type. This was reflected by a significantly increased percentage of CD4+CD25+Foxp3+ regulatory T cells and significantly increased expression of IL-10 and TGF-β within this cell population. Proteomic analysis of TsGal-1-like isolate by mass spectrometry identified nineteen proteins, seven with annotated function after blast analysis against a database for T. spiralis and the UniProt database. To our surprise, none of the identified proteins possesses homology with known galectin family members. Nevertheless, the isolated components of ES L1 possess certain galectin-1 properties, such as specific lactose binding and the potential to elicit a regulatory immune response, so it would be worth further investigating the structure of sugar binding within isolated proteins and its biological significance.

Trichinella spiralis excretory-secretory products downregulate MMP-9 in Dark Agouti rats affected by experimental autoimmune encephalomyelitis

Matrix metalloproteinases (MMPs), are implicated in the pathogenesis of multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). Our aim was to investigate whether amelioration of EAE in Dark Agouti (DA) rats, induced by Trichinella spiralis muscle larvae excretory-secretory products (ES L1), could be related to the level and activity of gelatinases, MMP-9 and MMP-2. Serum levels of MMP-9, MMP-2, NGAL/MMP-9, TIMP-1, and cytokines, evaluated by gel-zymography or ELISA, as well as gelatinases and TIMP-1 expression in the spinal cord (SC), were determined in: i) EAE induced, ii) ES L1-treated EAE induced animals. Milder clinical signs in ES L1-treated EAE induced DA rats were accompanied with lower serum levels of MMP-9 and NGAL/MMP-9 complex. However, the correlation between the severity of EAE and the level of serum MMP-9 was found only in the peak of the disease, with MMP-9/TIMP-1 ratio higher in EAE animals without ES L1 treatment. Lower expression of MMP-9 in SC of ES L1-treated, EAE induced rats, correlated with the reduced number of SC infiltrating cells. In SC infiltrates, in the effector and the recovery phase, production of anti-inflammatory cytokines IL-4 and IL-10 was higher in animals treated with ES L1 prior to EAE induction, compared to untreated EAE animals. Reduced expression of MMP-9 in SC tissue, which correlated with the reduced number of infiltrating cells, might be ascribed to regulatory mechanisms, among which is IL-10.

Regulation of human THP-1 macrophage polarization by Trichinella spiralis

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1β and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1β and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1β, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

Trichinella spiralis muscle larvae excretory/secretory products trigger apoptosis and S-phase arrest of the non-small-cell lung cancer line A549

Trichinella spiralis (T. spiralis) muscle larvae (ML) excretory/secretory products (ESPs) are antitumor substances extracted from the culture medium of T. spiralis ML. The ESPs inhibit tumor growth and induce tumor cell apoptosis. To explore the effects of these products on the non-small-cell lung cancer (NSCLC) line A549, logarithmically growing A549 cells were co-cultured with different concentrations of T. spiralis ML ESPs for 24, 36 and 48 h. Our results showed that T. spiralis ML ESPs significantly inhibited A549 cells proliferation, which was dose-and time-dependent. To evaluate the inhibition by T. spiralis ML ESPs of the growth of A549 cells, we assayed their apoptosis and cell-cycle distribution by flow cytometry (FCM). To determine whether ESPs induced apoptosis of A549 cells via the mitochondrial pathway, we evaluated the levels of mitochondrion-related factors by Western blotting. The FCM indicated a clear trend toward apoptosis of A549 cells co-cultured with ESPs for 24 h. The cells were blocked in S-phase. Western blotting revealed that the expression levels of the genes encoding Bax, caspase-3, and caspase-9 increased (compared to a control group), and the Bcl-2 gene expression level decreased. Our results suggest that T. spiralis ML ESPs induce apoptosis of the NSCLC line A549 via the mitochondrial pathway; the cells become arrested in S-phase. This may explain the antineoplastic activity of T. spiralis ML ESPs.

NLRP3 played a role in Trichinella spiralis-triggered Th2 and regulatory T cells response

Trichinella spiralis maintains chronic infections within its host. Muscle larvae excretory-secretory products (MLES) typically induce parasite-specific immune responses such as the Th2 response and regulatory T cells (Tregs) by modulating dendritic cell (DC) phenotype via the recognition of pattern recognition receptors (PRRs), such as Nod-like receptors (NLRs). We aimed to investigate the role of NLRP3 in T. spiralis-triggered immune response. We found that larvae burden was increased in NLRP3−/− mice compared to wild type (WT) mice. Administration of MLES induced higher levels of IL-4, IL-10, TGF-β and population of Tregs in WT mice than in NLRP3−/− mice. In vitro, we showed that increased expression of CD40 on the surface of MLES-treated DCs was inhibited after NLRP3 knockout. Increased production of IL-1β, IL-18, IL-10 and TGF-β, but not IL-12p70, was significantly diminished in the absence of NLRP3. Furthermore, our results demonstrated that MLES-treated DCs induced higher levels of IL-4, IL-10 and TGF-β and populations of Tregs in vitro. These inductions were abolished by NLRP3 deficiency in DCs, suggesting that NLRP3 in MLES-treated DCs plays a role in promoting the Th2 and Treg response. Taken together, we identified for the first time the involvement of NLRP3 in host defences against T. spiralis.

Antibodies in sera from multiple sclerosis patients recognize Trichinella spiralis muscle larvae excretory-secretory antigens.

Helminths, as complex pathogens, possess a large number of different epitopes, some of which may be similar to the epitopes of the host. Besides being the cause for the activation of self-reactive immune cells, molecular mimicry may also be the cause for the expansion of regulatory T cells, crucial for the host tolerance of self-antigens. Amelioration of experimental autoimmune encephalomyelitis (EAE), animal model of multiple sclerosis (MS), caused by Trichinella spiralis infection or application of its muscle larvae excretory-secretory products (ES L1), was achieved through activation of Th2 and regulatory responses. The present study aimed to reveal whether the cause of observed immunomodulation could be the existence of shared epitopes between ES L1 antigens and auto-antigens. Serum samples from 92 MS patients were tested in Western blot for the reactivity toward components of ES L1. Immunoglobulins from the sera of MS patients recognized several ES L1 components, but 45, 49 and 58 kDa proteins dominated others by the frequency of interaction. According to the logistic regression analysis, these interactions were statistically significantly associated with MS, regardless of the disease phenotype or severity. Selected molecules might share homology with self-antigens and as such are worthy of further investigation in terms of potential immunomodulatory capacity and involvement in the parasite's provoked amelioration of EAE.

Trichinella spiralis Excretory–Secretory Products Stimulate Host Regulatory T Cell Differentiation through Activating Dendritic Cells

Trichinella spiralis maintains chronic infections within its host, involving a variety of immunomodulatory properties, the mechanisms of which have not been completely elucidated. In this study, we found that T. spiralis infection induced strong regulatory T cell responses through parasite excretory–secretory (ES) products, characterized by increase of CD4+CD25+Foxp3+ and CD4+CD25−Foxp3+ Treg cells accompanied by high levels of IL-10 and TGF-β. T. spiralis adult worm excretory–secretory products (AES) and muscle larvae excretory–secretory products (MES) were both able to activate BMDCs in vitro to facilitate their maturation and to create regulatory cytokines IL-10 and TGF-β. The T. spiralis AES- and MES-pulsed dendritic cells (DCs) possessed abilities not only to present antigens to sensitized CD4+ T cell to stimulate their proliferation but also to induce naive CD4+ T cells to differentiate to Treg cells secreting IL-10 and TGF-β. The passive transfer of T. spiralis AES- and MES-pulsed bone marrow-derived dendritic cells (BMDCs) conferred the naive mice to acquire the differentiation of Treg cells. T. spiralis AES possesses a better ability to induce Treg cells than did MES, although the latter has the ability to induce CD4+CD25−Foxp3+ Treg cells. The results obtained in this study suggested that T. spiralis ES products stimulate the differentiation of host Treg cells possibly through activating dendritic cells to create a regulatory environment that benefits the survival of the parasite in the host.

Trichinella spiralis Excretory-Secretory Products Induce Tolerogenic Properties in Human Dendritic Cells via Toll-Like Receptors 2 and 4.

Trichinella spiralis, as well as its muscle larvae excretory–secretory products (ES L1), given either alone or via dendritic cells (DCs), induce a tolerogenic immune microenvironment in inbred rodents and successfully ameliorate experimental autoimmune encephalomyelitis. ES L1 directs the immunological balance away from T helper (Th)1, toward Th2 and regulatory responses by modulating DCs phenotype. The ultimate goal of our work is to find out if it is possible to translate knowledge obtained in animal model to humans and to generate human tolerogenic DCs suitable for therapy of autoimmune diseases through stimulation with ES L1. Here, the impact of ES L1 on the activation of human monocyte-derived DCs is explored for the first time. Under the influence of ES L1, DCs acquired tolerogenic (semi-matured) phenotype, characterized by low expression of HLA-DR, CD83, and CD86 as well as moderate expression of CD40, along with the unchanged production of interleukin (IL)-12 and elevated production of IL-10 and transforming growth factor (TGF)-β, compared to controls. The interaction with DCs involved toll-like receptors (TLR) 2 and 4, and this interaction was mainly responsible for the phenotypic and functional properties of ES L1-treated DCs. Importantly, ES L1 potentiated Th2 polarizing capacity of DCs, and impaired their allo-stimulatory and Th1/Th17 polarizing properties. Moreover, ES L1-treated DCs promoted the expansion of IL-10- and TGF-β- producing CD4+CD25hiFoxp3hi T cells in indolamine 2, 3 dioxygenase (IDO)-1-dependent manner and increased the suppressive potential of the primed T cell population. ES L1-treated DCs retained the tolerogenic properties, even after the challenge with different pro-inflammatory stimuli, including those acting via TLR3 and, especially TLR4. These results suggest that the induction of tolerogenic properties of DCs through stimulation with ES L1 could represent an innovative approach for the preparation of tolerogenic DC for treatment of inflammatory and autoimmune disorders.

Serum IgE and IgG4 against muscle larva excretory-secretory products during the early and late phases of human trichinellosis.

In human trichinellosis, the relevance of the presence and persistence of specific serum IgE and IgG4 during the early and late phases of infection is still controversial.The aim of this work was to determine the percentage of human sera presenting IgE and IgG4 against Trichinella spiralis muscle larvae excretory-secretory products as well as their levels during the early and late phases of the infection. The antigen recognition pattern by serum total immunoglobulins (IgGAM), IgE, and IgG4 was assessed over time. Serum samples during early and late phases were analyzed by ELISA and immunoelectrotransfer blot (IETB).Results showed that (a)-IgE and IgG4 are present at constant levels in both phases; (b)-IgE recognized the glycoproteins of ~ 45 and ~ 55kDa and IgG4 only the ~ 45kDa;

Secretory Products of Trichinella spiralis Muscle Larvae and Immunomodulation: Implication for Autoimmune Diseases, Allergies, and Malignancies.

Trichinella spiralis has the unique ability to make itself “at home” by creating and hiding in a new type of cell in the host body that is the nurse cell. From this immunologically privileged place, the parasite orchestrates a long-lasting molecular cross talk with the host through muscle larvae excretory-secretory products (ES L1). Those products can successfully modulate parasite-specific immune responses as well as responses to unrelated antigens (either self or nonself in origin), providing an anti-inflammatory milieu and maintaining homeostasis. It is clear, based on the findings from animal model studies, that T. spiralis and its products induce an immunomodulatory network (which encompasses Th2- and Treg-type responses) that may allow the host to deal with various hyperimmune-associated disorders as well as tumor growth, although the latter still remains unclear. This review focuses on studies of the molecules released by T. spiralis, their interaction with pattern recognition receptors on antigen presenting cells, and subsequently provoked responses. This paper also addresses the immunomodulatory properties of ES L1 molecules and how the induced immunomodulation influences the course of different experimental inflammatory and malignant diseases.

Inhibition of mammalian muscle differentiation by excretory secretory products of muscle larvae of Trichinella spiralis in vitro

The excretory-secretory products (ESP) released by muscle stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ESP modulate nurse cell formation remain unclear. In the present study, the ability of ESP of muscle larvae of T. spiralis (ML-ESP) to influence the proliferation and differentiation of murine myoblasts and the mechanisms were evaluated in vitro using C2C12 myoblast cell line, which were incubated for various times under grow or differentiation culture medium containing various concentrations of ML-ESP. The results indicated that ML-ESP promoted myoblast proliferation in a dose-dependent manner and increased the expression of the cell-cycle regulator cyclin D1 as well as that of proliferating cell nuclear antigen (PCNA). Conversely, ML-ESP inhibited the differentiation of these cells, which was evidenced by a reduction in the levels of MHC and MRFs expression (MyoD and myogenin) as well as that of p21. In addition, ML-ESP also inhibited the phosphorylation of p38 MAPK in differentiating C2C12 myoblast. Taken together, these results imply that certain critical mediators contained in ML-ESP inhibit myogenesis through enhancing skeletal myoblasts proliferation and down-regulating the expression of MRFs as well as involving p38 MAPK signalling pathway, which provides insight into the mechanisms utilised by T. spiralis to interfere normal wound repair in infected muscle cells and affect nurse cell formation.

Immunoparasitological parameters of the intestinal phase of trichinellosis in rats.

Enzyme-linked immunosorbent assays (ELISAs) were developed in order to detect coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes (FIC) in rats experimentally infected with Trichinella spiralis. The usefulness of these assays was compared to that of a conventional ELISA for detection of serum antibodies (Abs) to muscle larvae excretory-secretory products (ML-ESP). The ELISA for CAgs was the first parameter to give a positive result but the detection was limited only to day 2 p.i. CAbs against ML-ESP and adult worm excretory-secretory products (AW-ESP) was first positive on day 4 p.i. Anti-ML-ESP remained positive until day 12 p.i. while CAbs against AW-ESP remained positive throughout the study period. Specific IgE and IgA were found. FIC were detected between days 2 and 8 p.i. Serum Abs began to appear on day 10 p.i. Therefore, the ELISA for CAbs was a suitable assay for the detection of the enteral and early phases of the infection.

Ultrastructure, Antigenicity, and Histochemistry of Stichocyte Granules of Adult Trichinella spiralis

The stichosome of adult Trichinella spiralis was studied to determine its ultrastructural, antigenic, and histochemical characteristics. Stichocytes of adult worms had 2 types of granules, type I and type II, the ultrastructure of which was different from those of muscle larvae. Both types of granules consisted of a membrane surrounding a homogeneous matrix, and type I granules were rounder than type II granules. Sera from C3H mice immunized against excretory-secretory products of muscle larvae produced positive immunostaining of type I but not type II granules. Differences in antigenicity were observed between larval and adult stichocyte granules; monoclonal antibodies against alpha-granules of muscle larvae failed to label the adult granules. Azan staining revealed a histochemical difference between larval and adult stichocytes; adult stichocytes stained yellow, whereas larval stichocytes are known to stain red or blue. Thus, the present contribution revealed the existence of 2 distinct types of stichocyte granules in adult T. spiralis and showed them to differ profoundly from those characterized previously in muscle larvae.

Peripheral blood mononuclear cell subsets during Trichinella spiralis infection in pigs

The immune response of 'Yugoslav meat breed' pigs inoculated with low doses of Trichinella spiralis muscle larvae was followed over two to nine weeks of primary infection, by analysing changes in peripheral blood mononuclear cell subsets, the development of a humoral antibody response and muscle larvae burden. During the course of the infection, infected animals showed a persistent elevation of both CD4+ and CD8+ T cell subsets from days 15 to 60 after the parasite exposure. During this time, the number of peripheral blood mononuclear cells expressing major histocompatibility complex class II antigens was also increased, while no significant differences were found in the number of circulating monocytes/macrophages and B cells over time. Humoral antibody responses to muscle larvae excretory-secretory products were evident as early as 41 days after infection, while the muscle larvae were recovered as early as 27 days after infection. The increased levels of CD4+ and CD8+ T cell subsets, as well as cells expressing major histocompatibility complex class II antigens in pigs exposed to T spiralis, may be indicative of some considerable alterations in cell subsets that are involved in the regulation of the swine immune response to this parasite.