Fasciola hepatica GST mu-class suppresses the cytokine storm induced by E. coli-lipopolysaccharide, whereas it modulates the dynamic of peritoneal macrophages in a mouse model and suppresses the classical activation of macrophages.

IMPORTANCE: Sepsis is the consequence of a systemic bacterial infection that exacerbates the immune cell's activation via bacterial products, resulting in the augmented release of inflammatory mediators. A critical factor in the pathogenesis of sepsis is the primary component of the outer membrane of Gram-negative bacteria known as lipopolysaccharide (LPS), which is sensed by TLR4. For this reason, scientists have aimed to develop antagonists able to block TLR4 and, thereby the cytokine storm. We report here that a mixture of mu-class isoforms from the F. hepatica GST protein family administered intraperitoneally 1 h prior to a lethal LPS injection can modulate the dynamics and abundance of large peritoneal macrophages in the peritoneal cavity of septic mice while significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock. These results suggest that native F. hepatica glutathione S-transferase is a promising candidate for drug development against endotoxemia and other inflammatory diseases.

Fasciola hepatica GST mu-class suppresses the cytokine storm induced by E. coli -lipopolysaccharide whereas modulates the dynamic of peritoneal macrophages in a mouse model and suppresses the classical activation of macrophages.

The helminth Fasciola hepatica is known as a master of immunomodulation. It suppresses antigen specific Th1 responses in concurrent bacterial infections while promoting the Th2/Treg regulatory responses, thus demonstrating its anti-inflammatory ability in vivo . We have recently demonstrated that a single intraperitoneal injection with native F. hepatica Glutathione S -Transferase (nFhGST), mostly comprised of mu-class isoforms, can suppresses the cytokine storm and increasing the survival rate in a mouse model of septic shock (1). Knowing that the peritoneal macrophages in response to microbial stimuli play essential roles in the defense, tissue repairment, and maintenance of homeostasis, the present study aimed to determine whether nFhGST could modulate the amount and dynamic of these cells concurrently to the suppression of pro-inflammatory cytokines. The remarkable findings described in this article are, (i) nFhGST suppresses serum IL-12, TNF-α, and IFN-γ in BALB/c mice challenged with a lethal dose of LPS, (ii) Although nFhGST does not elicit IL-10, it was able to significantly suppress the high levels of LPS-induced IL-10, which is considered a key cytokine in the pathophysiology of sepsis (2). iii) nFhGST prevent the disappearance of large peritoneal macrophages (LPM) whereas significantly increasing this population in the peritoneal cavity (PerC) of LPS treated animals, (iv) nFhGST promotes the alternative activation of macrophages whereas suppress the classical activation of macrophages in vitro by expressing high levels of Ym-1, a typical M2-type marker, secreting the production of IL-37, and preventing the production of TNF-α, iNOS2 and nitric oxide, which are typical markers of M1-type macrophages, (v) nFhGST suppress the bacterial phagocytosis of macrophages, a role that plays both, M1-and M2-macrophages, thus partially affecting the capacity of macrophages in destroying microbial pathogens. These findings present the first evidence that nFhGST is an excellent modulator of the PerC content in vivo, reinforcing the capacity of nFhGST as an anti-inflammatory drug against sepsis in animal models. Importance: Sepsis is an infection that can lead to a life-threatening complication. Sepsis is the consequence of a systemic bacterial infection that exacerbates the immune cells' activation by bacterial products, resulting in the augmented release of inflammatory mediators. A critical factor in the pathogenesis of sepsis is the primary component of the outer membrane of Gram-negative bacteria known as lipopolysaccharide (LPS), which is sensed by toll-like receptor 4 (TLR4). For this reason, scientists aimed to develop antagonists able to block the cytokine storm by blocking TLR4. We report here that a mixture of mu-class isoforms from the F. hepatica glutathione S-transferase (nFhGST) protein family administered intraperitoneally 1 h after a lethal LPS injection, is capable of significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock whereas modulate the dynamic and abundance of large peritoneal macrophages in the peritoneal cavity of septic mice. These results suggest that nFhGST is a prominent candidate for drug development against endotoxemia and other inflammatory diseases.

TSLP as a Potential Therapy in the Treatment of CRLF2 B Cell Acute Lymphoblastic Leukemia.

Cytokine receptor-like factor 2 B-cell acute lymphoblastic leukemia (CRLF2 B-ALL) is a high-risk subtype characterized by CRLF2 overexpression with poor survival rates in children and adults. CRLF2 and interleukin-7 receptor alpha (IL-7Rα) form a receptor for the cytokine thymic stromal lymphopoietin (TSLP), which induces JAK/STAT and PI3K/AKT/mTOR pathway signals. Previous studies from our group showed that low TSLP doses increased STAT5, AKT, and S6 phosphorylation and contributed to CRLF2 B-ALL cell survival. Here we investigated the role of TSLP in the survival and proliferation of CRLF2 B-ALL cells in vitro and in vivo. We hypothesized that high doses of TSLP increase CRLF2 signals and contribute to increased proliferation of CRLF2 B-ALL cells in vitro and in vivo. Interestingly, we observed the opposite effect. Specifically, high doses of TSLP induced apoptosis in human CRLF2 B-ALL cell lines in vitro, prevented engraftment of CRLF2 B-ALL cells, and prolonged the survival of +TSLP patient-derived-xenograft mice. Mechanistically, we showed that high doses of TSLP induced loss of its receptor and loss of CRLF2 signals in vitro. These results suggest that high doses of TSLP could be further investigated as a potential therapy for the treatment of CRLF2 B-ALL.

Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells.

In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1ß, tumor necrosis factor (TNF)-α and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naïve T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFN-γ production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.

Fasciola hepatica GST downregulates NF-κB pathway effectors and inflammatory cytokines while promoting survival in a mouse septic shock model.

Parasitic helminths and helminth-derived molecules have demonstrated to possess powerful anti-inflammatory properties and confirmed therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress specific Th1 specific immune responses induced by concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. In this study, we demonstrate that native F. hepatica glutathione S-transferase (nFhGST), a major parasite excretory-secretory antigen, majorly comprised of Mu-class GST isoforms, significantly suppresses the LPS-induced TNFα and IL1ß of mouse bone-marrow derived macrophages in vitro and the pro-inflammatory cytokine/chemokine storm within C57BL/6 mice exposed to lethal doses of LPS increasing their survival rate by more than 85%. Using THP1-Blue CD14 cells, a human monocyte cell line, we also demonstrate that nFhGST suppresses NF-κB activation in response to multiple TLR-ligands, including whole bacteria clinical isolates and this suppression was found to be dose-dependent and independent of the timing of exposure. Moreover, the suppressive effect of nFhGST on NF-κB activation was shown to be independent of enzyme activity or secondary structure of protein. As part of its anti-inflammatory effect nFhGST target multiple proteins of the canonic and non-canonic NF-κB signaling pathway as well as also JAK/STAT pathway. Overall, our results demonstrate the potent anti-inflammatory properties of nFhGST and its therapeutic potential as an anti-inflammatory agent.

Fh15 Blocks the Lipopolysaccharide-Induced Cytokine Storm While Modulating Peritoneal Macrophage Migration and CD38 Expression within Spleen Macrophages in a Mouse Model of Septic Shock.

Sepsis caused by Gram-negative bacteria is the consequence of an unrestrained infection that continuously releases lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to multiorgan failure and death. After scrutinizing the immune modulation exerted by a recombinant Fasciola hepatica fatty acid binding protein termed Fh15, our group demonstrated that addition of Fh15 to murine macrophages 1 h prior to LPS stimulation significantly suppresses the expression of proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL1-ß). The present study aimed to demonstrate that Fh15 could exert a similar anti-inflammatory effect in vivo using a mouse model of septic shock. Among the novel findings reported in this article, (i) Fh15 suppressed numerous serum proinflammatory cytokines/chemokines when injected intraperitoneally 1 h after exposure of animals to lethal doses of LPS, (ii) concurrently, Fh15 increased the population of large peritoneal macrophages (LPMs) in the peritoneal cavity (PerC) of LPS-injected animals, and (iii) Fh15 downregulated the expression on spleen macrophages of CD38, a cell surface ectoenzyme with a critical role during inflammation. These findings present the first evidence that the recombinant parasitic antigen Fh15 is an excellent modulator of the PerC cell content and in vivo macrophage activation, endorsing Fh15's potential as a drug candidate against sepsis-related inflammatory response.IMPORTANCE Sepsis is a potentially life-threatening complication of an infection. Sepsis is mostly the consequence of systemic bacterial infections leading to exacerbated activation of immune cells by bacterial products, resulting in enhanced release of inflammatory mediators. Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is a critical factor in the pathogenesis of sepsis, which is sensed by Toll-like receptor 4 (TLR4). The scientific community highly pursues the development of antagonists capable of blocking the cytokine storm by blocking TLR4. We report here that a recombinant molecule of 14.5 kDa belonging to the Fasciola hepatica fatty acid binding protein (Fh15) is capable of significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock when administered by the intraperitoneal route 1 h after a lethal LPS injection. These results suggest that Fh15 is an excellent candidate for drug development against endotoxemia.

Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2.

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500 µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

Recombinant Fasciola hepatica fatty acid binding protein suppresses toll-like receptor stimulation in response to multiple bacterial ligands.

Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1ß and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.

Development of two antibody detection enzyme-linked immunosorbent assays for serodiagnosis of human chronic fascioliasis.

Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories.