Ana M. Espino

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by dysbiosis of the gut microbiota. Helminth infections are known to modulate host immunity and intestinal microbial composition; however, the therapeutic use of live parasites poses safety challenges. The recombinant Fasciola hepatica fatty acid-binding protein Fh15 is a helminth-derived molecule with anti-inflammatory effects in models of septic shock and dextran sulfate sodium (DSS)-induced colitis. Whether Fh15 also influences gut microbial composition during colitis remains unknown. Male C57BL/6 mice received 4% DSS in drinking water for 7 days to induce colitis and were treated intraperitoneally with Fh15 (2 mg/kg) on days 1, 3, and 5. Fecal samples were collected on days 2, 4, and 7 for 16S rRNA gene sequencing. Standard microbiota pipelines were used to evaluate community diversity. Acute DSS treatment disrupted gut microbial diversity and community structure compared with non-colitic controls. Fh15 treatment partially restored early microbial balance by shifting microbial composition toward that of healthy mice and reducing microbial dispersion, indicating enhanced community stability despite severe dysbiosis. Although alpha diversity did not return to control levels, Fh15 mitigated the expansion of pro-inflammatory genera (Enterococcus and Turicibacter) and preserved beneficial taxa, including Adlercreutzia.

Sepsis caused by Gram-negative bacteria is a severe systemic infection characterized by an excessive inflammatory response triggered by lipopolysaccharide (LPS), a potent endotoxin of these bacteria, often leading to organ failure and high mortality. Despite improved outcomes with antibiotic therapy, controlling persistent inflammation remains a major therapeutic challenge. Interestingly, helminth-derived molecules, such as the recombinant protein Fh15 from Fasciola hepatica , have been demonstrated to suppress inflammatory responses in experimental models of sepsis. This study aimed to evaluate the capacity of Fh15 to effectively attenuate cytokine storms and improve survival in mice, alongside analyzing its effects on macrophage phenotype and function to better understand its potential as an anti-inflammatory biotherapeutic. Our results demonstrate that a single intraperitoneal injection of an optimized dose of 100 μg Fh15 (~5 mg/kg body weight), administered 1 h after a lethal (LD100) LPS challenge, significantly improved survival and markedly suppressed the cytokine storm in mice. This treatment was associated with a substantial reduction in C-reactive protein (CRP) levels, along with an increase in IL-37. In addition, a 100 μg Fh15 dose (~5 mg/kg body weight [BW]) effectively controlled LPS-induced IL-10 overproduction, potentially preventing immunosuppression that may contribute to mortality. Fh15 also significantly reduced the expression of macrophage activation markers, including CD14, tumor necrosis factor-alpha (TNF-α), nitric oxide synthase-2 (NOS2), CD38, and IL-12p70, potentially impairing macrophage maturation, phagocytic activity, and their capacity to activate Th1 cells. These findings demonstrate the potential of Fh15 to modulate key inflammatory pathways involved in sepsis and support its potential as a novel recombinant protein-based anti-inflammatory therapy. IMPORTANCE: Sepsis remains a leading cause of mortality worldwide, primarily driven by an overwhelming inflammatory response triggered by bacterial components such as lipopolysaccharide (LPS). This excessive immune activation, characterized by cytokine storms and macrophage overactivation, leads to organ failure and death. Current therapeutic strategies, including antibiotic treatment, often fail to address the persistent inflammation that contributes to sepsis severity. In this study, we proposed Fh15, a recombinant protein derived from Fasciola hepatica , as a potent immunomodulatory agent capable of attenuating the cytokine storm, improving survival, and modulating macrophage activity in a mouse model of septic shock. These findings underscore the potential of Fh15 as a novel anti-inflammatory agent, offering a promising therapeutic approach to manage endotoxemia and reduce sepsis-related mortality.

There is a present need to develop alternative biotherapeutic drugs to mitigate the exacerbated inflammatory immune responses characteristic of sepsis. The potent endotoxin lipopolysaccharide (LPS), a major component of Gram-negative bacterial outer membrane, activates the immune system via Toll-like receptor 4 (TLR4), triggering macrophages and a persistent cascade of inflammatory mediators. Our previous studies have demonstrated that Fh15, a recombinant member of the Fasciola hepatica fatty acid binding protein family, can significantly increase the survival rate by suppressing many inflammatory mediators induced by LPS in a septic shock mouse model. Although Fh15 has been proposed as a TLR4 antagonist, the specific mechanisms underlying its immunomodulatory effect remained unclear. In the present study, we employed a quantitative proteomics approach using tandem mass tag (TMT) followed by LC-MS/MS analysis to identify and quantify differentially expressed proteins that participate in signaling pathways downstream TLR4 of macrophages, which can be dysregulated by Fh15. Data are available via ProteomeXchange with identifier PXD065520. Based on significant fold change (FC) cut-off of 1.5 and p-value ≤ 0.05 criteria, we focused our attention to 114 proteins that were upregulated by LPS and downregulated by Fh15. From these proteins, TNFα, IL-1α, Lck, NOS2, SOD2 and CD36 were selected for validation by Western blot on murine bone marrow-derived macrophages due to their relevant roles in the NF-κB, iNOS, oxidative stress, and phagosome signaling pathways, which are closely associated with sepsis pathogenesis. These results suggest that Fh15 exerts a broad spectrum of action by simultaneously targeting multiple downstream pathways activated by TLR4, thereby modulating various aspects of the inflammatory responses during sepsis.

There is a present need to develop alternative biotherapeutic drugs to mitigate the exacerbated inflammatory immune responses characteristic of sepsis. The potent endotoxin lipopolysaccharide (LPS), a major component of Gram-negative bacterial outer membrane, activates the immune system via Toll-like receptor 4 (TLR4), triggering macrophages and a persistent cascade of inflammatory mediators. Our previous studies have demonstrated that Fh15, a recombinant member of the Fasciola hepatica fatty acid binding protein family, can significantly increase the survival rate by suppressing many inflammatory mediators induced by LPS in a septic shock mouse model. Although Fh15 has been proposed as a TLR4 antagonist, the specific mechanisms underlying its immunomodulatory effect remained unclear. In the present study we employed a quantitative proteomics approach using tandem mass tag (TMT) followed by LC-MS/MS analysis to identify and quantify differentially expressed proteins that participate in signaling pathways downstream TLR4 of macrophages, which can be dysregulated by Fh15. Based on significant fold change (FC) cutoff of 1.5 and p-value ≤ 0.05 criteria, we focused our attention to 114 proteins that were upregulated by LPS and downregulated by Fh15. From these proteins, TNFα, IL-1α, Lck, NOS2, SOD2 and CD36 were selected for validation by Western blot on murine bone marrow derived macrophages due to their relevant roles in the NF-κB, iNOS, oxidative stress, and phagosome signaling pathways, which are closely associated to sepsis pathogenesis. These results suggest that Fh15 exerts a broad spectrum of action by simultaneously targeting multiple downstream pathways activated by TLR4, thereby modulating various aspects of the inflammatory responses during sepsis.

Ulcerative colitis (UC) is the most prevalent inflammatory bowel disease (IBD) in the USA. Current treatments present clinical limitations, underscoring the need for innovative therapeutics that promote an anti-inflammatory immune response. This study evaluates the anti-inflammatory potential of Fh15, a recombinant Fasciola hepatica fatty acid binding protein, in a DSS-induced UC mouse model. Our results demonstrated that Fh15 treatment significantly ameliorated the severity of colitis by reducing the disease activity index (DAI) and histopathological scores. Moreover, Fh15 also decreased the serum levels of myeloperoxidase (MPO) and chitinase-3-like protein 1 (CHI3L1), and the expression of S100A9, a calcium and zinc binding protein, which is an important marker for the pathogenesis of UC. Furthermore, Fh15 downregulated pro-inflammatory cytokines TNFα and IL-1β in the distal colon, suggesting modulation of macrophage activity. Immunohistochemistry analysis revealed significantly reduced neutrophil and macrophage infiltration in UC Fh15-treated mice. These findings highlight the therapeutic potential of Fh15 for UC, as it modulates inflammatory responses, reduces leukocyte infiltration, and preserves colon integrity.

Background/Objectives: In the past, FhSAP-2, an 11.5 kDa recombinant protein belonging to the Fasciola hepatica saposin-like/NK-lysin family, has been shown to induce over 60% partial protection in immunized rabbits and mice when challenged with F. hepatica metacercariae. However, despite FhSAP-2 being a promising vaccine candidate, its hydrophobic nature has made its purification a challenging process. The present study aimed to determine whether a modified 9.8 kDa variant of protein (mFhSAP-2), lacking a string of 16 hydrophobic amino acids at the amino terminus and a dominant Th1 epitope, could retain its immunogenic and Th1-inducing properties. Methods: RAW264.7 cells were stimulated with mFhSAP-2, and TNFα levels were determined. C57BL/6 mice were immunized with mFhSAP-2 alone or emulsified with Montanide ISA50. Total anti-mFhSAP-2 IgG subtypes, along with their avidity and titers, were measured using ELISA. The T cell proliferation index and levels of CD4+/CD8+ and IFNγ/IL-4 ratios were determined. Results: In vitro, mFhSAP-2 induced dose-dependent TNFα production in RAW264.7 cells. In vivo, mice immunized with mFhSAP-2 or mFhSAP-2+ISA50 developed high-avidity IgG2a and IgG2c antibodies at levels that were significantly higher than IgG1 antibody levels. However, the mFhSAP-2+ISA50 formulation induced higher and more homogenous antibody titers than mFhSAP-2, suggesting that an adjuvant may be required to enhance mFhSAP-2 immunogenicity. Immunization with mFhSAP-2+ISA50 also induced significantly higher activated CD4+/CD8+ T cell ratios and IFNγ/IL-4 ratios compared to naïve mice. Conclusions: Our results demonstrate that mFhSAP-2 retained its immunogenicity and Th1-polarizing properties, which were enhanced by the Montanide ISA50 adjuvant. The present study highlights the feasibility of inducing Th1-associated immune responses in mice using mFhSAP-2 as an antigen. Further studies are required to assess the potential application of the mFhSAP-2+ISA50 formulation as a vaccine against F. hepatica in natural hosts such as cattle and sheep, which could contribute to improved control and aid in the prevention and eradication of F. hepatica infection.

Background/Objectives: FhSAP-2 is a 11.5kDa recombinant protein belonging to the Fasciola hepatica saposin-like/NK-lysin protein family. It has shown to induce partial protection >60% (reduction in parasite burden and parasite egg output) against F. hepatica challenge infection when delivered subcutaneously in rabbits and mice, either emulsified in Freund’s adjuvant or as inclusion bodies. The protection induced by FhSAP-2 was associated with increased levels of IgG2a and IFNγ. However, despite FhSAP-2 being a promising vaccine candidate, its hydrophobic character has made its purification a challenged process. The present study aimed to determine whether a modified 9.8kDa variant of protein (mFhSAP-2), lacking the string of 15 hydrophobic amino acids at amino terminus containing a dominant Th1-epitope, could retain its immunogenic and Th1-inducing properties. Methods: RAW264.7 cells were stimulated with mFhSAP-2, and levels of TNFα were determined. C57BL6 mice were immunized with mFhSAP-2 alone or emulsified with Montanide ISA50. Levels of each specific anti-mFhSAP-2 IgG subtypes, their avidity and titers were measured by ELISA. T-cell proliferation index as well as levels of CD4+/CD8+ and IFNγ /IL-4 ratios were determined. Results: In vitro, mFhSAP-2 induced dose-dependent TNFα production in RAW264.7 cells. In vivo, mice immunized with mFhSAP-2 alone induced 1.76-fold higher than IgG2a and 1.28-fold higher than IgG1, whereas mice immunized with mFhSAP-2+ISA50 developed 2.16-fold more IgG2c than IgG2a and 1.49-fold more IgG2c than IgG1. All mice developed high-avidity IgG2a and IgG2c antibodies. Mice immunized with mFhSAP-2+ISA50 developed higher IgG2a and IgG2c titers (1:6,400 and 1:25,600, respectively) than those immunized with mFhSAP-2 alone (1:1,600 and 1:3,200, respectively). Immunization with mFhSAP-2+ISA50 also induced significantly high activated CD4+/CD8+ T-cells and IFNγ/IL-4 ratios. Conclusions: Our results showed that mFhSAP-2 retained its immunogenicity and Th1-polarizing properties, which were enhanced by Montanide ISA-50 adjuvant.

Ulcerative colitis (UC) is the most prevalent inflammatory bowel disease (IBD) in the USA. Current treatments present clinical limitations, underscoring the need for innovative therapeutics that promote an anti-inflammatory immune response. This study evaluates the anti-inflammatory potential of Fh15, a recombinant Fasciola hepatica fatty acid binding protein in a DSS-induced UC mouse model. Our results demonstrated that Fh15 treatment significantly ameliorated the severity of colitis by reducing the disease activity index (DAI) and histopathological scores. Moreover, Fh15 also decreased serum levels of myeloperoxidase (MPO), and chitinase-3-like protein 1 (CHI3L1), and the expression of S100A9, a calcium-and zinc binding protein, which is an important marker for the pathogenesis of UC. Furthermore, Fh15 downregulated pro-inflammatory cytokines TNFα and IL-1β in the distal colon, suggesting modulation of macrophage activity. Immunohistochemistry analysis revealed significantly reduced neutrophil and macrophage infiltration in UC Fh15-treated mice. These findings highlight the therapeutic potential of Fh15 for UC, as it modulates inflammatory responses, reduces leukocyte infiltration, and preserves colon integrity.

The coronavirus SARS-CoV-2 is the causative agent for the COVID-19 first registered in Wuhan, China and responsible for more than 6 million deaths worldwide. Currently, RT-PCR is the gold standard method for diagnosing COVID-19. However, serological tests are needed for screening acute disease diagnosis and screening large populations during the COVID-19 outbreak. Herein we described the development of an in-house enzyme-linked immunosorbent assay (ELISA) for detecting levels of anti-Spike-1-RBD IgM antibody in well-defined serum/plasma panel for screening and identifying subjects infected with SARS-CoV-2 in a Latin population. We found that, compared to the RT-PCR as standard method, the in-house CovIgM-ELISA displayed sensitivities of 96.15% and 93.22% for samples collected up to 30 or 60 days after infection, respectively, as well as 95.59% specificity with 97.3% accuracy. The agreement kappa value (κ) of our CovIgM-ELISA was substantial when compared to RT-PCR (κ =0.873) and to SCoV-2 DetectTM IgM ELISA (κ = 0.684). The IgM levels detected in the population positively correlate with the neutralizing activity against the Wild-type, Alpha and Delta variants but failed to neutralize Omicron. These data indicate that our in-house CovIgM-ELISA is a compatible performing assay for the detection of SARS-CoV-2 infection.