Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in different tissues from mice infected by Ascaris suum.

Human ascariasis is the most prevalent helminth infection, affecting 445 million people worldwide. To better understand the impact of the immune system on the pathophysiology of individuals infected with Ascaris suum, mice have been used as experimental models. The RT-qPCR technique is a critical auxiliary tool of investigation used to quantify mRNA levels. However, proper normalization using reference genes is essential to ensure reliable outcomes to avoid analytical errors and false results. Despite the importance of reference genes for experimental A. suum infection studies, no specific reference genes have been identified yet. Therefore, we conducted a study to assess five potential reference genes (GAPDH, 18s, ACTB, B2M, and HPRT1) in different tissues (liver, lungs, small and large intestines) affected by A. suum larval migration in C57BL/6j mice. Tissue collection was carried out to analyze parasite burden and confirm the presence of larvae during the peak of migration in each tissue. Upon confirmation, we analyzed different genes in the tissues and found no common gene with stable expression. Our results highlight the importance of analyzing different genes and using different software programs to ensure reliable relative expression results. Based on our findings, B2M was ranked as the ideal reference gene for the liver, while 18S was the most stable gene in the lung and small intestine. ACTB, or a combination of ACTB with GAPDH, was deemed suitable as reference genes for the large intestine due to their stable expression and less variation between the control and infected groups. To further demonstrate the impact of using different reference genes, we normalized the expression of a chemokine gene (CXCL9) in all tissues. Significant differences in CXCL9 expression levels were observed between different groups in all tissues except for the large intestine. This underscores the importance of selecting appropriate reference genes to avoid overestimating target gene expression levels and encountering normalization-related issues that can lead to false results. In conclusion, our study highlights the significance of using reliable reference genes for accurate RT-qPCR analysis, especially in the context of A. suum infection studies in different tissues. Proper normalization is crucial to ensure the validity of gene expression data and avoid potential pitfalls in interpreting results.

IL-17RA receptor signaling contributes to lung inflammation and parasite burden during Toxocara canis infection in mice.

IL-17 is a cytokine produced by innate and acquired immunity cells that have an action against fungi and bacteria. However, its action in helminth infections is unclear, including in Toxocara canis infection. Toxocariasis is a neglected zoonosis representing a significant public health problem with an estimated seroprevalence of 19% worldwide. In the present study, we describe the immunopathological action of IL-17RA in acute T. canis infection. C57BL/6j (WT) and IL-17RA receptor knockout (IL-17RA-/-) mice were infected with 1000 T. canis eggs. Mice were evaluated 3 days post-infection for parasite load and white blood cell count. Lung tissue was harvested for histopathology and cytokine expression. In addition, we performed multiparametric flow cytometry in the BAL and peripheral blood, evaluating phenotypic and functional changes in myeloid and lymphoid populations. We showed that IL-17RA is essential to control larvae load in the lung; however, IL-17RA contributed to pulmonary inflammation, inducing inflammatory nodular aggregates formation and presented higher pulmonary IL-6 levels. The absence of IL-17RA was associated with a higher frequency of neutrophils as a source of IL-4 in BAL, while in the presence of IL-17RA, mice display a higher frequency of alveolar macrophages expressing the same cytokine. Taken together, this study indicates that neutrophils may be an important source of IL-4 in the lungs during T. canis infection. Furthermore, IL-17/IL-17RA axis is important to control parasite load, however, its presence triggers lung inflammation that can lead to tissue damage.

Genetic Background Affects the Mucosal Secretory IgA Levels, Parasite Burden, Lung Inflammation, and Mouse Susceptibility to Ascaris suum Infection.

Ascariasis is a neglected tropical disease that is widespread in the world and has important socioeconomic impacts. The presence of various stages of worm development in the pulmonary and intestinal mucosae induces a humoral and cellular immune response. However, although there is much evidence of the protective role of mucosal immunity against various pathogens, including helminths, there is still a gap in the knowledge about the immune response and the mechanisms of action that are involved in protection against diseases, especially in the initial phase of ascariasis. Thus, the aim of this study was to evaluate the kinetic aspects of the immune parasitological parameters in intestinal and pulmonary mucosae in male mice with early ascariasis. Therefore, two mouse strains that showed different susceptibilities to ascariasis (BALB/c and C57BL/6J) when experimentally infected with 2,500 infective eggs of Ascaris suum from time point 0 were examined: the immune parasitological parameters were evaluated each 2 days after infection over a period of 12 days. The results were suggestive of a synergetic action of intestinal and pulmonary secretory IgA (S-IgA) contributing to protection against early ascariasis by reducing the amount of migrating larvae as well as the influx of leukocytes in the lung and the consequent impairment of pulmonary capacity.

Eosinophils mediate SIgA production triggered by TLR2 and TLR4 to control Ascaris suum infection in mice.

Human ascariasis is the most prevalent but neglected tropical disease in the world, affecting approximately 450 million people. The initial phase of Ascaris infection is marked by larval migration from the host's organs, causing mechanical injuries followed by an intense local inflammatory response, which is characterized mainly by neutrophil and eosinophil infiltration, especially in the lungs. During the pulmonary phase, the lesions induced by larval migration and excessive immune responses contribute to tissue remodeling marked by fibrosis and lung dysfunction. In this study, we investigated the relationship between SIgA levels and eosinophils. We found that TLR2 and TLR4 signaling induces eosinophils and promotes SIgA production during Ascaris suum infection. Therefore, control of parasite burden during the pulmonary phase of ascariasis involves eosinophil influx and subsequent promotion of SIgA levels. In addition, we also demonstrate that eosinophils also participate in the process of tissue remodeling after lung injury caused by larval migration, contributing to pulmonary fibrosis and dysfunction in re-infected mice. In conclusion, we postulate that eosinophils play a central role in mediating host innate and humoral immune responses by controlling parasite burden, tissue inflammation, and remodeling during Ascaris suum infection. Furthermore, we suggest that the use of probiotics can induce eosinophilia and SIgA production and contribute to controlling parasite burden and morbidity of helminthic diseases with pulmonary cycles.

ASCVac-1, a Multi-Peptide Chimeric Vaccine, Protects Mice Against Ascaris suum Infection.

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

Comorbidity associated to Ascaris suum infection during pulmonary fibrosis exacerbates chronic lung and liver inflammation and dysfunction but not affect the parasite cycle in mice.

Ascariasis is considered the most neglected tropical disease, and is a major problem for the public health system. However, idiopathic pulmonary fibrosis (IPF) is a result of chronic extracellular deposition of matrix in the pulmonary parenchyma, and thickening of the alveolar septa, which reduces alveolar gas exchange. Considering the high rates of ascariasis and pulmonary fibrosis, we believe that these two diseases may co-exist and possibly lead to comorbidities. We therefore investigated the mechanisms involved in comorbidity of Ascaris suum (A. suum) infection, which could interfere with the progression of pulmonary fibrosis. In addition, we evaluated whether a previous lung fibrosis could interfere with the pulmonary cycle of A. suum in mice. The most important findings related to comorbidity in which A. suum infection exacerbated pulmonary and liver injury, inflammation and dysfunction, but did not promote excessive fibrosis in mice during the investigated comorbidity period. Interestingly, we found that pulmonary fibrosis did not alter the parasite cycle that transmigrated preferentially through preserved but not fibrotic areas of the lungs. Collectively, our results demonstrate that A. suum infection leads to comorbidity, and contributes to the aggravation of pulmonary dysfunction during pulmonary fibrosis, which also leads to significant liver injury and inflammation, without changing the A. suum cycle in the lungs.

IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection.

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p < 0.001), CUT 59% (p < 0.001), and ExAD 51% (p < 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity.

Multiple Exposures to Ascaris suum Induce Tissue Injury and Mixed Th2/Th17 Immune Response in Mice.

Ascaris spp. infection affects 800 million people worldwide, and half of the world population is currently at risk of infection. Recurrent reinfection in humans is mostly due to the simplicity of the parasite life cycle, but the impact of multiple exposures to the biology of the infection and the consequences to the host's homeostasis are poorly understood. In this context, single and multiple exposures in mice were performed in order to characterize the parasitological, histopathological, tissue functional and immunological aspects of experimental larval ascariasis. The most important findings revealed that reinfected mice presented a significant reduction of parasite burden in the lung and an increase in the cellularity in the bronchoalveolar lavage (BAL) associated with a robust granulocytic pulmonary inflammation, leading to a severe impairment of respiratory function. Moreover, the multiple exposures to Ascaris elicited an increased number of circulating inflammatory cells as well as production of higher levels of systemic cytokines, mainly IL-4, IL-5, IL-6, IL-10, IL-17A and TNF-α when compared to single-infected animals. Taken together, our results suggest the intense pulmonary inflammation associated with a polarized systemic Th2/Th17 immune response are crucial to control larval migration after multiple exposures to Ascaris.