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.

The long-lasting Ascaris suum antigens in the lungs shapes the tissue adaptation modifying the pulmonary architecture and immune response after infection in mice.

Ascariasis is the most prevalent helminth affecting approximately 819 million people worldwide. The acute phase of Ascariasis is characterized by larval migration of Ascaris spp., through the intestinal wall, carried to the liver and lungs of the host by the circulatory system. Most of the larvae subsequently transverse the lung parenchyma leading to tissue injury, reaching the airways and pharynx, where they can be expectorated and swallowed back to the gastrointestinal tract, where they develop into adult worms. However, some larvae are trapped in the lung parenchyma inciting an inflammatory response that causes persistent pulmonary tissue damage long after the resolution of infection, which returns to tissue homeostasis. However, the mechanism by which chronic lung disease develops and resolves remains unknown. Here, using immunohistochemistry, we demonstrate that small fragments and larval antigens of Ascaris suum are deposited and retained chronically in the lung parenchyma of mice following a single Ascaris infection. Our results reveal that the prolonged presence of Ascaris larval antigens in the lung parenchyma contributes to the persistent immune stimulation inducing histopathological changes observed chronically following infection, and clearly demonstrate that larval antigens are related to all phases of tissue adaptation after infection: lung injury, chronic inflammation, resolution, and tissue remodeling, in parallel to increased specific humoral immunity and the recovery of lung function in mice. Additional insight is needed into the mechanisms of Ascaris antigen to induce chronic immune responses and resolution in the host lungs following larval migration.

Chronic infection by atypical Toxoplasma gondii strain induces disturbance in microglia population and altered behaviour in mice.

Toxoplasma gondii chronic infection is characterized by the establishment of tissue cysts in the brain and increased levels of IFN-γ, which can lead to brain circuitry interference and consequently abnormal behaviour in mice. In this sense, the study presented here sought to investigate the impact of chronic infection by two T. gondii strains in the brain of infection-resistant mice, as a model for studying the involvement of chronic neuroinflammation with the development of behavioural alterations. For that, male BALB/c mice were divided into three groups: non-infected (Ni), infected with T. gondii ME49 clonal strain (ME49), and infected with TgCkBrRN2 atypical strain (CK2). Mice were monitored for 60 days to establish the chronic infection and then submitted to behavioural assessment. The enzyme-linked immunosorbent assay was used for measurement of specific IgG in the blood and levels of inflammatory cytokines and neurotrophic factors in the brain, and the cell's immunophenotype was determined by multiparametric flow cytometry. Mice infected with ME49 clonal strain displayed hyperlocomotor activity and memory deficit, although no signs of depressive- and/or anxiety-like behaviour were detected; on the other hand, chronic infection with CK2 atypical strain induced anxiety- and depressive-like behaviour. During chronic infection by CK2 atypical strain, mice displayed a higher number of T. gondii brain tissue cysts and inflammatory infiltrate, composed mainly of CD3+ T lymphocytes and Ly6Chi inflammatory monocytes, compared to mice infected with the ME49 clonal strain. Infected mice presented a marked decrease of microglia population compared to non-infected group. Chronic infection with CK2 strain produced elevated levels of IFN-γ and TNF-ɑ in the brain, decreased NGF levels in the prefrontal cortex and striatum, and altered levels of fractalkine (CX3CL1) in the prefrontal cortex and hippocampus. The persistent inflammation and the disturbance in the cerebral homeostasis may contribute to altered behaviour in mice, as the levels of IFN-γ were shown to be correlated with the behavioural parameters assessed here. Considering the high incidence and life-long persistence of T. gondii infection, this approach can be considered a suitable model for studying the impact of chronic infections in the brain and how it impacts in behavioural responses.

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.

Concomitant experimental coinfection by Plasmodium berghei NK65-NY and Ascaris suum downregulates the Ascaris-specific immune response and potentiates Ascaris-associated lung pathology.

BACKGROUND: Ascariasis and malaria are highly prevalent parasitic diseases in tropical regions and often have overlapping endemic areas, contributing to high morbidity and mortality rates in areas with poor sanitary conditions. Several studies have previously aimed to correlate the effects of Ascaris-Plasmodium coinfections but have obtained contradictory and inconclusive results. Therefore, the present study aimed to investigate parasitological and immunopathological aspects of the lung during murine experimental concomitant coinfection by Plasmodium berghei and Ascaris suum during larvae ascariasis. METHODS: C57BL/6J mice were inoculated with 1 × 104 P. berghei strain NK65-NY-infected red blood cells (iRBCs) intraperitoneally and/or 2500 embryonated eggs of A. suum by oral gavage. P. berghei parasitaemia, morbidity and the survival rate were assessed. On the seventh day postinfection (dpi), A. suum lung burden analysis; bronchoalveolar lavage (BAL); histopathology; NAG, MPO and EPO activity measurements; haematological analysis; and respiratory mechanics analysis were performed. The concentrations of interleukin (IL)-1ß, IL-12/IL-23p40, IL-6, IL-4, IL-33, IL-13, IL-5, IL-10, IL-17A, IFN-γ, TNF and TGF-ß were assayed by sandwich ELISA. RESULTS: Animals coinfected with P. berghei and A. suum show decreased production of type 1, 2, and 17 and regulatory cytokines; low leukocyte recruitment in the tissue; increased cellularity in the circulation; and low levels of NAG, MPO and EPO activity that lead to an increase in larvae migration, as shown by the decrease in larvae recovered in the lung parenchyma and increase in larvae recovered in the airway. This situation leads to severe airway haemorrhage and, consequently, an impairment respiratory function that leads to high morbidity and early mortality. CONCLUSIONS: This study demonstrates that the Ascaris-Plasmodium interaction is harmful to the host and suggests that this coinfection may potentiate Ascaris-associated pathology by dampening the Ascaris-specific immune response, resulting in the early death of affected animals.

Detrimental role of IL-33/ST2 pathway sustaining a chronic eosinophil-dependent Th2 inflammatory response, tissue damage and parasite burden during Toxocara canis infection in mice.

Toxocariasis is a neglected disease that affects people around the world. Humans become infected by accidental ingestion of eggs containing Toxocara canis infective larvae, which upon reaching the intestine, hatch, penetrate the mucosa and migrate to various tissues such as liver, lungs and brain. Studies have indicated that Th2 response is the main immune defense mechanism against toxocariasis, however, there are still few studies related to this response, mainly the IL-33/ST2 pathway. Some studies have reported an increase in IL-33 during helminth infections, including T. canis. By binding to its ST2 receptor, IL-33 stimulating the Th2 polarized immune cell and cytokine responses. Thus, we aimed to investigate the role of the IL-33/ST2 pathway in the context of T. canis larval migration and the immunological and pathophysiological aspects of the infection in the liver, lungs and brain from Wild-Type (WT) BALB/c background and genetically deficient mice for the ST2 receptor (ST2-/-). The most important findings revealed that the IL-33/ST2 pathway is involved in eosinophilia, hepatic and cerebral parasitic burden, and induces the formation of granulomas related to tissue damage and pulmonary dysfunction. However, ST2-/- mice, the immune response was skewed to Th1/Th17 type than Th2, that enhanced the control of parasite burden related to IgG2a levels, tissue macrophages infiltration and reduced lung dysfunction. Collectively, our results demonstrate that the Th2 immune response triggered by IL-33/ST2 pathway mediates susceptibility to T. canis, related to parasitic burden, eosinophilia and granuloma formation in which consequently contributes to tissue inflammation and injury.

Immunological underpinnings of Ascaris infection, reinfection and co-infection and their associated co-morbidities.

Human ascariasis is the most common and prevalent neglected tropical disease and is estimated that ~819 million people are infected around the globe, accounting for 0.861 million years of disability-adjusted life years in 2017. Even with the existence of highly effective drugs, the constant presence of infective parasite eggs in the environment contribute to a high reinfection rate after treatment. Due to its high prevalence and broad geographic distribution Ascaris infection is associated with a variety of co-morbidities and co-infections. Here, we provide data from both experimental models and humans studies that illustrate how complex is the interaction of Ascaris with the host immune system, especially, in the context of reinfections, co-infections and associated co-morbidities.