Aspectos fisiopatológicos da inflamação crônica na infecção por Ascaris suum e o impacto na progressão do carcinoma de mama murino 4T1

Abstract

Infections by some helminths are among the risk factors for some cancers. Besides them being in constant contact with society due to socioeconomic and environmental conditions, these agents modulate the immune response and can enrich the tumor microenvironment. Ascaris spp., for example, is the most prevalent helminth in the world, but little is known about the effects of long term inflammation caused by this parasite and its impact on the progression of some cancers. In this study, we investigated the relationship between infection by A. suum and the impact on the progression of breast carcinoma (the most prevalent globally) in the 4T1 model in BALB/c mice, including respective metastases in the liver and lung. Experiments were standardized to validate reference genes for qPCR analyses in the infection model and in the 4T1 breast carcinoma model. In addition, we also standardized the use of auxiliary methodologies, such as the use of radioisotopes to monitor metastases, to be used in this study and in others involving the model. We also characterized the damage and the immune response profile present in animals infected up to 100 days post infection (dpi) with the parasite with different doses and frequencies of exposure to try to mimic the real infection scenario. For this, molecular biology techniques, cellular activity assays, and histopathological analyses were incorporated into this study after standardizations were performed. Our main results highlight that even in the absence of larvae, at 100 dpi by A. suum it is possible to visualize persistent damage to the lung and liver together with the predominance of inflammatory cytokines, especially IFN-γ, TNF, IL-5, IL-6, IL-10, IL-13, and TGF-β. High doses of infection caused more intense damage, but with faster repair, while lower doses, which better mimic the natural infection scenario, despite less damage led to slower repair mechanisms such as increased TGF-β expression. In addition, we observed that even at this time, the animals presented chronic lung disease, similar to Chronic Obstructive Pulmonary Disease (COPD) and liver disease suggestive of cholestatic disease. In both tissues, there was also the persistence of extensive areas of fibrosis. These findings contributed to our understanding of the possible relationship between the favoring of the tumor microenvironment for breast cancer metastases. Thus, we describe the impacts related to inflammation caused by the parasite at different doses of infection in a murine model. While the highest dose of infection contributed to a better prognosis for the animals, evidenced by the reduction in the number of metastatic foci in the lung and improvement in aspects related to tissue damage, the doses of 250 eggs proved to be an important risk factor for cancer progression and worsening of metastases, even resulting in early death of the animals. We believe that this scenario was the result of the distinct response profile that each dose resulted in. While the highest dose intensified a greater infiltration of cells, especially eosinophils, whose absence in deficient models accelerated mortality (pilot study conducted in GATA-1-/- animals), at the lowest dose there was a lower expression of inflammatory cytokines, probably due to the increase in TGF-β expression well above all groups and, thus, favoring tumor escape from the immune system and, thus, favoring cancer progression with worsening prognosis. Our findings highlight the importance of assessing the cytokine profile and cellular recruitment in the immune response and suggest that A. suum infection may exert crucial dose-dependent effects on tumor progression. Furthermore, they reinforce the need for control strategies for ascariasis, considering its relevance to public health, not only due to its association with cancer, but, as we have shown, due to the risk of developing other diseases such as COPD, Cholestatic Disease and persistent fibrosis in both the liver and lung. We believe that this work may open avenues for exploration in other cancer models besides breast cancer. It also highlights the potential to investigate the relationship between other parasitic diseases and the development of other types of cancer, expanding the understanding of the role of these agents in oncogenesis and prognosis.