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Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that has a heterogeneous population composed of a pool of strains with distinct characteristics, including variable levels of virulence. In previous work, transcriptome analyses of parasite genes after infection of human foreskin fibroblasts (HFF) with virulent (CL Brener) and non-virulent (CL-14) clones derived from the CL strain, revealed a reduced expression of genes encoding parasite surface proteins in CL-14 compared to CL Brener during the final steps of the intracellular differentiation from amastigotes to trypomastigotes. Here we analyzed changes in the expression of host genes during in vitro infection of HFF cells with the CL Brener and CL-14 strains by analyzing total RNA extracted from cells at 60 and 96 hours post-infection (hpi) with each strain, as well as from uninfected cells. Similar transcriptome profiles were observed at 60 hpi with both strains compared to uninfected samples. However, at 96 hpi, significant differences in the number and expression levels of several genes, particularly those involved with immune response and cytoskeleton organization, were observed. Further analyses confirmed the difference in the chemokine/cytokine signaling involved with the recruitment and activation of immune cells such as neutrophils upon T. cruzi infection. These findings suggest that infection with the virulent CL Brener strain induces a more robust inflammatory response when compared with the non-virulent CL-14 strain. Importantly, the RNA-Seq data also exposed an unexplored role of fibroblasts as sentinel cells that may act by recruiting neutrophils to the initial site of infection. This role for fibroblasts in the regulation of the inflammatory response during infection by T. cruzi was corroborated by measurements of levels of different chemokines/cytokines during in vitro infection and in plasma from Chagas disease patients as well as by neutrophil activation and migration assays. Author summary: Trypanosoma cruzi is the causative agent of Chagas disease, a debilitating and often life-threatening illness that affects 6 to 7 million people mainly in Latin America. The parasite, transmitted to humans by an insect vector, needs to invade different cells from the infected person in order to multiply and spread the infection to various organs, including the heart and the gut. In this study, we investigated how the host cell responds to the infection by analyzing changes in the expression of human genes in fibroblasts infected with the CL Brener and CL-14 strains, which are strains that present highly distinct virulent phenotypes during infection in mice. We showed that human fibroblasts build a strong immune response upon infection by T. cruzi and that this response is different depending on the parasite strain: infection with the virulent CL Brener strain induces a more robust inflammatory response compared with the infection with the avirulent CL-14 strain. We also showed that, in response to the infection, fibroblasts produce molecules that can recruit and activate neutrophils, which are important immune cells that controls the infection. [ABSTRACT FROM AUTHOR]