Abstract
Chagas disease, caused by Trypanosoma cruzi, exhibits a wide clinical spectrum, which is influenced by the parasite's extensive genetic diversity. Growing evidence indicates that human infections are often multiclonal, involving a dynamic population of parasite clones, collectively termed the "cruziome". The immunological consequences of these mixed infections, particularly at the initial host-parasite interface, remain poorly characterised. This study aimed to investigate how single versus co-infection with phylogenetically distinct T. cruzi strains modulates the innate immune response of murine macrophages. RAW 264.7 macrophages were infected with T. cruzi strains from Discrete Typing Unit (DTU) I (G strain) or DTU II (Y strain), either individually or in combination (co-infection). Assays for cellular invasion and intracellular multiplication were performed. The production of key cytokines (IL-1β, IL-18, IL-6, IL-10, IL-12) and nitric oxide (NO) was quantified at 24 to 96 h post-infection. The Y strain displayed significantly higher invasion and replication rates and induced a potent pro-inflammatory response, characterised by elevated levels of IL-1β, IL-18, IL-12, and NO. The G strain elicited a more regulatory profile, with a progressive increase in IL-10 production at later time points and lower levels of inflammatory mediators. Co-infection resulted in a distinct, hybrid immune profile, marked by intermediate levels of both pro-inflammatory and regulatory cytokines and a moderated NO output. Co-infection with phylogenetically distinct T. cruzi strains generates a unique immunomodulatory environment that is not merely an additive effect of the individual strains. These findings provide in vitro evidence supporting the hypothesis that the composition of the infecting parasite population shapes the host immune response from the earliest stages of infection. This balanced interplay between pro-inflammatory and regulatory signals may contribute to the clinical heterogeneity observed in Chagas disease and underscores the need to consider parasite diversity in pathogenic and therapeutic studies.