backend=biber, style=alphabetic, sorting=ynt ]biblatex 4. Discussion
We hypothesize that the mixed inflammatory process induced by ethanol could interfere with important mechanisms necessary to control the infection. A higher parasitic burden was observed during the prepatent period in mice pre-exposed to alcohol compared to the control group (not exposed to ethanol), suggesting potential alterations in immunological mechanisms.
As the immunopathogenesis of Chagas disease is associated with a cellular response profile and an imbalance in the network of interactions between cytokines, understanding this effector activity by innate and adaptive immunity cells is important in any study model of the disease. The analysis of cytokine production in stimulated cell cultures serves as a valuable strategy for assessing immunity by measuring the response to a specific stimulus under controlled conditions. This approach provides an initial, albeit preliminary, overview of the immune response profile. It is a fact that the effect of alcohol depends on the dose (high or low) and frequency (acute or chronic) of intake, but in general, there is a modulation in the activity of important antigen-presenting cells (APC), such as macrophages and dendritic cells, affecting the activation and type of response induced (Le Daré et al, 2019). Interestingly, in the present study model, in unstimulated cells from ethanol pre-exposed mice, macrophages and dendritic cells presented a modulatory or even suppressive effect, and in T CD8+ lymphocytes, a potentiating functional action was observed. In general, a polarized inflammatory profile is observed in T CD8 lymphocytes demonstrated by high IL-4 production and no alterations in the IL-10 and IFN-γ production. Accumulating scientific evidence highlights the impact of alcohol and its metabolites on the immune system, notably its tendency to induce a shift towards a type 2 immune response profile. This shift is characterized by an increased frequency of IL-4-producing cells, thereby influencing the induction of inflammatory responses crucial for the control of a wide range of processes, infectious or not (Andrade et al., 2006; Rendon et al., 2012; Ortiz & Wands, 2014; de Castro et al., 2021).
In T. cruzi infection, the reduction in pro-inflammatory cytokines productions such as IFN-γ by adaptive immune system may reflect a deficiency in macrophage activation, fundamental to control intracellular parasitism in acute phase. In fact, the combination of alcohol and infection generate a probably refractory effect in innate immunity, observed in our results by the low production of IL-10 and TNF-α in circulating monocytes. In turn, NK cells may be more resistant to cytokine modulation in the presence or induced by ethanol, as they do not significantly reduce the production of IFN-γ but also do not alter the secretion of a modulating cytokine such as IL-10. It remains to be seen whether this mixed inflammatory profile observed in animals double exposed to alcohol and T. cruzi would be sufficient to trigger protective immunological processes.
We assessed the frequencies of CD4+, CD8+ T and CD19+ B cells staining positive for IL-4, IL-10 and IFN-γ, after being incubated in vitro withT. cruzi soluble antigens. Interestingly, despite the limited sample size, this group of results demonstrated that when CD4+ T cells, from ethanol pre-exposed mice infected with T. cruzi were incubated in vitro to soluble antigens, they restore the capacity of global cytokine secretion, IL-4, IL-10 and IFN-γ, affected by only alcohol exposition. We propose that the soluble antigens led to the reactivation of memory T CD4+ lymphocytes, strengthening activating signals for expansion and their secretory function. The same behavior is observed in the T CD8+ cells, however, with more pronounced to IL-4 production. Is possible that this type II response can disruptive the efficacy of classical pathway of immune mechanisms acting in parasite control mediated by IFN-γ and TNF-α (Macaluso et al., 2023). It is not yet clear in this area of knowledge through which molecular pathways alcohol favors the synthesis of IL-4. However, Mörs and colleagues (2017) demonstrated that alcohol‘s anti-inflammatory effects are mediated via decreasing nuclear levels of activated p50-subunit and canonical NF-ĸB signaling pathway.
Is well known that cytokines coordinate both humoral and cellular immune responses to T. cruzi infection. Beyond their role as antibody producers, B cells secrete cytokines fundamental in activating Th1 cell activities which favor the control of parasite growth (Macaluso et al., 2023). In our model, B lymphocytes are partially affected, mainly by exposition to ethanol, reflecting the high production of IL-10, without alterations in IL-4 and IFN-γ, contributing to inhibits the induction of Th1 profile.
In our study, despite the stimulation of T lymphocytes in secreting cytokines in animals with combined exposition, alcohol and infection, this function in innate cells is highly downregulated. NK cells, macrophages and DC are strongly affected in their ability to secrete IL-4, IL-10, IFN-γ and TNF-α. Studies in humans suggest opposite impact of alcohol on immune system depending on frequency and dose of consumption. In vitro research has provided insights into the complex effects of ethanol on the inflammatory responses of innate immune cells. Short-term exposure, lasting only a few hours, tends to modulate the immune response by increasing the production of regulatory cytokines such as IL-10, while simultaneously decreasing the production of pro-inflammatory cytokines like TNF-α and IL-6 (Sureshchandra et al., 2019). Our experimental protocol of ethanol administration is considered short time, and can corroborate the negative impact on immunity, affecting regulatory and proinflammatory cytokines produced by innate immunity cells. Imbalance in the production of cytokines at a systemic level can considerably affect the inflammatory response in infectious diseases.
The stimulation index clearly demonstrates that T CD8+ lymphocytes is the most activated cell population in the combined situation of alcohol ingestion and infection, increasing all cytokines evaluated, including IL-4, IL-10 and IFN-γ in comparison with other groups. In T CD4+ lymphocytes there is a substantial increase in IFN-γ. Is well known that the anti-inflammatory cytokine, such as IL-4 and IL-10 which modulating the control of macrophage parasiticidal function mediated by IFN-γ and TNF-α. These pro-inflammatory cytokines generally regulate growth, cell activation, differentiation, and homing of the immune cells to the infection sites (Ramos-Rincon et al., 2024). In animal models, Th1 immune response is crucial in the acute phase of Chagas disease to control the parasitemia (Teixeira et al., 2002). Classical studies demonstrated that an imbalance of Th cell populations leading to the predominance of secreted IL-4 and IL-10 at the start of infection and the concomitant down-regulation of IFN-γ secretion reversed the host’s resistance to T. cruzi (Barbosa de Oliveira et al., 1996). In humans, IL-4 plays a dual role in Chagas disease, important to modulate the inflammatory response in chronic phase of disease and to induce cardiac fibrosis (Koh et al., 2023). Its negative influence on the host’s ability to eliminate the parasite and its contribution to fibrosis make its role complex and potentially harmful in the long term. In our experimental model, assessing the impact of changes in cytokines on adaptive immunity regarding the progression and establishment of the disease was not feasible. Nevertheless, there is a clear imbalance in cytokine production by adaptive immune responses, with IL-4 playing a pivotal role, in the acute phase of the infection.
It is important to highlight that there may be an even more powerful impairment of the inflammatory response, observed by the low functional secretory activity of innate immunity. NK and dendritic cells are more affected with very low production of IL-4, IL-10, IFN-γ and TNF-α in ethanol pre-exposed infected mice. Exposure to Trypanosoma cruzi parasites induces monocytes and macrophages to produce various endogenous mediators and is important in Chagas disease by the processes of inflammation and antigen presentation (Gomes et al., 2014), Interestingly, in turn, in the present study, monocytes have a dubious behavior, demonstrated by the low production of inflammatory cytokines and high production of IL-10. Interestingly, studies in humans indicate a predominantly pro-inflammatory profile of circulating monocytes in chronic excessive drinkers and monocyte-derived macrophages displaying a profile called “classical” or M1 profile, which is characterized by the secretion of pro-inflammatory cytokines and the generation of reactive species (Fernández-Regueras et al., 2023; García-Calvo et al., 2020). In the presence of pathogens, macrophages derived from monocytes exposed to alcohol tend to exhibit a suppressive effector function, which is associated with a polarization towards the M2 profile, with high IL-10 production (de Castro et al., 2021). Our data point to this effect of alcohol on macrophages.
Despite the limitations of being an animal model study and not evaluating a broad profile of cytokines and cellular functions, the present research highlights significant changes in immune behavior in the T. cruzi response in mice pre-exposed to ethanol. Further studies are needed to elucidate the molecular mechanisms underlying this behavior and to understand the implications of the alcohol and T. cruzi interaction in human infection.