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\title{Production}



\author[1]{Paulo Henrique Tolentino Moura}%
\author[2]{Beatriz Prado Noronha}%
\author[2]{Marcio Araújo}%
\author[2]{Marcela Lima Moreira}%
\author[1]{Diego Patrick Soares Lopes}%
\author[1]{Rodrigo Gentil Miquilino de Oliveira}%
\author[1]{Waldemar de Paula Junior}%
\author[1]{Marileia Chaves Andrade}%
\affil[1]{Universidade Estadual de Montes Claros Centro de Ciencias Biologicas e da Saude}%
\affil[2]{Fundacao Oswaldo Cruz Instituto Rene Rachou}%


\vspace{-1em}



  
  \date{April 25, 2026}


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\selectlanguage{english}
\begin{abstract}
backend=biber, style=alphabetic, sorting=ynt {]}biblatex
\textbf{Introduction:} \emph{Trypanosoma cruzi} infection, which causes
Chagas' disease, induces an immune response in the host whose efficiency
is important for the infection to persist or be eliminated. Alcohol
consumption produces a great impact on the immune system, inducing
alterations in the determination of T lymphocyte effector function,
directing the profile of these cells to tolerance or inflammation. Our
study aimed to evaluate, in C57BL/6 mice, the cytokine production in
splenic leukocytes from \emph{T. cruzi} infected and treated (EtOH) for
15 days and controls. \textbf{Methods:} Twenty-four mice were randomized
into four groups, with 12 animals each: (1) Non-Infected Control (NI),
(2) Control Infected (CI), (3) Experimental Non-Infected (EtOH
\textsubscript{-}NI), and (4) Experimental Infected (EtOH
\textsubscript{-}I). \textbf{Results:} Ethanol-pre-exposed infected mice
exhibited elevated parasitemia during the patent period compared to
controls. Adaptive immunity was characterized by increased IL-4, IL-10,
and IFN-\selectlanguage{greek}γ \selectlanguage{english}production by CD8+ T lymphocytes, while innate immunity showed
reduced cytokine production, particularly in NK cells and macrophages.
Ethanol amplified IL-10 and IFN-\selectlanguage{greek}γ \selectlanguage{english}responses in macrophages yet
suppressed TNF-\selectlanguage{greek}α \selectlanguage{english}production in dendritic cells and macrophages during
infection. \textbf{Conclusion:} These findings suggest ethanol modulates
the immune response by enhancing adaptive immunity while impairing
innate mechanisms, contributing to altered host-pathogen dynamics in
\emph{T. cruzi} infection.%
\end{abstract}%



\sloppy


\textbf{Alcohol Pre-exposure in \emph{T. cruzi-}Infected Mice Enhances
IL-4 Production by CD8+ T Cells and Modulates Innate Cytokines}

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \textbf{Running
Title: Alcohol Impacts IL-4 Production by CD8 T Cells in Chagas Disease}

Paulo Henrique Tolentino Moura\textsuperscript{1}, Beatriz Prado
Noronha\textsuperscript{2}, Marcio Sobreira Silva
Araujo\textsuperscript{2}, Marcela Lima Moreira\textsuperscript{2},
Diego Patrick Soares Lopes\textsuperscript{1}, Rodrigo Gentil Miquilino
de Oliveira\textsuperscript{1}, Waldemar de Paula
Junior\textsuperscript{1}, Marileia Chaves Andrade\textsuperscript{1,3}

\textsuperscript{1}Universidade Estadual de Montes Claros, UNIMONTES,
Montes Claros, Minas Gerais Brasil

\textsuperscript{2}Funda\selectlanguage{ngerman}ção Osvaldo Cruz, Instituto René Rachou,
FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brasil

\textsuperscript{3} Faculdade de Medicina de Itajubá, Grupo Afya,
Itajubá, Minas Gerais, Brasil

\textbf{Conflict of Interest Statement}

The authors declare no conflict of interest

\textbf{Funding Statement}

All research was carried out with resources from the Institutions
themselves

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \textbf{Data
Availability Statement}

The dataset supporting this article is not publicly available. It is
located in a database under the responsibility of the authors.

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \textbf{Author
Contribuitions}

Conceptualization and Study design, M.C.A. and M.S.S.A.; Methodology,
P.H.T.M., B.P.N, M.L.M.; Formal analysis, P.H.T.M., D.P.S.L.; Data
curation: M.C.A., W.P.J.; Writing original draft preparation: P.H.T.M.,
M.C.A., M.S.S.A.; Writing review and editing: W.P.J., R.G.M.;
Supervision and project administration, M.C.A. All authors have read and
agreed to the published version of the manuscript.

\textbf{Correspondence to:} Marileia Chaves Andrade, PhD

Lab. Epidemiologia e Biocontrole de Microrganismos, Cx. postal 126,
Montes Claros - MG Brasil, +55 38 3229-8189, MG, Brasil. E-mail:
marileia.andrade@unimontes.br

Tel: +55 (38) 3229-8189; +55 (38) 3229-8286

backend=biber, style=alphabetic, sorting=ynt {]}biblatex
\textbf{Abstract}

\textbf{Introduction:} \emph{Trypanosoma cruzi} \textbf{infection, which
causes Chagas' disease, induces an immune response in the host whose
efficiency is important for the infection to persist or be eliminated.
Alcohol consumption produces a great impact on the immune system,
inducing alterations in the determination of T lymphocyte effector
function, directing the profile of these cells to tolerance or
inflammation. Our study aimed to evaluate, in C57BL/6 mice, the cytokine
production in splenic leukocytes from \emph{T. cruzi} infected and
treated (EtOH) for 15 days and controls. Methods:} Twenty-four mice were
randomized into four groups, with 12 animals each: (1) Non-Infected
Control (NI), (2) Control Infected (CI), (3) Experimental Non-Infected
(EtOH\textsubscript{-}NI), and (4) Experimental Infected
(EtOH\textsubscript{-}I). \textbf{Results:} Ethanol-pre-exposed infected
mice exhibited elevated parasitemia during the patent period compared to
controls. Adaptive immunity was characterized by increased IL-4, IL-10,
and IFN-\selectlanguage{greek}γ \selectlanguage{english}production by CD8+ T lymphocytes, while innate immunity showed
reduced cytokine production, particularly in NK cells and macrophages.
Ethanol amplified IL-10 and IFN-\selectlanguage{greek}γ \selectlanguage{english}responses in macrophages yet
suppressed TNF-\selectlanguage{greek}α \selectlanguage{english}production in dendritic cells and macrophages during
infection. \textbf{Conclusion:} These findings suggest ethanol modulates
the immune response by enhancing adaptive immunity while impairing
innate mechanisms, contributing to altered host-pathogen dynamics
in\emph{T. cruzi} infection.

backend=biber, style=alphabetic, sorting=ynt {]}biblatex
\textbf{Keywords: Alcoholism. Chagas Disease. Cytokines. Interleukins.
Neglected Diseases. Tropical Medicina.}

\textbf{1. Introduction}

There are many studies in the literature, aiming elucidate the immune
response in protozoan infections (Costagliola et al., 2016; da
Silveira-Lemos et al., 2021; de Araujo et al., 2021; Tarannun et al.,
2023), associated with diseases with high morbidity and mortality in the
human population, such as those caused by members of the
Trypanosomatidae family (Echeverria and Morillo, 2019; Graepp-Fontoura
et al., 2020).

During the process of infection by the etiological agent of Chagas
disease, the immunological networking will define the inflammatory
profile and, consequently, the evolution of the disease. It is known
that the absence of Chagas pathology is mainly associated with the
infected host's ability to modulate the anti-\emph{T. cruzi} immune
response. However, this response that controls persistent parasitemia
can also contribute to the inflammatory damage that causes morbidity and
clinical manifestations of Chagas disease (Sathler-Avelar et al., 2021).

Is well known that innate immune system cells as dendritic cells,
monocytes and macrophages modulate the adaptive immune response, and
natural killer cells are majority producers of interferon-gamma (IFN-\selectlanguage{greek}γ\selectlanguage{english})
in the initial phases of \emph{T. cruzi} infection (Guilmot et al.,
2014). Phagocytes secrete cytokines that promote inflammation and
activation of other cells involved in immunity (Macaluso et al., 2023).
Despite the great importance of T cells in controlling parasitemia
during the acute phase of disease, it is known that these activated
cells are the main components of the inflammatory infiltrate observed in
cardiac and digestive patients. Their presence and the cytokine profile
secreted by them are related to the pathology during the chronic phase
(Natale et al., 2021; Castro-Eiro et al., 2022). Therefore, it is
consistently found in the literature that high expression and imbalance
of inflammatory cytokines, such as TNF-\selectlanguage{greek}α \selectlanguage{english}and IFN-\selectlanguage{greek}γ, \selectlanguage{english}is related to the
development of pathology, both in the cardiac and digestive clinical
forms (Dutra et al., 2009; Torres et al., 2024).

Given that Chagas disease has a chronic and debilitating evolution, the
consumption of alcoholic beverages could worsen clinical conditions.
Although alcoholism is a serious public health problem, there are no
studies in the literature that address the relationship between
alcoholism and infections by Trypanosomatidae protozoa (\emph{Leishmania
spp} . or \emph{T. cruzi} ) in human population. There are few studies
in the scientific literature investigating this relationship or impact
(Sandri et al., 2017; Falkowski-Temporini et al., 2017). In general,
these studies report that alcohol consumption is directly related to a
greater degree of morbidity from infection by these trypanosomatids.
However, is important to investigate whether immunological changes
resulting from ethanol use would influence the course of \emph{T. cruzi}
infection.

The consumption of alcohol, whether chronic or acute, results in
alterations in cellular components of the innate and adaptive immune
system (Gonz\selectlanguage{ngerman}ález-Reimers et al., 2012). Ethanol has been shown to
influence both subpopulations of CD4+ T cells, or helper T lymphocytes
(Th). In human alcoholics, there is an increase in Th1 responses and a
decrease in Th2 responses (Krolewiecki et al., 2001; González-Reimers et
al., 2014). A recent study demonstrated that ethanol impairs CD4+ T cell
immunometabolism and promoting CD4+ T cell differentiation to a
pro-inflammatory phenotype (MacTernan et al., 2022).

However, scientific evidence is accumulating that ethanol (EtOH)
modulates the function of immune cells that circulate in the blood until
recruited into tissues, in a dose and time dependent manner (Barr et al,
2016). For example, the administration of ethanol in the short term can
shift the class of immune response triggered towards a type 2
reactivity, promoting a quickly increase in serum levels of IgE (Alonso
et al., 2012). Our research group demonstrated that short-term high-dose
EtOH administration has differential impact on APC populations.
Highlighting the downregulating of splenic macrophages and DC activity
in contrast to up-regulating of B lymphocyte function as APC and
ultimately yielding a micro-environment that led to increased activation
of CD4+ T cells and induction of allergic biomarkers, such as IL-4 and
IgE (Andrade et al., 2009).

Given the significant alterations induced by alcohol consumption on the
immune system, and the fact that developing countries have a high
prevalence of neglected diseases, such as Chagas disease, it is highly
relevant to evaluate the relation between ethanol consumption and the
immune response in the face of \emph{T. cruzi} infection. This study
becomes even more important considering that the exact role of IL-4
in\emph{T. cruzi} infection is not yet fully defined, and the fact that
alcohol is a legal drug of abuse widely consumed throughout the world.

\textbf{2. Material and Methods}

\textbf{2.1. Study Design}

This experimental study was conducted at the Biomarkers for Diagnosis
and Monitoring Laboratory of the René Rachou Institute/Fiocruz-MG.
Animals were weighed daily, maintained under controlled temperature
(22.7\selectlanguage{english} +-\selectlanguage{english} 1.2\selectlanguage{english} degC), 12h/12h light/dark cycle, with filtered water and food
ad libitum. The study was approved by the Animal Use Committee of
FIOCRUZ (protocol P-23/14-6, LW-19/15).

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \textbf{2.2.
Experimental Groups}

Twenty-four C57/BL6 female mice (5--8 weeks old) were divided into four
groups (n=6/group): (1) Non-Infected Control (NI), (2) Control Infected
(CI), (3) Experimental Non-Infected (EtOH-NI), and (4) Experimental
Infected (EtOH-I). Day 0 corresponded to the infection day, 24h after
the last ethanol administration.

\textbf{2.3. Oral Administration of Ethanol (EtOH)}

EtOH groups received 0.2\selectlanguage{english} mL of 10\% (v/v) ethanol by gavage for 15
consecutive days. Control groups received filtered water. Infection
occurred 24h after the last EtOH dose.

\textbf{2.4. Experimental Infection with \emph{Trypanosoma cruzi} and
Parasitemia Curve}

Mice were infected intraperitoneally with 5x103 bloodstream
trypomastigotes (Y strain). Parasitemia was monitored daily from day 2
to day 13 post-infection, analyzing 5\selectlanguage{english} \selectlanguage{greek}µ\selectlanguage{english}L of tail blood under optical
microscopy. Results were expressed as trypomastigotes/0.1 mL blood.

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \textbf{2.5.
Obtention, Preparation and Counting of Splenic Cell Suspensions}

Fourteen days post-infection, mice were anesthetized (ketamine 50 mg/kg
and xylazine 25 mg/kg) and euthanized. Spleens were removed, macerated
in RPMI-1640 medium, and subjected to centrifugation (400\selectlanguage{ngerman}×g, 10\selectlanguage{english} min,
4degC). Red blood cells were lysed using sterile water and PBS 10X. After
washing and fat removal, leukocytes were resuspended in PBS with 5\%
FBS. Viable and non-viable cells were differentiated using Trypan Blue,
counted in a Neubauer chamber, and adjusted to 1x10\selectlanguage{english}7 cells/mL.

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \textbf{2.6.
Cultured Spleen Cells and Immunophenotyping to Flow cytometry for Cell
Subsets and Intracellular Cytokines Analysis}

Splenic cells (5\selectlanguage{ngerman}×10\selectlanguage{english}6) were cultured in RPMI-1640 for 12 h at 37 \selectlanguage{ngerman}°C with
5\% CO\selectlanguage{english}2, with or without Soluble Epimastigote Antigen (EPI) stimulation.
Brefeldin A (10 \selectlanguage{greek}µ\selectlanguage{english}g/mL) was added for the final 4 h. After EDTA treatment
and FACS buffer washing, cells were stained for surface markers (CD4,
CD8, CD19, NK1.1, MAC3, CD11c) using FITC-conjugated antibodies. After
fixation and permeabilization, intracellular cytokines (TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ,
\selectlanguage{english}IL-4, IL-10) were stained with PerCP, APC, or PE-labeled antibodies.
Flow cytometry was performed acquiring 30,000 events/sample on a
FACScalibur cytometer. Data were analyzed using FlowJo software v10.0.1.

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \emph{2.7.
Trypanosoma cruzi} \textbf{Epimastigote Antigen (EPI) Preparation}

EPI was prepared from stationary-phase Y strain epimastigotes cultivated
in LIT medium. After three freeze-thaw cycles and sonication, the lysate
was centrifuged (37,000\selectlanguage{ngerman}×g, 90\selectlanguage{english} min), dialyzed against PBS, sterilized
through 0.22\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}m filters, and stored at -70\selectlanguage{ngerman}°C. Protein content was
determined by Lowry's method (Lowry et al. 1951).

\textbf{2.8. Statistical Analyses}

Data distribution (parametric or non-parametric) was evaluated using
Minitab 11.0. Statistical analyses were performed with GraphPad Prism
5.0. ANOVA followed by Tukey's test was used for parametric data;
Kruskal-Wallis followed by Dunn's test was used for non-parametric data.

\textbf{3. Results}

\textbf{3.1. Assessment of Parasitemia}

The assessment of parasitemia curve shows first days of analysis, that
they were similar for the control (CI) and ethanol-pre-exposed infected
(EtOH\textsubscript{-}I) groups (Figure 1). However, animals in this
group (EtOH\textsubscript{-}I), presented a higher parasite counting
(trypomastigotes) in the blood from the beginning of the patent
period-PP (the seventh day after infection), until the eighth day
post-infection, when compared to the control infected group
(p\textless{}0.05). Interestingly, around the seventh-day
post-infection, an increase in the number of circulating parasites was
observed in both groups, with the level of parasitemia remaining higher
in the ethanol-pre-exposed \emph{T. cruzi} -infected mice (ETOH-I) than
in the control group until the seventh day (p\textless{}0.05). On the
ninth day, there was a peak of parasitemia for both groups, and no
difference in the level of parasitemia was observed until the end of the
experiment. Both groups presented the same dynamics of parasitemia
control, at least regarding the decrease of circulating parasites.

\textbf{3.2.} \textbf{CD8+ T Lymphocytes Dominate IL-4 Production in
Unstimulated Immune Cells, While Innate Immunity Exhibits Reduced
Cytokine Production in \emph{T. cruzi}-Infected Mice Pre-Exposed to
Ethanol}

The production of IL-4, IL-10, and IFN-\selectlanguage{greek}γ \selectlanguage{english}was evaluated in unstimulated
splenic CD4+ \selectlanguage{english}T lymphocytes, with no differences observed among groups
(Figure 2A). However, CD8+ \selectlanguage{english}T lymphocytes from ethanol-pre-exposed
and\emph{T. cruzi-} infected mice (EtOH-I) showed increased IL-4 levels
compared to all other groups (Figure 2B). Regarding IL-10, CD8+ \selectlanguage{english}T cells
from control-infected mice (CI) produced more of this cytokine,
indicating a modulatory response to infection, while EtOH-I CD8+ \selectlanguage{english}T cells
exhibited elevated IFN-\selectlanguage{greek}γ \selectlanguage{english}compared to non-infected controls (NI). For B
lymphocytes, \emph{T. cruzi} infection led to reduced IL-10 production
in both infected groups (CI and EtOH-I), regardless of ethanol exposure
(Figure 2C).

Innate immunity showed a distinct pattern. Ethanol-pre-exposed
uninfected mice displayed higher IL-10 and IFN-\selectlanguage{greek}γ \selectlanguage{english}levels in NK cells,
while only IFN-\selectlanguage{greek}γ \selectlanguage{english}was elevated in the EtOH-I group. For monocytes and
dendritic cells, IL-10 and TNF-\selectlanguage{greek}α \selectlanguage{english}production was assessed (Figure 3A).
Macrophages from ethanol-pre-exposed uninfected mice exhibited increased
IL-10 and TNF-\selectlanguage{greek}α, \selectlanguage{english}whereas EtOH-I mice showed a significant reduction
(Figure 3B). No differences were detected in cytokine production by
dendritic cells among groups (Figure 3C).

\textbf{3.3. Stimulation of Immune Cells with \emph{T. cruzi} Antigens
Reveals a Distinct Impact of Alcohol and Infection Combination on
Cytokine Production: Selective Enhanced of T CD8+ Lymphocytes Activation
and Reduction of Innate Immunity.}

The assessment of adaptive immunity cells reveals differential
activation profiles in response to combined alcohol and \emph{T. cruzi}
infection. In T CD4+ lymphocytes, elevated levels of IL-4 and IL-10
cytokines were observed in ethanol-pre-exposed and infected with\emph{T.
cruzi} compared to the uninfected group (Figure 4A). However, this
percentage of positive cells did not differ when compared to both
control groups that did not receive alcohol (NI and CI). In these cells,
the inflammatory cytokine, IFN-\selectlanguage{greek}γ, \selectlanguage{english}the elevation observed in the ETOH-I
group was also significant compared to the control not infected group.

Consistent with observations in unstimulated cells, B lymphocytes from
ethanol-pre-exposed mice, in stimulated culture, exhibited a significant
reduction in the proportion of IL-4-positive cells in the context of
infection (Figure 4C).

As presented in Figure 5 (A, B, and C), there appears to be a trend
towards a decrease in the percentage of positive innate immune cells
producing the evaluated cytokine profile in ethanol-pre-exposed \emph{T.
cruzi} -infected mice. Regarding NK cells (Figure 5A), while no
significant difference was observed compared to the infected group
without alcohol consumption, all cytokines (IL-4, IL-10, and IFN-\selectlanguage{greek}γ\selectlanguage{english}),
showed a reduction in the proportion of positive cells in the context of
alcohol and infection.

In macrophages (Figure 5B) this seems to be more evident, with a sudden
reduction in related to IL-10 and TNF-\selectlanguage{greek}α \selectlanguage{english}positive cells in
ethanol-pre-exposed \emph{T. cruzi} -infected mice as compared to
pre-exposed uninfected group (ETOH-NI) and control uninfected mice to
TNF-\selectlanguage{greek}α \selectlanguage{english}cytokine.

The percentage of dendritic cells (Figure 5C) positive to TNF is also
significantly reduced in mice infected with \emph{T. cruzi}
ethanol-pre-exposed compared to the control unexposed infected group
(CI).

\textbf{3.4. The stimulation index highlights the variances in the
impact of alcohol on both adaptive and innate immunity in animals
infected with \emph{T. cruzi}.}

The stimulation index (SI) is an important metric in experimental
designs assessing functional parameters in leukocytes following
stimulation with specific antigens. It estimates the degree to which
cytokine production varies, either increasing or decreasing, compared to
unstimulated control cultures. The results reiterate that ethanol
pre-administration in \emph{T. cruzi} infected mice, appears to amplify
the adaptive immunity response, especially mediated by CD8+ T
lymphocytes, reflecting a mixed profile of activation compared to other
groups, due to the high production of IL-4, IL-10 and IFN-\selectlanguage{greek}γ \selectlanguage{english}(Figure 6B).
In turn, for CD4+ T lymphocytes (Figure 6A) the most prominent results
are selective for IFN-\selectlanguage{greek}γ \selectlanguage{english}and B lymphocytes (Figure 6C) for the increase
in IL-10. Regarding innate immune cells, the SI confirms the apparent
suppression of the cytokine response in ethanol-pre-exposed mice
infected with \emph{T. cruzi} compared to all other groups, mainly NK
(Figure 6D) and dendritic cells (Figure 6F). In turn, IL-10 production
by macrophages (Figure 6E) is elevated in ethanol-pre-exposed mice
(infected or not) compared to control-infected mice. The main results
are observed in this study were summarized in Figure 7.

backend=biber, style=alphabetic, sorting=ynt {]}biblatex \textbf{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 Dar\selectlanguage{ngerman}é 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-\selectlanguage{greek}γ \selectlanguage{english}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 \emph{T. cruzi} infection, the reduction in pro-inflammatory
cytokines productions such as IFN-\selectlanguage{greek}γ \selectlanguage{english}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-\selectlanguage{greek}α \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}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 \emph{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-\selectlanguage{greek}γ, \selectlanguage{english}after being incubated in vitro
with\emph{T. 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 \emph{T. cruzi} were
incubated in vitro to soluble antigens, they restore the capacity of
global cytokine secretion, IL-4, IL-10 and IFN-\selectlanguage{greek}γ, \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}(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, M\selectlanguage{ngerman}örs and colleagues
(2017) demonstrated that alcohol`s anti-inflammatory effects are
mediated via decreasing nuclear levels of activated p50-subunit and
canonical NF-\selectlanguage{polish}ĸ\selectlanguage{english}B signaling pathway.

Is well known that cytokines coordinate both humoral and cellular immune
responses to \emph{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-\selectlanguage{greek}γ, \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α. \selectlanguage{english}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-\selectlanguage{greek}α \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}in comparison with other groups. In T CD4+
lymphocytes there is a substantial increase in IFN-\selectlanguage{greek}γ. \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}and
TNF-\selectlanguage{greek}α. \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}secretion reversed the host's
resistance to \emph{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-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}in
ethanol pre-exposed infected mice. Exposure to \emph{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 (Fern\selectlanguage{ngerman}ández-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 \emph{T.
cruzi} interaction in human infection.

\textbf{4. Acknowledgements}

This study was supported in part by the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a
Pesquisa de Minas Gerais (FAPEMIG) and the Universidade Estadual de
Montes Claros (UNIMONTES). The author MSSA thank to CNPq for their
research fellowship. To the Program for Technological Development in
Tools for Health-RPT-FIOCRUZ for using the flow cytometry facilities.

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\textbf{6. Figure Legends}

\textbf{Figure 1.} Parasitemia curve of mice infected
with\emph{Trypanosoma cruzi} that received prior ingestion of EtOH. Mice
(n=6/group) were infected intraperitoneally with 5×10³ with
trypomastigotes (Y strain) bloodstream twenty four hours after the last
intragastric ethanol administration (ETOH-I) or control group (C-I).
Parasitemia was monitored daily from day 2 to day 13 post-infection.
Results were expressed as trypomastigotes/0.1\selectlanguage{english} mL blood. Pre-patent
period (PPP): Period between inoculation and the first day of parasite
detection in the animal's peripheral blood; Patent period (PP): Period
between the first day of positive fresh examination and complete
negativity of parasitemia; Maximum peak of parasitemia (MPP): This
corresponds to the maximum parasitemia; Day of maximum peak of
parasitemia (DMPP): This corresponds to the day of maximum parasitemia
occurrence. \textsuperscript{*} Indicates statistically significant
difference between groups.

\textbf{Figure 2.} Evaluation of cytokine production (IL-4, IL-10,
IFN-g) by unstimulated splenic lymphocytes from mice infected
with\emph{T. cruzi} that received ethanol previously. For fifteen
consecutive days, C57BL/6 female mice (n = 6) were given 50\% (v/v)
ethanol (EtOH) or filtered water by gavage. After 24 hours of last
administration, the animals were infected with \emph{T. cruzi} and
fourteen days post-infection, spleens were removed and prepared for
immunophenotyping, by flow cytometry, of (A) T CD4+ Lymphocytes, (B) T
CD8+ Lymphocytes and (C) B Lymphocytes, performed acquiring 30,000
events/sample on a FACScalibur cytometer. Data were analyzed using
FlowJo software v10.0.1. \textsuperscript{*} Indicates statistically
significant difference (p \textless{}0.05) between groups submitted to
the same intervention (EtOH or control). Connecting lines indicate
statistical difference between groups of different intervention.

\textbf{Figure 3.} Evaluation of cytokine production (IL-4, IL-10,
IFN-g, TNF-a) by unstimulated innate immune splenic cells of mice
infected with \emph{T. cruzi} that received ethanol previously. For
fifteen consecutive days, C57BL/6 female mice (n = 6) were given 50\%
(v/v) ethanol (EtOH) or filtered water by gavage. After 24 hours of last
administration, the animals were infected with \emph{T. cruzi} and
fourteen days post-infection, spleens were removed and prepared for
immunophenotyping by flow cytometry of (A) NK cells, (B) Macrophages and
(C) Dendritic cells\textbf{,} performed acquiring 30,000 events/sample
on a FACScalibur cytometer. Data were analyzed using FlowJo software
v10.0.1. \textsuperscript{*} Indicates statistically significant
difference (p \textless{}0.05) between groups submitted to the same
intervention (EtOH or control). Connecting lines indicate statistical
difference between groups of different intervention.

\textbf{Figure 4.} Evaluation of cytokine production (IL-4, IL-10,
IFN-g) by stimulated splenic lymphocytes of mice infected with \emph{T.
cruzi} that received ethanol previously. For fifteen consecutive days,
C57BL/6 female mice (n = 6) were given 50\% (v/v) ethanol (EtOH) or
filtered water by gavage. After 24 hours of last administration, the
animals were infected with \emph{T. cruzi} and fourteen days
post-infection, spleens were removed and prepared, cultured and
stimulated with soluble epimastigote antigen during 12 hours. After
that, preparation for immunophenotyping was performed in a flow
cytometry for analysis of (A) T CD4+ Lymphocytes, (B) T CD8+ Lymphocytes
and (C) B Lymphocytes with the acquisition of 30,000 events/sample on a
FACScalibur cytometer. Data were analyzed using FlowJo software
v10.0.1.\textsuperscript{*} Indicates statistically significant
difference (p or control). Connecting lines indicate statistical
difference between groups of different intervention.

\textbf{Figure 5.} Evaluation of cytokine production (IL-4, IL-10,
IFN-g, TNF-a) by stimulated innate immune splenic cells of mice infected
with \emph{T. cruzi} that received ethanol previously. For fifteen
consecutive days, C57BL/6 female mice (n = 6) were given 50\% (v/v)
ethanol (EtOH) or filtered water by gavage. After 24 hours of last
administration, the animals were infected with \emph{T. cruzi} and
fourteen days post-infection, spleens were removed and prepared,
cultured and stimulated with soluble epimastigote antigen during 12
hours. After that, preparation for immunophenotyping was performed in a
flow cytometry for analysis of (A) NK cells, (B) Macrophages and (C)
Dendritic cells with the acquisition of 30,000 events/sample on a
FACScalibur cytometer. Data were analyzed using FlowJo software
v10.0.1.\textsuperscript{*} Indicates statistically significant
difference (p or control). Connecting lines indicate statistical
difference between groups of different intervention.

\textbf{Figure 6.} Determination of the stimulation index (SI) of
lymphocyte cultures and innate immune cells from the spleen of mice
infected with \emph{T. cruzi} that previously received ethanol. The SI
was calculated as the ratio between the immunophenotyping values
obtained in cultures stimulated with \emph{Trypanosoma cruzi} antigens
and the values obtained in unstimulated cultures (control). This
parameter quantifies the magnitude of the specific cytokine production
induced by exposure to \emph{T. cruzi} antigens in (A) T CD4+
Lymphocytes, (B) T CD8+ Lymphocytes, (C) B Lymphocytes, (D) NK cells,
(E) Macrophages, and (F) Dendritic cells.

\textbf{Figure 7.} Illustrative image showing the summary of the main
results obtained in the study. Top of the figure: In
ethanol-treated\emph{T. cruzi} infected-mice, CD4+ T and B splenic
lymphocytes, in unstimulated culture, showed reduced production of the
cytokine IL-4, while CD8+ T lymphocytes demonstrated an increase in this
cytokine. Macrophages showed reduction in the production of IL-10 and
TNF-a. Bottom of the figure: In ethanol-treated \emph{T. cruzi}
infected-mice, CD4+ and CD8+ T lymphocytes, in stimulated culture,
demonstrated an increase in IL-4 production, while B lymphocytes showed
reduction. Macrophages and NK cells showed reduction in the production
of IL-10, TNF-a and IL-4, IL-10 and TNF-a, respectively.\selectlanguage{english}
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