Trypanosoma cruzi is an intracellular protozoan parasite, the etiologic agent of Chagas disease, that affect 7 million people worldwide. It is a main public health problem in Latin America, which spreads to developing countries, mainly through blood transfusion from infected patients who migrates to non-endemic areas (de Sousa et al., 2024). The parasite induces the development of chronic megaviscera and/or cardiomyopathy in ∼30% of patients. Chronic Chagas cardiomyopathy is the most frequent and severe, and is a consequence of an intense, and permanent inflammation process throughout the infection caused by local parasite persistence (Garcia et al., 2005; Marin-Neto et al., 2007). It has been accepted that the parasitic load is responsible for triggering the inflammatory response (Tarleton and Zhang, 1999).

Galectins (Gals) are mammalian lectins containing conserved carbohydrate recognition domains (CRDs) that interact with ß-galactose-containing glycans displayed on different cells. Galectin-8 (Gal-8) together with Gal-4 and Gal-9, belong to the tandem-repeated group. Gal-8 contains two CRDs, with different glycan specificity, that are fused via a peptide linker (Bidon et al., 2001) and, unlike other Gals, is widely distributed in different tissues under normal conditions, as well as in tumors and synovial fluid (Tribulatti et al., 2020). It is also expressed in endothelium and released into the environment when tissue is damaged (Thijssen et al., 2008; Cattaneo et al., 2014) and has been involved in different events, both pathological and homeostatic (Elola et al., 2014). It has been communicated Gal-8 ability to bind to surface ligands of T. cruzi (Pineda et al., 2015) as well as to exposed glycans in damaged bacterial-infected vacuoles, acting as danger receptors (Thurston et al., 2012). Gal-8 plays both pro- or anti-inflammatory roles depending on the context (Reviewed in (Tribulatti et al., 2020), see also (Zick, 2022). We have reported the association of Gal-8 in inducing T cell expansion under both antigen-specific and nonspecific conditions and showed that this T cell co-stimulation is dependent on Gal-8 concentration (Tribulatti et al., 2009). On the other hand, an anti-inflammatory effect has been attributed to Gal-8 in autoimmune diseases such as rheumatoid arthritis (Eshkar Sebban et al., 2007), as well as in uveitis and encephalomyelitis models (Sampson et al., 2015; Pardo et al., 2017). In line with this, we demonstrated the anti-inflammatory role of Gal-8 during chronic T. cruzi infection by examining Gal-8-deficient (Gal-8KO) mice infected with the T. cruzi Ac strain (4 months post-infection, mpi) (Bertelli et al., 2020). While survival and parasitemia rates were similar between Gal-8KO-infected mice and wild-type (WT) C57BL/6J control mice, the absence of Gal-8 resulted in significant inflammation in the heart, skeletal muscle and liver, independently of parasite load. Remarkably, we observed an increased presence of neutrophils and macrophages in cardiac tissue of infected Gal-8KO (iGal-8KO) mice. The latter is associated with the absence of Gal-8-dependent preaparesis. These findings highlight the anti-inflammatory role that Gal-8 exerts during chronic T. cruzi infection (Bertelli et al., 2020).

During the early stages of infection, T. cruzi employs sophisticated strategies to evade and modulate the host immune response. Although unable to eliminate the parasite, the specific immune response allows the host survival and development of chronicity. Th1 effector mechanisms are relevant to parasite control (Kumar and Tarleton, 2001; Hoft and Eickhoff, 2002; Volta et al., 2021), but orchestrating a balanced Th1 and Th2 immune response is crucial in T. cruzi infection, since an excessive or dysregulated activation, can lead to host tissue damage (Ruiz Diaz et al., 2015; Llaguno et al., 2019). The role of Th17 in protection, in murine and human studies have been also communicated (Magalhaes et al., 2013; Cai et al., 2021; Duarte et al., 2025). CD8⁺ T cells play a central role in controlling the infection by targeting and eliminating the infected cells (Araujo Furlan et al., 2018). However, in the chronic phase, T. cruzi modulates the effectiveness of CD8⁺ T cell responses, leading to incomplete pathogen clearance and immune exhaustion (Albareda et al., 2013). Regulatory T (Treg) cells play a dual role by helping to suppress excessive immune activation that could damage host tissue, while also aiding tissue repair once the infection is controlled. A transient decrease in Treg cells, during the acute phase, allows for a stronger activation of CD8⁺ T cell responses, which is crucial for controlling the parasite (Araujo Furlan et al., 2018). B cell response and antibodies are important in targeting and controlling circulating parasites (Umekita and Mota, 2000; Gorosito Serran et al., 2017).

A prominent feature of acute T. cruzi infection is splenomegaly (Lopes et al., 1995; Cabrera et al., 2013; Dehesa-Rodriguez et al., 2022), driven by intense polyclonal proliferation of B cells, CD4⁺ and CD8⁺ T cells. This hyperactivation, which includes both antigen-specific and non-specific responses, contributes to immune dysregulation and facilitates parasite dissemination (Minoprio, 2001). Although the triggers for this massive lymphocyte expansion are not fully understood, parasite-derived components have been associated (Reina-San-Martin et al., 2000; Montes et al., 2002; Montes et al., 2006). Interestingly, splenomegaly typically resolves during the transition from acute to chronic infection, with the spleen regaining its normal size and function. Our study identifies Gal-8 as a critical regulator in this process. The absence of Gal-8 results in persistent splenomegaly during chronic T. cruzi infection. Given the importance of lymphocyte hyperactivation, often described as a polyclonal response during the acute phase, we conducted an in-depth analysis of the cellular events underlying chronic splenomegaly. Our findings highlight Gal-8 as a pivotal player in controlling splenic size and immune homeostasis.

The immune system is regulated by a complex network of interactors, including Gals. Currently, Gals are considered potential therapeutic targets to control chronic inflammatory processes in autoimmune or infectious diseases (Rabinovich and Toscano, 2009). In particular, the role of Gal-8 in the activation of the immune system is variable depending on the cellular context. While it promotes the activation of effector functions, such as inducing platelet activation (Romaniuk et al., 2010; Cattaneo et al., 2014), co-stimulation (Tribulatti et al., 2009) and T lymphocyte proliferation (Tribulatti et al., 2012), several studies have reported an immunosuppressive role for this galectin in Jurkat cells (Norambuena et al., 2009), in CD4⁺CD8⁺ thymocytes (Tribulatti et al., 2007) and in models of autoimmune diseases (Sampson et al., 2015; Pardo et al., 2017).

Our study on the role of Gal-8 in the inflammatory context induced during chronic T. cruzi infection showed its anti-inflammatory property (Bertelli et al., 2020). Here, we describe and analyze the persistence of splenomegaly in the chronic phase of T. cruzi infection in mice lacking Gal-8. Although there are several hypothesis and findings supporting the development of splenomegaly based in the high immune reactivity during the acute phase of the infection, there is scant information regarding the recovery of organ size. In our model, during the acute phase, the occurrence of splenomegaly, evaluated as organ weight, was similar between the infected groups ( Figure 1C ). After 2 mpi, and as it progresses towards the chronic phase, splenomegaly was reduced in iWT mice. However, in iGal-8KO mice this did not occurs ( Figures 1C-F ). At 4 mpi, we observed significant differences in the weight of the spleen and in the number of splenocytes in iGal-8KO animals compared to iWT ( Figures 1D-F ). This phenomenon is associated to the infection, since neither the weight nor the total number of splenocytes were altered by the absence of this galectin in uninfected mice. In human Chagas disease, splenomegaly has been described during the acute phase in infections acquired by different routes, including vectorial, congenital and organ transplants (Zaidenberg and Segovia, 1993; Moya et al., 2005; Bern et al., 2011; Cabrera et al., 2013; Fernandez-Villegas et al., 2014; Echeverria and Morillo, 2019). In all cases, splenomegaly resolves after the acute phase is controlled, as occurs in the mouse model. Therefore, this model could provide important clues to understanding the resolution of splenomegaly, the associated events of which are still unknown.

Lymphocytes B response to T. cruzi acquires an important role in controlling the spread and maintaining low levels of parasitemia in the chronic phase (Amezcua Vesely et al., 2012). At 4 mpi, the analysis of the expression of different phenotypic markers in B cells, allowed us to study the role of Gal-8 in the humoral immune response against the parasite. We observed an increased number of transitional B lymphocytes in infected mice ( Figure 3B ), that could be related with the known polyclonal activation of B cells (Amezcua Vesely et al., 2012). iGal-8KO mice showed reduced cell numbers in the MZ population ( Figure 3D ). It is possible that this population, that respond rapidly to stimuli circulating in the blood, quickly differentiate into ASC or GC B cells (Song and Cerny, 2003; Amezcua Vesely et al., 2012; Cerutti et al., 2013). Therefore, it is not surprising that a decrease in MZ B lymphocytes is associated with an increase in GC B cells. The observed reduction in the number of MZ B cells does not necessarily indicate that these cells have not exerted or are not exerting their function. Simultaneously, a significant increase in the number of mature B lymphocytes occurs, as consequence of cellular division ( Figures 3F, G ). Cytometry evaluation showed increased number of GC B lymphocytes and ASC in iGal-8KO vs iWT mice ( Figures 3F, G ). Surprisingly, the evaluation in serum of total IgGs level and IgG1, IgG2a, IgG2b specific anti-T. cruzi subclasses values were similar between iWT and iGal-8KO mice (data not shown) as well as sera antibodies avidity. However, immunohistochemical assays showed in iGal-8KO that GCs, identified as PNA⁺, were highly disorganized ( Figure 4 ).

Gals have been involved in the development of B cells. Evidence have suggested that they play an important role in their signaling and activation (Acosta-Rodriguez et al., 2004; Beccaria et al., 2018) then they can influence cell fate decisions by regulating the balance between differentiation into memory or plasma cells. During T. cruzi infection, it was shown that Gal-3 in collaboration with IL-4, promotes differentiation into memory B cells at the expense of plasma cell differentiation (Acosta-Rodriguez et al., 2004). The absence of Gal-3 generates a higher number of ASC in response to other parasites challenge (Brand et al., 2012; Toscano et al., 2012) and also drives lupus-like disease by promoting spontaneous germinal centers in aged mice (Beccaria et al., 2018). In addition, Gal-1 and Gal-8 have been involved in the development of plasma cells. Gal-1 is significantly increased by B cell differentiation and is directly regulated by Blimp-1, in murine and human B cells (Tsai et al., 2008). However, Gal-1 would not be strictly essential since Gal-8 can functionally compensate for its absence, being the antibody production not affected (Tsai et al., 2011). In our model, we cannot rule out that the absence of Gal-8 might be compensated by another Gals. For instance, Gal-9, that belongs to the same family as Gal-8 and is related to the promotion, formation, differentiation, and survival of B-lineage cells, could be involved in these events (Cao et al., 2018). On the other hand, since Tfh cells are involved in GC formation, somatic hypermutation and affinity maturation (Qi, 2016), the significant increase of Tfh could impact promoting B-lineage development in iGal-8KO mice resulting in uncontrolled GC reactions (Sage and Sharpe, 2015).

The study of DCs showed their significant increase in the spleen of iGal-8KO mice ( Figure 3E ). These cells have the capacity to activate and become potent antigen presenters. DCs are known to secrete chemokines that recruit different cell populations of the innate response. In addition, Gals can inhibit or increase the activation of these cells (Tribulatti et al., 2020), which could explain the increase in the total number of monocytes and neutrophils in this organ ( Figures 2B, C ). In turn, Gals can also modulate key cellular activities that leads to leukocyte recruitment, probably by affecting chemoattraction and/or cell adhesion, potentially through both extracellular and intracellular signaling pathways (Liu and Stowell, 2023). Furthermore, the increase in these myeloid populations could be associated with a possible extramedullary myelopoiesis in the spleen (Swirski et al., 2009; Robbins et al., 2012; Zick, 2022). This event has been described in other infectious processes such as those triggered by Plasmodium sp., Leishmania major and Ehrlichia muris (Mirkovich et al., 1986; MacNamara et al., 2011; Belyaev et al., 2013). The absence of Gal-8 could be favoring this phenomenon. As mentioned before, the most striking immunological event of acute T. cruzi infection is the polyclonal activation of lymphocytes, leading to immunosuppression and splenomegaly (Minoprio et al., 1989). An intense proliferative activity of either B cells, CD4⁺ and CD8⁺ T cells, specific or nonspecific (Minoprio et al., 1986) is unleashed. As the infection progresses, a decrease in splenomegaly and a specific immune response is developed that is essential to control parasite replication and the survival of the host. The spleen reduction was observed in iWT mice from day 60 pi, however iGal-8KO mice failed to carry out it ( Figure 1C ). CD4⁺ and CD8⁺ T populations were increased in iGal-8KO mice compared to their iWT counterpart ( Figure 5 ). Different authors have shown that Gal-8 is involved in Tregs differentiation modulating IL-2 and TGF-β signaling and thus, promoting cell death and inhibiting the proliferation of stimulated T cells through the increase of inhibitory molecules and the production of IL-10 (Sampson et al., 2016). This ability of Gal-8 generates in the murine model of autoimmune uveitis a decrease in the production of cytokines of the Th1 and Th17 pathways, favoring the decrease of the inflammatory process (Sampson et al., 2015). In addition, the role of Gal-8 as an immunosuppressor in experimental autoimmune encephalomyelitis was demonstrated. Mice deficient in this Gal show increased inflammatory levels leading to a more severe chronic phase due to a decrease in the regulatory population (Pardo et al., 2017). In addition, during T. cruzi infection, Tregs can induce a suppressive action on CD4⁺, CD8⁺ T lymphocytes and on other populations such as antigen-presenting cells (Araujo Furlan et al., 2018). In our T. cruzi chronic murine model Gal-8 showed anti-inflammatory properties (Bertelli et al., 2020), similar results were observed in a Gal-8 deficient murine model of autoimmunity (Pardo et al., 2017). However, we did not observe differences in the total numbers of regulatory T cells ( Figures 6A, B ) and IL-10 levels between infected groups ( Supplementary Figure 3 ). When analyzing phenotypic characteristics related to functional status of these regulatory cells, we found an increase in CTLA-4 and CD39 ( Figures 6C, D ), which suggests that the overexpression of these molecules in iGal-8KO mice could be a compensatory effect, since the same number of regulatory cells are involved in controlling twice the number of cells ( Figure 6B ).

The study of splenocytes proliferation showed that levels were indeed significantly increased in iGal-8KO mice ( Figure 7 ). Using Ki-67 we identified, by flow cytometry, that both CD4⁺ and CD8⁺ T-lineage populations were proliferating ( Figure 7 ). The status of this lineage showed that effector and memory cells were increased in the spleens of iGal-8KO mice ( Figure 5 ). Increased proliferation in Gal-8KO, was accompanied by an increase in the production of IL-6 ( Supplementary Figure 3 ). IL-6 is a pleiotropic cytokine (Hunter and Jones, 2015) that enhances the immune response and has a strong pro-inflammatory ability (Gao and Pereira, 2002). While Gal-8 is known to exacerbate IL-6 production by DC stimulation (Carabelli et al., 2017; Carabelli et al., 2018), in this study we show that iGal-8KO mice produce higher levels of IL-6 than their iWT counterpart, supporting its induction by infection-derived stimuli. Depending on the context, IL-6 might play pro- or anti-inflammatory roles that can be protective in infectious diseases it helps solve, but deleterious in autoimmune affections associated with the chronic presence of this cytokine (Hunter and Jones, 2015). IL-6 is crucial for host survival from T. cruzi infection, as its absence leads to increased parasitemia and earlier mortality, appearing to play a role as a direct mediator of inflammation (Gao and Pereira, 2002). In our study we observed sustained IL-6 presence in the chronic phase of infection. Considering the persistent splenomegaly in the absence of Gal-8 and the increased titter of IL-6, it appears that a chronic inflammatory effect can also be attributed to this cytokine. Additionally, Gal 8 seems to modulate IL 6 production. Thus, IL-6 appears to be involved in both stages of infection, playing a protective role during the acute phase, and a detrimental one in the chronic phase, similar to what occurs during its sustained presence in several autoimmune diseases.

Gal-8 has been reported to induce apoptosis via different pathways such as the accumulation of p21, a cyclin-dependent kinase inhibitor that exhibits strong anti-apoptotic activity (Hadari et al., 2000; Arbel-Goren et al., 2005). CD4^highCD8^high thymocytes enter apoptosis via caspases activation, after being stimulated with Gal-8 (Tribulatti et al., 2020). Gal-8 stimulation in Jurkat T cells induces apoptosis via the phosphatidic acid mediated ERK1/2 activation pathway (Norambuena et al., 2009). It is interesting to highlight that different groups have demonstrated that human peripheral blood mononuclear cells (PBMCs) must be activated either by PHA or anti-CD3/CD28, in order to be sensitive to apoptosis induced by Gal-8 (Norambuena et al., 2009; Cattaneo et al., 2011). Then, it was proposed that Gal-8, together with other galectins that are able to kill activated T cells, contribute to T cells homeostasis (Norambuena et al., 2009). Given that the persistent increased cellularity in the spleens from iGal-8KO mice corresponds mainly to the T cell population ( Figure 5 ), we could hypothesize that this excessive proliferation would be related to the inability to activate apoptosis pathways.

In our efforts to deepen our knowledge of the role of this galectin in the development of chronic splenomegaly, we extended our analysis to chronic T. gondii infection in Gal-8KO and the counterpart mice. The spleen weight and cytometry populations studies didn’t show differences between infected groups at different times of infection ( Supplementary Figure 4 ), indicating that the induced splenomegaly persistence in Gal-8KO mouse is not a general event but a result of the interaction with the T. cruzi infection process.

Another issue to take in consideration, is that the absence of Gal-8 didn’t modify parasitemia values nor mortality rate in T. cruzi-infected mice (Bertelli et al., 2020). Likewise, similar number of cerebral cysts and mortality values were observed in T. gondii-infected mice. Taken together, we can speculate that Gal-8 is not related to the events involved in protection, in the analyzed models. A compensatory mechanism by another galectin cannot be ruled out.

The Gal-8KO mouse model provides evidence supporting that the retraction of the acute splenomegaly is strongly related to Gal-8 participation during the T. cruzi infection, a parasite known as a stronger inducer of inflammation.

-The murine model used allowed us, for the first time, to associate a specific molecule, Gal-8, with the resolution of acute splenomegaly induced by Trypanosoma cruzi infection.

-The absence of Gal-8 did not modify parasitic load or mortality rate in either Trypanosoma cruzi- or Toxoplasma gondii-infected mice.

-The generation of germinal centers is hampered in the absence of Gal-8 during the infection with Trypanosoma cruzi.

-The absence of Gal-8 promotes increased proliferation of CD4⁺ and CD8⁺ T cells during Trypanosoma cruzi infection.

-Gal-8 plays an anti-inflammatory role in Trypanosoma cruzi infection.