B cells are the main effectors of the humoral immunity, which is mediated by their activation and secretion of different classes of immunoglobulins against the original antigen (Nutt et al., 2015) However, these cells exhibit other antibody-independent functions such as their performance as antigen-presenting-cell (APC) or cytokine secretion that modulate the entire immune response (Shen and Fillatreau, 2015; Getahun and Cambier, 2019).

The immune response associated with survival against infection by Trypanosoma cruzi, the agent of Chagas disease, infection relies on the generation of a CD4⁺ T helper 1 (Th1) profile and a strong CD8⁺ T response that is, however, unable to resolve the infection (Tarleton et al., 2000; Kumar and Tarleton, 2001). Although the T cell response is the one that carries out the effector action, several other cell types such as macrophages and dendritic cells are also involved and have been well studied (Acevedo et al., 2018). On the other hand, beyond the particular parasite-induced proliferative effects that have been observed, our knowledge on the effect of T. cruzi infection on B cells is still unclear. T. cruzi induces polyclonal lymphocytes proliferation and unspecific hypergammaglobulinemia during the early stage of the acute phase of the infection with an antigen-specific delayed response (Ortiz-Ortiz et al., 1980; Minoprio et al., 1988; Rottenberg et al., 1993; el Bouhdidi et al., 1994; Bermejo et al., 2011). This unspecific antibody response is characterized by their maturation as plasma cells and the secretion of parasite-unrelated antibodies, a response that is maintained all along the infectious process (D’Imperio Lima et al., 1986; Minoprio et al., 1988; Bermejo et al., 2011). This pathogenic manifestation is known as polyclonal expansion and is histologically characterized by an excessive generation of germinal centers (typical and atypical) accompanied by extensive generation of extra-follicular B cell response (Montes et al., 2007; Bermejo et al., 2011). Particularly, different cell extracts or antigen preparations from either epimastigotes (the insect replicative stage) or trypomastigotes (the mammal infective stage) induce a T cell-independent B cell proliferative response and even several components of the parasite are related to this effect, although just a few are molecularly identified such as trans-sialidase, malate or glutamate dehydrogenase and proline racemase (Montes et al., 1999; Reina-San-Martín et al., 2000; Montes et al., 2002; Gao et al., 2002; Montes et al., 2006). In contrast, studies with live trypomastigotes are scarce.

In several models, both infectious and non-infectious, the importance of B cells in the development of the T cell response has been addressed and it has been shown that B lymphocytes can act as regulators of the T response and play an important role in the effector activities of the cellular response (Candando et al., 2014; Gorosito Serrán et al., 2015; Lykken et al., 2015; Catalán et al., 2021). There is indirect evidence of the regulatory role of B cells in the development of the immune response against T. cruzi. Although there are some discrepancies, most of the evidence supports the idea that both, partial (Xid mouse) and absolute (μMT mouse) deficiencies in B cells generate important outcomes in the development of the infection, generally associated with a stronger inflammatory response presenting higher levels of IFNγ, IL-6, IL-18 and TNFα in serum (Minoprio et al., 1993; Cardillo et al., 2007; Gorosito Serrán et al., 2017; da Rocha et al., 2019). In the μMT mouse absolute depletion model, Gorosito Serrán et al. demonstrate that the increased of infected animals mortality was associated with an unconventional CD4⁺ T cell exacerbated response leading to an uncontrolled immune response and increased inflammation (Gorosito Serrán et al., 2017). Furthermore, it was reported that B cells help to develop the CD8⁺ response in mice infected with T. cruzi (Fiocca Vernengo et al., 2020). These results lead us to infer that B cells undoubtedly play a regulatory role in the overall immune response against the parasite. However, little is known about the mechanisms associated with this ability, and there is almost no direct cellular evidence that mechanistically interprets and supports these findings.

In this work, we analyze the effects of live trypomastigotes on B cells and, in turn, their regulation of the T cell compartment. In search for clues that could provide evidence, we followed the proliferative response of B cells induced by trypomastigotes, and we then analyzed their effects on T cells and their correlation with the in vivo findings.

Within the physiology of infection by T. cruzi, the relevance of B cells was circumscribed to the production of antibodies. Despite the humoral response as a whole is not sufficient to effectively eliminate the parasite (Acevedo et al., 2018), relevant antibodies are induced during the infection such as lytic antibodies directed against anti-α-gal epitopes, neutralizing the activity of trans-sialidase and TcDAF (Tambourgi et al., 1993; Almeida et al., 1994; Buschiazzo et al., 2012). On the other hand, there are defined parasite factors such as trans-sialidase, proline-racemase and glutamate dehydrogenase that can, directly or indirectly, induce cytokine secretion and proliferation in B cells (Reina-San-Martín et al., 2000; Gao et al., 2002; Montes et al., 2006).

Although B cell ability to secrete cytokines and to act as an antigen-presenting cell is well known, it was historically considered as a secondary role. During the last years, it demonstrated that B cells turned out to be more than producers of immunoglobulins. Effector B cells are capable of secreting IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, GM-CSF, and IFNγ, important cytokines in regulating the assembly and functionality of the immune system (Lund, 2008; Shen and Fillatreau, 2015). Furthermore, it was demonstrated that in T. cruzi infection, B cells are able to produce IL-17 via the action of trans-sialidase (Bermejo et al., 2013). In models of autoimmune diseases, chronic inflammatory processes and cancer, there are multiple reports of regulatory B cells (Gorosito Serrán et al., 2015; Sarvaria et al., 2017). Regulatory B cells (Breg) are a heterogeneous population that produces IL-10, IL-35 and/or TGFβ capable of inhibiting the Th1, Th17 and CD8⁺ responses, as well as the secretion of pro-inflammatory cytokines such as IFNγ or TNFα by macrophages or dendritic cells (Catalán et al., 2021). Furthermore, they could favor the development of regulatory T cells that, in turn, might impede the resolution of the pathology (Burdin et al., 1997; Candando et al., 2014; Lykken et al., 2015; Rosser and Mauri, 2015; Catalán et al., 2021). Other molecules have been associated in the generation of Breg such as APRIL and Blimp-1 (Fehres et al., 2019; Wang et al., 2019). In Figure 3 , we showed that in our model we could detect the up-regulation of APRIL transcript.

So far, multiple populations of Breg have been identified according to their surface markers and the cytokines they release. The reported B populations that can express IL-10 are T2-MZP (CD21⁺/CD23⁺), classical B10 (CD5⁺/CD1d⁺), MZ (CD21⁺/CD23^lo/-), plasma cells and plasmablasts (CD138⁺) (Rosser and Mauri, 2015). At present, the induction mechanism of these cells has not been fully elucidated and there is no known specific transcription factor that regulates this cell differentiation which might reflect the plasticity of these cells. However, some required pathways have been described. In an inflammatory context (IL-1β, IL-2, IL-6 and IFNα, among others) there are at least two known pathways that are involved in the generation of these cells, the B receptor-dependent (BCR) and the Toll-like receptors dependent. In the first one, it would appear that signaling through the BCR is critical. In this pathway, the Bregs would be antigen-specific and when presenting the antigen in the context of MHC-II to the lymphocyte T CD4⁺ they would be stimulated by it via CD40/CD40L and IL-21. In the second route, it was possible to determined that the TLR2/4/9 receptors are involved and their signaling via MyD88 plus IL-21 would induce the secretion of IL-10 by B cells (Yoshizaki et al., 2012; Horikawa et al., 2013; Lykken et al., 2015; Okada et al., 2018).

The presence of Breg cells and their regulatory activity on the T cell response have recently been described in the course of infectious diseases caused by pathogens of the genera Plasmodium, Listeria, Leishmania, and Schistosoma (Horikawa et al., 2013; Gorosito Serrán et al., 2015; Silva-Barrios et al., 2017). Within the parasitic infections, the B populations capable of developing this regulatory profile differ in each case and in several of them they were little studied. For example, in Leishmania donovani a CD19⁺/CD21⁺/CD23^lo (marginal zone phenotype) secreting IL-10 population was identified, while in the related Leishmania major, the identified population was CD19⁺/CD1d⁺/CD5^lo (B10 phenotype) (Ronet et al., 2010). On the other hand, in Plasmodium they are described as IL-10 secreting B220⁺ and in Schistosoma as IL-10 and TGF-β secreting CD19⁺/CD1d⁺ cells (Liu et al., 2013; Van Der Vlugt et al., 2014).

In T. cruzi infection, previous works with B cell-depleted Xid or µMT mice provided evidence supporting that B cells can regulate the T cell response (Minoprio et al., 1993; Gorosito Serrán et al., 2017; Fiocca Vernengo et al., 2020). However, the mechanisms and cell populations involved in the T CD4⁺ modulation process are still not understood.

In this study, we demonstrated that the infectious form of the parasite directly stimulated B cells that can regulate T CD4⁺ cells, impairing their proliferation and inducing an apoptotic process.

Notably, we determined that trypomastigotes directly induced naïve B cell proliferation accompanied by secretion of IL-6 and IL-10. These molecules are involved in B activation and cell proliferation. Although here we have shown that they are not involved in B cell division, both regulated the immunoglobulin secretion. There are several reports that support the induction of IL-6 and IL-10 via TLR4 (Greenhill et al., 2011; Sanin et al., 2015). It has also been reported that T. cruzi GIPLs are able to stimulate TLR4 (DosReis et al., 2002). Since B cells express TLR4 (Ma et al., 2015), the mechanism of induction of these cytokines might be explained in this way. Also, we detected the presence of APRIL and BAFF transcripts which are classically expressed by myeloid accessory cells, representing a hypothetic autocrine mechanism induced directly by trypomastigotes on B cells in the context of CD40 stimulation. Even these growth factors are canonically secreted by another cell source rather than B cells, evidence has emerged of its expression by B cells themselves both in vitro and in vivo (Mackay et al., 2003; Ng et al., 2005; Chu et al., 2007). Several reports state that toll-like receptors (TLRs) signaling could stimulate BAFF and APRIL expression in B cells and other antigen presenting cells (APCs) (Chu et al., 2007; Sakai and Akkoyunlu, 2017). In this line, pathogen-associated molecular patterns (PAMPs) from trypomastigotes have been identified, including GPI anchored mucins, membrane GIPL, DNA CpGs and parasite RNA (Rodrigues et al., 2012). Since B cells express variety and quantity of TLRs (Ma et al., 2015), the expression of BAFF and APRIL might be associated with their presence.

Bermejo et al. (2011) have reported that, in the context of T. cruzi infection, BAFF levels are elevated and it is related to polyclonal B cell response and antibody titers. Moreover, in another report of the same workgroup they observed that the T. cruzi antigen glutamate dehydrogenase induced B cells proliferation mediated by BAFF produced by CD11b⁺ cells. The authors hypothesized that these cells were macrophages but exist the possibility that they could be also CD11b⁺ expressing B cells (Montes et al., 2006).

In humans, circulating regulatory B10 results altered during the chronic stage and is associated with improved cardiac function in the indeterminate stage of Chagas disease (Passos et al., 2019; Girard et al., 2021). Also, since we observed a regulatory phenotype on parasite-stimulated B cells we tested for the presence of IL-21, a cytokine related to regulatory B10 cell generation (Horikawa et al., 2013). Surprisingly, given that IL-21 is paradigmatically mainly produced by CD4⁺ cells, we have found IL-21 expression in B cells during the experimental infection. This finding is noteworthy as IL-21 expression has previously been reported only in Hodgkin’s lymphoma (B-cell-related lineage) (Scheeren et al., 2008). It has been proved that IL-6 is able to induce the production of IL-21 in CD4⁺ cells (Dienz et al., 2009). It is also known that B cells are responsive to IL-6 and constitute a growth and differentiation factor. In our model, we showed that this cytokine is involved in IgG secretion ( Figure 2B ). Furthermore, NFAT, a downstream signaling molecule shared in the TCR and BCR cascade, has been shown to be involved in IL-21 secretion (Antony et al., 2004). Therefore, the combination of IL-6 and BCR signals in B cells could be plausible stimuli to induce IL-21 in these cells. Overall, the expression of B cell growth factors such as BAFF and APRIL plus IL-21 by B cells could represent a mechanism of T. cruzi to generate B-cell subsets that regulate the immune response against the pathogen.

We also showed that T. cruzi stimulated B cells damped T cell proliferation, the typically Th1-like cytokine secretion, and increased the apoptotic rate (Annexin V⁺/PI^-) of CD4⁺ activated T cells. Moreover, recent evidence from Gorosito-Serran et al. (2015) described that the absence of B cells results in an uncontrolled CD4⁺ inflammatory response. These results suggest that T. cruzi trypomastigotes are able to manipulate B cells to restrain the CD4⁺ T cell activation inhibiting cell proliferation and increasing apoptosis induction.

The stimulation of CD40 on B cells was necessary for most of the described features or the enhancement of B regulatory cells generation in this model. This opens two hypotheses about the CD40L source in vivo during T. cruzi infection: the first, according to the paradigm, is that CD4⁺ cells present in lymphoid tissues are the natural CD40L source. Upon stimulation with CD40L from these cells, regulatory B cells are generated and constitute a restraint of the immune response against the parasite, contributing to the prevention of pathogen clearance from the host. The other possibility is that CD40L stimuli proceed from another source, like platelets. It is well-known that in the course of an acute infection with T. cruzi, platelet blood count decreases dramatically generating thrombocytopenia (de Titto and Araujo, 1988). In a previous work from our laboratory, we demonstrated that trans-sialidase from the parasite alters platelet surface glycan profile and induces their removal from circulation, promoting the marked platelet count fall (Tribulatti et al., 2005). In addition to this phenomenon, platelet activation results in its own disassembling into smaller fragments (vesiculation) and can also cause thrombocytopenia (Heemskerk et al., 2002; Taus et al., 2019). During platelet activation, they release several components, including CD40L. Some reports claim that platelet-derived CD40L might represent a relevant source of the circulating form, which is crucial for the proper development of the B response (André et al., 2002; Viallard et al., 2002; Sprague et al., 2008; Aloui et al., 2014). Moreover, the injection of small molecules that mimic the functional trimeric CD40L can control parasitemia and mortality (Fournel et al., 2005; Habib et al., 2007). Further research needed to be performed to clarify these issues.

In the context of T. cruzi infection we have found that IL-10 production by B cells is elicited by different subpopulations. Surprisingly, we detected that follicular phenotype B cells (Fas⁺/GL7⁺) express IL-10 upon T. cruzi infection. A recent report of Leishmania amazonensis infection also concludes that B2 follicular cells are relevant on IL-10 production, indicating that both pathogens might share common mechanisms relying on their phylogenetic similarity (Firmino-Cruz et al., 2018). Also, we observed that newly formed B cells (NF-B) constitute an important source of IL-10 in T. cruzi infection ( Figure 5 ). It has been reported that NF-B are significant IL-10 producers in healthy individuals and expand in several viral (HIV, HCV) and bacterial infections, thus, they might represent a new regulating subset in parasitic infections, as Chagas disease (Giltiay et al., 2019). In addition, we observed the appearance of CD5⁺/CD1d IL-10⁺ B cells after infection according to several previous reports in different infectious processes, (Gorosito Serrán et al., 2015). This former phenotype is associated with the classical regulatory B cell subset, which shares molecular markers with B1 cells.

According to our in vitro results, the proliferation of purified murine B cells is induced by trypomastigotes per se, but a significant fraction of cells does not divide. However, upon CD40 stimulation the fraction of undivided cells was reduced. This fact reflects itself the heterogeneity of natural B subsets, each needing a different nature and combination of stimuli including TLR, BCR and CD40 among others. Moreover, it is well-known that T. cruzi induces a massive expansion of the B cell compartment, reflected in the characteristic splenomegaly, and a consequent polyclonal response. According to these facts, it seems reasonable that the expression of IL-10 by B cells is not confined to a unique subset but comes from different phenotypes with different ontogenesis.

All the evidence described here suggests that T. cruzi trypomastigotes are able to modulate the fate of B cells. In this condition, the stimulated B cells secrete various cytokines and immunoglobulins and module the response of T CD4⁺ cells by altering cytokine secretion, arresting cell division, and apoptosis induction. Our results add strong evidence of the central relevance of B cells during the pathogenesis of Chagas disease beyond the antibodies production.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Committee on the Ethics of Animal Experiments of the Universidad Nacional de San Martín (UNSAM).

MS, RV, OC, and JM were responsible for the study design and/or funding acquisition. MS, AB, CAP, and JM performing the experiments. MS, OC, and JM were responsible for results analysis. Project supervision was performed by OC and JM. MS, OC, and JM were responsible for writing the original manuscript draft. All authors contributed to the article and approved the submitted version.

This work was funded by grants from the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (ANPCyT), Argentina and from the National Institutes of Health, USA (grant R01AI104531 to OC). RV is supported by R01GM121595 from the National Institute of General Medical Sciences (NIH/NIGMS), 1R01CA233945 and from the National Cancer Institute of the National Institutes of Health (NIH/NCI) and the National Cancer Institute of the National Institutes of Health under Award Number P30CA240139. The funders had no role in the design of studies, data collection, and analysis, or in the decision to publish or the preparation of the manuscript.

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