As essential components of the adaptive immune system, B cells are mostly recognized by their role as mediators of humoral immune responses through antibody production (1). However, upon activation, B cells can also produce a wide range of cytokines (2) as well as present antigens to T cells, thus playing a critical role in orchestrating an immune response (3–5).

B cells represent a heterogeneous cell population that based on their ontogeny, anatomical location and cellular markers can be divided into three different subsets: B1 B cells, follicular B cells (FO), and marginal zone (MZ) B cells (6, 7). Particularly, B1 B cells originate mainly during embryonic development from precursors present in the yolk sac and fetal liver, and their numbers are maintained by cell division in adulthood (6, 8, 9). Although B1 B cells are mainly located in peritoneal and pleural cavities (10, 11), in mice (12), they can also be found in the spleen and can be distinguished from B2 B cells by their low expression of B220 (9, 13, 14). One of the main features of the B1 B cell population is the production of natural antibodies (NAbs), which are produced in the absence of antigenic stimulus (15). Albeit the molecular mechanism regulating the production of NAbs by B1 B cells is not fully understood, it appears to be associated with a down-regulation of CD1d (16). NAbs participate in many physiological processes, including regulation of homeostasis, prevention of inflammation, and infections (17). In addition, due to their polyreactivity, NAbs and B1 B cells are involved in autoimmunity (18). Besides, upon activation, B1 B cells produce large quantities of IL-10, a potent anti-inflammatory cytokine (19–22). In the context of pregnancy, the role of B1 B cells has been evaluated controversially. Indeed, it was proposed that B1 B cells can produce natural/polyreactive antibodies which may be involved in the onset of preeclampsia (23), a pregnancy-associated disorder with suspected autoimmune origin (24). On the other hand, B1 B cells were shown to control the differentiation of pro-inflammatory Th1 and Th17 cells, thus promoting a healthy gestation in mice (25). Furthermore, IL-10 producing B1 B cells protect the fetus from rejection after adoptive transfer into an abortion-prone mice model (26).

In the work presented here, we aimed to identify and functionally characterize the splenic B1 B cell population in pregnant mice.

In this study, we have demonstrated that during pregnancy in mice, splenic B1 B cells are expanded and acquire an anti-inflammatory profile.

Pregnancy in mammals represents a unique challenge for the maternal immune system as it should be able to tolerate the developing semi-allogenic fetus and prevent rejection reactions. This requires temporal modifications in the innate as well as the adaptive immunity (28). Among others, maternal B cell depletion has been proposed as a potential mechanism to avoid immune self-reactivity which could compromise pregnancy well-being (29). However, in contrast, the expansion of certain B cell populations, namely regulatory B cells and MZ B cells, has been previously described in pregnant mice. This expansion has been suggested to be part of the intricate mechanisms of immune tolerance induced during gestation (23, 25, 30). Similarly, here we have shown an in-silico analysis of a transcriptomic profile established in pure isolated total splenic B cells from P and NP mice, showing an up-regulation of genes and pathways associated with cell cycle and DNA replication in B cells during gestation. Interestingly, when we analyzed expression levels of the proliferation marker Ki-67 in main B cell populations in the spleen, only B1 B cells, but not FO or MZ B cells, showed an increased expression of Ki-67 in P mice compared to NP animals, suggesting that only B1 B cells are boosted to proliferate during gestation. Indeed, percentages of B1 B cells were significantly higher in the spleen of P mice as compared to NP animals. From a functional point of view, B-1 B cells were shown to regulate immunity by their special cytokine secretion pattern (15). B1 B cells constitutively produce IL-10, which seems to switch them towards regulatory functions (15, 31). Furthermore, B1 B cell-derived IL-10 production has been associated with attenuated responses to infections with Leishmania (32, 33) and atherosclerosis (34) and seems to enhance B1 B cell expansion via induction of proliferation (35). Here we have demonstrated that B1 B cells from P mice produced significantly higher levels of IL-10 compared to B1 B cells from NP animals, suggesting that during pregnancy, B1 B cells boost their immune regulatory capacity. In addition, the higher production of IL-10 by B1 B cells from P mice could also provide a mechanistic explanation for the higher proliferation rate as well as the expanded proportion of B1 B cells observed during pregnancy. The potent anti-inflammatory cytokine IL-10 is a critical component in the process of pregnancy tolerance as it regulates the inflammation elicited by paternal antigen recognition (36). Indeed, serum levels of IL-10 were shown to be significantly increased in women coursing normal pregnancies but not in those pregnancies destined for loss (37). On the other hand, the pro-inflammatory cytokine TNF-α undergoes the opposite kinetics, expressed at lower levels in women coursing normal pregnancies and at higher levels in those suffering pregnancy loss (37). Moreover, the IL-10/TNF-α ratio is increased in normal pregnancies but not in women with pregnancy failure (37). In this line, the present study did not only demonstrate a higher IL-10 and lower TNF-α expression in B1 B cells during pregnancy, but also an increased IL-10/TNF-α ratio, thus, positioning the B1 B cells as central components for the cytokine balance required for pregnancy wellbeing.

A major function of B1 B cells is the generation of natural antibodies (NAbs), mainly IgM and IgG₃, which are produced in the absence of antigen stimulation (15). The exact mechanisms that induce NAbs production remain incompletely understood. However, recently, it has been demonstrated that the suppression of CD1d expression on splenic B1 B cells is associated with NAbs production (16). In the present study, we found a significant reduction in the expression of CD1d in B1 B cells during normal pregnancy, suggesting an enhanced capacity of NAbs production during gestation. Supporting this idea, we have previously demonstrated that serum levels of both, IgM and IgG₃, are significantly higher in pregnant women compared to non-pregnant controls (38). Similarly, using a mouse model of pregnancy disorders, we also have demonstrated that serum levels of IgM are significantly higher in normal pregnant mice compared to non-pregnant and pregnant mice suffering pregnancy failures (39). It is worth mentioning that B1 B cells and natural/auto-antibodies they produce have previously been implicated in the onset of preeclampsia (PE), a pregnancy-associated syndrome with a strong inflammatory profile and suspected autoimmune origin (23). Nevertheless, in the mentioned work, the numbers, as well as the functionality of B1 B cells, were measured in diagnosed PE patients, opening the question of whether they are involved in the dysregulated mechanisms that trigger the onset of the disease or if its consequence. Further studies are demanded to better understand the role of B1 B cells in PE patients.

Although the presented experiments have been performed in mice and several substantial molecular differences between human and murine pregnancies have been reported (40), we expect transferability of our observations to humans.

Overall, data presented here position the B1 B cells as key players in cytokine balance required for the immunological equilibrium in pregnancy. Further studies are demanded to better understand the biological significance of B1 B cell expansion as well as NAbs production during pregnancy.

Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE174290.

The animal study was reviewed and approved by CICUAL, Medical Faculty, Buenos Aires University (CICUAL resolution number: 2248).

NV designed experiments, performed experiments, analyzed data, and wrote the manuscript. MSV collaborated in some experiments and helped to draft the manuscript; MD analyzed data and helped to draft the manuscript. UM analyzed data and critically reviewed and discussed the prefinal version of the manuscript. FJ designed experiments, analyzed data, contributed with reagents, supervised the work, and wrote the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica PICT (2016/004), PICT (2016-201-0151), and PICT-PRH (2016-004) to FJ.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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