Leishmania amazonensis Promastigotes or Extracellular Vesicles Modulate B-1 Cell Activation and Differentiation.

B-1 cells are considered an innate-like B cell population that participates in effective innate and adaptive responses to pathogens. B-1 cells produce immunoglobulins, cytokines, chemokines, migrate to inflammatory sites, and differentiate into mononuclear phagocyte-like cells. Murine B-1 cells phagocytosed Leishmaniain vitro and in vivo and participate in immunity against Leishmania. Our group showed that B-1 cells or their extracellular vesicles (EVs) led to a resistance to experimental infection by L. amazonensis. However, the B-1 cells' responses to Leishmania or EVs isolated from parasites are still poorly characterized. Studying the activation and differentiation of B-1 cells in vivo can contribute to a better understanding of how these cells participate in immunity to L. amazonensis. Thus, we evaluated the expression of myeloid (M-csfr, G-csfr, Spi-1) and lymphoid (EBF, E2A, IL-7R) lineage commitment factors, Toll-like receptors (TLRs), activation cell surface markers, nitric oxide (NO) and reactive oxygen species (ROS) production in murine peritoneal B-1 cells collected after 24 or 48 h post-infection with Leishmania (Leishmania) amazonensis promastigotes or EVs released by the parasites. Our results demonstrated that L. amazonensis infection did not stimulate the expression of CD40, CD80, CD86, F4/80, and MHC II in B-1 cells, but a significant decrease in the production of NO and ROS was observed. The infection induced a significantly higher arginase expression in B-1 cells, but the stimulation with EVs led to a decrease in this gene expression. TLR-2 and TLR-6 had significantly higher expression in B-1 cells from mice intraperitoneally stimulated with the parasite. The TLR-9 expression was higher in animals infected or stimulated for 48 h with EVs. Interestingly, in B-1 cells the stimulus with L. amazonensis led to a substantial increase in the expression of myeloid restricted transcription factors. Thus, our study suggests that the parasites or EVs differently modulated the activation and differentiation of B-1 cells.

Deregulation of Ikaros expression in B-1 cells: New insights in the malignant transformation to chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a chronic form of leukemia that originates from an abnormal expansion of CD5+ B-1 cells. Deregulation in the BCR signaling is associated with B-cell transformation. Contrariwise to B-2 cells, BCR engagement in B-1 cells results in low proliferation rate and increased apoptosis population, whereas overactivation may be associated with lymphoproliferative disorders. It has been demonstrated that several transcription factors that are involved in the B cell development play a role in the regulation of BCR function. Among them, Ikaros is considered an essential regulator of lymphoid differentiation and activation. Several reports suggest that Ikaros expression is deregulated in different forms of leukemia. Herein, we demonstrated that CLL cells show decreased Ikaros expression and abnormal cytoplasmic cell localization. These alterations were also observed in radioresistant B-1 cells, which present high proliferative activity, suggesting that abnormal localization of Ikaros could determine its loss of function. Furthermore, Ikaros knockdown increased the expression of BCR pathway components in murine B-1 cells, such as Lyn, Blnk, and CD19. Additionally, in the absence of Ikaros, B-1 cells become responsive to BCR stimulus, increasing cell proliferation even in the absence of antigen stimulation. These results suggested that Ikaros is an important controller of B-1 cell proliferation by interfering with the BCR activity. Therefore, altered Ikaros expression in CLL or radioresistant B-1 cells could determine a responsive status of BCR to self-antigens, which would culminate in the clonal expansion of B-1 cells.

B-1 cells and B-1 cell precursors prompt different responses to Wnt signaling.

Recently several studies demonstrated a role for the Wnt pathway in lymphocyte development and self-renewal of hematopoietic stem cells (HSCs). B-1 cells constitute a separate lineage of B lymphocytes, originating during fetal hematopoiesis, expressing lymphoid and myeloid markers and possessing self-renewal ability, similar to early hematopoietic progenitors and HSCs. A plethora of studies have shown an important role for the evolutionary conserved Wnt pathway in the biology of HSCs and T lymphocyte development. Our previous data demonstrated abundant expression of Wnt pathway components by B-1 cells, including Wnt ligands and receptors. Here we report that the canonical Wnt pathway is activated in B-1 cell precursors, but not in mature B-1 cells. However, both B-1 precursors and B-1 cells are able to respond to Wnt ligands in vitro. Canonical Wnt activity promotes proliferation of B-1 cells, while non-canonical Wnt signals induce the expansion of B-1 precursors. Interestingly, using a co-culture system with OP9 cells, Wnt3a stimulus supported the generation of B-1a cells. Taking together, these results indicate that B-1 cells and their progenitors are differentially responsive to Wnt ligands, and that the balance of activation of canonical and non-canonical Wnt signaling may regulate the maintenance and differentiation of different B-1 cell subsets.

Alteration in Ikaros expression promotes B-1 cell differentiation into phagocytes.

Ikaros is a broad transcription factor pointed as a critical regulator of lymphocyte development. Recent reports have emphasized that distinct isoforms of Ikaros control the dichotomy of the hematopoietic system into lymphoid and myeloid lineages. In addition, expression of dominant-negative isoforms of Ikaros is linked to abnormal hematopoiesis, which could culminate in hematological disorders due to loss of function of the protein. B-1 cells are an intriguing subtype of B-lymphocytes that preserves some myeloid characteristics. These cells are able to differentiate into phagocytes (B-1CDP - B-1 cell derived phagocytes) in vitro and in vivo. During such process, reprogramming of gene expression occurs: lymphoid genes are turned off, while expression of myeloid genes is increased. This study aims to investigate whether Ikaros could be related to the control of B-1 cell plasticity. Interestingly, Ikaros expression by B-1CDP cells was found to be relatively low, and the protein is abnormally localized in the cytoplasm. Moreover, the isoforms expressed by B-1 cells are different from those expressed by other lymphocytes, with expression of active isoforms being almost absent in B-1CDP. Based on these findings, Ikaros could be an important factor driving the differentiation and proliferation of B-1 cells.

Ikaros could be a key factor in the maintenance of "B-side" of B-1 cells.

Ikaros, a zinc finger transcription factor, is an important regulator of the hematopoietic system. Several studies have suggested the role of Ikaros in the development, maturation, activation and differentiation of lymphocytes. To elucidate this mechanism, it is important to understand how this transcription factor works in the dichotomy of the hematopoietic system, a topic that remains uncertain. Herein, we investigated the role of Ikaros in the control of the lymphomyeloid phenotype of B-1 lymphocytes. We found that Ikaros, as well as its target genes, are expressed in B-1 cells,. Moreover, Ikaros positively regulates the expression of Flt3, Gfi and Il7r, while it down-regulates PU.1. During the induction of differentiation of B-1 cells toward phagocytes, Ikaros transcription was reduced. Taken together, these data pointed to the relevance of Ikaros in the maintenance of the promiscuous gene profile of B-1 cells. It could be suggested that Ikaros functions as a guardian of B-1 lymphoid pattern, and that its absence directs the differentiation of B-1 cells into phagocytes.

B-1 cells produce insulin and abrogate experimental streptozotocin-induced diabetes.

The participation of B-1 cells in a murine model of spontaneous diabetes has been recently reported. Here, we describe the role of B-1 cells in streptozotocin (STZ) induced diabetes in mice. We demonstrated that XID (B-1 cell-deficient) mice are more susceptible to STZ treatment than WT mice, as evidenced by their higher blood glucose level in response to STZ. Unexpectedly, the XID mice that were i.p. transferred with purified B-1 cells, either before or after the STZ treatment, did not develop diabetes. These cell transfers provided long-lasting protection for the XID mice against STZ-induced diabetes, suggesting that B-1 cells play an important role in the experimental diabetes pathobiology. We also showed that B-1 cell culture supernatants were able to regulate the blood glucose level of the diabetic XID mice, and we identified insulin-producing cells when B-1 cells were differentiated in B-1 cell-derived phagocyte in vitro. These findings provide a novel role for B-1 cells in metabolic processes, presenting a new mechanism to explain the pathogenesis of diabetes and a possible therapeutical target.