B-1 cell response in immunity against parasites.

The peritoneal cavity has a microenvironment capable of promoting proliferation, differentiation, and activation of the resident cells and recruitment of blood cells through the capillary network involved in the peritoneum. Among the cells found in the peritoneal cavity, B-1 cells are a particular cell type that contains features that are not very well defined. These cells differ from conventional B lymphocytes (B-2) by phenotypic, functional, and molecular characteristics. B-1 cells can produce natural antibodies, migrate to the inflammatory focus, and have the ability to phagocytose pathogens. However, the role of B-1 cells in immunity against parasites is still not completely understood. Several experimental models have demonstrated that B-1 cells can affect the susceptibility or resistance to parasite infections depending on the model and species. Here, we review the literature to provide information on the peculiarities of B-1 lymphocytes as well as their interaction with parasites.

Long-Term In Vitro Passaging Had a Negligible Effect on Extracellular Vesicles Released by Leishmania amazonensis and Induced Protective Immune Response in BALB/c Mice.

Depending on Leishmania species and the presence/absence of virulence factors, Leishmania extracellular vesicles (EVs) can differently stimulate host immune cells. This work is aimed at characterizing and evaluating the protective role of EVs released by Leishmania amazonensis promastigotes under different maintenance conditions. Initially, using a control strain, we standardized 26°C as the best release temperature to obtain EVs with a potential protective role in the experimental leishmaniasis model. Then, long-term (LT-P) promastigotes of L. amazonensis were obtained after long-term in vitro culture (100 in vitro passages). In vivo-derived (IVD-P) promastigotes of L. amazonensis were selected after 3 consecutive experimental infections in BALB/c mice. Those strains developed similar lesion sizes except for IVD-P at 8 weeks post infection. No differences in EV production were detected in both strains. However, the presence of LPG between LT-P and IVD-P EVs was different. Groups of mice immunized with EVs emulsified in the adjuvant and challenged with IVD-P parasites showed decreased lesion size and parasitic load compared with the nonimmunized groups. The immunization regimen with two doses showed high IFN-γ and IgG2a titers in challenged mice with either IVD-P or LT-P EVs. IL-4 and IL-10 were detected in immunized mice, suggesting a mixed Th1/Th2 profile. EVs released by either IVD-P or LT-P induced a partial protective effect in an immunization model. Thus, our results uncover a potential protective role of EVs from L. amazonensis for cutaneous leishmaniasis. Moreover, long-term maintenance under in vitro conditions did not seem to affect EV release and their immunization properties in mice.

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.

Effects of extracellular vesicles released by peritoneal B-1 cells on experimental Leishmania (Leishmania) amazonensis infection.

B-1 cells are a B-lymphocyte subtype whose roles in immunity are not completely defined. These cells can produce cytokines (mainly IL-10) and natural and specific antibodies. Currently, extracellular vesicles (EVs) released by immune cells have emerged as new important entities in cell-cell communication. Immune cells release EVs that can activate and/or modulate other immune cells. Here, we characterized the EVs released by peritoneal B-1 cells infected or not with Leishmania (Leishmania) amazonensis. This Leishmania species causes cutaneous leishmaniasis and can infect macrophages and B-1 cells. Our results showed that peritoneal B-1 cells spontaneously release EVs, but the parasite stimulated an increase in EVs production by peritoneal B-1 cells. The treatment of BALB/c and C57BL/6 bone marrow-derived macrophages (BMDM) with EVs from infected peritoneal B-1 cells led to differential expression of iNOS, IL-6, IL-10, and TNF-α. Additionally, BALB/c mice previous treated with EVs released by peritoneal B-1 cells showed a significant lower lesion size and parasite burden. Thus, this study demonstrated that peritoneal B-1 cells could release EVs that can alter the functions of macrophages in vitro and in vivo these EVs altered the course of L. amazonensis infection. These findings represent the first evidence that EVs from peritoneal B-1 cells can act as a new mechanism of cellular communication between macrophages and B-1 cells, contributing to immunity against experimental leishmaniasis.

Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells.

The extracellular vesicles (EVs) released by Leishmania can contribute to the establishment of infection and host immunomodulation. In this study, we characterized the shedding of EVs from Leishmania (Leishmania) amazonensis promastigotes. This species is the causative agent of cutaneous leishmaniasis, and its role during interactions with bone marrow-derived macrophages (BMDMs) and peritoneal B-1 cells was evaluated. Leishmania amazonensis promastigotes cultivated in vitro at different times and temperatures spontaneously released EVs. EVs were purified using size-exclusion chromatography (SEC) and quantitated by nanoparticle tracking analysis (NTA). NTA revealed that the average size of the EVs was approximately 180 nm, with concentrations ranging from 1.8 × 108 to 2.4 × 109 vesicles/mL. In addition, the presence of LPG and GP63 were detected in EVs obtained at different temperatures. Naïve BMDMs stimulated with EVs exhibited increased IL-10 and IL-6 expression. However, incubating B-1 cells with parasite EVs did not stimulate IL-10 expression but led to an increase in the expression of IL-6 and TNFα. After 7 weeks post-infection, animals infected with L. amazonensis promastigotes in the presence of parasite EVs had significant higher parasite load and a polarization to Th2 response, as compared to the group infected with the parasite alone. This work demonstrated that EVs isolated from L. amazonensis promastigotes were able to stimulate macrophages and B-1 cells to express different types of cytokines. Moreover, the immunomodulatory properties of EVs probably contributed to an increase in parasite burden in mice. These findings suggest that the functionality of L. amazonensis EVs on immune system favor of parasite survival and disease progression.