Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors

Abstract

Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell–pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs) with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ΔiglC and ΔftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria’s internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

B Lymphocyte as a Target of Bacterial Infections

“…For the case of Francisella, evidence has demonstrated that Francisella is recognized by the B1-a cell by their BCRs alone; meanwhile, in the B1-band B2-cell subsets, bacterial recognition required simultaneous participation of BCR and complement receptors CR1 and CR2. F. tularensis was internalized by B cells at low rate, and internalized bacteria survive intracellularly [63]. In both studies the internalization process resembled more a cell membrane protrusion formation mechanism (like phagocytosis or macropinocytosis) rather than a clathrin-coated endocytosis mechanism.…”

“…Changes in CR expression on B lymphocytes are related with breaking of B-cell tolerance and increased susceptibility to bacterial infections [62]. CR participates in bacteria internalization; Francisella tularensis is engulfed by B lymphocytes through complement receptors [63].…”

“…Up today there are few studies on B-cell capabilities for bacterial internalization; there are descriptions of B-cell internalization of opsonized bacteria with immunoglobulin and with complement [63,69,74], but also there are descriptions that B cells can uptake non-opsonized bacteria [72]; so far there are no studies that clarified the role of small GTPases involved in bacterial internalization by B cells that could help to clarify if bacterial uptake by B cells is a phagocytic type I or type II process or if it is a unique endocytic mechanism for B cells.…”

Recreation of an antigen-driven germinal center in vitro by providing B cells with phagocytic antigen

“…Although the defect in RhoG does not lead to a blockade of B cell phagocytosis and GC formation in vitro, perhaps because of a possible redundancy with other Rho GTPases (Tzircotis et al, 2011), we previously showed that RhoG-deficient B cells are a valuable tool to determine the role of antigen phagocytosis by B cells in the humoral response in vivo (Martinez-Riano et al, 2018). Although it has been previously shown that B1 cells and to a lesser extent follicular B2 cells can phagocytose bacteria (Gao et al, 2012;Plzakova et al, 2015;Zhu et al, 2016), the prevalent view is that B cells acquire antigen from immune complexes retained at the surface of FDCs or macrophages (Avalos and Ploegh, 2014;Suzuki et al, 2009). Although our data do not oppose this view, they highlight the relevance of the phagocytic process for GC differentiation.…”

Beyond the antibody: B cells as a target for bacterial infection

“…F. tularensis was detected on the cell surface and interior of B cells by transmission electronic microscopy . Francisella entry into Balb/c mouse spleen B cells employs different receptors depending on the B cell subset: entry into B1a B cells requires BCR, whereas the entry into B1b and B2 B cells involves both BCR and CR1/2 . Once localized intracellularly, Francisella trafficking follows the endosomal pathway where F. tularensis induces death by activating caspase‐3, caspase‐8, caspase‐9, and the BH3 interacting‐domain death agonist (BID) in both the A20 and Ramos RA‐1 B cell lines.…”