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

Plzakova L, Krocova Z, Kubelkova K, Macela A - PLoS ONE (2015)

Bottom Line: 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.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.The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic.

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.

No MeSH data available.


Related in: MedlinePlus

F. tularensis infecting subsets of B cells in vitro.Subsets of B cells were infected for 3 h with unopsonized F. tularensis LVS/GFP (GFP), F. tularensis LVS/GFP opsonized with fresh un-inactivated serum (GFP+C) from naïve mice, and bacteria opsonized with heat-inactivated immune sera (GFP+Ab). The proportions of infected CD19+ cells from all measured cells and of infected B-1a, B-1b, and B-2 cells from CD19+ cells were measured by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between GFP and GFP+C and GFP+Ab (*** P < 0.001, ** P < 0.01, * P < 0.05). Results shown from one experiment are representative of three independent experiments.
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pone.0132571.g003: F. tularensis infecting subsets of B cells in vitro.Subsets of B cells were infected for 3 h with unopsonized F. tularensis LVS/GFP (GFP), F. tularensis LVS/GFP opsonized with fresh un-inactivated serum (GFP+C) from naïve mice, and bacteria opsonized with heat-inactivated immune sera (GFP+Ab). The proportions of infected CD19+ cells from all measured cells and of infected B-1a, B-1b, and B-2 cells from CD19+ cells were measured by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between GFP and GFP+C and GFP+Ab (*** P < 0.001, ** P < 0.01, * P < 0.05). Results shown from one experiment are representative of three independent experiments.

Mentions: The number of peritoneal CD19+ cells infected with unopsonized F. tularensis fluctuated around 5% (4.44 ± 0.74%). Opsonization of F. tularensis with fresh un-inactivated serum from naïve mice almost doubled the number of infected cells in culture (8.22 ± 1.02%). Opsonization of bacteria with antibodies–in contrast to opsonization of bacteria with murine fresh serum had no effect (4.28 ± 0.59%). If individual CD19+ cell subsets were monitored, then the B-1a cells (CD19+CD5+CD11b+ cells) comprised the dominant subset infected with Francisellae. B-1b (CD19+CD5-CD11b+) and B-2 (CD19+CD5-CD11b-) cells were also infected, but at lower frequency than were B-1a cells. Opsonization of bacteria with murine fresh serum or with antibodies in fact copied their effect demonstrated on the entire CD19+ cell population with the exception that in B-2 cells the effect of opsonization with fresh murine serum was insignificant (Fig 3). Isotype controls were used for IgG2a (CD19, CD5) and for IgG2b (CD11b). Mean fluorescent intensity for untreated peritoneal control cells was 2.69 ± 0.043, for isotype control IgG2a was 3.50 ± 0.158, for CD19+ (isotype IgG2a) was 454.18 ± 31.641 (t-test CD19+ vs IsoC IgG2a P = 3.58 x 10−5), for CD5+ (isotype IgG2a) was 115.07 ± 6.944 (t-test CD5+ vs IsoC IgG2a P = 2.16 x 10−5), for isotype control IgG2b was 3.54 ± 0.106, and for CD11b+ (IgG2b) was 539.18 ± 56.917 (t-test CD11b+ vs IsoC IgG2b P = 1.84 x 10−4).


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

Plzakova L, Krocova Z, Kubelkova K, Macela A - PLoS ONE (2015)

F. tularensis infecting subsets of B cells in vitro.Subsets of B cells were infected for 3 h with unopsonized F. tularensis LVS/GFP (GFP), F. tularensis LVS/GFP opsonized with fresh un-inactivated serum (GFP+C) from naïve mice, and bacteria opsonized with heat-inactivated immune sera (GFP+Ab). The proportions of infected CD19+ cells from all measured cells and of infected B-1a, B-1b, and B-2 cells from CD19+ cells were measured by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between GFP and GFP+C and GFP+Ab (*** P < 0.001, ** P < 0.01, * P < 0.05). Results shown from one experiment are representative of three independent experiments.
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pone.0132571.g003: F. tularensis infecting subsets of B cells in vitro.Subsets of B cells were infected for 3 h with unopsonized F. tularensis LVS/GFP (GFP), F. tularensis LVS/GFP opsonized with fresh un-inactivated serum (GFP+C) from naïve mice, and bacteria opsonized with heat-inactivated immune sera (GFP+Ab). The proportions of infected CD19+ cells from all measured cells and of infected B-1a, B-1b, and B-2 cells from CD19+ cells were measured by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between GFP and GFP+C and GFP+Ab (*** P < 0.001, ** P < 0.01, * P < 0.05). Results shown from one experiment are representative of three independent experiments.
Mentions: The number of peritoneal CD19+ cells infected with unopsonized F. tularensis fluctuated around 5% (4.44 ± 0.74%). Opsonization of F. tularensis with fresh un-inactivated serum from naïve mice almost doubled the number of infected cells in culture (8.22 ± 1.02%). Opsonization of bacteria with antibodies–in contrast to opsonization of bacteria with murine fresh serum had no effect (4.28 ± 0.59%). If individual CD19+ cell subsets were monitored, then the B-1a cells (CD19+CD5+CD11b+ cells) comprised the dominant subset infected with Francisellae. B-1b (CD19+CD5-CD11b+) and B-2 (CD19+CD5-CD11b-) cells were also infected, but at lower frequency than were B-1a cells. Opsonization of bacteria with murine fresh serum or with antibodies in fact copied their effect demonstrated on the entire CD19+ cell population with the exception that in B-2 cells the effect of opsonization with fresh murine serum was insignificant (Fig 3). Isotype controls were used for IgG2a (CD19, CD5) and for IgG2b (CD11b). Mean fluorescent intensity for untreated peritoneal control cells was 2.69 ± 0.043, for isotype control IgG2a was 3.50 ± 0.158, for CD19+ (isotype IgG2a) was 454.18 ± 31.641 (t-test CD19+ vs IsoC IgG2a P = 3.58 x 10−5), for CD5+ (isotype IgG2a) was 115.07 ± 6.944 (t-test CD5+ vs IsoC IgG2a P = 2.16 x 10−5), for isotype control IgG2b was 3.54 ± 0.106, and for CD11b+ (IgG2b) was 539.18 ± 56.917 (t-test CD11b+ vs IsoC IgG2b P = 1.84 x 10−4).

Bottom Line: 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.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.The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic.

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.

No MeSH data available.


Related in: MedlinePlus