Promotion of a functional B cell germinal center response after Leishmania species co-infection is associated with lesion resolution

Abstract

Co-infection of C3HeB/FeJ (C3H) mice with both Leishmania major and Leishmania amazonensis leads to a healed footpad lesion, whereas co-infection of C57BL/6 (B6) mice leads to non-healing lesions. This inability to heal corresponds to a deficiency in B cell stimulation of the macrophage-mediated killing of L. amazonensis in vitro and a less robust antibody response. The mechanism that leads to healing of these lesions is not completely known, although our studies implicate the B cell response as having an important effector function in killing L. amazonensis. To understand more completely this disparate clinical outcome to the same infection, we analyzed the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. There were more germinal center B cells, more antibody isotype-switched germinal center B cells, more memory B cells, and more antigen-specific antibody-producing cells in co-infected C3H mice compared to B6 mice as early as 2 weeks postinfection. Interleukin (IL)-21 production and IL-21 receptor expression in both mouse strains, however, were similar at 2 weeks, suggesting that the difference in the anti-Leishmania response in these mouse strains may be due to differences in T follicular cell commitment or intrinsic B cell differences. These data support the idea that functional B cells are important for healing L. amazonensis in this infectious disease model.

Figure 1

Simultaneous co-infection with both Leishmania major and L. amazonensis allows for lesion resolution in co-infection of C3HeB/FeJ (C3H), but not C57BL/6 (B6) mice at 12 weeks postinfection. Mice with a single infection were inoculated with L. amazonensis (La) or L. major (Lm) stationary phase promastigotes, whereas co-infected mice (Co) were inoculated with Lm and La in the left hind footpad. Mice were infected for 12 weeks with weekly monitoring of lesion size. The lesion size was determined by measuring the infected footpad and comparing that to the non-infected footpad. Data are represented as the mean ± SEM of three separate experiments. *P ≤ 0.05.

Figure 2

Increased number of total germinal center B cells and germinal center B cells undergoing isotype switching in co-infection of C3HeB/FeJ (C3H) mice. C3H and C57BL/6 (B6) mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 and 5 weeks postinfection. Cells were analyzed via fluorescence-activated cell sorting (FACS) and first gated on the B220⁺ population and analyzed for binding to peanut agglutinin (PNA) (A), PNA binding (B), surface expression of major histocompatibility complex (MHC) class II (C), and surface expression of CD86, as indicated by mean fluorescence intensity (MFI) (D). Cell number was determined based on the percentage of cells within the gated population and the total number of lymph node cells recovered. Data are represented as the mean ± SEM of three separate experiments. *P ≤ 0.05.

Figure 3

Increased number of germinal centers in the draining lymph node following co-infection of co-infection of C3HeB/FeJ (C3H) mice with Leishmania major and L. amazonensis. A: Photomicrographs of lymph node sections labeled with anti-mouse B220 and biotin-peanut agglutinin (PNA) from C3H and C57BL/6 (B6) mice co-infected for 2 weeks. Bar = 200 μm. Designated areas magnified in the insets which highlight PNA immunoreactive cells. Scale bars: 40 μm. B: Histological germinal center scores for PNA immunoreactivity at 2 weeks. Score is based on the number of PNA⁺ germinal centers within a single draining lymph node. Data are representative of two separate experiments ± SEM. *P = 0.0087.

Figure 4

Increased number of memory B cells (B220⁺, IgM⁻, and CD23⁻) in the draining lymph node of co-infected C3HeB/FeJ (C3H) mice. C3H and C57BL/6 (B6) mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 and 5 weeks postinfection. Via flow cytometry, surface marker expression was determined based on a CD19⁺ population. Surface expression of both IgM and CD23 was determined based on the percentage of cells within the gated population as compared to the total lymph node cells recovered. Data are representative of two separate experiments ± SEM. *P ≤ 0.05.

Figure 5

Increased number of antigen-specific IgG2a-producing cells following co-infection in C3HeB/FeJ (C3H) mice. C3H and C57BL/6 (B6) mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 and 5 weeks postinfection (please note scales). Number of IgG2a (C3H) and IgG2c (B6)-producing cells (A), and IgG1-producing cells (B), as determined by enzyme-linked immunosorbent spot (ELISPOT) analysis of total draining lymph node cells stimulated with freeze-thawed Leishmania promastigote antigen, as previously indicated. Data are represented as the mean ± SEM of three separate experiments. *P ≤ 0.05.

Figure 6

No difference in the number of IL-21 producing cells, IL-21 receptor expression, or site of IL-21 production in draining lymph node cells from co-infected C3HeB/FeJ (C3H) and C57BL/6 (B6) mice. A: Number of IL-21-producing cells was determined by enzyme-linked immunosorbent spot (ELISPOT) analysis of total draining lymph node cells stimulated with freeze-thawed Leishmania promastigote antigen. C3H and B6 mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 weeks postinfection. Data are represented as the mean ± SEM of two (for B6) or three (for C3H) separate experiments. B: Cells were first gated on the B220⁺ population and surface expression of IL-21 receptor was measured as indicated by mean fluorescence intensity (MFI). The MFI for each marker presented as fold increase over naïve control. Total draining lymph node cells were harvested at 2 weeks postinfection. Data are represented as the mean ± SEM of two separate experiments. C: Photomicrographs of lymph node sections labeled with anti-mouse IL-21 from C3H and B6 mice co-infected for 2 weeks. Bar = 50 μm.