Potential Role for Regulatory B Cells as a Major Source of Interleukin-10 in Spleen from Plasmodium chabaudi-Infected Mice

Abstract

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells were found to be induced in a variety of infectious diseases. However, its importance in the regulation of immune response to malaria is still unclear. Here, we investigated the dynamics, phenotype, and function of Breg cells using Plasmodium chabaudi chabaudi AS-infected C57BL/6 and BALB/c mice. BALB/c mice were more susceptible to infection and had a stronger IL-10 response in spleen than C57BL/6 mice. Analysis of the surface markers of IL-10-producing cells with flow cytometry showed that CD19⁺ B cells were one of the primary IL-10-producing populations in P. c. chabaudi AS-infected C57BL/6 and BALB/c mice, especially in the latter one. The Breg cells had a heterogeneous phenotype which shifted during infection. The well-established Breg subset, CD19⁺ CD5⁺ CD1d^hi cells, accounted for less than 20% of IL-10-producing B cells in both strains during the course of infection. Most Breg cells were IgG⁺ and CD138^- from day 0 to day 8 postinfection. Adoptive transfer of Breg cells to C57BL/6 mice infected with P. c. chabaudi AS led to a transient increase of parasitemia without an impact on survival rate. Our finding reveals that B cells play an active and important regulatory role in addition to mediating humoral immunity in immune response against malaria, which should be paid more attention in developing therapeutic or vaccine strategies against malaria involving stimulation of B cells.

Keywords: B10; IL-10; P. c. chabaudi AS; malaria; regulatory B cells.

FIG 1

Parasitemia, survival, and cytokine responses of BALB/c and C57BL/6 mice during P. c. chabaudi AS infection. BALB/c and C57BL/6 mice were administered with 1 × 10⁶ pRBC, and the parasitemia (A) and survival rate (B) were monitored daily. For evaluation of cytokine production, mice were sacrificed at the indicated time. Serum samples were prepared and total RNA was isolated from spleen. The mRNA level of IFN-γ (C) and IL-10 (D) were measured by real-time PCR. The serum concentration of IFN-γ (E) and IL-10 (F) was determined with ELISA. All data except survival rate are presented as arithmetic means ± SE. Single and double asterisks indicate P values of <0.05 and <0.01, respectively, compared with uninfected mice of the same strain. A pound sign means a P value of <0.05 compared with the other strain at the same time point. The P value for the difference in survival between the two strains was less than 0.01.

FIG 2

Dynamics of IL-10-producing cells and the frequency of IL-10-producing CD3⁺, CD19⁺, and CD11b⁺ cells in P. c. chabaudi AS-infected C57BL/6 and BALB/c mice. Mice were infected with P. c. chabaudi AS and sacrificed at the indicated time. (A) Spleen cells were isolated and counted. The percentages of IL-10-producing cells in spleen cells and their phenotypes were analyzed with flow cytometry. (B) The absolute numbers of total IL-10-producing splenocytes were calculated. The percentages of CD3⁺ (C), CD19⁺ (D), and CD11b⁺ (E) cells in IL-10-producing cells as well as the absolute number of CD19⁺ IL-10⁺ cells (F) in spleen from C57BL/6 and BALB/c mice are shown. Data are presented as arithmetic means ± SE. Single and double asterisks indicate P values of <0.05 and <0.01, respectively, compared with uninfected mice of the same strain. Single and double pound signs mean P values of <0.05 and <0.01, respectively, compared with the other strain at the same time point.

FIG 3

Dynamics of CD19⁺ CD5⁺ CD1d^hi cells and their contribution to IL-10 production during the course of P. c. chabaudi AS infection in C57BL/6 and BALB/c mice. (A) Spleen cells were isolated from mice and stained for membrane CD19, CD5, and CD1d as well as intracellular IL-10. CD19⁺ CD5⁺ CD1d^hi cells were gated as shown in the left and middle panels, and the subgating of IL-10⁺ cells in the CD19⁺ CD5⁺ CD1d^hi population is displayed on the right. (B and C) Change of frequency and absolute number of CD19⁺ CD5⁺ CD1d^hi cells in spleen from C57BL/6 and BALB/c mice. The percentage of IL-10-producing cells in CD19⁺ CD5⁺ CD1d^hi cells (D) and the proportion of CD5⁺ CD1d^hi B cells in CD19⁺ IL-10⁺ cells (E) were also analyzed. (F) Gating strategy used for panel E. Data are presented as arithmetic means ± SE. Single and double asterisks indicate P values of <0.05 and <0.01, respectively, compared with uninfected mice of the same strain. Single and double pound signs mean P values of <0.05 and <0.01, respectively, compared with the other strain at the same time point.

FIG 4

Effects of adoptive transfer of Breg cells to P. c. chabaudi AS-infected C57BL/6 mice. Breg cells (1 × 10⁶) and IL-10⁻ B cells were isolated from C57BL/6 mice 8 days p.i. and transferred intravenously to C57BL/6 mice on the fifth day of infection. (A) Parasitemia was monitored daily. The recipient mice were sacrificed on day 7 p.i. and the mRNA levels of IL-10 (B) and IFN-γ (C) of spleen were measured. Data are presented as arithmetic means ± SE. A pound sign means a P value of <0.05 compared with the other strain at the same time point. One data set out of two independent experiments with similar results is shown.