Rotavirus differentially infects and polyclonally stimulates human B cells depending on their differentiation state and tissue of origin

Abstract

We have shown previously that rotavirus (RV) can infect murine intestinal B220(+) cells in vivo (M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, and H. B. Greenberg, J. Virol. 80:5219-5232, 2006) and human blood B cells in vitro (M. C. Mesa, L. S. Rodriguez, M. A. Franco, and J. Angel, Virology 366:174-184, 2007). However, the effect of RV on B cells, especially those present in the human intestine, the primary site of RV infection, is unknown. Here, we compared the effects of the in vitro RV infection of human circulating (CBC) and intestinal B cells (IBC). RV infected four times more IBC than CBC, and in both types of B cells the viral replication was highly restricted to the memory subset. RV induced cell death in 30 and 3% of infected CBC and IBC, respectively. Moreover, RV induced activation and differentiation into antibody-secreting cells (ASC) of CBC but not IBC when the B cells were present with other mononuclear cells. However, RV did not induce these effects in purified CBC or IBC, suggesting the participation of other cells in activating and differentiating CBC. RV infection was associated with enhanced interleukin-6 (IL-6) production by CBC independent of viral replication. The infection of the anti-B-cell receptor, lipopolysaccharide, or CpG-stimulated CBC reduced the secretion of IL-6 and IL-8 and decreased the number of ASC. These inhibitory effects were associated with an increase in viral replication and cell death and were observed in polyclonally stimulated CBC but not in IBC. Thus, RV differentially interacts with primary human B cells depending on their tissue of origin and differentiation stage, and it affects their capacity to modulate the local and systemic immune responses.

FIG. 1.

RRV and Wa infection of CBC and IBC. Microbead-purified CBC (empty circles) and IBC (filled square) from different patients were mock treated or treated with psoralen-inactivated RRV (iRRV), RRV, or Wa RV (all at an MOI of 5). After 10 h, the cells were stained for surface markers and then permeabilized to detect intracellular viral proteins. (A) Summary of the expression of NSP2 in B cells. Lines represent the median. An asterisk indicates statistically significant differences between the percentage of CBC and IBC infected by RRV (P < 0.0001, Mann-Whitney test). (B) Coexpression of nonstructural (NSP2) and structural (VP6) proteins in paired CBC and IBC purified from the same patient. (C) RRV productively infects human CBC and IBC. Bead-purified CBC and IBC were mock treated or were treated with iRRV or RRV (MOI, 5) for 45 min. After infection, the cells were washed three times. For the first wash, a 1/1,000 dilution of neutralizing MAb (159; anti-VP7) was used. Supernatants at 2 and 24 h were collected, and viral replication was determined by the titration of infectious virus in MA104 cells. A supernatant of RV-infected MA104 cells was used as a positive control. The results of individual experiments are shown, and horizontal lines represent the median. An asterisk indicates statistically significant differences (P < 0.05, Wilcoxon test).

FIG. 2.

RV replication is highly restricted to mBC. (A) Flow cytometry assay to detect the frequency of RV-infected cells in sort-purified subsets of B cells. Microbead-purified CBC were mock infected or were infected with RRV (MOI, 5) for 45 min, stained, sorted by FACS, and cultured for 10 h. The expression of CD19, CD27, IgD, and intracellular NSP2 was evaluated after culture. One representative experiment of three performed is shown. (B) CBC and IBC were purified with beads and then were mock infected or were infected with RRV (MOI, 5). After 10 h, cells were stained with a viability marker, MAb against CD19, CD27, and IgD, and then permeabilized to detect NSP2. For IBC, MAb anti-CD20 and anti-CD38 were included to analyze ASC (data not shown). Dot plots are gated on lymphocytes by size and granularity, CD19⁺ CBC, or CD19⁺ CD20^+/− IBC. (C) Distribution of RRV- and Wa-infected cells in the B-cell subsets. Results from more than eight experiments for CBC and IBC are presented. Bars represented the medians and ranges. An asterisk indicates statistically significant differences between the frequencies of cells expressing NSP2 in IgD^+/− B-cell subsets (P < 0.003, Wilcoxon test).

FIG. 3.

RV-induced death in CBC and IBC. Bead-purified CBC and IBC were mock infected or were infected with iRRV, RRV, or Wa RV (MOI, 5) or with 80 μM etoposide as a positive control. The viability of B cells was evaluated by flow cytometry after 10 h. (A) Frequency of amine⁺ CBC and IBC. Medians and ranges from at least 12 experiments for CBC and IBC are shown. For Wa RV, six experiments are included. The double asterisk indicates statistically significant differences between results for mock- and RRV-infected B cells (P = 0.005, Wilcoxon test). An asterisk indicates statistically significant differences between iRRV- and RRV-infected B cells or between RRV- and Wa-infected cells (P = 0.01, Wilcoxon test). ns, nonsignificant differences. (B) CBC were mock infected or were infected with iRRV, RRV (MOI, 5), or 80 μM etoposide. After 10 h, the expression of PI, annexin V, and NSP2 was evaluated by flow cytometry in B cells. For RRV-infected cells, gates on NSP2⁺ and NSP2⁻ cells are shown. One representative experiment of three performed is presented.

FIG. 4.

RRV-induced expression of CD69 and ASC differentiation in CBC or IBC. (A) Purified B cells (open bars) derived from either blood (upper) or intestine (lower) and whole mononuclear cells from either blood or the intestine (stippled bars) were mock infected or were infected with iRRV, RRV (MOI, 5), LPS (50 μg/ml), or CpG 2006 (3 μg/ml). After overnight stimulation, the expression of CD69 was analyzed by flow cytometry. The medians and ranges from seven experiments are shown. *, significant differences between mock infection and positive control, iRRV-stimulated, and RRV-stimulated PBMC (P ≤ 0.01, Mann-Whitney test); &, statistically significant differences between purified B cells and B cells as a component of whole mononuclear cells (P < 0.04, Wilcoxon test). (B) PBMC (filled circles; upper) and purified CBC (open circles, lower) were mock infected or were infected with iRRV and RRV (MOI, 5) for 5 days. The numbers of total IgA and IgG ASC were analyzed by two-color ELISPOT assay. Individual paired experiments are shown. Horizontal lines represent the median. An asterisk indicates statistically significant differences between mock-, iRRV-, and RRV-stimulated cells (P ≤ 0.046, Wilcoxon test). (C) PBMC and CBC were treated with LPS (50 μg/ml) or CpG (3 μg/ml) in the presence of mock (open circles) or RRV (filled circles) infection. After 5 days, the frequency of total IgA and IgG ASC was evaluated by ELISPOT assay. An asterisk indicates statistical differences between mock- and RRV-infected CBC (P ≤ 0.016, Wilcoxon test).

FIG. 5.

RV induces IL-6 secretion in human CBC but not in IBC. Microbead-purified CBC (upper) and IBC (low) were subjected to mock, iRRV, RRV, anti-BCR plus mock, or anti-BCR plus RRV infection. After 72 h of culture, the supernatants were collected and the secretion of IL-6, IL-8, IL-10, and TNF-α was analyzed by CBA. Bars represent the medians. *, statistically significant differences between mock- and iRRV- or RRV-infected cells (P ≤ 0.036, Wilcoxon test); &, significant decrease in the cytokine production between mock- and RRV-infected anti-BCR-treated cells (P ≤ 0.03, Wilcoxon test).

FIG. 6.

Polyclonal stimulation induces increases in RRV infection and cell death in CBC but not in IBC. (A) Purified CBC and IBC were treated with 10 μg/ml of anti-BCR antibodies before RRV infection (MOI, 5). Viability and CD19 and NSP2 expression were assessed by flow cytometry at 10 hpi. A representative of 14 experiments is shown. (B) Enhancement in viral replication after polyclonal stimulation is presented as the fold increase. The bars show the medians and ranges from 8 to 12 experiments for CBC and 6 to 12 experiments for IBC. For Wa RV, five experiments are presented. * and **, statistically significant differences between RRV and anti-BCR plus RRV (P = 0.0009) and between RRV and LPS plus RRV or Wa and anti-BCR plus Wa (P = 0.03, Mann-Whitney test), respectively. (C) Purified CBC and IBC were infected with RRV or RRV plus polyclonal stimulation and then cultured for 10 h. Cells were stained to evaluate the coexpression of amine viability marker and NSP2. Bars represent the median frequency of double-positive (NSP2⁺ and amine⁺) CBC (empty bars) and IBC (stippled bars). P values (Mann-Whitney test) are shown (n = 6 to 12 experiments).

FIG. 7.

Circulating and intestinal B cells are not critical antigen-presenting cells for RV-specific T cells. Whole (open circles) or B-cell-depleted (filled circles) PBMC or IMC were mock stimulated or stimulated with RRV or SEB, as described in Materials and Methods. After 10 h of culture (brefeldin A was added for the last 5 h), the cells were stained with a viability marker, MAb against CD3, CD4, CD8, CD69, and IFN-γ. The frequency of RV-specific IFN-γ-producing T cells was analyzed by flow cytometry. Dead cells were excluded from the analysis by viability staining. Individual experiments are shown. Lines represent the medians. An asterisk indicates significant differences between mock- and RV- or SEB-stimulated cells (P ≤ 0.04, Wilcoxon test).