Impaired germinal center responses and suppression of local IgG production during intracellular bacterial infection

Abstract

Germinal centers (GCs) are specialized microenvironments in secondary lymphoid organs that facilitate the development of high-affinity, isotype-switched Abs, and immunological memory; consequently, many infections require GC-derived IgG for pathogen clearance. Although Ehrlichia muris infection elicits a robust expansion of splenic, IgM-secreting plasmablasts, we detected only very low frequencies of isotype-switched IgG-secreting cells in mouse spleens, until at least 3 wk postinfection. Instead, Ag-specific IgG was produced in lymph nodes, where it required CD4 T cell help. Consistent with these findings, organized GCs and phenotypically defined splenic GC B cells were found in lymph nodes, but not spleens. Ehrlichial infection also inhibited spleen IgG responses against a coadministered T cell-dependent Ag, hapten 4-hydroxy-3-nitrophenyl acetyl (NP)-conjugated chicken gamma globulin in alum. NP-specific B cells failed to undergo expansion and differentiation into GC B cells in the spleen, Ab titers were reduced, and splenic IgG production was inhibited nearly 10-fold when the Ag was administered during infection. Our data provide a mechanism whereby an intracellular bacterial infection can compromise local immunity to coinfecting pathogens or antigenic challenge.

FIGURE 1

E. muris infection inhibits the production of splenic, Ag-specific IgG-secreting cells. E. muris OMP-19–specific IgM and IgG (A), and day 28 postinfection isotype-switched IgG (B), were measured in the serum of C57BL/6 mice by ELISA. C, The frequencies and numbers of Ag-specific IgM-secreting (●) and IgG-secreting (■) cells in the spleen (top panels) and lymph nodes (bottom panels) were determined by ELI-SPOT analysis. Spots produced by cells from mock-infected control mice, which were negligible, were subtracted from the number of spots obtained from the infected mice. The data are representative of three independent experiments in which three mice were analyzed at each time point.

FIGURE 2

CD4 T cells are required for both IgM and IgG production in the LNs. Serum titers of OMP-19 IgM (A) and IgG (B) were assessed on day 16 postinfection in MHC class II-deficient and TCRβ-deficient mouse strains and compared with titers obtained in infected C57BL/6 mice. C, An ELISPOT analysis was used to determine the frequencies and numbers of OMP-19 IgM-secreting (●) and IgG-secreting (■) cells in the spleen (top panels) and LNs (bottom panels) on day 16 postinfection. Spots produced by cells isolated from mock-infected control mice of each of the strains, which were negligible, were enumerated and subtracted from the spots measured in the infected mice. The data are representative of two independent experiments in which four mice were analyzed at each time point. *p ≤ 0.05.

FIGURE 3

Infection inhibits the differentiation of splenic GC B cells. A, To determine the kinetics of the onset of the GC response, cells from the spleen (top row) and lymph nodes (bottom row) of C57BL/6 mice were stained with Abs directed against GL7 and CD19, on the indicated days postinfection. The frequencies (B) and numbers (C) of GL7-positive B cells are shown. D, To further characterize GC B cells, the GL7+ CD19+ cells were analyzed for their expression of CD38 and CD95. The frequencies and numbers of GC B cells (i.e., CD95+CD38^lo cells), within the GL7−, CD19-positive flow cytometry gate, are shown in E and F. The data are representative of three independent experiments in which three mice were analyzed at each time point. *p ≤ 0.05.

FIGURE 4

GC formation was observed in the LNs, but not in the spleens, of infected mice. Frozen sections from spleen tissue (top row) and an inguinal lymph node (bottom row) from mock-infected and day 16-infected C57BL/6 mice were stained with Abs specific for B220 (red), Thy1.2 (blue), and PNA (green). Colocalization of the PNA-positive cells with the B and T cells was detected in the LNs, but not in the spleens, of infected mice. Original magnification ×200.

FIGURE 5

E. muris infection suppressed the IgG response to an irrelevant Ag. E. muris-infected mice and uninfected control C57BL/6 mice were immunized i.p. with NP-CGG in alum on the indicated days postinfection. To assess the NP-specific IgG response, serum and spleen tissue were harvested on day 12 postimmunization and were analyzed by ELISA (A) and ELISPOT analysis (B, C) in mice immunized with NP-CGG only (●) and in E. muris-infected, NP-CGG–immunized mice (■). The frequency (B) and number (C) of splenic NP-specific IgG-secreting cells was determined by ELISPOT analysis after subtracting background spots obtained using cells from mock-infected control mice. D, Day 9 E. muris-infected and mock-infected mice were immunized s.c. with NP-CGG in alum, and the NP-specific IgG response was analyzed at day 21 postinfection by ELISPOT analysis in the spleen (top row) and pooled brachial and axillary draining LNs (bottom row). The data are representative of three independent experiments in which three mice were analyzed at each time point. *p ≤ 0.05.

FIGURE 6

The differentiation of spleen GC B cells was suppressed during infection. On day 2 after E. muris infection, infected (m+s)Ig B cell transgenic mice and mock-infected mice were immunized with NP-CGG and analyzed 12 d later. A, Splenocytes were stained for cell surface expression of NIP and CD19, to detect Ag-specific B cells. The frequencies of NIP-specific B cells among the total B cells (B) and the numbers of Ag-specific B cells (C) were determined. D, The NIP+ CD19+ B cells were analyzed for cell surface expression of GL7 and CD38 to identify GC B cells. The frequencies (E) and numbers (F) of GC B cells among the total CD19-positive B cells were determined. The data are representative of three independent experiments in which three mice were analyzed at each time point. *p ≤ 0.05.