Figure 1.
Tick-borne infection with B. burgdorferi causes systemic lymphadenopathy in mice.
C3H/HeN mice (n = 30) were each exposed to five B. burgdorferi-infected nymphal Ixodes scapularis ticks (or non-infected ticks). At indicated times after tick-attachment, groups of five mice were necropsied and (A) cellularity of four indicated lymph nodes and (B) total antibody-forming cells (AFC) of these lymph nodes were assessed by hemocytometer count and ELISPOT analysis, respectively. Shown are mean values ± SD per timepoint for each lymph node type. ELISPOT analysis of lymph nodes from sham-infected mice showed no significant induction of antibody production (data not shown).
Figure 2.
Infection with host-adapted B. burgdorferi induces lymphadenopathy near the site of infection.
Lymphadenopathy of the right inguinal lymph node of C57BL/6 mice 10 days after infection via subcutaneous transplantation of small pieces of ear from B. burgdorferi-infected (A) or non-infected (B) congenic SCID mice into their right dorsal tarsal region. (C) Shown are mean cell numbers ± SD of right inguinal lymph nodes from groups of four mice per timepoint. (D) Comparison of lymph node celluarity (mean ± SD) obtained from draining lymph nodes in (C) and from the axillary lymph nodes of tick-infected mice as shown in Figure 1.
Table 1.
B. burgdorferi presence correlates with lymphadenopathy in mice infected with host-adapted spirochetes.
Figure 3.
B. burgdorferi localizes to the subcapsular sinus of lymph nodes.
(A, B) Immunohistochemistry with polyclonal B. burgdorferi-specific rabbit serum, demonstrates the presence of extracellular B. burgdorferi spirochete (arrow) in the subcapsular sinus of the right inguinal lymph node of C57BL/6 mice infected by tissue-transplant eight days prior to analysis. (C) Shown are mean cell numbers of right inguinal lymph nodes of C57BL/6 mice (n = 2 per timepoint) either infected via injection of 104 culture-grown live (live) or heat-killed and sonicated B. burgdorferi in PBS. “+” identifies positive B. burgdorferi cultures of lymph nodes (n = 2 per timepoint) from the same group of mice, “−” shows lack of B. burgdorferi growth in culture. Results are from one of two independent experiments that gave similar results. (D–F) Comparison of mice infected as in (C) but injected with 106 inactivated B. burgdorferi for 8 days prior to analysis (D) Total numbers of cells recovered from lymph nodes. Each symbol represents results from one animal; horizontal bars indicate the mean for the group. (E) Data shown are total numbers CD4, CD8 T cells and CD19 B cells calculated from total cell counts and frequencies of cell subsets as determined by flow cytometry. (F) Shown are frequencies of IgG antibody-secreting cells in lymph nodes of mice given live or inactivated B. burgdorferi. Note data are normalized to B cell numbers. Each symbol represents the results from one mouse; the horizontal line indicates the mean for the group.
Figure 4.
B. burgdorferi-induced lymphadenopathy is caused by a massive proliferation and differentiation of lymph node B cells.
(A–C) H.E. staining of right inguinal lymph node from a C57BL/6 mouse (A) sham-infected or (B) infected by tissue-transplantation with host-adapted spirochetes 10 days prior. (C) Close-up image of (B). Note the complete lack of follicular and extrafollicular structure. (D) Immunohistochemistry on lymph nodes as in (B, C) reveals the presence of large numbers of Ki67+ proliferating cells in the lymph node cortex. (E) Three-color immunofluorescence identifies the Ki67+ (red) cells as B220+ (green) B cells. Blue color indicates nuclei as stained with DAPI. (F) Large numbers CD138+ plasma cells accumulate in the medullary cords of lymph nodes of infected mice as (in B/C) (G) Shown are mean cell numbers ± SD from 4 C57BL/6 mice per time point of CD4, CD8 T cells and CD19+ B cells in inguinal lymph nodes before (white) and at day 10 (black) of infection. Data were calculated from flow cytometric evaluation of live cell frequencies and total live cell counts by hemocytometer with trypan blue exclusion. Results are from one of >3 experiments performed that gave similar results. Statistical analysis was conducted by comparing results from each cell population using Student's t test; n.s. not significant. (H) Shown are mean numbers of antibody-secreting cells (AFC) ± SD for total (grey bars) and Borrelia-lysate (black bars)-specific antibodies in inguinal lymph nodes from 4 C57BL/6 mice per timepoint at the indicated times after infection with host-adapted B. burgdorferi. Horizontal bar indicates threshold levels of detection.
Figure 5.
Strong induction of B. burgdorferi-specific antibodies following infection.
(A) Indicated recombinant B. burgdorferi antigens and lysate were tested as coating reagents for ELISPOT to enumerate B. burgdoferi-specific antibody-secreting cells (AFC) in the inguinal lymph nodes of C57BL/6 mice infected for 14 days with host-adapted B. burgdorferi. Shown are mean numbers of antibody-secreting cells (AFC) ± SD from 5 mice. (B) A pool of recombinant proteins (DbpA, OspC, Arp and BmpA) was used to determine the frequencies of B. burgdorferi-specific antibody-secreting cells (black bars) by ELISPOT in C57BL/6 mice at indicated times after infection. Grey bars indicate the total number of IgG-AFC per lymph node. For each timepoint two infected mice and two sham-infected control mice were analyzed. Shown are the mean numbers B. burgdorferi-specific antibody-secreting cells ± SD assessed for the individual mice from which cell spots seen in sham-infected mice were subtracted. SD was calculated from titration curves of 3 replicates for each of the two mice analyzed. (C) Shown are the mean frequencies ± SD of B. burgdorferi-specific antibody-secreting cells expressing the indicated Ig-isoytypes or total Ig (black) on days 8 and 14 after infection. Data are from 4 mice per group and timepoint.
Figure 6.
Lymph nodes of B. burgdorferi-infected mice contain large numbers borrelia-specific B cells.
Shown are the results of three independent hybridoma fusion experiments. (A) Shown are stacked bars with the total number of antibody-secreting cells (black) and the number of lines generating B. burgdorferi-specific antibody as assessed by ELISA with four recombinant B. Burgdorferi antigens. Cells for the fusion were from indicated lymph nodes and times after infection with host-adapted spirochetes. (B) Indicated are the Ig-isotype distribution among all B. burgdorferi-specific hybridoma lines generated.
Figure 7.
Lymphadenopathy and lymph node B cell activation are independent of MyD88-signaling.
Control C57BL/6 (wildtype) and congenic MyD88−/− mice (n = 6 per group) were infected with host-adapted B. burgdorferi for 10 days. Lymph nodes were harvested and compared for (A) total cellularity and (B) numbers of CD4, CD8 and CD19 as assessed by flow cytometry. ELISPOT analysis was conducted to determine numbers of (C) borrelia-specific IgM or (D) all Ig-isotype secreting borrelia-specific cells. For scatter plots, each symbol represents the result from an individual animal. Lines indicate mean of the group. Bar chart shows the mean values ± SD. Results are pooled from two independent experiments. Statistical analysis for (A) (C) (D) was conducted by Student's t test and (B) by two-way ANOVA. None of the data showed significant differences between control and MyD88−/− mice.