B cells and antibodies are required for resistance to the parasitic gastrointestinal nematode Trichuris muris

Abstract

Previous studies using cell transfers and antibody receptor knockout mice have shown that B cells and antibodies are not essential components of the expulsion mechanism in Trichuris muris infections. Serum transfer experiments have given mixed results regarding the importance of antibodies in this infection model, and the role of B cells in initiating or maintaining T-cell responses has not been addressed. We used B-cell-deficient muMT mice to determine if B cells play a role in anti-T. muris immune responses. In contrast to wild-type C57BL/6 mice, muMT mice were susceptible to infection. Antigen-restimulated mesenteric lymph node cells from infected muMT mice produced only naive levels of Th2-associated cytokines but had increased levels of gamma interferon. However, these mice appeared capable of mounting a Th2-dependent mucosal mastocytosis, though this was significantly delayed compared to that seen in wild-type mice. Resistance to T. muris was restored following reconstitution with naive C57BL/6 splenic B cells, as was in vitro Th2 cytokine production in response to parasite antigen. Treatment of muMT mice with anti-interleukin-12 monoclonal antibody during the first 2 weeks of infection also restored immunity, suggesting that muMT mice can be manipulated to expel worms at the time of T-cell priming. Additionally, treatment of muMT mice with parasite-specific immunoglobulin G1 purified from the serum of resistant NIH mice prevented worm establishment, suggesting an important role for antibodies. Our results as a whole describe the first detailed report of a critical role for B cells in resistance to an intestinal nematode.

FIG. 1

T. muris worm burdens (mean ± SE) during the course of infection in B-cell-deficient μMT mice and wild-type C57BL/6 mice. Mice were infected with approximately 100 infective embryonated T. muris eggs on day 0 (n = 4 per group). ∗, significant difference in worm numbers between C57BL/6 and μMT mice (P = 0.0193).

FIG. 2

Cytokine analyses of MLNC supernatants from μMT mice and C57BL/6 mice. MLNC from naive mice and T. muris-infected mice (day 21 p.i. [d21p.i.]) were restimulated in vitro with parasite antigen (50 μg/ml). Supernatants were harvested after 48 h and analyzed by sandwich ELISA for the presence of IL-4 (A), IL-5 (B), IL-9 (C), and IFN-γ (D). Results are shown as means ± SE (n = 4 per group). ∗, significant increase above naive levels (P < 0.05).

FIG. 3

Mucosal mast cell numbers (A) and serum MMCP-1 levels (B) in naive and T. muris-infected μMT and C57BL/6 mice. Naive levels are indicated at day 0. Mast cells were counted from 20 crypt units (c.u.) in toluidine blue-stained cecal sections, and results are presented as mean mast cell numbers per 20 crypt units ± SE. Serum MMCP-1 levels were determined by ELISA and presented as means ± SE. ∗, significant difference between C57BL/6 and μMT mice (P < 0.05).

FIG. 4

T. muris worm burdens recovered from C57BL/6 (C57), μMT (muMT), and B-cell-reconstituted μMT (muMTB) mice at day 35 p.i. (d35). B-cell-reconstituted μMT mice received 2.5 × 10⁷ naive splenic C57BL/6 B cells intravenously 1 day before infection. Mice were infected with approximately 175 infective embryonated T. muris eggs (C57 d11) (n = 5 per group) at day 35 p.i. ∗, significant difference in worm numbers between C57BL/6 day 35 p.i. and B-cell-reconstituted μMT day 35 p.i. (P = 0.0088).

FIG. 5

Cytokine analyses of B-cell-reconstituted μMT mice. MLNC from naive and T. muris-infected C57BL/6 (C57), μMT (muMT), and B cell-reconstituted μMT (muMTB) mice at day 22 p.i. (d22) were cultured as described for Fig. 2. Supernatants were tested for the presence of IL-4 (A), IL-5 (B), IL-9 (C), and IFN-γ (D) by sandwich ELISA. Results are shown as means ± SE. Naive groups are represented by pooled cells from infected groups (n = 2 except muMT d22 [n = 1]).

FIG. 6

Serum was collected from naive and T. muris-infected C57BL/6 (C57), μMT (muMT), and B cell-reconstituted μMT (muMTB) mice and tested for the presence of parasite-specific IgG1 (A) and IgG2a (B) by ELISA. Data shown compare mean optical densities (od) ± SE obtained from serum at a nonsaturating dilution of 1:160 (n = 4 for naive and day 35 p.i. [d35] groups). ∗, significant increase above naive levels (P < 0.05).

FIG. 7

T. muris worm burdens (mean ± SE) for μMT and AKR mice treated with either anti-IL-12 monoclonal antibody C17.8 or rat IgG (rIg) on days 0, 5, 9, and 14 p.i. (1 mg of antibody per intraperitoneal injection). Infected C57BL/6 mice were included as controls. Mice were infected with approximately 150 infective embryonated T. muris eggs on day 0, and worm burdens were counted in groups of four mice per strain at the time points shown. ∗, significant difference in worm burdens between rat IgG- and C17.8-treated groups of the same mouse strain (P < 0.05).

FIG. 8

T. muris worm burdens (mean ± SE) for μMT and AKR mice treated with either parasite-specific IgG1 (pIgG1) or nonspecific IgG (nIgG) on days 0, 1, and 3 p.i. (1 mg of antibody per intraperitoneal injection). Mice were infected with approximately 175 infective embryonated T. muris eggs on day 0 (n = 4 per group). ∗, significant difference between parasite-specific and nonspecific IgG-treated groups of the same mouse strain (P < 0.05). d11, day 11 p.i. group; d35, day 35 p.i. group.