Reduced severity of peanut-induced anaphylaxis in TLR9-deficient mice is associated with selective defects in humoral immunity

Abstract

Signaling through the innate immune system can promote or suppress allergic sensitization. Toll-like receptor 9 (TLR9) has modulatory effects on the mucosal immune system, and we hypothesized that TLR9 would influence susceptibility to allergic sensitization to foods. We observed that TLR9-/- mice were resistant to peanut-induced anaphylaxis. This was associated with a significant impairment in total immunoglobulin E (IgE) and peanut-specific IgE and IgA, but not IgG1 or Th2 cytokine production. TLR9-/- mice had reduced development of Peyer's patches, but resistance to sensitization was not restricted to oral routes. Rag1-deficient mice were reconstituted with TLR9+/+ or -/- B cells plus CD4+ T cells. TLR9-/- B cells regained the ability to produce IgE in the presence of a wild-type environment. Our results demonstrate that TLR9 on an unknown cell type is required for the development of IgE-producing B cells, and we conclude that TLR9 signaling indirectly shapes the immune response for optimal IgE production.

Figure 1

Impact of TLR9 deficiency on peanut-induced anaphylaxis and antibody and cytokine responses. TLR9 +/+ or −/− mice were orally sensitized to peanut with cholera toxin (PN/CT), followed by intraperitoneal challenge with crude peanut extract. (A) Body temperature measured 30 min after peanut challenge. Peanut (PN)-specific IgE (B), IgG1 (C) and IgA (D) as measured by ELISA in serum obtained 1-2 days prior to challenge. Cytokine secretion (E) measured after re-stimulation of spleen cells with peanut extract. Spleen cells were harvested immediately after allergen challenge. A is combined data from 2 independent experiments. B-E are from one representative experiment of 2, with n = 10 (+/+) and 8 (−/−). *** p < 0.001, ** p < 0.01, * p < 0.05 comparing the indicated groups.

Figure 2

Detection of IgE+ B cells. Mice were left naïve as control, or sensitized with PN/CT for 6 weeks. Sensitization was verified by measurement of peanut-specific IgE in serum. Mice were not allergen challenged prior to collection of tissues. (A) Representative staining in bone marrow (BM) and spleen cells. Live CD19+ cells were gated, and plots show IgE versus CD19 in TLR9+/+ and TLR9−/− mice orally sensitized to peanut (PN/CT) or naive. (B) Median fluorescence intensity (MFI) of IgE in IgE+ B cells gated as above. N = 3/group (spleen) and 2/group (BM). *p < 0.05 (C) Total IgE measured by ELISA in serum of naïve or peanut-sensitized (PN/CT) TLR9+/+ or −/− mice. ** p < 0.01, n = 8-15 mice/group.

Figure 3

Impact of TLR9 deficiency on mast cells. (A) Peritoneal cells were harvested from naïve or orally peanut-sensitized (PN/CT) TLR9+/+ or −/− mice. Peritoneal cells were harvested from sensitized un-challenged mice. Mast cells were gated based on c-kit and IgE staining, and the level of IgE positivity was compared in naïve and sensitized +/+ and −/− mice. (B) Naïve TLR9+/+ or −/− mice were passively sensitized in the ear with serum from peanut-sensitized mice or OVA-sensitized mice as controls. The next day, mice were intravenously injected with Evans Blue and peanut extract, and extravasation in each ear was quantified by spectrophotometry. * p < 0.05, ns = not significant. (n = 5/group).

Figure 4

Impact of TLR9 deficiency on Peyer’s patches. The entire small intestine from naïve TLR9+/+ or −/− mice was removed and the total number of visible Peyer’s patches per mouse was counted (A). Cells were isolated by collagenase digestion and the yield of cells per mouse was determined (B). Segments of intestine containing a Peyer’s patch were fixed, embedded, and cross-sections stained with hematoxylin and eosin. The number of follicles per Peyer’s patch was counted (C). ** p < 0.01.

Figure 5

Sensitization to peanut by non-oral routes. TLR9+/+ and −/− mice were sensitized to peanut by intraperitoneal injection with alum (IP), or by repeated epicutaneous exposure (skin). A) Body temperature measured 30 min after intraperitoneal challenge with 500 g of peanut extract. B) IgE, C) IgA, and D) IgG1 antibodies were measured in serum obtained 1-2 days prior to challenge. * p < 0.05, ** P < 0.01.

Figure 6

Reconstitution of Rag1-deficient mice with TLR9+/+ or TLR9−/− B cells. Rag1−/− mice were reconstituted with B cells from naïve TLR9 +/+ or −/− mice together with CD4+ T cells from TLR9+/+ mice primed with peanut. (n = 4/group, and 2 controls receiving no CD4⁺ T cells) (A) Total (left) and peanut-specific (right) IgE was measured 4 weeks after cell transfer (pre-sensitization) and after 6 weeks of oral sensitization with peanut plus CT. (B) Mice were challenged by the intraperitoneal route with peanut extract, and rectal temperature measured before and 30 min after challenge to assess anaphylaxis severity. ** p < 0.01 using a paired T test.