B-cell adaptor for PI3K (BCAP) negatively regulates Toll-like receptor signaling through activation of PI3K

Abstract

Toll-like receptors (TLRs) recognize pathogens and their components, thereby initiating immune responses to infectious organisms. TLR ligation leads to the activation of NF-κB and MAPKs through well-defined pathways, but it has remained unclear how TLR signaling activates PI3K, which provides an inhibitory pathway limiting TLR responses. Here, we show that the signaling adapter B-cell adaptor for PI3K (BCAP) links TLR signaling to PI3K activation. BCAP-deficient macrophages and mice are hyperresponsive to TLR agonists and have reduced PI3K activation. The ability of BCAP to inhibit TLR responses requires its capacity to bind PI3K. BCAP is constitutively phosphorylated and associated with the p85 subunit of PI3K in macrophages. This tyrosine-phosphorylated BCAP is transiently enriched in the membrane fraction in response to LPS treatment, suggesting a model whereby TLR signaling causes the phosphorylation of the small amount of BCAP that is associated with membranes in the resting state or the translocation of phosphorylated BCAP from the cytoplasm to the membrane. This accumulation of tyrosine-phosphorylated BCAP at the membrane with its associated PI3K would then allow for the catalysis of Ptd Ins P2 to Ptd Ins P3 and downstream PI3K-dependent signals. Therefore, BCAP is an essential activator of the PI3K pathway downstream of TLR signaling, providing a brake to limit potentially pathogenic excessive TLR responses.

Fig. 1.

BCAP negatively regulates TLR-induced inflammatory cytokine production in vitro and in vivo. (A and B) BM-derived macrophages from WT or BCAP-deficient (BCAP KO) mice were incubated with the indicated concentrations of LPS (TLR4), Imiquimod (TLR7), or CpG DNA (TLR9) for 16 h. Supernatants were collected, and the amounts of IL-12 p40, IL-6, and TNF (A) or IL-10 (B) were measured by ELISA. Data are representative of four independent experiments and are expressed as the mean ± SD of triplicate wells. *P < 0.05. ND, not detected. (C) WT or BCAP-deficient mice were injected i.p. with 1 μg/g of LPS, and plasma IL-12 p40 concentrations at 1, 2, 4, and 6 h postinjection were measured. Data are from one of three independent experiments and are expressed as the mean ± SD with n = 4 mice per group, with each mouse assayed in triplicate. *P < 0.05; **P < 0.01.

Fig. 2.

BCAP deficiency has minimal effects on TLR-induced MAPK activation and IκBα degradation. BM-derived macrophages were stimulated with 1 ng/mL LPS for the indicated times, after which cells were lysed. Cytoplasmic extracts were analyzed by Western blot using antibodies specific for p38 MAPK, p42/44 ERK, and JNK or for phosphorylated versions of these proteins (A) or for IκBα with β-actin as a loading control (B). Data are representative of three independent experiments.

Fig. 3.

PI3K activity is reduced in BCAP-deficient macrophages. (A) Macrophages were stimulated with 1 ng/mL LPS for the indicated time and then lysed. Cytoplasmic extracts were analyzed by Western blot using antibodies specific for phospho-Akt (Ser473 or Thr308) or Akt. (B and C) Macrophages were stimulated for 16 h with CpG DNA (25 nM) after 30 min of pretreatment with vehicle control or wortmannin. (B) Secretion of IL-12 p40 and IL-6 was measured by ELISA after treatment with 250 nM wortmannin. (C) Results are expressed as fold change compared with CpG DNA-treated, no wortmannin control, and they show the mean ± SD of triplicate wells. Data are representative of five (A) and three (B and C) experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

BCAP is constitutively tyrosine-phosphorylated and associated with p85 PI3K in macrophages. (A) WT or BCAP-deficient macrophages were lysed and immunoprecipitated with anti-BCAP monoclonal antibody, separated by SDS-PAGE and immunoblotted for phosphotyrosine or BCAP. (B) Macrophages were treated with 1 ng/mL LPS for the indicated times and then lysed and analyzed as in A. (C) WT macrophages were treated with 1 ng/mL LPS for the indicated times, and cytosolic or membrane protein fractions were then extracted and subjected to immunoprecipitation with anti-BCAP monoclonal antibody and analyzed as in A. Cell fractions were also analyzed by Western blot for pan-Cadherin or GAPDH to examine the extraction efficiency. Data are representative of eight (A), six (B), and three (C) experiments.

Fig. 5.

BCAP negatively regulates TLR-induced cytokine production through binding to p85 subunit of PI3K. (A and B) WT or BCAP-deficient macrophages were transduced with an empty vector (control) retrovirus or retrovirus encoding WT BCAP. The macrophages were activated with CpG DNA (25 nM) in the presence of Brefeldin A for 6 h, and cytokine production was assessed by flow cytometry. Transduced cells were gated based on GFP fluorescence, and the percentage of IL-12 p40 or TNF-producing cells was determined. (C and D) BM-derived macrophages from BCAP-deficient mice were transduced with an empty vector (Control) retrovirus or retrovirus encoding WT BCAP (BCAP WT) or a BCAP tyrosine mutant (BCAP Y4F). Cytokine production was measured as in A. Gray histograms show staining of unstimulated cells. Data are representative of four independent experiments and are expressed as the mean ± SD of triplicate wells. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.