TRAF5 negatively regulates TLR signaling in B lymphocytes

Abstract

The cytoplasmic adaptor proteins TNFR-associated factor (TRAF)3 and TRAF6 are important mediators of TLR signaling. To our knowledge, we show in this study for the first time that another TRAF family member, TRAF5, is a negative regulator of TLR signaling. B lymphocytes from TRAF5(-/-) mice produced more IL-6, IL-12p40, IL-10, TNF-α, and IgM than did wild-type B cells after TLR stimulation. Consistent with these data, exogenous overexpression of TRAF5 in B cells inhibited TLR-mediated cytokine and Ab production. TLR stimulation of TRAF5-deficient B cells did not affect cell survival, proliferation, or NF-κB activation but resulted in markedly enhanced phosphorylation of the MAPKs ERK1/2 and JNK. TRAF5 negatively regulated TLR signaling in a cell-specific manner, because TRAF5(-/-) macrophages and dendritic cells showed less dramatic differences in TLR-mediated cytokine production than B cells. Following TLR stimulation, TRAF5 associated in a complex with the TLR adaptor protein MyD88 and the B cell-specific positive regulator of TLR signaling TAB2. Furthermore, TRAF5 negatively regulated the association of TAB2 with its signaling partner TRAF6 after TLR ligation in B cells. To our knowledge, these data provide the first evidence that TRAF5 acts as a negative regulator of TLR signaling.

Figure 1. Effect of TRAF5 status on TLR-mediated cytokine production by B lymphocytes

Splenic B cell isolation and detection of cytokine production were performed as described in Materials and Methods. (A) Splenic B cells were isolated from TRAF5 KO and TRAF5 WT mice and cultured with the indicated TLR ligands for 48 hours. Cytokine production from TRAF5 KO B cells (filled bars) was compared to TRAF5 WT B cells (open bars). (B) CH12.LX B cells were cultured for 20 hours with IPTG to induce overexpression of TRAF5. Cytokine production after 48 hours of TLR stimulation from these cells (filled bars) was compared to CH12.LX cells that were not induced with IPTG (open bars). Data represent the mean values ± SD of three experiments. Statistical analysis was conducted using Student’s t test. Asterisks denote a p value of less than 0.05.

Figure 2. Effect of TRAF5 deficiency on TLR-mediated cytokine production by bone marrow-derived dendritic cells

BMDC (A) and BMM (B) differentiation was performed as described in Materials and Methods. BMDC and BMM derived from TRAF5 WT and TRAF5 KO mice were cultured with the indicated TLR ligands for 24 hours. Cytokine production from TRAF5 KO BMDC or BMM (filled bars) was compared to TRAF5 WT BMDC or BMM (open bars). Data represent the mean values ± SD of three experiments. Statistical analysis was conducted using Student’s t test. Asterisks denote a p value of less than 0.05.

Figure 3. Effect of TRAF5 status on TLR-induced antibody production

Antibody production was measured as described in Materials and Methods. (A) CH12.LX B cells were cultured for 20 hours with IPTG to induce overexpression of TRAF5 (CH12-TRAF5). IgM production after TLR ligation from these cells (filled bars) was compared to CH12.LX cells that were not induced with IPTG (open bars). (B) Splenic B cells were isolated from TRAF5 KO and TRAF5 WT mice and cultured with the indicated TLR ligands for five days. IgM production from TRAF5 KO B cells (filled bars) was compared to TRAF5 WT B cells (open bars). Data represent the mean values ± SD of three experiments. Statistical analysis was conducted using Student’s t test. Asterisks denote a p value of less than 0.05.

Figure 4. TLR-induced MAPK activation in TRAF5-deficient B lymphocytes

Splenic B cells were isolated from TRAF5 KO and WT mice as described in Materials and Methods and stimulated with TLR7 agonist R848 for the indicated times. Preparation of whole cell lysates and Western blotting were as described in Materials and Methods. Western blots shown are representative of three experiments, while the quantitation provided combines three separate experiments.

Figure 5. TLR-induced NF-κB activation in TRAF5-deficient B lymphocytes

Splenic B cells were isolated from TRAF5 KO and WT mice as described in Materials and Methods and stimulated with TLR7 agonist R848 for the indicated times. Preparation of whole cell lysates and Western blotting were as described in Materials and Methods. Western blots shown are representative of three experiments, while the quantitation provided combines three separate experiments.

Figure 6. TLR-induced association of TRAF5 with TLR signaling proteins

CH12.LX B cells (A, B, D) were cultured for 20 hours with IPTG to induce expression of FLAG-tagged TRAF5. Splenic B cell isolation was performed as described in Materials and Methods (C). Cells were stimulated with TLR7 agonist R848 for the indicated times. Immunoprecipitation of FLAG (A), MyD88 (B), TRAF6 (C, D) and Western blotting was performed as described in Materials and Methods. Quantitation in D is shown below the blots as percent of maximum association.