Proinflammatory TLR signalling is regulated by a TRAF2-dependent proteolysis mechanism in macrophages

Abstract

Signal transduction from toll-like receptors (TLRs) is important for innate immunity against infections, but deregulated TLR signalling contributes to inflammatory disorders. Here we show that myeloid cell-specific ablation of TRAF2 greatly promotes TLR-stimulated proinflammatory cytokine expression in macrophages and exacerbates colitis in an animal model of inflammatory bowel disease. TRAF2 deficiency does not enhance upstream signalling events, but it causes accumulation of two transcription factors, c-Rel and IRF5, known to mediate proinflammatory cytokine induction. Interestingly, TRAF2 controls the fate of c-Rel and IRF5 via a proteasome-dependent mechanism that also requires TRAF3 and the E3 ubiquitin ligase cIAP. We further show that TRAF2 also regulates inflammatory cytokine production in tumour-associated macrophages and facilitates tumour growth. These findings demonstrate an unexpected anti-inflammatory function of TRAF2 and suggest a proteasome-dependent mechanism that limits the proinflammatory TLR signalling.

Figure 1. TRAF2 negatively regulates experimental colitis

WT and Traf2-MKO (T2-MKO) mice were treated with 3% (a, c-h) or 3.5% (b) DSS (in drinking water) for 6 days and then supplied with normal drinking water. (a) IB analysis of TRAF2 and TRAF3 in FACS sorted spleen (SM), peritoneal (PM), and colon (CM) macrophages (CD11b⁺F4/80⁺) from naïve (H₂O treated) and day 8 DSS-treated WT mice. (b,c) Survival rate (b) and body weight loss (c) of DSS-treated Traf2-MKO and WT mice. (d,e) Colon Length (d) and H&E histology staining (e) from day 8 DSS-treated Traf2-MKO and WT mice. (Scale bar, 100μm) (f,g) FACS analysis of the total immune cells (CD45⁺) and the indicated subsets, presented as a representative plot (f) and mean ± SD values of multiple mice (n=3 mice/genotype) (g). (h) qRT-PCR analysis of the indicated genes (presented as fold relative to Actin mRNA level) using FACS-sorted colon macrophages (CD11b⁺F4/80⁺) from day 8 DSS-treated WT and Traf2-MKO mice. Data shown are representative of at least three independent experiments. The error bars represent standard deviation (SD). Differences between experimental and control groups were determined by Student's t test. For animal survival analysis, the Kaplan-Meier method was adopted to generate graphs, and the survival curves were analyzed with log-rank analysis. *P < 0.05; **P < 0.01.

Figure 2. TRAF2 negatively regulates proinflammatory cytokine induction

(a,b) qRT-PCR analysis of the indicated genes using BMDMs (A) or peritoneal macrophages (b) derived from WT or Traf2-MKO mice at 0, 2 and 6 hours after LPS stimulation. Data are presented as fold relative to the Actin mRNA level. (c) ELISA of the indicated cytokines in the supernatants of the WT and Traf2-MKO BMDMs, which were either not treated (0) or stimulated with LPS for 24 h. Data are presented as mean ± SEM values and representative of at least three independent experiments. The error bars represent SD, and differences between experimental and control groups were determined by Student's t test. *P < 0.05; **P < 0.01.

Figure 3. TRAF2 negatively regulates proinflammatory cytokine induction independently of NIK

(a) IB analysis of the indicated phosphorylated (P-) and total proteins in the whole-cell lysates of LPS-stimulated BMDMs derived from WT or Traf2-MKO (MKO) mice crossed to NIK^+/+ or NIK^–/– background. (b) qRT-PCR analysis of the indicated mRNAs in LPS-stimulated BMDMs derived from the WT and Traf2-MKO mice crossed to NIK^+/+ or NIK^–/– background. The mRNA level was calculated as fold relative to an internal control, Actb. Data are mean ± SEM based on multiple samples and representative of three independent experiments. The error bars represent SD, and differences between experimental and control groups were determined by Student's t test. *P<0.05.

Figure 4. TRAF2 deficiency attenuates ubiquitin-dependent degradation of c-Rel and IRF5 and promotes their function

(a) IKK kinase assay (KA) and IB assays using whole-cell lysates of LPS-stimulated BMDMs derived from WT and Traf2-MKO mice. (b-c) IB analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of WT and Traf2-MKO BMDMs stimulated with LPS. (d) Immunoblot analysis of the indicated proteins in the cytoplasmic (CE) and nuclear (NE) extracts of WT and Traf2-MKO BMDMs either not treated (0) or stimulated with LPS for 3 h. (e) ChIP analysis of c-Rel and IRF5 at the promoter of the indicated genes using WT or Traf2-MKO BMDMs that were either not treated (0) or stimulated for 3 h with LPS. Data are presented as fold relative to the total input DNA and representative of three or more independent experiments. *P < 0.05. (f) IRF5 and c-Rel were isolated by IP (under denaturing conditions) from whole-cell lysates of DMSO or MG132-treated WT and Traf2-MKO BMDMs and subjected to IB assays using anti-ubiquitin (top panels) or anti-K48-linked ubiquitin (middle panels) antibodies. Protein lysates were also subjected to direct IB (bottom panels). Data are representative of at least three independent experiments. The error bars represent SD, and differences between experimental and control groups were determined by Student's t test. *P<0.05.

Figure 5. TRAF3 controls the fate of c-Rel and IRF5 and negatively regulates inflammation

(a) IB analysis of the indicated proteins in whole-cell lysates of LPS-stimulated Traf3^fl/flCre^ER or control Traf3^+/+Cre^ER BMDMs differentiated in the presence of the Cre inducer tamoxifen. (b) IB analysis using whole-cell lysates of M-CSF treated bone marrow cells from tamoxifen-injected Traf3^fl/flCre^ER or control Traf3^+/+Cre^ER mice. (c) qRT-PCR analysis of the indicated mRNAs in LPS- or poly(I:C)-stimulated Traf3^+/+Cre^ER and Traf3^fl/flCre^ER BMDMs differentiated in the presence of the Cre inducer tamoxifen. (d-h) Traf3-MKO and WT control mice were challenged with 3.5% (d) or 3% (E-H) DSS (in drinking water) for 6 days. The mice were monitored for survival (d) and weight loss (e) calculation or sacrificed on day 8 for H&E histology analysis (Scale bar, 100 μm) (f), colon length measuring (g), or flow cytometry analysis of colon macrophages (h). Data are representative of at least three independent experiments. The error bars represent SD, and differences between experimental and control groups were determined by Student's t test. For animal survival analysis, the Kaplan-Meier method was adopted to generate graphs, and the survival curves were analyzed with log-rank analysis. *P < 0.05.

Figure 6. cIAP is involved in the regulation of IRF5/c-Rel degradation and proinflammatory cytokine induction

(a) IB analysis of the indicated proteins in the whole-cell lysates of WT and Traf2-MKO BMDMs, treated for 12 hr with a Smac mimic (SM) compound or solvent control DMSO (DM). (b,c) BMDMs from WT and Traf2-MKO mice (b) or tamoxifen-treated TRAF3^+/+Cre^ER and TRAF3^fl/flCre^ER mice (c) were pretreated with MG132. Whole-cell lysates were subjected to IP using antibodies for IRF5 or c-Rel or a control rabbit IgG (Ig), followed by detecting the precipitated IRF5, c-Rel, and their associated cIAP2 by IB. Cell lysates were also subjected to direct IBs (Bottom panels). (d) qRT-PCR analysis of the indicated mRNAs in WT and Traf2-MKO BMDMs, pre-treated for 12 h with a Smac mimic (SM) or DMSO and then stimulated with LPS. Data are mean ± SEM of 3 replicate samples and representative of three independent experiments. The error bars represent SD, and differences between experimental and control groups were determined by Student's t test. *P<0.05; **P < 0.01.

Figure 7. TRAF2 functions in myeloid cells to regulate antitumor immunity

WT or Traf2-MKO were injected with 2 × 10⁵ (a,b and d-h) or 5 × 10⁵ (c) B16 melanoma cells. (a-c) Tumor growth curve (A), a representative picture of day 18 tumors (b), and survival curve (c). (d) Flow cytometric analysis of immune cells (CD45⁺) infiltrated to day 18 tumors and the frequency of CD4⁺ T cells, CD8⁺ T cells, macrophages (CD11b⁺F4/80⁺), and myeloid derived suppressor cells (MDSCs) (CD11b⁺Gr-1⁺) within the CD45⁺ cell population. (e) Summary of flow cytometry data of D based on multiple animals, showing the frequency of the indicated cell populations within total tumor cells. (f,g) ICS and flow cytometric analysis of IFNγ+ CD4⁺ and CD8⁺ T cells, presented as a representative plot (f) and a summary graph based on multiple animals with day 18 tumors (g). (h) qRT-PCR analysis of the indicated mRNAs in TAMs derived from day 18 tumors of WT or Traf2-MKO (MKO) mice. Data are mean ± SEM value of multiple animals (each circle or square represents a mouse) and representative of three independent experiments. The error bars represent SD, and differences between experimental and control groups were determined by Student's t test. *P<0.05; **P < 0.01.