High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor

Abstract

The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam₃CysSerLys₄ (Pam₃CSK₄), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam₃CSK₄ complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain-containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain-containing adaptor-inducing interferon-β [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam₃CSK₄. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.

Figure 1

HMGB1 in complex with LPS or Pam₃CSK₄ induces cytokines via TLR4 and TLR2, respectively, but not via RAGE signaling. Peritoneal macrophages from wild-type, RAGE^−/−, TLR2^−/− or TLR4^−/− mice were stimulated with preformed HMGB1-LPS or HMGB1-Pam₃CSK₄ complexes, or with individual components separately. Stimulations with 10μg/mL LPS or Pam₃CSK₄ were used as positive controls. HMGB1-LPS (A) and HMGB1-Pam₃CSK₄ (B) complexes both gave enhanced IL-6 production compared with stimulation with the same dose of LPS or Pam₃CSK₄ alone. (A, B): Results of one representative experiment. Four to five individual experiments were performed with each stimulation. The stimulatory capacity of the complexes (fold change) were calculated by normalizing the raw data in each experiment against 10 ng/mL LPS (n = 5) (C) or Pam₃CSK₄ (n = 4) (D). All values are expressed as mean ± SD. *P < 0.05, **P < 0.01. ND, not detected; ns, not significant; (A, C)▨, unstimulated;□, LPS (10 ng/mL); , HMGB1 (1 μg/mL);■, HMGB1 (1 μg/mL) + LPS (10 ng/mL); , LPS (1 μg/mL); (B, D) ▨, unstimulated;□, Pam₃CSK₄ (10 ng/mL); , HMGB1 (20 μg/mL);■, HMGB1 (20 μg/mL) + Pam₃CSK₄ (10 ng/mL); , Pam₃CSK₄ (10 μg/mL).

Figure 2

Effect of TIRAP, MyD88, TRIF and TRAM deficiency on HMGB1 complex stimulation. Peritoneal macrophages from wild-type, MyD88^−/−, TIRAP^−/−, TRIF^−/− or TRAM^−/− mice were stimulated with preformed HMGB1-LPS (A) or HMGB1-Pam₃CSK₄ (B) complexes or individual components separately. Stimulations with 1 μg/mL LPS or 10 μg/mL Pam₃CSK₄ were used as positive controls. The results of one representative experiment out of three to seven individual experiments performed for each stimulation are shown: wild-type (n = 7), TRAM^−/− (n = 3), TIRAP^−/− (n = 4), TRIF^−/− (n = 3) and MyD88^−/− (n = 3). All values are expressed as mean ± SD. ND, not detected; (A) ▨, unstimulated;□, LPS (1 ng/mL); , HMGB1 (1 μg/mL);■, HMGB1 (1 μg/mL) + LPS (1 ng/mL); , LPS (1 μg/mL); (B) ▨, unstimulated;□, Pam₃CSK₄ (10 ng/mL); , HMGB1 (20 μg/mL);■, HMGB1 (20 μg/mL) + Pam₃CSK₄ (10 ng/mL); , Pam₃CSK₄ (10 μg/mL).

Figure 3

Deficiency of TIRAP, MyD88, TRAM or TRIF does not influence the enhancing effect of HMGB1 complexes on IL-6 production. Peritoneal macrophages from wild-type, TRIF^−/−, TRAM^−/−, TIRAP^−/− or MyD88^−/− mice were stimulated with preformed HMGB1-LPS or HMGB1-Pam₃CSK₄ complexes or individual components separately. Stimulations with 1 μg/mL LPS or 10 μg/mL Pam₃CSK₄ were used as positive controls. Three to seven individual experiments were performed for each stimulation: wild-type (n = 7), TRAM^−/− (n = 3), TIRAP^−/− (n = 4), TRIF^−/− (n = 3) and MyD88^−/− (n = 3). Raw data in each experiment were normalized to 1 μg/mL LPS (A) or 10 μg/mL Pam₃CSK₄ (B) stimulation to generate fold change. All values are expressed as mean ± SD. ND, not detected; ns, not significant. (A)□, LPS (1 ng/mL);■, HMGB1 (1 μg/mL) + LPS (1 ng/mL);(B)□, Pam₃CSK₄ (10 ng/mL);■, HMGB1 (20 μg/mL) + Pam₃CSK₄ (10 ng/mL).