Thyroxine is a potential endogenous antagonist of macrophage migration inhibitory factor (MIF) activity

Abstract

Abnormally low plasma concentrations of thyroid hormones during sepsis often occur in the absence of thyroidal illness; however, the mechanisms involved in the "euthyroid sick syndrome" remain poorly understood. Here, we describe a previously unrecognized interaction between the thyroid hormone thyroxine (T(4)) and the proinflammatory cytokine macrophage migration inhibitory factor (MIF), together with its clinical relevance in sepsis. We found that in both patients with severe sepsis, and our rodent model, low plasma T(4) concentrations were inversely correlated with plasma MIF concentrations. The MIF molecule contains a hydrophobic pocket that is important for many of its proinflammatory activities. Binding of L-T(4) (or its hormonally inert isomer D-T(4)) significantly, and dose-dependently, inhibited the catalytic activity of this pocket. Moreover, administration of exogenous D-T(4) significantly improved survival in mice with severe sepsis. To examine the specificity of the MIFT(4) interaction, wild-type and MIF knockout mice were subjected to the carrageenan-air pouch model of inflammation and then treated with D-T(4) or vehicle. D-T(4) significantly inhibited leukocyte infiltration in wild-type mice but not in MIF knockout mice, providing evidence that in vivo T(4) may influence MIF-mediated inflammatory responses via inhibition of its hydrophobic proinflammatory pocket. These findings demonstrate a new physiological role for T(4) as a natural inhibitor of MIF proinflammatory activity. The data may also, in part, explain the low plasma T(4) concentrations in critically ill, euthyroid patients and suggest that targeting the imbalance between MIF and T(4) may be beneficial in improving outcome from sepsis.

Fig. 1.

Plasma T4 levels decrease and MIF levels increase in the course of sepsis. (A) MIF levels (log ₁₀) are negatively correlated with free T₄ levels in plasma of patients with sepsis (n = 26, Corr Coeff: -0.71, p = 0.0001). (B) Plasma-free T₄ levels (solid squares) decrease, whereas MIF levels (open circles) increase (n = 5) during CLP-induced severe sepsis in rats [represented as percentage of vehicle control (n = 6)].

Fig. 2.

T4 binds to MIF hydrophobic cavity. (A) Chemical structures of L thyroxine (L-T₄), D thyroxine (D-T₄), triiodothyronine (T₃), and ISO-1. (B) Pose of T₄ in the binding site of MIF (chains A and C from crystal structure 1LJT) with highlight of H bonds (purple dashed lines) and I⋯O distances within reach for halogen bonding (plain green lines). (C) Highlight of contact preferences for hydrophobic moieties (green meshed surface) and for lone pair acceptors (purple meshed surface) overlaid to T₄ posed in MIF. Ile 64 has not been represented for clarity in C and D. (D) Close view of bound T₄ within MIF. The analytical solvent-accessible Connolly surface accenting the pocket is colored based on physico-chemical properties: hydrophobic (blue), hydrophilic (red), and solvent-exposed (gray). Lys 32 and Pro 33 have not been surfaced for clarity.

Fig. 3.

L-T₄ (solid circles), D-T₄ (open circles), and ISO-1 (open triangles), but not T₃ (solid triangles) produce dose-dependent inhibition of the MIF active site (tautomerase), which is associated with its proinflammatory activities.

Fig. 4.

D-T₄ administration reduces local inflammation and improves survival in murine sepsis. (A) D-T₄ inhibition of MIF-induced localized inflammation. D-T₄ (solid bars) significantly suppressed leukocyte infiltration in wild-type mice (n = 17) in a carrageenan-induced airpouch model of localized inflammation compared to vehicle-treated controls (open bars). However, D-T₄ administration had no effect on leukocyte migration in MIF knockout mice (n = 18). (B) D-T₄ improves survival in murine sepsis. Administration of D-T₄ (open circles) starting at 24 h after the onset of CLP-induced sepsis in mice significantly improves survival (n = ○17/•17, p < 0.01) compared with vehicle-treated controls (solid circles). (C) D-T₄ improves survival in thyroidectomized mice subjected to CLP sepsis. Male C57BL/6 mice underwent thyroidectomy and parathyroidectomy as described in Materials and Methods and were then subject to CLP surgery. Mice were administered DMSO vehicle control (solid circles, n = 16) or D-T₄ (solid triangles, n = 17), and survival was then monitored over two weeks. The survival, which was significantly increased in the D-T₄ group, was 13 and 53%, respectively (p < 0.05).