The physiological regulation of toll-like receptor expression and function in humans

Abstract

Eleven mammalian toll-like receptors (TLRs 1-11) have been identified to date and are known to play a crucial role in the regulation of immune responses; however, the factors that regulate TLR expression and function in vivo are poorly understood. Therefore, in the present study, we investigated the physiological regulation of TLR expression and function in humans. To examine the influence of diurnal rhythmicity on TLR expression and function, peripheral venous blood samples were collected from healthy volunteers (n = 8) at time points coinciding with the peak and nadir in the endogenous circulating cortisol concentration. While no diurnal rhythmicity in the expression of TLRs 1, 2, 4 or 9 was observed, the upregulation of costimulatory (CD80 and CD86) and antigen-presenting (MHC class II) molecules on CD14(+) monocytes following activation with specific TLR ligands was greater (P < 0.05) in samples obtained in the evening compared with the morning. To examine the influence of physical stress on TLR expression and function, peripheral venous blood samples were collected from healthy volunteers (n = 11) at rest and following 1.5 h of strenuous exercise in the heat (34 degrees C). Strenuous exercise resulted in a decrease (P < 0.005) in the expression of TLRs 1, 2 and 4 on CD14(+) monocytes. Furthermore, the upregulation of CD80, CD86, MHC class II and interleukin-6 by CD14(+) monocytes following activation with specific TLR ligands was decreased (P < 0.05) in samples obtained following exercise compared with at rest. These results demonstrate that TLR function is subject to modulation under physiological conditions in vivo and provide evidence for the role of immunomodulatory hormones in the regulation of TLR function.

Figure 1. The effect of circadian rhythmicity on toll-like receptor (TLR) cell surface expression and activation

Peripheral blood samples were obtained at 07.00 h and 17.00 h from eight healthy donors. A and B, samples were labelled with specific TLR monoclonal antibodies or isotype controls, and examined by flow cytometry. C–F, samples were incubated with media only (unstimulated), lipopolysaccharide (LPS; TLR4 ligand), zymosan (TLR2 and 6 ligand) or polyinosine-polycytidylic acid (poly(I:C); TLR3 ligand) for either 6 (D, E and F) or 24 h (C), and expression of costimulatory molecules and intracellular cytokines was examined by flow cytometry. All data represent means ± s.e.m.*Statistically significant difference as determined by paired-samples t test. ND, not detected.

Figure 2. The effect of exercise on TLR expression on CD14⁺ monocytes

Peripheral blood samples were obtained from 11 healthy volunteers before, immediately after, and following 2 h of resting recovery from 90 min of exercise at 55% maximal work rate (W_max) in the heat (34°C). A, B, C and D, samples were labelled with specific TLR monoclonal antibodies or isotype controls, and examined by flow cytometry. All data represent means ± s.e.m.†Statistically significant difference (P < 0.005) from pre-exercise. GMFI, geometric mean fluorescence intensity.

Figure 3. The effect of exercise on CD86 and MHC class II expression on CD14⁺ monocytes

Peripheral blood samples were obtained from 11 healthy volunteers before, immediately after, and following 2 h of resting recovery from 90 min of exercise at 55%W_max in the heat (34°C). A and B, samples were incubated with culture media only for 6 h, and the expression of CD86 and MHC class II was examined by flow cytometry. All data represent means ± s.e.m.†Statistically significant difference (P < 0.05) from pre-exercise.

Figure 4. The effect of exercise on CD86 and MHC class II expression on CD14⁺ monocytes

Peripheral blood samples were obtained from 11 healthy volunteers before, immediately after, and following 2 h of resting recovery from 90 min of exercise at 55%W_max in the heat (34°C). A–F, samples were incubated with LPS (TLR4 ligand; A and B), zymosan (TLR2 and 6 ligand; C and D) or poly(I:C) (TLR3 ligand; E and F) for 6 h, following which the expression of CD86 and MHC class II was examined by flow cytometry. All data represent means ± s.e.m.†Statistically significant difference (P < 0.05) from pre-exercise. *Statistically significant difference (P < 0.05) from post-exercise.

Figure 5. The effect of exercise on CD80 expression on CD14⁺ monocytes

Peripheral blood samples were obtained from nine healthy volunteers before, immediately after, and following 2 h of resting recovery from 90 min of exercise at 55%W_max in the heat (34°C). A–C, samples were incubated with LPS (TLR4 ligand; A), zymosan (TLR2 and 6 ligand; B) or poly(I:C) (TLR3 ligand; C) for 24 h, following which the expression of CD80 was examined by flow cytometry. All data represent means ± s.e.m.†Statistically significant (P < 0.01) difference from pre-exercise during the placebo trial.

Figure 6. The effect of exercise on intracellular interleukin (IL)-6 expression in CD14⁺ monocytes

Peripheral blood samples were obtained from 10 healthy volunteers before, immediately after, and following 2 h of resting recovery from 90 min of exercise at 55%W_max in the heat (34°C). Samples were incubated with LPS (TLR4 ligand), zymosan (TLR2 and 6 ligand), or with culture media only for 6 h, following which monocyte intracellular IL-6 expression was examined by flow cytometry. All data represent means ± s.e.m.†Statistically significant (P < 0.01) difference from pre-exercise.