α7 nicotinic acetylcholine receptor (α7nAChR) expression in bone marrow-derived non-T cells is required for the inflammatory reflex

Abstract

The immune response to infection or injury coordinates host defense and tissue repair, but also has the capacity to damage host tissues. Recent advances in understanding protective mechanisms have found neural circuits that suppress release of damaging cytokines. Stimulation of the vagus nerve protects from excessive cytokine production and ameliorates experimental inflammatory disease. This mechanism, the inflammatory reflex, requires the α7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel expressed on macrophages, lymphocytes, neurons and other cells. To investigate cell-specific function of α7nAChR in the inflammatory reflex, we created chimeric mice by cross-transferring bone marrow between wild-type (WT) and α7nAChR-deficient mice. Deficiency of α7nAChR in bone marrow-derived cells significantly impaired vagus nerve-mediated regulation of tumor necrosis factor (TNF), whereas α7nAChR deficiency in neurons and other cells had no significant effect. In agreement with recent work, the inflammatory reflex was not functional in nude mice, because functional T cells are required for the integrity of the pathway. To investigate the role of T-cell α7nAChR, we adoptively transferred α7nAChR-deficient or WT T cells to nude mice. Transfer of WT and α7nAChR-deficient T cells restored function, indicating that α7nAChR expression on T cells is not necessary for this pathway. Together, these results indicate that α7nAChR expression in bone marrow-derived non-T cells is required for the integrity of the inflammatory reflex.

Figure 1

Effect of α7nAChR deficiency in non–BM-derived cells and BM-derived cells on vagus nerve–mediated TNF suppression in endotoxemia. Mice with α7nAChR deficiency either in non–BM-derived (nBMα7⁰) (n = 6) (A) or BM-derived cells (BMα7⁰) (n = 5) (B) were subjected to vagus nerve stimulation followed by intraperitoneal endotoxin injection. Serum TNF was measured 90 min after endotoxin administration. Median TNF levels in pg/mL (lower quartile–upper quartile) were Sham, 476 (279–521), and vagus nerve stimulation (VNS), 159 (114–287), in nBMα7⁰ mice and Sham, 275 (265–400), and VNS, 508 (428–594), in BMα7⁰ mice. Results in diagrams are expressed as the median TNF as percent of sham (lower quartile–upper quartile). *P < 0.05.

Figure 2

Effect of transfer of α7nAChR-deficient T cells to nude mice on vagus nerve–mediated TNF suppression in endotoxemia. Nude mice were subjected to vagus nerve stimulation (VNS) followed by endotoxemia in the presence or absence of functional T cells. TNF was measured in serum 90 min after endotoxin administration. (A) Nude mice (n = 4). Median TNF levels in pg/mL [lower quartile–upper quartile]) were Sham, 324 (170–483), and VNS, 621 (512–816) (P = 0.08). (B) Nude mice after transfer of WT T cells (Twt) (n = 5 sham and n = 6 VNS). TNF levels were Sham, 1,120 (1,070–1,290), and VNS, 630 (589–974) (P = 0.03). (C) Nude mice after transfer of α7nAChR-deficient T cells (Tα7°) (n = 6). TNF levels were Sham, 831 (715–1,030), and VNS, 556 (346–665) (P = 0.04). Results in diagrams are expressed as median TNF as percent of sham (lower quartile–upper quartile). *P < 0.05.

Figure 3

Purity of isolated murine spleno-cyte fractions. Cells were stained with an anti-TCRβ antibody and analyzed by flow cytometry. T-cell–depleted splenocytes from WT (A) and α7nAChR KO mice (B) are shown. T-cell fraction from WT mice (C) and α7nAChR KO mice (D) are shown. The numbers in the graph show fractions of TCRβ⁻and TCRβ⁺ cells.

Figure 4

Effect of T-cell α7nAChR deficiency on suppression of endotoxin-induced TNF production in vitro. T cells from WT or α7nAChR-deficient mice were cocultured with T cell–depleted WT splenocytes, stimulated with endotoxin and TNF measured in the supernatant. Results are shown as median TNF in pg/mL (lower quartile–upper quartile). NT, T cell depleted; α7⁰, α7nAChR KO.