The vagus nerve as a modulator of intestinal inflammation - TI Pharma

Vagal anti-inflammatory pathway mediated via JAK2-STAT3 activation
the anti-inflammatory effect of nicotine on human and mouse monocyte-derived
macrophages 2,4,5 .
Given the crucial function of STAT3 in anti-inflammatory responses 6,7 , we
hypothesized that activation of STAT3 and its gp130-binding regulatory protein
SOCS3 may be involved in the anti-inflammatory effect of nicotine. Consistent with
that hypothesis, we found that nicotine treatment activated STAT3 as well as SOCS3
in resting and LPS-stimulated primary peritoneal macrophages in a dose- and timedependent
way (fig. 1b,c). Nicotine activated STAT3 directly, as phosphorylation
of STAT3 was not affected by the protein synthesis inhibitors actinomycin D and
cycloheximide (Fig.1d). In contrast, interferon-γ-induced STAT1 activation was not
effected by nicotine (Fig. 1e). Thus, nicotine reduced the production of proinflammatory
cytokines and activated STAT3 as well as SOCS3 in stimulated macrophages.
Deactivation by nicotine requires STAT3 transactivation
We next sought to determine whether the anti-inflammatory effect of nicotine
depended on nuclear transactivation of phosphorylated STAT3. We overexpressed
a dominant negative form of STAT3 (STAT3D) in primary peritoneal macrophages.
Dimerized STAT3D is altered in its ability to bind DNA response elements and
induce transcription of target genes16,17 . Nicotine failed to reduce LPS-induced TNF
release in LPS-stimulated macrophages transfected with STAT3D but not those
transfected with the STAT3 wild-type construct (Fig. 2a). Thus, the nicotine-induced
inhibition of TNF release is dependent on STAT3 DNA transactivation. To evaluate
whether SOCS3 expression is crucial to the nicotinic anti-inflammatory effect, we
abrogated SOCS3 expression in peritoneal macrophages with SOCS3-specific small
interfering RNA (Fig. 1b). SOCS3 expression was substantially decreased in response
to nicotine (less than 10% of that expressed in control transfected cells), whereas
STAT3 activation was not affected (transfection efficiency was more than 90%; Fig.
2b). In macrophages with reduced SOCS3, however, nicotine was still able to decrease
endotoxin-induced production of IL-6 (data not shown) and TNF in a concentrationdependent
way, although the reduction was less pronounced than that in control
transfected cells (Fig. 2c). Thus, blockade of STAT3 transactivation counteracted the
anti-inflammatory effects of nicotine, whereas blockade of SOCS3 expression did not.
These results indicate that SOCS3 expression is not strictly required for the reduction
in macrophage TNF release by nicotine.
STAT3 phosphorylation depends on α7 nAChR activation
To determine whether STAT3 activation by nicotine was mediated by nAChR,
we pretreated cells with nAChR antagonists. The nonselective antagonists
hexamethonium and d-tubocurarine prevented the STAT3 phosphorylation induced
by nicotine (Fig. 3a). In addition, the α7 nAChR−selective antagonists α-bungarotoxin
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