Distinct Roles of α7 nAChRs in Antigen-Presenting Cells and CD4+ T Cells in the Regulation of T Cell Differentiation

It is now apparent that immune cells express a functional cholinergic system and that α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating T cell differentiation and the synthesis of antigen-specific antibodies and proinflammatory cytokines. Here, we investigated the specific function α7 nAChRs on T cells and antigen presenting cells (APCs) by testing the effect of GTS-21, a selective α7 nAChR agonist, on differentiation of CD4+ T cells from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice activated with OVA or OVA peptide323−339 (OVAp). GTS-21 suppressed OVA-induced antigen processing-dependent development of CD4+ regulatory T cells (Tregs) and effector T cells (Th1, Th2, and Th17). By contrast, GTS-21 up-regulated OVAp-induced antigen processing-independent development of CD4+ Tregs and effector T cells. GTS-21 also suppressed production of IL-2, IFN-γ, IL-4, IL-17, and IL-6 during OVA-induced activation but, with the exception IL-2, enhanced their production during OVAp-induced activation. In addition, during antigen-nonspecific, APC-independent anti-CD3/CD28 antibody-induced CD4+ polyclonal T cell activation in the presence of respective polarizing cytokines, GTS-21 promoted development of all lineages, which indicates that GTS-21 also acts via α7 nAChRs on T cells. These results suggest 1) that α7 nAChRs on APCs suppress CD4+ T cell activation by interfering with antigen presentation through inhibition of antigen processing; 2) that α7 nAChRs on CD4+ T cells up-regulate development of Tregs and effector T cells; and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement

TRPV1 and TRPV4 play pivotal roles in delayed onset muscle soreness.

Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes tenderness and movement related pain after some delay (delayed-onset muscle soreness, DOMS). We previously demonstrated that nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are up-regulated in exercised muscle through up-regulation of cyclooxygenase (COX)-2, and they sensitized nociceptors resulting in mechanical hyperalgesia. There is also a study showing that transient receptor potential (TRP) ion channels are involved in DOMS. Here we examined whether and how TRPV1 and/or TRPV4 are involved in DOMS. We firstly evaluated a method to measure the mechanical withdrawal threshold of the deep tissues in wild-type (WT) mice with a modified Randall-Selitto apparatus. WT, TRPV1-/- and TRPV4-/- mice were then subjected to LC. Another group of mice received injection of murine NGF-2.5S or GDNF to the lateral gastrocnemius (LGC) muscle. Before and after these treatments the mechanical withdrawal threshold of LGC was evaluated. The change in expression of NGF, GDNF and COX-2 mRNA in the muscle was examined using real-time RT-PCR. In WT mice, mechanical hyperalgesia was observed 6-24 h after LC and 1-24 h after NGF and GDNF injection. LC induced mechanical hyperalgesia neither in TRPV1-/- nor in TRPV4-/- mice. NGF injection induced mechanical hyperalgesia in WT and TRPV4-/- mice but not in TRPV1-/- mice. GDNF injection induced mechanical hyperalgesia in WT but neither in TRPV1-/- nor in TRPV4-/- mice. Expression of NGF and COX-2 mRNA was significantly increased 3 h after LC in all genotypes. However, GDNF mRNA did not increase in TRPV4-/- mice. These results suggest that TRPV1 contributes to DOMS downstream (possibly at nociceptors) of NGF and GDNF, while TRPV4 is located downstream of GDNF and possibly also in the process of GDNF up-regulation

Measurement of muscular mechanical withdrawal threshold with Randall-Selitto apparatus in mice.

<p>(<b>A</b>) Schedule for testing effects of EMLA cream treatment on the withdrawal threshold. More than 12 h before these measurements, inflammation was induced by injecting carrageenan into the LGC muscle. VFT: von Frey hair test, RST: Randall-Sellito test. (<b>B</b>) Change in VFT threshold (tip diameter: 0.25 mm) by surface anesthesia. (i) Vehicle cream (n = 9) did not change the threshold. (ii) EMLA treatment (n = 10) significantly raised the threshold compared with before the treatment. Median and interquartile range (IQR) are shown. *** <i>p</i><0.001 for pre- and post-cream treatment comparison by Mann-Whitney test. Note that pre values are decreased ones after induction of inflammation (same in C). (<b>C</b>) Change after surface anesthesia by EMLA cream treatment in withdrawal threshold measured by RST with a self-made larger probe (tip diameter: 2.6 mm). Filled circles: EMLA treatment (n = 10), open square: vehicle cream treatment (n = 9). EMLA cream did not significantly change the threshold, the same as vehicle cream. Mean ± S.E.M. (n = 6–10 for each group). S.E.M.s are hardly seen because they are small.</p

Muscular mechanical hyperalgesia induced by lengthening contraction in mice.

<p>(<b>A</b>) Schema of lengthening contraction (LC) application to the lower hindleg flexors, mainly the lateral gastrocnemius (LGC) muscle. LC was induced by electrical stimulation through a pair of needle electrodes inserted near the tibial and sciatic nerves. The ankle joint was dorsi-flexed in synchrony with muscle contraction, and then returned to the starting position over a 3 s resting period. This cycle was repeated 300 times. (<b>B</b>) Change in withdrawal thresholds by RST in WT mice that received LC or sham (stretch only) exercise (n = 9 for each group, mean ± S.E.M.). Vertical axis: withdrawal threshold in mN, horizontal axis: time after exercise. There was a significant difference between the groups, and the threshold decreased 6 to 36 h after exercise in LC group, but not in sham group. ** <i>p</i><0.01, *** <i>p</i><0.001 compared with −1 day in LC group; # <i>p</i><0.05, ### <i>p</i><0.001 compared with sham group on each time point, two-way repeated measures ANOVA with Bonferroni t-test.</p

Muscular mechanical hyperalgesia did not develop after LC in TRPV1−/− and TRPV4−/− mice.

<p>(<b>A</b>) Change in the withdrawal thresholds after LC measured by RST in TRPV1−/− (crosses) and TRPV4−/− (open squares) mice. Vertical axis: difference in the threshold from −1 d in mN, horizontal axis: time after LC. (<b>B</b>) Changes in the mechanical hyperalgesia by intramuscular injection of HC-067047, a TRPV4 selective antagonist (100 mg/kg; crosses) or DMSO (open squares) in WT mice. Mean ± S.E.M. (n = 6–10 for each group). ** <i>p</i><0.01, *** <i>p</i><0.001 compared with −1 d in WT,+<i>p</i><0.05 compared with −1 d in TRPV4−/−, ## <i>p</i><0.01, ### <i>p</i><0.001 compared with 14 h in HC-067047 group; two-way repeated measures ANOVA with Bonferroni t-test.</p

NGF-β mRNA was up-regulated in all three genotypes.

<p>(<b>A</b>) Time course of NGF-β mRNA expression in LC-exercised LGC muscle in WT mice. (<b>B</b>) Up-regulation of NGF-β mRNA 3 h after LC in the muscle of three genotypes. Median and interquartile range (IQR). All values were normalized with β-actin mRNA. n = 3–8 for each group (shown in the parentheses under each column). * <i>p</i>≤0.05 compared with pre, and n.s. not different from WT (Kruskal-Wallis one-way analysis of variance on ranks test followed by the Dunn's test).</p