Interleukin-17A increases neurite outgrowth from adult postganglionic sympathetic neurons

Abstract

Inflammation can profoundly alter the structure and function of the nervous system. Interleukin (IL)-17 has been implicated in the pathogenesis of several inflammatory diseases associated with nervous system plasticity. However, the effects of IL-17 on the nervous system remain unexplored. Cell and explant culture techniques, immunohistochemistry, electrophysiology, and Ca2+ imaging were used to examine the impact of IL-17 on adult mouse sympathetic neurons. Receptors for IL-17 were present on postganglionic neurons from superior mesenteric ganglia (SMG). Supernatant from activated splenic T lymphocytes, which was abundant in IL-17, dramatically enhanced axonal length of SMG neurons. Importantly, IL-17-neutralizing antiserum abrogated the neurotrophic effect of splenocyte supernatant, and incubation of SMG neurons in IL-17 (1 ng/ml) significantly potentiated neurite outgrowth. The neurotrophic effect of IL-17 was accompanied by inhibition of voltage-dependent Ca2+ influx and was recapitulated by incubation of neurons in a blocker of N-type Ca2+ channels (ω-conotoxin GVIA; 30 nM). IL-17-induced neurite outgrowth in vitro appeared to be independent of glia, as treatment with a glial toxin (AraC; 5 μM) did not affect the outgrowth response to IL-17. Moreover, application of the cytokine to distal axons devoid of glial processes enhanced neurite extension. An inhibitor of the NF-κB pathway (SC-514; 20 μM) blocked the effects of IL-17. These data represent the first evidence that IL-17 can act on sympathetic somata and distal neurites to enhance neurite outgrowth, and identify a novel potential role for IL-17 in the neuroanatomical plasticity that accompanies inflammation.

Figure 1.

Neurons from SMG express the receptor for IL-17. A, Immunofluorescent labeling of cryostat sections of SMG revealed abundant IL-17 receptor expression within neuronal perikarya. Receptor immunoreactivity was also observed in cultured SMG neurons B, IL-17 receptor expression was localized to cell bodies, neurites, and growth cones. Scale bars, 50 μm.

Figure 2.

Supernatants from cultured splenocytes exerted an IL-17-dependent neurotrophic effect on SMG neurons. Concanavalin A-stimulated splenocyte supernatant was added to distal compartments of SMG neurons in compartmented cultures and markedly enhanced levels of neurite extension relative to neurites cultured in the vehicle control for 5 d. Blocking IL-17 activity with a neutralizing antibody had no effect on neurite outgrowth when added alone but eliminated the ability of splenocyte supernatants to promote sympathetic axon outgrowth. An isotype-matched control antiserum did not block the neurotrophic effect of splenocyte supernatant. N > 40 lanes per treatment group. ***p < 0.0001 versus all other treatment groups except for supernatant plus control IgG; ^#p < 0.05 versus all other treatment groups except for supernatant; Kruskal–Wallis test with Dunn's multiple comparison posttest.

Figure 3.

IL-17 enhanced neurite outgrowth and branching. A, B, Representative images of control (A) and IL-17 (1 ng/ml)-treated (B) SMG cultures at Day 7 in vitro. Cultures were fixed and immunostained for HuD (a neuronal marker, green) and SNAP-25 (an axonal marker, red). Scale bars, 25 μm. C, Cultures incubated in IL-17 had significantly more neurites per neuron compared with controls after 3 d in culture. *p < 0.05, Kruskal–Wallis test plus Dunn's multiple comparison posttest; N = 6 coverslips from at least three mice per treatment group. D, Sholl analysis of neurite morphology after 5 d of IL-17 treatment revealed that neurite arborization was significantly greater in neurons incubated in IL-17 compared with controls. N = 40–60 neurons per treatment group. *p < 0.05; **p < 0.01; ***p < 0.001, two-way ANOVA with Bonferroni's posttest.

Figure 4.

Neurotrophic effects of IL-17 did not depend on glial cells. When the glial cell population was depleted with AraC (5 μm), IL-17 still significantly enhanced the number of neurites per neuron after 3 and 7 d in culture. N = at least 6 coverslips per treatment group, *p < 0.05 AraC versus AraC plus IL-17; ^#p < 0.05 AraC versus IL-17; Kruskal–Wallis test with Dunn's multiple comparison posttest.

Figure 5.

IL-17 inhibited voltage-gated Ca²⁺ current. A, Representative voltage-clamp recordings of I_Ca from a control neuron and a neuron incubated in IL-17 overnight. The voltage-clamp protocol is illustrated in inset. B, Mean ± SEM current voltage relations of control and IL-17-treated neurons. Incubation of neurons in 1 ng/ml IL-17 significantly reduced I_Ca between −10 and 10 mV. N = 21 control cells, 13 IL-17; *p < 0.05, two-way ANOVA with Bonferroni's posttest. C, Ca²⁺-imaging data demonstrate a significant decrease in the peak intracellular Ca²⁺ transient during a 40 mm KCl-induced depolarization in neurons incubated in IL-17 compared with controls. N = 34 cells per treatment group; ***p < 0.0001, Mann–Whitney test. D, IL-17 significantly attenuated depolarization-induced Ca²⁺ influx in SMG neuronal growth cones. N = 8 cells for control, 10 cells for IL-17; *p < 0.05, Mann–Whitney test. E, Treatment of SMG neurons with the N-type Ca²⁺ channel blocker ω-conotoxin GVIA (30 nm) significantly enhanced the ratio of neurites to neurons compared with controls. *p < 0.05 relative to control; two-way ANOVA with Bonferroni's post hoc test; N = 6 coverslips per treatment group.

Figure 6.

Inhibition of NF-κB signaling blocked the effects of IL-17 on I_Ca and neurite outgrowth. A, Current voltage curve depicting the effect of blocking the NF-κB pathway on I_Ca inhibition by IL-17. With SC-514 pretreatment, control and experimental values were not significantly different (N = 21 for control, 13 for IL-17, 12 for SC-514; p > 0.05 between control and SC-514, two-way ANOVA; p < 0.05 between control and IL-17 from −10 to +10 mV; Fig. 4). B, Pretreatment with SC-514 prevented the enhanced neurite outgrowth caused by IL-17. In SC-514-treated coverslips, the ratio of neurites to neurons in the presence of IL-17 was not significantly different from control (*p > 0.05, Mann–Whitney test).

Figure 7.

Neurites of SMG neurons grew preferentially toward sources of IL-17. Analysis of β3-tubulin immunoreactivity in SMG explants cultures revealed enhanced neurite outgrowth in the direction of an IL-17-soaked bead relative to control (N = 5 per group). Bead location is marked with a circle on the representative micrographs. A, B, Control (HBSS-soaked) bead (A) and IL-17 (100 ng/ml)-soaked bead (B). Scale bars, 25 μm. C, Mean ± SEM ratio of fluorescence from regions of interest in the quadrant of the micrograph closest to the bead relative to the quadrant on the opposite side of the micrograph (opposite quadrants not shown in A and B). *p < 0.05, Mann–Whitney test. D, Dissociated SMG neurons were added to compartmented cultures to examine the effect of adding IL-17 (1 ng/ml) to either the cell bodies and PAx, or the DAx. Application of IL-17 to distal axon compartments significantly enhanced axonal extension after 5 d relative to neurons cultured in control medium and neurons exposed to the cytokine in the cell body compartment (***p < 0.0001 vs control; ^###p < 0.0001 vs PAx; Kruskal–Wallis test with Dunn's multiple vomparison posttest; N > 30 lanes per treatment).