Circadian clock-dependent increase in salivary IgA secretion modulated by sympathetic receptor activation in mice

Abstract

The salivary gland is rhythmically controlled by sympathetic nerve activation from the suprachiasmatic nucleus (SCN), which functions as the main oscillator of circadian rhythms. In humans, salivary IgA concentrations reflect circadian rhythmicity, which peak during sleep. However, the mechanisms controlling this rhythmicity are not well understood. Therefore, we examined whether the timing of parasympathetic (pilocarpine) or sympathetic (norepinephrine; NE) activation affects IgA secretion in the saliva. The concentrations of saliva IgA modulated by pilocarpine activation or by a combination of pilocarpine and NE activation were the highest in the middle of the light period, independent of saliva flow rate. The circadian rhythm of IgA secretion was weakened by an SCN lesion and Clock gene mutation, suggesting the importance of the SCN and Clock gene on this rhythm. Adrenoceptor antagonists blocked both NE- and pilocarpine-induced basal secretion of IgA. Dimeric IgA binds to the polymeric immunoglobulin receptor (pIgR) on the basolateral surface of epithelial cells and forms the IgA-pIgR complex. The circadian rhythm of Pigr abundance peaked during the light period, suggesting pIgR expression upon rhythmic secretion of IgA. We speculate that activation of sympathetic nerves during sleep may protect from bacterial access to the epithelial surface through enhanced secretion of IgA.

Figure 1

The circadian rhythm dynamics of salivary IgA secretion. (a) Salivary IgA secretion rhythms in the case of administration of either pilocarpine (control) or a mixture of pilocarpine and norepinephrine (NE) (n = 8–10). (b) Saliva flow rhythms in control versus NE groups (n = 8–10). (c) Salivary IgA volume rhythms. Data were calculated by multiplying the results from Fig. 1a and b (n = 8–10). (d) Salivary IgA concentration rhythms in control versus NE groups (control, n = 4; NE, n = 9–10). (e) Total protein concentration rhythms in saliva (control, n = 4; NE, n = 9–10). (f) Salivary IgA concentration rhythms were normalized to total protein concentration (control, n = 4; NE, n = 9–10). (g) Salivary IgA concentration rhythms in mice fasted for 24 hours (n = 9–12). Values are shown as the means ± SEM. (a,c,d) **p < 0.01, NE group ZT6 vs. ZT18 (one-way ANOVA with Tukey post-hoc test). (f) *p < 0.05, NE group ZT6 vs. ZT18 (Kruskal-Wallis test with Dunn post-hoc test). (g) ***p < 0.001 **p < 0.01, NE group ZT0, ZT6 vs. ZT18 (one-way ANOVA with Tukey post-hoc test), ^#p < 0.05, cont. group ZT6 vs. ZT18 (Kruskal-Wallis test with Dunn post-hoc test).

Figure 2

The circadian rhythms of salivary IgA secretion in SCN-lesioned mice. (a,b) Representative double-plotted actograms of locomotor activity for one sham mouse and one SCNX mouse after pilocarpine (a) or NE administration (b). (c,d) Representative Chi-squared periodogram analysis of behavioral data for one sham mouse and one SCNX mouse after pilocarpine (c) or NE (d) administration. Straight lines in each figure indicate statistical significance. (e,f) Salivary IgA concentration rhythms in non-SCN-lesioned (sham) and SCN-lesioned (SCNX) after pilocarpine (e) or NE administration (f) (n = 4–6). Values are shown as mean ± SEM. (f) ^$$p < 0.01, sham group (one-way ANOVA). **p < 0.01, *p < 0.05 vs. sham ZT18. ^#p < 0.05 vs. sham ZT0.

Figure 3

Effects of sympathetic nerve agonists/antagonists on salivary IgA secretion. (a) Salivary IgA concentrations after application of the sympathetic nervous system agonist (norepinephrine 1 mg/kg) and/or antagonist (prazosin 1 mg/kg, propranolol 1 mg/kg, or 10 mg/kg) with pilocarpine. “+” indicates the reagent that was mixed and administered to mice, “−” indicates the reagent that was not administered to mice. (b,c) Salivary IgA concentrations when the α receptor (phenylephrine, 5 mg/kg; the numbers in parentheses are the number of samples) (b) or β receptor (isoproterenol, 5 mg/kg; n = 4–5) (c) were stimulated at ZT6 or ZT18 (n = 3–5). Values were shown as means ± SEM. (a) ***p < 0.001, **p < 0.01, vs. the NE(+)-Prazosin(−)-Propranolol(−) group (one-way ANOVA with Tukey post-hoc test). (b) *p < 0.05, ZT18 group cont. vs. PE (Mann-Whitney test). (c) **p < 0.01, iso group ZT6 vs. ZT18 (Student’s t-test).

Figure 4

Sensitivity of the submandibular gland clock to sympathetic nerve stimulation, as evaluated by IVIS in PER2::LUC mice. (a) Experimental schedule. White and black bars indicate 12 hours of light and dark. Triangles under the white and black bars indicate the time at which the reagents were administered intraperitoneally. Bi-directional arrows under the white and black bars indicate the period of measurement using IVIS. (b,c) Average peaks of phase of PER2::LUC rhythms (b) and PER2::LUC expression rhythms (c) in submandibular glands after mice were administered the reagent at ZT4 for 3 days (n = 3–6). (d,e) Average phase peaks of PER2::LUC rhythms (d) and PER2::LUC expression rhythms (e) in submandibular glands after mice were administered the reagent at ZT16 for 3 days (n = 4–8). Values were shown as the means ± SEM. (b) **p < 0.01, control vs. NE 2 mg/kg (one-way ANOVA with Tukey post-hoc test).

Figure 5

Circadian rhythm for Pigr gene abundance in submandibular glands in intact SCN-lesioned and Clock ^−/− mice. (a) Relative mRNA abundance rhythms of Pigr (n = 4) in the submandibular gland. (b) The relative mRNA abundance of Pigr in SCN-lesioned mice compared to that in sham-operated mice at ZT6 and ZT18 (n = 6–7). (c,d) The relative mRNA abundance of Per2 (c) and Pigr (d) in Clock ^−/− mice compared to that in wild-type (WT) mice at ZT6 and ZT18 (n = 4 for each group). Values are shown as means ± SEM. (a) *p < 0.05, ZT1, ZT5 vs. ZT13 (Kruskal-Wallis test with Dunn post-hoc test), (b–d) *p < 0.05, ZT6 vs. ZT18 or WT vs. Clock ^−/− (Mann-Whitney test).

Figure 6

Effects of adrenal gland lesions on salivary IgA secretion. (a,b) Serum concentrations of corticosterone in the adrenal gland lesion (ADX) group compared to the sham-operated group for pilocarpine-only (a) and pilocarpine/NE-treated mice (b) (n = 3–6). (c,d) The rhythm of salivary IgA concentrations in the ADX group compared to that in the sham-operated mice for pilocarpine-only (c) and pilocarpine/NE-treated mice (d) (n = 3–6). (a) p = 0.0952 (Mann-Whitney test), (b) ***p < 0.001 (Student’s t-test), (c) sham, p = 0.0538 (Friedman test); ADX, p = 0.0882 (Friedman test), (d) sham, p = 0.0678 (Friedman test); ADX, **p < 0.01 (Friedman test).