Elevated levels of Rorc and IL17A mRNA and serum IL-17A in RORγt Tg mice
Th17 cells are constitutively present in lamina propria of the gut [16, 17], and differentiate from naive T cells upon stimulation with IL-6 and TGF-β . mRNA levels of Rorc (the gene encoding RORγt) and IL17A were determined by qPCR and compared between RORγt Tg and wild-type (WT) mice (Fig. 1A). In the colon, levels of both Rorc and IL17A mRNA were significantly higher in RORγt Tg mice compared with WT mice (Fig. 1A, n = 4, p < 0.05). Consistently, IL-17A concentrations were significantly higher in RORγt Tg mice compared with WT mice (Fig. 1B, 2.72 ± 1.11 vs. 9.54 ± 1.77 pg/mL, respectively; p < 0.05); however, levels of IL-6 were unaltered (Fig. 1B, 3.91 ± 0.11 vs. 4.63 ± 0.49 pg/mL, respectively). These results indicate significantly elevated serum levels of IL-17A in RORγt Tg mice, which occurred concomitantly with upregulation of Rorc and IL-17A mRNA in the bowel.
Microglia and astrocytes in the hippocampus of RORγt Tg mice
IL-17A binds to a receptor complex of IL-17 receptor subunit A (IL-17RA) and IL-17RC . In the mouse brain, IL-17RA is expressed in microglia [20, 21]. To investigate potential cellular alterations induced by overexpression of RORγt in the mouse brain, we visualized microglia and astrocytes using immunohistochemistry in RORγt Tg mice (Fig. 2A and 2D).
Fluorescence intensity of Iba1, a marker of microglia, was significantly decreased in the DG of RORγt Tg mice compared with WT mice (Fig. 2B, 16571.93 ± 1150.53 vs. 25387.14 ± 1706.29, respectively; p < 0.05). However, no significant difference in fluorescence intensities was observed in the cornu ammonis 1 (CA1) region of the hippocampus between RORγt Tg and WT mice (Fig. 2B, 15245.00 ± 1302.7 vs. 17636.00 ± 817.87, respectively; p = 0.18). Accordingly, the density of Iba1+ cells was significantly decreased in the DG of RORγt Tg mice compared with WT mice (Fig. 2C, 4.71 ± 0.27 vs. 7.71 ± 0.36 /104 µm2, respectively; p < 0.05), but not in the CA1 (6.11 ± 0.51 vs. 5.47 ± 0.52 /104 µm2, respectively; p = 0.17), without any marked morphological changes. Thus, the activity and density of Iba1+ microglia was specifically reduced in the DG of RORγt Tg mouse brains.
Immunofluorescence intensity of GFAP, a marker of astrocyte activation, was unaltered in the hippocampal DG and CA1 of RORγt Tg mice compared with WT mice (Fig. 2E, DG: 49249.33 ± 202.19 vs. 48719.25 ± 4324.98, respectively; p = 0.93; CA1: 25387.14 ± 1706.29 vs. 16571.93 ± 1150.53, respectively; p = 0.17). There was also no difference in the density of GFAP+ astrocytes between WT and RORγt Tg mice (Fig. 2F, DG: 11.56 ± 0.63 vs. 10.67 ± 0.57 /104 µm2, respectively; p = 0.93; CA1: 6.11 ± 0.51 vs. 6.67 ± 0.29 104/µm2, respectively; p = 0.31). No substantial changes were observed in the activity or density of GFAP+ astrocytes in these hippocampal regions of RORγ Tg mice despite constitutively high levels of serum IL-17A.
Activation of microglia causes a decrease in the number and activity of stem cells, thereby reducing neurogenesis in the DG [22, 23]. To evaluate the potential effects of altered glial-cell activity on neurogenesis in the brains of RORγ Tg mice, we performed immunohistochemistry with an antibody against DCX, a marker of immature neurons (Fig. 2G). The density of DCX+ neurons was almost the same in WT and RORγt Tg mice (Fig. 2H, 23.88 ± 2.34 vs. 31.63 ± 4.79 /104 µm2, respectively; p = 0.157), indicating that reduced microglial activity does not affect neurogenesis in the DG of adult RORγ Tg mice.
Hippocampal levels of synaptic molecules were maintained in RORγt Tg mice
To examine potential changes in synaptic molecules in response to a constitutive increase of IL-17A in RORγt Tg mice, western blot analysis was performed using hippocampal tissues (Fig. 2I). Protein levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, which are important for synaptic plasticity , and postsynaptic density proteins PSD93 and PSD95 were compared between RORγt Tg and WT mice. There was no significant difference in protein expression of these four synaptic molecules between RORγt Tg and WT mice (NR2A: p = 0.130; NR2B: p = 0.211; PSD93: p = 0.101; and PSD95: p = 0.964).
Object location recognition testing revealed no difference in preference between WT and RORγt Tg mice
To examine whether increased IL-17A and microglial alterations in RORγt Tg mice influenced hippocampus-dependent brain function, an object location recognition test was performed . RORγt Tg mice exhibited preference to the displaced object to the same extent as WT mice (Fig. 3, p = 0.0006 and p = 0.020, respectively). These results suggest that RORγt Tg mice have normal hippocampus-dependent spatial memory function.