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. 2011 May 4;134(5):1293–1314. doi: 10.1093/brain/awr074

Figure 7.

Figure 7

Tregs suppress toxic microglial responses through a mechanism involving the upregulation of IL-4. Wild-type (WT) and mSOD1 primary microglia (mc) were harvested from 130-day-old mice. Wild-type and mSOD1 T lymphocytes were obtained from 100-day-old mice. (A) Co-culturing mSOD1 Teffs with mSOD1 microglia increased NOX2 messenger RNA expression compared with wild-type microglia co-cultured with wild-type Teffs (*P = 0.021). Co-culturing wild-type microglia with wild-type Tregs did not change the expression of NOX2 compared with wild-type microglia co-cultured with wild-type Teffs (#P ≥ 0.05). mSOD1 Tregs obtained from stable disease phase mSOD1 mice reduced the NOX2 messenger RNA expression from mSOD1 microglia to levels detected in wild-type microglia/Tregs co-cultures. mSOD1 microglia/Tregs expressed less NOX2 messenger RNA than mSOD1 microglia/Teffs (**P = 0.022). (B) IL-4 levels in the supernatant from wild-type Teffs/microglia were not different than the supernatant IL-4 levels from mSOD1 Teffs / microglia co-cultures (#P ≥ 0.05). Co-culturing stable phase mSOD1 Tregs with mSOD1 microglia increased the amount of IL-4 in the supernatant compared with wild-type microglia/Tregs co-cultures (*P = 0.007). (C) Although anti-IL-4 blocking antibodies (Ab) did not alter the expression levels on NOX2 messenger RNA in mSOD1 microglia/Teffs co-cultures, they increased the NOX2 messenger RNA expression in mSOD1 microglia/Tregs compared with untreated mSOD1 microglia/Tregs co-cultures (*P = 0.0008) and were not different from mSOD1 microglia/Teffs co-cultures treated with anti-IL-4 antibodies (##P ≥ 0.05), suggesting that the suppressive effects of mSOD1 Tregs was mediated through an IL-4 mechanism (Zhao et al., 2006). (D) IL-10 messenger RNA was increased in mSOD1 microglia/Tregs co-cultures compared with wild-type microglia/Tregs (*P = 0.032) or mSOD1 microglia/Teffs co-cultures (**P = 0.004). (E) Both wild-type Tregs and mSOD1 Tregs had increased IL-10 messenger RNA levels compared with wild-type Teffs (*P < 0.001 and **P = 0.008) or mSOD1 Teffs (&P = 0.0004 and &&P = 0.008). (F) Again, mSOD1 Tregs reduced the level of NOX2 messenger RNA expression in mSOD1 microglia/Tregs co-cultures compared with mSOD1 microglia/Teffs (*P < 0.001), but the anti-IL-10 blocking antibodies did not reverse NOX2 messenger RNA expression in mSOD1 microglia/Tregs compared with untreated mSOD1 microglia/Tregs co-cultures (#P ≥ 0.05), suggesting that the suppressive effects of mSOD1 Tregs did not involve IL-10.