IL-6 and Cognitive Changes Figure 4. PV Interneurons are decreased with aging, but spared in IL-6 -/- mice or mice treated chronically with an SOD mimetic, C3. (a) Images, depicting the regions analyzed for PV-interneuron immunostaining in the prelimbic region (between Bregma 2.2 and 1.9), and dorsal hippocampus (between Bregmas 21.2 and 21.7). Four consecutive slices were analyzed for each animal. All cells in the regions were counted, and counts per slice were calculated as described in Methods (b) PV-cell counts in the regions of CA1, CA3 and dentate gyrus (DG) of old and old-C3 treated animals are expressed as % of their control. Aging was accompanied by a statistically significant decrease in PV-interneuron number in all regions analyzed and treatment of animals from middle age with the SOD-mimetic C3 (Old-C3) prevented the reduction of PV-interneuron numbers in CA1 and CA3, but not in DG. (c) The loss of PV neurons in all of the studied regions was partially rescued in IL-6 KO mice. * indicates statistical significance with respect to young animals (P,0.05) and # indicates statistical significance with respect to old animals (P,0.05) as determined by ANOVA followed by Tukey’s test. YM and OM: n = 9 animals per group; OM + C3: n = 7 animals. doi:10.1371/journal.pone.0005518.g004 treatment for CA3: F(2,22) = 9.3, P = 0.003). No tissue was available for analysis of PV-interneurons in PFC. Nuclear translocation of NFkB is increased in aged brain, and mediates IL-6 induction and activation of Nox2 in neurons The activation and nuclear translocation of the transcription factor NFkB is involved in signaling mechanisms that lead to increased inflammatory cytokine expression. Conversely, IL-6 has recently been shown in an epithelial tumor cell line to activate NFkB signaling through non-canonical binding of unphosphory- lated STAT3 (U-STAT3) to NFkB, releasing inhibition by IkB. The U-STAT3-NFkB dimer then translocates to the nucleus and activates a subset of NFkB-dependent genes[36]. Although both Nox2 and its p22phox subunit are NFkB target genes, the question of whether IL-6 can act through NFkB to induce Nox2 or p22phox has not been previously addressed. We therefore analyzed whether nuclear translocation of NFkB was elevated in forebrains of old mice, by detection of the p65 subunit using a commercially available EMSA kit (TransAM). Mouse forebrain nuclear extracts showed increased content of the p65 subunit when compared to similar extracts prepared from young animals (Fig. 5a). The age- related increase in nuclear translocation of NFkB was absent in old IL-6-/- mice, suggesting that NFkB is involved in the CNS effects of IL-6 (Fig. 5a). To further understand the relationship between nuclear translocation of NFkB and the activation of superoxide production by Nox2, we utilized a primary neuronal culture PLoS ONE | www.plosone.org 6 May 2009 | Volume 4 | Issue 5 | e5518