Interleukin-6 trans-signaling in the senescent mouse brain is involved in infection-related deficits in contextual fear conditioning

Abstract

Excessive production of pro-inflammatory cytokines in the senescent brain in response to peripheral immune stimulation is thought to induce behavioral pathology, however, few studies have examined if the increase in pro-inflammatory cytokines is accompanied by an increase in cytokine signaling. Here, we focused on IL-6 as a prototypic pro-inflammatory cytokine and used phosphorylated STAT3 as a marker of IL-6 signaling. In an initial study, IL-6 mRNA and the magnitude and duration of STAT3 activation were increased in the hippocampus of senescent mice compared to adults after i.p. injection of LPS. The LPS-induced increase in STAT3 activity was ablated in aged IL-6(-/-) mice, suggesting IL-6 is a key driver of STAT3 activity in the aged brain. To determine if IL-6 activated the classical or trans-signaling pathway, before receiving LPS i.p., aged mice were injected ICV with sgp130, an antagonist of the trans-signaling pathway. Importantly, the LPS-induced increases in both IL-6 and STAT3 activity in the hippocampus were inhibited by sgp130. To assess hippocampal function, aged mice were injected ICV with sgp130 and i.p. with LPS immediately after the acquisition phase of contextual fear conditioning, and immobility was assessed in the retention phase 48h later. LPS reduced immobility in aged mice, indicating immune activation interfered with memory consolidation. However, sgp130 blocked the deficits in contextual fear conditioning caused by LPS. Taken together, the results suggest IL-6 trans-signaling is increased in the senescent brain following peripheral LPS challenge and that sgp130 may protect against infection-related neuroinflammation and cognitive dysfunction in the aged.

Figure 1. Activated STAT3 in the hippocampus of adult and aged mice after peripheral injection of LPS

Adult and aged BALB/c mice were injected i.p. with LPS and hippocampal tissue was collected at various time points after injection to measure phosphorylated STAT3. The upper panel shows representative western blots and the bar graph shows mean STAT3 phosphorylation (± SEM) from 8-9 treatment replicates at each time point for each age (because STAT3 activity was not affected in saline controls over time, data from saline treated mice at different time points was pooled. Means with different letters are significantly different (P<0.05).

Figure 2. Plasma IL-6 and IL-6 mRNA in the hippocampus in aged and adult mice 6 h after LPS injection

Adult and aged BALB/c mice were injected i.p. with LPS and 6 h later, blood and brain tissue were collected for analysis. Bars represent the mean ± SEM (n = 7-8). Means with different letters are significantly different (P<0.05).

Figure 3. Phosphorylated STAT3 in hippocampus of adult and aged IL6^+/+ and IL-6^−/− mice after injection of LPS

Adult and aged IL-6^+/+ and IL-6^−/− mice were injected i.p. with LPS and hippocampal tissue was collected to measure phosphorylated STAT3. The upper panel shows representative western blots and the bar graph shows mean STAT3 phosphorylation (± SEM) from 6-7 treatment replicates. Means with different letters are significantly different (P<0.05).

Figure 4. Effects of sgp130 on LPS-induced STAT3 activity in the hippocampus of aged mice

Aged BALB/c mice were injected ICV with sgp130 and i.p. with LPS and hippocampal tissue was collected 6 h later. The upper panel shows representative western blots and the bar graph shows mean STAT3 phosphorylation (± SEM) from 8-9 treatment replicates. Means with different letters are significantly different (P<0.05).

Figure 5. Effects of sgp130 on LPS-induced IL-6 in the hippocampus of aged mice

Aged BALB/c mice were injected ICV with sgp130 and i.p. with LPS and hippocampal tissue was collected 6 h later for determination of IL-6. The bar graph shows mean IL-6 concentration (± SEM) from 8-9 treatment replicates. Means with different letters are significantly different (P<0.05).

Figure 6. Effects of sgp130 on the LPS-induced deficit in contextual fear conditioning in aged mice

Aged BALB/c mice were injected ICV with sgp130 and i.p. with LPS immediately after the acquisition phase of contextual fear conditioning. Forty-eight hours later immobility in the retention phase was assessed. Bars represent the mean ± SEM (n = 12-15). Means with different letters are significantly different (P < 0.05).