Production of proinflammatory cytokines and chemokines during neuroinflammation: novel roles for estrogen receptors alpha and beta

Abstract

Neuroinflammation is a common feature of many neurological disorders, and it is often accompanied by the release of proinflammatory cytokines and chemokines. Estradiol-17β (E2) exhibits antiinflammatory properties, including the suppression of proinflammatory cytokines, in the central nervous system. However, the mechanisms employed by E2 and the role(s) of estrogen receptors (ERs) ERα and ERβ are unclear. To investigate these mechanisms, we employed an in vivo lipopolysaccharide (LPS) model of systemic inflammation in ovariectomized (OVX) and OVX and E2-treated (OVX+E2) mice. Brain levels of proinflammatory cytokines (IL-1β, IL-6, and IL-12p40) and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, and CXCL1/KC) were quantified in mice at 0 (sham), 3, 6, 12, and 24 h after infection using multiplex protein analysis. E2 treatment inhibited LPS-induced increases in all cytokines. In contrast, E2 treatment only suppressed CCL/RANTES chemokine concentrations. To determine whether ERα and ERβ regulate brain cytokine and chemokine levels, parallel experiments were conducted using ERα knockout and ERβ knockout mice. Our results revealed that both ERα and ERβ regulated proinflammatory cytokine and chemokine production through E2-dependent and E2-independent mechanisms. To assess whether breakdown of the blood-brain barrier is an additional target of E2 against LPS-induced neuroinflammation, we measured Evan's blue extravasation and identified distinct roles for ERα and ERβ. Taken together, these studies identify a dramatic cytokine- and chemokine-mediated neuroinflammatory response that is regulated through ERα- and ERβ-mediated ligand-dependent and ligand-independent mechanisms.

Figure 1

Brain proinflammatory cytokines are suppressed by E₂ after a peripheral LPS stimulus in WT mice. Mice were injected with LPS (ip), and brain levels of cytokines were measured using a multiplex cytokine assay in sham (0 h) animals and at 3, 6, 12, and 24 h after LPS injection. Low physiological levels of E₂ significantly decreased production of (A) IL-1β [interaction between time and hormone (F_(1,51) = 3.11, P < 0.03), time (F_(4,51) = 14.69, P < 0.0001), and hormone (F_(1,51) = 6.57, P < 0.02)]; (B) IL-6 [time (F_(4,50) = 21.61, P < 0.0001) and hormone (F_(1,50) = 4.13, P < 0.05)]; and (C) IL-12p40 [interaction between time and hormone (F_(4,43) = 9.49, P < 0.0001), time (F_(4,43) = 44.07, P < 0.0001), and hormone (F_(1,43) = 21.51, P < 0.0001)]. *, P < 0.05 and ***, P < 0.0001 indicates significance between oil and E₂; n = 5–9 mice/hormone group/time point.

Figure 2

ERα and ERβ mediate the antiinflammatory effects of E₂ on proinflammatory cytokine production. ERαKO and ERβKO mice were injected with LPS (ip) and brain levels of cytokines measured using a multiplex cytokine assay at 6 h (A, C, and E) and 24 h (B, D, and F) after LPS injection. A, IL-1β production was higher in both ERαKO and ERβKO mice compared with WT mice [F_(2,26) = 24.01, P < 0.03]. C, IL-6 production was also significantly higher in ERαKO and ERβKO mice compared with WT mice [F_(2,29) = 20.36, P < 0.03]. E, IL-12p40 production was suppressed by E₂ treatment [F_(2,28) = 23.7, P < 0.003] in WT and ERβKO mice; this effect was abolished in ERαKO mice. There were no significant differences at 24 h for (B) IL-1β, (D) IL-6, and (F) IL-12p40. *, P < 0.05 indicates significance between oil and E₂; #, P < 0.05 indicates significance between WT and ERαKO or ERβKO; ERαKO, n = 4–7/time point; ERβKO, n = 5–8/time point.

Figure 3

Peripheral LPS induces brain proinflammatory chemokines. Mice were injected with LPS (ip) and brain levels of cytokines measured using a multiplex cytokine assay in sham (0 h) animals and at 3, 6, 12, and 24 h after LPS-injection. A, CCL5 [F_(4,49) = 29.17, P < 0.0001] was induced by LPS and suppressed by E₂ [hormone (F_(1,49) = 12.28, P < 0.002)]; B, CCL2 [F_(4,52) = 28.4, P < 0.0001]; C, CCL3 [F_(4,46) = 12.08, P < 0.0001]; and D, CXCL1 [F_(4,51) = 29.87, P < 0.0001] were induced by LPS but unaffected by E₂. *, P < 0.05 indicates significance between oil and E₂; n = 5–9 mice/hormone group/time point.

Figure 4

Brain chemokine induction is regulated by both ERα and ERβ. ERαKO and ERβKO mice were injected with LPS (ip), and brain levels of chemokines were measured using a multiplex cytokine assay after LPS-injection. CCL5 levels were measured at 12 (A) and 24 h (B) after injury. A significant interaction between ER genotype and hormone was observed at 12 h [F_(2,23) = 10.83, P < 0.001], as well as an effect of genotype [F_(2,23) = 10.24, P < 0.001] and hormone [F_(1,23) = 7.973, P < 0.01]. The interaction between ER genotype and hormone persisted at 24 h [F_(2,25) = 3.958, P < 0.04], in addition to an effect of genotype [F_(2,25) = 3.533, P < 0.05]. In contrast, chemokine production was significantly higher after 24 h in both ERαKO and ERβKO mice as measured by a significant effect of ER genotype for (C) CCL2 [F_(2,33) = 11.12, P < 0.001], (D) CCL3 [F_(2,27) = 12.16, P < 0.001], and (E) CXCL1 [F_(2,26) = 11.46, P < 0.001]. *, P < 0.05 indicates significance between oil and E₂; #, P < 0.05 and **, P < 0.01 indicates significance between WT and ERαKO or ERβKO; ERαKO, n = 4–7/time point; ERβKO, n = 5–8/time point.

Figure 5

ERα mediates E₂-dependent suppression of BBB permeability. A, EB permeability is decreased in OVX+E₂-treated WT mice at 6 h after LPS injection [F_(2,33) = 5.627, P < 0.01]; n = 5–8. B, E₂-mediated suppression of EB permeability is lost in ERαKO mice and retained in ERβKO mice as shown by a significant interaction between genotype and hormone [F_(2,29) = 3.763, P < 0.05] and an effect of genotype [F_(2,29) = 18.9, P < 0.001)]. *, P < 0.05 indicates significance between oil and E₂; #, P < 0.05 and ###, P < 0.0001 indicates significance between WT and ERαKO or ERβKO; ERαKO and ERβKO, n = 5–7.