Trained Innate Immunity by Repeated Low-Dose Lipopolysaccharide Injections Displays Long-Term Neuroprotective Effects

Abstract

Disrupted immune response is an important feature of many neurodegenerative conditions, including sepsis-associated cognitive impairment. Accumulating evidence has demonstrated that immune memory occurs in microglia, which has a significant impact on pathological hallmarks of neurological diseases. However, it remains unclear whether immune memory can cause subsequent alterations in the brain immune response and affect neurobehavioral outcomes in sepsis survivors. In the present study, mice received daily intraperitoneal injection of low-dose lipopolysaccharide (LPS, 0.1 mg/kg) for three consecutive days to induce immune memory (immune tolerance) and then were subjected to sham operation or cecal ligation and puncture (CLP) 9 months later, followed by a battery of neurobehavioral and biochemical studies. Here, we showed that repeated low-dose LPS injection-induced immune memory protected mice from sepsis-induced cognitive and affective impairments, which were accompanied by significantly decreased brain proinflammatory cytokines and immune response. In conclusion, our study suggests that modulation of brain immune responses by repeated LPS injections confers neuroprotective effects by preventing overactivated immune response in response to subsequent septic insult.

Figure 1

Timeline of the experimental procedure in mice (a). Effects of CLP and repeated LPS injections on survival rate (b). LPS: lipopolysaccharide; CLP: cecal ligation and puncture; Veh: vehicle.

Figure 2

Effects of repeated LPS injections and CLP on neurobehavioral outcomes. CLP induced a significantly decreased freezing time to context, immobility time, and sucrose preference which were observed in the CLP group, whereas repeated LPS injections prevented the decreased freezing time to context and sucrose preference. Data represent mean ± SEM; n = 12/group. ^∗P < 0.05 vs. sham + Veh group; #P < 0.05 vs. CLP + Veh group. LPS: lipopolysaccharide; CLP: cecal ligation and puncture; Veh: vehicle.

Figure 3

Serum cytokine changes following LPS injection. ELISA showed increased TNF-α, IL-1β, and IL-6 levels following one dose of LPS injection. However, repeated LPS injections abolished TNF-α, IL-1β, and IL-6 production. Data represent mean ± SEM; n = 6/group. ^∗P < 0.05 vs. NS group. LPS: lipopolysaccharide; NS: normal saline.

Figure 4

Effects of repeated LPS injections and CLP on hippocampal cytokines. Tissue ELISA showed increased IL-1β and IL-6 levels in the hippocampus following CLP, which were prevented by repeated LPS injections. Data represent mean ± SEM; n = 6/group. ^∗P < 0.05 vs. sham + Veh group; #P < 0.05 vs. CLP + Veh group. LPS: lipopolysaccharide; CLP: cecal ligation and puncture; Veh: vehicle.

Figure 5

Effects of repeated LPS injections and CLP on prefrontal cortex cytokines. Tissue ELISA showed the increased IL-6 expression in the prefrontal cortex following CLP, which was not prevented by repeated LPS injections. Data represent mean ± SEM; n = 6/group. ^∗P < 0.05 vs. sham + Veh group; #P < 0.05 vs. CLP + Veh group. LPS: lipopolysaccharide; CLP: cecal ligation and puncture; Veh: vehicle.

Figure 6

Effects of repeated LPS injections and CLP on hippocampal inflammatory mediators. There were significantly increased hippocampal pNF-κB p65, IL-1β, and IL-6 levels following CLP. However, repeated LPS injections were able to prevent the increased pNF-κB p65 and IL-1β levels. Data represent mean ± SEM; n = 5/group. ^∗P < 0.05 vs. sham + Veh group; #P < 0.05 vs. CLP + Veh group. LPS: lipopolysaccharide; CLP: cecal ligation and puncture; Veh: vehicle.

Figure 7

Effects of repeated LPS injections and CLP on IBA1-positive cells. Immunohistochemistry showed increased intensity of IBA1-positive in the CA1 and CA3 following CLP, which were partially prevented by repeated LPS injections. Data represent mean ± SEM; n = 5/group. ^∗P < 0.05 vs. sham + Veh group; #P < 0.05 vs. CLP + Veh group. LPS: lipopolysaccharide; CLP: cecal ligation and puncture; Veh: vehicle; PFC: prefrontal cortex; CA1: cornu ammonis 1; CA3: cornu ammonis 3; DG: dentate gyrus. Red indicates IBA1; blue indicates DAPI; scale bar = 100 μm.