Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance

Background: Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1β (IL-1β) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1β production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1β source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice. Methods: We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1β-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1β production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1β expression in the SFO perivascular macrophages. Results: We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1β in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1β expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1β, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space. Conclusions: The current data indicate that perivascular macrophages enable the SFO to produce IL-1β in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.