Anna Eskilsson

Immune-Induced Fever Is Mediated by IL-6 Receptors on Brain Endothelial Cells Coupled to STAT3-Dependent Induction of Brain Endothelial Prostaglandin Synthesis

The cytokine IL-6, which is released upon peripheral immune challenge, is critical for the febrile response, but the mechanism by which IL-6 is pyrogenic has remained obscure. Here we generated mice with deletion of the membrane bound IL-6 receptor α (IL-6Rα) on neural cells, on peripheral nerves, on fine sensory afferent fibers, and on brain endothelial cells, respectively, and examined its role for the febrile response to peripherally injected lipopolysaccharide. We show that IL-6Rα on neural cells, peripheral nerves, and fine sensory afferents are dispensable for the lipopolysaccharide-induced fever, whereas IL-6Rα in the brain endothelium plays an important role. Hence deletion of IL-6Rα on brain endothelial cells strongly attenuated the febrile response, and also led to reduced induction of the prostaglandin synthesizing enzyme Cox-2 in the hypothalamus, the temperature-regulating center in the brain, as well as reduced expression of SOCS3, suggesting involvement of the STAT signaling pathway. Furthermore, deletion of STAT3 in the brain endothelium also resulted in attenuated fever. These data show that IL-6, when endogenously released during systemic inflammation, is pyrogenic by binding to IL-6Rα on brain endothelial cells to induce prostaglandin synthesis in these cells, probably in concerted action with other peripherally released cytokines.

Deletion of Prostaglandin E2 Synthesizing Enzymes in Brain Endothelial Cells Attenuates Inflammatory Fever

Fever is a hallmark of inflammatory and infectious diseases. The febrile response is triggered by prostaglandin E2 synthesis mediated by induced expression of the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1). The cellular source for pyrogenic PGE2 remains a subject of debate; several hypotheses have been forwarded, including immune cells in the periphery and in the brain, as well as the brain endothelium. Here we generated mice with selective deletion of COX-2 and mPGES1 in brain endothelial cells. These mice displayed strongly attenuated febrile responses to peripheral immune challenge. In contrast, inflammation-induced hypoactivity was unaffected, demonstrating the physiological selectivity of the response to the targeted gene deletions. These findings demonstrate that PGE2 synthesis in brain endothelial cells is critical for inflammation-induced fever.

Acetaminophen reduces lipopolysaccharide-induced fever by inhibiting cyclooxygenase-2.

Acetaminophen is one of the worlds most commonly used drugs to treat fever and pain, yet its mechanism of action has remained unclear. Here we tested the hypothesis that acetaminophen blocks fever through inhibition of cyclooxygenase-2 (Cox-2), by monitoring lipopolysaccharide induced fever in mice with genetic manipulations of enzymes in the prostaglandin cascade. We exploited the fact that lowered levels of a specific enzyme make the system more sensitive to any further inhibition of the same enzyme. Mice were immune challenged by an intraperitoneal injection of bacterial wall lipopolysaccharide and their body temperature recorded by telemetry. We found that mice heterozygous for Cox-2, but not for microsomal prostaglandin E synthase-1 (mPGES-1), displayed attenuated fever, indicating a rate limiting role of Cox-2. We then titrated a dose of acetaminophen that did not inhibit the lipopolysaccharide-induced fever in wild-type mice. However, when the same dose of acetaminophen was given to Cox-2 heterozygous mice, the febrile response to lipopolysaccharide was strongly attenuated, resulting in an almost normalized temperature curve, whereas no difference was seen between wild-type and heterozygous mPGES-1 mice. Furthermore, the fever to intracerebrally injected prostaglandin E₂ was unaffected by acetaminophen treatment. These findings reveal that acetaminophen, similar to aspirin and other non-steroidal anti-inflammatory drugs, is antipyretic by inhibiting cyclooxygenase-2, and not by inhibiting mPGES-1 or signaling cascades downstream of prostaglandin E₂.

Interleukin-1β induced activation of the hypothalamus-pituitary-adrenal axis is dependent on interleukin-1 receptors on non-hematopoietic cells.

The proinflammatory cytokine interleukin-1β (IL-1β) plays a major role in the signal transduction of immune stimuli from the periphery to the central nervous system, and has been shown to be an important mediator of the immune-induced stress hormone release. The signaling pathway by which IL-1β exerts this function involves the blood-brain-barrier and induced central prostaglandin synthesis, but the identity of the blood-brain-barrier cells responsible for this signal transduction has been unclear, with both endothelial cells and perivascular macrophages suggested as critical components. Here, using an irradiation and transplantation strategy, we generated mice expressing IL-1 type 1 receptors (IL-1R1) either in hematopoietic or non-hematopoietic cells and subjected these mice to peripheral immune challenge with IL-1β. Following both intraperitoneal and intravenous administration of IL-1β, mice lacking IL-1R1 in hematopoietic cells showed induced expression of the activity marker c-Fos in the paraventricular hypothalamic nucleus, and increased plasma levels of ACTH and corticosterone. In contrast, these responses were not observed in mice with IL-1R1 expression only in hematopoietic cells. Immunoreactivity for IL-1R1 was detected in brain vascular cells that displayed induced expression of the prostaglandin synthesizing enzyme cyclooxygenase-2 and that were immunoreactive for the endothelial cell marker CD31, but was not seen in cells positive for the brain macrophage marker CD206. These results imply that activation of the HPA-axis by IL-1β is dependent on IL-1R1s on non-hematopoietic cells, such as brain endothelial cells, and that IL-1R1 on perivascular macrophages are not involved.

Distribution of microsomal prostaglandin E synthase-1 in the mouse brain

Previous studies in rats have demonstrated that microsomal prostaglandin E synthase‐1 (mPGES‐1) is induced in brain vascular cells that also express inducible cyclooxygenase‐2, suggesting that such cells are the source of the increased PGE2 levels that are seen in the brain following peripheral immune stimulation, and that are associated with sickness responses such as fever, anorexia, and stress hormone release. However, while most of what is known about the functional role of mPGES‐1 for these centrally evoked symptoms is based on studies on genetically modified mice, the cellular localization of mPGES‐1 in the mouse brain has not been thoroughly determined. Here, using a newly developed antibody that specifically recognizes mouse mPGES‐1 and dual‐labeling for cell‐specific markers, we report that mPGES‐1 is constitutively expressed in the mouse brain, being present not only in brain endothelial cells, but also in several other cell types and structures, such as capillary‐associated pericytes, astroglial cells, leptomeninges, and the choroid plexus. Regional differences were seen with particularly prominent labeling in autonomic relay structures such as the area postrema, the subfornical organ, the paraventricular hypothalamic nucleus, the arcuate nucleus, and the preoptic area. Following immune stimulation, mPGES‐1 in brain endothelial cells, but not in other mPGES‐1‐positive cells, was coexpressed with cyclooxygenase‐2, whereas there was no coexpression between mPGES‐1 and cyclooxygenase‐1. These data imply a widespread synthesis of PGE2 or other mPGES‐1‐dependent products in the mouse brain that may be related to inflammation‐induced sickness symptom as well as other functions, such as blood flow regulation. J. Comp. Neurol. 522:3229–3244, 2014.

Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells

Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll‐like and IL‐1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell‐specific deletions. We found that MyD88‐knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)‐induced anorexia, displayed anorexia when transplanted with wild‐type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS‐induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild‐type mice. Furthermore, in a model for cancer‐induced anorexia‐cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88‐dependent signaling within the brain is not required for eliciting inflammation‐induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory‐driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.—Ruud, J., Wilhelms, D. B., Nilsson, A., Eskilsson, A., Tang, Y.‐J., Ströhle, P., Caesar, R., Schwaninger, M., Wunderlich, T., Bäckhed, F., Engblom, D., Blomqvist, A. Inflammation‐ and tumor‐induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells. FASEB J. 27, 1973–1980 (2013). www.fasebj.org

Interleukin-6 primarily produced by non-hematopoietic cells mediates the lipopolysaccharide-induced febrile response

Interleukin-6 (IL-6) is critical for the lipopolysaccharide (LPS)-induced febrile response. However, the exact source(s) of IL-6 involved in regulating the LPS-elicited fever is still to be identified. One known source of IL-6 is hematopoietic cells, such as monocytes. To clarify the contribution of hematopoietically derived IL-6 to fever, we created chimeric mice expressing IL-6 selectively either in cells of hematopoietic or, conversely, in cells of non-hematopoietic origin. This was performed by extinguishing hematopoietic cells in wild-type (WT) or IL-6 knockout (IL-6 KO) mice by whole-body irradiation and transplanting them with new stem cells. Mice on a WT background but lacking IL-6 in hematopoietic cells displayed normal fever to LPS and were found to have similar levels of IL-6 protein in the cerebrospinal fluid (CSF) and in plasma and of IL-6 mRNA in the brain as WT mice. In contrast, mice on an IL-6 KO background, but with intact IL-6 production in cells of hematopoietic origin, only showed a minor elevation of the body temperature after peripheral LPS injection. While they displayed significantly elevated levels of IL-6 both in plasma and CSF compared with control mice, the increase was modest compared with that seen in LPS injected mice on a WT background, the latter being approximately 20 times larger in magnitude. These results suggest that IL-6 of non-hematopoietic origin is the main source of IL-6 in LPS-induced fever, and that IL-6 produced by hematopoietic cells only plays a minor role.

Immune-Induced Fever Is Dependent on Local But Not Generalized Prostaglandin E2 Synthesis in the Brain

Fever occurs upon binding of prostaglandin E2 (PGE2) to EP3 receptors in the median preoptic nucleus of the hypothalamus, but the origin of the pyrogenic PGE2 has not been clearly determined. Here, using mice of both sexes, we examined the role of local versus generalized PGE2 production in the brain for the febrile response. In wild-type mice and in mice with genetic deletion of the prostaglandin synthesizing enzyme cyclooxygenase-2 in the brain endothelium, generated with an inducible CreERT2 under the Slco1c1 promoter, PGE2 levels in the CSF were only weakly related to the magnitude of the febrile response, whereas the PGE2 synthesizing capacity in the hypothalamus, as reflected in the levels of cyclooxygenase-2 mRNA, showed strong correlation with the immune-induced fever. Histological analysis showed that the deletion of cyclooxygenase-2 in brain endothelial cells occurred preferentially in small- and medium-sized vessels deep in the brain parenchyma, such as in the hypothalamus, whereas larger vessels, and particularly those close to the neocortical surface and in the meninges, were left unaffected, hence leaving PGE2 synthesis largely intact in major parts of the brain while significantly reducing it in the region critical for the febrile response. Furthermore, injection of a virus vector expressing microsomal prostaglandin E synthase-1 (mPGES-1) into the median preoptic nucleus of fever-refractive mPGES-1 knock-out mice, resulted in a temperature elevation in response to LPS. We conclude that the febrile response is dependent on local release of PGE2 onto its target neurons and not on the overall PGE2 production in the brain. SIGNIFICANCE STATEMENT By using mice with selective deletion of prostaglandin synthesis in brain endothelial cells, we demonstrate that local prostaglandin E2 (PGE2) production in deep brain areas, such as the hypothalamus, which is the site of thermoregulatory neurons, is critical for the febrile response to peripheral inflammation. In contrast, PGE2 production in other brain areas and the overall PGE2 level in the brain do not influence the febrile response. Furthermore, partly restoring the PGE2 synthesizing capacity in the anterior hypothalamus of mice lacking such capacity with a lentiviral vector resulted in a temperature elevation in response to LPS. These data imply that the febrile response is dependent on the local release of PGE2 onto its target neurons, possibly by a paracrine mechanism.

Involvement of interleukin-1 type 1 receptors in lipopolysaccharide-induced sickness responses

Sickness responses to lipopolysaccharide (LPS) were examined in mice with deletion of the interleukin (IL)-1 type 1 receptor (IL-1R1). IL-1R1 knockout (KO) mice displayed intact anorexia and HPA-axis activation to intraperitoneally injected LPS (anorexia: 10 or 120µg/kg; HPA-axis: 120µg/kg), but showed attenuated but not extinguished fever (120µg/kg). Brain PGE2 synthesis was attenuated, but Cox-2 induction remained intact. Neither the tumor necrosis factor-α (TNFα) inhibitor etanercept nor the IL-6 receptor antibody tocilizumab abolished the LPS induced fever in IL-1R1 KO mice. Deletion of IL-1R1 specifically in brain endothelial cells attenuated the LPS induced fever, but only during the late, 3rd phase of fever, whereas deletion of IL-1R1 on neural cells or on peripheral nerves had little or no effect on the febrile response. We conclude that while IL-1 signaling is not critical for LPS induced anorexia or stress hormone release, IL-1R1, expressed on brain endothelial cells, contributes to the febrile response to LPS. However, also in the absence of IL-1R1, LPS evokes a febrile response, although this is attenuated. This remaining fever seems not to be mediated by IL-6 receptors or TNFα, but by some yet unidentified pyrogenic factor.