Christoph Rummel

Signaling the brain in systemic inflammation: role of sensory circumventricular organs.

The sensory circumventricular organs (CVOs) are specialized brain regions that lack a tight blood-brain barrier. A role for these brain structures in signaling the brain during systemic inflammation is based on the following sets of observations. In spite of some conflicting data from literature, lesions of CVOs have been shown to block several components of brain controlled illness responses (i.e. fever or neuroendocrine modifications). Receptors for inflammatory cytokines and for bacterial fragments are constitutively expressed in cells within the sensory CVOs. The expression of most of these receptors is upregulated under conditions of systemic inflammation. Cellular responses in theses brain areas can be recorded and documented after stimulation of these respective receptors. Such responses include changes in electrical activity of neurons, induction of transcription factors leading to modifications in gene expression during inflammation and to a localized release of secondary signal molecules. These molecules may influence or even gain access to neural structures inside the blood-brain barrier, which can normally not directly be reached by circulating cytokines or bacterial fragments.

Nuclear STAT3 translocation in guinea pig and rat brain endothelium during systemic challenge with lipopolysaccharide and interleukin-6

During systemic inflammation, cytokines are released by immune‐competent cells into the circulation, which in turn signal the brain to mediate brain‐controlled signs of illness. Cytokine‐responsive brain cells can be mapped by histological analysis of cytokine‐induced transcription factors or transcription factor‐associated molecules revealing different cell phenotypes that respond to activation of the immune system. Critical sites mediating cytokine‐dependent immuneffector functions can be divided into two groups, one group of responding cells situated along a tight blood–brain barrier (BBB), and a second cell group in structures with an open BBB, e.g., the sensory circumventricular organs (CVOs). Previous reports from our group suggest that activation of the signal transducer and activator of transcription factor 3 (STAT3) during lipopolysaccharide (LPS)‐induced systemic inflammation is mediated by interleukin‐6 (IL‐6) and occurs in astrocytes of the rat CVOs. Here we show in the guinea pig a time‐dependent marked LPS‐induced STAT3 activation within astrocytes and endothelial cells of the CVOs, within astrocytes located in brain structures with a functional BBB and within the brain endothelium of the entire brain. In addition, systemic treatment of rats with either rat recombinant IL‐6 or LPS induced STAT3 activation in brain endothelial cells in a similar way as observed in the guinea pig brain, stressing the involvement of IL‐6 in this phenomenon in a more generalized way. The STAT3‐activated brain cells are located in critical target structures mediating cytokine action during LPS‐induced inflammation. STAT3‐controlled transcriptional activation with yet unknown cell‐specific functional consequences seems to be involved in this process. J. Comp. Neurol. 491:1–14, 2005.

Selective contribution of interleukin-6 and leptin to brain inflammatory signals induced by systemic LPS injection in mice.

This study aimed to address the relative contributions of the proinflammatory cytokine interleukin‐6 (IL‐6) and the cytokine‐like hormone leptin to the genomic activation of brain cells during lipopolysaccharide (LPS)‐induced systemic inflammation. Wildtype and IL‐6KO mice were injected with LPS (50 μg/kg, intraperitoneally) and the brains analyzed by immunohistochemistry and reverse‐transcriptase polymerase chain reaction (RT‐PCR). LPS induced a pronounced nuclear translocation of the signal transducer and activator of transcription (STAT3) throughout the brains of wildtype mice, an effect that was significantly diminished, but not abolished, in the IL‐6KOs. The remnant STAT3‐activation, although still observed within some of the same areas activated by IL‐6, was most intense in ependymal and meningial cells and along distinct blood vessels throughout the brain. This expression was almost totally abolished in the presence of an anti‐leptin antiserum. Interestingly, the induction of cyclooxygenase 2 and microsomal prostaglandin E synthase (mPGES), the rate‐limiting enzymes for synthesis of PGE2 by LPS, was diminished to a degree that correlated with the absence of IL‐6 but not entirely with leptin. These results demonstrate that the induction of the inflammatory pathway in the brain is mediated by both IL‐6 and leptin, which appear to work in tandem. Unlike IL‐6, however, the contribution of leptin to this response was limited to distinct cell types/brain areas and STAT3‐responsive target genes implicated in the brain‐controlled sickness‐type response. The physiological significance of leptins action on meningeal and endothelial cells remains to be clarified but might reflect a role in LPS‐induced immune cell infiltration into the brain. J. Comp. Neurol. 511:373–395, 2008.

Nuclear translocation of the transcription factor STAT3 in the guinea pig brain during systemic or localized inflammation.

The purpose of the present study was to investigate a possible lipopolysaccharide (LPS)‐induced activation of brain cells that is mediated by the pleiotropic cytokine interleukin‐6 (IL‐6) and its transcription factor STAT3 during systemic or localized inflammation. In guinea pigs, intra‐arterial (i.a., 10 μg kg−1) or intraperitoneal (i.p., 30 μg kg−1) injections of bacterial LPS cause a systemic inflammatory response which is accompanied by a robust fever. A febrile response can also be induced by administration of LPS into artificial subcutaneously implanted Teflon chambers (s.c. 100 or 10 μg kg−1), which reflects an experimental model that mimics local tissue inflammation. Baseline plasma levels of bioactive IL‐6 determined 60 min prior to injections of LPS or vehicle amounted to 35–80 international units (i.u.) ml−1. Within 90 min of LPS injection, plasma IL‐6 rose about 1000‐fold in the groups injected i.a. or i.p., about 50‐fold in the group injected s.c. with 100 μg kg−1 LPS, and only 5‐fold in guinea pigs injected with the lower dose of LPS (10 μg kg−1). At this time point, a distinct nuclear translocation pattern of the transcription factor STAT3 became evident in several brain structures. Amongst those, the sensory circumventricular organs known to lack a tight blood–brain barrier such as the area postrema, the vascular organ of the lamina terminalis and the subfornical organ, as well as the hypothalamic supraoptic nucleus showed intense nuclear STAT3 signals in the i.a. or i.p. injected groups. In contrast a moderate (s.c. group, 100 μg kg−1), or even no (s.c. group, 10 μg kg−1), nuclear STAT3 translocation occurred in response to s.c. injections of LPS. These results suggest that STAT3‐mediated genomic activation of target gene transcription in brain cells occurred only in those cases in which sufficiently high concentrations of circulating IL‐6 were formed during systemic (i.a.. and i.p. groups) or localized (s.c. group, 100 μg kg−1) inflammation.

Leptin regulates leukocyte recruitment into the brain following systemic LPS-induced inflammation.

The appetite suppressing hormone leptin has emerged as an important modulator of immune function and is now considered to be a critical link between energy balance and host defense responses to pathogens. These ‘adaptive’ responses can, in situations of severe and sustained systemic inflammation, lead to adverse effects including brain damage that is partly mediated by neutrophil recruitment into the brain. We examined the contribution of leptin to this process in leptin-deficient (ob/ob), -resistant (db/db) and wild-type (WT) mice injected intraperitoneally with a septic dose of lipopolysaccharide (LPS). This treatment induced a dramatic increase in the number of neutrophils entering the brain of WT mice, an effect that was almost totally abolished in the mutant mice and correlated with a significant reduction in the mRNA levels of interleukin-1β, intracellular adhesion molecule-1 and neutrophil-specific chemokines. These effects were reversed with leptin replenishment in ob/ob mice leading to recovery of neutrophil recruitment into the brain. Moreover, 48 h food deprivation in WT mice, which decreased circulating leptin levels, attenuated the LPS-induced neutrophil recruitment as did a single injection of an anti-leptin antiserum 4 h before LPS treatment in WT mice. These results provide the first demonstration that leptin has a critical role in leukocyte recruitment to the brain following severe systemic inflammation with possible implications for individuals with altered leptin levels such as during obesity or starvation.

Spatiotemporal nuclear factor interleukin-6 expression in the rat brain during lipopolysaccharide-induced fever is linked to sustained hypothalamic inflammatory target gene induction

Rats injected with lipopolysaccharide (LPS) show brain‐controlled sickness symptoms, including fever. In these animals, early genomic activation of brain cells was previously monitored by immunohistochemical detection of transcription factors such as nuclear factor (NF)‐κB or signal transducer and activator of transcription (STAT)3 and was linked to the initiation or maintenance of the febrile response. To investigate whether NF‐IL6 might be another important transcription factor implicated in this kind of immune‐to‐brain signaling, rats were injected with LPS (100 μg/kg, intraperitoneally) or phosphate‐buffered saline, and brains were analyzed by immunohistochemistry, real‐time PCR, or Western blot 4, 6, 8, and 10 hours later. Moderate to strong LPS‐induced nuclear NF‐IL6 immunoreactivity (IR) occurred in a time‐dependent manner within circumventricular organs, namely, the vascular organ of the lamina terminalis, the subfornical organ, the area postrema, and the median eminence, brain structures with a leaky blood–brain barrier. Furthermore, nuclear NF‐IL6‐IR was observed in the pituitary gland, the choroid plexus, and the meninges as well as blood vessels throughout the entire brain. Endothelial, microglial, and ependymal cells, astrocytes, perivascular macrophages, and neurons exhibited LPS‐induced nuclear NF‐IL6‐IR; mRNA levels of NF‐IL6, responsive inflammatory genes, and NF‐IL6 protein levels were significantly elevated. As opposed to observations on STAT3 or NFκB, the percentage of NF‐IL6‐reactive cells increased in parallel to late phases of the febrile response. In conclusion, these results suggest a potential role for NF‐IL6 in the maintenance or possibly the termination of LPS‐induced fever. Moreover, we propose NF‐IL6 to be a delayed brain cell activation marker. J. Comp. Neurol. 519:480–505, 2011.

Parthenolide attenuates LPS-induced fever, circulating cytokines and markers of brain inflammation in rats

Parthenolide, a sesquiterpene lactone, has been reported to exhibit a variety of anti-inflammatory and immunomodulatory effects. To test the effect of parthenolide on brain inflammatory responses, brain oxidative stress and fever, we treated rats with parthenolide (1 mg/kg), simultaneously or 1 h prior to a systemic (i.p.) challenge with a moderate dose (100 μg/kg) of lipopolysaccharide (LPS). The initial hypothermia was exaggerated; the second phase of the biphasic LPS-induced fever and circulating interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were significantly attenuated only in parthenolide-pretreated animals. In the hypothalamus, markers of NFκB/NF-IL6 pathway activation (inhibitor κBα, NF-IL6 and the serin/threonin kinase-like protein mRNA expression) and markers of oxidative stress (including nuclear respiratory factor 1) and NFκB immunoreactivity were significantly reduced while NF-IL6 immunoreactivity and suppressor of cytokine signaling 3 mRNA expression remained unaltered, 8 h after LPS-stimulation with parthenolide-pretreatment. Importantly, this response was accompanied by decreased mRNA expression of the rate limiting enzyme in prostaglandin synthesis, cyclooxygenase 2 (COX2), known for its critical role in fever induction pathways. A direct action of parthenolide on brain cells was also confirmed in a primary neuro-glial cell culture of the vascular organ of the lamina terminalis a pivotal brain structure for fever manifestation with a leaky blood-brain barrier. In summary, pretreatment with parthenolide attenuates the febrile response during LPS-induced systemic inflammation by reducing circulating IL-6 and TNFα and decreasing hypothalamic NFκB/NF-IL6 activation, oxidative stress and expression of COX2. Thus parthenolide appears to have the potential to reduce brain inflammation.

Fever, sickness behavior, and expression of inflammatory genes in the hypothalamus after systemic and localized subcutaneous stimulation of rats with the toll-like receptor 7 agonist imiquimod

The Toll-like receptor 7 (TLR7) agonist imiquimod is used for topical treatment of skin cancers. We studied the consequences of injections of imiquimod into a subcutaneous (s.c.) air pouch or of intraperitoneal (i.p.) injections on the manifestation of fever, sickness behavior, and the peripheral and brain-intrinsic induction of a variety of inflammatory molecules. Rats were given imiqimod s.c. or i.p. (1 or 5 mg/kg). Body temperature, motor activity, and food and water intake were recorded by telemetric devices. Peripheral and brain-intrinsic induction of inflammatory mediators was analyzed by real-time polymerase chain reaction (RT-PCR), bioassays, enzyme-linked immunosorbent assays (ELISAs), and immunohistochemistry. Imiquimod is the first TLR-agonist to produce more potent effects with s.c. than i.p. administration. Peripheral induction of interferons (IFNs) and putative circulating pyrogens corresponded to the magnitude of the illness responses. In the brain, an expression of cytokines (TNFα, IL-1β, and IL-6) and inducible forms of enzymes for prostaglandin E2 synthesis (COX-2 and mPGES) occurred, which was accompanied by a moderate activation of the transcription factors NFκB and STAT3, and a strong activation of the transcription factor NF-IL6, in cells of specific areas with an open blood-brain barrier. These inflammatory responses noted within the brain were more marked after s.c. administration, than i.p. administration of imiquimod. At a dose of 5 mg/kg, imiquimod causes rather moderate brain-inflammatory responses, which are related to peripheral IFN-expression and possibly mediated by brain-intrinsic activation of NF-IL6 and induction of a proinflammatory cocktail. The lack of a septic-like state in imiquimod-treated rats reinforces the therapeutic use of this drug.

Volume reduction of the jugular foramina in Cavalier King Charles Spaniels with syringomyelia

BackgroundUnderstanding the pathogenesis of the chiari-like malformation in the Cavalier King Charles Spaniel (CKCS) is incomplete, and current hypotheses do not fully explain the development of syringomyelia (SM) in the spinal cords of affected dogs. This study investigates an unconventional pathogenetic theory for the development of cerebrospinal fluid (CSF) pressure waves in the subarachnoid space in CKCS with SM, by analogy with human diseases. In children with achondroplasia the shortening of the skull base can lead to a narrowing of the jugular foramina (JF) between the cranial base synchondroses. This in turn has been reported to cause a congestion of the major venous outflow tracts of the skull and consequently to an increase in the intracranial pressure (ICP). Amongst brachycephalic dog breeds the CKCS has been identified as having an extremely short and wide braincase. A stenosis of the JF and a consequential vascular compromise in this opening could contribute to venous hypertension, raising ICP and causing CSF jets in the spinal subarachnoid space of the CKCS. In this study, JF volumes in CKCSs with and without SM were compared to assess a possible role of this pathologic mechanism in the development of SM in this breed.ResultsComputed tomography (CT) scans of 40 CKCSs > 4 years of age were used to create three-dimensional (3D) models of the skull and the JF. Weight matched groups (7–10 kg) of 20 CKCSs with SM and 20 CKCSs without SM were compared. CKCSs without SM presented significantly larger JF -volumes (median left JF: 0.0633 cm3; median right JF: 0.0703 cm3; p < 0.0001) when compared with CKCSs with SM (median left JF: 0.0382 cm3; median right JF: 0.0434 cm3; p < 0.0001). There was no significant difference between the left and right JF within each group. Bland-Altman analysis revealed excellent reproducibility of all volume measurements.ConclusionA stenosis of the JF and consecutive venous congestion may explain the aetiology of CSF pressure waves in the subarachnoid space, independent of cerebellar herniation, as an additional pathogenetic factor for the development of SM in this breed.

Fever and circulating cytokines induced by double-stranded RNA in guinea pigs: dependence on the route of administration and effects of repeated injections.

Aims:  The aim of this study was to characterize the properties of synthetic double‐stranded RNA to induce fever and circulating cytokines in guinea pigs with special emphasis on the route of administration and on a putative development of tolerance to this pyrogen.