Sylvain Nadeau

How the Blood Talks to the Brain Parenchyma and the Paraventricular Nucleus of the Hypothalamus During Systemic Inflammatory and Infectious Stimuli

There are exciting new developments regarding the molecular mechanisms involved in the influence of circulating proinflammatory molecules within cells of the blood-brain barrier (BBB) during systemic immune challenges. These molecules, when present in the circulation, have the ability to trigger a series of events in cascade, leading to either the mitogen-activated protein (MAP) kinases/nuclear factor kappa B (NF-kappaB) or the janus kinase (JAK)/signal transducer and activator of transcription (STAT) transduction pathways in vascular-associated cells of the central nervous system (CNS). The brain blood vessels exhibit both constitutive and induced expression of receptors for different proinflammatory ligands that have the ability to stimulate these signaling molecules. Depending on the challenges and the cytokines involved, the transduction signal(s) solicited in cells of the BBB may orient the neuronal activity in a very specific manner in activating the transcription and production of soluble factors, such as prostaglandins (PGs). It is interesting to note that cytokines as well as systemic localized inflammation stimulate the cells of the BBB in a nonselective manner (i.e., within both large blood vessels and small capillaries across the brain). This nonselectivity raises several questions with regard to the localized neuronal activation induced by different experimental models of inflammation and cytokines. It is possible that the selectivity of the neuronal response is a consequence of the fine interaction between nonparenchymal synthesis of soluble mediators and expression of specific receptors for these ligands within parenchymal elements of different brain nuclei. This review will present the recent developments on this concept and the mechanisms that take place in cells of the BBB, which lead to the neuronal circuits involved in restoring the bodys homeostasis during systemic immunogenic challenges. The induction of fever, the hypothalamic-pituitary adrenal (HPA) axis, and other autonomic functions are among the physiological outcomes necessary for the protection of the mammalian organism in the presence of foreign material.

Functional Recovery after Peripheral Nerve Injury is Dependent on the Pro-Inflammatory Cytokines IL-1β and TNF: Implications for Neuropathic Pain

IL-1β and TNF are potential targets in the management of neuropathic pain after injury. However, the importance of the IL-1 and TNF systems for peripheral nerve regeneration and the mechanisms by which these cytokines mediate effects are to be fully elucidated. Here, we demonstrate that mRNA and protein levels of IL-1β and TNF are rapidly upregulated in the injured mouse sciatic nerve. Mice lacking both IL-1β and TNF, or both IL-1 type 1 receptor (IL-1R1) and TNF type 1 receptor (TNFR1), showed reduced nociceptive sensitivity (mechanical allodynia) compared with wild-type littermates after injury. Microinjecting recombinant IL-1β or TNF at the site of sciatic nerve injury in IL-1β- and TNF-knock-out mice restored mechanical pain thresholds back to levels observed in injured wild-type mice. Importantly, recovery of sciatic nerve function was impaired in IL-1β-, TNF-, and IL-1β/TNF-knock-out mice. Notably, the infiltration of neutrophils was almost completely prevented in the sciatic nerve distal stump of mice lacking both IL-1R1 and TNFR1. Systemic treatment of mice with an anti-Ly6G antibody to deplete neutrophils, cells that play an essential role in the genesis of neuropathic pain, did not affect recovery of neurological function and peripheral axon regeneration. Together, these results suggest that targeting specific IL-1β/TNF-dependent responses, such as neutrophil infiltration, is a better therapeutic strategy for treatment of neuropathic pain after peripheral nerve injury than complete blockage of cytokine production.

Regulation of the Gene Encoding Tumor Necrosis Factor Alpha (TNF-α) in the Rat Brain and Pituitary in Response to Different Models of Systemic Immune Challenge

Tumor necrosis factor (TNF)-alpha is usually referred to as a proinflammatory cytokine that plays a central role in initiating the cascade of other cytokines and factors for an appropriate immune response to infection. Like systemic phagocytes, recent studies have reported that specific cellular populations of the CNS have the ability to express and release the proinflammatory cytokine in response to peripheral administration of the bacterial endotoxin lipopolysaccharide (LPS). Whether such phenomenon represents a general mechanism of systemic immunogenic stimuli and how the severity of the challenge may influence TNF-alpha transcription in the brain has yet to be defined. Adult male rats were sacrificed 1, 3, 6, 12, 24 and 48 hours (h) after intraperitoneal (IP) injection of LPS (25-250 microg/100 g) or intramuscular (IM) injection of turpentine. Brains and pituitary glands were removed, cut, and TNF-alpha mRNA assayed by in situ hybridization using a full-length rat cRNA probe. The results show no positive signal under basal conditions or following sterile inflammation into the left hind limb. Systemic LPS caused a profound increase in the expression of the gene encoding TNF-alpha in the leptomeninges, choroid plexus (chp) and all sensorial circumventricular organs (CVOs). Interestingly, a migratory-like pattern of TNF-alpha-positive cells became apparent around the sensorial CVOs at 3 h, while a ubiquitous-like positive signal was found throughout the brain 6 h after the injection with the highest dose of LPS. The IP LPS injection also stimulated TNF-alpha transcription in the anterior pituitary lobe; the signal was maximal 1 h after the injection and returned gradually to basal levels at 12 h, whereas the mRNA encoding the cytokine was detected later in the neurohypophysis, i.e. 3 and 6 h post challenge. Dual-labeling procedure provided the evidence of an LPS-dependent induction of TNF-alpha in different phagocytic cellular populations of the brain, including parenchymal microglial cells during severe endotoxemia. The fact that these myeloid-derived cells have the ability to express the LPS receptor CD14 in the brain may well explain the transcriptional activation of the cytokine in response to the bacterial endotoxin, but not to systemic localized inflammation.

Endotoxemia Prevents the Cerebral Inflammatory Wave Induced by Intraparenchymal Lipopolysaccharide Injection: Role of Glucocorticoids and CD14

There is a robust and transient innate immune response in the brain during endotoxemia, which is associated with a cascade of NF-κB signaling events and transcriptional activation of genes that encode TNF-α and the LPS receptor CD14. The present study investigated whether circulating LPS has the ability to modulate the cerebral innate immune response caused by an intrastriatal (IS) injection of the endotoxin. We also tested the possibility that CD14 plays a role in these effects and male rats received an intracerebroventricular injection with an anti-CD14 before the IS LPS administration. The single LPS bolus into the striatum caused a strong and time-dependent transcriptional activation of TNF-α, IκBα, CD14, and monocyte chemoattractant protein-1 mRNA in microglial cells ipsilateral to the site of injection. Surprisingly, this wave of induced transcripts was essentially abolished by the systemic endotoxin pretreatment. Such anti-inflammatory properties of circulating LPS are mediated via plasma corticosterone, because exogenous corticoids mimicked while glucocorticoid receptor antagonist RU486 prevented the effects of systemic endotoxin challenge. Of interest is the partial involvement of CD14 in LPS-induced neuroinflammation; the anti-CD14 significantly abolished the microglial activity at day 3, but not at times earlier. The inflammatory response provoked by an acute intraparenchymal LPS bolus was not associated with convincing neurodegenerative processes. These data provide compelling evidence that systemic inflammation, through the increase in circulating glucocorticoids, has the ability to prevent the cerebral innate immune reaction triggered by an IS endotoxin injection. This study also further consolidates the existence of such system in the brain, which is finely regulated and its transient activation is not harmful for the neuronal elements.

The complement system is an integrated part of the natural innate immune response in the brain

The complement system consists of a group of proteins that play essential roles in coordinating the host defense to infection. It can be activated by two primary pathways, namely the classical and the alternative. This study aimed to determine the cellular distribution and the regulation of the genes encoding the proteins that are essential in guiding these pathways in the central nervous system (CNS) during innate immune recognition in mice. The results show a low‐to‐moderate C3 mRNA signal in few nonneuronal structures under basal conditions, whereas a robust C5 expression level was found in numerous populations of neuronal and nonneuronal cells. However, hybridization signal for the gene encoding the anaphylatoxin C5a receptor (C5aR) was low in the brain of vehicle‐administered mice. The constitutive C5 mRNA levels remained unaltered during endotoxemia, but a strong and transient de novo expression of the other members of the complement protein family was found in the brain of mice that received a single systemic bolus of lipopolysaccharide (LPS). Indeed, transcriptional activation C3 and factor B genes occurred in the circumventricular organs and a wave of C3aR‐expressing cells took place from the regions devoid of blood‐brain barrier to deeper brain parenchyma. The C5aR mRNA levels also increased in the cerebral endothelium at 1 h post‐LPS challenge, and the signal became gradually positive in microglial cells surrounding the capillaries and thereafter across the brain parenchyma. The present data provide evidence of an elegant pattern of expression and de novo transcription of key members of the complement system in the CNS, which underlies a sophisticated innate immune system that is triggered by circulating cell wall components of gram‐negative bacteria.

Development of a Novel Noncompetitive Antagonist of IL-1 Receptor

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed “101.10”), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC50 ≈ 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.

Clinical Outcome of Adjuvant Treatment of Endometrial Cancer Using Aperture-Based Intensity-Modulated Radiotherapy

PURPOSE To assess disease control and acute and chronic toxicity with aperture-based intensity-modulated radiotherapy (AB-IMRT) for postoperative pelvic irradiation of endometrial cancer. METHODS AND MATERIALS Between January and July 2005, after hysterectomy for endometrial cancer, 15 patients received 45 Gy to the pelvis using AB-IMRT. The AB-IMRT plans were generated by an in-house treatment planning system (Ballista). The AB-IMRT plans were used for treatment and were dosimetrically compared with three other approaches: conventional four-field, enlarged four-field, and beamlet-based IMRT (BB-IMRT). Disease control and toxicity were prospectively recorded and compared with retrospective data from 30 patients treated with a conventional four-field technique. RESULTS At a median follow-up of 27 months (range, 23-30), no relapse was noted among the AB-IMRT group compared with five relapses in the control group (p = 0.1). The characteristics of each group were similar, except for the mean body mass index, timing of brachytherapy, and applicator type used. Patients treated with AB-IMRT experienced more frequent Grade 2 or greater gastrointestinal acute toxicity (87% vs. 53%, p = 0.02). No statistically significant difference was noted between the two groups regarding the incidence or severity of chronic toxicities. AB-IMRT plans significantly improved target coverage (93% vs. 76% of planning target volume receiving 45 Gy for AB-IMRT vs. conventional four-field technique, respectively). The sparing of organs at risk was similar to that of BB-IMRT. CONCLUSION The results of our study have shown that AB-IMRT provides excellent disease control with equivalent late toxicity compared with the conventional four-field technique. AB-IMRT provided treatment delivery and quality assurance advantages compared with BB-IMRT and could reduce the risk of second malignancy compared with BB-IMRT.

Proinflammatory signal transduction pathways in the CNS during systemic immune response

Abstract Circulating lipopolysaccharide (LPS) causes a rapid transcriptional activation of its transmembrane receptor mCD14 within the circumventricular organs (CVOs), brain regions that contain a rich vascular plexus with specialized arrangements of the blood vessels. Parenchymal cells located in the anatomical boundaries of the CVOs exhibit a delayed response, which is followed by a positive signal for CD14 transcript in microglia across the brain parenchyma. The constitutive expression of the toll-like receptor 4 (TLR4) in the CVOs is likely to be a key element allowing the proinflammatory signal transduction pathways (MyD88/IRAK/NIK/NF-κB) to take place rapidly in these organs in response to circulating LPS. These results strongly suggest that the endotoxin first reaches organs devoid of the blood brain barrier (BBB) to induce the transcription of its own receptor and thereafter increases CD14 biosynthesis within parenchymal structures surrounding the CVOs and then the entire brain of severely challenged animals. Brain CD14 expression may be a key step in the transcription of proinflammatory cytokines primarily within accessible structures from the blood and subsequently through scattered parenchymal cells during severe sepsis. However, CD14 synthesis in parenchymal cells of the brain is also dependent on the production of proinflammatory cytokines. Of interest is the data that systemic injection of the bacterial endotoxin induces a strong expression of CD14 mRNA in a pattern that is closely related to the induction of tumor necrosis factor alpha (TNF-α) transcript with a rapid and delayed response. Although there is a large body of evidence that CD14 (and now TLR4) is necessary for the role of LPS on the induction of cytokine transcription from different myeloid cells, the possibility remains that the cytokine itself acts as an autocrine and paracrine factor to up regulate the LPS receptor. The binding of TNF to its type I receptor (p55) leads to the activation and translocation of p50/65 NF-κB into the nucleus, which seems a key player in activating CD14 transcription in the CNS. Central injection of recombinant rat TNF-α causes a robust expression of the genes encoding IκBα, TNF-α and CD14 in microglial cells of the brain parenchyma. The time-related induction of these transcripts suggested a potential role of NF-κB in mediating TNF-induced transcriptional activation of the LPS receptor. Systemic injection with the endotoxin LPS provoked a similar microglial activation that was prevented in inhibiting the biological activity of the proinflammatory cytokine in the CNS. Together these data provide the evidence that centrally-produced TNF-α plays an essential autocrine/paracrine role in triggering parenchymal microglial cells during severe endotoxemia. These events may be determinant for orchestrating the neuroinflammatory responses that take place in a well coordinated manner to activate the resident phagocytic population of cells in the brain. The physiological outcomes of this innate immune response of the CNS are likely to include a rapid elimination of LPS particles via an increased opsonic activity of the transmembrane CD14 receptor to prevent potential detrimental consequences on neuronal elements during blood sepsis.