Steve Lacroix

Proinflammatory cytokine synthesis in the injured mouse spinal cord: Multiphasic expression pattern and identification of the cell types involved

We have studied the spatial and temporal distribution of six proinflammatory cytokines and identified their cellular source in a clinically relevant model of spinal cord injury (SCI). Our findings show that interleukin‐1β (IL‐1β) and tumor necrosis factor (TNF) are rapidly (<5 and 15 minutes, respectively) and transiently expressed in mice following contusion. At 30–45 minutes post SCI, IL‐1β and TNF‐positive cells could already be seen over the entire spinal cord segment analyzed. Multilabeling analyses revealed that microglia and astrocytes were the two major sources of IL‐1β and TNF at these times, suggesting a role for these cytokines in gliosis. Results obtained from SCI mice previously transplanted with green fluorescent protein (GFP)‐expressing hematopoietic stem cells confirmed that neural cells were responsible for the production of IL‐1β and TNF for time points preceding 3 hours. From 3 hours up to 24 hours, IL‐1β, TNF, IL‐6, and leukemia inhibitory factor (LIF) were strongly upregulated within and immediately around the contused area. Colocalization studies revealed that all populations of central nervous system resident cells, including neurons, synthesized cytokines between 3 and 24 hours post SCI. However, work done with SCI‐GFP chimeric mice revealed that at least some infiltrating leukocytes were responsible for cytokine production from 12 hours on. By 2 days post‐SCI, mRNA signal for all the above cytokines had nearly disappeared. Notably, we also observed another wave of expression for IL‐1β and TNF at 14 days. Overall, these results indicate that following SCI, all classes of neural cells initially contribute to the organization of inflammation, whereas recruited immune cells mostly contribute to its maintenance at later time points. J. Comp. Neurol. 500:267–285, 2007.

Effect of acute systemic inflammatory response and cytokines on the transcription of the genes encoding cyclooxygenase enzymes (COX-1 and COX-2) in the rat brain

Abstract: The aim of this study was to investigate the influence of the acute‐phase response and the proinflammatory cytokines on the transcription of the genes encoding the limiting enzymes for the production of prostaglandins, cyclooxygenase (COX)‐1 and COX‐2, in the rat brain. The bacterial endotoxin lipopolysaccharide (intravenous and intraperitoneal) and turpentine (intramuscular) were used as different models of inflammation in adult male rats. Animals were also killed at various times after intravenous administration of interleukin‐1β, tumor necrosis factor‐α, and interleukin‐6, and mRNAs encoding COX‐1 and COX‐2 were assayed by in situ hybridization histochemistry. A profound transcriptional activation of the gene encoding COX‐2 was detected over blood vessels of the entire brain microvasculature, choroid plexus, and leptomeninges of lipopolysaccharide‐challenged rats. Injection of the endotoxin intravenously also increased COX‐2 gene expression within parvocellular division of the hypothalamic paraventricular nucleus. It is interesting that intramuscular turpentine injection stimulated transcription of COX‐2 along endothelium of brain capillaries, and the signal of this transcript paralleled the inflammation of the left hind limb. A robust COX‐2 mRNA signal was detected rapidly in the brain microvessels of interleukin‐1β‐injected rats, whereas tumor necrosis factor‐α administration caused a modest but significant induction of this transcript. In contrast, intravenous injection of interleukin‐6 did not alter genetic expression of COX‐2, and none of the above described models affected the synthesis of COX‐1 gene in the rat brain. These results indicate that specific cell populations, in particular vascular‐ and/or perivascular‐associated cells, are responsible for the central production of prostaglandins during systemic inflammation, and circulating interleukin‐1β is likely to be a potent mediator of this response.

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.

The Bacterial Endotoxin Lipopolysaccharide has the Ability to Target the Brain in Upregulating Its Membrane CD14 Receptor Within Specific Cellular Populations

Systemic injection of the bacterial endotoxin lipopolysaccharide (LPS) provides a very good mean for increasing the release of proinflammatory cytokines by circulating monocytes and tissue macrophages. There is now considerable evidence that LPS exerts its action on mononuclear phagocytes via the cell surface receptor CD14. The aim of the present study was to verify the hypothesis that the brain has also the ability to express the gene encoding the LPS receptor, which may allow a direct action of the endotoxin onto specific cellular populations during blood sepsis. Adult male Sprague‐Dawley rats were sacrificed 1, 3, 6 and 24 h after systemic (i.v. or i.p.) injection of LPS or the vehicle solution. Brains were cut from the olfactory bulb to the medulla in 30‐μm coronal sections and mRNA encoding rat CD14 was assayed by in situ hybridization histochemistry using a specific 35S‐labeled riboprobe. The results show low levels of CD14 mRNA in the leptomeninges, choroid plexus and along blood vessels of the brain microvasculature under basal conditions. Systemic injection of the bacterial endotoxin caused a profound increase in the expression of the gene encoding CD14 within these same structures as well as in the circumventricular organs (CVOs) the organum vasculosum of the lamina terminalis, subfornical organ, median eminence and area postrema. In most of these structures, the signal for CD14 mRNA was first detected at 1 h, reached a peak at 3 h post‐injection, declined at 6 h, and return to basal levels 24 h after LPS treatment. Quite interestingly, a migratory‐like pattern of CD14 positive cells was observed from all sensorial CVOs to deeper parenchymal brain 3 and 6 h after LPS injection. At 6 h post‐challenge, small positive cells were found throughout the entire parenchymal brain and dual‐labeling procedure indicated that different cells of myeloid origin have the ability to express CD14 in response to systemic LPS. These included CVO microglia, choroid plexus and leptomeninge macrophages, parenchymal and perivascular‐associated microglial cells, although specific nonmyeloid cells were also positive for the LPS receptor. These results provide the very first evidence of a direct role of LPS on specific cell populations of the central nervous system, which is likely to be responsible for the transcription of proinflammatory cytokines; first within accessible structures from the blood and thereafter through scattered parenchymal cells during severe sepsis.

Toll-Like Receptor Signaling Is Critical for Wallerian Degeneration and Functional Recovery after Peripheral Nerve Injury

Toll-like receptors (TLRs) bind specific components conserved among microorganisms as well as endogenous ligands produced by necrotic cells, injured axons, and the extracellular matrix. Here, we investigated whether TLRs are involved in regulating the immune response, Wallerian degeneration (WD), and nerve regeneration after sciatic nerve lesion. Early expression of interleukin-1β and monocyte chemoattractant protein-1 was compromised in the sciatic nerve distal stump of mice deficient in TLR signaling. In addition, significantly fewer macrophages were recruited and/or activated in the sciatic nerve distal stump of TLR2-, TLR4-, and MyD88-deficient mice compared with wild-type littermates, whereas WD, axonal regeneration, and recovery of locomotor function were impaired. In contrast, animals that received a single microinjection of TLR2 and TLR4 ligands at the site of sciatic nerve lesion had faster clearance of the degenerating myelin and recovered earlier than saline-injected control rats. Finally, rats that had altered innate immune response through dexamethasone treatment exhibited three times more myelin debris in their sciatic nerve distal stump and a significant delay in recovery of locomotor function. Our results provide strong evidence that TLR signaling plays a critical role in orchestrating the innate immune response leading to efficient and rapid clearance of inhibitory myelin debris and nerve regeneration.

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.

Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation

Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.

Requirement of myeloid cells for axon regeneration.

The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TKmt-30 mice expressing a mutated HSV-1 thymidine kinase (TK) gene regulated by the myeloid-specific CD11b promoter. Continuous delivery of ganciclovir at a sciatic nerve lesion site greatly decreased the number of granulocytes/inflammatory monocytes and macrophages in the distal stump of CD11b-TKmt-30 mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TKmt-30 mice. This was caused by an unsuitable growth environment rather than an altered regeneration capacity of neurons. In absence of CD11b+ cells, the clearance of inhibitory myelin debris was prevented, neurotrophin synthesis was abolished, and blood vessel formation/maintenance was severely compromised in the sciatic nerve distal stump. Spinal cord-injured axons also failed to regenerate through peripheral nerve grafts in the absence of CD11b+ cells. Therefore, myeloid cells support axonal regeneration and functional recovery by creating a growth-permissive milieu for injured axons.

NT-3 gene delivery elicits growth of chronically injured corticospinal axons and modestly improves functional deficits after chronic scar resection.

Nervous system growth factors promote axonal growth following acute spinal cord injury. In the present experiment, we examined whether delivery of neurotrophic factors after chronic spinal cord injury would also promote axonal growth and influence functional outcomes. Adult Fischer 344 rats underwent mid-thoracic spinal cord dorsal hemisection lesions. Three months later, primary fibroblasts genetically modified to express human neurotrophin-3 (NT-3) were placed in, and distal to, the lesion cavity. Upon sacrifice 3 months later (6 months following the initial lesion), NT-3-grafted animals exhibited significant growth of corticospinal axons up to 15 mm distal to the lesion site and showed a modest but significant 1.5-point improvement in locomotor scores (P < 0.05) on the BBB scale, compared to control-grafted animals. Thus, growth factor gene delivery can elicit growth of corticospinal axons in chronic stages of injury and improves functional outcomes compared to non-growth-factor-treated animals.

Functional circuitry in the brain of immune-challenged rats: partial involvement of prostaglandins.

This study investigated the role of prostaglandins (PGs) on the neuronal activity and the transcription of corticotropin‐releasing factor (CRF) in the brain of conscious immune‐challenged rats. Intravenous (i.v.) administration of indomethacin, an inhibitor of PG synthesis, was performed prior to and after the intraperitoneal (i.p.) injection of different doses [250 μg, 25 μg, and 2.5 μg/100 g body weight (b.w.)] of the immune activator lipopolysaccharide (LPS). Systemic administration of the high and middle doses of LPS caused a robust and widespread induction of both immediate‐early genes (IEGs), c‐fos and nerve growth factor‐inducible gene B (NGFI‐B) mRNAs, whereas injection of the low dose selectively triggered c‐fos expression within the sensorial circumventricular organs. Pretreatment with indomethacin did not prevent c‐fos transcription in the rat brains challenged with the high dose of LPS at 3 hours postinjection. Inhibition of PG formation was more effective for interruption of the neuronal activation in animals injected with 25 μg LPS/100 g b.w., although the influence depended on the structures and the groups of activated cells. Indeed, PG inhibition significantly altered LPS‐induced c‐fos mRNA expression in the medial preoptic area/organum vasculosum of the lamina terminalis, the periventricular nucleus, the paraventricular nucleus of the hypothalamus (PVN), and the ventrolateral medulla (VLM) but not in many other regions, including the subfornical organ, the central nucleus of the amygdala, the arcuate nucleus/median eminence, the parabrachial nucleus, the choroid plexus, and the nucleus of the solitary tract (NTS). In the hypothalamic PVN, inhibition of both c‐fos and NGFI‐B transcripts by indomethacin was also associated to an abolished influence of the endotoxin on the transcription of neuroendocrine CRF; induction of CRF primary transcript by the middle dose of LPS was selective to the PVN and was completely blocked by pretreatment with indomethacin. Moreover, a large number of tyrosine hydroxylase (TH)‐immunoreactive neurons of the VLM (A1/C1) and the NTS (A2/C2) were positive for c‐fos mRNA in immune‐challenged rats, an effect that was largely prevented by indomethacin in the VLM but not in the NTS. These results indicate that the role of PGs in mediating the stimulatory influence of the acute‐phase response depends on the severity of the systemic stressful situation, the brain regions, and the cell groups as well as the activated target genes. J. Comp. Neurol. 387:307–324, 1997.