Steve Lacroix

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.

The role of microglia in spinal cord injury (SCI) remains poorly understood and is often confused with the response of macrophages. Here, we use specific transgenic mouse lines and depleting agents to understand the response of microglia after SCI. We find that microglia are highly dynamic and proliferate extensively during the first two weeks, accumulating around the lesion. There, activated microglia position themselves at the interface between infiltrating leukocytes and astrocytes, which proliferate and form a scar in response to microglia-derived factors, such as IGF-1. Depletion of microglia after SCI causes disruption of glial scar formation, enhances parenchymal immune infiltrates, reduces neuronal and oligodendrocyte survival, and impairs locomotor recovery. Conversely, increased microglial proliferation, induced by local M-CSF delivery, reduces lesion size and enhances functional recovery. Altogether, our results identify microglia as a key cellular component of the scar that develops after SCI to protect neural tissue.

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-1beta (IL-1beta) 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-1beta 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-1beta 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-1beta and TNF for time points preceding 3 hours. From 3 hours up to 24 hours, IL-1beta, 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-1beta 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.

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.

When released into the bloodstream, proinflammatory cytokines have the ability to trigger the transcription of different genes in cells of the blood-brain barrier (BBB), including members of the nuclear factor kappa B (NF-kappaB) family and cyclooxygenase-2 (COX-2), the limiting enzyme for the formation of prostaglandins (PGs). The present study investigated the possibility that interleukin-1beta (IL-1beta) plays an essential role in these events during a systemic inflammatory response. Both wild-type and IL-1beta-deficient mice were killed at different times after two different immunogenic stimuli, i.e., intraperitoneal lipopolysaccharide (LPS) injection and intramuscular turpentine injection, used here as a model of systemic localized inflammatory insult. The inhibitory factor kappaBalpha (IkappaBalpha, index of NF-kappaB activity) and COX-2 transcripts were detected throughout the brain by means of in situ hybridization. Systemic LPS injection caused a strong and rapid expression of IkappaBalpha in endothelial cells lining the BBB of large and small blood vessels and thereafter within parenchymal microglia across the brain. This treatment also provoked a transient expression of COX-2 along cells of the vascular system, and the expression pattern and intensity of the signal for both transcripts were essentially the same in wild-type and IL-1beta-deficient animals. In contrast, the induction of these genes that was quite selective to the cells of the BBB in response to intramuscularly turpentine insult was completely abolished in IL-1beta-deficient mice. Indeed, a late and prolonged expression of IkappaBalpha and COX-2 mRNAs was found along the cerebral blood vessels in response to the sterile and localized inflammation in wild-type mice, whereas such induction was absent in the brain of IL-1beta-deficient animals. These results indicate that IL-1beta has an obligatory role in the activation of NF-kappaB molecules and PGs within endothelial cells of the BBB in an experimental model of intramuscularly turpentine-induced inflammation but not during endotoxemia.