S100 calcium-binding proteins A8/A9 initiate the early inflammatory program in injured peripheral nerve
ABSTRACT: To shed light on the early processes of immune response to peripheral nerve injury, we first used genome-wide transcriptional profiling and bioinformatics (Ingenuity, NextBio) pathway analyses of the proximal (P; regenerating) and distal (D; degenerating) nerve stumps at day 1 in the sciatic nerve axotomy model in rats. We identified a number of specific immunomodulatory genes and pathways that were regulated shortly post-injury in both the P and D segments, including all members of the interleukin (IL), chemokine, tumor necrosis factor (TNF), matrix metalloproteinase (MMP), toll-like receptor (TLR), tissue inhibitor of metalloproteinase (TIMP), ion channel and myosin families. Immunomodulatory calcium-binding S100A8 and S100A9 were the top up-regulated genes in both the D and P segments. In cultured Schwann cells stimulated with the purified S100A8/A9 heterodimer we recorded a high level of similarity of the activated genes and pathways with that of the injured nerve, especially in the activation of the chemokine and cytokine gene networks that support agranulocyte and granulocyte chemotaxis, adhesion, transmigration and rolling signaling pathways. We also confirmed activation of multiple cell death related gene networks supporting TNFR1, natural killer cell receptor and death receptor apoptosis signaling in the D stump, and the gluconeogenesis/glycolysis and cytoskeletal motility signaling in the P stump, corroborated by activation of ERK, PI3K and JNK kinase pathways. As compared to the D segment, multiple additional pathways were more efficiently upregulated in the P stump, including the IL-6 and -17, MMP-9, calcium, activated agranulocyte, leukocyte rolling and glutathione-mediated detoxification signaling pathways. These data suggest that shortly after nerve injury, upregulation of S100A8/A9 is responsible for the expression and release of chemokines and cytokines by Schwann cells, necessary to generate the initial chemotactic gradient and guide the hematogenous immune cells into the injury site. Gene expression profiling of total RNAs extracted from injured and non-injured rat sciatic nerves, and primary rat Schwann cells stimulated with S100A8/A9 proteins
Project description:To shed light on the early immune response processes in severed peripheral nerves, we performed genome-wide transcriptional profiling and bioinformatics analyses of the proximal (P, regenerating) and distal (D, degenerating) nerve stumps on day 1 in the sciatic nerve axotomy model in rats. Multiple cell death-related pathways were activated in the degenerating D stump, whereas activation of the cytoskeletal motility and gluconeogenesis/glycolysis pathways was most prominent in the P stump of the axotomized nerve. Our bioinformatics analyses also identified the specific immunomodulatory genes of the chemokine, IL, TNF, MHC, immunoglobulin-binding Fc receptor, calcium-binding S100, matrix metalloproteinase, tissue inhibitor of metalloproteinase, and ion channel families affected in both the P and D segments. S100a8 and S100a9 were the top up-regulated genes in both the P and D segments. Stimulation of cultured Schwann cells using the purified S100A8/A9 heterodimer recapitulated activation of the myeloid cell and phagocyte chemotactic genes and pathways, which we initially observed in injured nerves. S100A8/A9 heterodimer injection into the intact nerve stimulated macrophage infiltration. We conclude that, following peripheral nerve injury, an immediate acute immune response occurs both distal and proximal to the lesion site and that the rapid transcriptional activation of the S100a8 and S100a9 genes results in S100A8/A9 hetero- and homodimers, which stimulate the release of chemokines and cytokines by activated Schwann cells and generate the initial chemotactic gradient that guides the transmigration of hematogenous immune cells into the injured nerve.
Project description:ChIP-seq of H3K4me3 in rat peripheral nerve was used to identify transcription start sites associated with Schwann cell-expressed genes. The analysis was performed in injured and control nerve to identify injury-responsive changes in Schwann cells. H3K4me3 ChIP samples were prepared from rat sciatic nerve at 1 day post-transection using both the distal stump of the injured nerve and the contralateral (sham) nerve.
Project description:BACKGROUND: Bacterial products add to mechanical ventilation in enhancing lung injury. The role of endogenous triggers of innate immunity herein is less well understood. S100A8/A9 proteins are released by phagocytes during inflammation. The present study investigates the role of S100A8/A9 proteins in ventilator-induced lung injury. METHODS: Pulmonary S100A8/A9 levels were measured in samples obtained from patients with and without lung injury. Furthermore, wild-type and S100A9 knock-out mice, naive and with lipopolysaccharide-induced injured lungs, were randomized to 5 hours of spontaneously breathing or mechanical ventilation with low or high tidal volume (VT). In addition, healthy spontaneously breathing and high VT ventilated mice received S100A8/A9, S100A8 or vehicle intratracheal. Furthermore, the role of Toll-like receptor 4 herein was investigated. RESULTS: S100A8/A9 protein levels were elevated in patients and mice with lung injury. S100A8/A9 levels synergistically increased upon the lipopolysaccharide/high VT MV double hit. Markers of alveolar barrier dysfunction, cytokine and chemokine levels, and histology scores were attenuated in S100A9 knockout mice undergoing the double-hit. Exogenous S100A8/A9 and S100A8 induced neutrophil influx in spontaneously breathing mice. In ventilated mice, these proteins clearly amplified inflammation: neutrophil influx, cytokine, and chemokine levels were increased compared to ventilated vehicle-treated mice. In contrast, administration of S100A8/A9 to ventilated Toll-like receptor 4 mutant mice did not augment inflammation. CONCLUSION: S100A8/A9 proteins increase during lung injury and contribute to inflammation induced by HVT MV combined with lipopolysaccharide. In the absence of lipopolysaccharide, high levels of extracellular S100A8/A9 still amplify ventilator-induced lung injury via Toll-like receptor 4.
Project description:The close homolog of L1 (CHL1) is a cell adhesion molecule involved in regulation of neuronal differentiation and survival, neurite outgrowth and axon guidance during development. In the mature nervous system, CHL1 regulates synaptic activity and plasticity. The aim of the present study was to evaluate the influence of CHL1 on peripheral nerve regeneration after trauma. Using the established model of mouse femoral nerve regeneration, CHL1 knock-out mice were investigated in comparison to the wild type littermates. First, non-injured mice of both genotypes were compared regarding the synaptic phenotypes in the corresponding spinal cord segment. While no differences in phenotypes were detectable in the femoral nerve, corresponding segments in the spinal cord were observed to differ in that inhibitory perisomatic innervation of motor neurons was increased in CHL1-deficient mice, and numbers of perisomatic cholinergic synapses on motor neuronal somata were reduced. Regarding the femoral nerve after injury, CHL1-deficient mice demonstrated preferential motor axon regrowth into the saphenous vs. quadriceps branch after nerve transection upstream of the nerve bifurcation by 8 weeks after transection, indicating decreased preferential motor re-innervation. Furthermore, in injured wild-type mice, enhanced CHL1 expression was observed in regenerating axons in the proximal nerve stump upstream of the bifurcation at days 1, 3, 5, 7 and 14, and in the distal stump at days 7 and 14 after injury, when compared to non-injured mice. Injury-related upregulation of CHL1 expression was more pronounced in axons than in Schwann cells. Despite a more pronounced capacity for preferential motor axon regrowth in wild-type vs. mutant mice, only a tendency for difference in recovery of motor functions was observed between genotypes, without statistical significance Taken together, these results indicate that CHL1 is involved in peripheral nerve regeneration, because it guides regrowing axons into the appropriate nerve branch and regulates synaptic coverage in the spinal cord.
Project description:Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylyl Cyclase Activating Peptide (PACAP) are regeneration-associated neuropeptides, which are up-regulated by neurons following peripheral nerve injury. So far, they have only been studied for their roles as autocrine signals for both neuronal survival and axon outgrowth during peripheral nerve regeneration. In this report, we examined VIP and PACAP's paracrine effects on Schwann cells and macrophages in the distal nerve stump during peripheral nerve regeneration. We show that VPAC1, VPAC2, and PAC1 are all up-regulated in the mouse distal nerve following peripheral nerve injury and are highly expressed in Schwann cells and macrophages within the distal sciatic nerve. We further investigated the effect of VIP and PACAP on cultured rat Schwann cells, and found that VIP and PACAP can not only promote myelin gene expression in Schwann cells but can also inhibit the release of pro-inflammatory cytokines by Schwann cells. Furthermore, we show that VIP and PACAP inhibit the release of pro-inflammatory cytokines and enhance anti-inflammatory cytokine expression in sciatic nerve explants. Our results provide evidence that VIP and PACAP could have important functions in the distal nerve stump following injury to promote remyelination and regulate the inflammatory response. Thus, VIP and PACAP receptors appear as important targets to promote peripheral nerve repair following injury.
Project description:Studies using bone marrow chimeric mice revealed that S100A8/A9 expression on myeloid cells is essential for development of colon tumors. Our results thus reveal a novel role for myeloid-derived S100A8/A9 in activating specific downstream genes associated with tumorigenesis and in promoting tumor growth and metastasis. Subconfluent cultures of MC38 cells were serum-starved for 16 hrs and activated with 10ug/mL S100A8/A9 for 6 hrs. Total RNA was extracted from unactivated or activated cells. 2 replicates each per stimulated cells, unstimulated cells, and control cells.
Project description:Injured peripheral nerves regenerate their lost axons but functional recovery in humans is frequently disappointing. This is so particularly when injuries require regeneration over long distances and/or over long time periods. Fat replacement of chronically denervated muscles, a commonly accepted explanation, does not account for poor functional recovery. Rather, the basis for the poor nerve regeneration is the transient expression of growth-associated genes that accounts for declining regenerative capacity of neurons and the regenerative support of Schwann cells over time. Brief low-frequency electrical stimulation accelerates motor and sensory axon outgrowth across injury sites that, even after delayed surgical repair of injured nerves in animal models and patients, enhances nerve regeneration and target reinnervation. The stimulation elevates neuronal cyclic adenosine monophosphate and, in turn, the expression of neurotrophic factors and other growth-associated genes, including cytoskeletal proteins. Electrical stimulation of denervated muscles immediately after nerve transection and surgical repair also accelerates muscle reinnervation but, at this time, how the daily requirement of long-duration electrical pulses can be delivered to muscles remains a practical issue prior to translation to patients. Finally, the technique of inserting autologous nerve grafts that bridge between a donor nerve and an adjacent recipient denervated nerve stump significantly improves nerve regeneration after delayed nerve repair, the donor nerves sustaining the capacity of the denervated Schwann cells to support nerve regeneration. These reviewed methods to promote nerve regeneration and, in turn, to enhance functional recovery after nerve injury and surgical repair are sufficiently promising for early translation to the clinic.
Project description:The striking PNS regenerative response to injury rests on the plasticity of adult Schwann cells and their ability to transit between differentiation states, a highly unusual feature in mammals. Using mice with inactivation of Schwann cell c-Jun, we show that the injury response involves c-Jun dependent natural reprograming of differentiated cells to generate a distinct Schwann cell state specialized to promote regeneration. Transected distal stumps of c-Jun mutants show 172 disregulated genes, resulting in abnormal expression of growth factors, adhesion molecules and cytoskeletal changes that lead to neuronal death, inhibition of axon growth and striking failures of functional repair after injury. These observations provide a molecular basis for understanding Schwann cell plasticity and nerve regeneration. They offer conclusive support for the notion that Schwann cells control repair in the PNS, using dedicated transcriptional controls to generate a distinct repair cell, a transition that shows similarities to transdifferentiation seen in other systems. Total RNA was purified from a 10mm segment of the distal stump and uninjured contralateral nerve from c-Jun mutants and control mice 7 days after nerve cut. For each condition (injured/uninjured) and genotype (control/ knock-out) 2 independent samples (replicates) were generated from pooled nerves of 4/6 mice resulting in a total of 8 samples: CTRL.cut.R1, CTRL.cut.R2, CTRL.uncut.R1, CTRL.uncut.R2, KO.cut.R1, KO.cut.R2, KO.uncut.R1,KO.uncut.R2.
Project description:The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors. Although we previously reported that Slit1-3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system, how this expression changes after injury has not been well studied. Herein, we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve. At 14 days after injury, quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1-3 and Robo1-2 in L4-5 spinal cord and dorsal root ganglia, as well as the sciatic nerve. Immunohistochemical analysis was performed to examine Slit1-3, Robo1-2, neurofilament heavy chain, F4/80, and vimentin in L4-5 spinal cord, L4 dorsal root ganglia, and the sciatic nerve. Co-expression of Slit1-3 and Robo1-2 in L4 dorsal root ganglia was detected by in situ hybridization. In addition, Slit1-3 and Robo1-2 protein expression in L4-5 spinal cord, L4 dorsal root ganglia, and sciatic nerve were detected by western blot assay. The results showed no significant changes of Slit1-3 or Robo1-2 mRNA expression in the spinal cord within 14 days after injury. In the dorsal root ganglion, Slit1-3 and Robo1-2 mRNA expression were initially downregulated within 4 days after injury; however, Robo1-2 mRNA expression returned to the control level, while Slit1-3 mRNA expression remained upregulated during regeneration from 4-14 days after injury. In the sciatic nerve, Slit1-3 and their receptors Robo1-2 were all expressed in the proximal nerve stump; however, Slit1, Slit2, and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury. Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury. Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge. Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury. Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration, and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury. This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No. 30/3203) on April 12, 2014.
Project description:The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury; however, the potential for full repair following a transection injury is much less. Currently, the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps, which prevent regenerating axons reaching the distal nerve. Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins, Slits, Ephrins and Semaphorins. Several recent studies have indicated key roles of Netrin1, Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury. Inside the nerve bridge, nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2. EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump. Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1; within Schwann cells, Robo1 expression is also Sox2-dependent. Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge. In addition to the Slit3/Robo1 signalling system, migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor. It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap. Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps. Therefore, understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.