IL-17 is a neuromodulator of Caenorhabditis elegans sensory responses.

Interleukin-17 (IL-17) is a major pro-inflammatory cytokine: it mediates responses to pathogens or tissue damage, and drives autoimmune diseases. Little is known about its role in the nervous system. Here we show that IL-17 has neuromodulator-like properties in Caenorhabditis elegans. IL-17 can act directly on neurons to alter their response properties and contribution to behaviour. Using unbiased genetic screens, we delineate an IL-17 signalling pathway and show that it acts in the RMG hub interneurons. Disrupting IL-17 signalling reduces RMG responsiveness to input from oxygen sensors, and renders sustained escape from 21% oxygen transient and contingent on additional stimuli. Over-activating IL-17 receptors abnormally heightens responses to 21% oxygen in RMG neurons and whole animals. IL-17 deficiency can be bypassed by optogenetic stimulation of RMG. Inducing IL-17 expression in adults can rescue mutant defects within 6 h. These findings reveal a non-immunological role of IL-17 modulating circuit function and behaviour.

Combining single-cell RNA-sequencing with a molecular atlas unveils new markers for Caenorhabditis elegans neuron classes.

Single-cell RNA-sequencing (scRNA-seq) of the Caenorhabditis elegans nervous system offers the unique opportunity to obtain a partial expression profile for each neuron within a known connectome. Building on recent scRNA-seq data and on a molecular atlas describing the expression pattern of ∼800 genes at the single cell resolution, we designed an iterative clustering analysis aiming to match each cell-cluster to the ∼100 anatomically defined neuron classes of C. elegans. This heuristic approach successfully assigned 97 of the 118 neuron classes to a cluster. Sixty two clusters were assigned to a single neuron class and 15 clusters grouped neuron classes sharing close molecular signatures. Pseudotime analysis revealed a maturation process occurring in some neurons (e.g. PDA) during the L2 stage. Based on the molecular profiles of all identified neurons, we predicted cell fate regulators and experimentally validated unc-86 for the normal differentiation of RMG neurons. Furthermore, we observed that different classes of genes functionally diversify sensory neurons, interneurons and motorneurons. Finally, we designed 15 new neuron class-specific promoters validated in vivo. Amongst them, 10 represent the only specific promoter reported to this day, expanding the list of neurons amenable to genetic manipulations.

Fractionation Coupled to Molecular Networking: Towards Identification of Anthelmintic Molecules in Terminalia leiocarpa (DC.) Baill.

Terminalia leiocarpa is a medicinal plant widely used in ethnoveterinary medicine to treat digestive parasitosis whose extracts were shown to be active against gastrointestinal nematodes of domestic ruminants. The objective of our study was to identify compounds responsible for this activity. Column fractionation was performed, and the activity of the fractions was assessed in vitro on Haemonchus contortus and Caenorhabditis elegans as well as their cytotoxicity on WI38 fibroblasts. Two fractions were the most active on both nematode models and less cytotoxic. LC-MS/MS analysis and manual dereplication coupled to molecular networking allowed identification of the main compounds: ellagic acid and derivatives, gallic acid, astragalin, rutin, quinic acid, and fructose. Other potentially identified compounds such as shikimic acid, 2,3-(S)-hexahydroxydiphenoyl-D-glucose or an isomer, quercetin-3-O-(6-O-galloyl)-ß-D-galactopyranoside or an isomer, and a trihydroxylated triterpenoid bearing a sugar as rosamultin are reported in this plant for the first time. Evaluation of the anthelmintic activity of the available major compounds showed that ellagic and gallic acids were the most effective in inhibiting the viability of C. elegans. Their quantification in fractions 8 and 9 indicated the presence of about 8.6 and 7.1 µg/mg ellagic acid and about 9.6 and 2.0 µg/mg gallic acid respectively. These concentrations are not sufficient to justify the activity observed. Ellagic acid derivatives and other compounds that were found to be positively correlated with the anthelmintic activity of the fractions may have additive or synergistic effects when combined, but other unidentified compounds could also be implicated in the observed activity.

Multidimensional phenotyping predicts lifespan and quantifies health in Caenorhabditis elegans.

Ageing affects a wide range of phenotypes at all scales, but an objective measure of ageing remains challenging, even in simple model organisms. To measure the ageing process, we characterized the sequence of alterations of multiple phenotypes at organismal scale. Hundreds of morphological, postural, and behavioral features were extracted from high-resolution videos. Out of the 1019 features extracted, 896 are ageing biomarkers, defined as those that show a significant correlation with relative age (age divided by lifespan). We used support vector regression to predict age, remaining life and lifespan of individual C. elegans. The quality of these predictions (age R2 = 0.79; remaining life R2 = 0.77; lifespan R2 = 0.72) increased with the number of features added to the model, supporting the use of multiple features to quantify ageing. We quantified the rate of ageing as how quickly animals moved through a phenotypic space; we quantified health decline as the slope of the declining predicted remaining life. In both ageing dimensions, we found that short lived-animals aged faster than long-lived animals. In our conditions, for isogenic wild-type worms, the health decline of the individuals was scaled to their lifespan without significant deviation from the average for short- or long-lived animals.

Genetic dissection of neuropeptide cell biology at high and low activity in a defined sensory neuron.

Neuropeptides are ubiquitous modulators of behavior and physiology. They are packaged in specialized secretory organelles called dense core vesicles (DCVs) that are released upon neural stimulation. Unlike synaptic vesicles, which can be recycled and refilled close to release sites, DCVs must be replenished by de novo synthesis in the cell body. Here, we dissect DCV cell biology in vivo in a Caenorhabditis elegans sensory neuron whose tonic activity we can control using a natural stimulus. We express fluorescently tagged neuropeptides in the neuron and define parameters that describe their subcellular distribution. We measure these parameters at high and low neural activity in 187 mutants defective in proteins implicated in membrane traffic, neuroendocrine secretion, and neuronal or synaptic activity. Using unsupervised hierarchical clustering methods, we analyze these data and identify 62 groups of genes with similar mutant phenotypes. We explore the function of a subset of these groups. We recapitulate many previous findings, validating our paradigm. We uncover a large battery of proteins involved in recycling DCV membrane proteins, something hitherto poorly explored. We show that the unfolded protein response promotes DCV production, which may contribute to intertissue communication of stress. We also find evidence that different mechanisms of priming and exocytosis may operate at high and low neural activity. Our work provides a defined framework to study DCV biology at different neural activity levels.

Neuropeptide-Driven Cross-Modal Plasticity following Sensory Loss in Caenorhabditis elegans.

Sensory loss induces cross-modal plasticity, often resulting in altered performance in remaining sensory modalities. Whereas much is known about the macroscopic mechanisms underlying cross-modal plasticity, only scant information exists about its cellular and molecular underpinnings. We found that Caenorhabditis elegans nematodes deprived of a sense of body touch exhibit various changes in behavior, associated with other unimpaired senses. We focused on one such behavioral alteration, enhanced odor sensation, and sought to reveal the neuronal and molecular mechanisms that translate mechanosensory loss into improved olfactory acuity. To this end, we analyzed in mechanosensory mutants food-dependent locomotion patterns that are associated with olfactory responses and found changes that are consistent with enhanced olfaction. The altered locomotion could be reversed in adults by optogenetic stimulation of the touch receptor (mechanosensory) neurons. Furthermore, we revealed that the enhanced odor response is related to a strengthening of inhibitory AWC→AIY synaptic transmission in the olfactory circuit. Consistently, inserting in this circuit an engineered electrical synapse that diminishes AWC inhibition of AIY counteracted the locomotion changes in touch-deficient mutants. We found that this cross-modal signaling between the mechanosensory and olfactory circuits is mediated by neuropeptides, one of which we identified as FLP-20. Our results indicate that under normal function, ongoing touch receptor neuron activation evokes FLP-20 release, suppressing synaptic communication and thus dampening odor sensation. In contrast, in the absence of mechanosensory input, FLP-20 signaling is reduced, synaptic suppression is released, and this enables enhanced olfactory acuity; these changes are long lasting and do not represent ongoing modulation, as revealed by optogenetic experiments. Our work adds to a growing literature on the roles of neuropeptides in cross-modal signaling, by showing how activity-dependent neuropeptide signaling leads to specific cross-modal plastic changes in neural circuit connectivity, enhancing sensory performance.

Pulp fibroblasts synthesize functional complement proteins involved in initiating dentin-pulp regeneration.

The complement system is an efficient plasma immune surveillance system that controls tissue injury and infection. Although the liver constitutes the primary circulating complement protein synthesis site, extrahepatic synthesis is known to optimize local tissue inflammatory reaction. Because dentin-pulp regeneration is known to be regulated locally, we investigated activation of the local complement system within the dental pulp and its role in initiating the regeneration process. Membrane attack complex (C5b-9) formation and Gram's staining revealed that complement activation is correlated with the presence of Gram-positive bacteria in carious human teeth. RT-PCR analysis demonstrated that cultured human pulp fibroblasts stimulated with lipoteichoic acid produce all the proteins required for efficient complement activation. This was demonstrated in vitro by C5b-9 formation and C5a active fragment production in the absence of plasma proteins. Finally, the dynamic migration assays performed in µ-Slide chemotaxis chambers and use of a C5aR-specific antagonist (W54011) demonstrated that the activation of complement proteins synthesized by pulp fibroblasts and the subsequent release of C5a specifically induced pulp progenitor cell recruitment. Our study reveals human pulp fibroblasts as the first nonimmune cell type capable of synthesizing all complement proteins. These fibroblasts cells contribute significantly to tissue regeneration by recruiting pulp progenitors via complement activation, which suggests to a potential therapeutic strategy of targeting pulp fibroblasts in dentin-pulp regeneration.

Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans.

Behaviours evolve by iterations of natural selection, but we have few insights into the molecular and neural mechanisms involved. Here we show that some Caenorhabditis elegans wild strains switch between two foraging behaviours in response to subtle changes in ambient oxygen. This finely tuned switch is conferred by a naturally variable hexacoordinated globin, GLB-5. GLB-5 acts with the atypical soluble guanylate cyclases, which are a different type of oxygen binding protein, to tune the dynamic range of oxygen-sensing neurons close to atmospheric (21%) concentrations. Calcium imaging indicates that one group of these neurons is activated when oxygen rises towards 21%, and is inhibited as oxygen drops below 21%. The soluble guanylate cyclase GCY-35 is required for high oxygen to activate the neurons; GLB-5 provides inhibitory input when oxygen decreases below 21%. Together, these oxygen binding proteins tune neuronal and behavioural responses to a narrow oxygen concentration range close to atmospheric levels. The effect of the glb-5 gene on oxygen sensing and foraging is modified by the naturally variable neuropeptide receptor npr-1 (refs 4, 5), providing insights into how polygenic variation reshapes neural circuit function.

Cilia-Derived Extracellular Vesicles in Caenorhabditis Elegans: In Vivo Imaging and Quantification of Extracellular Vesicle Release and Capture.

Caenorhabditis elegans is a microscopic model nematode characterized by body transparency and ease of genetic manipulation. Release of extracellular vesicles (EVs) is observed from different tissues; of particular interest are the EVs released by the cilia of sensory neurons. C. elegans ciliated sensory neurons produce EVs that are environmentally released and/or captured by neighboring glial cells. In this chapter, we describe a methodological approach to image the biogenesis, release, and capture of EVs by glial cells in anesthetized animals. This method will allow the experimenter to visualize and quantify the release of ciliary-derived EVs.

A lipid transfer protein ensures nematode cuticular impermeability.

The cuticle of C. elegans is impermeable to chemicals, toxins, and pathogens. However, increased permeability is a desirable phenotype because it facilitates chemical uptake. Surface lipids contribute to the permeability barrier. Here, we identify the lipid transfer protein GMAP-1 as a critical element setting the permeability of the C. elegans cuticle. A gmap-1 deletion mutant increases cuticular permeability to sodium azide, levamisole, Hoechst, and DiI. Expressing GMAP-1 in the hypodermis or transiently in the adults is sufficient to rescue this gmap-1 permeability phenotype. GMAP-1 protein is secreted from the hypodermis to the aqueous fluid filling the space between collagen fibers of the cuticle. In vitro, GMAP-1 protein binds phosphatidylserine and phosphatidylcholine while in vivo, GMAP-1 sets the surface lipid composition and organization. Altogether, our results suggest GMAP-1 secreted by hypodermis shuttles lipids to the surface to form the permeability barrier of C. elegans.

Ectocytosis prevents accumulation of ciliary cargo in C. elegans sensory neurons.

Cilia are sensory organelles protruding from cell surfaces. Release of extracellular vesicles (EVs) from cilia was previously observed in mammals, Chlamydomonas, and in male Caenorhabditis elegans. Using the EV marker TSP-6 (an ortholog of mammalian CD9) and other ciliary receptors, we show that EVs are formed from ciliated sensory neurons in C. elegans hermaphrodites. Release of EVs is observed from two ciliary locations: the cilia tip and/or periciliary membrane compartment (PCMC). Outward budding of EVs from the cilia tip leads to their release into the environment. EVs' budding from the PCMC is concomitantly phagocytosed by the associated glial cells. To maintain cilia composition, a tight regulation of cargo import and removal is achieved by the action of intra-flagellar transport (IFT). Unbalanced IFT due to cargo overexpression or mutations in the IFT machinery leads to local accumulation of ciliary proteins. Disposal of excess ciliary proteins via EVs reduces their local accumulation and exports them to the environment and/or to the glia associated to these ciliated neurons. We suggest that EV budding from cilia subcompartments acts as a safeguard mechanism to remove deleterious excess of ciliary material.

The prolactin-releasing peptide antagonizes the opioid system through its receptor GPR10.

Prolactin-releasing peptide (PrRP) and its receptor G protein-coupled receptor 10 (GPR10) are expressed in brain areas involved in the processing of nociceptive signals. We investigated the role of this new neuropeptidergic system in GPR10-knockout mice. These mice had higher nociceptive thresholds and stronger stress-induced analgesia than wild-type mice, differences that were suppressed by naloxone treatment. In addition, potentiation of morphine-induced antinociception and reduction of morphine tolerance were observed in mutants. Intracerebroventricular administration of PrRP in wild-type mice promoted hyperalgesia and reversed morphine-induced antinociception. PrRP administration had no effect on GPR10-mutant mice, showing that its effects are mediated by GPR10. Anti-opioid effects of neuropeptide FF were found to require a functional PrRP-GPR10 system. Finally, GPR10 deficiency enhanced the acquisition of morphine-induced conditioned place preference and decreased the severity of naloxone-precipitated morphine withdrawal syndrome. Altogether, our data identify the PrRP-GPR10 system as a new and potent negative modulator of the opioid system.

BioRoot RCS Extracts Modulate the Early Mechanisms of Periodontal Inflammation and Regeneration.

INTRODUCTION: The balance between periapical tissue inflammation and regeneration after the removal of necrotic/infected tissues is pivotal in determining the success of endodontic treatment. This study was designed to investigate the effect of silicate-based root canal sealer BioRoot RCS (BRCS; Septodont, Saint-Maur-des-Fossés, France) on modulating the inflammatory mechanisms and early steps of regeneration initiated by human periodontal ligament (PDL) fibroblasts. METHODS: Samples of BRCS and Pulp Canal Sealer (PCS; SybronEndo, Orange, CA) were incubated in culture medium to obtain material extracts. To simulate bacterial infection and endodontic sealer use, PDL fibroblasts were stimulated with lipopolysaccharides and cultured with material extracts. The secretion of proinflammatory cytokine (interleukin 6) and growth factor (transforming growth factor beta 1) were quantified by enzyme-linked immunosorbent assay. Inflammatory cell recruitment sequence was investigated using a human inflammatory monocytic cell line (THP-1) that can be activated into macrophage-like cells. The adhesion of THP-1 to endothelial cells (human umbilical vein endothelial cells) was studied using fluorescent THP-1, their migration using Boyden chambers, and their activation into macrophage-like cells using a cell adhesion assay. The proliferation of PDL fibroblasts was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas the migration of PDL stem cells was investigated using Boyden chambers after immunofluorescence and reverse transcription polymerase chain reaction characterization. RESULTS: Interleukin 6 secretion decreased with BRCS, whereas it increased with PCS. Transforming growth factor beta 1 secretion significantly increased only with BRCS. The material extracts did not affect THP-1 adhesion to human umbilical vein endothelial cells, but only BRCS inhibited their migration. Moreover, activation of THP-1 decreased with BRCS and to a lesser extent with PCS. Finally, BRCS increased PDL fibroblast proliferation without affecting PDL stem cell migration. By contrast, PCS decreased PDL fibroblast proliferation and PDL stem cell migration. CONCLUSIONS: This work shows that the endodontic sealers modulate the PDL inflammatory and regeneration potentials in vitro. It demonstrates that BRCS has anti-inflammatory effects and the potential to promote tissue regeneration.

Pulp capping materials modulate the balance between inflammation and regeneration.

The interrelations between inflammation and regeneration are of particular significance within the dental pulp tissue inextensible environment. Recent data have demonstrated the pulp capacity to respond to insults by initiating an inflammatory reaction and dentin pulp regeneration. Different study models have been developed in vitro and in vivo to investigate the initial steps of pulp inflammation and regeneration. These include endothelial cell interaction with inflammatory cells, stem cell interaction with pulp fibroblasts, migration chambers to study cell recruitment and entire human tooth culture model. Using these models, the pulp has been shown to possess an inherent anti-inflammatory potential and a high regeneration capacity in all teeth and at all ages. The same models were used to investigate the effects of tricalcium silicate-based pulp capping materials, which were found to modulate the pulp anti-inflammatory potential and regeneration capacity. Among these, resin-containing materials such as TheraCal® shift the pulp response towards the inflammatory reaction while altering the regeneration process. On the opposite, resin-free materials such as Biodentine™ have an anti-inflammatory potential and induce the pulp regeneration capacity. This knowledge contradicts the new tendency of developing resin-based calcium silicate hybrid materials for direct pulp capping. Additionally, it would allow investigating the modulatory effects of newly released pulp capping materials on the balance between tissue inflammation and regeneration. It would also set the basis for developing future capping materials targeting these processes.

Induction of specific cell responses to a Ca(3)SiO(5)-based posterior restorative material.

OBJECTIVES: A Ca(3)SiO(5)-based cement has been developed to circumvent the shortcomings of traditional filling materials. The purpose of this work was to evaluate its genotoxicity, cytotoxicity and effects on the target cells' specific functions. METHODS: Ames' test was applied on four Salmonella typhimurium strains. The micronuclei test was studied on human lymphocytes. The cytotoxicity (MTT test), the Comet assay and the effects on the specific functions by immunohistochemistry were performed on human pulp fibroblasts. RESULTS: Ames' test did not show any evidence of mutagenicity. The incidence of lymphocytes with micronuclei and the percentage of tail DNA in the Comet assay were similar to the negative control. The percentage of cell mortality with the new cement as performed with the MTT test was similar to that of biocompatible materials such as mineral trioxide aggregate (MTA) and was less than that obtained with Dycal. The new material does not affect the target cells' specific functions such as mineralization, as well as expression of collagen I, dentin sialoprotein and Nestin. SIGNIFICANCE: The new cement is biocompatible and does not affect the specific functions of target cells. It can be used safely in the clinic as a single bulk restorative material without any conditioning treatment. It can be used as a potential alternative to traditionally used posterior restorative materials.

Tricalcium Silicate Capping Materials Modulate Pulp Healing and Inflammatory Activity In Vitro.

INTRODUCTION: On stimulation by lipoteichoic acid or by a physical injury, fibroblasts have been shown to play a major role in the initiation of the pulp inflammatory reaction and healing through secretion of complement proteins and growth factors. The application of direct pulp-capping materials on these cells may interfere with the inflammatory and the healing processes within the pulp's inextensible environment. This work was designed to study in vitro the effects of silicate-based materials on pulp fibroblast modulation of the initial steps of pulp inflammation and healing. METHODS: The effects of Biodentine, TheraCal, and Xeno III eluates were studied on lipoteichoic acid-stimulated and physically injured fibroblasts. Cytokine secretion (interleukin 6, vascular endothelial growth factor, fibroblast growth factor-2, and transforming growth factor-ß1) was quantified by enzyme-linked immunosorbent assay. Inflammatory THP-1 adhesion to endothelial cells and their migration and activation were studied in vitro. Human pulp fibroblast proliferation was investigated with the MTT test, and their migration to the injury site was studied with the scratch healing assay. RESULTS: Interleukin 6 and vascular endothelial growth factor secretion increased with all materials but to a lesser extent with Biodentine. Fibroblast growth factor-2 and transforming growth factor-ß1 secretion was significantly higher with Biodentine than with all other materials. THP-1 cell adhesion to endothelial cells and their activation were reduced by Biodentine and TheraCal. However, their migration decreased only with Biodentine. Fibroblast proliferation significantly increased with Biodentine but significantly decreased with Xeno III after day 6. Finally, only Biodentine induced fibroblast migration to the injury site in the scratch assay. CONCLUSIONS: These results confirm that pulp-capping materials affect the early steps of pulp inflammation and healing. They show that Biodentine had the highest pulp healing and anti-inflammatory potential when compared with the resin-containing materials. This highlights the interest of the material choice for direct pulp-capping.

Light-cured Tricalcium Silicate Toxicity to the Dental Pulp.

INTRODUCTION: Numerous studies reported dentin bridge formation after pulp capping with tricalcium silicates. By contrast, pulp capping with resins leads to pulp toxicity and inflammation. Hybrid materials made up of tricalcium silicates and resins have also been developed to be used in direct pulp capping. This work was designed to study the consequences of adding resins to tricalcium silicates by investigating TheraCal (BISCO, Lançon De Provence, France) and Biodentine (Septodont, Saint Maur des Fosses, France) interactions with the dental pulp. METHODS: Media conditioned with the biomaterials were used to analyze pulp fibroblast proliferation using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test and proinflammatory cytokine interleukin 8 (IL-8) secretion using the enzyme-linked immunosorbent assay. The effects of conditioned media on dentin sialoprotein (DSP) and nestin expression by dental pulp stem cells (DPSCs) were investigated by immunofluorescence. The materials' interactions with the vital pulp were investigated using the entire tooth culture model. RESULTS: TheraCal-conditioned media significantly decreased pulp fibroblast proliferation, whereas no effect was observed with Biodentine. When DPSCs were cultured with Biodentine-conditioned media, immunofluorescence showed an increased expression of DSP and nestin. This expression was lower with TheraCal, which significantly induced proinflammatory IL-8 release both in cultured fibroblasts and entire tooth cultures. This IL-8 secretion increase was not observed with Biodentine. Entire tooth culture histology showed a higher mineralization with Biodentine, whereas significant tissue disorganization was observed with TheraCal. CONCLUSIONS: Within the limits of these preclinical results, resin-containing TheraCal cannot be recommended for direct pulp capping.

Characterization and angiogenic potential of xenogeneic bone grafting materials: Role of periodontal ligament cells.

Adequate revascularization is a prerequisite for successful healing of periodontal bone defects. This study characterized three different xenogeneic bone grafting materials: Gen-Os of equine and porcine origins, and anorganic Bio-Oss. We also investigated their angiogenic potential. All materials were composed of poorly crystalline calcium oxide phosphate, with Bio-Oss exhibiting a carbonated phase and larger particle size and both Gen-Os showing the presence of collagen. Both Gen-Os materials significantly enhanced vascular endothelial growth factor (VEGF) secretion by PDL cells. A significant increase in endothelial cell proliferation was observed in cultures with both Gen-Os conditioned media, but not with that of Bio-Oss. Finally, angiogenesis was stimulated by both Gen-Os conditioned media as demonstrated by an increased formation of capillary-like structures. Taken together, these findings indicate an enhanced angiogenic potential of both Gen-Os bone grafting materials when applied on PDL cells, most likely by increasing VEGF production.

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Complement C3a Mobilizes Dental Pulp Stem Cells and Specifically Guides Pulp Fibroblast Recruitment.

INTRODUCTION: Complement activation is considered a major mechanism in innate immunity. Although it is mainly involved in initiating inflammation, recent data reported its involvement in other processes such as tissue regeneration. In the dental pulp, complement C5a fragment has been shown to be involved in the recruitment of dental pulp stem cells (DPSCs). This study sought to investigate the possible role of C3a, another complement fragment, in the early steps of dentin-pulp regeneration. METHODS: Expression of C3a receptor (C3aR) was investigated by immunofluorescence and reverse transcriptase polymerase chain reaction on cultured pulp fibroblasts, STRO-1-sorted DPSCs, as well as on human tooth sections in vivo. The effect of C3a on proliferation of both DPSCs and pulp fibroblasts was investigated by MTT assay. Cell migration under a C3a gradient was investigated by using microfluidic chemotaxis chambers. RESULTS: C3aR was expressed in vivo as well as in cultured pulp fibroblasts co-expressing fibroblast surface protein and in DPSCs co-expressing STRO-1. Addition of recombinant C3a induced a significant proliferation of both cell types. When subjected to a C3a gradient, DPSCs were mobilized but not specifically recruited, whereas pulp fibroblasts were specifically recruited following a C3a gradient. CONCLUSIONS: These results provide the first demonstration of C3aR expression in the dental pulp and demonstrate that C3a is involved in increasing DPSCs and fibroblast proliferation, in mobilizing DPSCs, and in specifically guiding fibroblast recruitment. This provides an additional link to the tight correlation between inflammation and tissue regeneration.