A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis

A new formulation of poly(MAOTIB) nanoparticles as an efficient contrast agent for in vivo X-ray imaging

Polymeric nanoparticles (PNPs) are gaining increasing importance as nanocarriers or contrasting material for preclinical diagnosis by micro-CT scanner. Here, we investigated a straightforward approach to produce a biocompatible, radiopaque, and stable polymer-based nanoparticle contrast agent, which was evaluated on mice. To this end, we used a nanoprecipitation dropping technique to obtain PEGylated PNPs from a preformed iodinated homopolymer, poly(MAOTIB), synthesized by radical polymerization of 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) monomer (MAOTIB). The process developed allows an accurate control of the nanoparticle properties (mean size can range from 140 nm to 200 nm, tuned according to the formulation parameters) along with unprecedented important X-ray attenuation properties (concentration of iodine around 59 mg I/mL) compatible with a follow-up in vivo study. Routine characterizations such as FTIR, DSC, GPC, TGA, 1H and 13C NMR, and finally SEM were accomplished to obtain the main properties of the optimal contrast agent. Owing to excellent colloidal stability against physiological conditions evaluated in the presence of fetal bovine serum, the selected PNPs suspension was administered to mice. Monitoring and quantification by micro-CT showed that iodinated PNPs are endowed strong X-ray attenuation capacity toward blood pool and underwent a rapid and passive accumulation in the liver and spleen

Innervation of bioengineered teeth implanted in Nude mice.

<p>Bioengineered teeth germs were co-implanted with trigeminal ganglia in adult Nude mice <b>(A–J)</b> for 1 <b>(A–C)</b> or 2 weeks <b>(D–J)</b>. Nerve fibers and blood vessels in dental pulp and peridental tissues of bioengineered teeth were analysed immunohistochemically by using specific antibodies for peripherin and CD31. Blood vessels were present in peridental tissues and could enter in the dental pulp and reach odontoblasts already after 1 week of implantation <b>(A–C)</b>. The staining for peripherin showed that nerve fibers entered in the dental pulp <b>(A, D)</b> and extend in the pulp <b>(B, E)</b> after 1 <b>(A–C)</b> and 2 <b>(D–J)</b> weeks. After 1 week of implantation, nerve fibers did not reach the odontoblasts <b>(C)</b>. This was achieved only after 2 weeks of implantation <b>(F, G, I, J)</b>. Double staining for peripherin <b>(G–I)</b>, CD31 <b>(G)</b>, CD34 <b>(H)</b> and CD146 <b>(I)</b> showed associations between nerve fibers and blood vessels in the dental pulp <b>(H)</b> and subodontoblastic layer <b>(G, I)</b>. After 2 weeks of implantation, nerve fibers, visualized by anti-peripherin antibody, had reached the odontoblast (positive for nestin) layer <b>(J)</b>. Am, ameloblasts; D, dentin; DP, dental pulp; E, enamel; Od, odontoblasts; PDM, peridental mesenchyme; TG, trigeminal ganglia.</p

Transmission electronic microscopy of bioengineered teeth implanted for 2 weeks in cyclosporin A-treated ICR mice.

<p>Odontoblasts, dentinogenesis <b>(A–D, G)</b> and ameloblasts-enamel <b>(E, F, H)</b> in bioengineered teeth implanted for 2 weeks in CsA-treated ICR mice were analysed by transmission electron microscopy. Elongated odontoblasts showed a polarized position of the nucleus <b>(A)</b>. A desmosome was observed between two odontoblasts <b>(B: arrows and insert)</b>. In the cytoplasm of odontoblasts, the rough endoplasmic reticulum, mitochondria and secretory vesicles <b>(arrowheads)</b> were present in the supra-nuclear area <b>(C)</b>. The insert in <b>C</b> showed a zonula adherens between two odontoblasts. Odontoblast processes were surrounded by collagen fibers from predentin <b>(D)</b>. Ameloblasts were elongated and their nuclei were distant from the secretory pole <b>(E)</b>, which contained numerous vesicles <b>(F, arrowheads and insert)</b>. The junction between predentin and mineralized dentin was clearly visible <b>(G)</b>. The insert in <b>G</b> showed the typical periodic striation of collagen fibers. The dental-enamel junction showed typical organization of enamel <b>(E and insert)</b> and dentin <b>(H)</b>. Am, ameloblast; D, dentin; DEJ, dentin-enamel junction; E, enamel; Od, odontoblast; m, mitochondria; N, nucleus; OP, odontoblast processes; Pd, predentin; RER, rough endoplasmic reticulum.</p

Innervation of bioengineered teeth implanted in cyclosporin A-treated ICR mice by transmission electron microscopy.

<p>Transmission electron microscopy (TEM) of trigeminal ganglia <b>(A)</b> showed the presence of myelinated and unmyelinated axons surrounded by Schwann cells <b>(A)</b>. TEM of dental pulp of epithelial and mesenchymal cell-cell re-associations co-implanted for 2 weeks with trigeminal ganglia in CsA-treated ICR mice showed the presence of unmyelinated axons <b>(B)</b>. These axons were surrounded by a Schwann cell and located near fibroblasts, which secreted collagen <b>(B)</b>. In <b>C</b>, one unmyelinated axon was surrounded by a Schwann cell with a developed rough endoplasmic reticulum. Neurofilaments <b>(D, E)</b>, numerous secretory vesicles <b>(arrowheads in F and insert)</b> and mitochondria <b>(F)</b> were present in the axons. A typical structure of a pre-synapse with numerous mitochondria and synaptic vesicles <b>(arrowheads)</b> was observed <b>(F)</b>. Thickening of the membrane suggested presence of synaptic contacts <b>(arrows in E and F)</b>. Ax, myelinated axon; DP, dental pulp; F, fibroblasts; m, mitochondria; N, nucleus; NF, neurofilaments; My, myelin; RER, rough endoplasmic reticulum; SC, Schwann cells; *, unmyelinated axon.</p

Protocol for tooth organ engineering.

<p>The mandibular first molars were dissected from ICR mouse embryos at embryonic day (ED) 14 (cap stage) <b>(A)</b>. Then, the dental epithelium <b>(B)</b> and ecto-mesenchyme <b>(C)</b> were separated by using a mixture of 0.25% trypsin and 1.2 U/mL dispase in DMEM-F12 (preheated to 37°C) at room temperature during 15 min. Each tissue was dissociated into single cells, which were then re-associated <b>(D)</b> and grown on semi-solid cultured medium <b>(E)</b>. After 7 days <i>in vitro</i>, each re-association was co-cultured overnight with trigeminal ganglia from ICR newborn mice <b>(F)</b>. The eighth day <b>(G)</b>, bioengineered tooth unit and trigeminal ganglia were co-implanted between skin and muscles behind the ears in adult ICR mice (mice a), CsA-treated ICR mice (mice b) and Nude mice (mice c) for 1 week or 2 weeks <b>(H)</b>. BT, bioengineered tooth; TG, trigeminal ganglia.</p

Innervation of bioengineered teeth implanted in ICR mice.

<p>Bioengineered teeth germs were co-implanted with trigeminal ganglia in adult ICR mice <b>(A–G)</b> for 1 <b>(A–C)</b> or 2 weeks <b>(D–G).</b> Nerve fibers and blood vessels in dental pulp and peridental tissues of bioengineered tooth were analysed immunohistochemically by using specific antibodies for peripherin (red) and CD31 (green). Blood vessels were present in peridental tissues and could enter in the dental pulp and reach odontoblasts already after 1 week of implantation <b>(A–C)</b>. Nerve fibers were detected in peridental tissues, in peridendal mesenchyme <b>(F)</b> and dental pulp but never in the dental pulp after 1 week <b>(A–C)</b> or even 2 weeks <b>(D–G)</b> of implantation. D, dentin; DP, dental pulp; E, enamel; Od, odontoblasts; PDM, peridental mesenchyme; TG, trigeminal ganglia.</p

Histology of bioengineered teeth implanted for 2 weeks in cyclosporin A-treated ICR mice.

<p>Bioengineered teeth implanted for 2 weeks under the skin in CsA-treated ICR mice were analysed by histology. Crown development was identical to that observed for Nude mice <b>(A)</b>. Blood vessels could enter in the dental pulp <b>(A)</b>, migrated in the pulp <b>(A)</b> and reached odontoblasts <b>(B)</b>. Odontoblasts were elongated and polarized by the position of the nucleus, opposite to the secretory pole <b>(B)</b>. Thus the secretion of predentin/dentin was polarized <b>(B)</b>. As well as to Nude mice, ameloblasts of bioengineered teeth implanted in CsA-treated ICR mice, were elongated, polarized and secreted enamel <b>(C)</b>. In contact with ameloblasts stratum intermedium could be observed <b>(D)</b>. In the predentin/dentin, dentinal tubules were present and reached the dentin-enamel junction <b>(E)</b>. After 2 weeks of implantation, root was developed <b>(F)</b> and newly formed bone was present in dental mesenchyme <b>(G)</b> as well as in Nude mice. In contact with the external surface of dentin, cementoblasts were observed <b>(I)</b>. These cells were functional and deposited cementum <b>(H)</b>. Periodontal ligament was attached to the root by cementum and extended until reaching newly formed bone <b>(G, H)</b>. Am, ameloblast; B, bone; Bv, blood vessel; Cb, cementoblast; Ce, cementum; D, dentin; DEJ, dentin-enamel junction; DP, dental pulp; DT, dentinal tubule; E, enamel; Od, odontoblast; Pd, predentin; PDL, periodontal ligament; SI, stratum intermedium.</p

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

International audiencePhagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis

Silver Nanoparticle‐Decorated Reduced Graphene Oxide Nanomaterials Exert Membrane Stress and Induce Immune Response to Inhibit the Early Phase of HIV‐1 Infection

Abstract Graphene‐based 2D nanomaterials exhibit unique physicochemical, electric, and optical properties that facilitate applications in a wide range of fields including material science, electronics, and biotechnology. Recent studies have shown that graphene oxide (GO) and reduced graphene oxide (rGO) exhibit antimicrobial effects on bacteria and viruses. While the bactericidal activity of graphene‐based nanomaterials is related to mechanical and oxidative damage to bacterial membranes, their antiviral activity has been less explored. Currently available experimental data are limited and suggest mechanical disruption of viral particles prior to infection. In this study, the antiviral properties of reduced GO‐based nanocomposites decorated with Ag nanoparticles (rGO‐Ag) are evidenced against human immunodeficiency virus‐1 pseudovirus used as an enveloped virus model. By combining biochemical and original single virus imaging approaches, it is shown that rGO‐Ag induces peroxidation of pseudoviral lipid membrane and that consequent alteration of membrane properties leads to a reduction in cell entry. In addition, rGO‐Ag is found to be efficiently internalized in the host cell leading to the elevated expression of pro‐inflammatory cytokines. Altogether, the presented results shed new light on the mechanisms of rGO‐Ag antiviral properties and confirm the high potential of graphene derivatives as an antimicrobial material for biomedical applications