Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion.

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

Therapeutic anti-glioma effect of the combined action of PCSK inhibitor with the anti-tumoral factors secreted by Poly (I:C)-stimulated macrophages.

Macrophages plasticity is a key feature in cancer progression. Neoplastic cells can alter their immune functions and orient them into a pro-tumoral phenotype. In this context, we developed a new therapeutic strategy to switch macrophages phenotype and reactivate their anti-tumoral functions. We showed a dual activity of a proprotein convertases inhibitor as anti-glioma drug and anti-tumoral macrophages' reactivation drug. Proprotein convertases are proteases that cleave proteins into functional proteins. Several of their substrates are involved in tumorigenesis and immunosuppression. We combine here proprotein convertases inhibitor with Poly (I:C), a TLR3 ligand, to increase the anti-tumoral activity of macrophages. With mass spectrometry-based proteomics, system biology, combined with biological assays, we established that a stimulation of macrophages with Poly (I:C) increased their secretion of pro-inflammatory cytokines and anti-tumoral factors. 3D invasion assay showed the efficacy of these anti-tumoral factors against mixed glioma cells and macrophages spheroids. Besides, immunofluorescence and proliferation assays showed an additive effect of the proprotein convertases inhibitor and the anti-tumoral factors secreted by Poly (I:C)-treated macrophages on both anti-glioma activity and macrophages anti-tumoral orientation directly in tumor microenvironment, leading to an innovative glioma therapy.

The Role of Proprotein Convertases in the Regulation of the Function of Immune Cells in the Oncoimmune Response.

Proprotein convertases (PC) are a family of 9 serine proteases involved in the processing of cellular pro-proteins. They trigger the activation, inactivation or functional changes of many hormones, neuropeptides, growth factors and receptors. Therefore, these enzymes are essential for cellular homeostasis in health and disease. Nine PC subtilisin/kexin genes (PCSK1 to PCSK9) encoding for PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P and PCSK9 are known. The expression of PC1/3, PC2, PC5/6, Furin and PC7 in lymphoid organs such as lymph nodes, thymus and spleen has suggested a role for these enzymes in immunity. In fact, knock-out of Furin in T cells was associated with high secretion of pro-inflammatory cytokines and autoantibody production in mice. This suggested a key role for this enzyme in immune tolerance. Moreover, Furin through its proteolytic activity, regulates the suppressive functions of Treg and thus prevents chronic inflammation and autoimmune diseases. In macrophages, Furin is also involved in the regulation of their inflammatory phenotype. Similarly, PC1/3 inhibition combined with TLR4 stimulation triggers the activation of the NF-κB signaling pathway with an increased secretion of pro-inflammatory cytokines. Factors secreted by PC1/3 KD macrophages stimulated with LPS exert a chemoattractive effect on naive auxiliary T lymphocytes (Th0) and anti-tumoral activities. The link between TLR and PCs is thus very important in inflammatory response regulation. Furin regulates TL7 and TLR8 processing and trafficking whereas PC1/3 controls TLR4 and TLR9 trafficking. Since PC1/3 and Furin are key regulators of both the innate and adaptive immune responses their inhibition may play a major role in oncoimmune therapy. The role of PCs in the oncoimmune response and therapeutic strategies based on PCs inhibition are proposed in the present review.

Epigenetic Studies Revealed a Ghost Proteome in PC1/3 KD Macrophages under Antitumoral Resistance Induced by IL-10.

Our previous investigation on macrophages has allowed us to show that the inhibition of the enzyme proprotein convertase (PC1/3) controls the activation of macrophages. We demonstrated that PC1/3 knockdown (KD) in macrophages exhibits an increased secretion of proinflammatory and antitumoral factors. In this biological context, we assessed the presence of histone modifications and the presence and contribution of a "ghost proteome" in these macrophages. We identified a set of alternative proteins (AltProts) that have a key role in the regulation of various signaling pathways. In this study, to further investigate the underlying mechanisms involved in the resistance of PC1/3-KD macrophages to anti-inflammatory stimuli, we have conducted a proteomic system biology study to assess the epigenome variation, focusing on histone modifications. Results from our study have indicated the presence of significant variations in histone modifications along with the identification of 28 AltProts, which can be correlated with antitumoral resistance under IL-10 stimulation. These findings highlight a key role of altered epigenome histone modifications in driving resistance and indicate that like the reference proteins, AltProts can have a major impact in the field of epigenetics and regulation of gene expression, as shown in our results.

The Role of a Proprotein Convertase Inhibitor in Reactivation of Tumor-Associated Macrophages and Inhibition of Glioma Growth.

Tumors are characterized by the presence of malignant and non-malignant cells, such as immune cells including macrophages, which are preponderant. Macrophages impact the efficacy of chemotherapy and may lead to drug resistance. In this context and based on our previous work, we investigated the ability to reactivate macrophages by using a proprotein convertases inhibitor. Proprotein convertases process immature proteins into functional proteins, with several of them having a role in immune cell activation and tumorigenesis. Macrophages were treated with a peptidomimetic inhibitor targeting furin, PC1/3, PC4, PACE4, and PC5/6. Their anti-glioma activity was analyzed by mass spectrometry-based proteomics and viability assays in 2D and 3D in vitro cultures. Comparison with temozolomide, the drug used for glioma therapy, established that the inhibitor was more efficient for the reduction of cancer cell density. The inhibitor was also able to reactivate macrophages through the secretion of several immune factors with antitumor properties. Moreover, two proteins considered as good glioma patient survival indicators were also identified in 3D cultures treated with the inhibitor. Finally, we established that the proprotein convertases inhibitor has a dual role as an anti-glioma drug and anti-tumoral macrophage reactivation drug. This strategy could be used together with chemotherapy to increase therapy efficacy in glioma.

PC1/3 KD Macrophages Exhibit Resistance to the Inhibitory Effect of IL-10 and a Higher TLR4 Activation Rate, Leading to an Anti-Tumoral Phenotype.

During tumorigenesis, macrophages are recruited by tumors and orientated towards a pro-tumoral phenotype. One of the main anti-tumoral immunotherapy consists of their re-polarization in an anti-tumoral phenotype. We have demonstrated that the inhibition of proprotein convertase 1/3 combined with TLR4 activation in macrophages is a promising strategy. These macrophages display pro-inflammatory and anti-tumoral phenotypes. A hallmark is a stronger activation of the pro-inflammatory NFKB pathway. We believe that this can be explained by a modification of TLR4 expression at the cell surface or MYD88 cleavage since it exhibits a potential cleavage site for proprotein convertases. We tested these hypotheses through immunofluorescence and Western blot experiments. A proteomics study was also performed to test the sensitivity of these macrophages to IL-10. We demonstrated that these macrophages treated with LPS showed a quicker re-expression of TLR4 at the cell surface. The level of MYD88 was also higher when TLR4 was internalized. Moreover, these macrophages were resistant to the pro-tumoral effect of IL-10 and still produced pro-inflammatory factors. This established that the sensitivity to anti-inflammatory molecules and the length of TLR4 desensitization were reduced in these macrophages. Therefore, during antitumoral immunotherapy, a repeated stimulation of TLR4 may reactivate PC1/3 inhibited macrophages even in an anti-inflammatory environment.

3D MALDI mass spectrometry imaging reveals specific localization of long-chain acylcarnitines within a 10-day time window of spinal cord injury.

We report, for the first time, the detection and specific localization of long-chain acylcarnitines (LC ACs) along the lesion margins in an experimental model of spinal cord injury (SCI) using 3D mass spectrometry imaging (MSI). Acylcarnitines palmitoylcarnitine (AC(16:0)), palmitoleoylcarnitine (AC(16:1)), elaidic carnitine (AC(18:1)) and tetradecanoylcarnitine (AC(14:1)) were detected as early as 3 days post injury, and were present along the lesion margins 7 and 10 days after SCI induced by balloon compression technique in the rat. 3D MSI revealed the heterogeneous distribution of these lipids across the injured spinal cord, appearing well-defined at the lesion margins rostral to the lesion center, and becoming widespread and less confined to the margins at the region located caudally. The assigned acylcarnitines co-localize with resident microglia/macrophages detected along the lesion margins by immunofluorescence. Given the reported pro-inflammatory role of these acylcarnitines, their specific spatial localization along the lesion margin could hint at their potential pathophysiological roles in the progression of SCI.

Deciphering molecular consequences of the proprotein convertase 1/3 inhibition in macrophages for application in anti-tumour immunotherapy.

During tumour development, macrophages are recruited to the tumour site and orientated towards an anti-inflammatory phenotype. Due to their immunosuppressive function, tumour associated macrophages (TAMs) are recognized as major components in tumour progression. Changing these macrophages to a pro-inflammatory phenotype is thus extensively studied as a potential means for developing novel anti-tumour therapy. In this context, we found that the Proprotein convertase 1/3 (PC1/3) is a relevant target. Proteomic analysis reveals that PC1/3 knockdown (KD) macrophages present all the characteristic of activated pro-inflammatory macrophages. Moreover, in PC1/3 KD macrophages, TLR4 and TLR9 signaling pathways can be enhanced leading to the secretion of pro-inflammatory factors and anti-tumour factors. To develop an efficient anti-tumour immunotherapy, we may (i) target TAMs directly inside the tumour site for PC1/3 inhibition and TLR activation and used them as "Trojan macrophages" or (ii) directly take advantage of PC1/3 inhibited macrophages and use them as "drone macrophages" by activating them "at distance" with a TLR ligand. Therefore, PC1/3 inhibited macrophages constitute an innovative cell therapy to treat tumours efficiently.

Paclitaxel Treatment and Proprotein Convertase 1/3 (PC1/3) Knockdown in Macrophages is a Promising Antiglioma Strategy as Revealed by Proteomics and Cytotoxicity Studies.

High grade gliomas are the most common brain tumors in adult. These tumors are characterized by a high infiltration in microglial cells and macrophages. The immunosuppressive tumor environment is known to orient immune cells toward a pro-tumoral and anti-inflammatory phenotype. Therefore, the current challenge for cancer therapy is to find a way to reorient macrophages toward an antitumoral phenotype. Previously, we demonstrated that macrophages secreted antitumoral factors when they were invalidated for the proprotein converstase 1/3 (PC1/3) and treated with LPS. However, achieving an activation of macrophages via LPS/TLR4/Myd88-dependent pathway appears yet unfeasible in cancer patients. On the contrary, the antitumor drug Paclitaxel is also known to activate the TLR4 MyD88-dependent signaling pathway and mimics LPS action. Therefore, we evaluated if PC1/3 knock-down (KD) macrophages could be activated by Paclitaxel and efficient against glioma. We report here that such a treatment of PC1/3 KD macrophages drove to the overexpression of proteins mainly involved in cytoskeleton rearrangement. In support of this finding, we found that these cells exhibited a Ca2+ increase after Paclitaxel treatment. This is indicative of a possible depolymerization of microtubules and may therefore reflect an activation of inflammatory pathways in macrophages. In such a way, we found that PC1/3 KD macrophages displayed a repression of the anti-inflammatory pathway STAT3 and secreted more pro-inflammatory cytokines. Extracellular vesicles isolated from these PC1/3 KD cells inhibited glioma growth. Finally, the supernatant collected from the coculture between glioma cells and PC1/3 KD macrophages contained more antitumoral factors. These findings unravel the potential value of a new therapeutic strategy combining Paclitaxel and PC1/3 inhibition to switch macrophages toward an antitumoral immunophenotype.

Proprotein convertase 1/3 inhibited macrophages: A novel therapeutic based on drone macrophages.

We demonstrated here thanks to proteomic, that proprotein convertase 1/3 knockdown macrophages present all the characteristic of activated pro-inflammatory macrophages. TLR4 and TLR9 signaling pathways can be enhanced leading to the secretion of pro-inflammatory factors and antitumor factors. We can control their activation by controlling one enzyme, PC1/3. In a tumor context, PC1/3 inhibition in macrophages may reactivate them and lead to a cytokine storm after stimulation "at distance" with a TLR ligand. Therefore, we name these proprotein convertase inhibited macrophages the "drone macrophages". They constitute an innovative cell therapy to treat efficiently tumors.

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study.

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.

Characterization of GnRH-related peptides from the Pacific oyster Crassostrea gigas.

Gonadotropin-releasing hormone (GnRH), a key neuropeptide regulating reproduction in vertebrates has now been characterized in a number of non-vertebrate species. Despite the demonstration of its ancestral origin, the structure and the function of this family of peptides remain poorly known in species as distant as lophotrochozoans. In this study, two GnRH-related peptides (Cg-GnRH-a and CgGnRH-G) were characterized by mass spectrometry from extracts of the visceral ganglia of the Pacific oyster Crassostrea gigas. These peptides showed a high degree of sequence identity with GnRHs of other mollusks and annelids and to a lesser extent with those of vertebrates or with AKH and corazonins of insects. Both the mature peptides and the transcript encoding the precursor protein were exclusively expressed in the visceral ganglia. Significant differences in transcriptional activity of Cg-GnRH encoding gene were recorded in the ganglia along the reproductive cycle and according to trophic conditions with a higher level in fed animals compared to starved animals. This suggests the involvement of Cg-GnRHs as synchronizers of nutritional status with energy requirements during reproduction in oyster. Evidence for a role of Cg-GnRHs as neuroregulators and as neuroendocrine factors in bivalve is discussed.