The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection.

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.

Fetal Muse-based therapy prevents lethal radio-induced gastrointestinal syndrome by intestinal regeneration.

BACKGROUND: Human multilineage-differentiating stress enduring (Muse) cells are nontumorigenic endogenous pluripotent-like stem cells that can be easily obtained from various adult or fetal tissues. Regenerative effects of Muse cells have been shown in some disease models. Muse cells specifically home in damaged tissues where they exert pleiotropic effects. Exposition of the small intestine to high doses of irradiation (IR) delivered after radiotherapy or nuclear accident results in a lethal gastrointestinal syndrome (GIS) characterized by acute loss of intestinal stem cells, impaired epithelial regeneration and subsequent loss of the mucosal barrier resulting in sepsis and death. To date, there is no effective medical treatment for GIS. Here, we investigate whether Muse cells can prevent lethal GIS and study how they act on intestinal stem cell microenvironment to promote intestinal regeneration. METHODS: Human Muse cells from Wharton's jelly matrix of umbilical cord (WJ-Muse) were sorted by flow cytometry using the SSEA-3 marker, characterized and compared to bone-marrow derived Muse cells (BM-Muse). Under gas anesthesia, GIS mice were treated or not through an intravenous retro-orbital injection of 50,000 WJ-Muse, freshly isolated or cryopreserved, shortly after an 18 Gy-abdominal IR. No immunosuppressant was delivered to the mice. Mice were euthanized either 24 h post-IR to assess early small intestine tissue response, or 7 days post-IR to assess any regenerative response. Mouse survival, histological stainings, apoptosis and cell proliferation were studied and measurement of cytokines, recruitment of immune cells and barrier functional assay were performed. RESULTS: Injection of WJ-Muse shortly after abdominal IR highly improved mouse survival as a result of a rapid regeneration of intestinal epithelium with the rescue of the impaired epithelial barrier. In small intestine of Muse-treated mice, an early enhanced secretion of IL-6 and MCP-1 cytokines was observed associated with (1) recruitment of monocytes/M2-like macrophages and (2) proliferation of Paneth cells through activation of the IL-6/Stat3 pathway. CONCLUSION: Our findings indicate that a single injection of a small quantity of WJ-Muse may be a new and easy therapeutic strategy for treating lethal GIS.

Interplay between FACT subunit SPT16 and TRIM33 can remodel chromatin at macrophage distal regulatory elements.

BACKGROUND: Cell type-specific use of cis-acting regulatory elements is mediated by the combinatorial activity of transcription factors involved in lineage determination and maintenance of cell identity. In macrophages, specific transcriptional programs are dictated by the transcription factor PU.1 that primes distal regulatory elements for macrophage identities and makes chromatin competent for activity of stimuli-dependent transcription factors. Although the advances in genome-wide approaches have elucidated the functions of these macrophage-specific distal regulatory elements in transcriptional responses, chromatin structures associated with PU.1 priming and the underlying mechanisms of action of these cis-acting sequences are not characterized. RESULTS: Here, we show that, in macrophages, FACT subunit SPT16 can bind to positioned nucleosomes directly flanking PU.1-bound sites at previously uncharacterized distal regulatory elements located near genes essential for macrophage development and functions. SPT16 can interact with the transcriptional co-regulator TRIM33 and binds to half of these sites in a TRIM33-dependent manner. Using the Atp1b3 locus as a model, we show that FACT binds to two positioned nucleosomes surrounding a TRIM33/PU.1-bound site in a region, located 35 kb upstream the Atp1b3 TSS, that interact with the Atp1b3 promoter. At this - 35 kb region, TRIM33 deficiency leads to FACT release, loss of the two positioned nucleosomes, RNA Pol II recruitment and bidirectional transcription. These modifications are associated with higher levels of FACT binding at the Atp1b3 promoter, an increase of RNA Pol II recruitment and an increased expression of Atp1b3 in Trim33-/- macrophages. CONCLUSIONS: Thus, sequestering of SPT16/FACT by TRIM33 at PU.1-bound distal regions might represent a new regulatory mechanism for RNA Pol II recruitment and transcription output in macrophages.

TRIM33 deficiency in monocytes and macrophages impairs resolution of colonic inflammation.

BACKGROUND: Mature myeloid cells play a crucial role in Crohn's disease (CD) but the molecular players that regulate their functions in CD are not fully characterized. We and others have shown that TRIM33 is involved in the innate immune response and in the inflammatory response but TRIM33 role in intestinal inflammation is not known. In this study, we investigated the role of TRIM33 in myeloid cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: We study the role of TRIM33 during DSS-induced colitis which mimics intestinal inflammation using mice deleted for Trim33 only in mature myeloid cells (Trim33-/- mice) FINDINGS: We first show that Trim33 mRNA level is decreased in CD patient's blood monocytes suggesting a role of TRIM33 in CD. Using Trim33-/- mice, we show that these mice display an impaired resolution of colonic inflammation with an increased number of blood and colon monocytes and a decreased number of colonic macrophages. Trim33-/- monocytes are less competent for recruitment and macrophage differentiation. Finally, during resolution of inflammation, Trim33-/- colonic macrophages display an impaired M1/M2 switch and express a low level of membrane-bound TNF that is associated with an increased number of colonic neutrophils. INTERPRETATION: Our study shows an important role of TRIM33 in monocytes/macrophages during DSS-induced colitis and suggests that the decreased expression of TRIM33 in CD patient's blood monocytes might not be a consequence but might be involved in CD progression. FUND: La Ligue contre le Cancer (équipe labelisée), INSERM, CEA, Université Paris-Diderot, Université Paris-Sud.

Isolation and Phenotyping of Intestinal Macrophages.

Macrophages are one of the most abundant leucocytes in the intestinal mucosa where they are essential for maintaining homeostasis. However they are also implicated in the pathogenesis of disorders such as inflammatory bowel disease (IBD), offering potential targets for novel therapies.Tissue macrophages are a heterogeneous population of immune cells that fulfill tissue-specific and niche-specific functions. These unique phenotypes likely reflect the heterogeneity of tissue macrophage origins and influence the tissue environment in which they reside. Here we describe how we can characterize and isolate the colonic macrophages.

Macrophage production and activation are dependent on TRIM33.

The tripartite motif (TRIM) family of proteins plays important roles in innate immunity and antimicrobial infection. None of these proteins has been shown to directly regulate transcription of genes in monocyte/macrophage except TRIM33 that we have recently shown to be a macrophage specific transcriptional inhibitor of Ifnb1. Using ChIP-seq analyses, we now report that TRIM33 is bound to two fold more genes in immature than in mature myeloid cell lines. When located near the same genes, TRIM33 is bound to different sequences in the two cell lines suggesting a role of TRIM33 in both immature and mature myeloid cells. Accordingly, expression of TRIM33 in immature myeloid cells is necessary for efficient production of small peritoneal macrophages, monocytes and bone marrow derived macrophage (BMDM) and TRIM33 targets a subset of genes involved in the inflammatory response only in mature myeloid cells. Functionally, this targeting is associated with impaired repression of pathways regulating the late phases of lipopolysaccharide (LPS) activation of BMDM and a high sensitivity to LPS in vivo when the trim33 gene is inactivated in mature myeloid cells. These findings pinpoint TRIM33 as an important transcriptional actor of monocyte/macrophage mediated inflammation.

A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes.

BACKGROUND: Hemocyanins are respiratory proteins with multiple functions. In diverse crustaceans hemocyanins can release histidine-rich antimicrobial peptides in response to microbial challenge. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of hemocyanins. METHODS: The three-dimensional structure of the antifungal peptide PvHCt from Litopenaeus vannamei was determined by NMR. Its mechanism of action against the shrimp pathogen Fusarium oxysporum was investigated using immunochemistry, fluorescence and transmission electron microscopy. RESULTS: PvHCt folded into an amphipathic α-helix in membrane-mimicking media and displayed a random conformation in aqueous environment. In contact with F. oxysporum, PvHCt bound massively to the surface of fungal hyphae without being imported into the cytoplasm. At minimal inhibitory concentrations, PvHCt made the fungal membrane permeable to SYTOX-green and fluorescent dextran beads of 4 kDa. Higher size beads could not enter the cytoplasm. Therefore, PvHCt likely creates local damages to the fungal membrane. While the fungal cell wall appeared preserved, gradual degeneration of the cytoplasm most often resulting in cell lysis was observed in fungal spores and hyphae. In the remaining fungal cells, PvHCt induced a protective response by the formation of daughter hyphae. CONCLUSION: The massive accumulation of PvHCt at the surface of fungal hyphae and subsequent insertion into the plasma membrane disrupt its integrity as a permeability barrier, leading to disruption of internal homeostasis and fungal death. GENERAL SIGNIFICANCE: The histidine-rich antimicrobial peptide PvHCt derived from shrimp hemocyanin is a strictly antifungal peptide, which adopts an amphipathic α-helical structure, and selectively binds to and permeabilizes fungal cells.

TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation.

Despite its importance during viral or bacterial infections, transcriptional regulation of the interferon-ß gene (Ifnb1) in activated macrophages is only partially understood. Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts. In macrophages, TRIM33 is recruited by PU.1 to a conserved region, the Ifnb1 Control Element (ICE), located 15 kb upstream of the Ifnb1 transcription start site. ICE constitutively interacts with Ifnb1 through a TRIM33-independent chromatin loop. At late phases of lipopolysaccharide activation of macrophages, TRIM33 is bound to ICE, regulates Ifnb1 enhanceosome loading, controls Ifnb1 chromatin structure and represses Ifnb1 gene transcription by preventing recruitment of CBP/p300. These results characterize a previously unknown mechanism of macrophage-specific regulation of Ifnb1 transcription whereby TRIM33 is critical for Ifnb1 gene transcription shutdown.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging reveals cholesterol overload in the cerebral cortex of Alzheimer disease patients.

Although cholesterol has been involved in the pathophysiology of Alzheimer disease (AD), its distribution in the cerebral cortex over the course of AD is unknown. We describe an original method to quantify cholesterol distribution using time-of-flight secondary ion mass spectrometry imaging. Cholesterol was unevenly distributed along the cortical thickness, being more abundant close to the white matter, in both control and AD cases. However, the mean cholesterol signal was significantly higher in the lower half of the cortex in AD samples compared to controls. This increase, when converted into cortical layers, was statistically significant for layers III and IV and did not reach significance in layers V + VI, the variability being too high at the interface between grey and white matter. The density of neurofibrillary tangles and of senile plaques was not statistically linked to the abundance of cholesterol. Cholesterol overload thus appears a new and independent alteration of AD cerebral cortex. The structure in which cholesterol accumulates and the mechanism of this accumulation remain to be elucidated.

MALDI imaging mass spectrometry of lipids by adding lithium salts to the matrix solution.

Mass spectrometry imaging of lipids using MALDI-TOF/TOF mass spectrometers is of growing interest for chemical mapping of organic compounds at the surface of tissue sections. Many efforts have been devoted to the best matrix choice and deposition technique. Nevertheless, the identification of lipid species desorbed from tissue sections remains problematic. It is now well-known that protonated, sodium- and potassium-cationized lipids are detected from biological samples, thus complicating the data analysis. A new sample preparation method is proposed, involving the use of lithium salts in the matrix solution in order to simplify the mass spectra with only lithium-cationized molecules instead of a mixture of various cationized species. Five different lithium salts were tested. Among them, lithium trifluoroacetate and lithium iodide merged the different lipid adducts into one single lithium-cationized species. An optimized sample preparation protocol demonstrated that the lithium trifluoroacetate salt slightly increased desorption of phosphatidylcholines. Mass spectrometry images acquired on rat brain tissue sections by adding lithium trifluoroacetate showed the best results in terms of image contrast. Moreover, more structurally relevant fragments were generated by tandem mass spectrometry when analyzing lithium-cationized species.

Multimodal spectroscopy combining time-of-flight-secondary ion mass spectrometry, synchrotron-FT-IR, and synchrotron-UV microspectroscopies on the same tissue section.

Mass spectrometry and spectroscopy-based approaches can provide an overview of the chemical composition of a tissue sample. This opens up the possibility to investigate in depth the subtle biochemical changes associated with pathological tissues. In this study, time-of-flight secondary ion mass spectrometry (TOF-SIMS) and synchrotron-FT-IR and -UV imaging were applied to the same tissue section by using the same sample holder. The tested sample involved liver cirrhosis, which is characterized by regeneration nodules surrounded by annular fibrosis. A tissue section from a cirrhotic liver was deposited on a gold coated glass slide and was initially analyzed by FT-IR microspectroscopy in order to image the distribution of lipids, proteins, sugars, and nucleic acids. This technique has identified collagen enrichment in fibrosis whereas esters were mostly distributed into the cirrhotic nodules. The exact same section was investigated using TOF-SIMS demonstrating that some molecular lipid species were differentially distributed into the fibrosis areas or cirrhotic nodules. Spectra of UV microspectroscopy obtained from the same section allowed visualizing high autofluorescence from fibrous septa confirming the presence of collagen. Altogether, these results demonstrated that TOF-SIMS and FT-IR/UV microspectroscopy analyses can be successfully performed on the same tissue section.

Insights into structure-activity relationships in the C-terminal region of divercin V41, a class IIa bacteriocin with high-level antilisterial activity.

Divercin V41 (DvnV41) is a class IIa bacteriocin with potent antilisterial activity isolated from Carnobacterium divergens V41. Previously, we expressed from a synthetic gene, in Escherichia coli Origami, a recombinant DvnV41 designated DvnRV41, which possesses four additional amino acids (AMDP) in the N-terminal region that result from enzymatic cleavage and retains the initial DvnV41 activity. To unravel the relationship between the structure of DvnRV41 and its particularly elevated activity, we produced by site-directed mutagenesis eight variants in which a single amino acid replacement was specifically introduced into the sequence. The point mutations were designed to change either conserved residues in class IIa bacteriocins or residues specific to DvnV41 located mainly in the C-terminal region. The fusion proteins were purified from the cytosoluble fractions by immobilized affinity chromatography. DvnRV41 and its variants were released from the fusion proteins by enzymatic cleavage, using enterokinase. The purity of DvnRV41 and of the variants was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and mass spectrometry. The antibacterial activity of DvnRV41 and its variants was assessed using different indicator strains, including Listeria monocytogenes EGDe and Enterococcus faecalis JH2-2. The activity of all of the variants appeared to be less than the activity of DvnRV41. The decrease in activity did not appear to be related to a global conformational change, as determined by circular dichroism. Overall, the variants of DvnRV41 produced in the present study provide interesting insights into structure-activity relationships of class IIa bacteriocins.

Collision induced dissociation-based characterization of nucleotide peptides: fragmentation patterns of microcin C7-C51, an antimicrobial peptide produced by Escherichia coli.

Covalent protein-nucleic acid conjugates form an original class of compounds that occur in nature or can be generated in vitro through cross-linking to investigate domains involved in protein/nucleic acid interactions. Their mass spectrometry fragmentation patterns are poorly characterized. We have used electrospray-ionization mass spectrometry (ESI-MS) combined with collision-induced dissociation (CID) to characterize microcin C7-C51, an antimicrobial nucleotide peptide that targets aspartyl-tRNA synthetase and inhibits translation. The fragments of microcin C7-C51 were analyzed in positive- and negative-ion modes and compared with those of the corresponding unmodified heptapeptide and to the derived aspartyl-adenylate. The positive- and negative-ion mode fragments of microcin C7-C51 provided information on both the nucleotide and peptide moieties. Accurate mass measurement obtained using an LTQ Orbitrap instrument was a key factor for a comprehensive interpretation of the fragments. The experimental results obtained permitted the proposal of stepwise fragmentation pathways involving ion-dipole complexes. The data provide a better understanding of nucleotide peptide fragmentation in the gas phase.

Structural and functional diversity of microcins, gene-encoded antibacterial peptides from enterobacteria.

Microcins are a peculiar class of gene-encoded low-molecular-mass antibacterial peptides secreted by enterobacteria. They contribute to the regulation of microbial competitions within the intestinal microbiota. The genetic systems involved in microcin biosynthesis share a conserved organization. Similar to bacteriocins of Gram-positive bacteria, microcins exert potent antibacterial activity directed against phylogenetically-related bacterial strains, with minimal inhibitory concentrations in the nanomolar range. In contrast to bacteriocins, they display a great structural diversity among the few representatives well characterized until now, that makes difficult the description of microcin subclasses. This review focuses on three microcins, MccE492m that carries a C-terminal posttranslational modification containing a catechol-type siderophore, MccJ25, a cyclic peptide with a unique 'lasso-type' structure and MccC7 or C51, with a common N-formylated heptapeptide-nucleotide structure. We show these microcins exhibit 'Trojan horse' mechanisms of antibacterial activity: either (i) the microcin structure is a mime of an essential element, permitting its recognition by outer membrane receptors used for vital functions in bacteria and further translocation into the periplasmic space, or (ii) it is secreted as a harmless molecule and further processed in susceptible bacteria to form the toxic entity. When inside target bacteria, microcins bind essential enzymes or interact with the inner membrane to form a bacterial killing structure.