Endothelial AHR activity prevents lung barrier disruption in viral infection.

Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.

Wiskott-Aldrich Syndrome Interacting Protein Deficiency Uncovers the Role of the Co-receptor CD19 as a Generic Hub for PI3 Kinase Signaling in B Cells.

Humans with Wiskott-Aldrich syndrome display a progressive immunological disorder associated with compromised Wiskott-Aldrich Syndrome Interacting Protein (WIP) function. Mice deficient in WIP recapitulate such an immunodeficiency that has been attributed to T cell dysfunction; however, any contribution of B cells is as yet undefined. Here we have shown that WIP deficiency resulted in defects in B cell homing, chemotaxis, survival, and differentiation, ultimately leading to diminished germinal center formation and antibody production. Furthermore, in the absence of WIP, several receptors, namely the BCR, BAFFR, CXCR4, CXCR5, CD40, and TLR4, were impaired in promoting CD19 co-receptor activation and subsequent PI3 kinase (PI3K) signaling. The underlying mechanism was due to a distortion in the actin and tetraspanin networks that lead to altered CD19 cell surface dynamics. In conclusion, our findings suggest that, by regulating the cortical actin cytoskeleton, WIP influences the function of CD19 as a general hub for PI3K signaling.

New structural insights into anomeric carbohydrate recognition by frutalin: an α-d-galactose-binding lectin from breadfruit seeds.

Frutalin (FTL) is a multiple-binding lectin belonging to the jacalin-related lectin (JRL) family and derived from Artocarpus incisa (breadfruit) seeds. This lectin specifically recognizes and binds α-d-galactose. FTL has been successfully used in immunobiological research for the recognition of cancer-associated oligosaccharides. However, the molecular bases by which FTL promotes these specific activities remain poorly understood. Here, we report the whole 3D structure of FTL for the first time, as determined by X-ray crystallography. The obtained crystals diffracted to 1.81 Å (Apo-frutalin) and 1.65 Å (frutalin-d-Gal complex) of resolution. The lectin exhibits post-translational cleavage yielding an α- (133 amino acids) and ß-chain (20 amino acids), presenting a homotetramer when in solution, with a typical JRL ß-prism. The ß-prism was composed of three 4-stranded ß-sheets forming three antiparallel Greek key motifs. The carbohydrate-binding site (CBS) involved the N-terminus of the α-chain and was formed by four key residues: Gly25, Tyr146, Trp147 and Asp149. Together, these results were used in molecular dynamics simulations in aqueous solutions to shed light on the molecular basis of FTL-ligand binding. The simulations suggest that Thr-Ser-Ser-Asn (TSSN) peptide excision reduces the rigidity of the FTL CBS, increasing the number of interactions with ligands and resulting in multiple-binding sites and anomeric recognition of α-d-galactose sugar moieties. Our findings provide a new perspective to further elucidate the versatility of FTL in many biological activities.

Action mechanism of naphthofuranquinones against fluconazole-resistant Candida tropicalis strains evidenced by proteomic analysis: The role of increased endogenous ROS.

The increased incidence of candidemia in terciary hospitals worldwide and the cross-resistance frequency require the new therapeutic strategies development. Recently, our research group demonstrated three semi-synthetic naphthofuranquinones (NFQs) with a significant antifungal activity in a fluconazole-resistant (FLC) C. tropicalis strain. The current study aimed to investigate the action's preliminary mechanisms of NFQs by several standardized methods such as proteomic and flow cytometry analyzes, comet assay, immunohistochemistry and confocal microscopy evaluation. Our data showed C. tropicalis 24 h treated with all NFQs induced an expression's increase of proteins involved in the metabolic response to stress, energy metabolism, glycolysis, nucleosome assembly and translation process. Some aspects of proteomic analysis are in consonance with our flow cytometry analysis which indicated an augmentation of intracellular ROS, mitochondrial dysfunction and DNA strand breaks (neutral comet assay and γ-H2AX detection). In conclusion, our data highlights the great contribution of ROS as a key event, probably not the one, associated to anti-candida properties of studied NFQs.

A panel of protein candidates for comprehensive study of Caprine Arthritis Encephalitis (CAE) infection.

The caprine arthrite encephalite (CAE) is a disease that affects especially dairy goat. The virus shows compartmentalization features, that allows it to hide at certain times during the course of the disease, making it difficult to control. The present study was conducted to identify the major seminal plasma protein profile of goats infected by CAE and its associations with seroconversion using Western blotting. Two groups containing five males each, were used in this experiment. The first group was composed by seropositive animals and the control by seronegative confirmed by Western blotting and PCR. The semen was collected through artificial vagina and after that, two-dimensional electrophoresis and MALDI-TOF MS were used. Seventy-five spots were identified in the goat seminal plasma gels, equivalent to 13 different proteins with more expression. The similar proteins found in both groups and related to reproduction were spermadhesin Z13-like, bodhesin and bodhesin-2, Lipocalin, protein PDC-109-like, and albumin. In infected goats, proteases such as arisulfatase A have been identified, whose function probably is related to metabolism control of sulfatides, involved to virus control. The other ones were bifunctional ATP-dependent dihydroxyacetone kinase/FAD-AMP lyase, cathepsin F isoform X1, disintegrin and metalloproteinase domain-containing protein 2-like isoform X1, clusterin, carbonic anhydrase 2, electron transfer flavoprotein subunit beta, and epididymal secretory glutathione peroxidase. The results of this study show the reaction of the innate immune system against chronic infection of goats by CAE.

B-cell-independent lymphoid tissue infection by a B-cell-tropic rhadinovirus.

Lymphocytes provide gammaherpesviruses with a self-renewing substrate for persistent infection and with transport to mucosal sites for host exit. Their role in the initial colonization of new hosts is less clear. Murid herpesvirus 4 (MuHV-4), an experimentally accessible, B-cell-tropic rhadinovirus (gamma-2 herpesvirus), persistently infects both immunocompetent and B-cell-deficient mice. A lack of B-cells did not compromise MuHV-4 entry into lymphoid tissue, which involved myeloid cell infection. However, it impaired infection amplification and MuHV-4 exit from lymphoid tissue, which involved myeloid to B-cell transfer.

Subcapsular sinus macrophages limit acute gammaherpesvirus dissemination.

Lymphocyte proliferation, mobility and longevity make them prime targets for virus infection. Myeloid cells that process and present environmental antigens to lymphocytes are consequently an important line of defence. Subcapsular sinus macrophages (SSMs) filter the afferent lymph and communicate with B-cells. How they interact with B-cell-tropic viruses is unknown. We analysed their encounter with murid herpesvirus-4 (MuHV-4), an experimentally accessible gammaherpesvirus related to Kaposi's sarcoma-associated herpesvirus. MuHV-4 disseminated via lymph nodes, and intranasally or subcutaneously inoculated virions readily infected SSMs. However, this infection was poorly productive. SSM depletion with clodronate-loaded liposomes or with diphtheria toxin in CD169-diphtheria toxin receptor transgenic mice increased B-cell infection and hastened virus spread to the spleen. Dendritic cells provided the main route to B-cells, and SSMs slowed host colonization, apparently by absorbing virions non-productively from the afferent lymph.

BAFF receptor deficiency limits gammaherpesvirus infection.

UNLABELLED: Lymphocyte colonization by gammaherpesviruses (γHVs) is an important target for cancer prevention. However, how it works is not clear. Epstein-Barr virus drives autonomous B cell proliferation in vitro but in vivo may more subtly exploit the proliferative pathways provided by lymphoid germinal centers (GCs). Murid herpesvirus 4 (MuHV-4), which realistically infects inbred mice, provides a useful tool with which to understand further how a γHV colonizes B cells in vivo. Not all γHVs necessarily behave the same, but common events can with MuHV-4 be assigned an importance for host colonization and so a potential as therapeutic targets. MuHV-4-driven B cell proliferation depends quantitatively on CD4(+) T cell help. Here we show that it also depends on T cell-independent survival signals provided by the B cell-activating factor (BAFF) receptor (BAFF-R). B cells could be infected in BAFF-R(-/-) mice, but virus loads remained low. This corresponded to a BAFF-R-dependent defect in GC colonization. The close parallels between normal, antigen-driven B cell responses and virus-infected B cell proliferation argue that in vivo, γHVs mostly induce infected B cells into normal GC reactions rather than generating large numbers of autonomously proliferating blasts. IMPORTANCE: γHVs cause cancers by driving the proliferation of infected cells. B cells are a particular target. Thus, we need to know how virus-driven B cell proliferation works. Controversy exists as to whether viral genes drive it directly or less directly orchestrate the engagement of normal, host-driven pathways. Here we show that the B cell proliferation driven by a murid γHV requires BAFF-R. This supports the idea that γHVs exploit host proliferation pathways and suggests that interfering with BAFF-R could more generally reduce γHV-associated B cell proliferation.

Glycoprotein B cleavage is important for murid herpesvirus 4 to infect myeloid cells.

Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many-but not all-herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this, we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation, or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a postendocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless.

A heparan-dependent herpesvirus targets the olfactory neuroepithelium for host entry.

Herpesviruses are ubiquitous pathogens that cause much disease. The difficulty of clearing their established infections makes host entry an important target for control. However, while herpesviruses have been studied extensively in vitro, how they cross differentiated mucus-covered epithelia in vivo is unclear. To establish general principles we tracked host entry by Murid Herpesvirus-4 (MuHV-4), a lymphotropic rhadinovirus related to the Kaposi's Sarcoma-associated Herpesvirus. Spontaneously acquired virions targeted the olfactory neuroepithelium. Like many herpesviruses, MuHV-4 binds to heparan sulfate (HS), and virions unable to bind HS showed poor host entry. While the respiratory epithelium expressed only basolateral HS and was bound poorly by incoming virions, the neuroepithelium also displayed HS on its apical neuronal cilia and was bound strongly. Incoming virions tracked down the neuronal cilia, and either infected neurons or reached the underlying microvilli of the adjacent glial (sustentacular) cells and infected them. Thus the olfactory neuroepithelium provides an important and complex site of HS-dependent herpesvirus uptake.

Myeloid infection links epithelial and B cell tropisms of Murid Herpesvirus-4.

Gamma-herpesviruses persist in lymphocytes and cause disease by driving their proliferation. Lymphocyte infection is therefore a key pathogenetic event. Murid Herpesvirus-4 (MuHV-4) is a rhadinovirus that like the related Kaposi's Sarcoma-associated Herpesvirus persists in B cells in vivo yet infects them poorly in vitro. Here we used MuHV-4 to understand how virion tropism sets the path to lymphocyte colonization. Virions that were highly infectious in vivo showed a severe post-binding block to B cell infection. Host entry was accordingly an epithelial infection and B cell infection a secondary event. Macrophage infection by cell-free virions was also poor, but improved markedly when virion binding improved or when macrophages were co-cultured with infected fibroblasts. Under the same conditions B cell infection remained poor; it improved only when virions came from macrophages. This reflected better cell penetration and correlated with antigenic changes in the virion fusion complex. Macrophages were seen to contact acutely infected epithelial cells, and cre/lox-based virus tagging showed that almost all the virus recovered from lymphoid tissue had passed through lysM(+) and CD11c(+) myeloid cells. Thus MuHV-4 reached B cells in 3 distinct stages: incoming virions infected epithelial cells; infection then passed to myeloid cells; glycoprotein changes then allowed B cell infection. These data identify new complexity in rhadinovirus infection and potentially also new vulnerability to intervention.

Proteomic analysis of salt stress and recovery in leaves of Vigna unguiculata cultivars differing in salt tolerance.

KEY MESSAGE: Cowpea cultivars differing in salt tolerance reveal differences in protein profiles and adopt different strategies to overcome salt stress. Salt-tolerant cultivar shows induction of proteins related to photosynthesis and energy metabolism. Salinity is a major abiotic stress affecting plant cultivation and productivity. The objective of this study was to examine differential proteomic responses to salt stress in leaves of the cowpea cultivars Pitiúba (salt tolerant) and TVu 2331 (salt sensitive). Plants of both cultivars were subjected to salt stress (75 mM NaCl) followed by a recovery period of 5 days. Proteins extracted from leaves of both cultivars were analyzed by two-dimensional electrophoresis (2-DE) under salt stress and after recovery. In total, 22 proteins differentially regulated by both salt and recovery were identified by LC-ESI-MS/MS. Our current proteome data revealed that cowpea cultivars adopted different strategies to overcome salt stress. For the salt-tolerant cultivar (Pitiúba), increase in abundance of proteins involved in photosynthesis and energy metabolism, such as rubisco activase, ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19), glycine decarboxylase (EC 1.4.4.2) and oxygen-evolving enhancer (OEE) protein 2, was observed. However, these vital metabolic processes were more profoundly affected in salt-sensitive cultivar (TVu), as indicated by the down-regulation of OEE protein 1, Mn-stabilizing protein-II, carbonic anhydrase (EC 4.2.1.1) and Rubisco (EC 4.1.1.39), leading to energy reduction and a decline in plant growth. Other proteins differentially regulated in both cultivars corresponded to different physiological responses. Overall, our results provide information that could lead to a better understanding of the molecular basis of salt tolerance and sensitivity in cowpea plants.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Murid herpesvirus-4 exploits dendritic cells to infect B cells.

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells.

Insights on the phytochemical profile (cyclopeptides) and biological activities of Calotropis procera latex organic fractions.

Calotropis procera is a medicinal plant whose pharmacological properties are associated with its latex. Here, the Calotropis procera latex fractions were investigated in an attempt to trace its phytochemical profile and measure its anti-inflammatory and toxicity activity. The crude latex was partitioned, yielding five fractions (49.4% hexane, 5.2% dichloromethane, 2.0% ethyl acetate, 2.1% n-butanol, and 41.1% aqueous). Phytochemical screening and spectroscopy analysis revealed that dichloromethane is the most chemically diverse fraction. Triterpenes were detected in both the hexane and dichloromethane fractions, while flavonoids were detected in the dichloromethane and ethyl acetate fractions. These fractions were cytotoxic to cancer cell lines (LD50 0.05 to 3.9 µ g/mL) and lethal to brine shrimp (LD50 10.9 to 65.7 µ g/mL). Reduced neutrophil migration in rats was observed in carrageenan-induced peritonitis for the dichloromethane (67%), ethyl acetate (56%), and aqueous (72%) fractions. A positive reaction with tolidine and ninhydrin suggested that cyclopeptides are in the ethyl acetate fraction. It is therefore concluded that Calotropis procera latex dichloromethane and ethyl acetate fractions exhibit both in vitro and in vivo activities as well as anti-inflammatory properties. Cyclopeptide detection is especially interesting because previous attempts to investigate these low-molecular cyclic amino acid sequences in C. procera have failed.

SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cells.

Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ T cells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ T cells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity.

DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential.

Cells with latent stem ability can contribute to mammalian tissue regeneration after damage. Whether the central nervous system (CNS) harbors such cells remains controversial. Here, we report that DNGR-1 lineage tracing in mice identifies an ependymal cell subset, wherein resides latent regenerative potential. We demonstrate that DNGR-1-lineage-traced ependymal cells arise early in embryogenesis (E11.5) and subsequently spread across the lining of cerebrospinal fluid (CSF)-filled compartments to form a contiguous sheet from the brain to the end of the spinal cord. In the steady state, these DNGR-1-traced cells are quiescent, committed to their ependymal cell fate, and do not contribute to neuronal or glial lineages. However, trans-differentiation can be induced in adult mice by CNS injury or in vitro by culture with suitable factors. Our findings highlight previously unappreciated ependymal cell heterogeneity and identify across the entire CNS an ependymal cell subset wherein resides damage-responsive neural stem cell potential.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.

Tissue clonality of dendritic cell subsets and emergency DCpoiesis revealed by multicolor fate mapping of DC progenitors.

Conventional dendritic cells (cDCs) are found in all tissues and play a key role in immune surveillance. They comprise two major subsets, cDC1 and cDC2, both derived from circulating precursors of cDCs (pre-cDCs), which exited the bone marrow. We show that, in the steady-state mouse, pre-cDCs entering tissues proliferate to give rise to differentiated cDCs, which themselves have residual proliferative capacity. We use multicolor fate mapping of cDC progenitors to show that this results in clones of sister cDCs, most of which comprise a single cDC1 or cDC2 subtype, suggestive of pre-cDC commitment. Upon infection, a surge in the influx of pre-cDCs into the affected tissue dilutes clones and increases cDC numbers. Our results indicate that tissue cDCs can be organized in a patchwork of closely positioned sister cells of the same subset whose coexistence is perturbed by local infection, when the bone marrow provides additional pre-cDCs to meet increased tissue demand.