Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity

Natural killer (NK) cells and dendritic cells (DCs) are, respectively, central components of innate and adaptive immune responses. We describe here a third DC lineage, termed interferon-producing killer DCs (IKDCs), distinct from conventional DCs and plasmacytoid DCs and with the molecular expression profile of both NK cells and DCs. They produce substantial amounts of type I interferons (IFN) and interleukin (IL)-12 or IFN-gamma, depending on activation stimuli. Upon stimulation with CpG oligodeoxynucleotides, ligands for Toll-like receptor (TLR)-9, IKDCs kill typical NK target cells using NK-activating receptors. Their cytolytic capacity subsequently diminishes, associated with the loss of NKG2D receptor (also known as Klrk1) and its adaptors, Dap10 and Dap12. As cytotoxicity is lost, DC-like antigen-presenting activity is gained, associated with upregulation of surface major histocompatibility complex class II (MHC II) and costimulatory molecules, which formally distinguish them from classical NK cells. In vivo, splenic IKDCs preferentially show NK function and, upon systemic infection, migrate to lymph nodes, where they primarily show antigen-presenting cell activity. By virtue of their capacity to kill target cells, followed by antigen presentation, IKDCs provide a link between innate and adaptive immunity.     

Sandfly Fever Sicilian Virus-Leishmania major co-infection modulates innate inflammatory response favoring myeloid cell infections and skin hyperinflammation

BackgroundThe leishmaniases are a group of sandfly-transmitted diseases caused by species of the protozoan parasite, Leishmania. With an annual incidence of 1 million cases, 1 billion people living in Leishmania-endemic regions, and nearly 30,000 deaths each year, leishmaniasis is a major global public health concern. While phlebotomine sandflies are well-known as vectors of Leishmania, they are also the vectors of various phleboviruses, including Sandfly Fever Sicilian Virus (SFSV).Cutaneous leishmaniasis (CL), caused by Leishmania major (L. major), among other species, results in development of skin lesions on the infected host. Importantly, there exists much variation in the clinical manifestation between individuals. We propose that phleboviruses, vectored by and found in the same sandfly guts as Leishmania, may be a factor in determining CL severity. It was reported by our group that Leishmania exosomes are released into the gut of the sandfly vector and co-inoculated during blood meals, where they exacerbate CL skin lesions. We hypothesized that, when taking a blood meal, the sandfly vector infects the host with Leishmania parasites and exosomes as well as phleboviruses, and that this viral co-infection results in a modulation of leishmaniasis.Methodology/Principal findingsIn vitro, we observed modulation by SFSV in MAP kinase signaling as well as in the IRF3 pathway that resulted in a pro-inflammatory phenotype. Additionally, we found that SFSV and L. major co-infection resulted in an exacerbation of leishmaniasis in vivo, and by using endosomal (Toll-like receptor) TLR3, and MAVS knock-out mice, deduced that SFSV’s hyperinflammatory effect was TLR3- and MAVS-dependent. Critically, we observed that L. major and SFSV co-infected C57BL/6 mice demonstrated significantly higher parasite burden than mice solely infected with L. major. Furthermore, viral presence increased leukocyte influx in vivo. This influx was accompanied by elevated total extracellular vesicle numbers. Interestingly, L. major displayed higher infectiveness with coincident phleboviral infection compared to L. major infection alone.Conclusion/SignificanceOverall our work represents novel findings that contribute towards understanding the causal mechanisms governing cutaneous leishmaniasis pathology. Better comprehension of the potential role of viral co-infection could lead to treatment regimens with enhanced effectiveness.     

Computer-aided X-ray screening for tuberculosis and HIV testing among adults with cough in Malawi (the PROSPECT study): A randomised trial and cost-effectiveness analysis

BackgroundSuboptimal tuberculosis (TB) diagnostics and HIV contribute to the high global burden of TB. We investigated costs and yield from systematic HIV-TB screening, including computer-aided digital chest X-ray (DCXR-CAD).Methods and findingsIn this open, three-arm randomised trial, adults (≥18 years) with cough attending acute primary services in Malawi were randomised (1:1:1) to standard of care (SOC); oral HIV testing (HIV screening) and linkage to care; or HIV testing and linkage to care plus DCXR-CAD with sputum Xpert for high CAD4TBv5 scores (HIV-TB screening). Participants and study staff were not blinded to intervention allocation, but investigator blinding was maintained until final analysis. The primary outcome was time to TB treatment. Secondary outcomes included proportion with same-day TB treatment; prevalence of undiagnosed/untreated bacteriologically confirmed TB on day 56; and undiagnosed/untreated HIV. Analysis was done on an intention-to-treat basis. Cost-effectiveness analysis used a health-provider perspective. Between 15 November 2018 and 27 November 2019, 8,236 were screened for eligibility, with 473, 492, and 497 randomly allocated to SOC, HIV, and HIV-TB screening arms; 53 (11%), 52 (9%), and 47 (9%) were lost to follow-up, respectively. At 56 days, TB treatment had been started in 5 (1.1%) SOC, 8 (1.6%) HIV screening, and 15 (3.0%) HIV-TB screening participants. Median (IQR) time to TB treatment was 11 (6.5 to 38), 6 (1 to 22), and 1 (0 to 3) days (hazard ratio for HIV-TB versus SOC: 2.86, 1.04 to 7.87), with same-day treatment of 0/5 (0%) SOC, 1/8 (12.5%) HIV, and 6/15 (40.0%) HIV-TB screening arm TB patients (p = 0.03). At day 56, 2 SOC (0.5%), 4 HIV (1.0%), and 2 HIV-TB (0.5%) participants had undiagnosed microbiologically confirmed TB. HIV screening reduced the proportion with undiagnosed or untreated HIV from 10 (2.7%) in the SOC arm to 2 (0.5%) in the HIV screening arm (risk ratio [RR]: 0.18, 0.04 to 0.83), and 1 (0.2%) in the HIV-TB screening arm (RR: 0.09, 0.01 to 0.71). Incremental costs were US$3.58 and US$19.92 per participant screened for HIV and HIV-TB; the probability of cost-effectiveness at a US$1,200/quality-adjusted life year (QALY) threshold was 83.9% and 0%. Main limitations were the lower than anticipated prevalence of TB and short participant follow-up period; cost and quality of life benefits of this screening approach may accrue over a longer time horizon.ConclusionsDCXR-CAD with universal HIV screening significantly increased the timeliness and completeness of HIV and TB diagnosis. If implemented at scale, this has potential to rapidly and efficiently improve TB and HIV diagnosis and treatment.Trial registrationclinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03519425","term_id":"NCT03519425"}}NCT03519425.

In a randomised trial, Peter MacPherson and colleagues investigate the costs, timeliness, and completeness of computer-aided X-ray screening for tuberculosis and HIV testing in adults with cough in Malawi.     

Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.

An improved virus-induced gene silencing approach led to the discovery of the alkaloid biosynthetic enzyme serpentine synthase.     

Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.     

A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.     

Defective trafficking and localization of mutated transferrin receptor 2: implications for type 3 hereditary hemochromatosis

Transferrin receptor 2 (TfR2), a homologue of transferrin receptor 1 (TfR1), is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 result in iron overload with similar features to HFE-associated hereditary hemochromatosis. The precise role of TfR2 in iron metabolism and the functional consequences of disease-causing mutations have not been fully determined. We have expressed wild-type and various mutant forms of TfR2 that are associated with human disease in a mouse liver cell line. Intracellular and surface analysis shows that all the TfR2 mutations analyzed cause the intracellular retention of the protein in the endoplasmic reticulum, whereas the wild-type protein is expressed in endocytic structures and at the cell surface. Our results indicate that the majority of mutations that cause type 3 hereditary hemochromatosis are a consequence of the defective localization of the protein.