SUMMARY Autophagy is known to be important in presentation of cytosolic antigens on MHC class II (MHC II). However, the role of autophagic process in antigen presentation in vivo is unclear. Mice with dendritic cell (DC)-conditional deletion in Atg5, a key autophagy gene, showed impaired CD4+ T cell priming after herpes simplex virus infection and succumbed to rapid disease. The most pronounced defect of Atg5−/− DCs was the processing and presentation of phagocytosed antigens containing Toll-like receptor stimuli for MHC class II. In contrast, cross-presentation of peptides on MHC I was intact in the absence of Atg5. Although induction of metabolic autophagy did not enhance MHC II presentation, autophagic machinery was required for optimal phagosome-to-lysosome fusion and subsequent processing of antigen for MHC II loading. Thus, our study revealed that DCs utilize autophagic machinery to optimally process and present extracellular microbial antigens for MHC II presentation.

Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0267-5) contains supplementary material, which is available to authorized users.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, in some cases leading to gastrointestinal disorders 1 – 4 . Here we report that viral community assembly in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium/early stool samples show few or no particles, but by one month of life particle numbers achieve 10 9 per gram, and these numbers appear to persist through life 5 – 7 . We investigated the origin of these viral populations using shotgun metagenomic sequencing of viral-enriched preparations and whole microbial communities, and followed up with targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut, and by one month prophage induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies exclusively fed formula versus those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections 8 – 10 . Phage populations also differed associated with breastfeeding. Evidently colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells, with the second phase modulated by breastfeeding.

BackgroundHistorically, the human womb has been thought to be sterile in healthy pregnancies, but this idea has been challenged by recent studies using DNA sequence-based methods, which have suggested that the womb is colonized with bacteria. For example, analysis of DNA from placenta samples yielded small proportions of microbial sequences which were proposed to represent normal bacterial colonization. However, an analysis by our group showed no distinction between background negative controls and placenta samples. Also supporting the idea that the womb is sterile is the observation that germ-free mammals can be generated by sterile delivery of neonates into a sterile isolator, after which neonates remain germ-free, which would seem to provide strong data in support of sterility of the womb.ResultsTo probe this further and to investigate possible placental colonization associated with spontaneous preterm birth, we carried out another study comparing microbiota in placenta samples from 20 term and 20 spontaneous preterm deliveries. Both 16S rRNA marker gene sequencing and shotgun metagenomic sequencing were used to characterize placenta and control samples. We first quantified absolute amounts of bacterial 16S rRNA gene sequences using 16S rRNA gene quantitative PCR (qPCR). As in our previous study, levels were found to be low in the placenta samples and indistinguishable from negative controls. Analysis by DNA sequencing did not yield a placenta microbiome distinct from negative controls, either using marker gene sequencing as in our previous work, or with shotgun metagenomic sequencing. Several types of artifacts, including erroneous read classifications and barcode misattribution, needed to be identified and removed from the data to clarify this point.ConclusionsOur findings do not support the existence of a consistent placental microbiome, in either placenta from term deliveries or spontaneous preterm births.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0575-4) contains supplementary material, which is available to authorized users.

Monocytes patrol various tissues for signs of infection and inflammation. Inflammatory monocytes enter peripheral tissues at sites of microbial infection and differentiate into dendritic cells and macrophages. Here, we examined the importance of monocytes in primary mucosal infection with herpes simplex virus 2 (HSV-2), and demonstrate that monocyte-derived APCs are required to elicit IFN-γ secretion from effector Th1 cells to mediate antiviral protection. However, monocyte-derived APCs were dispensable for the generation of Th1 immunity and for the restimulation of memory Th1 cells during secondary viral challenge. These results demonstrate that distinct APC subsets are dedicated for CD4 T cell priming, elicitation, and memory recall responses to a given viral pathogen within the same mucosal tissue and reveal a specialized role for monocyte-derived APCs in the emergency response to infection. migration | sexually transmitted infection | antigen presentation