Kouji Kobiyama

Autophagy is an essential process for physiological homeostasis, but its role in viral infection is only beginning to be elucidated. We show here that the Atg5-Atg12 conjugate, a key regulator of the autophagic process, plays an important role in innate antiviral immune responses. Atg5-deficient mouse embryonic fibroblasts (MEFs) were resistant to vesicular stomatitis virus replication, which was largely due to hyperproduction of type I interferons in response to immunostimulatory RNA (isRNA), such as virus-derived, double-stranded, or 5'-phosphorylated RNA. Similar hyperresponse to isRNA was also observed in Atg7-deficient MEFs, in which Atg5-Atg12 conjugation is impaired. Overexpression of Atg5 or Atg12 resulted in Atg5-Atg12 conjugate formation and suppression of isRNA-mediated signaling. Molecular interaction studies indicated that the Atg5-Atg12 conjugate negatively regulates the type I IFN production pathway by direct association with the retinoic acid-inducible gene I (RIG-I) and IFN-beta promoter stimulator 1 (IPS-1) through the caspase recruitment domains (CARDs). Thus, in contrast to its role in promoting the bactericidal process, a component of the autophagic machinery appears to block innate antiviral immune responses, thereby contributing to RNA virus replication in host cells.

Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis. Methods and Results: Using single-cell RNA-sequencing of aortic leukocytes from chow diet– and Western diet–fed Apoe −/− and Ldlr −/− mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe −/− and Ldlr −/− mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type–specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.