From our data, we cannot determine whether this is due to preferential infection by HCMV of monocyte lineage committed cells within the HSPC compartment or whether HCMV infects multipotent cells, which are then either preferentially skewed towards the monocytic lineage or viral gene expression is initiated only when cells start to differentiate in the monocytes lineage. However since it was previously demonstrated that multipotent cells are infected with HCMV Goodrum et al.
Previous studies demonstrated that HCMV infection leads to an increase in monocyte markers Zhu et al. Our results are in line with these studies and show directly and in an unbiased manner that several days post infection, viral gene expression can be found only in cells in the monocyte lineage.
This is especially interesting as it suggests that regardless of the developmental stage in which HCMV infects, the end point, a few days after infection, are cells with very similar characteristics.
Overall, we use here single cell data to pinpoint the characteristics of the latently infected cells in an unbiased manner. These findings shed light on the characteristics of the latent reservoir, which may help in the effort of developing strategies to eradicate the latently infected cells. The cells were cultured in X-Vivo15 media Lonza supplemented with 2. All experiments were conducted when there was a shift in GFP intensity of the entire population following infection, indicating all cells were infected.
HCMV latency was validated by absence of GFP positive plaques on fibroblasts incubated with infected monocytes cell lysate. For reactivation assays, infected monocytes were counted, plated and co-cultured with primary fibroblasts immediately or following differentiation into dendritic cells DCs. Release of infectious virions was assayed by quantification of GFP positive plaques on the fibroblasts monolayer. Ruxolitinib Ruxo was added at a concentration of 4 uM, either immediately after infection 3hpi or at later time points 1, 2, or 3dpi.
Monocytes were washed to remove residual Ruxo before co-culturing with fibroblasts. RNA 2. The limit of detection was 3 events per sample, with accuracy improved at 10 copies and higher Figure 5—figure supplement 1. RNA-Seq libraries pooled at equimolar concentration were performed in duplicates and sequenced using NextSeq Illumina , with read parameters: Read1: 72 cycles and Read2: 15 cycles.
Reads for gene expression and correlation analyses were normalized using DEseq2. All the analyses in this paper include only the remaining 3, cells. For calculating the Spearman correlation between host genes and either viral expression levels or dpi, in each cell, the sum of viral reads was normalized to the total number of reads in the cell, and the number of reads of each host gene was normalized to the total number of host reads in the cell.
The correlation was calculated across 1, cells for genes. Cells with no viral reads, or with less than different host genes expressed, were omitted. Genes with total number of reads less than 20 were ignored. The Z-score was calculated based on the mean and the standard deviation calculated over all genes.
Figure 7C, G, H plots for expression levels of groups of genes was calculated according to the average relative expression as follows: First, genes with low level of expression, that is expressed in less than 50 cells, were omitted. Next, the number of host reads per cell was normalized so that all cells will include the same number of reads.
Finally, for each cell the average l o g 2 relative expression over all genes in the group was calculated. The differential expression analysis was done with DESeq2 version 1. The genes sets that were used were GO biological process c5. To calculate differential expression and pathway enrichment within the distinct group of cells that expressed monocyte priming markers, we used the graph-based clustering of the Cell Ranger software, stratifying this group of cells into two distinct clusters and GSEA version 3.
Differential expression and enrichment analysis were done on these two clusters. The cells used in this analysis were filtered to have at least expressed genes and not more than 10, UMIs, while the genes were filtered to have mean expression greater than 0.
All fresh peripheral blood samples were obtained after approval of protocols by the Weizmann Institutional Review Board IRB application 92—1.
Informed consent was obtained from all study participants prior to enrolment in accordance with the Declaration of Helsinki. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. The human pathogen Human Cytomegalovirus HCMV causes a lifelong infection and achieves this through effective modulation of the host's immune response and establishment of a dormant state called latency.
Reactivation from latency can cause life threatening disease. Our understanding of the molecular mechanisms of HCMV latency is far from complete. This study comprehensively characterizes, by applying single cell RNA-seq analysis, latency in monocytes and hematopoietic stem and progenitor cells HSPCs. The authors identify monocyte cell surface markers that enable enrichment of latent cells which harbor higher viral transcript levels and can reactivate more efficiently.
Importantly, these cells are characterized by reduced intrinsic immune responses, which is important for viral gene expression. For HSPCs latently infected with HCMV, viral transcripts could only be detected in monocyte progenitors, and these were also associated with reduced immune responses. In conclusion, this study shows that HCMV drives hematopoietic cells towards a weaker immune-responsive state, which is crucial for the virus to eventually reactivate and cause disease.
Thank you for submitting your article "Single cell analysis reveals human cytomegalovirus drives latently infected cells towards an anergic-like monocyte state" for consideration by eLife. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Overall, the reviewers were very impressed with the data and how it was presented. In their manuscript entitled "Single cell analysis reveals human cytomegalovirus drives latently infected cells towards an anergic-like monocyte state", the authors employ scRNA-seq to analyze determinants that are associated with HCMV latency.
They go on to show that these differences are not due to differences in virus entry but are rather induced upon virus entry. They show that cells exhibiting higher viral transcript levels support more efficient reactivation. The same phenotype was observed in HSPC-derived monocytes indicating that HCMV infection drives an immunologically anergic-like state of monocytic cells.
This would also support the conclusion from Figure 2, especially if the viral load could also be measured and would show lower values in cells with higher CD74 surface expression. As many viral genes act as immune evasins, it is not surprising that cells that express higher levels of these viral genes show signs of a counter-regulated IFNgamma response, namely a reduction in MHCII and CD74 surface levels.
Furthermore, it is not surprising that this also augments virus reactivation. The million-dollar-question, however, is how it is possible that viral gene expression so closely matches the profile which is observed in late lytic infection despite no apparent DNA replication.
The data obtained using ruxolitinib to inhibit IFN signaling provide strong evidence that the observed viral gene expression indeed results from de novo transcriptional activity and not from virion-associated RNA the authors may want to state this in the Discussion. The key open question, however, is at what time after infection this viral gene expression occurs.
It could either occur immediately after virus entry and then be more and more efficiently silenced, or it could be rather continuous thereby reflecting "true latent" viral gene expression. To clarify this important question, the authors should infect monocytes with HCMV and apply ruxolitinib at different time of infection and analyze the extent and expression profile of viral genes at 6dpi.
This could e. Furthermore, they should apply ruxolitinib for the first three days and then replace the media with conditioned media from HCMV-infected monocytes. In case viral gene expression only occurs within the first days post infection and is then efficiently silenced, treatment with ruxolitinib later on should not make any difference.
The distribution of CD74 surface expression in infected cells does not significantly differ from what is seen in uninfected cells nor changed along infection. These results thus do not support two or several biologically distinct groups, rather a gradient of expression of CD74 which following infection is most likely affected by the innate immune response to infection which increase CD74 expression and by the effects of the virus which counteracts this increase.
Our results indeed show that the viral load both genomes and transcripts are lower in cells with higher CD74 surface expression Figure 2B, D. As suggested by the reviewer, we conducted the proposed experiment. This indeed supports, as the reviewer said, that there is continuous viral gene expression but the reduced effect of ruxolitinib also points that viral gene expression is gradually repressed.
The results of applying the drug at the beginning of infection and then removing it 3dpi showed decreased effect on viral transcript levels compared to adding a new dose of ruxolitinib also at 72hpi, further supporting there is continuous expression of viral transcripts. However, since replacing the media also for untreated cells led to decreased viral transcripts levels, as shown in Author response image 1 , these results are more complicated for interpretation and we did not include them in the manuscript.
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We thank the members of the Stern-Ginossar lab for critical reading of the manuscript.
NS-G is incumbent of the skirball career development chair in new scientist. Human subjects: All fresh peripheral blood samples were obtained after approval of protocols by the Weizmann Institutional Review Board IRB application Informed written consent was obtained from all volunteers, and all experiments were carried out in accordance with the approved guidelines. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.
Article citation count generated by polling the highest count across the following sources: Scopus , Crossref , PubMed Central.
Short-chain fatty acids SCFAs acetate, propionate, and butyrate are produced in large quantities by the gut microbiome and contribute to a wide array of physiological processes. Here, we systematically investigate how SCFAs alter the epigenome. Using quantitative proteomics of histone modification states, we identified rapid and sustained increases in histone acetylation after the addition of butyrate or propionate, but not acetate.
While decades of prior observations would suggest that hyperacetylation induced by SCFAs are due to inhibition of histone deacetylases HDACs , we found that propionate and butyrate instead activate the acetyltransferase p For many intracellular pathogens, the phagosome is the site of events and interactions that shape infection outcome. Phagosomal membrane damage, in particular, is proposed to benefit invading pathogens.
To define the innate immune consequences of this damage, we profiled macrophage transcriptional responses to wild-type Mycobacterium tuberculosis Mtb and mutants that fail to damage the phagosomal membrane. We identified a set of genes with enhanced expression in response to the mutants.
These genes represented a late component of the TLR2-dependent transcriptional response to Mtb, distinct from an earlier component that included Tnf. Expression of the later component was inherent to TLR2 activation, dependent upon endosomal uptake, and enhanced by phagosome acidification.
Canonical Mtb virulence factors that contribute to phagosomal membrane damage blunted phagosome acidification and undermined the endosome-specific response. Profiling cell survival and bacterial growth in macrophages demonstrated that the attenuation of these mutants is partially dependent upon TLR2.
Further, TLR2 contributed to the attenuated phenotype of one of these mutants in a murine model of infection. These results demonstrate two distinct components of the TLR2 response and identify a component dependent upon endosomal uptake as a point where pathogenic bacteria interfere with the generation of effective inflammation.
This interference promotes TB pathogenesis in both macrophage and murine infection models. Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like Escherichia coli colony biofilms grown on agar-solidified media.
Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during E. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. CMV eventually becomes latent within T lymphocytes, endothelial cells, and macrophages. Suppression of cell-mediated immunity e.
The virus has the ability to induce immunosuppression during primary infections and reactivation of latent infections. This virus can also produce an IL analogue that inhibits TH1 protective responses. Diagnosis of a CMV infection can be confirmed by detection of CMV-induced large inclusion bodies present in urine sediment. The owl's eye appearance of CMV-infected cells can easily be seen in tissue or organ preparations from any part of the body.
Cells are enlarged and contain intranuclear and intracytoplasmic inclusions and peripheralized chromatin. An atypical lymphocytosis is also present in a complete blood count. The ability to culture CMV from the patient is the most reliable diagnosis. CMV can be found in most body fluids and cultured on eukaryotic diploid fibroblast cells. Viral growth in tissue culture can be visualized 16—36 hours after inoculation by applying monoclonal antibodies and immunofluorescent staining.
Patients infected with CMV who have symptoms of mononucleosis i. Viral antigens can be detected with an immunofluorescence assay. Ganciclovir, valganciclovir, cidofovir, and foscarnet have all been approved to treat diseases caused by CMV in immunocompromised patients. Hyperimmune human anti-CMV immunoglobulin has been used in combination with anti-viral medications to reduce CMV disease associated with renal and bone marrow transplantations. Safe-sex practices as well as blood and tissue screening will help limit the spread of CMV.
Send comments and email to Dr. Neal R. The human cytomegalovirus HCMV can infect a remarkably broad cell range within its host, including parenchymal cells and connective tissue cells of virtually any organ and various hematopoietic cell types. Epithelial cells, endothelial cells, fibroblasts and smooth muscle cells are the predominant targets for virus replication. The pathogenesis of acute HCMV infections is greatly influenced by this broad target cell range.
Infection of epithelial cells presumably contributes to inter-host transmission.
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