Systemic Virus Infections Differentially Modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo

Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants

Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections

Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.Andreas Meyerhans and Javier P. Martinez were supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER grant no. SAF2016-75505-R (AEI/MINEIC/FEDER, UE). Mark Brönstrup, Andreas Meyerhans and Javier P. Martinez would like to acknowledge a networking contribution from the COST Action CM1407 “Challenging organic syntheses inspired by nature – from natural products chemistry to drug discovery”. Martin Messerle and Thomas F. Schulz were supported by funding from DZIF (project 07.802 TTU IICH). Christine Goffinet, Thomas Pietschmann and Mark Brönstrup were supported by a grant provided by “Innovationsfonds der Helmholtz-Zentren”. Christine Goffinet was supported by a DFG grant within German African Cooperation Projects in Infectiology (GO2153/3-1) and by funding of the Helmholtz Center for Infection Research (HZI) and of Berlin Institute of Health (BIH). Sergej Franz was supported by the Infection Biology international PhD program of Hannover Biomedical Research Schoo

MCMV activates Ly49H⁺ NK cells and expands Ag-specific CD8⁺ T cells from Siglec-H^−/− and wt mice with similar viral clearance in the primary and secondary organs.

<p>Siglec-H^−/− and wt chimeric mice were infected with 5×10⁴ PFU of wt MCMV or mock treated with PBS. (<b>A, B</b>) IFNγ serum concentrations were determined at 36 h p.i. and the MFI of CD69 expression on NK1.1⁺ blood NK cells was quantified by flow cytometry. (<b>C, D</b>) Representative histogram overlays and quantification of KLRG-1 expression on splenic NK1.1⁺ Ly49H⁺ cells at day 8 p.i. (<b>E</b>) Representative FACS plots for H-2D^b M45 tetramer staining and CD62L expression among CD8⁺ splenocytes. (<b>F, G</b>) Quantification of frequencies and absolute numbers of tetramer⁺ CD8⁺ T cells from the tetramer staining shown in (E). (<b>H</b>) Shows frequencies of IFNγ⁺ cells among CD8⁺ T cells after restimulation with the indicated concentrations of H-2D^b M45 peptide. (<b>I</b>) Viral load was measured in the spleen, liver and salivary glands of MCMV infected Siglec-H^−/− and wt mice at day 3, 6 and 8 p.i. Dashed line indicates the limit of detection. Data shown are pooled from 2–3 individual experiments. **<i>p</i><0.01 Students t-test, ns = not significant, nd = not detectable.</p

Siglec-H is downregulated upon MCMV infection <i>in vivo</i>.

<p>pDCre x RFP mice were infected with 5×10⁴ PFU MCMV <i>in vivo</i>. (<b>A</b>) Representative FACS plots show CD11c versus Siglec-H of live splenocytes at 36 h p.i. (<b>B</b>) Gating strategy showing exclusion of B-, T-, NK-T, and NK cells by CD19, CD3ε, NK1.1 in a default channel. RFP⁺ pDCs were gated as CD11c^int B220⁺ MHCII^int to exclude MHCII⁻ DC precursors. Histogram overlays show RFP⁺ pDCs from mock (bold line) and MCMV infected (dashed line) pDCre x RFP mice at 36 h p.i. Isotype staining is displayed as grey histogram. Data are representative of 2 independent experiments using 4–5 mice with comparable results.</p

Siglec-H receptor does not play a role in pDC infection.

<p>Flt3-L derived CD11c⁺ B220⁺ pDCs and CD11c⁺ B220⁻ cDCs were sorted from BMDC cultures of wt and Siglec-H^−/− mice. DCs were mock treated with PBS or MCMV-GFP infected at MOI 2. (<b>A</b>) Representative FACS plots show Siglec-H expression and MCMV-GFP expression at 24 h p.i. (<b>B</b>) Quantification of the viral titers per 10⁶ DCs or MEFs at 24 h p.i. (<b>C</b>) Representative histogram plots of CD86 expression on wt and Siglec-H^−/− sorted pDCs (live cell gate) at 24 h p.i. (gray bar = isotype control, gray dashed line = mock treatment, gray solid line = CpG-A treatment, dark bold line = MCMV infection). Data are from one of three individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p

IFNα serum levels are elevated in the absence of Siglec-H upon MCMV infection.

<p>Lethally irradiated CD45.1⁺ wt mice were reconstituted with CD45.2⁺ wt or Siglec-H^−/− BM followed by infection with 5×10⁴ PFU of wt MCMV. (<b>A</b>) FACS plots show efficient donor reconstitution in the blood eight weeks after BM transfer (upper panel). Siglec-H and B220 stainings of splenocytes confirm the lack of Siglec-H expression in wt mice reconstituted with Siglec-H^−/− BM (lower panel). (<b>B</b>) The kinetics of serum IFNα levels 1.5, 3 and 6 days p.i. compared between Siglec-H^−/− and wt mice, n = 4–5 mice/group. Data are from one of two individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p

Characterization of the pDCre x RFP reporter line shows terminal targeting of pDCs.

<p>(<b>A, B</b>) BM and spleen cells from pDCre x RFP mice were stained for Siglec-H, CD11c, MHCII, CD3ε, CD19 and NK1.1 (<b>A</b>) Characterization of the reporter expression (RFP) by different cell types: pDCs were gated as Siglec-H⁺, B cells as Siglec-H⁻ CD19⁺ CD3ε⁻ NK1.1⁻, T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁻, NK-T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁺, and NK cells as Siglec-H⁻ CD19⁻ CD3ε⁻ CD11c^int NK1.1⁺. FACS plots are representative for two individual experiments. (<b>B</b>) Characterization of the RFP reporter expression by CD11c^hi MHCII^hi cDCs in spleen and CD11c^int CD19⁻ CD3ε⁻ NK1.1⁻ Siglec-H⁻ cells in BM. (<b>C</b>) Quantification of (A, B) showing pooled data from 2 independent experiments using 4–5 mice/group. (<b>D</b>) Phenotypic comparison of pDC markers expressed by RFP⁻ and RFP⁺ pDCs from BM (top panel) and spleen (bottom panel). pDCs were gated as Siglec-H⁺ CD11c^int. Histogram overlays display the isotype controls as dashed line, and the marker expression by RFP⁻ pDCs as grey filled histogram and by RFP⁺ pDCs as bold line. Data shown are from one representative experiment out of two using 4 mice/group. (<b>E</b>) Splenic <i>ex vivo</i> pDCs were purified by FACS sorting from B16-Flt3L treated pDCre x RFP mice and incubated with the indicated MOIs of MCMV <i>in vitro</i>. IFNα/TNFα concentrations were quantified in the supernatants after 24 h incubation by ELISA or cytometric bead assay. Data shown are from one representative experiment out of two using a pool of 3 mice. The differences between RFP⁻ and RFP⁺ pDCs were not significant as calculated by Students t-test. Data displayed in (D, E) are from one out of two individual experiments with similar results. Data are displayed as mean ± SD.</p

Evidence for PTGER4 ,PSCA, and MBOAT7 as risk genes for gastric cancer on the genome and transcriptome level

Genetic associations between variants on chromosome 5p13 and 8q24 and gastric cancer (GC) have been previously reported in the Asian population. We aimed to replicate these findings and to characterize the associations at the genome and transcriptome level. We performed a fine-mapping association study in 1926 GC patients and 2012 controls of European descent using high dense SNP marker sets on both chromosomal regions. Next, we performed expression quantitative trait locus (eQTL) analyses using gastric transcriptome data from 143 individuals focusing on the GC associated variants. On chromosome 5p13 the strongest association was observed at rs6872282 (P = 2.53 x 10(-04)) and on chromosome 8q24 at rs2585176 (P = 1.09 x 10(-09)). On chromosome 5p13 we found cis-eQTL effects with an up-regulation of PTGER4 expression in GC risk allele carrier (P = 9.27 x 10(-11)). On chromosome 8q24 we observed cis-eQTL effects with an upregulation of PSCA expression in GC risk allele carrier (P = 2.17 x 10(-47)). In addition, we found trans-eQTL effects for the same variants on 8q24 with a downregulation of MBOAT7 expression in GC risk allele carrier (P = 3.11 x 10(-09)). In summary, we confirmed and refined the previously reported GC associations at both chromosomal regions. Our data point to shared etiological factors between Asians and Europeans. Furthermore, our data imply an upregulated expression of PTGER4 and PSCA as well as a downregulated expression of MBOAT7 in gastric tissue as risk-conferring GC pathomechanisms