• A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells.

      Dag, Franziska; Weingärtner, Adrien; Butueva, Milada; Conte, Ianina; Holzki, Julia; May, Tobias; Adler, Barbara; Wirth, Dagmar; Cicin-Sain, Luka; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2013-06-17)
      The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood.
    • Block of death-receptor apoptosis protects mouse cytomegalovirus from macrophages and is a determinant of virulence in immunodeficient hosts.

      Ebermann, Linda; Ruzsics, Zsolt; Guzmán, Carlos A; van Rooijen, Nico; Casalegno-Garduño, Rosaely; Koszinowski, Ulrich; Cičin-Šain, Luka; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2012-12)
      The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC(-/-)), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system.
    • The chromatin remodeling factor SPOC1 acts as a cellular restriction factor against human cytomegalovirus by repressing the major immediate-early promoter.

      Reichel, Anna; Stilp, Anne-Charlotte; Scherer, Myriam; Reuter, Nina; Lukassen, Sören; Kasmapour, Bahram; Schreiner, Sabrina; Cicin-Sain, Luka; Winterpacht, Andreas; Stamminger, Thomas; et al. (2018-05-09)
      The cellular protein SPOC1 (survival time-associated PHD finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and DNA damage response. It binds H3K4me2/3 containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of HCMV replication. We show that expression of the immediate-early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection SPOC1 is downregulated in a GSK-3β-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed augmented initiation of viral IE gene expression. This occurs in a MOI-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection while a later upregulation had no negative impact suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate-early promoter (MIEP) strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and subsequent recruitment of heterochromatin building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections.
    • The chromatin remodeling factor SPOC1 acts as a cellular restriction factor against human cytomegalovirus by repressing the major immediate-early promoter.

      Reichel, Anna; Stilp, Anne-Charlotte; Scherer, Myriam; Reuter, Nina; Lukassen, Sören; Kasmapour, Bahram; Schreiner, Sabrina; Cicin-Sain, Luka; Winterpacht, Andreas; Stamminger, Thomas; et al. (2018-05-09)
      The cellular protein SPOC1 (survival time-associated PHD finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and DNA damage response. It binds H3K4me2/3 containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of HCMV replication. We show that expression of the immediate-early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection SPOC1 is downregulated in a GSK-3β-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed augmented initiation of viral IE gene expression. This occurs in a MOI-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection while a later upregulation had no negative impact suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate-early promoter (MIEP) strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and subsequent recruitment of heterochromatin building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections. Accumulating evidence indicates that during millennia of co-evolution host cells have developed a sophisticated compilation of cellular factors to restrict cytomegalovirus infections. Defining this equipment is important to understand cellular barriers against viral infection and to develop strategies to utilize these factors for antiviral approaches. So far, constituents of PML nuclear bodies and the interferon gamma inducible protein 16 (IFI16) were known to mediate intrinsic immunity against HCMV. In this study, we identify the chromatin modulator SPOC1 as a novel restriction factor against HCMV. We show that pre-existing high SPOC1 protein levels mediate a silencing of HCMV gene expression via a specific association with an important viral -regulatory element, the major immediate-early promoter. Since SPOC1 expression varies between cell-types, this factor may play an important role in the tissue-specific defense against HCMV.
    • Circulating levels of the anti-oxidant indoleproprionic acid are associated with higher gut microbiome diversity.

      Menni, Cristina; Hernandez, Marisa Matey; Vital, Marius; Mohney, Robert P; Spector, Tim D; Valdes, Ana M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2019-04-29)
      The gut microbiome has recently emerged as an important regulator of insulin resistance and abdominal obesity. The tryptophan metabolite generated by the gut microbiome, indoleproprionic acid (IPA) has been shown to predict the onset of type 2 diabetes. IPA is a metabolite produced by gut microbes from dietary tryptophan that exhibits a high degree of inter-individual variation. The microbiome composition parameters that are associated with circulating levels of this potent anti-oxidant have however not been investigated to date in human populations. In 1018 middle-aged women from the TwinsUK cohort, we assessed the relationship between serum IPA levels and gut microbiome composition targeting the 16S rRNA gene. Microbiome alpha-diversity was positively correlated with serum indoleproprionic acid levels (Shannon Diversity: Beta[95%CI] = 0.19[0.13;0.25], P = 6.41 × 10-10) after adjustment for covariates. Sixteen taxa and 12 operational taxonomic units (OTUs) associated with IPA serum levels. Among these are positive correlations with the butyrate-producing Faecalibacterium prausnitzii, the class Mollicutes and the order RF39 of the Tenericutes, and Coprococcus Negative correlations instead were observed with Eubacterium dolichum previously shown to correlate with visceral fat mass and several genera in the Lachnospiraceae family such as Blautia and Ruminococcus previously shown to correlate with obesity. Microbiome composition parameters explained ~20% of the variation in circulating levels of IPA, whereas nutritional and host genetic parameters explained only ~4%. Our data confirm an association between IPA circulating levels and metabolic syndrome parameters and indicate that gut microbiome composition influences IPA levels.
    • CMV and Immunosenescence: from basics to clinics.

      Solana, Rafael; Tarazona, Raquel; Aiello, Allison E; Akbar, Arne N; Appay, Victor; Beswick, Mark; Bosch, Jos A; Campos, Carmen; Cantisán, Sara; Cicin-Sain, Luka; et al. (2012)
      Alone among herpesviruses, persistent Cytomegalovirus (CMV) markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates "immunosenescence". This contention remains controversial and was the primary subject of the Third International Workshop on CMV & Immunosenescence which was held in Cordoba, Spain, 15-16th March, 2012. Discussions focused on several main themes including the effects of CMV on adaptive immunity and immunosenescence, characterization of CMV-specific T cells, impact of CMV infection and ageing on innate immunity, and finally, most important, the clinical implications of immunosenescence and CMV infection. Here we summarize the major findings of this workshop.
    • CMV immune evasion and manipulation of the immune system with aging.

      Jackson, Sarah E; Redeker, Anke; Arens, Ramon; van Baarle, Debbie; van den Berg, Sara P H; Benedict, Chris A; Čičin-Šain, Luka; Hill, Ann B; Wills, Mark R; Helmholtz Centre for infection research, Inhoffenstr. 7., 38124 Braunschweig, Germany. (2017-06-24)
      Human cytomegalovirus (HCMV) encodes numerous proteins and microRNAs that function to evade the immune response and allow the virus to replicate and disseminate in the face of a competent innate and acquired immune system. The establishment of a latent infection by CMV, which if completely quiescent at the level of viral gene expression would represent an ultimate in immune evasion strategies, is not sufficient for lifelong persistence and dissemination of the virus. CMV needs to reactivate and replicate in a lytic cycle of infection in order to disseminate further, which occurs in the face of a fully primed secondary immune response. Without reactivation, latency itself would be redundant for the virus. It is also becoming clear that latency is not a totally quiescent state, but is characterized by limited viral gene expression. Therefore, the virus also needs immune evasion strategies during latency. An effective immune response to CMV is required or viral replication will cause morbidity and ultimately mortality in the host. There is clearly a complex balance between virus immune evasion and host immune recognition over a lifetime. This poses the important question of whether long-term evasion or manipulation of the immune response driven by CMV is detrimental to health. In this meeting report, three groups used the murine model of CMV (MCMV) to examine if the contribution of the virus to immune senescence is set by the (i) initial viral inoculum, (ii) inflation of T cell responses, (iii) or the balance between functionally distinct effector CD4+ T cells. The work of other groups studying the CMV response in humans is discussed. Their work asks whether the ability to make immune responses to new antigens is compromised by (i) age and HCMV carriage, (ii) long-term exposure to HCMV giving rise to an overall immunosuppressive environment and increased levels of latent virus, or (iii) adapted virus mutants (used as potential vaccines) that have the capacity to elicit conventional and unconventional T cell responses.
    • The Contribution of Cytomegalovirus Infection to Immune Senescence Is Set by the Infectious Dose.

      Redeker, Anke; Remmerswaal, Ester B M; van der Gracht, Esmé T I; Welten, Suzanne P M; Höllt, Thomas; Koning, Frits; Cicin-Sain, Luka; Nikolich-Žugich, Janko; Ten Berge, Ineke J M; van Lier, René A W; et al. (2017)
      The relationship between human cytomegalovirus (HCMV) infections and accelerated immune senescence is controversial. Whereas some studies reported a CMV-associated impaired capacity to control heterologous infections at old age, other studies could not confirm this. We hypothesized that these discrepancies might relate to the variability in the infectious dose of CMV occurring in real life. Here, we investigated the influence of persistent CMV infection on immune perturbations and specifically addressed the role of the infectious dose on the contribution of CMV to accelerated immune senescence. We show in experimental mouse models that the degree of mouse CMV (MCMV)-specific memory CD8+ T cell accumulation and the phenotypic T cell profile are directly influenced by the infectious dose, and data on HCMV-specific T cells indicate a similar connection. Detailed cluster analysis of the memory CD8+ T cell development showed that high-dose infection causes a differentiation pathway that progresses faster throughout the life span of the host, suggesting a virus-host balance that is influenced by aging and infectious dose. Importantly, short-term MCMV infection in adult mice is not disadvantageous for heterologous superinfection with lymphocytic choriomeningitis virus (LCMV). However, following long-term CMV infection the strength of the CD8+ T cell immunity to LCMV superinfection was affected by the initial CMV infectious dose, wherein a high infectious dose was found to be a prerequisite for impaired heterologous immunity. Altogether our results underscore the importance of stratification based on the size and differentiation of the CMV-specific memory T cell pools for the impact on immune senescence, and indicate that reduction of the latent/lytic viral load can be beneficial to diminish CMV-associated immune senescence.
    • Cytomegalovirus infection impairs immune responses and accentuates T-cell pool changes observed in mice with aging.

      Cicin-Sain, Luka; Brien, James D; Uhrlaub, Jennifer L; Drabig, Anja; Marandu, Thomas F; Nikolich-Zugich, Janko; Department of Vaccinology and Applied Microbiology, Helmholtz Center for Infection Research, Braunschweig, Germany. (2012-08)
      Prominent immune alterations associated with aging include the loss of naïve T-cell numbers, diversity and function. While genetic contributors and mechanistic details in the aging process have been addressed in multiple studies, the role of environmental agents in immune aging remains incompletely understood. From the standpoint of environmental infectious agents, latent cytomegalovirus (CMV) infection has been associated with an immune risk profile in the elderly humans, yet the cause-effect relationship of this association remains unclear. Here we present direct experimental evidence that mouse CMV (MCMV) infection results in select T-cell subset changes associated with immune aging, namely the increase of relative and absolute counts of CD8 T-cells in the blood, with a decreased representation of the naïve and the increased representation of the effector memory blood CD8 T-cells. Moreover, MCMV infection resulted in significantly weaker CD8 responses to superinfection with Influenza, Human Herpes Virus I or West-Nile-Virus, even 16 months following MCMV infection. These irreversible losses in T-cell function could not be observed in uninfected or in vaccinia virus-infected controls and were not due to the immune-evasive action of MCMV genes. Rather, the CD8 activation in draining lymph nodes upon viral challenge was decreased in MCMV infected mice and the immune response correlated directly to the frequency of the naïve and inversely to that of the effector cells in the blood CD8 pool. Therefore, latent MCMV infection resulted in pronounced changes of the T-cell compartment consistent with impaired naïve T-cell function.
    • Demarcated thresholds of tumor-specific CD8 T cells elicited by MCMV-based vaccine vectors provide robust correlates of protection.

      Beyranvand Nejad, Elham; Ratts, Robert B; Panagioti, Eleni; Meyer, Christine; Oduro, Jennifer D; Cicin-Sain, Luka; Früh, Klaus; van der Burg, Sjoerd H; Arens, Ramon; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2019-01-31)
      The capacity of cytomegalovirus (CMV) to elicit long-lasting strong T cell responses, and the ability to engineer the genome of this DNA virus positions CMV-based vaccine vectors highly suitable as a cancer vaccine platform. Defined immune thresholds for tumor protection and the factors affecting such thresholds have not well been investigated in cancer immunotherapy. We here determined using CMV as a vaccine platform whether critical thresholds of vaccine-specific T cell responses can be established that relate to tumor protection, and which factors control such thresholds. We generated CMV-based vaccine vectors expressing the E7 epitope and tested these in preclinical models of HPV16-induced cancer. Vaccination was applied via different doses and routes (intraperitoneal (IP), subcutaneous (SC) and intranasal (IN)). The magnitude, kinetics and phenotype of the circulating tumor-specific CD8 Immunization with CMV-based vaccines via the IP or SC route eliciting vaccine-induced CD8 This study highlight the effectiveness of CMV-based vaccine vectors, and shows that demarcated thresholds of vaccine-specific T cells could be defined that correlate to tumor protection. Together, these results may hold importance for cancer vaccine development to achieve high efficacy in vaccine recipients.
    • Early primed KLRG1- CMV-specific T cells determine the size of the inflationary T cell pool.

      Baumann, Nicolas S; Welten, Suzanne P M; Torti, Nicole; Pallmer, Katharina; Borsa, Mariana; Barnstorf, Isabel; Oduro, Jennifer D; Cicin-Sain, Luka; Oxenius, Annette; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-05-01)
      Memory T cell inflation is a process in which a subset of cytomegalovirus (CMV) specific CD8 T cells continuously expands mainly during latent infection and establishes a large and stable population of effector memory cells in peripheral tissues. Here we set out to identify in vivo parameters that promote and limit CD8 T cell inflation in the context of MCMV infection. We found that the inflationary T cell pool comprised mainly high avidity CD8 T cells, outcompeting lower avidity CD8 T cells. Furthermore, the size of the inflationary T cell pool was not restricted by the availability of specific tissue niches, but it was directly related to the number of virus-specific CD8 T cells that were activated during priming. In particular, the amount of early-primed KLRG1- cells and the number of inflationary cells with a central memory phenotype were a critical determinant for the overall magnitude of the inflationary T cell pool. Inflationary memory CD8 T cells provided protection from a Vaccinia virus challenge and this protection directly correlated with the size of the inflationary memory T cell pool in peripheral tissues. These results highlight the remarkable protective potential of inflationary CD8 T cells that can be harnessed for CMV-based T cell vaccine approaches.
    • Expression of S100A8/A9 in HaCaT keratinocytes alters the rate of cell proliferation and differentiation.

      Voss, Andreas; Bode, Günther; Sopalla, Claudia; Benedyk, Malgorzata; Varga, Georg; Böhm, Markus; Nacken, Wolfgang; Kerkhoff, Claus; Institute of Immunology, University of Muenster, Muenster, Germany. (2011-01-21)
      S100A8/A9 promotes NADPH oxidase in HaCaT keratinocytes and subsequently increases NFκB activation, which plays important roles in the balance between epidermal growth and differentiation. S100A8/A9-positive HaCaT cells present with a significantly reduced rate of cell division and greater expression of two keratinocyte differentiation markers, involucrin and filaggrin, than control cells. S100A8/A9 mutants fail to enhance NFκB activation, TNFα-induced IL-8 gene expression and NFκB p65 phosphorylation, and S100A8/A9-positive cells demonstrate better cell survival in forced suspension culture than mutant cells. S100A8/A9 is induced in epithelial cells in response to stress. Therefore, S100A8/A9-mediated growth arrest could have implications for tissue remodeling and repair.
    • The human cytomegalovirus UL51 protein is essential for viral genome cleavage-packaging and interacts with the terminase subunits pUL56 and pUL89.

      Borst, Eva Maria; Kleine-Albers, Jennifer; Gabaev, Ildar; Babic, Marina; Wagner, Karen; Binz, Anne; Degenhardt, Inga; Kalesse, Markus; Jonjic, Stipan; Bauerfeind, Rudolf; et al. (2013-02)
      Cleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.
    • Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines.

      Becker, Jennifer; Kinast, Volker; Döring, Marius; Lipps, Christoph; Duran, Veronica; Spanier, Julia; Tegtmeyer, Pia-Katharina; Wirth, Dagmar; Cicin-Sain, Luka; Alcamí, Antonio; et al. (2018-01-01)
      Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-β. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.
    • IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

      Popovic, Branka; Golemac, Mijo; Podlech, Jürgen; Zeleznjak, Jelena; Bilic-Zulle, Lidija; Lukic, Miodrag L; Cicin-Sain, Luka; Reddehase, Matthias J; Sparwasser, Tim; Krmpotic, Astrid; et al. (2017-04)
      Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection.
    • Leishmania promastigotes lack phosphatidylserine but bind annexin V upon permeabilization or miltefosine treatment.

      Weingärtner, Adrien; Kemmer, Gerdi; Müller, Frederic D; Zampieri, Ricardo Andrade; Gonzaga dos Santos, Marcos; Schiller, Jürgen; Pomorski, Thomas Günther; Institute of Biology, Humboldt-Universität zu Berlin, Berlin, Germany. (2012)
      The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and (31)P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.
    • Life-long control of cytomegalovirus (CMV) by T resident memory cells in the adipose tissue results in inflammation and hyperglycemia.

      Contreras, Nico A; Sitnik, Katarzyna M; Jeftic, Ilija; Coplen, Christopher Patrick; Čičin-Šain, Luka; Nikolich-Žugich, Janko; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-06-01)
      Cytomegalovirus (CMV) is a ubiquitous herpesvirus infecting most of the world's population. CMV has been rigorously investigated for its impact on lifelong immunity and potential complications arising from lifelong infection. A rigorous adaptive immune response mounts during progression of CMV infection from acute to latent states. CD8 T cells, in large part, drive this response and have very clearly been demonstrated to take up residence in the salivary gland and lungs of infected mice during latency. However, the role of tissue resident CD8 T cells as an ongoing defense mechanism against CMV has not been studied in other anatomical locations. Therefore, we sought to identify additional locations of anti-CMV T cell residency and the physiological consequences of such a response. Through RT-qPCR we found that mouse CMV (mCMV) infected the visceral adipose tissue and that this resulted in an expansion of leukocytes in situ. We further found, through flow cytometry, that adipose tissue became enriched in cytotoxic CD8 T cells that are specific for mCMV antigens from day 7 post infection through the lifespan of an infected animal (> 450 days post infection) and that carry markers of tissue residence. Furthermore, we found that inflammatory cytokines are elevated alongside the expansion of CD8 T cells. Finally, we show a correlation between the inflammatory state of adipose tissue in response to mCMV infection and the development of hyperglycemia in mice. Overall, this study identifies adipose tissue as a location of viral infection leading to a sustained and lifelong adaptive immune response mediated by CD8 T cells that correlates with hyperglycemia. These data potentially provide a mechanistic link between metabolic syndrome and chronic infection.
    • The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus.

      Kutle, Ivana; Sengstake, Sarah; Templin, Corinna; Glaß, Mandy; Kubsch, Tobias; Keyser, Kirsten A; Binz, Anne; Bauerfeind, Rudolf; Sodeik, Beate; Čičin-Šain, Luka; et al. (2017-11-14)
      Cell rounding is a hallmark of the cytopathic effect induced by cytomegaloviruses. By screening a panel of deletion mutants of mouse cytomegalovirus (MCMV) a mutant was identified that did not elicit cell rounding and lacked the ability to form typical plaques. Altered cell morphology was assigned to the viral M25 gene. We detected an early 2.8 kb M25 mRNA directing the synthesis of a 105 kDa M25 protein, and confirmed that a late 3.1 kb mRNA encodes a 130 kDa M25 tegument protein. Virions lacking the M25 tegument protein were of smaller size because the tegument layer between capsid and viral envelope was reduced. The ΔM25 mutant did not provoke the rearrangement of the actin cytoskeleton observed after wild-type MCMV infection, and isolated expression of the M25 proteins led to cell size reduction, confirming that they contribute to the morphological changes. Yields of progeny virus and cell-to-cell spread of the ΔM25 mutant in vitro were diminished and replication in vivo was impaired. The identification of an MCMV gene involved in cell rounding provides the basis for investigating the role of this cytopathic effect in CMV pathogenesis.
    • Mouse CMV infection delays antibody class switch upon an unrelated virus challenge.

      Marandu, Thomas F; Finsterbusch, Katja; Kröger, Andrea; Čičin-Šain, Luka; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014-06)
      Poor immune protection upon vaccination is a critical determinant of immunosenescence. Latent Cytomegalovirus (CMV) infection has been associated with poor antibody responses to vaccination, but a causative role for CMV in the poor immune response requires experimental evidence and thus could not be confirmed in clinical studies. To test the hypothesis that latent CMV infection causes poor antibody responses, we infected young or adult mice with mouse CMV and challenged them with Vesicular stomatitis virus (VSV) at 15 or 18months of age. Latent, but not primary infection with mouse CMV resulted in diminished neutralizing titers of the serum IgG fraction at day 7 post challenge, which recovered by day 14 post challenge. This phenomenon was specific for mice infected with mouse CMV, but not mice infected with other herpesviruses, like murine herpesvirus-68 or herpes simplex virus type 1, or mice infected with non-persistent viruses, such as influenza or Vaccinia virus. Hence, our data indicate a delay in IgG class-switch that was specific for the CMV infection. Herpesviral infections did not change the B-cell memory compartment, and increased the size of the effector-memory subset of blood CD4 T-cells only when administered in combination. Furthermore, CD4 T-cell response to VSV infection was maintained in latently infected mice. Therefore, our results argue that latent CMV infection impairs B-cell, but not T-cell responses to a challenge with VSV and delays antibody class-switch by a mechanism which may be independent of T-cell help.
    • Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

      Zheng, Xiaoyan; Oduro, Jennifer D; Boehme, Julia D; Borkner, Lisa; Ebensen, Thomas; Heise, Ulrike; Gereke, Marcus; Pils, Marina C; Krmpotic, Astrid; Guzmán, Carlos A; et al. (PLOS, 2019-09-01)
      Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.