Stanford Advisors

All Publications

  • Sequential MAVS and MyD88/TRIF signaling triggers anti-viral responses of tick-borne encephalitis virus-infected murine astrocytes JOURNAL OF NEUROSCIENCE RESEARCH Ghita, L., Breitkopf, V., Mulenge, F., Pavlou, A., Gern, O., Duran, V., Prajeeth, C., Kohls, M., Jung, K., Stangel, M., Steffen, I., Kalinke, U. 2021


    Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is typically transmitted upon tick bite and can cause meningitis and encephalitis in humans. In TBEV-infected mice, mitochondrial antiviral-signaling protein (MAVS), the downstream adaptor of retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling, is needed to induce early type I interferon (IFN) responses and to confer protection. To characterize the brain-resident cell subset that produces protective IFN-β in TBEV-infected mice, we isolated neurons, astrocytes, and microglia from mice and exposed these cell types to TBEV in vitro. Under such conditions, neurons showed the highest percentage of infected cells, whereas astrocytes and microglia were infected to a lesser extent. In the supernatant (SN) of infected neurons, IFN-β was not detectable, while infected astrocytes showed high and microglia low IFN-β expression. Transcriptome analyses of astrocytes implied that MAVS signaling was needed early after TBEV infection. Accordingly, MAVS-deficient astrocytes showed enhanced TBEV infection and significantly reduced early IFN-β responses. Nevertheless, at later time points, moderate amounts of IFN-β were detected in the SN of infected MAVS-deficient astrocytes. Transcriptome analyses indicated that MAVS deficiency negatively affected the induction of early anti-viral responses, which resulted in significantly increased TBEV replication. Treatment with MyD88 and TRIF inhibiting peptides reduced only late IFN-β responses of TBEV-infected WT astrocytes and blocked entirely IFN-β responses of infected MAVS-deficient astrocytes. Thus, upon TBEV exposure of brain-resident cells, astrocytes are important IFN-β producers showing biphasic IFN-β induction that initially depends on MAVS and later on MyD88/TRIF signaling.

    View details for DOI 10.1002/jnr.24923

    View details for Web of Science ID 000676133300001

    View details for PubMedID 34296786

  • Fucosylated lipid nanocarriers loaded with antibiotics efficiently inhibit mycobacterial propagation in human myeloid cells. Journal of controlled release : official journal of the Controlled Release Society Durán, V., Grabski, E., Hozsa, C., Becker, J., Yasar, H., Monteiro, J. T., Costa, B., Koller, N., Lueder, Y., Wiegmann, B., Brandes, G., Kaever, V., Lehr, C. M., Lepenies, B., Tampé, R., Förster, R., Bošnjak, B., Furch, M., Graalmann, T., Kalinke, U. 2021; 334: 201-212


    Antibiotic treatment of tuberculosis (TB) is complex, lengthy, and can be associated with various adverse effects. As a result, patient compliance often is poor, thus further enhancing the risk of selecting multi-drug resistant bacteria. Macrophage mannose receptor (MMR)-positive alveolar macrophages (AM) constitute a niche in which Mycobacterium tuberculosis replicates and survives. Therefore, we encapsulated levofloxacin in lipid nanocarriers functionalized with fucosyl residues that interact with the MMR. Indeed, such nanocarriers preferentially targeted MMR-positive myeloid cells, and in particular, AM. Intracellularly, fucosylated lipid nanocarriers favorably delivered their payload into endosomal compartments, where mycobacteria reside. In an in vitro setting using infected human primary macrophages as well as dendritic cells, the encapsulated antibiotic cleared the pathogen more efficiently than free levofloxacin. In conclusion, our results point towards carbohydrate-functionalized nanocarriers as a promising tool for improving TB treatment by targeted delivery of antibiotics.

    View details for DOI 10.1016/j.jconrel.2021.04.012

    View details for PubMedID 33865899

  • Reprogramming of Small Noncoding RNA Populations in Peripheral Blood Reveals Host Biomarkers for Latent and Active Mycobacterium tuberculosis Infection. mBio de Araujo, L. S., Ribeiro-Alves, M., Leal-Calvo, T., Leung, J., Durán, V., Samir, M., Talbot, S., Tallam, A., Mello, F. e., Geffers, R., Saad, M. H., Pessler, F. 2019; 10 (6)


    In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB (n = 8), latent TB infection (LTBI; n = 21), and treated LTBI (LTBItt; n = 6) and in uninfected exposed controls (ExC; n = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of Mycobacterium tuberculosis infection on chest radiographs. SNORD104 expression decreased during M. tuberculosis infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like (P = 0.03) but not M1-like (P = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB.IMPORTANCE Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active M. tuberculosis infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection.

    View details for DOI 10.1128/mBio.01037-19

    View details for PubMedID 31796535

    View details for PubMedCentralID PMC6890987

  • Preferential uptake of chitosan-coated PLGA nanoparticles by primary human antigen presenting cells. Nanomedicine : nanotechnology, biology, and medicine Durán, V., Yasar, H., Becker, J., Thiyagarajan, D., Loretz, B., Kalinke, U., Lehr, C. M. 2019; 21: 102073


    Biodegradable polymeric nanoparticles (NP) made from poly (lactid-co-glycolide) acid (PLGA) and chitosan (CS) hold promise as innovative formulations for targeted delivery. Since interactions of such NP with primary human immune cells have not been characterized, yet, here we assessed the effect of PLGA or CS-PLGA NP treatment on human peripheral blood mononuclear cells (PBMC), as well as on monocyte-derived DC (moDC). Amongst PBMC, antigen presenting cells (APC) showed higher uptake of both NP preparations than lymphocytes. Furthermore, moDC internalized CS-PLGA NP more efficiently than PLGA NP, presumably because of receptor-mediated endocytosis. Consequently, CS-PLGA NP were delivered mostly to endosomal compartments, whereas PLGA NP primarily ended up in lysosomes. Thus, CS-PLGA NP confer enhanced delivery to endosomal compartments of APC, offering new therapeutic options to either induce or modulate APC function and to inhibit pathogens that preferentially infect APC.

    View details for DOI 10.1016/j.nano.2019.102073

    View details for PubMedID 31376570

  • Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines. Virulence Becker, J., Kinast, V., Döring, M., Lipps, C., Duran, V., Spanier, J., Tegtmeyer, P. K., Wirth, D., Cicin-Sain, L., Alcamí, A., Kalinke, U. 2018; 9 (1): 1669-1684


    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.

    View details for DOI 10.1080/21505594.2018.1535785

    View details for PubMedID 30403913

    View details for PubMedCentralID PMC7000197

  • Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway. EBioMedicine Lirussi, D., Ebensen, T., Schulze, K., Trittel, S., Duran, V., Liebich, I., Kalinke, U., Guzmán, C. A. 2017; 22: 100-111


    Cyclic di-nucleotides (CDN) are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA) is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8+ cytotoxic T lymphocytes (CTL) is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α) is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation.

    View details for DOI 10.1016/j.ebiom.2017.07.016

    View details for PubMedID 28754303

    View details for PubMedCentralID PMC5552247

  • Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration. PLoS pathogens González-Motos, V., Jürgens, C., Ritter, B., Kropp, K. A., Durán, V., Larsen, O., Binz, A., Ouwendijk, W. J., Lenac Rovis, T., Jonjic, S., Verjans, G. M., Sodeik, B., Krey, T., Bauerfeind, R., Schulz, T. F., Kaufer, B. B., Kalinke, U., Proudfoot, A. E., Rosenkilde, M. M., Viejo-Borbolla, A. 2017; 13 (5): e1006346


    Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.

    View details for DOI 10.1371/journal.ppat.1006346

    View details for PubMedID 28542541

    View details for PubMedCentralID PMC5444840

  • Antigen presenting cell-selective drug delivery by glycan-decorated nanocarriers. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V Frenz, T., Grabski, E., Durán, V., Hozsa, C., Stępczyńska, A., Furch, M., Gieseler, R. K., Kalinke, U. 2015; 95 (Pt A): 13-7


    Targeted drug delivery systems hold promise for selective provision of active compounds to distinct tissues or cell subsets. Thus, locally enhanced drug concentrations are obtained that would confer improved efficacy. As a consequence adverse effects should be diminished, as innocent bystander cells are less affected. Currently, several controlled drug delivery systems based on diverse materials are being developed. Some systems exhibit material-associated toxic effects and/or show low drug loading capacity. In contrast, liposomal nanocarriers are particularly favorable because they are well tolerated, poorly immunogenic, can be produced in defined sizes, and offer a reasonable payload capacity. Compared with other immune cells, professional antigen-presenting cells (APCs) demonstrate enhanced liposome uptake mediated by macropinocytosis, phagocytosis and presumably also by clathrin- and caveolae-mediated endocytosis. In order to further enhance the targeting efficacy toward APCs, receptor-mediated uptake appears advisable. Since APC subsets generally do not express single linage-specific receptors, members of the C-type lectin receptor (CLR) family are compelling targets. Examples of CLR expressed by APCs include DEC-205 (CD205) expressed by myeloid dendritic cells (DC) and monocytes, the mannose receptor C type 1 (MR, CD206) expressed by DC, monocytes and macrophages, DC-SIGN (CD209) expressed by DC, and several others. These receptors bind glycans, which are typically displayed by pathogens and thus support pathogen uptake and endocytosis. Further research will elucidate whether glycan-decorated liposomes will not only enhance APCs targeting but also enable preferential delivery of their payload to discrete subcellular compartments.

    View details for DOI 10.1016/j.ejpb.2015.02.008

    View details for PubMedID 25701806