CMMC: FP 2020-2022 - von Stebut-Borschitz, Esther

Introduction

Treatment options against leishmaniasis range from local to systemic options, some having severe side effects or being cost-intensive (with limited access in countries at risk). Despite the development of antigen-specific, life-long immunity in immunocompetent hosts, a vaccine has not been developed yet. Identification of determinants for antigenicity and underlying factors influencing efficient immunity is of high importance for successful (preventive and therapeutic) vaccine development.

Leishmaniasis is a parasitic disease with a high disease burden worldwide. Macrophages (MF) and dendritic cells (DCs) are the major host cells for Leishmania parasites, and these cells are essential for control or susceptibility to the parasite. Under healthy, non-pathological conditions, the skin harbours at least five known MF and DC subsets, which differ phenotypically, functionally and ontologically. To better understand the diverse role these unique myeloid subsets play during anti-Leishmania immunity, we now aim to analyse these cells in more detail. Here, we propose to analyse the role of selected myeloid cell subsets during Leishmania infection in detail, and to characterize the genes and protein expression profile of the different skin myeloid cells involved in anti-Leishmania immunity. We opt to use CyTOF & multi-parameter myeloid cell FACS panel to identify and characterize the different subsets. Next, using our own established protocols, we will isolate myeloid subsets and perform proteome and transcriptome analysis to link the subsets molecular and genetic signature to their function during Leishmania infection. In light of vaccine development, knowledge about immune modulating functions of skin DCs and other myeloid (inflammatory) cells is essential.

Our Aims

1. In vivo analysis of the contribution of myeloid cells to anti-Leishmania immunity
Identify and characterize the different skin-derived MF and DC subsets found in healthy vs. Leishmania-infected tissue.

2. Identification of specific myeloid subsets regulating anti-Leishmania immunity
Performing proteomic and transcriptomic analysis of selected/isolated skin-derived myeloid subsets, during the different phases of Leishmania-immunity.

3. Specific targeting (vaccine) of selected myeloid subsets to induce long-term anti-Leishmania protection

Previous Work

Protective immunity against leishmaniasis is mediated by Leishmania-specific, IFNg-producing Th1/Tc1 cells, whereas Th2/(Th17)/Treg promote parasite susceptibility ADDIN EN.CITE.DATA (1-15). Previously, we have shown that epidermal Langerhans cells regulate anti-Leishmania immunity by inducing peripheral regulatory T cells (Treg), yet fail to induce Th1/Tc1 cells (16). Dermal DCs (dDCs), in contrast, appear to promote protection from leishmaniasis (Fig. 1) ADDIN EN.CITE.DATA (10,12,17-31).

Figure 1

Fig. 1: Role of epidermal LC and different dermal DC subsets in cutaneous leishmaniasis. Mice were infected intradermally with physiological low dose inocula of 103 L. major and disease score was monitored over the indicated time (weeks). A, DT-treated Langerin-DTR and bone marrow chimera (C57BL/6 bone marrow in Langerin-DTR hosts) permitted LC-specific depletion. B, DT-treated Langerin-DTR bone marrow in C57BL/6 hosts for depletion of dDC. A+B, DT was applied as indicated. Lesion development was monitored over time (mean±SEM, n≥3).

Previous work of our lab revealed that in addition to the above-mentioned epidermal and dermal DC subsets, cells other than DCs also contribute to protection (Fig. 2). The role of these subsets will be further addressed here. Several genetic mouse models will be utilized to permit selective targeting of specific myeloid cell population. In addition, a mixed bone-marrow chimeric system will be used to address the contribution of DC-derived molecules (e.g. IL-12p40) to anti-Leishmania immunity (Fig. 3).

Figure 2

Fig. 2: Phenotype of CD11b+ myeloid cells in lesions of resistant vs. susceptible mice. C57BL/6 (black bars) and BALB/c (white bars) mice were infected intradermally with 103 L. major metacyclic promastigotes. A, Lesional inflammatory cells were characterized by pre-gating on CD11b+ cells and further separation based on Ly6G versus Ly6C expression. B, Percentage of Ly6G/Cneg MФ (pink), Ly6Ghigh neutrophils (green), and Ly6Chigh pro-/anti-inflammatory monocytes over nine-weeks are depicted (mean±SEM, n=5).

Figure 3

Fig. 3: Overview of the different skin myeloid cell subsets and cell-specific depletion models. A. DC depletion models (inducible or genetic) using DT/DTR system as depicted. Bone marrow (BM) chimeras are generated by lethal irradiation followed by BM reconstitution of e.g. CD11c-DTR mice. 6 weeks post BM reconstitution; the host (radio-sensitive) hematopoietic compartment is fully replaced by donor-derived cells. B. Other myeloid cell-specific depletion models (inducible or genetic) using DT/DTR system as depicted.

Project Related Publications

Belkaid, Y., Piccirillo, C. A., Mendez, S., Shevach, E. M., and Sacks, D. L. (2002) CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502-507

von Stebut, E., Ehrchen, J. M., Belkaid, Y., Kostka, S. L., Molle, K., Knop, J., Sunderkotter, C., and Udey, M. C. (2003) Interleukin 1alpha promotes Th1 differentiation and inhibits disease progression in Leishmania major-susceptible BALB/c mice. The Journal of experimental medicine 198, 191-199

von Stebut, E., and Udey, M. C. (2004) Requirements for Th1-dependent immunity against infection with Leishmania major. Microbes and infection 6, 1102-1109

Maurer, M., Lopez Kostka, S., Siebenhaar, F., Moelle, K., Metz, M., Knop, J., and von Stebut, E. (2006) Skin mast cells control T cell-dependent host defense in Leishmania major infections. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 20, 2460-2467

Lopez Kostka, S., Dinges, S., Griewank, K., Iwakura, Y., Udey, M. C., and von Stebut, E. (2009) IL-17 promotes progression of cutaneous leishmaniasis in susceptible mice. Journal of immunology 182, 3039-3046

Ehrchen, J. M., Roebrock, K., Foell, D., Nippe, N., von Stebut, E., Weiss, J. M., Munck, N. A., Viemann, D., Varga, G., Muller-Tidow, C., Schuberth, H. J., Roth, J., and Sunderkotter, C. (2010) Keratinocytes determine Th1 immunity during early experimental leishmaniasis. PLoS pathogens 6, e1000871

Dudeck, A., Suender, C. A., Kostka, S. L., von Stebut, E., and Maurer, M. (2011) Mast cells promote Th1 and Th17 responses by modulating dendritic cell maturation and function. European journal of immunology 41, 1883-1893

Fischer, M. R., John, D., Kautz-Neu, K., Schermann, A. I., Schwonberg, K., and von Stebut, E. (2013) Animal model for cutaneous leishmaniasis. Methods in molecular biology 961, 389-402

Ngouateu, O. B., Weller, K., Brohl, K., Kamtchouing, P., Same-Ekobo, A., Dondji, B., Maurer, M., and von Stebut, E. (2015) Impaired T-cell-dependent protection against Leishmania major infection in HIV-positive patients is associated with worsened disease outcome. Experimental dermatology 24, 302-304

Dietze-Schwonberg, K., Lorenz, B., Lopez Kostka, S., Waisman, A., and von Stebut, E. (2016) Parasite Clearance in Leishmaniasis in Resistant Animals Is Independent of the IL-23/IL-17A Axis. The Journal of investigative dermatology 136, 1906-1908

Dietze-Schwonberg, K., Grewe, B., Brosch, S., Kuharev, J., van Zandbergen, G., Rammensee, H. G., Tenzer, S., and von Stebut, E. (2017) In silico prediction of Leishmania major-specific CD8(+) epitopes. Experimental dermatology 26, 838-840

Dietze-Schwonberg, K., Kautz-Neu, K., Lorenz, B., Clausen, B. E., and von Stebut, E. (2017) In cutaneous leishmaniasis, induction of retinoic acid in skin-derived Langerhans cells is not sufficient for induction of parasite persistence-mediating regulatory T cells. Journal of dermatological science 87, 307-309

Dietze-Schwonberg, K., Lorenz, B., Kostka, S. L., Schumak, B., Gessner, A., and von Stebut, E. (2018) Insufficient generation of Th17 cells in IL-23p19-deficient BALB/c mice protects against progressive cutaneous leishmaniasis. Experimental dermatology 27, 101-103

Dietze-Schwonberg, K., Lopez Kostka, S., Lorenz, B., Regen, T., Waisman, A., and von Stebut, E. (2019) IL-17A/F in Leishmania major-resistant C57BL/6 mice. Experimental dermatology 28, 321-323

Mukherjee, S., Sengupta, R., Mukhopadhyay, D., Braun, C., Mitra, S., Roy, S., Kanti Das, N., Chatterjee, U., von Stebut, E., and Chatterjee, M. (2019) Impaired activation of lesional CD8(+) T-cells is associated with enhanced expression of Programmed Death-1 in Indian Post Kala-azar Dermal Leishmaniasis. Scientific reports 9, 762

Kautz-Neu, K., Noordegraaf, M., Dinges, S., Bennett, C. L., John, D., Clausen, B. E., and von Stebut, E. (2011) Langerhans cells are negative regulators of the anti-Leishmania response. The Journal of experimental medicine 208, 885-891

von Stebut, E., Belkaid, Y., Nguyen, B., Wilson, M., Sacks, D. L., and Udey, M. C. (2002) Skin-derived macrophages from Leishmania major-susceptible mice exhibit interleukin-12- and interferon-gamma-independent nitric oxide production and parasite killing after treatment with immunostimulatory DNA. The Journal of investigative dermatology 119, 621-628

Woelbing, F., Kostka, S. L., Moelle, K., Belkaid, Y., Sunderkoetter, C., Verbeek, S., Waisman, A., Nigg, A. P., Knop, J., Udey, M. C., and von Stebut, E. (2006) Uptake of Leishmania major by dendritic cells is mediated by Fcgamma receptors and facilitates acquisition of protective immunity. The Journal of experimental medicine 203, 177-188

Nigg, A. P., Zahn, S., Ruckerl, D., Holscher, C., Yoshimoto, T., Ehrchen, J. M., Wolbing, F., Udey, M. C., and von Stebut, E. (2007) Dendritic cell-derived IL-12p40 homodimer contributes to susceptibility in cutaneous leishmaniasis in BALB/c mice. Journal of immunology 178, 7251-7258

von Stebut, E. (2007) Immunology of cutaneous leishmaniasis: the role of mast cells, phagocytes and dendritic cells for protective immunity. European journal of dermatology : EJD 17, 115-122

Kautz-Neu, K., Meyer, R. G., Clausen, B. E., and von Stebut, E. (2010) Leishmaniasis, contact hypersensitivity and graft-versus-host disease: understanding the role of dendritic cell subsets in balancing skin immunity and tolerance. Experimental dermatology 19, 760-771

Zahn, S., Kirschsiefen, P., Jonuleit, H., Steinbrink, K., and Von Stebut, E. (2010) Human primary dendritic cell subsets differ in their IL-12 release in response to Leishmania major infection. Experimental dermatology 19, 924-926

Kautz-Neu, K., Kostka, S. L., Dinges, S., Iwakura, Y., Udey, M. C., and von Stebut, E. (2011) A role for leukocyte-derived IL-1RA in DC homeostasis revealed by increased susceptibility of IL-1RA-deficient mice to cutaneous leishmaniasis. The Journal of investigative dermatology 131, 1650-1659

von Stebut, E. (2011) Research in practice: Different dendritic cell types in skin with various functions - important implications for intradermal vaccines. Journal der Deutschen Dermatologischen Gesellschaft = Journal of the German Society of Dermatology : JDDG 9, 506-509

Kautz-Neu, K., Schwonberg, K., Fischer, M. R., Schermann, A. I., and von Stebut, E. (2012) Dendritic cells in Leishmania major infections: mechanisms of parasite uptake, cell activation and evidence for physiological relevance. Medical microbiology and immunology 201, 581-592

Girard-Madoux, M. J. H., Kautz-Neu, K., Lorenz, B., Ober-Blobaum, J. L., von Stebut, E., and Clausen, B. E. (2015) IL-10 signaling in dendritic cells attenuates anti-Leishmania major immunity without affecting protective memory responses. The Journal of investigative dermatology 135, 2890-2894

Fischer, M. R., Kunz, C., Dietze-Schwonberg, K., Lorenz, B., and von Stebut, E. (2016) Myeloid cells do not contribute to gender-dependent differences in disease outcome in murine cutaneous leishmaniasis. Cellular immunology 308, 13-18

von Stebut, E. (2017) Parasites Dampen Dendritic Cell Activation to Ensure Their Survival. Trends in parasitology 33, 78-80

von Stebut, E., and Tenzer, S. (2018) Cutaneous leishmaniasis: Distinct functions of dendritic cells and macrophages in the interaction of the host immune system with Leishmania major. International journal of medical microbiology : IJMM 308, 206-214

Fischer, M. R., Schermann, A. I., Twelkmeyer, T., Lorenz, B., Wegner, J., Jonuleit, H., and von Stebut, E. (2019) Humanized mice in cutaneous leishmaniasis-Suitability analysis of human PBMC transfer into immunodeficient mice. Experimental dermatology 28, 1087-1090

Bohn, T., Rapp, S., Luther, N., Klein, M., Bruehl, T. J., Kojima, N., Aranda Lopez, P., Hahlbrock, J., Muth, S., Endo, S., Pektor, S., Brand, A., Renner, K., Popp, V., Gerlach, K., Vogel, D., Lueckel, C., Arnold-Schild, D., Pouyssegur, J., Kreutz, M., Huber, M., Koenig, J., Weigmann, B., Probst, H. C., von Stebut, E., Becker, C., Schild, H., Schmitt, E., and Bopp, T. (2018) Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages. Nature immunology 19, 1319-1329

Publications until 11/2022

Dermicik F, Lopez Kostka S, Tenzer S, Waisman A, Von Stebut E. Cross-reactive, natural IgG recognizing L. major promote parasite internalization by dendritic cells and promote protective immunity. J Mol Med (Berl). 2022 Mar;100(3):451-460.

Lopez Kostka S, Kautz-Neu K, Yogev N, Lukas D, Holzmann B, Waisman A, Clausen BE, von Stebut E. Exclusive Expression of MyD88 on Dendritic Cells Is Sufficient to Induce Protection against Experimental Leishmaniasis. J Invest Dermatol. 2022 Apr;142(4):1230-1233.

Yogev N, Bedke T, Kobayashi Y, Brockmann L, Lukas D, Regen T, Croxford AL, Nikolav A, Hovelmeyer N, von Stebut E, Prinz M, Ubeda C, Maloy KJ, Gagliani N, Flavell RA, Waisman A, and Huber S (2022). CD4(+) T-cell-derived IL-10 promotes CNS inflammation in mice by sustaining effector T cell survival. Cell Rep38, 110565. doi:10.1016/j.celrep.2022.110565.

Publications 2021

de Freitas E Silva R, von Stebut E. Unraveling the Role of Immune Checkpoints in Leishmaniasis. Front Immunol. 2021 Mar 11;12:620144.

Lukas D, Dietze-Schwonberg K, Kautz-Neu K, Lorenz B, Yogev N, Clausen BE, von Stebut E. Induction of Regulatory T Cells in Leishmania major‒Infected BALB/c Mice Does Not Require Langerin+ Dendritic Cells. J Invest Dermatol. 2021 Apr;141(4):936-938.

Publications 2019

Fischer MR, Schermann AI, Twelkmeyer T, Lorenz B, Wegner J, Jonuleit H, von Stebut E. Humanized mice in cutaneous leishmaniasis-Suitability analysis of human PBMC transfer into immunodeficient mice. Exp Dermatol. 2019 Sep;28(9):1087-1090.