P. Bartsch

• 
• 

Staphylococcus epidermidis innate immune evasion during implant infections

Project leader: Prof. Dr. Holger Rohde

Affiliation: Institute for Medical Microbiology, Virology and Hygiene

Background and preliminary data: Staphylococcus epidermidis is a leading pathogen in implant-associated infections. Biofilm formation protects S. epidermidis from innate immune effector mechanisms, and plays a key role for persistence in in a hostile environment. The underlying molecular mechanisms are not yet understood. Previous work from our lab has provided evidence that biofilm formation interferes with phagocytosis, bacterial killing and pro-inflammatory macrophage activation. More recently, using S. epidermidis wild-type strains and specific mutants in combination with live cell imaging approaches, evidence was obtained that S. epidermidis is able survive within primary human macrophages. While an isogenic biofilm-negative mutant was readily degraded and almost completely killed with 12 hours after up-take, the biofilm forming S. epidermidis wild type strain was able to persist within macrophages for up to 72 hours. So far, it is a common opinion that S. epidermidis is a strictly extracellular pathogen and lacks the ability to survive intracellularly. Therefore, our finding add an important novel and so far unrecognized piece of evidence to our picture of S. epidermidis pathogenicity and the pathogenesis of chronic implant infections.

Hypothesis: Building on own preliminary observations we hypothesize that S. epidermidis employs defined mechanisms that facilitate intracellular pathogen survival in macrophages.

Aims and Work Program: 1. Dissection of molecular events during up-take and intracellular fate of S. epidermidis in human macrophages. 2. Identification of S. epidermidis factors relevant to within macrophage survival. 3. Importance of intracellular survival for the pathogensis of implant infections.

Aim #1 Work package 1 aims at dissecting the molecular events and cellular pathways contributing to S. epidermidis phagocytosis. Using a recently developed model to study macrophage - biofilm interactions, to this end S. epidermidis wildtype 1457 and a series of fluorescence labeled derivatives will be employed in live cell imaging experiments focusing on the very early phase of pathogen-macrophage contact (0 - 20 h). To specifically characterize the contribution of biofilm forming ability, a biofilm-negative mutant shall be used as a comparator. Imaging and quantification of S. epidermidis uptake as well as identification of cellular compartments involved in pathogen handling will be substantially aided by the close collaboration with the UKE microscope imaging facility Umif and infrastructure from RTG Humans and Microbes (M. Aepfelbacher, Stefan Linder, Thomas Braulke). Making use of their established methods, a major goal will be to draw a detailed functional picture of relevant host cell events during S. epidermidis up-take. In extension to imaging driven analysis, transcriptomics shall be employed to more precisely define host pathways engaged by biofilm forming S. epidermidis. Importantly, by parallel approaching host and pathogen transcriptomes using a dualSeq strategy, macrophage responses will be linked to adaptive transcriptional S. epidermidis events. Transcriptomics essentially depend on well-established collaborations with in-house bioinformatics resources (Jiabin Huang) and infrastructures from the SFB 1192 (Christian Krebs). Using biofilm-negative mutant 1457-M10, the contribution of biofilm forming ability to evade macrophage clearance can be estimated.

In Aim #2 Preliminary evidence supports the idea that S. epidermidis engages defined mechanisms to persist in macrophages. A two-tier approach shall be employed to identify those functionally involved pathogen factors. Firstly, a high density Bursa aurealis Tn-mutant library of S. epidermidis 1457 will be established. By testing the library in a macrophage infection model and a high-throughput FACS assay to quantify intracellular S. epidermidis, mutations will be selected that are associated with decreased intracellular S. epidermidis numbers. Results will feed into experiments in which specific mutations of identified genes are being generated using a recently developed CRISPRr/Cpf1 based genome editing system. CRISPR/Cpf1-based genome editing is also used in the second complementary approach to Tn mutagenesis. Making use of results from dualSeq experiments, genes regulated after macrophage contact will be inactivated. Mutants from Tn mutagenesis and CRISPR/Cpf editing will then be tested using imaging approaches as used in WP1 to nail down their specific contribution for S. epidermidis handling by macrophages.

In Aim #3, So far, evidence for intracellular S. epidermidis survival of S. epidermidis is limited to in vitro experiments. Currently, our group is involved in establishment of a mouse implant-infection model (collaboration with Johannes Keller, experimentelle Unfallchirurgie). The model shall be used to quantify survival of S. epidermidis wildtype 1457 and corresponding biofilm-negative mutant 1457-M10. Relying on available antisera to stain for S. epidermidis, ex vivo material will be investigated with a special focus on characterizing and enumerating intra- and extracellular bacteria. Similarly, we will make use of human ex vivo material from PJI (collaboration Helios ENDO-Klinik) to characterize the presence of intracellular S. epidermidis using microscopic and FACS approaches. Finally, the infection model will serve to test the in vivo relevance of S. epidermidis genes identified in aim 2 for pathogenesis of S. epidermidis implant infections.

Project-related publications:

1. Stamm J, Weißelberg S, Both A, Failla AV, Nordholt G, Büttner H, Linder S, Aepfelbacher M, Rohde H. 2022. Development of an artificial synovial fluid useful for studying Staphylococcus epidermidis joint infections. Front Cell Infect Microbiol. 2022 Jul 29;12:948151. doi: 10.3389/fcimb.2022.948151. eCollection 2022.

2. Bartsch P, Kilian C, Hellmig M, Paust HJ, Borchers A, Sivayoganathan A, Enk L, Zhao Y, Shaikh N, Büttner H, Wong MN, Puelles VG, Wiech T, Flavell R, Huber TB, Turner JE, Bonn S, Huber S, Gagliani N, Mittrücker HW, Rohde H, Panzer U, Krebs CF.. 2022. Th17 cell plasticity towards a T-bet-dependent Th1 phenotype is required for bacterial control in Staphylococcus aureus infection. PLoS Pathog. 2022 Apr 21;18(4):e1010430. doi: 10.1371/journal.ppat.1010430. eCollection 2022 Apr.

3. Both A, Huang J, Qi M, Lausmann C, Weißelberg S, Büttner H, Lezius S, Failla AV, Christner M, Stegger M, Gehrke T, Baig S, Citak M, Alawi M, Aepfelbacher M, Rohde H. 2021. Distinct clonal lineages and within-host diversification shape invasive Staphylococcus epidermidis populations PLoS Pathog. 2021 Feb 5;17(2):e1009304. doi: 10.1371/journal.ppat.1009304. eCollection 2021 Feb. PMID: 33544760

4. Du X, Larsen J, Li M, Walter A, Slavetinsky C, Both A, Sanchez Carballo PM, Stegger M, Lehmann E, Liu Y, Liu J, Slavetinsky J, Duda KA, Krismer B, Heilbronner S, Weidenmaier C, Mayer C, Rohde H, Winstel V, Peschel A.. 2021. Staphylococcus epidermidis clones express Staphylococcus aureus-type wall teichoic acid to shift from a commensal to pathogen lifestyle. Nat Microbiol. 2021 Jun;6(6):757-768. doi: 10.1038/s41564-021-00913

5. Büttner H, Perbandt M, Kohler T, Kikhney A, Wolters M, Christner C, Heise M, Wilde J, Weißelberg S, Both A, Betzel C, Hammerschmidt S, Svergun D, Aepfelbacher M, Rohde H. 2020. A giant extracellular matrix binding protein of Staphylococcus epidermidis binds surface-immobilized fibronectin via a novel mechanism. mBio. 2020 Oct 20;11(5):e01612-20. doi: 10.1128/mBio.01612-20

C. Kilian

• 
• 

Analysis of the role of CMV-reactive tissue-resident T cells on intestinal inflammation in IBD

Project leader: Prof. Dr. Samuel Huber

Affiliation: I. Department of Medicine, University Medical Center Hamburg-Eppendorf

Background and preliminary data:

Inflammatory bowel diseases (IBD) are immune-mediated diseases characterized by recurrent inflammation of the intestinal tract. The etiology of those chronic disorders is not completely understood. It is postulated that lifestyle factors and the microbiota can lead to a dysregulation of the mucosal immune system in genetically susceptible individuals. Interestingly, clinical studies suggest that viral infections can also affect the disease course in IBD patients. Compared to healthy individuals, IBD patients have a higher risk of the development of CMV (cytomegalovirus) colitis. Interestingly, CMV colitis is also associated with disease activity and steroid resistance in patients with ulcerative colitis. Indeed, infectious disease are known to be able to precede and aggravate various autoimmune and immune-mediated diseases. While the mechanisms underlying this phenomenon are largely unknown, several studies suggest that the induction of tissue resident memory T cells (TRM) might play a crucial role in this setting. These cells are categorized as a distinct memory T cell subset that usually does not take part in cell migration but adheres to a specific tissue. TRM cells can occur after infection and are considered to provide a protective function against reinfection. Phenotypically, they are characterized by the expression of high levels of CD69 and low levels of S1PR1. Interestingly, recent publications have shown that the induction of TRM cells upon infection with pathogens can aggravate the subsequent course of immune-mediated diseases even after clearance of the initial pathogen.

Based on these findings, we aim to test whether CMV infection can affect the course of IBD by inducing tissue-resident T cells in the intestine.

Hypothesis:

We hypothesize that CMV induces tissue-resident memory cells in the intestine and thereby contributes to an exacerbated intestinal inflammation during in IBD.

Aims and Work Programme:

We want to test this hypothesis using a translational approach and by studying both IBD/CMV colitis in IBD patients and by using murine CMV (MCMV) as a mouse model for CMV infection. Specifically, we want to pursue the following aims:

• Aim #1: Study of the immune cell composition and the clonal expansion of tissue-resident memory T cells in patients with IBD/CMV colitis
• Aim #2 Using a murine model for CMV Infection (MCMV), we aim to test whether there is a causal link between MCMV infection and a dysregulated mucosal immune response, which in turn leads to an exacerbation of colitis.

In Aim #1, we plan to collect intestinal biopsies from IBD patients with active colitis. Specifically, our aim is to compare patients with active IBD and CMV colitis (determined by positive CMV PCR in the biopsy specimens) compared to active IBD without CMV colitis. To this end, we will compare the immune cell composition in those two groups using flow cytometry and our staining panel will be focused on the in depth-characterization of effector and memory T cells. Furthermore, we plan to perform single-cell sequencing and TCR-sequencing of isolated mucosal T cells. Based on ligand-receptor interactions, we can thus identify CMV-reactive T cells in the mucosa of patients with active IBD vs. IBD/CMV colitis. Furthermore, we will be able to monitor, whether a clonal expansion of CMV-reactive T cells can be observed in this setting and whether those clonally expanding cells indeed have tissue-resident characteristics. Finally, we will correlate our flow cytometry results with our single-cell sequencing data in order to test, whether those clonally-expanding, tissue-resident CMV-reactive T cells are indeed contributing to a strongly dysregulated immune response in IBD/CMV colitis. Those results will be incremental to understand the mechanism of why CMV colitis is associated with a complicated disease course in IBD patients.

In Aim #2, we aim to test the hypothesis that latent MCMV infection leads to a dysregulated mucosal T cell-composition, which in turn leads to an exacerbation of colitis. To test this, we aim to induce a latent MCMV infection in mice. In the next step, we will use different murine models for colitis (DSS-colitis as a model of chemically induced colitis and Il10-/- mice as a genetic colitis model) in order to test, whether latent MCMV infection leads to an exacerbation of colitis in experimental colitis models. The use of those animal models (together with the MCMV infection) has already been approved by the responsible authorities (Genehmigung eines Tierversuchsvorhabens nach § 8 Abs. 1 des Tierschutzgesetzes). Furthermore, we plan to perform an in-depth study of the mucosal immune cell composition in those murine models. Specifically, we aim to analyze the T cell composition by FACS and single-cell RNA as well as TCR sequencing, as described in Aim #1. This will allow us to determine, whether tissue-resident, MCMV-reactive T cells are clonally expanding after the induction of experimental colitis in mice. Since IBD (in the case of Crohn’s Disease) can also affect the small intestine, we aim to further test the role of latent CMV infection in a murine model of small intestinal inflammation that is already established in our lab (the aCD3-model of small intestinal inflammation which is induced by the injection of CD3-specific antibodies). This model would allow us to study the functional role of tissue-resident immune cells after CMV infection in more detail by performing small intestinal transplantation after the CMV infection. Our long-term collaborator Dr. Giannou has learned rodent small intestinal transplantation and would therefore enable us to study the role of CMV-reactive tissue-resident cells using a highly sophisticated state-of-the-art method. Overall, we believe that our animal studies can pave to the way towards a novel understanding of how viral infections can induce tissue-resident T cells which then play a causal role in controlling the course of immune-mediated disorders.

In conclusion, we believe that this project will offer novel insights into how CMV infection can induce tissue-resident intestinal T cells, which in turn exacerbate colitis in IBD patients. Understanding the mechanisms underlying this phenomenon could pave the way towards novel treatments for IBD.

Project-related publications:

1. Kempski J*, Giannou AD, Riecken K, Zhao L, …, Huber S. IL22BP Mediates the Antitumor Effects of Lymphotoxin Against Colorectal Tumors in Mice and Humans. Gastroenterology. 2020;159(4):1417-1430.e3.

2. Perez LG, Kempski J, McGee HM, Pelzcar P, …, Huber S. TGF-β signaling in Th17 cells promotes IL-22 production and colitis-associated colon cancer. Nat Commun. 2020;11(1):2608.

3. Brockmann L, Soukou S, Steglich B, Czarnewski P, …, Huber S. Molecular and functional heterogeneity of IL-10 producing CD4+ T cells. Nat Commun. 2018 May 25;11(1):2608; 9(1):5457

4. Pelczar P*, Witkowski M*, Perez LG*, Kempski J, …, Huber S. A pathogenic role for T cell-derived IL-22BP in inflammatory bowel disease. Science. 2016;354(6310):358-362.

B. Zecher

• 
• 

Effect of KIR3DS1/3DL1 co-expression and copy number variation on anti-viral activity of NK cells

Project leader: Prof. Dr. Marcus Altfeld

Affiliation: Leibniz Institute of Virology, Hamburg

Background and preliminary data:

KIR3DS1 and KIR3DL1 belong to the family of killer cell immunoglobulin like receptors (KIRs) that play an important role in modulating NK cell function, including control of viral infections. Both KIR3DS1 and KIR3DL1 have been described to be involved in the control of viral infections such as human immunodeficiency virus (HIV)1, human adenovirus (hAdV)2, BK polyomavirus (BKV)3 and hepatitis C virus (HCV)4 infection. This protective effect has been observed in individuals expressing both KIR3DS1 and KIR3DL1, but not expressing KIR3DS1 alone. However, to date the mechanisms of NK cell mediated control of viral infection requiring both KIR3DS1 and KIR3DL1 remain unclear.KIRs are divided into two groups according to their function, which is linked to the intracytoplasmic domain. Activating KIRs have a short intracytoplasmic domain with an ITAM motif (KIRxDSx), while inhibitory KIRs have a long intracytoplasmic domain with an ITIM motif (KIRxDLx). Recognition of self-MHC class I molecules by inhibitory KIRs promotes tolerance towards endogenous cells in a process termed NK cell education. In consequence, downregulation of self MHC-class I molecules in malignant transformed or virus-infected cells leads to missing self-recognition by NK cells.KIR3DS1 and KIR3DL1, representing an activating and inhibitory receptor respectively, are encoded at the same locus and therefore represent alleles of one gene. Individuals can thus be homozygous or heterozygous for KIR3DS1 and KIR3DL1. Despite the high homology in the amino acid sequence, KIR3DS1 and KIR3DL1 have different ligands5. While KIR3DL1 binds to HLA-Bw4 molecules, the non-classical HLA-class I molecule HLA-F, that is upregulated e.g. on virus infected cells, serves as ligand for KIR3DS1 (Garcia-Beltran WF et al., Nat Immunol 2016). However, to date it is not clear whether KIR3DS1 and KIR3DL1 are co-expressed by the same NK cell on a single cell level, and how the expression pattern of KIR3DS1 and KIR3DL1 shapes the anti-viral activity of NK cells. Furthermore, the KIR3DS1/KIR3DL1 locus underlies copy number variations (CNVs). The total allele count for KIR3DS1/KIR3DL1 has been described to vary between one and three copies. In the third part of the project, we will therefore investigate the influence of CNV on KIR3DS1 and KIR3DL1 surface expression and subsequently the impact on anti-viral activity of the NK cells.

Hypothesis:

The co-expression of KIR3DS1 and KIR3DL1 influences the anti-viral activity of NK cells on a single cell level

Aims and Work Programme:

1. Determine (co-)expression of KIR3DS1/DL1 on a single cell level

2. Assess the functional impact of KIR3DS1/3DL1 co-expression on the anti-viral activity of NK cells

3. Modulation of anti-viral activity of NK cells through KIR3DS1/KIR3DL1 copy number variations

Work programme aim #1:

Healthy donors will be typed for HLA- and KIR alleles. Co-expression patterns of KIR3DS1/KIR3DL1 will be determined on NK cells of donors being heterozygous for these alleles. Representing two alleles of the same gene, KIR3DS1 and KIR3DL1 have a homology of >95% in their extracellular domain. Consequently, there are currently only KIR3DS1 monospecific and KIR3DS1/3DL1 bispecific antibodies available. Via flow cytometry, it is therefore only possible to distinguish between KIR3DS1-/KIR3DL1-, KIR3DS1+/KIR3DL1- andKIR3DL1+ cells that may or may not co-express KIR3DS1. To determine the co-expression pattern of KIR3DS1 and KIR3DL1, individual single NK cells of KIR3DS1/KIR3DL1 heterozygous, healthy individuals will be sorted via fluorescence activated cell sorting (FACS). Subsequently, the expression of KIR3DL1 and KIR3DS1 on an mRNA level will be determined via single-cell RT-qPCR (Fluidigm Technology).

Work programme aim #2:

To determine the functional impact of KIR3DS1/KIR3DL1 co-expression on the anti-viral activity of NK cells, NK cell clones from single cell sorted primary NK cells of healthy donors heterozygous for KIR3DS1 and KIR3DL1 will be generated. KIR3DS1 and KIR3DL1 expression of the clones will be determined via flow cytometry and RT-qPCR, as described above. The selected donors will be HLA-Bw4 positive or negative. This will allow us to differentiate between KIR3DL1/HLA-Bw4 educated or non-educated NK cells. NK cell clones expressing KIR3DS1 and/or KIR3DL1 of HLA-Bw4 positive or negative donors will be incubated with the respective ligands HLA-Bw4 and HLA-F (protein-coated beads and transfected cell lines). NK cell activation in will be determined by flow cytometric analysis of degranulation and cytokine expression.After dissecting the required KIR3DL1/KIR3DL1 expression pattern and genetic background for effective NK cell activation, the impact on control of viral infections will be assessed. We will use infection models that are well established in our lab and for which an influence of viral control by KIR3DS1 and KIR3DL1 has been demonstrated, such as HIV or hAdV infection. Antiviral activity of the NK cell clones will be determined via assessment of NK cell degranulation and killing of virus-infected cells, as well as inhibition of viral replication.

Work programme aim #3:

To assess anti-viral NK cell activity in the context of CNV, KIR3DS1/KIR3DL1 copy numbers will be determined via digital droplet PCR. NK cell clones will be generated from individuals with different copy numbers of KIR3DS1 and KIR3DL1, and the consequences for KI3DS1 and KIR3DL1 expression on the single cell level will be determined, as described in aim #1. The effect of CNV on the control of viral infection will be assessed, as described in aim #2.

Project-related publications:

1. Pelak K, Need AC, Fellay J, Shianna KV, Feng S, Urban TJ, Ge D, De Luca A, Martinez-Picado J, Wolinsky SM, Martinson JJ, Jamieson BD, Bream JH, Martin MP, Borrow P, Letvin NL, McMichael AJ, Haynes BF, Telenti A, Carrington M, Goldstein DB, Alter G; NIAID Center for HIV/AIDS Vaccine Immunology. Copy number variation of KIR genes influences HIV-1 control. PLoS Biol. 2011 Nov;9(11):e1001208.

2. Jung JM, Ching W, Baumdick ME, Hofmann-Sieber H, Bosse JB, Koyro T, Möller KJ, Wegner L, Niehrs A, Russu K, Ohms M, Zhang W, Ehrhardt A, Duisters K, Spierings E, Hölzemer A, Körner C, Jansen SA, Peine S, Königs I, Lütgehetmann M, Perez D, Reinshagen K, Lindemans CA, Altfeld M, Belderbos M, Dobner T, Bunders MJ. KIR3DS1 directs NK cell-mediated protection against human adenovirus infections. Sci Immunol. 2021 Sep 17

3. Koyro TF, Kraus E, Lunemann S, Hölzemer A, Wulf S, Jung J, Fittje P, Henseling F, Körner C, Huber TB, Grundhoff A, Wiech T, Panzer U, Fischer N, Altfeld M. Upregulation of HLA-F expression by BK polyomavirus infection induces immune recognition by KIR3DS1-positive natural killer cells. Kidney Int. 2021 May;99(5):1140-1148.

4. Lunemann S, Schöbel A, Kah J, Fittje P, Hölzemer A, Langeneckert AE, Hess LU, Poch T, Martrus G, Garcia-Beltran WF, Körner C, Ziegler AE, Richert L, Oldhafer KJ, Schulze Zur Wiesch J, Schramm C, Dandri M, Herker E, Altfeld M. Interactions Between KIR3DS1 and HLA-F Activate Natural Killer Cells to Control HCV Replication in Cell Culture. Gastroenterology. 2018 Nov;155(5):1366-1371.e3.

5. O'Connor GM, Guinan KJ, Cunningham RT, Middleton D, Parham P, Gardiner CM. Functional polymorphism of the KIR3DL1/S1 receptor on human NK cells. J Immunol. 2007 Jan 1;178(1):235-41.

I. Grewe

• 
• 

Dissecting the Role of T follicular helper cells in vaccination against Betacoronaviruses: Clonal evolution and TCR specificity

Project leader: Prof. Dr. med. Marylyn M. Addo, MSc, DTM&H

Affiliation: Director, Institute for Infection Research and Vaccine Development (IIRVD) Center for Internal Medicine (ZIM), UKE

Email: m.addo@uke.de, fon: 040-7410-51102

Background and preliminary data:

Emerging infectious diseases: Emerging infectious diseases (EID) represent a major threat to public health worldwide and were recently named among the top ten threats to global health by the World Health Organization (WHO). The Ebola virus disease (EVD) crisis in West Africa and the current coronavirus disease (COVID)-19 pandemic demonstrated that the world community was ill prepared and challenged by developing effective interventions for the control of severe EID outbreaks in a timely manner. In response to the current COVID-19 pandemic of historic scale, the speed of vaccine development against SARS-CoV-2 and implementation of clinical trials have been unprecedented. However critical knowledge gaps remain to be addressed to evaluate vaccine candidates at an early stage of vaccine development.

Challenges in the development of vaccines against emerging infectious diseases: Vaccines are among the most successful public health interventions. Current licensed vaccines have been mostly generated by empiric strategies and the exact mechanisms that mediate vaccine-induced protection remain inadequately understood. In light of strategic vaccine development against EID, there is a critical need to better understand how vaccines work and how correlates of vaccine-induced immunogenicity and protection can be assessed at an early stage (i) to facilitate decision-making on the next stages of vaccine development, (ii) to evaluate vulnerable cohorts (iii), and to improve the design of novel vaccine candidates. Phase I clinical trials in humans are thus a critical step in vaccine development as through intensive sampling and comprehensive analysis of study subjects, they allow for critical insight into both safety and immunogenicity of new vaccine constructs.

Vaccine-induced immunogenicity: Adaptive immunity induced by vaccines is critical for vaccine efficacy. While analysis of antigen-specific T and B cells as well as antibodies is a standard approach to evaluate vaccine-induced immunogenicity, T follicular helper (TFH) cells have been understudied to date. TFH cell populations play a key role in B cell selection and antibody affinity maturation during GC reactions and are essential for generation of protective B cell responses. Thus, induction of a strong antigen-specific TFH response upon immunization represents a key goal for the design of vaccines and may emerge as a predictive marker and/or a correlate for protection against infection or disease.

Preliminary work: Clinical trials to evaluate vaccine candidates against MERS and COVID-19: As part of the TTU Emerging Infections of the German Center for Infection Research (DZIF), our group has a specific expertise in early phase clinical testing of vaccine candidates against emerging viruses. We recently performed investigator-initiated (IIT) vaccine trials evaluating the viral-vector vaccine candidates VSV-EBOV, MVA-MERS-S and MVA-SARS-2-S/ST against Ebola virus disease, MERS and COVID-19, respectively. Multiple samples from healthy vaccine volunteers pre- and early post-vaccination were collected to gain comprehensive insights into innate and adaptive immune mechanisms induced by vaccination. Analysis include early gene expression profiles associated with the candidate vaccine, as well as humoral responses and detailed T and B cell analyses in response to vaccination.

Hypothesis: We hypothesize that early induction of TFH cells populations results in strong neutralizing and non-neutralizing humoral immunity. Detailed longitudinal analyses of TFH cell populations will provide critical insight into molecular and cellular mechanisms related to the kinetics of TFH cell populations and optimal induction of B cell help.

Aims and Work Programme: Human TFH cell populations will be comprehensively analyzed to understand mechanisms of antigen-delivery systems that confer protective immunity. We will take advantage of longitudinally collected human samples originating from the Phase I/Ib clinical trials testing MVA-MERS-S and COVID-19 vaccine studies. Longitudinal samples from SARS-CoV-2-positive individuals will be included as controls.

WP 1: Profiling of antigen-specific TFH cell populations following natural infection and vaccination. Characterize TFH cell responses to MERS-CoV/SARS-CoV-2 spike, analyzed from participants of different vaccine trials and compare responses with samples from infected individuals. Classical assays based on flow cytometry and Luminex/Legendplex will be applied to identify TFH1, TFH2 and TFH17 cell populations and their activation status. The aim is to longitudinally investigate TFH cell populations induced by different vaccine candidates against coronaviruses (MVA-based vaccines, mRNA-based vaccines), and to compare data between vaccinees and infected individuals.

WP 2: TCR repertoire analysis of TFH cell population. A subgroup of each cohort will be included in the analysis of the TCR gene. Flowcytometric cell sorting will be applied to isolate PD1highICOS+ TFH cells from specific timepoints following vaccination or infection. The aim is to sequence the TRB and TRA genes and to investigate the clonal expansion of TFH cells following immunization or infection.

WP3:Comparison of TFH cell signatures with antibody responses induced by vaccine candidates or infection. Our cohorts were comprehensively analyzed for humoral responses. These data will be used to associate TFH cell populations with antibody responses.

Project-related publications:

1. Agnandji ST#, Huttner A#, Zinser ME#, Njuguna P#, Dahlke C, …., Lohse AW, …, Ramharter M, …, Bejon P#, Kremsner PG#, Siegrist CA#, Addo MM#. Phase I Trials of rVSV Ebola Vaccine in Africa and Europe. N Engl J Med 2016 374:1647-60

2. Dahlke C#, Kasonta R#, Lunemann S#, …, Lohse AW, Becker S, Addo MM; VEBCON Consortium. Dose-dependent T-cell Dynamics and Cytokine Cascade Following rVSV-ZEBOV Immunization. EBioMedicine 2017;19:107-18.

3. Koch T#, Dahlke C#, Fathi A, …, Sutter G, Becker S, Addo MM. Safety and immunogenicity of a modified vaccinia virus Ankara vector vaccine candidate for Middle East respiratory syndrome: an open-label, phase 1 trial. Lancet Infect Dis 2020;20:827-38.

4. Weskamm M, …., Dahlke C#, Addo MM#. Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine. Cell Rep Med 2022, Jul 19;3(7):100685

5. Fathi A, Dahlke C, …, Haagmans BL, Sutter G, Becker S, Addo MM. Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome. Nat Commun 2022 Jul 19;13(1):4182. ^#equally contributing authors

A. Harberts

• 
• 

Trajectories of immune dysfunction between the liver, the intestine andluminal flora in advanced chronic liver disease

Project leader: PD Dr. med. Peter Huebener

Affiliation: Department of Medicine, Ist Medical Clinic and Polyclinic, University MedicalCenter Hamburg-Eppendorf

Background and preliminary data:

Prevalences of chronic liver diseases areincreasing worldwide, and infections aremajor drivers of morbidity and mortality inthis patient cohort. A dysfunctionalinterplay between the liver, the intestinalmucosa and the luminal microbiota -three anatomically and functionallyclosely intertwined biological systems - isbelieved to favor infections, but also toprovide aims for prophylactic andtherapeutic medical interventions (1).

Here, we aim to determine dysfunctional trajectories of these three biological entities duringinfections, and to characterize their therapeutic accessibility. We plan 3 major project goals includinganimal experiments, clinical observations as well as interventional studies in human subjects.

Hypothesis:

Dysregulated immune responses to infections in advanced chronic liver diseases (ACLD) are driven by defective inner- and inter-system crosstalk between the liver, the gut and theintestinal microbiota, which may be exploited for prophylactic or therapeutic interventions.

Aims and Work Programme:

Aim 1: Patterns of pathogen-induced immune dysregulation in ACLD: We aim for a comprehensive phenotypic and molecular characterization of immune dysregulation asa function of progressive liver diseases.

In Aim 1A) of the proposal, the effects of well-controlled infectious stimuli are to be examined againstthe background of pathogenetically diverse and differently advanced liver diseases in rodents. Twomouse models of chronic liver damage are primarily used:

• the Tak1Δhep model of chronic progressive cell death-dependent hepatopathy (2)

• the Mdr2-/- model of progressive cholestatic liver disease

By using two genetic models of liver damage, hepatopathy-specific and overarching mechanisms ofimmune dysfunction in liver diseases can be identified. Experimental animals (and controls) areexposed to bacterial pathogens (Str. pneumoniae, E. coli) systemically (i.v.) or locally (i.t., i.p.) atdifferent stages of liver disease. Target variables are a) cellular compositions of the liver, spleen,intestine, bone marrow, regional lymph nodes and circulating blood, b) biochemical analyses of theimmune response including multiplex ELISA, bulk and single cell-sequencing, Cytof-analyses,immunostaining), c) infection-associated alterations of the intestinal flora, permeability of theintestinal wall and epithelial cell death processes, and are supplemented by d) in vitro experimentalsetups of primary cell cultures from the corresponding experimental animals.

Aim 2: Intestinal dysbiosis as a modifiable effector system of ACLD-immune dysregulation. ACLD isassociated with characteristic alterations of the intestinal flora, whose pathophysiological roles areunclear. Here, we aim to characterize the consequences of intestinal dysbiosis on 1) intestinalinflammation and permeability, 2) systemic immune responses to acute infections, and 3) theprogression of liver disease.

Part (2A) The influence of chronic liver diseases will be characterized using stool samples from testanimals from application part 1A).

Animals: Tak1Δhep and Tak1f/f per n=10 mice aged 4 weeks, 16 weeks and 48 weeks, respectively.Samples from the deep small intestine, proximal colon and stool pellets as well as from test animalswith infections (see part 1A of the application) before/after targeted antibiotic therapies.The Tak1Δhep mouse samples offer - since the genetic defect is limited to the liver - the advantageof a longitudinal observation of microbial flora alterations that may exacerbate with progressive liverdisease and are otherwise largely free of confounding factors - even in regions that are difficult toaccess in patients, i.e. the small intestine. Separate housing of the animals prevents homogenizationof the microbiota through coprophagy. The focus of the analyses is on the composition of theintestinal flora by 16S sequencing and shotgun metagenomics on selected, representative samples.

In sub-project 2B), potential pathogenetic properties of intestinal dysbiosis are characterized. Usingfecal microbiome transfer (FMT), the microbiome of ACLD, septic or ACLF experimental animalsfrom Aim 1A) are transplanted into germ-free experimental animals. The pathophysiologicalconsequences of intestinal dysbiosis on frequent complications of advanced liver diseases can thusbe examined experimentally (cf. Figure 1).

Experimental setup

• 
• 

Figure 1) experimental setupfor aim 2). Fecal microbiometransfer (FMT) from healthycontrols, infected animals,ACLD and ACLF animals,respectively, into healthyrecipients. The impact ofdysbiotic FMT on inflammationand liver disease progressionwill then be tested in FMTrecipients in injury models.GFM = germ-free mouse. LPS= lipopolysaccharide. DSS =dextran sodium sulfate.

To this end, we will test the hypothesis that ACLD-associated dysbiosis is an independently causaland therapeutically modifiable agent in the pathogenesis of ACLD-associated immune dys-function.Animals with an intestinal xenobiota are first generated by replacing the microbiota of healthyrecipient animals via FMT from Tak1Δhep animals - and vice versa as, a proof of concept.Subsequently, using models of acute colitis (induced by dextran sodium sulfate), septic shock(induced by lipopolysaccharide i.v.) or bacterial peritonitis (induced by E.coli i.p., analogous tospontaneous bacterial peritonitis in humans), we will assess the impact of FMT of eu- or dysbioticmicrobiota, respectively, on the host response to the corresponding immunological stimuli. Finally,we will examine the effects of the infection-associated pathobiota on the progression of liver diseasein the CCl4 model of inflammation-associated liver fibrosis, since acute extrahepatic inflammatorycomplications may promote the progression of ACLD and further worsen patients´ prognosis (3).

(3) The intestinal barrier as interface and target of intervention of the disrupted hostpathobiome interaction

In this part of the application, the focus will be on the (dys-)functionality of the intestinal barrier,mediating multi-layered interactions between host and microbiota, in the context of luminal dysbiosis.For this purpose, investigations on intestinal permeability, production of barrier-promoting factors(mucus, antimicrobial peptides), cell death processes in the epithelial layer and the release ofdamage-associated molecular patterns will be carried out in animals from parts 1A, 2A and 2B. Inaddition, we are planning FACS analyses of the intestinal mucosa at rest and under infectionconditions, with a focus on innate and adaptive immune cells, as well as sequencing analyzes of theintestinal mucosa and interactome analyzes of microbiota and intestinal immune cells in an unbiasedapproach. The aim of the investigations is to identify cellular effectors and signal cascades via whichthe pathobiota mediates its cross-border effects in liver diseases. Depending on the outcome of theinvestigations, targeted interventions that modulate immune cell recruitment (e.g. through integrinantagonists) or aberrant signaling of the pathobiota (e.g. Janus kinase inhibitors) are to beinvestigated in subsequent steps, and with regard to their suitability as therapeutic strategies incorresponding situations.

Project-related publications:

1. Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: Pathophysiological basis for therapy. J Hepatol. 2020Mar;72(3):558-577.

2. Bettermann K, Vucur M, Haybaeck J, et al. TAK1 suppresses a NEMO-dependent but NF-kappaB-independent pathway toliver cancer. Cancer Cell. 2010 May 18;17(5):481-496.

3. Engelmann C, Claria J, Szabo G, et al. Pathophysiology of decompensated cirrhosis: Portal hypertension, circulatorydysfunction, inflammation, metabolism and mitochondrial dysfunction. J Hepatol. 2021 Jul;75 Suppl 1:S49-S66.

T. Omansen

• 
• 

Lassa fever Pathogenesis

Project leader: Prof. Dr. med. univ. Michael Ramharter

Affiliation: Department of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine & I. Dep. of Medicine, University Medical Center Hamburg-Eppendorf

Background and preliminary data:

Lassa virus (LSAV) is an enveloped, negative-strand RNA virus causing Lassa fever (LF), a viral hemorrhagic fever (VHF). The disease is occurring in different regions in West Africa, with Nigeria being the hotspot. Hospitalized patients most commonly exhibit a sepsis-like disease phenotype with high inflammatory parameters, multi-organ failure and ultimately cardiovascular shock or a secondary neurological phenotype with seizures, meningitis and encephalopathy while kidney and liver function tests remain only moderately altered. To date it is unknown how LASV causes pathology in kidney, other organs and the central nervous system (CNS).

Within this research project we aim to decipher the pathogenesis of LF in a cohort of acutely infected patients in Nigeria and to test what the driving factors in the pathology of LF is. To this end, we recruit a prospective, observational cohort (n=300) of RT-PCR confirmed LF cases at our partner hospital in Nigeria. In addition, patients exhibiting neurologic or renal complications will be studied in detail with further non-invasive diagnostic tools such as EEG and ultrasound. The analysis for hypothesis testing will include measurement of inflammatory and hemostaseological biomarkers and analysis of the specific T-cell response.

This will be supplemented by a postmortem study on specimens collected with minimal invasive tissue sampling (MITS). MITS is a technique where tissue is collected using ultrasound-guided biopsies on the deceased. It offers the possibility to recover postmortem tissue for histopathological and other studies as conventional autopsy is not possible due to the biohazard in the case of a risk group 4 pathogen. In addition, MITS is culturally easier acceptable.

The study is carried out in close collaboration with the Department of Virology at the BNITM (further laboratory diagnostics and biosafety level 4 laboratory) and the Institute for Legal Medicine at UKE (MITS).

Hypothesis:

Severe & fatal Lassa fever is caused by specific dysregulation of the inflammatory response and coagulopathy

Aims and Work Programme:

In Aim #1, we will recruit a prospective, observational cohort of Lassa fever patients at our partner hospital in Edo State, Nigeria during outbreaks (n=200-300). Besides standard laboratory parameters and clinical examinations, blood samples will be taken every two days for the period of the hospitalization for further testing including of inflammatory and hemostasis markers. In addition, PBMCs are prepared on-site for further analysis of the specific T-cell response. Laboratory findings will be correlated to the disease severity and clinical phenotype to test which host response is associated with sever (and fatal) LF.

In Aim #2, postmortem tissue from all relevant organs and bodily fluids will be collected in a specialized facility within the isolation ward in Nigeria using the MITS protocol, which has recently been established on-site. Standard histopathological analysis is carried out in Nigeria while both fixated and native tissue is shipped to Hamburg for further analysis including immunohistochemistry, electron microscopy, and (spatial) transcriptomics.

Project-related publications:

Erameh et al. Prospective observational study on the pharmacokinetic properties of the Irrua ribavirin regimen used in routine clinical practice in patients with Lassa fever in Nigeria. BMJ Open . 2020 Apr 16;10(4):e036936. doi: 10.1136/bmjopen-2020-036936.

Groger et al. Pharmacokinetics of ribavirin in the treatment of Lassa fever: An observational clinical study at the Irrua Specialist Teaching Hospital, Edo State, Nigeria. Clin Infect Dis . 2022 Jul 26;ciac578. doi: 10.1093/cid/ciac578.

Strampe et al. Factors associated with progression to death in patients with Lassa fever in Nigeria: an observational study. Lancet Infect Dis . 2021 Jun;21(6):876-886. doi: 10.1016/S1473-3099(20)30737-4.

Akpede et al. Caseload and Case Fatality of Lassa Fever in Nigeria, 2001-2018: A Specialist Center's Experience and Its Implications. Front Public Health . 2019 Jun 25;7:170. doi: 10.3389/fpubh.2019.00170.

Eberhardt et al. Ribavirin for the treatment of Lassa fever: A systematic review and meta-analysis. Int J Infect Dis . 2019 Oct;87:15-20. doi: 10.1016/j.ijid.2019.07.015.

F. Heinrich

• 
• 

Analysis of the prevalence of respiratory viruses in deceased and investigation of a possible association with the cause of death - a representative cross-sectional study in the Hamburg area

Project leaders: Prof. Dr. Benjamin Ondruschka, Institute of Legal Medicine, UKE; Dr. Marc Lütgehetmann, Institute of Medical Microbiology, Virology and Hygiene, UKE.

Background and preliminary data: Respiratory tract infections (RTIs) contribute significantly to the global burden of infectious diseases. Key to understanding the morbidity and mortality of RTIs is also the involvement of organs beyond the respiratory tract, induced e.g., by systemic spread of the pathogens and associated with induction of severe inflammatory or septic processes (1). Robust data on the overall contribution of respiratory pathogens to mortality in the general population in Germany are scarce and probably underestimated because many deaths in residential and care settings are not investigated in this regard. A wide range of viruses can infect cells of the upper and/or lower respiratory tract, including epithelial-, endothelial-, tissue- and immune cells. Viral tropism depends on the expression of different receptors and co-receptors. E.g., for SARS-CoV-2, the receptor ACE2 and the co-factor TMPRTSS2 are essential, but several co-receptors, including vimentin, further promote viral entry. Interestingly, vimentin is upregulated in tissues in response to cell death and inflammation, further enhancing SARS-CoV-2 infection (1). Therefore, data on the tissue distribution of respiratory viruses in humans is important for understanding their pathophysiology, but currently knowledge in humans beyond influenza A and SARS-CoV-2 is very limited.

During the SARS-CoV-2 pandemic, we established high-throughput molecular workflows for the detection of respiratory viruses and bacteria in respiratory materials such as nasopharyngeal swabs, and bronchoalveolar lavage (2), allowing sensitive and cost effective screening of large cohorts. During the pandemic, we used these methods to investigate all cadavers (nasopharyngeal swabs) at the Institute of Legal Medicine Hamburg for the presence of SARS-CoV-2 RNA (1*). Positive cases were further analyzed through scientific autopsies and extensive tissue examination using molecular and histological methods. This approach has been shown to provide important and novel insights into the pathophysiology of this disease (2*-3*). The projects based on SARS-CoV-2 were funded by the German Research Network (NUM) within the projects DEFEAT PANDEMIcs and NATON. Current NUM funding covers exclusively infrastructure to enable future preparedness.

In order to go beyond SARS-CoV-2 epidemiology, we have screened (from 10/2021 to 12/2022) for 16 relevant respiratory viruses in a population-based approach, amounting to 35% of the death population in Hamburg (n = 7533 cases). Surprisingly, the positivity rate of samples with at least one respiratory virus detected was 21%. SARS-CoV-2 was still the dominant virus (12%), followed by rhino-/enterovirus (2%) and others with less than 1% (data unpublished). However, the extent to which virus detection represents a clinically relevant infection is an open question, and additional information on the cause of death - either anamnestic or biological - is needed to allow further differentiation. Based on the data and results of the previous work, we propose the following study.

Hypothesis: The number of respiratory infections as a cause of death is greatly underestimated in Germany.

Project and Goals: We will generate prevalence data of respiratory viruses in a representative cross-section of deceased people in Hamburg, evaluate virus-associated RTIs as a possible cause of death in this population, and analyze pathophysiological features, e.g., the tissue tropism and damage of the different viruses. This will re-evaluate the relevance of RTIs as cause of death and provide new insights into the pathophysiology of virus induced RTIs. We will address these objectives in three different work packages (WP).

- WP1: Estimation of the prevalence rates of different respiratory viruses in a population-based cross-sectional study of the deceased population in the metropolitan area of Hamburg (2023-2025).

- WP2: Determination of cause of death using death certificate documentation and, in positive cases, verbal autopsy.

- WP3: Determination of viral tissue tropism and damage as a potential cause of death by minimally invasive- and/or conventional autopsy in a subset of cases.

In WP1, we will use nasopharyngeal swabs (Universal transport medium, Copan) to screen corpses in the Hamburg area (Hamburg Crematoria and Institute for Legal Medicine, Hamburg, Germany). Based on the 2021/2022 data, we plan to screen at least 10% of the Hamburg deceased population (n = 1500 cases/year) starting in the 2023/24 winter season. Samples will be analyzed using established protocols for multiplex qPCR on highly automated systems (Cobas 6800/8800, Roche), allowing screening for 16 different respiratory viruses (ADV, Bocavirus, Influenza A/B, RSV, hMPV, PIV1-4, Rhino/Enterovirus, hCOV (229E, OC43, NL63, HKU1), SARS-CoV-2 using 3 qPCR reactions). Pooling protocols as described (4*) will be used to reduce material and financial costs.

In WP2, we plan to determine the plausible cause of death in all cases by 1) analyzing death certificate documentation and 2) additionally, in cases with positive virus detection (n= approx. 300/year), conducting a verbal autopsy (VA) with relatives of the deceased. The VA is a standardised WHO structured questionnaire used to determine and attribute causes of death. The process could be guided by the SmartVA software tool, which takes VA interview data as input and produces estimates of causes of death at the individual and population level (2).

In WP3, we plan to perform scientific autopsies in selected cases with detection of relevant amounts of virus NA (approximately 5-10 cases/for Influenza A_H1N1, Influenza A_H3N2, RSV, PIV1-4, hCOVs, Rhino/Enterovirus, ADV) after consent of the relatives using minimally invasive and/or conventional autopsy (as described in 5*). In addition to a rigorous review of the cause of death, a comprehensive sampling of various organs will be performed (pharynx, bronchus, lung, heart, liver, kidney, brain, colon, blood, vessels). Multiple samples per site will be collected (stored in liquid nitrogen for future projects by other scientists) and the tissue sample/organ will be analyzed by standard histology and by quantitative qPCR or digital PCR for virus RNA/DNA detection as previously described (2*-3*). The distribution of viral RNA/DNA in the tissues will be investigated by RNA in situ hybridisation (RNAscope) and viral protein by immunohistochemical staining (as described in 2*-3*).

Project related publications:

1. Heinrich F, Huter T, Mertens S, Lange P, Vering J, Heinemann A, Nörz DS, Hoffmann A, Aepfelbacher M, Ondruschka B*, Krasemann S*, Lütgehetmann M*. New Postmortem Perspective on Emerging SARS-CoV-2 Variants of Concern, Germany. Emerg Infect Dis. 2023;29(3):652-656.

2. Puelles VG*, Lütgehetmann M*, Lindenmeyer MT*, Sperhake JP*, Wong MN, Allweiss L, Chilla S, Heinemann A, Wanner N, Liu S, Braun F, Lu S, Pfefferle S, Schröder AS, Edler C, Gross O, Glatzel M, Wichmann D, Wiech T, Kluge S, Püschel K, Aepfelbacher M, Huber TB. Multiorgan and Renal Tropism of SARS-CoV-2. N Engl J Med. 2020;383(6):590-592.

3. Braun F*, Lütgehetmann M*, Pfefferle S*, Wong MN, Carsten A, Lindenmeyer MT, Nörz D, Heinrich F, Meißner K, Wichmann D, Kluge S, Gross O, Püschel K, Schröder AS, Edler C, Aepfelbacher M, Puelles VG, Huber TB. SARS-CoV-2 renal tropism associates with acute kidney injury. Lancet. 2020;396(10251):597-598.

4. Olearo F*, Nörz D*, Hoffman A, Grunwald M, Gatzemeyer K, Christner M, Both A, Campos CEB, Braun P, Andersen G, Pfefferle S, Zapf A, Aepfelbacher M, Knobloch JKM, Lütgehetmann M. Clinical performance and accuracy of a qPCR-based SARS-CoV-2 mass-screening workflow for healthcare-worker surveillance using pooled self-sampled gargling solutions: A cross-sectional study. J Infect. 2021;83(5):589-593.

5. Fitzek A, Schädler J, Dietz E, Ron A, Gerling M, Kammal AL, Lohner L, Falck C, Möbius D, Goebels H, Gerberding AL, Schröder AS, Sperhake JP, Klein A, Fröb D, Mushumba H, Wilmes S, Anders S, Kniep I, Heinrich F, Langenwalder F, Meißner K, Lange P, Zapf A, Püschel K, Heinemann A, Glatzel M, Matschke J, Aepfelbacher M, Lütgehetmann M, Steurer S, Thorns C, Edler C, Ondruschka B. Prospective postmortem evaluation of 735 consecutive SARS-CoV-2-associated death cases. Sci Rep. 2021;11(1):19342.

References:

1. Eslami N, Aghbash PS, Shamekh A, Entezari-Maleki T, Nahand JS, Sales AJ, Baghi HB. SARS-CoV-2: Receptor and co-receptor tropism probability. Curr Microbiol. 2022;79(5):133.

2. Pfefferle S, Reucher S, Nörz D, Lütgehetmann M. Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system. Euro Surveill. 2020;25(9):2000152.

3. Hart JD, de André PA, de André CDS, Adair T, Barroso LP, Valongueiro S, et al. Validation of SmartVA using conventional autopsy: A study of adult deaths in Brazil. Lancet Reg. Health-Am. 2021;5:100081.

• 
• 

Mechanisms underlying sex differences in HIV reservoir composition andsize in people living with HIV-1 on antiretroviral therapy

Project leader: Marcus Altfeld

Affiliation: Leibniz Institute of Virology, Hamburg

Background and preliminary data: Manifestations of HIV-1 infection differ between females and males. Previous studies haveshown that cis-gender women living with HIV-1 (WLWH) control viral replication better thancis-gender men living with HIV-1 (MLWH) in acute HIV-1 infection (Meditz et al., JID 2011). Incontrast, WLWH experience faster loss of CD4+ T cells and faster progression to AIDS duringuntreated chronic HIV-1 infection after controlling for the level of viral replication (Sterling etal., NEJM 2001). Increasing data indicate that these sex differences in the manifestations ofHIV-1 disease are mediated by sex-specific differences in antiviral immunity.

Please follow the link to download the project proposal.

• 
• 

Analysis of the influence of gastrointestinal infections on host behavior via alteration of the gut microbiota, immune system and bacterial metabolites

Project leader: Samuel Huber and Matthias Kneussel

Affiliation: I. Department of Medicine, University Medical Center Hamburg-Eppendorf and Institute of Molecular Genetics, ZMNH

Background and preliminary data:

The gut-brain axis is a bidirectional communication network between the gut and the brain. It involves the gut microbiota, enteric nervous system, and immune system, influencing brain function including sociability, anxiety, and cognition. It has been shown that changes in the microbiota modulate host behaviour such as anxiety-related responses, social behaviour, and cognition. However, little is known about the influence of gastrointestinal infections on the infected individuals’ behaviour. Interestingly, one publication has postulated that infection with Citrobacter rodentium can cause anxiety-like symptoms that are probably mediated via vagal sensory neurons. However, a more recent study has shown that chronic inflammatory infections with Trichus muris induce anxiety-like behaviour in mice, which was present even after vagotomy. Furthermore, recent studies show that the immune system corresponds directly with the brain and modulates behaviour, and suggest that this might be mediated by altered cytokine production. For instance, maternal immune activation as a rodent model for autism induced a systemic release of IL-17a by Th17 cells in pregnant dams, resulting in impaired cortical patch development and behavioural abnormalities in the offspring. These effects were significantly increased in dams containing segmented filamentous bacteria which are known to potently induce Th17 cells in the intestine. Indeed, the phenotype could be reversed upon injection of IL-17A blocking antibodies. We have been studying the impact of the intestinal microbiota on the immune system and inflammatory diseases for years. Thus, as a first step and proof of concept, we found that different SPF microbiota are associated with a different intestinal immune cell composition (e.g. Th17 cell frequencies) and behaviour (e.g. anxiety) (unpublished data). Overall, those data suggest a crucial role of the immune system in mediating behavioural changes in the context of gastrointestinal infections. Whether this is actually the case and what mechanism(s) underlie this is, however, unclear and will be investigated in this project.

Hypothesis:

We hypothesize that gastrointestinal infections alter mice behaviour via modulating the intestinal microbiota and the immune system. Specifically, we hypothesize that infections promote anxiety, reduce sociability and cognition via microbiota alterations.

Our aim is to test this hypothesis and to decipher the underlying mechanism(s) in order to build the basis for targeted interventions for mental health conditions. To test the above-mentioned hypothesis, we aim to pursue the following objectives:

Aims and Work Programme:

• Analysis of behaviour including sociability, anxiety, and cognition of mice after infection with C. rodentium (model of bacterial gastrointestinal infection), and murine Cytomegalovirus (MCMV, viral model)
• Deciphering the functional role of the microbiota and bacterial metabolites on the immune system and host’s behaviour

Aim 1A: we will administer C. rodentium (a mouse model for E. coli infections) orally to 8 weeks old C57BL/6 wild-type mice. After 6 days (peak of disease) and 23 days (clearance of the pathogen) we will perform behavioural experiments including anxiety-like behaviour, sociability, and the cognition tests including a control group.

First, anxiety-like behaviour will be tested in an elevated-plus-maze with four arms, two closed and two open. A camera is used to measure the time spent in the open arms, indicating less anxious behaviour. Second, differences between the two groups in sociability are quantified in a social recognition test. After habituation in an empty three-chamber apparatus, mice are observed with an unknown intruder under one pencil cup while the other pencil cup is empty. The subject then encounters the now known intruder and a second unknown intruder. The number of entrances and the time spent in the different chambers, as well as the time spent sniffing each pencil cup, is recorded. Mice, which are normally social, prefer to spend time with conspecifics and are naturally more interested in novel objects or intruders, indicating social novelty and cognitive function. Deficits are seen, for example, in autism-like behaviour. Third, to further assess possible differences in cognition tasks, we perform an object recognition test. After several behavioural tests, the mice get used to the tests and tend to be less anxious in new situations, which is an advantage in cognitive tasks. The mice are placed in an open arena on the first day for habituation. On the training day, two similar objects are placed in different corners of the arena and the mouse is carefully placed in the middle to explore the arena. On the test day, one object is exchanged for a new, different object and the mouse explores the arena again. A camera is used to record the number of contacts and the time. Rodents tend to explore novel objects and this experiment is used to assess long-term recognition memory. Finally, we will also perform this experiment using a viral intestinal infection using MCMV to determine possible differences due to bacterial vs. viral gastrointestinal infections.

Aim 1B: we aim to decipher the role of the immune system in mediating the changes in behaviour upon a gastrointestinal infection. Specifically, we hypothesize that gastrointestinal infections can either directly or indirectly (via changes in the microbiota) promote the systemic release of proinflammatory cytokines and/or metabolites which are responsible for behavioural changes. To test this hypothesis, we will collect in the experiments of aim 1A intraepithelial lymphocytes and mesenteric lymph nodes, meninges, CNS, and blood. We will analyze the immune cell repertoire using flow cytometry (FACS) and defined cytokines as well as inflammatory markers (such as LPS) by using Enzyme-linked Immunosorbent Assay (ELISA). After separation of immune cells from tissues using Percoll gradient, quantitative determination of cell number is performed using automated cell counters (BioRad). This is followed by flow cytometry (FACS Fortessa, allows simultaneous measurement of 18 parameters) with extra-and intracellular staining for markers and cytokines (including for instance CD3, CD4, Foxp3, IL-10, IL-17A, IFN-γ, IL-4, IL-22, F4/80, CD11c, CD11b, CD14, Ly6G, Ly6C). The methods are already established in the laboratory. Furthermore, we will analyze the intestinal microbiota composition using metagenome sequencing. This analysis will be complemented by the metabolome analysis using biocrates.

Aim 2: we will use germ-free mice, which will be engrafted with the intestinal microbiota of the mice in aim 1 upon clearance of infection (A) and of patients upon clearance of a gastrointestinal infection (e.g. Campylobacter) (B). We will further study behaviour, immune cell composition, and metabolites essentially as described in aim 1B.

Long-term aim: to decipher the specific impact of metabolites, immune cells and cytokines on the observed effects to pave the way for targeted therapies.

Project-related publications (*own publications):

Bercik P et al (2010) Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry in mice. Gastroenterology 139(6):2102–2112 e1

Lyte M et al (2006) Induction of anxiety-like behavior in mice during the initial stages of infection with the agent of murine colonic hyperplasia Citrobacter rodentium. Physiol Behav 89(3):350–357

Lammert C et al (2018) Cutting Edge: Critical Roles for Microbiota-Mediated Regulation of the Immune System in a Prenatal Immune Activation Model of Autism. The Journal of Immunology 201 (3):845–850

Reed M et al. (2020) IL-17a promotes sociability in mouse models of neurodevelopmental disorders. Nature 577 (7789):249–253

*Tintelnot et al (2023) Microbiota-derived 3-IAA influences chemotherapy efficacy in pancreatic cancer. Nature 615(7950):168-174

xxx

xxx

xxx

J. Malsy

• 
• 

Innate immune response to human norovirus infection in human intestinal organoids

Project leader: Prof. Dr. Marcus Altfeld

Affiliation: Leibniz Institute of Virology

Background and preliminary data: Human Noroviruses (hNVs) are the leading cause of non-bacterial gastroenteritis worldwide and a major factor of diarrhea-associated deaths in children in developing countries. During each winter, up to 5000 cases of hNV infections per week are reported in Germany. Pathogenesis and immunologic responses to hNV infection remain largely unknown and no causal therapy is currently available. The lack of a robust experimental model for studying hNVs pathogenesis has hindered the development of effective therapeutic interventions. Recently, the landscape of hNV research has changed importantly with the initial demonstration in 2016 that hNVs can replicate in human intestinal organoids¹ and the subsequent demonstration in July 2022 that hNV can also replicate in a primary salivary cell line². These novel in vitro models will now enable to investigate hNV biology and innate host responses using human cell systems.

Pattern recognition receptors (PRRs) play a key role in the early immune response to viral infection, inducing innate Type I Interferon (IFN) responses and initiating subsequent adaptive immune responses. During murine NV infections, nucleotide-binding and oligomerization domain like receptor (NLR) activation and stimulation of the cGAS-STING-pathway have been described; however, the specific PRRs that can recognize hNV infection remain unknown. In Aims #1 and #2 of this project, human intestinal organoids and salivary cells will be used to identify the PRRs that sense hNV infection, and the mechanisms by which PRR sensing of hNV induces antiviral Type I IFN responses. Aim #3 will build on previous studies of the Altfeld lab demonstrating that antiviral Type I IFN responses differ between the sexes, with consequences for the clinical manifestations of viral infections3,4. Sex differences in hNV infection have also emerged from the case numbers reported to the Robert Koch Institute in Germany. While incidences of hNV infections are higher in male children, hNV incidences after puberty are higher in females. In Aim #3, we plan to investigate whether PRR sensing of hNV induces sex-specific differences in Type I IFN responses, and whether these differences are underlying the sex-differences in reported hNV cases and manifestations.

The Altfeld laboratory has extensive experience in investigating innate and adaptive immune responses against viral infections5, including the use of human intestinal organoid models. Preliminary studies have furthermore demonstrated successful hNV infection of human intestinal monolayers and indicated NLR activation (see below).

Left image: HNV Capsid (VP1) positive cells from human intestinal monolayers, identified using flow cytometry, after 1 hour past NV infection (blue) and 48 hours past infection (orange), indicating hNV replication. Right image: ASC speck formation in a human intestinal monolayers as a surrogate for NLR activation after incubation with hNV. Scale bar 20 μm.

Aims and Work-Programme:

Aim #1 – Identification of PRR expression pattern during the early immune response to hNV infection

Hypothesis: Specific PRR pathways are activated in human intestinal organoids during early hNV infection, through sensing of hNVs and/or cell damage.

Approach: Upon infection of human intestinal organoids (HIO) and infection of salivary primary cell lines, expression of PRRs and of key regulators of PRR signaling will be assessed, using qRT-PCR and multiparameter flow cytometry. Specifically, expression of TLR3-4, TLR7-9, NLRs, RIG-1, MDA5, cGAS, STING, P2X7, MyD88, Caspase-1, NFkB, IRF7 and IRF3 will be assessed during early and late acute infection.

Aim #2 – Assessing the consequences of PRR signaling during hNV infection

Hypothesis: PRR signaling influences infection rates and replication of hNVs in HIOs.

Approach: Induction of Type I IFNs and other antiviral cytokines (TNF, Il-1, Il-6, Il-18) will be quantified following infection. We will generate HIOs knockouts, using the CRISPR-Cas9 platform in our laboratory, to inactivate the specific PRRs identified in Aim 1 in order to determine their contribution to antiviral effector functions. Knockouts and naïve HIOs will be infected with hNVs and cell viability, organelle damage, infection rates and hNV replication will be assessed to identify effects of the specific PRRs during hNV infection.

Aim #3 – The role of sex hormones in regulating hNV infection and PRR signaling

Hypothesis: Sex hormones influence hNV infection through regulation of PRR expression.

Approach: To determine whether increased levels of estrogens, testosterone and/or progesterone influence hNV infection and viral replication of hNVs, HIOs will be incubated with the respective hormones before infection. We will investigate whether sex-hormones alter expression of PRRs identified in Aims 1 & 2, or whether they impact downstream signaling.

Project-related publications:

[1] Ettayebi, K., Crawford, S. E., Murakami, K., Broughman, J. R., Karandikar, U., Tenge, V. R., Neill, F. H., Blutt, S. E., Zeng, X.-L., Qu, L., Kou, B., Opekun, A. R., Burrin, D., Graham, D. Y., Ramani, S., Atmar, R. L., & Estes, M. K. (2016). Replication of human noroviruses in stem cell-derived human enteroids. Science, 353(6306), 1387–1393.

[2] Ghosh, S., Kumar, M., Santiana, M., Mishra, A., Zhang, M., Labayo, H., Chibly, A. M., Nakamura, H., Tanaka, T., Henderson, W., Lewis, E., Voss, O., Su, Y., Belkaid, Y., Chiorini, J. A., Hoffman, M. P., & Altan-Bonnet, N. (2022). Enteric viruses replicate in salivary glands and infect through saliva. Nature, 607.

[3] Hagen, S. H., Henseling, F., Hennesen, J., Savel, H., Delahaye, S., Richert, L., Ziegler, S. M., & Altfeld, M. (2020). Heterogeneous Escape from X Chromosome Inactivation Results in Sex Differences in Type I IFN Responses at the Single Human pDC Level. Cell Reports, 33(10).

[4] Ziegler, S. M., & Altfeld, M. (2017). Human immunodeficiency virus 1 and type I interferons-where sex makes a difference. Frontiers in Immunology, 8(Sep).

[5] Jung, J. M., Ching, W., Baumdick, M. E., Hofmann-Sieber, H., Bosse, J. B., Koyro, T., Möller, K. J., Wegner, L., Niehrs, A., Russu, K., Ohms, M., Zhang, W., Ehrhardt, A., Duisters, K., Spierings, E., Hölzemer, A., Körner, C., Jansen, S. A., Peine, S., … Bunders, M. J. (2021). KIR3DS1 directs NK cell–mediated protection against human adenovirus infections. Science Immunology, 6(63).