Stephan Halle

Introduction: Intact kidney function is essential for fluid and electrolyte homeostasis, with renal tubular epithelial cells providing critical functions in the reabsorption or secretion of numerous metabolites. Cellular senescence of the renal epithelial cells can lead to functional deficits. Therefore, immune-cell-mediated clearance of senescent epithelial cells can be an important factor for the maintenance of kidney function.Methods: In this study, we established a model to directly monitor natural killer (NK) cell-mediated clearance of senescent renal tubular cells. To this end we used primary cell co-cultures with different read-outs and life cell imaging.Results and Discussion: We observed that the clustering of NK cells on senescent renal epithelial cells could be used to detect senescence-triggered NK cell activation. Also, we found that NKG2D signaling and perforin-dependent lysis of senescent renal epithelial cells were crucial steps in the lysis of senescent cells. In addition, NK cell-mediated attack of senescent renal epithelial cells could be dampened by the addition of cyclosporine A and was augmented by the addition of interleukin 7. Together, these data show that NK cells can efficiently mediate the clearance of senescent renal tubular epithelial cells involving NK cell activation by NKG2D signaling.

Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murine ex vivo explant model using gentle depilation without further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently tagged capsid protein VP26, and observed the formation of nuclear capsid assembly sites and nuclear capsid egress and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By using electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinical ex vivo infection model. IMPORTANCE: This study describes a novel murine ex vivo skin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers increasing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.

Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murine ex vivo explant model using gentle depilation but no further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy studies showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently-tagged capsid protein VP26, and monitored the formation of nuclear capsid assembly sites, nuclear capsid egress, and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinical ex vivo infection model. IMPORTANCE: This study describes a novel murine ex vivo skin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers growing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.

Immediately after entry into host cells, viruses are sensed by the innate immune system, leading to the activation of innate antiviral effector mechanisms including the type I interferon (IFN) response and natural killer (NK) cells. This innate immune response helps to shape an effective adaptive T cell immune response mediated by cytotoxic T cells and CD4⁺ T helper cells and is also critical for the maintenance of protective T cells during chronic infection. The human gammaherpesvirus Epstein-Barr virus (EBV) is a highly prevalent lymphotropic oncovirus that establishes chronic lifelong infections in the vast majority of the adult population. Although acute EBV infection is controlled in an immunocompetent host, chronic EBV infection can lead to severe complications in immunosuppressed patients. Given that EBV is strictly host-specific, its murine homolog murid herpesvirus 4 or MHV68 is a widely used model to obtain in vivo insights into the interaction between gammaherpesviruses and their host. Despite the fact that EBV and MHV68 have developed strategies to evade the innate and adaptive immune response, innate antiviral effector mechanisms still play a vital role in not only controlling the acute infection but also shaping an efficient long-lasting adaptive immune response. Here, we summarize the current knowledge about the innate immune response mediated by the type I IFN system and NK cells, and the adaptive T cell-mediated response during EBV and MHV68 infection. Investigating the fine-tuned interplay between the innate immune and T cell response will provide valuable insights which may be exploited to design better therapeutic strategies to vanquish chronic herpesviral infection.

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediatorRbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

In vivoimaging of cytotoxic T lymphocyte (CTL) killing activity revealed that infected cells have a higher observed probability of dying after multiple contacts with CTLs. We developed a three-dimensional agent-based model to discriminate different hypotheses about how infected cells get killed based on quantitative 2-photonin vivoobservations. We compared a constant CTL killing probability with mechanisms of signal integration in CTL or infected cells. The most likely scenario implied increased susceptibility of infected cells with increasing number of CTL contacts where the total number of contacts was a critical factor. However, when allowingin silicoT cells to initiate new interactions with apoptotic target cells (zombie contacts), a contact history independent killing mechanism was also in agreement with experimental datasets. The comparison of observed datasets to simulation results, revealed limitations in interpreting 2-photon data, and provided readouts to distinguish CTL killing models.

Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.

In vivo imaging of cytotoxic T lymphocyte (CTL) killing activity revealed that infected cells have a higher observed probability of dying after multiple contacts with CTLs, suggesting memory effect in CTLs or infected cells. We developed a three-dimensional agent-based model of CTL killing activity to discriminate different hypotheses about how infected cells get killed based on quantitative 2-photon in vivo observations. We compared a constant CTL killing probability with mechanisms of signal integration in CTL or infected cells. The most likely scenario implied increased susceptibility of infected cells with increasing number of CTL contacts where the total number of contacts was a critical factor as opposed to signal integration over many contacts. However, when allowing in silico T cells to interact with apoptotic target cells (zombie contacts), a contact history independent killing mechanism was also in agreement with the experimental datasets. We showed that contacts that take place between CTLs and dying infected cells impact the observed killing dynamics because even in absence of modulation of cell properties, we saw an increase of the observed probability of killing infected cells with more interactions. The duration taken by an infected cell to die and the per capita killing rate (PCKR) of CTLs, parameters hard to measure directly, were determined from the model and turned out predictive to distinguish the different CTL killing models in future experiments. The comparison of observed datasets to simulation results, revealed limitations in interpreting 2-photon data, and provided prediction for additional measurements to distinguish CTL killing models. Highlights: Killing of infected cells by cytotoxic T cells typically involves more than a single contact. Cytotoxic T cells or infected cells integrate signals from multiple interactions. T cell contacts with dying infected cells have a major impact on in vivo data interpretation. Significance Statement: Despite having a clear understanding of cytotoxic T lymphocyte (CTL) mediated cytotoxicity mechanisms, the quantitative dynamics remain unexplored at a cellular level. We developed an agent-based model to compare different hypotheses for mechanisms of CTL mediated cytotoxicity that could lead to an increase in observed probability of killing infected cells at higher interactions with CTLs as seen in vivo . We showed that this behaviour can be explained by modulation of properties by infected cells or CTLs with increasing number of contacts. For the modulation, we compared two modes of signal integration and showed that time is not a relevant parameter in signal integration. We also studied the impact of contacts between CTLs and apoptotic infected cells on observed killing properties.

Bacterial infections of the central nervous system (CNS) remain a major cause of mortality in the neonatal population. Commonly used parenteral infection models, however, do not reflect the early course of the disease leaving this critical step of the pathogenesis largely unexplored. Here, we analyzed nasal exposure of 1-day-old newborn mice to Listeria monocytogenes (Lm). We found that nasal, but not intragastric administration, led to early CNS infection in neonate mice. In particular, upon bacterial invasion of the olfactory epithelium, Lm subsequently spread along the sensory neurons entering the brain tissue at the cribriform plate and causing a significant influx of monocytes and neutrophils. CNS infection required listeriolysin for penetration of the olfactory epithelium and ActA, a mediator of intracellular mobility, for translocation into the brain tissue. Taken together, we propose an alternative port of entry and route of infection for neonatal neurolisteriosis and present a novel infection model to mimic the clinical features of late-onset disease in human neonates.