Person:

von Andrian-Werburg, Ulrich

After host entry through mucosal surfaces, HIV-1 disseminates to lymphoid tissues to establish a generalized infection of the immune system. The mechanisms by which this virus spreads among permissive target cells locally during early stages of transmission, and systemically during subsequent dissemination are not known1. In vitro studies suggest that formation of virological synapses (VSs) during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer2. It is unclear, however, if T cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial3 and lymphoid tissues4. Here, using multiphoton intravital microscopy (MP-IVM), we examined the dynamic behavior of HIV-infected T cells in lymph nodes (LNs) of humanized mice. We found that most productively infected T cells migrated robustly, resulting in their even distribution throughout the LN cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope (Env)-dependent cell fusion. Both uncoordinated motility of syncytia as well as adhesion to CD4+ LN cells led to the formation of long membrane tethers, increasing cell lengths to up to 10 times that of migrating uninfected T cells. Blocking the egress of migratory T cells from LNs into efferent lymph, and thus interrupting T cell recirculation, limited HIV dissemination and strongly reduced plasma viremia. Thus, we have found that HIV-infected T cells are motile, form syncytia, and establish tethering interactions that may facilitate cell-to-cell transmission through VSs. While their migration in LNs spreads infection locally, T cell recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection.

Background: The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. Objectives: To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. Methodology/Principal Findings: Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-κB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-κB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. Conclusions/Significance: We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness.

To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance.

Heterogeneity and functional specialization among skin-resident macrophages are incompletely understood. In this study, we describe a novel subset of murine dermal perivascular macrophages that extend protrusions across the endothelial junctions in steady-state and capture blood-borne macromolecules. Unlike other skin-resident macrophages that are reconstituted by bone marrow-derived progenitors after a genotoxic insult, these cells are replenished by an extramedullary radio-resistant and UV-sensitive Bmi1+ progenitor. Furthermore, they possess a distinctive anti-inflammatory transcriptional profile, which cannot be polarized under inflammatory conditions, and are involved in repair and remodeling functions for which other skin-resident macrophages appear dispensable. Based on all their properties, we define these macrophages as Skin Transendothelial Radio-resistant Anti-inflammatory Macrophages (STREAM) and postulate that their preservation is important for skin homeostasis. DOI: http://dx.doi.org/10.7554/eLife.15251.001

iNKT cells are CD1d-restricted lipid-sensing innate T cells that express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, and their targets in adipose tissue are unknown. Here we report that adipose tissue iNKT cells have a unique transcriptional program and produce interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lack PLZF, but express the transcription factor E4BP4, which controls their IL-10 production. Adipose iNKT cells are a tissue resident population that induces an anti-inflammatory phenotype in macrophages and, through production of IL-2, controls the number, proliferation and suppressor function of adipose regulatory T (Treg) cells. Thus, adipose tissue iNKT cells are unique regulators of immune homeostasis in this tissue.

Abstract Natural killer (NK) cells are critical players against tumors. The outcome of anti‐tumor vaccination protocols depends on the efficiency of NK‐cell activation, and efforts are constantly made to manipulate them for immunotherapeutic approaches. Thus, a better understanding of NK‐cell activation dynamics is needed. NK‐cell interactions with accessory cells and trafficking between secondary lymphoid organs and tumoral tissues remain poorly characterized. Here, we show that upon triggering innate immunity with lipopolysaccharide (LPS), NK cells are transiently activated, leave the lymph node, and infiltrate the tumor, delaying its growth. Interestingly, NK cells are not actively recruited at the draining lymph node early after LPS administration, but continue their regular homeostatic turnover. Therefore, NK cells resident in the lymph node at the time of LPS administration become activated and exert anti‐tumor functions. NK‐cell activation correlates with the establishment of prolonged interactions with dendritic cells (DCs) in lymph nodes, as observed by two‐photon microscopy. Close DC and NK‐cell contacts are essential for the localized delivery of DC‐derived IL‐18 to NK cells, a strict requirement in NK‐cell activation.

Vibrio cholerae, the agent of cholera, is a motile non-invasive pathogen that colonizes the small intestine (SI). Most of our knowledge of the processes required for V. cholerae intestinal colonization is derived from enumeration of wt and mutant V. cholerae recovered from orogastrically infected infant mice. There is limited knowledge of the distribution of V. cholerae within the SI, particularly its localization along the villous axis, or of the bacterial and host factors that account for this distribution. Here, using confocal and intravital two-photon microscopy to monitor the localization of fluorescently tagged V. cholerae strains, we uncovered unexpected and previously unrecognized features of V. cholerae intestinal colonization. Direct visualization of the pathogen within the intestine revealed that the majority of V. cholerae microcolonies attached to the intestinal epithelium arise from single cells, and that there are notable regiospecific aspects to V. cholerae localization and factors required for colonization. In the proximal SI, V. cholerae reside exclusively within the developing intestinal crypts, but they are not restricted to the crypts in the more distal SI. Unexpectedly, V. cholerae motility proved to be a regiospecific colonization factor that is critical for colonization of the proximal, but not the distal, SI. Furthermore, neither motility nor chemotaxis were required for proper V. cholerae distribution along the villous axis or in crypts, suggesting that yet undefined processes enable the pathogen to find its niches outside the intestinal lumen. Finally, our observations suggest that host mucins are a key factor limiting V. cholerae intestinal colonization, particularly in the proximal SI where there appears to be a more abundant mucus layer. Collectively, our findings demonstrate the potent capacity of direct pathogen visualization during infection to deepen our understanding of host pathogen interactions.

The skin has a dual function as a barrier and a sensory interface between the body and the environment. To protect against invading pathogens, the skin harbors specialized immune cells, including dermal dendritic cells (DDCs) and interleukin (IL)-17 producing γδ T cells (γδT17), whose aberrant activation by IL-23 can provoke psoriasis-like inflammation1–4. The skin is also innervated by a meshwork of peripheral nerves consisting of relatively sparse autonomic and abundant sensory fibers. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear5,6. Here, we have exposed the skin of mice to imiquimod (IMQ), which induces IL-23 dependent psoriasis-like inflammation7,8. We show that a subset of sensory neurons expressing the ion channels TRPV1 and NaV1.8 is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of DDCs, the principal source of IL-23, is in close contact with these nociceptors. Upon selective pharmacological or genetic ablation of nociceptors9–11, DDCs failed to produce IL-23 in IMQ exposed skin. Consequently, the local production of IL-23 dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were dramatically reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with DDCs and restored the inflammatory response12. These findings indicate that TRPV1+NaV1.8+ nociceptors, by interacting with DDCs, regulate the IL-23/IL-17 pathway and control cutaneous immune responses.

Lrrc8a is a ubiquitously expressed gene that encodes a leucine-rich repeat (LRR)–containing protein detected at higher levels on the surface of thymocytes than on other immune cells. We generated Lrrc8a−/− mice to investigate the role of LRRC8A in lymphocyte development and function. Lrrc8a−/− mice had increased prenatal and postnatal mortality, growth retardation, and multiple tissue abnormalities. Lrrc8a−/− mice displayed a modest block in B cell development but intact intrinsic B cell function. In contrast, both Lrrc8a−/− mice and Lrrc8a−/−→Rag2−/− bone marrow chimeras exhibited a severe cell-intrinsic block in early thymic development, with decreased proliferation and increased apoptosis of thymocytes, and impaired peripheral T cell function. Thymic epithelial cells expressed an LRRC8A ligand that was critical for double-negative to double-positive thymocyte differentiation and survival in vitro. LRRC8A constitutively associated with the GRB2–GAB2 complex and lymphocyte-specific protein tyrosine kinase (LCK) in thymocytes. LRRC8A ligation activated AKT via the LCK–ZAP–70–GAB2–PI3K pathway, and AKT phosphorylation was markedly reduced in the thymus of Lrrc8a−/− mice. These findings reveal an essential role for LRRC8A in T cell development, survival, and function.

Phosphatidylinositol 3,4,5-trisphosphate ((PIP_3)) is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. (PIP_3) is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced (PIP_3) production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-α intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers.