Two-photon intravital imaging is used here to define the regulation of interstitial neutrophil migration at local sites of cell death upon sterile tissue injury and infection; leukotriene B4 (LTB4) is shown to act between neutrophils as a signal relay molecule that acts to enhance the radius of neutrophil recruitment within the inflamed interstitium, and also to control, in concert with integrin receptors, dense neutrophil clustering for tight wound seal formation. Neutrophils, key components of the innate immune defence system, also help in wound healing after tissue damage. How these cells leave the bloodstream to reach sites of infection or damage has been well characterized, but less is known about how they find their way outside the bloodstream and move to the site of infection or damage. Using intravital dynamic two-photon imaging Ronald Germain and colleagues examine the behaviour of neutrophils in a new model of skin sterile cell damage. The study shows that neutrophil production of the lipid leukotriene B4 (LTB4) is required for long-distance neutrophil recruitment and initial cluster formation. Subsequent dense cluster formation and the rearrangement of the collagen fibre network at centre of the wound, generating a collagen-free zone and sealing off the wound from surrounding tissue, requires LTB4 as well as integrins. Neutrophil recruitment from blood to extravascular sites of sterile or infectious tissue damage is a hallmark of early innate immune responses, and the molecular events leading to cell exit from the bloodstream have been well defined1,2. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects3,4,5,6,7,8,9,10,11. The molecular players that direct this response at the single-cell and population levels within the complexity of an inflamed tissue are unknown. Using two-photon intravital microscopy in mouse models of sterile injury and infection, we show a critical role for intercellular signal relay among neutrophils mediated by the lipid leukotriene B4, which acutely amplifies local cell death signals to enhance the radius of highly directed interstitial neutrophil recruitment. Integrin receptors are dispensable for long-distance migration12, but have a previously unappreciated role in maintaining dense cellular clusters when congregating neutrophils rearrange the collagenous fibre network of the dermis to form a collagen-free zone at the wound centre. In this newly formed environment, integrins, in concert with neutrophil-derived leukotriene B4 and other chemoattractants, promote local neutrophil interaction while forming a tight wound seal. This wound seal has borders that cease to grow in kinetic concert with late recruitment of monocytes and macrophages at the edge of the displaced collagen fibres. Together, these data provide an initial molecular map of the factors that contribute to neutrophil swarming in the extravascular space of a damaged tissue. They reveal how local events are propagated over large-range distances, and how auto-signalling produces coordinated, self-organized neutrophil-swarming behaviour that isolates the wound or infectious site from surrounding viable tissue.

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host. Chikungunya virus (CHIKV) is a reemerging alphavirus responsible for an unprecedented epidemic in countries of the Indian Ocean region, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The most recent epidemic reemergences were documented in Kinshasa, (50,000 estimated cases in 1999–2000), in Indonesia (2001–2003), the Indian Ocean islands of Mayotte, Mauritius, Réunion, and the Seychelles (270,000 cases in 2005–2006 in La Réunion island), and in India (1.4 to 6.5 million estimated cases in 2006–2007). There is a critical lack of knowledge on the biology of CHIKV. In particular, virtually nothing is known about the interaction of CHIKV (and of most alpahaviruses) with human primary cells. We have studied the replication characteristics and the tropism of clinical CHIKV strains from La Réunion. We designed various assays and reagents to follow viral replication, and we report here that adherent cells (epithelial and endothelial cells, primary fibroblasts), as well as macrophages, are sensitive to infection. In contrast, blood cells did not allow viral replication. We also characterized viral entry pathways and sensitivity to interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host. This paper is the result of a collaborative effort between numerous teams from Institut Pasteur, the Groupe Hospitalier Sud Réunion, and other institutions. Our aim was to establish a task force with multiple and complementary expertise on virology, immunology, and cell biology in order to characterize this enigmatic virus.

Chikungunya (CHIK) virus is a mosquito-transmitted alphavirus that causes in humans an acute infection characterised by fever, polyarthralgia, head-ache, and myalgia. Since 2005, the emergence of CHIK virus was associated with an unprecedented magnitude outbreak of CHIK disease in the Indian Ocean. Clinically, this outbreak was characterized by invalidating poly-arthralgia, with myalgia being reported in 97.7% of cases. Since the cellular targets of CHIK virus in humans are unknown, we studied the pathogenic events and targets of CHIK infection in skeletal muscle. Immunohistology on muscle biopsies from two CHIK virus-infected patients with myositic syndrome showed that viral antigens were found exclusively inside skeletal muscle progenitor cells (designed as satelllite cells), and not in muscle fibers. To evaluate the ability of CHIK virus to replicate in human satellite cells, we assessed virus infection on primary human muscle cells; viral growth was observed in CHIK virus-infected satellite cells with a cytopathic effect, whereas myotubes were essentially refractory to infection. This report provides new insights into CHIK virus pathogenesis, since it is the first to identify a cellular target of CHIK virus in humans and to report a selective infection of muscle satellite cells by a viral agent in humans.

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

The blood–brain barrier (BBB), which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1) Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans), both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies. The blood–brain barrier (BBB) forms the interface between the blood and the central nervous system (CNS). BBB disruption is considered to be a key event in the pathogenesis of retroviral-associated neurological diseases. The present paper deals with the susceptibility of the endothelial cells (i.e., one of the main cellular components of BBB) to retroviral infection, and with the impact of infection in BBB function. This study focuses on the Human T-Lymphotropic Virus (HTLV-1), which infects 20 million people worldwide, and is the etiological agent of a neurodegenerative disease called HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). We first demonstrated that the cerebral endothelial cells express the receptors for the retrovirus in vitro, and on spinal cord autopsy sections from non-infected and HAM/TSP patients. We found on these latter that vascular-like structures were infected and confirmed in vitro that the endothelial cells could be productively infected by HTLV-1. We demonstrated that such an infection impairs BBB properties in vitro, as well as tight junctions, that are cell adhesion structures. This study is the first to demonstrate the impact of HTLV-1 infection on human BBB integrity; such a susceptibility has to be considered in the design of future therapeutics strategies.