Edward Seung

Lipid-Cytokine-Chemokine Cascade Drives Neutrophil Recruitment in a Murine Model of Inflammatory Arthritis

A large and diverse array of chemoattractants control leukocyte trafficking, but how these apparently redundant signals collaborate in vivo is still largely unknown. We previously demonstrated an absolute requirement for the lipid chemoattractant leukotriene B(4) (LTB(4)) and its receptor BLT1 for neutrophil recruitment into the joint in autoantibody-induced arthritis. We now demonstrate that BLT1 is required for neutrophils to deliver IL-1 into the joint to initiate arthritis. IL-1-expressing neutrophils amplify arthritis through the production of neutrophil-active chemokines from synovial tissue cells. CCR1 and CXCR2, two neutrophil chemokine receptors, operate nonredundantly to sequentially control the later phase of neutrophil recruitment into the joint and mediate all neutrophil chemokine activity in the model. Thus, we have uncovered a complex sequential relationship involving unique contributions from the lipid mediator LTB(4), the cytokine IL-1, and CCR1 and CXCR2 chemokine ligands that are all absolutely required for effective neutrophil recruitment into the joint.

Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice

ABSTRACT The generation of humanized BLT mice by the cotransplantation of human fetal thymus and liver tissues and CD34+ fetal liver cells into nonobese diabetic/severe combined immunodeficiency mice allows for the long-term reconstitution of a functional human immune system, with human T cells, B cells, dendritic cells, and monocytes/macrophages repopulating mouse tissues. Here, we show that humanized BLT mice sustained high-level disseminated human immunodeficiency virus (HIV) infection, resulting in CD4+ T-cell depletion and generalized immune activation. Following infection, HIV-specific humoral responses were present in all mice by 3 months, and HIV-specific CD4+ and CD8+ T-cell responses were detected in the majority of mice tested after 9 weeks of infection. Despite robust HIV-specific responses, however, viral loads remained elevated in infected BLT mice, raising the possibility that these responses are dysfunctional. The increased T-cell expression of the negative costimulator PD-1 recently has been postulated to contribute to T-cell dysfunction in chronic HIV infection. As seen in human infection, both CD4+ and CD8+ T cells demonstrated increased PD-1 expression in HIV-infected BLT mice, and PD-1 levels in these cells correlated positively with viral load and inversely with CD4+ cell levels. The ability of humanized BLT mice to generate both cellular and humoral immune responses to HIV will allow the further investigation of human HIV-specific immune responses in vivo and suggests that these mice are able to provide a platform to assess candidate HIV vaccines and other immunotherapeutic strategies.

HIV-infected T cells are migratory vehicles for viral dissemination

After host entry through mucosal surfaces, human immunodeficiency virus-1 (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 the early stages of transmission and systemically during subsequent dissemination are not known. In vitro studies suggest that the formation of virological synapses during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer. It is unclear, however, whether T-cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial and lymphoid tissues. Here, using multiphoton intravital microscopy, we examine the dynamic behaviour of HIV-infected T cells in the lymph nodes of humanized mice. We find that most productively infected T cells migrate robustly, resulting in their even distribution throughout the lymph node cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope-dependent cell fusion. Both uncoordinated motility of syncytia and adhesion to CD4+ lymph node cells led to the formation of long membrane tethers, increasing cell lengths to up to ten times that of migrating uninfected T cells. Blocking the egress of migratory T cells from the lymph nodes into efferent lymph vessels, and thus interrupting T-cell recirculation, limited HIV dissemination and strongly reduced plasma viraemia. 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 virological synapses. Migration of T cells in lymph nodes therefore spreads infection locally, whereas their recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection.

Evolutionarily conserved recognition and innate immunity to fungal pathogens by the scavenger receptors SCARF1 and CD36

Receptors involved in innate immunity to fungal pathogens have not been fully elucidated. We show that the Caenorhabditis elegans receptors CED-1 and C03F11.3, and their mammalian orthologues, the scavenger receptors SCARF1 and CD36, mediate host defense against two prototypic fungal pathogens, Cryptococcus neoformans and Candida albicans. CED-1 and C03F11.1 mediated antimicrobial peptide production and were necessary for nematode survival after C. neoformans infection. SCARF1 and CD36 mediated cytokine production and were required for macrophage binding to C. neoformans, and control of the infection in mice. Binding of these pathogens to SCARF1 and CD36 was β-glucan dependent. Thus, CED-1/SCARF1 and C03F11.3/CD36 are β-glucan binding receptors and define an evolutionarily conserved pathway for the innate sensing of fungal pathogens.

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras.

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4⁺ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.

CD11b+ Myeloid Cells Are the Key Mediators of Th2 Cell Homing into the Airway in Allergic Inflammation

STAT6-mediated chemokine production in the lung is required for Th2 lymphocyte and eosinophil homing into the airways in allergic pulmonary inflammation, and thus is a potential therapeutic target in asthma. However, the critical cellular source of STAT6-mediated chemokine production has not been defined. In this study, we demonstrate that STAT6 in bone marrow-derived myeloid cells was sufficient for the production of CCL17, CCL22, CCL11, and CCL24 and for Th2 lymphocyte and eosinophil recruitment into the allergic airway. In contrast, STAT6 in airway-lining cells did not mediate chemokine production or support cellular recruitment. Selective depletion of CD11b+ myeloid cells in the lung identified these cells as the critical cellular source for the chemokines CCL17 and CCL22. These data reveal that CD11b+ myeloid cells in the lung help orchestrate the adaptive immune response in asthma, in part, through the production of STAT6-inducible chemokines and the recruitment of Th2 lymphocytes into the airway.

BLT1-mediated T cell trafficking is critical for rejection and obliterative bronchiolitis after lung transplantation

Leukotriene B4 is a lipid mediator that recently has been shown to have potent chemotactic activity for effector T lymphocytes mediated through its receptor, BLT1. Here, we developed a novel murine model of acute lung rejection to demonstrate that BLT1 controls effector CD8+ T cell trafficking into the lung and that disruption of BLT1 signaling in CD8+ T cells reduces lung inflammation and mortality in the model. In addition, we used BLT1-deficient mice and a BLT1 antagonist in two tracheal transplant models of lung transplantation to demonstrate the importance of BLT1 for the recruitment of T cells into tracheal allografts. We also show that BLT1-mediated CD8+ T cell recruitment plays an important role in the development of airway fibroproliferation and obliteration. Finally, in human studies of lung transplant recipients, we found that BLT1 is up-regulated on T lymphocytes isolated from the airways of patients with obliterative bronchiolitis. These data demonstrate that BLT1 contributes to the development of lung rejection and obliterative bronchiolitis by mediating effector T lymphocyte trafficking into the lung. This is the first report that describes a pathologic role for BLT1-mediated T lymphocyte recruitment in disease and identifies BLT1 as a potential therapeutic target after lung transplantation.

Rapid Evolution of HIV-1 to Functional CD8+ T Cell Responses in Humanized BLT Mice

Humanized BLT mice accurately develop human HIV-specific CD8+ T cell responses capable of rapidly selecting for CTL escape mutations. Mirror, Mirror One limitation of using animal models of disease is that there’s no magic mirror to tell you which one best reflects human disease. Instead, most animal disease models mimic some aspects of the human condition, but may not recapitulate the disease in its entirety. This limitation is especially true for HIV infection because the virus does not naturally infect mice—the model of choice for biomedical research. Attempts to “humanize” immunodeficient mice through grafting of human immune cells may reconfigure the mouse from a distorting funhouse mirror into a well-lit vanity one. Now, Dudek et al. use humanized BLT (brain, liver, thymus) mice to study human immune responses to HIV. The authors found that HIV-1–specific immune responses in BLT mice mimicked those in humans in terms of specificity, kinetics, and dominant target. Importantly, HIV adapted to the immune responses in these mice just as it does in humans, evolving rapidly to escape from the selective pressure. Indeed, an HLA allele that is protective in humans induced similar protective immune responses in these mice. Although no animal model may perfectly reflect human disease, for HIV infection, humanized BLT mice may be one of the fairest of them all. The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1–specific immune responses and viral evolution during the acute phase of infection. HIV-1–specific CD8+ T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8+ T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen–specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.

PD-1 Blockade in Chronically HIV-1-Infected Humanized Mice Suppresses Viral Loads

An estimated 34 million people are living with HIV worldwide (UNAIDS, 2012), with the number of infected persons rising every year. Increases in HIV prevalence have resulted not only from new infections, but also from increases in the survival of HIV-infected persons produced by effective anti-retroviral therapies. Augmentation of anti-viral immune responses may be able to further increase the survival of HIV-infected persons. One strategy to augment these responses is to reinvigorate exhausted anti-HIV immune cells present in chronically infected persons. The PD-1-PD-L1 pathway has been implicated in the exhaustion of virus-specific T cells during chronic HIV infection. Inhibition of PD-1 signaling using blocking anti-PD-1 antibodies has been shown to reduce simian immunodeficiency virus (SIV) loads in monkeys. We now show that PD-1 blockade can improve control of HIV replication in vivo in an animal model. BLT (Bone marrow-Liver-Thymus) humanized mice chronically infected with HIV-1 were treated with an anti-PD-1 antibody over a 10-day period. The PD-1 blockade resulted in a very significant 45-fold reduction in HIV viral loads in humanized mice with high CD8+ T cell expression of PD-1, compared to controls at 4 weeks post-treatment. The anti-PD-1 antibody treatment also resulted in a significant increase in CD8+ T cells. PD-1 blockade did not affect T cell expression of other inhibitory receptors co-expressed with PD-1, including CD244, CD160 and LAG-3, and did not appear to affect virus-specific humoral immune responses. These data demonstrate that inhibiting PD-1 signaling can reduce HIV viral loads in vivo in the humanized BLT mouse model, suggesting that blockade of the PD-1-PD-L1 pathway may have therapeutic potential in the treatment of patients already infected with the AIDS virus.

CXCR3 and Its Ligands in a Murine Model of Obliterative Bronchiolitis: Regulation and Function

Lung transplantation remains the only effective therapy for patients with end-stage lung disease, but survival is limited by the development of obliterative bronchiolitis (OB). The chemokine receptor CXCR3 and two of its ligands, CXCL9 and CXCL10, have been identified as important mediators of OB. However, the relative contribution of CXCL9 and CXCL10 to the development of OB and the mechanism of regulation of these chemokines has not been well defined. In this study, we demonstrate that CXCL9 and CXCL10 are up-regulated in unique patterns following tracheal transplantation in mice. In these experiments, CXCL9 expression peaked 7 days posttransplant, while CXCL10 expression peaked at 1 day and then again 7 days posttransplant. Expression of CXCL10 was also up-regulated in a novel murine model of lung ischemia, and in bronchoalveolar lavage fluid taken from human lungs 24 h after lung transplantation. In further analysis, we found that 3 h after transplantation CXCL10 is donor tissue derived and not dependent on IFN-γ or STAT1, while 24 h after transplantation CXCL10 is from recipient tissue and regulated by IFN-γ and STAT1. Expression of both CXCL9 and CXCL10 7 days posttransplant is regulated by IFN-γ and STAT1. Finally, we demonstrate that deletion of CXCR3 in recipients reduces airway obliteration. However, deletion of either CXCL9 or CXCL10 did not affect airway obliteration. These data show that in this murine model of obliterative bronchiolitis, these chemokines are differentially regulated following transplantation, and that deletion of either chemokine alone does not affect the development of airway obliteration.