PAD4-Mediated Neutrophil Extracellular Trap Formation Is Not Required for Immunity against Influenza Infection

During an inflammatory response, neutrophils migrate to the site of infection where they can kill invading pathogens by phagocytosis, secretion of anti-microbicidal mediators or the release of neutrophil extracellular traps (NETs). NETs are specialized anti-microbial structures comprised of decondensed chromatin decorated with microbicidal agents. Increased amount of NETs have been found in patients suffering from the chronic lung inflammatory disease cystic fibrosis, correlating with increased severity of pulmonary obstruction. Furthermore, acute lung inflammation during influenza A infection is characterized by a massive influx of neutrophils into the lung. The role of NETs during virus-mediated lung inflammation is unknown. Peptidylarginine deiminase 4 (PAD4)-mediated deimination of histone H3 and H4 is required for NET formation. Therefore, we generated a PAD4-deficient mouse strain that has a striking inability to form NETs. These mice were infected with influenza A/WSN, and the disease was monitored at the level of leukocytic lung infiltration, lung pathology, viral replication, weight loss and mortality. PAD4 KO fared comparable to WT mice in all the parameters tested, but they displayed slight but statistically different weight loss kinetics during infection that was not reflected in enhanced survival. Overall, we conclude that PAD4-mediated NET formation is dispensable in a mouse model of influenza A infection

The convective storm initiation project

Copyright @ 2007 AMSThe Convective Storm Initiation Project (CSIP) is an international project to understand precisely where, when, and how convective clouds form and develop into showers in the mainly maritime environment of southern England. A major aim of CSIP is to compare the results of the very high resolution Met Office weather forecasting model with detailed observations of the early stages of convective clouds and to use the newly gained understanding to improve the predictions of the model. A large array of ground-based instruments plus two instrumented aircraft, from the U.K. National Centre for Atmospheric Science (NCAS) and the German Institute for Meteorology and Climate Research (IMK), Karlsruhe, were deployed in southern England, over an area centered on the meteorological radars at Chilbolton, during the summers of 2004 and 2005. In addition to a variety of ground-based remote-sensing instruments, numerous rawin-sondes were released at one- to two-hourly intervals from six closely spaced sites. The Met Office weather radar network and Meteosat satellite imagery were used to provide context for the observations made by the instruments deployed during CSIP. This article presents an overview of the CSIP field campaign and examples from CSIP of the types of convective initiation phenomena that are typical in the United Kingdom. It shows the way in which certain kinds of observational data are able to reveal these phenomena and gives an explanation of how the analyses of data from the field campaign will be used in the development of an improved very high resolution NWP model for operational use.This work is funded by the National Environment Research Council following an initial award from the HEFCE Joint Infrastructure Fund

ADAM17-Mediated Processing of TNF-α Expressed by Antiviral Effector CD8+ T Cells Is Required for Severe T-Cell-Mediated Lung Injury

Influenza infection in humans evokes a potent CD8+ T-cell response, which is important for clearance of the virus but may also exacerbate pulmonary pathology. We have previously shown in mice that CD8+ T-cell expression of TNF-a is required for severe and lethal lung injury following recognition of an influenza antigen expressed by alveolar epithelial cells. Since TNF-a is first expressed as a transmembrane protein that is then proteolytically processed to release a soluble form, we sought to characterize the role of TNF-a processing in CD8+ T-cell-mediated injury. In this study we observed that inhibition of ADAM17-mediated processing of TNF-a by CD8+ T cells significantly attenuated the diffuse alveolar damage that occurs after T-cell transfer, resulting in enhanced survival. This was due in part to diminished chemokine expression, as TNF-aprocessing was required for lung epithelial cell expression of CXCL2 and the subsequent inflammatory infiltration. We confirmed the importance of CXCL2 expression in acute lung injury by transferring influenza-specific CD8+ T cells into transgenic mice lacking CXCR2. These mice exhibited reduced airway infiltration, attenuated lung injury, and enhanced survival. Theses studies describe a critical role for TNF-a processing by CD8+ T cells in the initiation and severity of acute lung injury, which may have important implications for limiting immunopathology during influenza infection and other human infectious or inflammatory diseases

Type I Interferon Signaling Regulates Ly6Chi Monocytes and Neutrophils during Acute Viral Pneumonia in Mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1−/−) mice develop significant defects in the infiltration of Ly6Chi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6Chi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6Cint monocytes of Ifnar1−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6Chi monocytes. By using BM chimeric mice (WT BM into Ifnar1−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6Chi monocytes. Of note, WT BM reconstituted Ifnar1−/− chimeric mice with increased numbers of Ly6Chi monocytes survived longer than influenza-infected Ifnar1−/− mice. In contrast, WT mice that received Ifnar1−/− BM cells with alternative Ly6Cint monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung

A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections

The Critical Role of Notch Ligand Delta-like 1 in the Pathogenesis of Influenza A Virus (H1N1) Infection

Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFNα-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-γ. In addition, we blocked Notch signaling by using γ-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-γ in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-γ levels from CD4+and CD8+T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection

Host Genetic Background Strongly Influences the Response to Influenza A Virus Infections

The genetic make-up of the host has a major influence on its response to combat pathogens. For influenza A virus, several single gene mutations have been described which contribute to survival, the immune response and clearance of the pathogen by the host organism. Here, we have studied the influence of the genetic background to influenza A H1N1 (PR8) and H7N7 (SC35M) viruses. The seven inbred laboratory strains of mice analyzed exhibited different weight loss kinetics and survival rates after infection with PR8. Two strains in particular, DBA/2J and A/J, showed very high susceptibility to viral infections compared to all other strains. The LD50 to the influenza virus PR8 in DBA/2J mice was more than 1000-fold lower than in C57BL/6J mice. High susceptibility in DBA/2J mice was also observed after infection with influenza strain SC35M. In addition, infected DBA/2J mice showed a higher viral load in their lungs, elevated expression of cytokines and chemokines, and a more severe and extended lung pathology compared to infected C57BL/6J mice. These findings indicate a major contribution of the genetic background of the host to influenza A virus infections. The overall response in highly susceptible DBA/2J mice resembled the pathology described for infections with the highly virulent influenza H1N1-1918 and newly emerged H5N1 viruses

Loss of anti-contractile effect of perivascular adipose tissue in offspring of obese rats

RATIONALE: Maternal obesity pre-programmes offspring to develop obesity and associated cardiovascular disease. Perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the vasculature, which is reduced in hypertension and obesity. OBJECTIVE: The objective of this study was to determine whether maternal obesity pre-programmes offspring to develop PVAT dysfunction in later life. METHODS: Female Sprague–Dawley rats were fed a diet containing 10% (control) or 45% fat (high fat diet, HFD) for 12 weeks prior to mating and during pregnancy and lactation. Male offspring were killed at 12 or 24 weeks of age and tension in PVAT-intact or -denuded mesenteric artery segments was measured isometrically. Concentration–response curves were constructed to U46619 and norepinephrine. RESULTS: Only 24-week-old HFD offspring were hypertensive (P<0.0001), although the anti-contractile effect of PVAT was lost in vessels from HFD offspring of each age. Inhibition of nitric oxide (NO) synthase with 100 μM l-NMMA attenuated the anti-contractile effect of PVAT and increased contractility of PVAT-denuded arteries (P<0.05, P<0.0001). The increase in contraction was smaller in PVAT-intact than PVAT-denuded vessels from 12-week-old HFD offspring, suggesting decreased PVAT-derived NO and release of a contractile factor (P<0.07). An additional, NO-independent effect of PVAT was evident only in norepinephrine-contracted vessels. Activation of AMP-activated kinase (with 10 μM A769662) was anti-contractile in PVAT-denuded (P<0.0001) and -intact (P<0.01) vessels and was due solely to NO in controls; the AMPK effect was similar in HFD offspring vessels (P<0.001 and P<0.01, respectively) but was partially NO-independent. CONCLUSIONS: The diminished anti-contractile effects of PVAT in offspring of HFD dams are primarily due to release of a PVAT-derived contractile factor and reduced NO bioavailability

A Role for the Chemokine RANTES in Regulating CD8 T Cell Responses during Chronic Viral Infection

RANTES (CCL5) is a chemokine expressed by many hematopoietic and non-hematopoietic cell types that plays an important role in homing and migration of effector and memory T cells during acute infections. The RANTES receptor, CCR5, is a major target of anti-HIV drugs based on blocking viral entry. However, defects in RANTES or RANTES receptors including CCR5 can compromise immunity to acute infections in animal models and lead to more severe disease in humans infected with west Nile virus (WNV). In contrast, the role of the RANTES pathway in regulating T cell responses and immunity during chronic infection remains unclear. In this study, we demonstrate a crucial role for RANTES in the control of systemic chronic LCMV infection. In RANTES−/− mice, virus-specific CD8 T cells had poor cytokine production. These RANTES−/− CD8 T cells also expressed higher amounts of inhibitory receptors consistent with more severe exhaustion. Moreover, the cytotoxic ability of CD8 T cells from RANTES−/− mice was reduced. Consequently, viral load was higher in the absence of RANTES. The dysfunction of T cells in the absence of RANTES was as severe as CD8 T cell responses generated in the absence of CD4 T cell help. Our results demonstrate an important role for RANTES in sustaining CD8 T cell responses during a systemic chronic viral infection

Bordetella pertussis Infection Exacerbates Influenza Virus Infection through Pertussis Toxin-Mediated Suppression of Innate Immunity

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers