Die Wechselbeziehung zwischen autoreaktiven T Zellen und alveolaren Typ II Epithelzellen in Entzündungs- und Toleranzreaktionen

It is well established, that the lung is an organ with tolerogenic properties to avoid an immunological collapse due to infection and inflammation. However, little is known about the mechanisms underlying peripheral tolerance induction in the lung. To dissect the immunological and molecular mechanisms underlying autoimmune-mediated lung disease, CD4+ T cell reactivity to a lung-specific antigen was studied by transgenic expression of hemagglutinin (HA) in alveolar type II epithelial cells. Concomitant expression of HA and a MHC class II-restricted T cell receptor specific for HA in double transgenic mice resulted in a severe immune-mediated interstitial lung disease. Despite aggressive and rapid progression of inflammation in lung of young mice, the pulmonary inflammation reached a plateau state in elder mice, suggesting the induction of peripheral tolerance mechanisms. Extensive immunological characterization of self-reactive CD4+ T cells isolated from the inflamed lung and global gene expression profiling suggested the induction of functional regulatory T cells in the site of inflammation. Establishing a new protocol to isolate highly pure and vital alveolar type II epithelial cells ex vivo, enabled us to characterize the role of alveolar type II epithelial cells in disease progression and immune regulation. We show that alveolar type II epithelial cells are capable to induce antigen-specific T cell proliferation. Moreover, alveolar type II epithelial cells from diseased mice have a reduced capacity to stimulate naïve and pre-activated self-reactive CD4+ T cells and exhibit broad changes in their gene expression profile in comparison to alveolar type II epithelial cells derived from healthy control mice. Furthermore, alveolar type II epithelial cells from diseased mice secrete soluble factors that down modulate T cell proliferation. Data obtained by extensive functional and molecular characterization suggest an important role of alveolar type II epithelial cells for the induction of T cell tolerance and immune regulation in the lung.Immer mehr Anzeichen deuten darauf hin, dass die Lunge ein Organ mit tolerogenen Eigenschaften ist, um den immunologischen Kollaps, der durch Infektionen und Entzündungen verursacht werden könnte, zu vermeiden. Über die Mechanismen, die zu einer peripheren Toleranzinduktion in der Lunge führen ist allerdings wenig bekannt. Um die immunologischen und molekularen Mechanismen einer autoimmunvermittelten Lungenerkrankung zu untersuchen, wurde die CD4+ T-Zellreaktivität gegenüber des Hämagglutinins (HA) untersucht, welches in den alveolaren Typ II Epithelzellen transgen exprimiert wird. Die gleichzeitige Expression von HA und einem MHC Klasse-II-restringierten T-Zellrezeptor spezifisch für das HA in doppeltransgenen Mäusen führt zu einer schwerwiegenden T-Zellvermittelten interstitiellen Lungenerkrankung. Trotz der schnell fortschreitenden und aggressiven Entzündung der Lungen in jungen Mäusen, stagniert diese in älteren Tieren und lässt auf die Induktion von peripheren Toleranzmechanismen schließen. Intensive immunologische Untersuchungen der selbstreaktiven CD4+ T-Zellen aus den erkrankten Lungen und das Erstellen eines Genexpressionsprofils dieser Zellen ließen den Schluss zu, dass während des Entzündungsprozesses funktionelle, regulatorische T-Zellen induziert werden. Die Etablierung eines neuen Protokolls zur Isolierung reiner und vitaler alveolar Typ II Epithelzellen ex vivo ermöglicht uns die Rolle der Typ II Pneumozyten während der Entzündungsprozesse und Immunregulation zu charakterisieren. Wir zeigen hier, dass alveolare Typ II Epithelzellen aus gesunden Lungen die Fähigkeit haben eine antigenspezifische T-Zellantwort auszulösen, aber im Gegenzug dazu die Typ II Pneumozyten aus den erkrankten Lungen eine verminderte Stimulationsfähigkeit aufweisen und Faktoren sekretieren, die die T-Zellproliferation hemmen können. Weitere vergleichende, molekulare und funktionelle Analysen der alveolaren Typ II Epithelzellen aus gesunden und erkrankten Lungen deuten auf eine wichtige Rolle der alveloar Typ II Epithelzellen bei der Induktion von T-Zelltoleranz und der Immunregulation der Lunge hin

First Genomic Analysis of Dendritic Cells from Lung and Draining Lymph Nodes in Murine Asthma

Asthma is the consequence of allergic inflammation in the lung compartments and lung-draining lymph nodes. Dendritic cells initiate and promote T cell response and drive it to immunity or allergy. However, their modes of action during asthma are poorly understood. In this study, an allergic inflammation with ovalbumin was induced in 38 mice versus 42 control animals. After ovalbumin aerosol challenge, conventional dendritic cells (CD11c/MHCII/CD8) were isolated from the lungs and the draining lymph nodes by means of magnetic cell sorting followed by fluorescence-activated cell sorting. A comparative transcriptional analysis was performed using gene arrays. In general, many transcripts are up- and downregulated in the CD8− dendritic cells of the allergic inflamed lung tissue, whereas few genes are regulated in CD8+ dendritic cells. The dendritic cells of the lymph nodes also showed minor transcriptional changes. The data support the relevance of the CD8− conventional dendritic cells but do not exclude distinct functions of the small population of CD8+ dendritic cells, such as cross presentation of external antigen. So far, this is the first approach performing gene arrays in dendritic cells obtained from lung tissue and lung-draining lymph nodes of asthmatic-like mice

CD8+ T Cells Responding to Alveolar Self-Antigen Lack CD25 Expression and Fail to Precipitate Autoimmunity

Although the contribution of CD8+ T cells to the pathogenesis of noncommunicable lung diseases has become increasingly appreciated, our knowledge about the mechanisms controlling self-reactive CD8+ T cells in the respiratory tract remains largely elusive. The outcome of the encounter between pulmonary self-antigen and naive CD8+ T cells, in the presence or absence of inflammation, was traced after adoptive transfer of fluorescence- labeled CD8+ T cells specific for the neo–self-antigen influenza A hemagglutinin into transgenic mice expressing hemagglutinin specifically in alveolar type II epithelial cells in order: to study the outcome of alveolar antigen encounter in the steady state and under inflammatory conditions; to define the phenotype and fate of CD8+ T cells primed in the respiratory tract; and, finally, to correlate these findings with the onset of autoimmunity in the lung. We found that CD8+ T cells remain ignorant in the steady state, whereas transient proliferation of self-reactive CD8+ T cells is induced by forced maturation or licensing of dendritic cells, increases in the antigenic threshold, and targeted release of alveolar self-antigen by epithelial injury. However, these cells fail to acquire effector functions, lack the expression of the high-affinity IL-2 receptor CD25, and do not precipitate autoimmunity in the lung. We conclude that inadvertent activation of CD8+ T cells in the lung is prevented in the absence of “danger signals,” whereas tissue damage after infection or noninfectious inflammation creates an environment that allows the priming of previously ignorant T cells. Failure in effector cell differentiation after abortive priming, however, precludes the establishment of self-perpetuating autoimmunity in the lung

Evaluation of PRRSv specific, maternally derived and induced immune response in Ingelvac PRRSFLEX EU vaccinated piglets in the presence of maternally transferred immunity.

In this study, we analyzed PRRS virus (PRRSv) specific lymphocyte function in piglets vaccinated with Ingelvac PRRSFLEX EU® at two and three weeks of age in the presence of homologous maternal immunity. Complete analysis of maternal immunity to PRRSv was evaluated postpartum, as well as passive transfer of antibodies and T cells to the piglet through colostrum intake and before and after challenge with a heterologous PRRSv at ten weeks of age. Maternal-derived antibodies were detected in piglets but declined quickly after weaning. However, vaccinated animals restored PRRSv-specific antibody levels by anamnestic response to vaccination. Cell analysis in colostrum and milk revealed presence of PRRSv-specific immune cells at suckling with higher concentrations found in colostrum than in milk. In addition, colostrum and milk contained PRRSv-specific IgA and IgG that may contribute to protection of newborn piglets. Despite the presence of PRRSv-specific Peripheral Blood Mononuclear cells (PBMCs) in colostrum and milk, no PRRSv-specific cells could be detected from blood of the piglets at one or two weeks of life. Nevertheless, cellular immunity was detectable in pre-challenged piglets up to 7 weeks after vaccination while the non-vaccinated control group showed no interferon (IFN) γ response to PRRSv stimulation. After challenge, all piglets developed a PRRSv-specific IFNγ-response, which was more robust at significantly higher levels in vaccinated animals compared to the primary response to PRRSv in non-vaccinated animals. Cytokine analysis in the lung lumen showed a reduction of pro-inflammatory responses to PRRSv challenge in vaccinated animals, especially reduced interferon (IFN) α levels. In conclusion, vaccination of maternally positive piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX EU induced a humoral and cellular immune response to PRRSv and provided protection against virulent, heterologous PRRSv challenge

Alveolar Type II Epithelial Cells Contribute to the Anti-Influenza A Virus Response in the Lung by Integrating Pathogen- and Microenvironment-Derived Signals

ABSTRACT Influenza A virus (IAV) periodically causes substantial morbidity and mortality in the human population. In the lower lung, the primary targets for IAV replication are type II alveolar epithelial cells (AECII), which are increasingly recognized for their immunological potential. So far, little is known about their reaction to IAV and their contribution to respiratory antiviral immunity in vivo . Therefore, we characterized the AECII response during early IAV infection by analyzing transcriptional regulation in cells sorted from the lungs of infected mice. We detected rapid and extensive regulation of gene expression in AECII following in vivo IAV infection. The comparison to transcriptional regulation in lung tissue revealed a strong contribution of AECII to the respiratory response. IAV infection triggered the expression of a plethora of antiviral factors and immune mediators in AECII with a high prevalence for interferon-stimulated genes. Functional pathway analyses revealed high activity in pathogen recognition, immune cell recruitment, and antigen presentation. Ultimately, our analyses of transcriptional regulation in AECII and lung tissue as well as interferon I/III levels and cell recruitment indicated AECII to integrate signals provided by direct pathogen recognition and surrounding cells. Ex vivo analysis of AECII proved a powerful tool to increase our understanding of their role in respiratory immune responses, and our results clearly show that AECII need to be considered a part of the surveillance and effector system of the lower respiratory tract. IMPORTANCE In order to confront the health hazard posed by IAV, we need to complete our understanding of its pathogenesis. AECII are primary targets for IAV replication in the lung, and while we are beginning to understand their importance for respiratory immunity, the in vivo AECII response during IAV infection has not been analyzed. In contrast to studies addressing the response of AECII infected with IAV ex vivo , we have performed detailed gene transcriptional profiling of AECII isolated from the lungs of infected mice. Thereby, we have identified an exceptionally rapid and versatile response to IAV infection that is shaped by pathogen-derived as well as microenvironment-derived signals and aims at the induction of antiviral measures and the recruitment and activation of immune cells. In conclusion, our study presents AECII as active players in antiviral defense in vivo that need to be considered part of the sentinel and effector immune system of the lung

Increased susceptibility for superinfection with Streptococcus pneumoniae during influenza virus infection is not caused by TLR7-mediated lymphopenia.

Influenza A virus (IAV) causes respiratory tract infections leading to recurring epidemics with high rates of morbidity and mortality. In the past century IAV induced several world-wide pandemics, the most aggressive occurring in 1918 with a death toll of 20-50 million cases. However, infection with IAV alone is rarely fatal. Instead, death associated with IAV is usually mediated by superinfection with bacteria, mainly Streptococcus pneumoniae. The reasons for this increased susceptibility to bacterial superinfection have not been fully elucidated. We previously demonstrated that triggering of TLR7 causes immune incompetence in mice by induction of lymphopenia. IAV is recognized by TLR7 and infections can lead to lymphopenia. Since lymphocytes are critical to protect from S. pneumoniae it has long been speculated that IAV-induced lymphopenia might mediate increased susceptibility to superinfection. Here we show that sub-lethal pre-infections of mice with IAV-PR8/A/34 strongly increased their mortality in non-lethal SP infections, surprisingly despite the absence of detectable lymphopenia. In contrast to SP-infection alone co-infected animals were unable to control the exponential growth of SP. However, lymphopenia forced by TLR7-triggering or antibody-mediated neutropenia did not increase SP-susceptibility or compromise the ability to control SP growth. Thus, the immune-incompetence caused by transient lympho- or leukopenia is not sufficient to inhibit potent antibacterial responses of the host and mechanisms distinct from leukodepletion must account for increased bacterial superinfection during viral defence

Attenuation of immune-mediated influenza pneumonia by targeting the inducible co-stimulator (ICOS) molecule on T cells.

Inducible Co-stimulator (ICOS) plays a critical role in mediating T cell differentiation and function and is considered a key player in balancing T effector and T regulatory (Treg) cell responses. Here we show that activation of the ICOS signalling pathway during acute influenza A virus (IAV) infection by application of an agonistic ICOS antibody reduced the frequency of CD8+ T cells in the respiratory tract of IAV infected animals and delayed pathogen elimination. In line with this, immune-mediated influenza pneumonia was significantly ameliorated in mice that received ICOS agonist as indicated by significantly reduced alveolar infiltrations and bronchointerstitial pneumonia, while at the same time virus-related pathology remained unaffected. Importantly, ICOS agonist treatment resulted in expansion of CD4+Foxp3+ Tregs in IAV infected mice, which was associated with elevated levels of the immunosuppressive cytokine IL-10 in the alveolar space. Together, our findings suggest a prominent role of ICOS signaling during acute IAV infection by increasing the Treg/CD8+ T cell ratio with beneficial outcome on immune-mediated pneumonia and underline the suitability of ICOS as potential therapeutic target for immune intervention in those infectious conditions characterized by strong immunopathology rather than virus-mediated cytopathic effects