The underdogs: How small immune cell populations shape lung macrophage development

Der erste Atemzug eines Neugeborenen stellt eine Herausforderung für das Lungengewebe dar: Die Lungenbläschen entfalten sich und sind dabei zum ersten Mal der äußeren Umgebung ausgesetzt. Die dadurch verursachten strukturellen, aber auch zellulären Änderungen, sind bisher wenig erforscht. In den zwei in der Thesis enthaltenen Publikationen geht es um das Zusammenspiel von Struktur- und Immunzellen während den verschiedenen Phasen der Lungenentwicklung. Wir konnten dabei zeigen, dass die Interaktion zwischen Epithelzellen und den angeborenen Typ-2-Lymphozyten (ILC2s), sowie zwischen Epithelzellen und Basophilen zur Produktion von Interleukinen und Zytokinen führt, die für die Entwicklung von Alveolarmakrophagen (AM) wichtig sind. In der ersten Studie konnten wir in dem Lungengewebe von neugeborenen Mäusen einen deutlichen Anstieg des Zyotkins Interleukin-33 (IL-33) beobachten. IL-33 ist bekannt dafür, dass es als Antwort auf mechanischen Stress von Strukturzellen gebildet wird. Kurz darauf folgt eine Aktivierung der sich im Gewebe teilenden ILC2 Population und dem parallel einhergehenden Auftreten von AMs. Das von ILC2 produzierte Interleukin-13 (IL-13) führt zur Polarisierung der AMs in eine entzündungshemmende Richtung. Dieser sogenannte M2-Phäntotyp ist später im voll entwickelten Gewebe wichtig um unnötige Entzündungsreaktionen zu vermeiden. Es führt jedoch auch dazu, dass AMs aus WT Mäusen eine verzögerte Immunreaktion im Vergleich zu AMs aus IL-13 oder ILC2-defizienten Mäusen aufweisen, sobald sie mit einem bakteriellen Erreger konfrontiert werden. Im zweiten Teil der Thesis geht es um Basophile, einer bisher wenig beachteten Population in der Lunge. Durch Einzelzell-Untersuchungen auf RNA Ebene konnten wir einen „Entwicklungsatlas der Lunge“ erstellen, der von sehr frühen Stadien im Embryo bis zum Erwachsenenalter reicht. Dabei haben wir mögliche Interaktionen zwischen Zellpopulationen verglichen und nicht nur die vorher beschriebenen Signalwege zwischen Epithelzellen und ILC2s bestätigt, sondern auch eine Lungen-spezifische Basophilen Population entdeckt, die ebenfalls IL-33 abhängig ist. In einem Antikörper basierten Versuch konnten wir eine signifikante Verminderung der Basophilen im Mausmodell erreichen, was zu einem unreifen AM Phänotyp führte. Wenn man ein genetisches Modell verwendet, findet man weniger AMs in einer bronchoalveolären Lavage von Basophil-defizienten Mäusen im Vergleich zu WT Mäusen. Zudem weisen die AMs reduzierte phagozytotische Eigenschaften auf. Die Studien, die in dieser Thesis präsentiert werden, ermöglichen erstmals einen genauen Blick auf die Lungenentwicklung kurz vor und kurz nach der Geburt zu werfen, einer Zeit, die vor allem in frühgeborenen Babys eine wichtige Rolle spielt. Die Hoffnung ist, dass durch ein besseres Verständnis der involvierten Signalwege und Immunzellpopulationen, ein Schritt in die Richtung verbesserter Therapieansätze gemacht wurde.With the first breath, the lung is suddenly exposed to the outer environment leading to substantial structural and cellular changes. It is not completely understood how immune and non-immune cells in the lung develop and how essential interactions between the two niches are for these processes. Here we show in two publications that microenvironmental factors derived from non-immune cells lead to the activation of two small immune cell populations of the lung, innate lymphoid cells type 2 (ILC2s) and basophils. Factors released by these cells shape the identity of the tissue-resident macrophages of the lung, alveolar macrophages (AM). In the first study we showed that the alarmin interleukin-33 (IL-33), an epithelial-derived cytokine, is up-regulated shortly after birth due to mechanical stress. This subsequently leads to the activation of tissue resident ILC2s. The resulting expansion of ILC2s around postnatal day 3 is accompanied by the arrival of AMs that obtain a rather anti-inflammatory phenotype, which is driven by ILC2-derived IL-13. This M2-like polarization state of AMs is required to secure tissue homeostasis by preventing excessive inflammation that otherwise would be induced by the constant exposure to airborne particles and pathogens. This comes at the expense of a delayed response in WT mice when challenged with a bacterial infection like pneumococcal pneumonia while IL-13 deficient or ILC2 deficient mice show an increased bacterial clearance due to a more pro-inflammatory phenotype of AMs. In the second part we studied lung development on a single-cell level from early fetal development until adulthood. In addition to the lung cell atlas we generated, we went on to investigate possible interaction ligand and receptor pairs. Next to the ILC2-AM axis described in the previous paper, we found a population of basophils that had a lung-specific expression pattern that was IL-33 dependent. Deletion of these lung-specific basophils in vivo resulted in a premature monocyte-like gene expression profile of AMs. In a genetic model of adult basophil-deficient mice we found a decreased number of alveolar macrophages in the bronchoalveolar lavage, which in addition had a reduced phagocytic capacity. Both studies provide new insights into lung development shortly before and after birth, a time that was shown to be critical in premature babies and infants. By broadening our understanding of the pathways involved in the early events of immune cell maturation, we hope to get a few steps closer to understand how lung immune and non-immune cells behave in health and disease.submitted by Anna-Dorothea GorkiAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018(VLID)354993

Lipocalin 2 modulates dendritic cell activity and shapes immunity to influenza in a microbiome dependent manner.

Lipocalin 2 (LCN2) is a secreted glycoprotein with roles in multiple biological processes. It contributes to host defense by interference with bacterial iron uptake and exerts immunomodulatory functions in various diseases. Here, we aimed to characterize the function of LCN2 in lung macrophages and dendritic cells (DCs) using Lcn2-/- mice. Transcriptome analysis revealed strong LCN2-related effects in CD103+ DCs during homeostasis, with differential regulation of antigen processing and presentation and antiviral immunity pathways. We next validated the relevance of LCN2 in a mouse model of influenza infection, wherein LCN2 protected from excessive weight loss and improved survival. LCN2-deficiency was associated with enlarged mediastinal lymph nodes and increased lung T cell numbers, indicating a dysregulated immune response to influenza infection. Depletion of CD8+ T cells equalized weight loss between WT and Lcn2-/- mice, proving that LCN2 protects from excessive disease morbidity by dampening CD8+ T cell responses. In vivo T cell chimerism and in vitro T cell proliferation assays indicated that improved antigen processing by CD103+ DCs, rather than T cell intrinsic effects of LCN2, contribute to the exacerbated T cell response. Considering the antibacterial potential of LCN2 and that commensal microbes can modulate antiviral immune responses, we speculated that LCN2 might cause the observed influenza phenotype via the microbiome. Comparing the lung and gut microbiome of WT and Lcn2-/- mice by 16S rRNA gene sequencing, we observed profound effects of LCN2 on gut microbial composition. Interestingly, antibiotic treatment or co-housing of WT and Lcn2-/- mice prior to influenza infection equalized lung CD8+ T cell counts, suggesting that the LCN2-related effects are mediated by the microbiome. In summary, our results highlight a novel regulatory function of LCN2 in the modulation of antiviral immunity

First-Breath-Induced Type 2 Pathways Shape the Lung Immune Environment

From birth onward, the lungs are exposed to the external environment and therefore harbor a complex immunological milieu to protect this organ from damage and infection. We investigated the homeostatic role of the epithelium-derived alarmin interleukin-33 (IL-33) in newborn mice and discovered the immediate upregulation of IL-33 from the first day of life, closely followed by a wave of IL-13producing type 2 innate lymphoid cells (ILC2s), which coincided with the appearance of alveolar macrophages (AMs) and their early polarization to an IL-13-dependent anti-inflammatory M2 phenotype. ILC2s contributed to lung quiescence in homeostasis by polarizing tissue resident AMs and induced an M2 phenotype in transplanted macrophage progenitors. ILC2s continued to maintain the M2 AM phenotype during adult life at the cost of a delayed response to Streptococcus pneumoniae infection in mice. These data highlight the homeostatic role of ILC2s in setting the activation threshold in the lung and underline their implications in anti-bacterial defenses.(VLID)456113

Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions

International audienceHemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders